Toxoplasma gondii forms latent cysts in the central nervous system (CNS) and persists for the lifetime of the host. This cyst is cloaked with a glycosylated structure called the cyst wall. Previously, we demonstrated that a mucin-like glycoprotein, CST1, localizes to the cyst wall and confers structural rigidity on brain cysts in a mucin-like domain-dependent manner. The mucin-like domain of CST1 is composed of 20 units of threonine-rich tandem repeats that are O-GalNAc glycosylated. A family of enzymes termed polypeptide N-acetylgalactosaminyltransferases  initiates O-GalNAc glycosylation. To identify which isoforms of ppGalNAc-Ts are responsible for the glycosylation of the CST1 mucin-like domain and to evaluate the function of each ppGalNAc-T in the overall glycosylation of the cyst wall, all five ppGalNAc-T isoforms were deleted individually from the T. gondii genome. The ppGalNAc-T2 and -T3 deletion mutants produced various glycosylation defects on the cyst wall, implying that many cyst wall glycoproteins are glycosylated by T2 and T3. Both T2 and T3 glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the cyst wall. We established that T2 is required for the initial glycosylation of the mucin-like domain and that T3 is responsible for the sequential glycosylation on neighboring acceptor sites, demonstrating hierarchical glycosylation by two distinct initiating and filling-in ppGalNAc-Ts in an intact organism.The protozoan intracellular parasite  Toxoplasma gondii is an obligate intracellular parasite that infects a third of the world’s population. It can cause severe congenital disease and devastating encephalitis in immunocompromised individuals. We identified two glycosyltransferases, ppGalNAc-T2 and -T3, which are responsible for glycosylating cyst wall proteins in a hierarchical fashion. This glycosylation confers structural rigidity on the brain cyst. Our studies provide new insights into the mechanisms of O-GalNAc glycosylation in T. gondii. Toxoplasma gondii is an intracellular protozoan parasite that infects up to a third of the world’s population  that are found in cat feces and which can contaminate food or water. The persistence and reactivation of bradyzoites in tissue cysts are a critical factor in the progression of disease in patients with congenital infection as well as in patients who are immunocompromised (–pulation . The aseue cysts . Developpromised . Diseasepromised –5. Due tDolichos biflorus lectin (DBA), which binds to GalNAcα1-3GalNAc, and succinylated wheat germ lectin (s-WGA), which binds to terminal GlcNAc (TGME49_264660) and T. gondii proteophosphoglycan 1  have both been demonstrated to be glycosylated proteins . These enzymes  transfer GalNAc from UDP-GalNAc to the hydroxyl group of serine or threonine of polypeptide chains to form GalNAcα1-O-serine/threonine. In the T. gondii genome, there are five putative isoforms of ppGalNAc-T . All of the T. gondii ppGalNAc-Ts have conserved glycosyltransferase domains that contain a GT-A motif and a DxH Mn2+ ion binding motif . Of these genes, ppGalNAc-T1, -T2, and -T3 are constitutively expressed; T4 expression is present only in the cat enterocyte stage (merozoite) exclusively; and T5 expression is highly upregulated in the oocyst stage and also present in the cat stage. All expression values were taken from the publicly available RNA-seq data in ToxoDB deposited by the previous studies . As phenotypes were seen for both the ppGalNAc-T2 and -T3 gene deletions (see the results below), complementation of the Δt3 mutant (Δt3::t3) was performed at the uracil phosphoribosyltransferase (UPRT) locus using the 1.5-kb endogenous putative promoter to maintain the native expression pattern of T3. Complementation of the Δt2 mutant (Δt2::t2) was performed at the endogenous ppGalNAc-T2 locus (using a pyrimethamine-resistant dihydrofolate reductase [DHFR] selectable marker), as complementation of the Δt2 mutant at the UPRT locus was not successful.Previous studies have demonstrated that e system , 25, 26.i strain  utilizini strain . All of 10.1128/mBio.02048-16.2FIG S2 T. gondii genomic DNA separated by agarose gel electrophoresis demonstrating the successful generation of the various ppGalNAc-T null mutants. The upper left gel illustrates the integration of the Δt1 fragment into the T1 locus. The upper right is the detection of the T1 gene, indicating that the T1 gene is deleted. The middle left gel illustrates the integration of the Δt2 fragment into the T2 locus. T2 complementation was done at the T2 locus, resulting in the lack of the Δt2 fragment in the Δt2::t2 mutant. The middle center gel shows detection of the T2 locus. This primer set is specific to the T2 genomic DNA of T2 and will not amplify the T2 cDNA. The middle right gel demonstrates detection of the T2 cDNA copy, indicating complementation of the Δt2 strain. The lower left gel illustrates detection of the Δt3 fragment at the T3 locus. The lower center gel demonstrates detection of T3. The lower right gel demonstrates detection of the T3 cDNA copy in the complemented strain. Download FIG S2, TIF file, 47.2 MB.Generation of ppGalNAc-T deletion mutants. These are the diagnostic PCRs of Copyright © 2017 Tomita et al.2017Tomita et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the O-GalNAc glycans require a ppGalNAc-T for their initial glycosylation step. Therefore, the deletion of any of the identified T. gondii ppGalNAc-T genes could potentially disrupt the parasite glycoproteome. However, as described in mammalian studies, ppGalNAc-Ts have significant overlaps of substrate specificity among the multiple isoforms that have been identified, and this has limited the identification of the key ppGalNAc-Ts involved in this initial glycosylation in many eukaryotic systems . Download TABLE S1, DOCX file, 0.01 MB.List of lectins used for lectin survey. This is the list of lectins used for the lectin overlay and immunocytochemistry assays. The glycan specificities described in this file are from glycoarray data at the Consortium of Functional Glycomics gateway database , VVA (Vicia villosa lectin), HPA (Helix pomatia lectin), DBA (Dolichos biflorus lectin), concanavalin A (ConA) , s-WGA (succinylated wheat germ lectin), SNA-I (Sambucus nigra lectin I), and GSL-1 (Griffonia simplicifolia lectin I). Jacalin labeling identified five specific glycoprotein bands at 37, 49, 95, 150, and >180 kDa that are present in the wild type but absent in the ppGalNAc-Δt2 and -Δt3 parasites (t2 and Δt3 (Δt2 are still present in Δt3 (blue arrows). Complementation of T2 restored all of these lectin-binding bands, and complementation of T3 restored all of these bands except the 37-kDa band. β-elimination confirmed that the bands recognized by these lectins were most likely O-glycosylated proteins  membrane, and probed with the following lectins: jacalin  and core 3  polypeptides. Other lectins that bind N-linked glycans, such as ConA, did not display any visible differential glycoprotein band patterns on examination of lectin blots of wild type and transferase deletion mutants (data not shown), consistent with a role for ppGalNAc-Ts in the posttranslational modification of O-GalNAc glycosylation but not N-glycosylation.Jacalin has binding specificity to α-linked GalNAc  with the CSer/Thr) . VVA binSer/Thr) , 37. TheUsing this lectin overlay approach, we could not appreciate a difference in the binding patterns of parasites purified from cultures grown under either tachyzoite or bradyzoite conditions (see 10.1128/mBio.02048-16.3FIG S3 T. gondii strains cultivated under bradyzoite (pH 8.1 with 0.1% CO2) or tachyzoite (pH 7.1 with 5% CO2) culture conditions were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. The membrane was then incubated with either biotin-conjugated jacalin (A) or VVA (B). This blot demonstrates that multiple T. gondii glycoproteins are similarly expressed between the bradyzoite and tachyzoite stages of this parasite. Download FIG S3, TIF file, 10.4 MB.Comparison of jacalin and VVA binding proteins between tachyzoite stage and bradyzoite stage. Purified lysates from Copyright © 2017 Tomita et al.2017Tomita et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro cysts with the above mentioned lectins. T. gondii probed with jacalin, VVA, and HPA lectins. Jacalin cyst wall staining is absent in Δt2 and Δt3 parasites. In contrast, VVA cyst wall staining is absent only in Δt2 parasites. While HPA cyst wall staining is absent in Δt2 parasites, the punctate cytosolic HPA staining remained intact. These defects in lectin staining of the cyst wall in the ppGalNAc-T deletion mutants indicate that there are various cyst wall-associated glycoproteins that require ppGalNAc-T2 and -T3 activity for posttranslational O-GalNAc glycosylation.Since the parasite purification step removed the majority of parasitophorous vacuole-localized proteins, we assessed the glycosylation status of the parasitophorous vacuole of the ppGalNAc-T mutants by staining t2 lectin overlay and the major bands absent in ppGalNAc-Δt3 were absent in ppGalNAc-Δt2 . Δt2, and Δt2::t2 parasites. Surprisingly, 51% of proteins fell into an intersection between all three genotypes. This could be due to the relatively low affinity of lectin-binding . Therefore, to identify highly probable ppGalNAc-T2 substrate candidates, the semiquantitative criteria that proteins have more than 5 peptides mapped in wild type and more than a 2.5-fold-increased detection in wild type over the Δt2 mutant were used to select potential ppGalNAc-T2 substrates. In addition, nuclear and nonsecreted proteins (which do not transverse the endoplasmic reticulum [ER]/Golgi complex) were removed from these lists. Δt2 parasite lysate. Proteomic analysis of the corresponding gel band at 37 kDa showed that it contained dense granular proteins GRA3 and GRA7. The band at 49 kDa contained a lysine decarboxylase family protein (TGME49_236570). The 95-kDa band contained the perforin-like protein 1 (PLP1). The 150-kDa band contained a hypothetical protein that is rich in serine (TGME49_230940). The high-molecular-weight band contained CST1, vacuolar protein sorting 13 (VPS13)-like protein (TGME49_291180), Sec7 domain-containing protein (TGME49_232190), and a large hypothetical protein with numerous serine-rich tandem repeats (TGME49_287480).To identify some of the potential glycoproteins which were likely to have been modified by ppGalNAc-T2, we employed the two lectins that had differential binding patterns in lectin overlay, namely, jacalin and VVA, for glycoprotein affinity purification. Comparison of lectin overlay and immunofluorescence assay (IFA) suggested that ppGalNAc-T2 activity was required for ppGalNAc-T3 activity: more bands were absent in ppGalNAc-ΔlNAc-Δt2  and B. T jacalin ), result10.1128/mBio.02048-16.4FIG S4 O-GalNAc sites in ppGalNAc-T2 substrate candidates compared with those found in the entire T. gondii proteome. Venn diagrams of binary overlaps for proteins identified with each genotype that were affinity purified with either jacalin (A) or VVA (B) or both jacalin and VVA (C). Panel D is a kernel density graph of the distribution of the number of predicted O-GalNAc glycosylation sites per protein for ppGalNAc-T2-specific substrate candidates identified by capture with jacalin (green) or VVA (blue) or found in the entire T. gondii proteome (red). The distribution of the number of the glycosylation sites was statistically significant between jacalin-captured candidates and the entire proteome . Download FIG S4, TIF file, 5.5 MB.Predicted Copyright © 2017 Tomita et al.2017Tomita et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the T. gondii proteins . Δt2, and Δt2::t2 parasites. Similarly to jacalin, VVA also had large overlap between all three genotypes. Therefore, we used the same stringent semiquantitative criteria to select T2 substrate candidates. The same approach was used with the lectin VVA, and mass spectrometric analysis identified a total of 355 O-GalNAc posttranslational modification by ppGalNAc-T2, all identified proteins as well as the predicted T. gondii proteome  were used for an in silico prediction of O-GalNAc modifications using the support vector machine prediction algorithm NetOGlyc 4.0 , the VVA-affinity-purified T2 substrate candidate proteins (n = 13), or the whole T. gondii proteome . A substantial fraction of T2-specific proteins have a high number (>150) of predicted glycosylation sites per protein compared with the frequency in the entire T. gondii proteome. The predicted average O-GalNAc glycosylation sites are 93.1 in the jacalin-affinity-purified ppGalNAc-T2-specific proteins and 51.7 in the VVA-affinity-purified ppGalNAc-T2-specific proteins. The number of potential O-GalNAc glycosylation sites is higher in both affinity-purified proteins than the calculated mean of 38.0 sites per protein for all T. gondii proteins. The distributions of glycosylation sites per protein (P < 0.05) but not between the whole proteome and the VVA-captured T2 candidates . In particular, CST1, VPS13-like, and Sec7 domain-containing proteins are present in both the jacalin and VVA eluates in a ppGalNAc-T2-dependent manner and have a large number of predicted serine or threonine potential O-GalNAc modification sites . The presence of these proteins with large numbers of predicted O-GalNAc glycosylation sites suggests that the assay and analysis worked as expected. These lectin-based surveys in the context of ppGalNAc-Ts revealed a large variety of T. gondii proteins that are likely to be glycosylated by ppGalNAc-T2 and -T3 under both bradyzoite and tachyzoite conditions, suggesting that this glycosylation pathway is important in the function of many proteins in this ubiquitous eukaryotic pathogen.To confirm that the identified proteins are reasonable candidates for an Glyc 4.0 . Figure  protein  were staO-GalNAc glycans  did not change the presence of these high-molecular-mass proteins. The ppGalNAc-Δt2 parasites display the ~50-kDa major band (green), but the higher-molecular-mass glycoproteins (yellow and red) are absent. As expected, complementation of the ppGalNAc-T2 strain (Δt2::t2) restored the high-molecular-mass proteins, consistent with the high-molecular-mass proteins having multiple complex O-GalNAc glycan modifications that are dependent on the presence of functional ppGalNAc-T2. Interestingly, the ppGalNAc-Δt3 parasites produced the intermediate-molecular-mass bands  but not the very-high-molecular-mass  bands. This lack of very-high-molecular-mass bands is rescued by the complementation of ppGalNAc-T3 (Δt3::t3). This suggests that the very-high-molecular-mass bands  are due to O-GalNAc modifications by ppGalNAc-T3 but that these modifications require the initial ppGalNAc-T2 activity. The ppGalNAc-Δt3 mutant displays accumulation of the 60- to 80-kDa bands (yellow), which are likely to be products of the ppGalNAc-T2 reaction that cannot be further modified by ppGalNAc-T3. Collectively, these data strongly support the hypothesis that ppGalNAc-T2 and -T3 transfer GalNAc to their substrates in a hierarchical fashion where ppGalNAc-T2 initiates O-GalNAc glycosylation on a threonine residue of an unglycosylated mucin-like domain. Subsequently, T3 recognizes this initial O-GalNAc and glycosylates the adjacent acceptor threonine sites, filling in the rest of the glycosylation sites on the threonine-rich tandem repeat  tag driven by a strong constitutive GRA1 promoter was created . This mum repeat .T. gondii infection mouse model . Cysts from complemented strains that had a cDNA copy of ppGalNAc-Ts (Δt2::t2 and Δt3::t3) resulted in brain cysts with rigidity similar to that of the wild type. This suggests that the rigidity of the brain cyst is dependent on the presence of both ppGalNAc-T2 and -T3. Although both ppGalNAc-T2 and -T3 glycosylate the mucin-like domain of CST1 and other parasite proteins, the cyst wall rigidity phenotype is most likely due to the reduced glycosylation of the CST1 mucin-like domain, as our previous data revealed that deletion of the mucin-like domain from CST1 was sufficient in itself to cause the fragile cyst phenotype . Itis model , 43.10.1128/mBio.02048-16.5FIG S5 O-GalNAc glycosylation of the CST1 mucin-like domain. Represented is a single unit of the threonine-tandem repeat of CST1 mucin-like domain. One unit has 5 to 11 threonine residues, and the mucin domain has 20 repeats of the unit. The process proceeds in four stages. (1) Initiation: the furthest C-terminal end threonine gets O-GalNAc glycosylated by T. gondii ppGalNAc-T2. (2) Filling-in: following initiation, ppGalNAc-T3 binds to the O-GalNAc on the peptide and transfers another O-GalNAc to the threonine at the −1 position. (3) Sequential glycosylation: ppGalNAc-T3 then binds to the newly added O-GalNAc and sequentially glycosylates adjacent threonine residues in the N-terminal direction of the peptide until the end of the repeat is reached. (4) Extension: once the O-GalNAc is in position, other glycosyltransferases add sugars to form core 5 structure (yellow) and fully extended glycan modifications. Download FIG S5, TIF file, 1.1 MB.A model of hierarchical Copyright © 2017 Tomita et al.2017Tomita et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the O-GalNAc glycosylation of the CST1 mucin-like domain by sequential ppGalNAc-T glycosylation occurs in an intact organism and that this modification is biologically significant. The highly redundant nature of ppGalNAc-Ts , an as-yet-unknown glycosyltransferase adds additional GalNAc to the Tn antigen to form the core 5 structure  that is detected by the DBA lectin in Database ). Very lO-GalNAc glycosylation on an adjacent threonine at the +1 position relative to the acceptor threonine residue of peptide substrates . Based on the size of the bands observed in our experiments, there are likely additional extensions of that glycan by two to seven more sugars to form the higher-molecular-mass bands that are 16 to 28 kDa larger than the unglycosylated CST1 mucin-like domain. It is probable that ppGalNAc-T3 transfers GalNAc to threonine on the −1 position relative to the initial O-linked GalNAc modified by ppGalNAc-T2  Δku80 Δhxgprt T. gondii strain as the parental strain  were developed using the pUC19 vector and In-Fusion system (TaKaRa Clontech) in which the following vector fragments were assembled into the plasmid: a 1-kb 5′ upstream homologous arm region fragment, a 2-kb hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) cassette fragment, and a 1-kb 3′ downstream homologous arm region fragment with a 15-nucleotide overlap. The fragments were amplified using Phusion DNA polymerase (Thermo Fisher) according to the manufacturer’s protocol. The homologous arms were designed to completely remove each targeted ppGalNAc-T gene from its respective start codon to its stop codon. Transfections were performed in Cytomix, and parasites were selected using 25 µg/ml mycophenolic acid and 50 µg/ml xanthine as previously described  in Dulbecco modified Eagle medium (DMEM) (Gibco Life Technologies) with 10% fetal bovine serum  and penicillin-streptomycin (Gibco Life Technologies) at 5% COl strain   containing cOmplete EDTA-free protease inhibitor (Thermo Fisher), and the lysates were cleared by centrifugation. The supernatants were boiled with sample buffer and separated by 4 to 15% SDS-PAGE (15 µg/lane), and the separated proteins were transferred to either a nitrocellulose or a PVDF membrane. The membrane was incubated with each lectin (at 10 µg/ml) for 1 h at room temperature. After washing three times with PBS, the membranes were incubated with streptavidin-horseradish peroxidase (HRP) at 1:20,000, and subsequently, the signal was detected using SuperSignal West Femto maximum-sensitivity substrate (Thermo Fisher). Biotin-conjugated jacalin, ConA, DBA, s-WGA, SNA-I, GS-I, VVA, and HPA (EY Laboratories) were evaluated for their ability to bind to the SDS-PAGE-resolved protein bands. β-elimination of duplicate membranes was performed, to confirm O-glycosylation, using the previously described methods  for 3 days in 5% CO methods : e.g., tT. gondii in pH 8.1 DMEM containing 1% FBS and 50 mM HEPES at atmospheric CO2 (0.1%) for 3 days. The cells were fixed with 4% paraformaldehyde in PBS for 30 min and then permeabilized with 0.2% Triton X-100 in 3% BSA in PBS for 20 min. The cells were washed with PBS three times and blocked with 3% BSA in PBS at 4°C overnight. Cover glasses with cultured, fixed, permeabilized cells were then incubated with different biotin-conjugated lectins  at 10 µg/ml in PBS for 90 min at 37°C and washed with PBS three times. Afterward, a rabbit antibiotin antibody at a 1:500 dilution was incubated with the cover glasses for 90 min at 37°C and they were washed with PBS three times. Anti-rabbit Alexa Fluor 488 at a 1:1,000 dilution was incubated with cover glasses for 90 min at 37°C, and they were washed with PBS three times. Following this washing procedure, each cover glass was mounted on a slide using ProLong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher). Each cover glass was examined using a confocal fluorescence microscope .HFF cells cultured on cover glasses were infected with T. gondii (2 × 1010 parasites) was lysed in 1 ml PBS  and then incubated for 2 h on ice. The samples were centrifuged at 13,600 × g at 4°C for 16 min, and the collected supernatants were applied to either a jacalin- or a VVA-agarose bead affinity column (EY Laboratories). The columns were washed with 300 μl of phosphate buffer three times and incubated for 15 min with 200 μl of elution buffer, and the eluate was collected. The eluates were separated by SDS-PAGE on a 4 to 20% gradient gel and visualized with Coomassie blue staining. Each lane was cut into 24 bands, and the bands were processed for proteomic analysis as described previously (m/z survey scan will undergo MS/MS (or MS2) and the top two product ions from the MS2 scan will undergo MS3 fragmentation. MSn was performed using an isolation width of 2 m/z, normalized collision energy of 35%, and activation time of 30 ms. Protein identification was performed with the Mascot protein search engine (Matrix Science) using these parameters: fragment ion mass tolerance of 0.80 Da and parent ion tolerance of 2.0 Da. Carbamidomethyl of cysteine was specified in Mascot as a fixed modification. Glu→pyro-Glu of the N terminus, Gln→pyro-Glu of the N terminus, deamidated of asparagine and glutamine, and oxidation of methionine were specified in Mascot as variable modifications. Scaffold (Proteome Software) was used for protein peptide validation at 99% and 95% minimum protein and peptide, respectively, with a minimum number of 2 peptides. A decoy database was employed to obtain false discovery rates which were 1% and 0.06% at the protein and peptide levels, respectively. Kernel density plot Venn diagrams were generated using ggplot2 and venneuler packages, respectively. Kolmogorov-Smirnov statistical analysis was performed using R with default parameters.Parasites were cultured under tachyzoite conditions, harvested, and filter purified as described above. Purified eviously . BrieflyT. gondii ME49 strain. After the sequence was verified, PCR fragments spanning from the GRA1 promoter to the 3′ untranslated region (UTR) were generated using Q5 DNA polymerase (NEB). Five micrograms of the PCR fragments was transfected to the ppGalNAc-T deletion mutants using the Amaxa Nucleofector system with basic parasite kit 1 (Lonza) and the U-33 program. Parasites, cultured under the tachyzoite condition, were harvested 20 h after transfection by scraping, including host HFF cells. Cells were pelleted, lysed in 100 µl of SDS sample buffer, and boiled for 10 min. For each lane, 40 µl of sample was run on an SDS-PAGE gel. After transfer to the PVDF membrane, the membrane was blocked with 5% milk in PBS and incubated with HRP-conjugated rat anti-HA antibody at 1:1,000 overnight. Signal was detected using the Odyssey Fc system (Li-Cor).A mucin-like domain-containing construct in a pSMART-BAC2 bacmid (Lucigen) was generated using the HiFi DNA assembly kit (New England BioLabs [NEB]). From the 5′ direction, this vector contains a GRA1 promoter, CST1 signal peptide sequence, tandem strep-II tag, CST1 mucin-like domain, and then a 3×HA tag sequence .The brain cyst fragility assay was done as described previously . Brieflyild type , were inGuide for the Care and Use of Laboratory Animals  and the National Research Council  Animals . Animals


A need exists to develop bioinformatics for predicting differences in protein function, especially for members of a domain family who share a common fold, yet are found in a diverse array of proteins. Many domain families have been conserved over large evolutionary spans and representative genomic data during these periods are now available. This allows a simple method for grouping domain sequences to reveal common and unique/specific binding residues. As such, we hypothesize that sequence alignment analysis of the yeast SH3 domain family across ancestral species in the fungal kingdom can determine whether each member encodes specific information to bind unique peptide targets. With this approach, we identify important specific residues for a given domain as those that show little conservation within an alignment of yeast domain family members  but are conserved in an alignment of its direct relatives (orthologs). We find most of the yeast SH3 domain family members have maintained unique amino acid conservation patterns that suggest they bind peptide targets with high intrinsic specificity through varying degrees of non-canonical recognition. For a minority of domains, we predict a less diverse binding surface, likely requiring additional factors to bind targets specifically. We observe that our predictions are consistent with high throughput binding data, which suggests our approach can probe intrinsic binding specificity in any other interaction domain family that is maintained during evolution. To achieve the correct responses from signaling pathways, the protein-protein interactions involved must be specific, and not potentiate inappropriate activation of off-target pathways. This requisite precision can be readily achieved by proteins that possess high “intrinsic specificity”, directly binding their intended targets much more tightly than any other protein. For protein-DNA interactions, this can involve differences of three orders of magnitude or more in K binding . For exaf 1.5 μM –5. For epartners . The intificity” . Contextificity” , 8, and β-sheet in their secondary structure. SH3 domains generally bind short proline-rich peptides containing the core consensus sequences +xxPxxP (class I) or PxxPx+ motifs (class II), where x can be a variety of residues while + is a Lys or Arg residue [μM) [The bulk of protein interactions within signaling pathways are mediated by small modular domains, which are found within larger proteins . SH3 dom residue –22. The due [μM) .Early studies with short PxxP-containing peptides that bind predominantly to SI, showed SH3 domains have similar target peptide affinities –28, suggDespite these examples, it has been shown that the peptide residues flanking the PxxP motif can play crucial roles in mediating binding, and provide support for intrinsic specificity in SH3 domain interactions –37. Theset al. show peptide target conservation is correlated with peptide binding specificity for the complete yeast SH3 domain family [As such, we hypothesize that the analysis of domain sequence alignments can predict whether each yeast SH3 domain binds its targets with intrinsic and/or contextual specificity, which will be compared to published binding and structural data. Our approach is motivated by the numerous fungal genomes that have been sequenced  and the n family . Thus, tWe compare the sequences of every yeast SH3 domain in a paralog alignment, which reveals the key conserved features for any SH3 domain (basic/common fold and function). We also compare the conservation of each domain family member with its direct relatives in an ortholog alignment, to highlight the key conserved and unique features for each family member. Those positions that are conserved in both paralog and ortholog alignments indicate the housekeeping residues for essential SH3 domain structure and function. However, positions conserved in the ortholog alignment, but not conserved in the paralog alignment, have high specific conservation (SC) and suggest they provide uniqueness to that domain. We find that most SH3 domains in yeast have high SC in SII, suggesting they have the capacity to bind with intrinsic specificity. Additionally, some of the studied SH3 domains have high SC in alternative regions suggesting other important binding surfaces may exist. Comprehensive sequence alignment analysis of the yeast SH3 domain family promises to provide greater understanding of how its members mediate interactions inside the cell.S.cerevisiae) and ortholog information (find direct relatives for a given domain). All alignments are outputted as formatted excel spreadsheets. In all alignments, the following 6 amino acid equivalency groups are used to calculate the entropy [pi is the fraction of residues that belong to that equivalency group . For positions in the alignment where there are too many gaps (defined by “% no gap threshold”), entropies are not calculated. The threshold is 0.64, which corresponds to residues present in at least ∼19 of 29 sequences.The National Center for Biotechnology Information (NCBI) protein database was queried to generate paralog information  and organism name (saccharomyces cerevisiae S228c). From this information, we extract/identify the domain family members  from the ENTREZ records and use ClustalW  to perfoS.cerevisiae using an e-value filter (measure of protein similarity) of less than 1e-5. In almost all cases, this filter selected true orthologs to the query protein. Next, we performed an ENTREZ text search for each protein to extract the protein length and the location of their SH3 domains. For yeast proteins that have multiple SH3 domains, a ClustalW alignment is performed between the known yeast domain and the multiple domains found in the given ortholog and the closest matching domain is retained. In some cases, manual adjustments were made after this procedure to ensure the relative order of the domains was maintained. We found ∼250 ortholog species for each yeast domain, and reduced redundancy by the following method. For every species containing SH3 domains, we counted the number of different domain family members found in that species and referred to this as the species paralog count (SPC). Some species had as few as 1 yeast SH3 domain direct relative, while many had SPC values close to 28, which is the number of domain family members in S.cerevisiae.We examine the evolution of each of the 28 yeast domain family members within the fungal kingdom by generating an ortholog alignment for each. We first perform a separate protein BLAST on the NCBI database for each of the 28 full length protein in S.cerevisaie SH3 domain , A.gossypii Bem1a (2 domains), C.albicans Bem1a (2 domains), S.pombe Hof1 (3 domains), S.cerevisiae Lsb1/Lsb2 (2 domains), S.cerevisiae Lsb3/Lsb4 (2 domains), S.cerevisiae Myo3/Myo5 (2 domains), S.cerevisiae Boi1/Boi2 (2 domains) and C.albicans Rvs167 (2 domains). For a given peptide i, binding to a given domain d1, from a given species j, we calculated a binding fraction (BF) using binding intensity (BI) data for peptide i to all domains in that species. As listed above, 4 domain pairs in S.cerevisiae are averaged and thus BI’s from 24 domains are considered (d1 to d24)). Binding data  between ∼300 peptides and the SH3 domain family members from 4 fungal species was recently collected and has been analyzed further . Before We do not calculate BF’s for any peptide that has a sum of intensities to all domains in a given species below 1000 units to ensure good signal over noise and minimize inflated proportions from small numbers in the calculation for PSSMs contain the number of occurrences for an equivalency group  at a parTo test our hypothesis, we constructed alignments for each yeast SH3 domain by systematically selecting representative species from the >800 fungal genomes sequenced . For eacy values . The 60 y values  and for We consider the SC values from every domain in the yeast family  and findWe calculated the average SC value for groups of residues that define surface I, II and the rest of the domain to address our hypothesis and probe peptide binding further. To test the accuracy of our binding specificity predictions from the alignments, we calculated a domain-species binding fraction  for eachS.cerevisiae. As expected the SI residues are highly conserved within each ortholog alignment (red color) and very similar between paralogous domains. W36 and P51 are essential to the SH3 domain as noted previously as the top 2 conserved SH3 domain residues [residues , and posresidues . As suchTo complement SC value analysis, we also represent the surfaces in our paralog and ortholog alignments using position specific scoring matrices (PSSMs) to provide additional insights into the family . As can To systematically explore the complete family, we cluster all PSSMs using residues that define SI and find 4 main branches in the resultant dendogram . As expePSSMs also allow the close examination of diversity in SII, whose residues are chiefly found in loops. As noted previously, SII provides an excellent platform for encoding unique specificity information for a given domain. Figs To complement this analysis, we examined specifically conserved residues for clustering on their 3D structures as well as consider conservation of positions N- and C-terminal of the canonical 60 residue domain boundaries. For 13 of the 28 domains, structures have been deposited in the protein structure database and the remaining 15 core domain structures are easily modeled. Currently, most of these structures await analysis as they were deposited as part of a structural genomics effort. Unfortunately, apart from the Abp1, Nbp2 and Bem1 SH3 domain-peptide complex structures , 49, 50,Our simple approach of grouping representative domain sequences into paralog and orthologs allowed us to comprehensively assess the degree of uniqueness for all members of the yeast SH3 domain family. We focused on the binding surfaces I and II, where most proline-rich target peptides bind and large amounts of binding data is available. We also made an initial exploration of other clusters within the domain and sequences outside the canonical boundaries. Our findings, which are supported by high-throughput binding data analysis, accurately predict that most of this family can bind peptide targets specifically via a unique SII , 43. In et al for functional diversity found over evolution for families of related proteins [Although an extended peptide interaction appears the most common specificity mechanism, we found additional SC clusters and conserved insertions at the loops and termini. This suggests a range of other structural and functional features contribute to the uniqueness of every domain. For example, alternative binding surfaces found in the region from the termini to the distal loop, far from SI and II, could provide contextual specificity mechanisms, especially for domains such as Bem1a and Cyk3. These domains have low average SC values in SII, although an additional interaction at this alternative surface, could facilitate multiple simultaneous target interactions and increased specificity. Furthermore, some domains may increase specificity through their conserved core-termini cluster, which involves a network from the peptide binding site through the core to the termini. For these domains, peptide binding at SI/SII may affect interactions at the termini via allostery, which could be important for specificity . We alsoproteins .Specific conservation and PSSM analysis has been central in addressing our hypothesis that concerns a known binding surface region. The approach has shown functional predictions can be made about protein-protein interaction modules in the absence of peptide binding data and has identified other regions of the domain that could have functional significance. SC values are comparable to positional entropy differences from a previous approach that performed saturation mutagenesis of the binding site between human growth hormone and its receptor . The autDifference evolutionary trace (ET) is an alternative to our approach that can also identify important unique specific residues for paralogs –68. DiffS1 TableSacharamyces cerevisiae has a group number of 0, all other saccharamycotina species have a group number of 1. Group numbers are indicated in parentheses (see eses see  and are (TIF)Click here for additional data file.S2 TableNumber of species available at each taxonomic level  for dire(TIF)Click here for additional data file.S1 FigA tree depicting the relationships between the fungal species groups  compared(TIF)Click here for additional data file.S2 FigDomains are sorted in descending order by their average SII SC value. Table shows number of additional conserved residues either N- and C- terminal to the SH3 domain or insertions in the 3 loops (highlighted in red) as well as significant SC found in the binding surface and other residue clusters (indicated by an X). The core-termini cluster usually involve residues 37,50 and 52 as well as residues in the termini, thus potentially connecting binding to changes near the termini via coupled conformational changes. The termini-distal cluster is a group of surface residues, typically involving residues 25 and 27 that connect the distal loop to the termini residues. The termini cluster includes the termini residues as well as other surface residues to form another binding surface. For some domains alternative names are provided in parentheses.(TIF)Click here for additional data file.S1 File(Stacked Domains Tab). Only SH3 domain residues that align with the S.cerevisiae domain are shown and are colored according to our residue equivalence groups defined for entropy and PSSM calculations. The species names end with a number that refers to their taxonomic group as seen in our tree (S.cerevisiae protein (fifth row). The paralog entropy is calculated from an alignment of the 28 SH3 domains in S.cerevisiae and is found at the bottom. . Full alignment of the yeast SH3 domain family. . Each yeast SH3 domain is aligned with its Fungal orthologs. Only SH3 domain residues that align with the S.cerevisiae domain are shown.our tree . The SC (XLSX)Click here for additional data file.S2 FileFull-length SH3 domain containing proteins are aligned. Each tab contains the alignment of one yeast SH3 domain containing protein and all of its direct relatives. The 1st row contains the alignment numbers, the 2nd row, the ortholog entropy, which is only calculated if the no gap proportion (3rd row) is above its threshold (0.64). The 4th row contains the standard domain number. The 5th row contains the residue number according to the yeast protein. Residues are colored according to our residue equivalence groups defined for entropy and PSSM calculations. The species names end with a number that refers to their taxonomic group as seen in our tree .(XLSX)Click here for additional data file.S3 FilePeptide sequences for a peptide that has a binding fraction > 0.5 in at least one species. Binding fractions <0.5 are shaded green. The top peptide for each domain has its average binding fraction highlighted yellow.(XLSX)Click here for additional data file.S4 FileTotal occurrence for each amino acid group for each position is indicated and colored as yellow  to red .(XLSX)Click here for additional data file.


Here, we characterized solution conformations of FUS and its five differentially dissected fragments, followed by detailed investigations on thermal unfolding, NMR dynamics and self-assembly of RRM. The results decipher: (1) the N- and C-terminal LC domains are intrinsically disordered, while RRM is folded. Intriguingly, well-dispersed HSQC peaks of RRM disappear in the full-length FUS, reminiscent of the previous observation on TDP-43. (2) FUS RRM is characteristic of irreversible unfolding. “Model-free” analysis of NMR relaxation data decodes that RRM has high ps-ns conformational dynamics even over some residues within secondary structure regions. (3) RRM spontaneously self-assembles into amyloid fibrils. Therefore, in addition to the well-established prion-like region, FUS RRM is also prone to self-assembly to form amyloid fibrils. Taken together, FUS RRM appears to play a crucial role in exaggerating the physiological/reversible self-assembly into pathological/irreversible fibrillization, thus contributing to manifestation of FUS cytotoxicity.526-residue FUS functions to self-assemble into reversible droplets/hydrogels, which could be further solidified into pathological fibrils. FUS is intrinsically prone to aggregation, composed of N-terminal low-sequence complexity (LC); RNA-recognition motif (RRM) and C-terminal LC domains. Intriguingly, previous  The FUS protein belongs to the FET protein family, which also includes Ewing RNA binding protein (EWS), and TATA-binding protein associated factor (encoded by TAF15)4. Although the precise physiological functions of FUS remain to be fully elucidated, growing evidence suggests that FUS is involved in various cellular processes, including cell proliferation, DNA repair, transcription regulation, and multiple levels of RNA and microRNA processing7. On the other hand, FUS is extensively involved in the pathology of neurodegenerative diseases. FUS aggregation has been observed in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), the polyglutamine diseases which include Huntington disease, spinocerebellar ataxia, and dentatorubropallidoluysian atrophy10. In addition, genetic variants in the FUS gene have been identified as causative or risk factors for ALS, essential tremor and rare forms of FTLD15. These findings suggest that FUS might have a general role in neurodegenerative diseases.Fused in Sarcoma/Translocated in Sarcoma (FUS) consisting 526 residues is encoded by a gene which was first identified as a fusion oncogene in human liposarcomas10, which is composed of an N-terminal low-sequence complexity (LC) domain (1–267) including a QGSY-rich prion-like region (1–165) and a G-rich region (166–267); an RNA-recognition motif (RRM: 285–371) capable of binding a large array of RNA and DNA17; and C-terminal LC domain (371–526) including a RGG repeat region and a highly conserved nonclassical nuclear localization signal  contain one or several RRM domains that mediate the direct interaction with nucleic acids to control both RNA processing and gene expression19. Noticeably, despite a large sequence variation from other RRMs, the RRM domain of FUS has been determined by NMR spectroscopy to adopt the same overall fold as other RRMs, which consists of a four-stranded β-sheet and two perpendicular α-helices. Nevertheless, the FUS RRM domain does own a unique, extra-long, and positively-charged “KK” loop essential for binding nucleic acids17. Very amazingly, previous in vivo studies revealed that RRM is required for manifesting FUS cytotoxicity but its underlying mechanism remains largely elusive20.FUS is a multi-domain protein intrinsically prone to aggregationnal Fig. . RRM is 22, we have successfully studied the ALS-causing and aggregation-prone TDP-43 N-terminal and C-terminal prion-like domains24. Here, by the same approach, we characterized conformations of the full-length FUS and its five dissected domains , “insoluble” proteins could only be solubilized in aqueous solution with minimized salt concentrationsins Fig. , all of 25 and interestingly, all residues have negative score, except for a small portion of residues within the RRM domain  1H-15N NMR HSQC spectra  ensional D 1H-15N tra Fig. , indicattra Fig. . In part1H dispersion of 1.25 ppm and 15N dispersion of 19.2 ppm  consisting of the N-terminal LC domain and RRM; as well as FUS (267–526) containing the C-terminal LC domain and RRM Fig. . NoticeaWe also acquired HSQC spectra for FUS (1–371) and FUS (267–526), and subsequently superimposed them respectively to HSQC spectrum of RRM collected at the same conditions (at a protein concentration 0 f 40 µM in 1 mM phosphate buffer at pH 6.8). Interestingly, while many HSQC peaks of RRM are superimposable in the isolated RRM and in FUS (1–371), a portion of HSQC peaks show significant shifts Fig. . Very in29.To further exclude the possibility that the disappearance of HSQC peaks of RRM in the full-length FUS is due to its purification under denaturing conditions, we also conducted the purification of RRM under denaturing conditions using exactly the same protocol for the full-length FUS, but the obtained RRM protein was also folded at two pH values, with HSQC peaks completely superimposable to those purified under native condition. Furthermore, FUS (1–371) and FUS (267–526) were in fact purified under denaturing conditions but the well-dispersed HSQC peaks of RRM could still be detected. This implies that the disappearance of the well-dispersed HSQC signals of RRM is most likely due to the presence of both N- and C-terminal LC domains. Very amazingly, we have previously observed the same phenomenon on another ALS/FTD-causing protein TDP-43. The well-dispersed HSQC peaks of two well-folded RRM domains also became disappeared in the contexts of the full-length TDP-43 as well as its fragment with the N-terminal domain deleted, which has been characterized to mainly result from µs-ms exchanges between the open and closed conformations mediated by dynamic inter-domain interactions15N-/13C-double labeled sample. Figure 30. In addition to the N-terminal residue Ser282, as well as Pro320, Pro344, Pro345 and Pro363, only Lys315, Glu336, Asp343 and Ser360 were not assigned due to overlap or undetectable resonance signals. Most (ΔCα-ΔCβ) chemical shifts of the present FUS RRM construct are almost the same as those (BMRB ID of 17635) over the identical region17.Previously, the FUS RRM domain was determined by NMR to adopt the classical fold as other RRMs with secondary structures of β1–α1–β2–β3–α2–β4 Figs  and 5A. We conducted the thermal unfolding of RRM by acquiring a series of far-UV CD spectra at temperatures ranging from 20 to 90 °C Fig. . By moniAs intrinsic UV fluorescence of Trp residues is very sensitive to the surrounding chemical environments, and FUS RRM only has one Trp353 residue Fig. , we furtWe also conducted the thermal unfolding of RRM by monitoring HSQC spectra at different temperatures. As shown in Fig. 15N NMR backbone relaxation and CPMG-based relaxation dispersion data in order to gain insights into the conformational dynamics of the FUS RRM domain on both ps-ns and µs-ms time scales. 15N NMR backbone relaxation data, including the longitudinal relaxation time T1, transverse relaxation time T2, and {1H}-15N steady-state NOE (hNOE) are sensitive indicators of protein dynamics on the ps-ns timescale35, and Fig. We further acquired and analyzed 31, with an average of 0.72 only, which is very similar to that (0.7) of the ubiquitin-like (UBL) fold adopted by the TDP-43 N-domain existing in equilibrium between the folded and unfolded states exchangeable at ~14 Hz23. To gain a quantitative insight, the NMR relaxation data were further analyzed by “Model-free” formalism35. This analysis generated squared generalized order parameters, S2, which reflects the conformational rigidity on ps-ns time scale. S2 values range from 0 for high internal motion to 1 for completely restricted motion in a molecular reference frame. The FUS RRM domain has a S2 average of 0.77, much smaller than that of EphA4 (0.86)31. As shown in Fig. 2 values smaller than the average. Noticeably, several residues located within β1, β2 and β3 strands also have S2 values smaller than the average. In particular, all C-terminal residues from Phe359 including the whole β4 strand also have S2 values smaller than the average  by three fluorescence probes and CD spectroscopy as well as EM imaging at different time points up to 12 days  and (1–42), lysozyme as well as tau39. We also found the gradual development of the intrinsic visible fluorescence during the self-assembly of the TDP-43 C-terminal domain (CTD) containing a hydrophobic fragment flanked by two prion-like regions24. Most remarkably this intrinsic visible fluorescence has been characterized to have its origin in the formation of special hydrogen bond networks involved in the backbone C=O and N-H atom groups of peptide bonds, which already have electron delocalization to some degree. The formation of the cross-β fibrillar structures with highly aligned hydrogen bonds will further enhance electron delocalization and thus allow low energy electronic transitions required for the manifestation of this intrinsic visible fluorescence39.Previously it has been found that β-rich structures could have intrinsic visible fluorescence in both crystal and solution36. As seen in Fig. Figure Figure 43. However, the high-resolution biophysical mechanism for switching reversibility to irreversibility remains almost unknown although it has been extensively recognized to lie at the heart of ALS and other age-related diseases51.RNA-binding proteins containing the prion-like LC regions such as FUS, TDP-43 and hnRNPA1 have been increasingly identified to be involved in pathological aggregation in ALS and FTD. Recent studies established that FUS can undergo phase separation to self-assemble into physiological and reversible liquid droplet/hydrogel states. On the other hand, many factors such as long incubation times and ALS/FTD-associated mutations have been identified to be sufficient to induce further phase transitions into pathological and irreversible fibrillar states24, we have successfully characterized the solution conformations of the full-length FUS and its five differentially dissected domains. The results revealed that both N- and C-terminal LC domains are intrinsically disordered and only the RRM domain is folded. However, likely due to the interactions of the RRM domains with both N- and C-terminal LC domains, in the full-length FUS the well-dispersed HSQC peaks of the folded RRM domain become undetectable due to significantly provoked µs-ms dynamics, or/and increased rotational tumbling time. This is reminiscent of our previous observation on another ALS-/FTD-causing protein TDP-4352. The well-dispersed HSQC peaks of two folded RRM domains also disappeared in the full-length TDP-43 and even its truncated fragment with the N-domain (1–101) deleted, which was characterized to result from µs-ms exchanges between the closed and open conformations mediated by dynamic inter-domain interactions coordinated by intrinsically-disordered prion-like domain29. Despite being very challenging, in the future it would be important to identify the exact regions involved in inter-domain interactions of FUS. It is also of fundamental interest to examine whether other RNA-binding proteins containing RRM and LC regions also share this feature. If yes, what is their functional relevance/advantage?In the present study, we attempted to explore this issue by an integrated use of various biophysical tools. First, as facilitated by our previous discovery15N backbone relaxation data decodes that it also has a relatively high backbone dynamics on ps-ns time scale. More precisely, in addition to terminal and loop residues, residues even within four β-strands also have high conformational dynamics  cells (Novagen). The recombinant proteins of the full-length FUS, FUS (1–267), FUS (1–371), FUS (267–526) and FUS (371–526) were found only in inclusion body while RRM was highly soluble in supernatant. As a result, for the full-length FUS and its four aggregation-prone fragments, the pellets were first dissolved in a phosphate buffer (pH 8.5) containing 8 M urea and subsequently purified by a Ni2+-affinity column (Novagen) under denaturing conditions in the presence of 8 M urea. The fractions containing the recombinant proteins were acidified by adding 10% acetic acid and subsequently purified by reverse-phase (RP) HPLC on a C4 column eluted by water-acetonitrile solvent system. The HPLC elution containing pure recombinant proteins were lyophilized.The DNA fragments encoding the full-length FUS and its five dissected domains Fig.  were amp2+-affinity column (Novagen) under native condition, followed by a further purification by FPLC on a gel-filtration column. However, to assess whether the purification under denaturing conditions would affect the folding of RRM, we also conducted purification under denaturing conditions using the same protocol as for the full-length FUS and its four aggregation-prone fragments.For RRM, the recombinant proteins were purified by a Ni15NH4)2SO4 for 15N labeling and (15NH4)2SO4/[13C]-glucose for double labelling31. The purity of the recombinant proteins was checked by SDS–PAGE gels and their molecular weights were verified by a Voyager STR matrix-assisted laser desorption ionization time-of-flight-mass spectrometer (Applied Biosystems). The concentration of protein samples was determined by the UV spectroscopic method in the presence of 8 M urea. Briefly, under the denaturing condition, the extinct coefficient at 280 nm of a protein can be calculated by adding up the contribution of Trp, Tyr and Cys residues56.The generation of the isotope-labelled proteins for NMR studies followed a similar procedure except that the bacteria were grown in M9 medium with the addition of  dissolved in Milli-Q water (pH 4.0) into 1 mM phosphate buffers to obtain samples at a protein concentration of 40 µM with final pH at pH 5.0 and 6.8 respectively.All circular dichroism (CD) experiments were performed on a Jasco J-1500 spectropolarimeter equipped with a thermal controller as previously described52. For characterizing the solution structure of the FUS RRM domain, a pair of triple-resonance experiments HNCACB, CBCA(CO)NH were collected for the sequential assignment on a 15N-/13C-double labelled sample of 400 µM. NMR data were processed with NMRPipe57 and analyzed with NMRView58.All NMR experiments were acquired on an 800 MHz Bruker Avance spectrometer equipped with pulse field gradient units as described previously24 at different time points of the incubations at 25 °C at a protein concentration of 40 µM in 1 mM phosphate buffer (pH 6.8). The rectangular fluorescence quartz cuvette has the pathlength dimension of 10 × 10 mm and the general settings are: PMT at low sensitivity and scan speed of medium speed (200 nm/min). For the intrinsic UV fluorescence, the emission spectra were measured with the excitation wavelength at 280 nm and slit widths: excitation at 5 nm and emission at 10 nm. For the intrinsic visible fluorescence, the emission spectra were measured with the excitation wavelength at 375 nm and slit widths: excitation at 20 nm and emission at 10 nm.For monitoring the self-assembly of the FUS RRM domain, fluorescence spectra were measured at 25 °C with a RF-5301 PC spectrophotometer  as previously described24.For Thioflavin-T (ThT) binding assay, a 2 mM ThT stock solution was prepared by dissolving ThT in milli-Q water and filtered through a 0.22 μm Millipore filter. The fresh working solution was prepared by diluting the stock solution into 1 mM phospate buffer (pH 6.8) to reach a final ThT concentration of 50 μM. A 10 μL aliquot of each incubation solution, or 10 μL aliquot of the incubation buffer (1 mM phosphate at pH 6.8) as the control, was mixed with 130 μL of the ThT working solution in the dark for 10 mins. The fluorescence emission spectra were acquired for three repeats with the excitation wavelength at 442 nm and slit widths: excitation at 5 nm and emission at 10 nm15N backbone T1 and T1ρ relaxation times and {1H}-15N steady state NOE intensities were collected on the 15N-labeled RRM domain at 25 °C at a concentration of 400 µM in 10 mM phosphate buffer at pH 6.8 on an Avance 800 MHz Bruker spectrometer with both an actively shielded cryoprobe and pulse field gradient units31. Relaxation time T1 was determined by collecting 7 points with delays of 10, 160, 400, 500, 640, 800 and 1000 ms using a recycle delay of 1 s, with a repeat at 400 ms. Relaxation time T1ρ was measured by collecting 7 points with delays of 1, 12, 30, 40, 50, 80, 100 ms. {1H}-15N steady-state NOEs were obtained by recording spectra with and without 1H presaturation, a duration of 3 s and a relaxation delay of 6 s at 800 MHz.35. Briefly, relaxation of protonated heteronuclei is dominated by the dipolar interaction with the directly attached 1H spin and by the chemical shift anisotropy mechanism. Relaxation parameters are given by:μ0 is the permeability of free space; h is Planck’s constant; γX, γH are the gyromagnetic ratios of 1H and the X spin (X = 13C or 15N) respectively; γXH is the X-H bond length; ωH and ωX are the Larmor frequencies of 1H and X spins, respectively; and Δσ is the chemical shift anisotropy of the X spin.NMR relaxation data were analyzed by “Model-Free” formalism with protein dynamics software DYNAMICS32 and further extended35, determines the amplitudes and time scales of the intramolecular motions by modeling the spectral density function, J(ω), asThe Model-Free formalism, as previously establishedτm is the isotropic rotational correlation time of the molecule, τs is the effective correlation time for internal motions, S2 = Sf2Ss2 is the square of the generalized order parameter characterizing the amplitude of the internal motions, and Sf2 and Ss2 are the squares of the order parameters for the internal motions on the fast and slow time scales, respectively.In which, S2 and τloc; extended model-free treatment, including both fast  and slow  reorientations for the NH bond (τfast << τslow < τc); and could also allow for slow, milli- to microsecond dynamics resulting in a conformational exchange contribution, Rex, to the linewidth. In DYNAMICS, there are eight models for local motions and each residue is fitted with different models. Subsequently goodness of fit will be checked and the best-fitted model will be selected59.In order to allow for diverse protein dynamics, several forms of the spectral density function, based on various models of the local motion, were utilized, which include the original Lipari-Szabo approach, assuming fast local motion characterized by the parameters 17 by isotropic, axially-symmetric and fully anisotropic models for the overall motion and the results were tested and then compared. According to the illustration of ROTDIF, axially-symmetric model was finally selected because of smallest Ch2/df value59, which has τc of 6.9 ns; Dx = Dy = 2.56 ± 0.15; Dz = 3.04 ± 0.46 (107 S−1) and alpha = 108 ± 24; beta = 48 ± 45 degree.The relaxation data of the FUS RRM domain were analyzed with the previously published NMR structure (pdb ID of 2LCW)15N transverse relaxation dispersion experiments were acquired on the 15N-labeled RRM domain on a Bruker Avance 800 spectrometer. A constant time delay (TCP = 50 ms) was used with a series of CPMG frequencies, ranging from 40 Hz, 80 Hz, 120 Hz (x2), 160 Hz, 200 Hz, 240 Hz, 320 Hz, 400 Hz, 480 Hz, 560 Hz, 640 Hz, 720 Hz, 800 Hz, and 960 Hz (x2 indicates repetition). A reference spectrum without the CPMG block was acquired to calculate the effective transverse relaxation rate by the following equation:CPMG) is the peak intensity on the difference CPMG frequency, I0 is the peak intensity in the reference spectra31.24.Incubation samples of the FUS RRM domain at 25 °C at a protein concentration of 40 µM were imaged at 3, 6 and 10 days of the incubation in 1 mM phosphate buffer (pH 6.8), by a TEM microscope  operating at an accelerating voltage of 200 kV as previously describedBriefly, for EM imaging, a 5 µl aliquot of the incubation or aggregate solutions was placed onto the Cu grids  and negatively stained with 5 μl of 2% neutral, phosphotungstic acid (PTA). This aliquot was allowed to settle on Cu grid for 30 sec before the excess fluid was drained away. The Cu grid was later air-dried for another 15 mins before being imaged.


Plasmodium falciparum infected erythrocytes (IE). Through interactions with various host molecules, PfEMP1 mediate IE sequestration in tissues and play a key role in the pathology of severe malaria. PfEMP1 is encoded by a diverse multi-gene family calledvar. Previous studies have shown that that expression of specific subsets ofvar genes are associated with low levels of host immunity and severe malaria. However, in most clinical studies to date, full-lengthvar gene sequences were unavailable and various approaches have been used to make comparisons betweenvar gene expression profiles in different parasite isolates using limited information. Several studies have relied on the classification of a 300 – 500 base-pair “DBLα tag” region in the DBLα domain located at the 5’ end of mostvar genes.PfEMP1 are variant parasite antigens that are inserted on the surface ofvar gene sequences. We compared these different sequence features in full-lengthvar gene from six fully sequenced laboratory isolates. We assessed the relationship between various DBLα tag classification methods, and sequence features that are only fully assessable through full-length DBLα sequence tag classification can provide functional information on important features of full-lengthvar genes. Notably, a specific subset of DBLα tags previously defined as “group A-like” is associated with CIDRα1 domains proposed to bind to endothelial protein C receptor. These comparisons show that despite a long history of recombination, This analysis helps to bring together different sources of data that have been used to assess var gene expression in clinical parasite isolates. The range ofvar genes expressed at any one time in the infecting parasite population varies according to the antibodies and otherin vivo selection pressures. 3) Analysis is further complicated by the high diversity of each domain subclass and lack of clear associations between specific adhesion phenotypes and classes of domains.Various cytoadhesive functions are encoded by specific PfEMP1 domain subsets. PfEMP1 molecules contain a combination of two to nine domains . Theups classification partitions the sequences into groups A–E based on the sequence similarity of the 500 base-pair 5’ flanking region and thevar chromosomal location  specific CIDRα1 domains often found in the context of domain cassette 8 (DC8) and 13 (DC13) have been found to bind to EPCR   information were removed, leaving 313 sequences.α sequences was obtained from Kilifi  (The block sharing group (BS) classification of DBLα tags came from a sequence network analysis approach that aimed to visualize how different sequences share blocks of polymorphic sequence. Analysis of fully connected components of a sequence network constructed from observing the sharing of 14 amino acid blocks within DBLα tag sequences from parasites from Kenyan children showed that the largest component, called “block sharing group 1” (BS1) contained predominantly known upsAReceiver operator curves (ROC) were used to visualise the sensitivity and specificity of using specific subsets of DBLα sequence tags in the prediction of upsA, DC8, DC13 and CIDR1α, as outlined inThe block sharing groups were originally defined using a global collection of sequences that included sequences from 3D7 and IT4 laboratory isolates  Analysis is further complicated by the high diversity of each domain subclass and lack of clear associations between specific adhesion phenotypes and classes of domains.” I would phrase this differently. Although many binding phenotypes have been proposed for iRBCs, well characterized interactions for PfEMP1 are more limited. In fact only few interactions are studied to the extent that these can be used to predict PfEMP1 function: CSA, CD36, EPCR and ICAM1. For these there is only a small uncertainly for determining ICAM1 binding domains. Binding to HABP1, PECAM1, IgM, etc is, as stated not clearly linked to specific domains or sequence traits. As parasite adhesion phenotypes cannot be investigated in vivo, "specific adhesion phenotypes" is defined from a combination of clear association of PfMEP1 domain type with binding to a specific receptor, as well as validation of this by iRBC binding assays; and thus observed iRBC binding to various receptors cannot be taken as a gold standard on its own.et al., 2010). For example, the DBLa domains were reclassified into 33 sub-domains . I suggest Correcting next paragraph to: Based on full-length sequences from seven laboratory isolates, each domain class was divided through global sequence alignment into further sub-classes , which plays a central role in placental malaria . I suggest changing references as: “ Quote: “It was initially proposed that DCs may act as functional units. However, clearly defined functions have only been assigned at the level of individual domain sub-classes. Therefore, though common combinations of domains exist, it is unclear whether they represent functional units.” I agree with this. I would even suggest that it is clear that most domain cassettes, although useful to define molecular tools, does not appear to reflect functional units. However, I do not think the examples given does not elude clearly to this. In line with my comment above, I think it should be made clear that the binding phenotypes we understand well today,  all are associated with and fully contained within single domains. However, some subsets of these domains appear to have co-evolved - like ICAM1 binding (DBLb) in group A is always found in CIDRa1 (EPCR binding) variants, (co-evolution clearly seen within the DC13 context); whereas ICAM1-binding DBLb are rarely found in CIDRa1 (EPCR binding) containing DC8; and DBLb are not specifically associated with any Domain subclass when found in group B.  The comment of DBLb domains being targets of cross reactive antibodies seems irrelevant in this context and confusing here. Paragraph. “Clinical and laboratory studies have reported…..”. The DC8 and DC13 are useful for understanding how these variable genes can be probed and detected iin vivo. But given the limited usefulness of the DCs to describe known binding phenotypes, and their relation clinical outcome; And the recent studies of var expression  - which are in line with previous work as described in two paragraphs before; And to simplify for those new to the field – I think it would be best to use this paragraph to describe the consensus from these studies, that CIDRa1 is the only common trait of var genes whose expression is associated with malaria pathology; regardless of symptomology. And EPCR+IMCA1 but not CD36+icam1 is found more frequently in CM . These findings does not mean that future studies relying on DBLa tag is not useful or needed. M&M section: “Definition of block sharing groups”et al., 2012).”. This is not relevant here. This is a more detailed (and required) description of the statement above on SB1, correct? Perhaps just refer back to this. It is unclear which part of the text refers to previous work or and which to the re-analysis performed here. Leave out the sentence: “and found that their expression was associated with cerebral malaria ……” “Similar to group A-like sequences, DC8 sequences are associated with severe malaria  a B/A recombination  - thus B from UPS to DBLa tag end, and A like in its DBLaS3 and downstream from there. This is important to understand, as the A vs B/C grouping is tied to the chromosomal localization of these genes which ensures that A does not normally recombine with B . This is probably why the DC8 is the best conserved domain cassette, and why DBLa tag analysis is particularly difficult (and important) to apply for these genes. “Sequences with DC4 cassettes are reported to be associated with…..”. The ICAM1 prediction has been refined considerable in Lennartz et al 2017. The authors should color in sequences which contain group B DBLb5s show/predicted to bind ICAM1 and the DBLb3/1 domaains found and predicted to bind ICAM1 .  “Sequences with DC5 cassettes were from different…” ..As expected from this C terminal group A domain cassette - also previously described as not associated with N terminal seq features . “However, such analyses will soon be replaced by methods such …” I disagree with this prediction. RNAseq may indeed prove useful if costs are reduced further to allow enough depth perform the required assembly of var genes, which are few and rare in RNA exptracted from blood in vivo; RNA seq may prove useful if the upcoming analysis of Sangers 1000 Pf genomes suggest so. Using MS analysis it is extraordinarily difficult to do de novo assembly of multiple rare polymorphic sequences in a patient sample, which is what is required to elude further to current knowledge. On the contrary I think this work lays the ground for a developing a much more cost effective var type prediction tool using DBLa tag expression analysis, once such a tool could be developed and validated from the ~1000 pf genomes to be released from Sanger.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. var genes of the malaria parasiteP. falciparum. Thevar genes encodePlasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). Due to the diverse, recombining nature of thevar, from non-laboratory stains it is typically only feasible to sample a very short tag region of about 125 amino acids from the relatively conservedvar DBLα domain. Therefore, most of thevar/PfEMP1 sequence variation currently available from the clinical setting consists of only this tag region. This region does not include most of the variable host endothelial binding sites that have been proposed in the literature, and which are potentially relevant to severe malaria disease. The careful mapping of the relationships between differentPfEMP1 classification schemes is therefore important for deciphering this protein’s multiple and variable binding functions, and the various disease manifestations that likely result as a consequence. Until it is possible to obtain the completevar/PfEMP1 sequences from large numbers of clinical isolates, this type of study is of useful for the progression of the field.A nice computational study comparing multiple methods of categorization for the ultra-diverse, biologically complex, and clinically important family ofvar/PfEMP1. var/PfEMP1 categorization. In that paper, the authors discuss the association between HB36 and cys4/cp4-6 sequences. The authors could maybe include a discussion of HB36 when they talk about predictingupsA/CIDRa1.This paper relies heavily on the results of Rask et al. 2010, which lay an extensive groundwork forPfEMP1.Throughout the manuscript, I believe PfEMP1 should be formatted with Pf in italics:var genes as follows:  “due to recombination betweenvar genes on non-homologous chromosomes, the overall architecture of PfEMP1 encoded by different parasite genotypes is extremely diverse and sequences are mosaics of many semi-conserved sequence blocks”. I think this is slightly overstating our current knowledge of the genetic mechanisms and evolutionary and ecological dynamics shaping and maintaining var diversity. Why the variants have a mosaic nature is likely party due to ectopic recombination (recombination betweenvars on non-homologous chromosomes), but also likely due to recombination betweenvars at thesame genetic location withinhomologous chromosomes.Var genes surely recombine in a homologous manner at least as frequently as they recombine ectopically, and due to the diversity among different parasites even at a singlevar locus, this more normal type of recombination is also likely to generate mosaicism. Another reason it is an overstatement/misstatement: if we were going to give a reason for why there is all thevar diversity, balancing selection is a much more direct explanation as opposed to non-homologous recombination. Balancing selection must be invoked to explain why so muchvar diversity is maintained within the population. In my view at least, the question of the “extreme diversity” of thevar genes is not really addressed at all by just invoking the immediate genetic mechanisms generating the variants. Given the above, I recommend simply removing the following clause from the sentence: “due to recombination betweenvar genes on non-homologous chromosomes”, and replacing it with: “due to rapid recombination among var genes, and likely balancing selection” .The authors explain the diversity and mosaicism ofvar genes are expressed in a strictly mutually exclusive manner, and it may not be obvious that this does not translate to strict mutually exclusive expression at the level of the host individual . I suggest rephrasing as follows: “multiple var genes are expressed simultaneously within the infecting parasite population” could be changed to “whilevar genes are expressed in a strictly mutually exclusive manner at the level of the individual cell, multiplevar genes are expressed simultaneously at the level of the infected host”; and “The range ofvar genes expressed at any one time in the infecting parasite population” could be changed to “The range ofvar genes expressed at any one time within a given host”.The authors use the phrase “the infecting parasite population” twice near the end of the second paragraph of the introduction, and in both cases I believe they specifically mean the parasites within an single, individual host. I think this phrasing is more confusing than it needs to be. For any system with population-level dynamics occurring within organisms there is the possibility for confusion about the level of hierarchy the dynamics are operating on. Specifically, in this case, I think some readers may think the authors mean the population of infective parasites rather than only those parasites that exist simultaneously within a given host individual. Another possible source of confusion is that some readers may be familiar with the fact that theStarting with the third paragraph of the introduction I felt the structure of the manuscript begins to get a bit confusing. I had to work too hard as a reader to follow where they were going with the introduction, and why they were presenting this information in this order. I therefore suggest simplifying the writing in the third, fourth and fifth paragraphs of the introduction, and giving the reader a bit more of an explicit “road map” indicating how the paragraphs are connected and where they are taking us. Specific suggestions follow.PfEMP1 functional specializations, which may in turn clarify the disease process; however, it remains unclear which aspects ofvar diversity are the most relevant for achieving this goal.”A topic sentence could be added to the very beginning of the third paragraph: “The hope has been that it may be possible to identify a limited set ofPfEMP1 domain classes have been classified through global sequence alignment into a large number of highly refined and specific domain sub-classes .”For simplicity, I suggest deleting “Based on full length sequences from seven laboratory isolates …”, adding the topic sentence suggested above, and changing the original first sentence of the paragraph (which would now be the second sentence of the paragraph) to the following: “AllThe second sentence of the third paragraph of the Introduction is not a grammatically well-formed sentence. Also it mentions 33 sub-domains, which implies regions smaller than a domain, but I believe that is not what the authors mean. I believe they mean domain sub-classes . I suggest changing the first part of the sentence to the following: “For example, the DBLα domain can be classified into 33 domain sub-classes….”var sequences—variousbroader classification methods have also been employed for thevar genes that use a sparser set of their sequence features.PfEMP1 genes can be classified into just five broad functional and recombination groups based on sequence similarity of their upstream promoter regions (ups) and chromosomal location. This classification partitions the sequences into groups A-E (citations).UpsE is associated exclusively withvar2CSA, which plays a central role in placental malaria (citation).UpsA var gene [note typo correction here] expression is associated with severe disease (citations) and rosetting (citations). Increased transcription ofupsB sequences have also been reported in cases of severe malaria (citation). And, while some research indicates thatupsC sequences are expressed at higher levels in asymptomatic cases (citations), it has also been reported thatupsC expression is associated with severe malaria (citation).”I suggest rewriting the fourth paragraph as follows: “In addition to the refined domain classification schemes—which were based on the handful of sequenced laboratory strains for which we have completeups groups .Choose a consistent notation forChoose whether to say “subclasses/subclass” or “sub-classes/sub-class”. Both are used in the manuscript, and consistency is the important thing. I prefer the word without the hyphen, but its just personal preference.examples of the initial statement.Again, mostly just for clarity, I suggest rewriting the fifth paragraph of the introduction as indicated below. I removed the “for example” and the numbering because, while the information is relevant, it is not clear to me that they are reallyPfEMP1 can also be described in terms of common configurations of different subclasses of domains. These common configurations are called “domain cassettes” (DCs) . Twenty-threevar DCs have been defined from full-length domain alignments of sequences from seven laboratory strains. It was initially proposed that DCs may act as functional units. However clearly defined functions have still only been assigned at the level of individual domain subclasses. Therefore, it remains unclear whether DCs represent functional units under natural selection, or whether they are just neutral artifacts of the recombinatoric diversification process. [Paragraph break here.] Research pertaining to DCs has revealed the following: Specific CIDRα1 domains, often found in the context of domain cassette 8 (DC8) and 13 (DC13), appear to bind EPCR (citation). Var genes containing DC8 cassettes seem to bind human endothelial cells from various organs, including—notably—those from brain endothelial cells (citations). DBLβ domains found within DC4 genes reportedly adhere to ICAM-1 and may be targets of broadly cross-reactive and adhesion-inhibitory IgG antibodies (citations). [Remove paragraph break that is currently here.] Clinical and laboratory studies have reported associations between DCs....” “Page 3, second column, fifth line from the bottom: I would remove the word “respectively”.Page 4, first column, first paragraph: “by simply partitioning tags to those with and those without two cysteins” does not make grammatical sense, and it implies 2 versus 0 cysteins, which I believe is not what the authors mean. This could be changed to: “by simply partitioning tags by whether they contain two cysteins or some other number of cysteins”.Page 4, first column, line 9: I would remove “Furthermore”.Page 4, first column: For clarity, I recommend changing the sentence “Furthermore, Lavstsen et al., 2012 have suggested that information on EPCR binding by CIDRα1 within DC8 and DC13 may be unavailable within the DBLα tag due to a recombination hotspot situated between the DBLα tag region and the CIDRα domain” to the following: “Lavstsen et al., 2012 have suggested that the DBLα domain tag may not be informative about whether its flanking CIDRα1 domain binds EPCR because there is a recombination hotspot situated between the DBLα tag region and the CIDRα1 domain” .When referring to a paper within a sentence  the authors sometimes use no parentheses: “Lavstsen et al., 2012 have suggested…” and other times use parentheses: “classification that was suggested by ”. The style should at least be consistent, and ideally also consistent with the journal’s formatting recommendations for this type of citation.var” that does not appear to belong: Within the first paragraph of “Results and discussion”, and within the 6th line up from the very end of the manuscript.At least in two places there is a weird comma after “Final line of the first paragraph of “Results and discussion”: I recommend changing “sensitivity, specificity analysis” to “sensitivity-specificity analysis”.th line up from the bottom.For consistency and clarity, use italics for “ups” or spell it out and don’t use the abbreviation. For example, page 4, second column, 7I believe there is an error in the title of the section “DBLα sub-domains are not all homogeneous groups”. I believe it should read “DBLα domain subclasses are not all homogeneous groups”. A “sub-domain” and a domain “sub-class” are completely different things. The first is physically smaller than a domain, the second is a smaller category of a complete domain. As far as I can tell, everywhere the authors use the term “sub-domain” they actually mean “domain sub-class” .First sentence of the section “DBLα sub-domains are not all homogeneous groups”: Grammatical errors. Insert “The” before “Domain classification that was suggested…”, and replace “were” with “was”.Page 5, second column: These two sentences feel pretty meaningless to me, plus it seems the meaning the authors are trying to convey is redundant with the sentences the precede and follow. Therefore I would just delete both of the sentences: “The domain classification used in  has therefore brought together distinct sequences within the same sequence classification. This suggests that the newly defined sub-domains do not always classify sequences into wholly genetically distinct groups.”I think “This discordance between methods of classification, employing global and local sequence comparisons…” would read more clearly as follows: “This discordance between methods of classification when employing global versus local sequence comparisons…”The above sentence continues by describing “a mode of diversification… that we might speculate leads to impaired recognition and clearance of PfEMP1 antigens by the immune system.” I think this sentence presents an interesting biological hypothesis that could be elaborated on more. To me it is not immediately obvious that the discordance should be interpreted in this biological manner. An alternative interpretation might be that some categorization methods are less informative about recombination patterns, or more noisy, for example. I simply find the hypothesis interesting and warranting of further discussion.Page 6, first column: I recommend changing: “recombination hotspot proposed to be situated 3’ of the DBLα tag region” to “recombination hotspot purportedly situated 3’ of the DBLα tag region”. the parts of the gene encoding important cytoadhesive regions potentially limits the predictive information available with the DBLα tag sequence”.Page 6, first column: I recommend changing: “DBLα tag region and parts of the genes encoding important cytoadhesive regions potentially limits the predictive information available with DBLα tag sequence” to “DBLα tag region andPage 6, first column: I recommend adding a colon to “multiple cys/PoLV groups CP2, CP3 and CP4” so it reads “multiple cys/PoLV groups: CP2, CP3 and CP4”. The research methods appear to be of high quality, and the paper is clear and well-written. However, there are some typos and areas where the writing could be improved. Due to the complex nature of the topic I have included many detailed suggested edits below. The majority of the suggested changes are simply to improve the clarity of the manuscript - something that is important for readers from outside the community of scientists who studyI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


N-methyl-d-aspartate receptors (NMDARs) has been implicated in the pathophysiology of SCZ. During development, the composition of synaptic NMDARs dramatically changes from NR2B-containing NMDARs to NR2A-containing NMDARs through the phosphorylation of NR2B S1480 or Y1472 by CDK5, CSNK2A1, and EphB2, which plays a pivotal role in the maturation of neural circuits. We hypothesized that the dysregulation of developmental change in NMDARs could be involved in the onset of SCZ. Using next-generation sequencing, we re-sequenced all the coding regions and splice sites of CDK5, CSNK2A1, and EphB2 in 474 patients with SCZ and 475 healthy controls. Variants on the database for human control subjects of Japanese origin were removed and all the nonsynonymous and nonsense variants were validated using Sanger sequencing. Four novel variants in CDK5 were observed in patients with SCZ but were not observed in controls. The total number of variants, however, was not significantly different between the SCZ and control groups (P=0.062). In silico analyses predicted P271T to be damaging. Further genetic research using a larger sample is required to examine whether CDK5 is involved in the pathophysiology of SCZ.The mechanism underlying the vulnerability to developing schizophrenia (SCZ) during adolescence remains elusive. Hypofunction of  SCZ is a neurodevelopmental disorder with a strong genetic component and is highly related to disturbances in brain/circuit maturation.d-aspartate receptors (NMDARs) is involved in the pathophysiology of SCZ.4–11 This hypothesis was initially based on clinical findings that the uncompetitive NMDAR antagonists, phencyclidine (PCP) and ketamine, induced psychotic features resembling symptoms seen in patients with SCZ. These features included both positive and negative symptoms, as well as cognitive dysfunction in healthy volunteers.5 In addition, PCP exacerbated these symptoms in patients with chronic SCZ.12 Further, GRIN2A, which encodes the NR2A subunit of NMDARs, has been consistently reported to be associated with SCZ 9–11 and has reached genome-wide significance in the largest genome-wide association study (GWAS) performed by the Psychiatric Genomics Consortium.13Accumulating evidence has demonstrated that hypofunction of N-methyl-de novo mutations in genes related to NMDARs and in the activity-regulated cytoskeleton-associated protein (ARC) complex, including GRIN2A and DLG.14 Purcell et al. showed additional evidence of NMDAR hypofunction by identifying rare, disruptive mutations in NMDAR and ARC gene sets through exome-sequencing of 2536 patients with SCZ.15A large exome-sequencing study using 617 trios also revealed that patients with sporadic SCZ harbored 17 Each NMDAR is a tetramer composed of two NR1 subunits and two NR2 or NR3 subunits.18 The number, localization and subunit composition of NMDARs are strictly regulated in an age-dependent manner so that they can exert their function precisely.18 The NR2 subtype, that is, NR2A-NR2D, defines the pharmacological and biophysical properties of the NMDAR.20 NR2A-containing NMDARs are considered to be more neuroprotective than NR2B-containing NMDARs depending on their Ca2+ permeability.22NMDARs play a crucial role in synaptic plasticity, neuronal development, learning, and memory.24 This NMDAR subunit switch (NMDAR switch) plays an important role in the maturation of neural circuits, especially in the GABAergic inhibitory circuit in the prefrontal cortex, in a synaptic activity-dependent manner.25During development, the composition of NMDARs dramatically changes from those that contain NR2B subunits to those that contain NR2A.26 These phenomena are induced by cyclin-dependent kinase 5 (CDK5), casein kinase 2 (CK2), and erythropoietin-producing hepatocellular carcinoma group B (EphB2), whose activities are also regulated in an age-dependent manner.26–30Recent evidence also revealed that the NMDAR subunit switch is mediated by the endocytosis of NR2B, which is induced by phosphorylation of NR2B S1480 or Y1472 during early development.32 Postmortem brain studies in SCZ patients have shown a reduction in the mRNA expression of GRIN2A, which encodes NR2A, on parvalbumin-positive interneurons in the prefrontal cortex, and an increase in the mRNA expression of GRIN2B, which encodes NR2B, in the dorsomedial thalamus, temporal cortex, and hippocampus.33–35 A decrease in CK2 expression and dysregulation of CDK5-P35/P25 has been reported.36–38 CDK5 has been shown to phosphorylate the dopamine D2 receptor, which is involved in the pathophysiology of SCZ.39 In addition, 7 microRNAs that commonly regulate the expression of CDK5 were reported to be potential peripheral biomarkers for SCZ.40 Aripiprazole increased the ratio of NR2A/NR2B in rats, while risperidone increased the activity of CK2.41 Furthermore, EphB2 activates kalirin-7, leading to activity-dependent synaptic formation; knockdown mice for this receptor showed SCZ-like behavioral abnormalities after the period of adolescence.42The effect of PCP on NMDARs differs according to the developmental stage and depends on the NR2 subunit composition.43 Identifying rare variants with a strong effect is another alternative strategy to reveal the genetic architecture of SCZ.44GWAS have successfully provided evidence that the common variants in genes related to synaptic function contribute to disease risk in SCZ. However, the problem of missing heritability has emerged because only 23% of the heritability can be explained by current GWAS findings.CDK5 (BC005115), CSNK2A1 (BC050036), and EphB2 (BC067861) to examine the genetic contribution of these genes, focusing on rare variants to examine the pathophysiology of SCZ.In the current study, we hypothesized that the dysfunction of adolescence-related neurodevelopmental events, especially the NMDAR subunit switch, which is regulated by phosphorylation of NR2B, S1480, or Y1472, might contribute to the onset of SCZ. Thus, we re-sequenced t-test). Inpatients and outpatients of the Department of Neuropsychiatry, University of Tokyo Hospital, and related hospitals and daycare facilities located around Tokyo were recruited for this study. Two or more senior psychiatrists diagnosed the patients through interviews and clinical records using the Diagnostic and Statistical Manual of Mental Disorders DSM-IV criteria (American Psychiatric Association). Patients with a history of drug addiction and alcohol abuse/dependence were excluded. Using the Mini-International Neuropsychiatric Interview45 or unstructured interviews, control subjects with current or lifetime mental illness, alcohol abuse/dependence, or a family history of any psychiatric disorder were also excluded. All participants were ethnically Japanese. The study was performed in accordance with the Declaration of Helsinki. The Research Ethics Committee of the Faculty of Medicine, University of Tokyo approved this study ). Written informed consent was obtained from all participants after they were provided an explanation of the study.A total of 474 patients with SCZ  and 475 control subjects  who were unrelated were analyzed in the current study. There were no significant differences in sex and age between the two groups ) of CDK5, CSNK2A1, and EphB2 was performed to identify missense/nonsense variants and small insertions/deletions using a uniquely customized design TruSeq Custom Amplicon  with MiSeq  according to the manufacturer’s instructions.47 Human CDK5, CSNK2A1, and EphB2 are located on chromosomes 7q36, 20q13, and 1p36.12, respectively. Briefly, 250 ng of total double-stranded DNA was used. Amplicons were designed using Design Studio (http://designstudio.illumina.com/), and 59 amplicons were available. After hybridization, ligation, and amplification, the samples were applied to the sequencer. Raw FASTQ files from the sequencer were retrieved, and short reads were aligned to the hg19 reference using the Burrows-Wheeler Aligner software (http://bio-bwa.sourceforge.net/).48 Picard tools were used to remove duplicates. Subsequently, SAMtools (http://samtools.sourceforge.net/) was used to convert to BAM files and for variant calling.49 GATK was used for base quality score recalibration and was also used to detect SNV and small indels. VCF files were ultimately obtained, and variants were extracted. Using IGV, we visualized the next-generation sequencing data. Sanger sequencing was performed to sequence the genomic region when no amplicon was available (CDK5 exon 1 (Chr7: 150754894–150754935), exon 4 (Chr7: 150753818–150753888), exon 5 (Chr7: 150753662–150753728), CSNK2A1 exon 4 (Chr20: 485757–485878), EphB2 exon 1 (Chr1: 23037476–23037541)) and was used to validate all genetic variants using a 3130XL Genetic Analyzer  after removal of the variants detected in a healthy database cohort of Japanese origin . The primers were designed using Primer 3 software (http://bioinfo.ut.ee/primer3-0.4.0/primer3/). Primer pairs used in Sanger sequencing are shown in Genomic DNA was isolated from leukocytes derived from whole blood using a DNA blood kit , as previously described.https://sites.google.com/site/jpopgen/dbNSFP), an integrated functional analysis database of SIFT, Polyphen2, LRT, Mutation Taster, and FATHMM.50We predicted the functional effect of the variants that were detected using the database for nonsynonymous single nucleotide polymorphisms’ functional predictions  and one splice region variant  were observed in patients with SCZ but not in controls  in exon 13 was observed in patients with SCZ, and two nonsynonymous variants (M339I in exon 13 and R134Q in exon 7) were observed in controls after removing the variants that were observed in the healthy cohort database.In EphB2, we observed five nonsynonymous variants  and four nonsynonymous variants  in six patients with SCZ and four controls. R569W was observed in a patient with SCZ and in a control.In CDK5: P=0.062, CSNK2A1: P=0.624, EphB2: P=1.000).The total number of variants in each gene was not significantly different between the two groups . The crystal structure of the cAMP-dependent protein kinase, including CDK5, has been shown to have an N-terminal domain (N lobe) that is composed of mainly ß-sheets, a helical C-terminal domain (C lobe), and a deep ATP-binding cleft between the two lobes.51 According to InterPro (https://www.ebi.ac.uk/interpro/), a database for predicting protein function, P271 is located at the helical C-terminus in CDK5 and belongs to the protein kinase domain that is crucial for its serine/threonine protein kinase activity. The tyrosine (Y1472) within the YEKL motif of NR2B was shown to be phosphorylated by the CDK5, serine/threonine protein kinase. Phosphorylation of this site inhibited the binding of AP-2 to YEKL motif, which is necessary for NR2B endocytosis, and promoted synaptic surface expression of NR2B-containing NMDARs instead of reducing the surface expression of NR2A-containing NMDARs.27 These findings raise the possibility that the P271T found in this study may affect the regulation of the developmental NMDARs switch. Another possibility is that P271T may disrupt the function of CDK5 by interrupting the formation of the CDK5–p25 complex, as the α-helix is a critical structure in the interaction between CDK5 and p25, a co-activator of CDK5.51–53 Furthermore, the C-terminus of the helix is a well-conserved region among spices54 and may lead to a highly damaging effect on its function.In http://research.mssm.edu/statgen/sweden/),15 a rare, nonsynonymous variant, R50W, in CDK5 was detected in patients with SCZ but not in controls. In the subsequent updated exome study of 4877 patients with SCZ and 6203 controls showed that there was one control subject with p. P170L (c.509 C>T) in CDK5 but no missense or nonsense variants in patients with SCZ. R50W was later found in the largest exome database, the Exome Aggregation Consortium. We further combined the data from the largest SCZ exome study and our study to examine whether the number of rare variants in CDK5 was significantly different between SCZ patients and controls, as both studies referred to rare variants with MAF<0.5%. The total number of rare variants within CDK5 was not statistically significant . Additional research using a larger sample size may be required, as the races and pipeline for detecting the rare variants were different between the studies.In a previous large exome study of 2536 patients with SCZ and 2543 controls (55 Thus, the dysregulation of the composition of NMDARs may affect the dopamine levels during early adolescence, thereby affecting synapse maturation.56 Therefore, the involvement of other novel switching regulator genes warrants further research.Recently, excessive dopamine D2 receptor activation during early adolescence was shown to prevent the development of dendritic spines and led to cognitive dysfunction, a core symptom of SCZ, in an NR2B-dependent manner.CDK5 in patients with SCZ but not in controls, although the difference did not show statistical significance. Additional research using a larger sample size or examining other switching regulator genes is warranted.In conclusion, we examined whether the developmental NMDAR subunit switch was genetically involved in the pathophysiology of SCZ by re-sequencing three genes that regulate the composition of NMDARs. We found four novel variants in Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.