The C-P4H α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain, whereas the β-subunit is identical to the enzyme protein disulfide isomerase (PDI). The structure of the N-terminal part of the α-subunit  is known, but the structures of the PSB-CAT linker region and the CAT domain as well as its mode of assembly with the β/PDI subunit, are unknown. Here, we report the crystal structure of the CAT domain of human C-P4H-II complexed with the intact β/PDI subunit, at 3.8 Å resolution. The CAT domain interacts with the a, b’, and a’ domains of the β/PDI subunit, such that the CAT active site is facing bulk solvent. The structure also shows that the C-P4H-II CAT domain has a unique N-terminal extension, consisting of α-helices and a β-strand, which is the edge strand of its major antiparallel β-sheet. This extra region of the CAT domain interacts tightly with the β/PDI subunit, showing that the CAT-PDI interface includes an intersubunit disulfide bridge with the a’ domain and tight hydrophobic interactions with the b’ domain. Using this new information, the structure of the mature C-P4H-II α2β2 tetramer is predicted. The model suggests that the CAT active-site properties are modulated by α-helices of the N-terminal dimerization domains of both subunits of the α2-dimer.Collagen prolyl 4-hydroxylases (C-P4H) are α The catalysis involves oxidative decarboxylation of 2-oxoglutarate to succinate and carbon dioxide and the hydroxylation of procollagen peptidyl prolines into 4-hydroxyprolines. This modification is needed to provide stability for the triple helical collagen molecules. Subsequently, the collagens are secreted into the extracellular matrix where they are further modified and assembled into various supramolecular structures, and where they are involved in, for example, cell adhesion, cell migration, and cell remodeling during growth, differentiation, and wound healing , localized in the lumen of the endoplasmic reticulum, catalyze a vital cotranslational and post-translational modification of procollagen polypeptides required for the assembly of the collagen triple helix , 4. C-P4 healing , 6. Coll healing , 8, 9. Cetramers . The α-setramers , and theetramers , 13. In etramers  14, 15)2 moleculetramers , 17  and α(II) share about 35% sequence identity with α(III). The C-P4H α-subunit of each of the isoforms consists of four parts: the N-terminal (dimerization) domain, the peptide-substrate-binding (PSB) domain (middle domain), a linker region , and the C-terminal catalytic domain  21, 22,,22, 23. Chlamydomonas reinhardtii P4H isoform 1 (Cr-P4H) , both in solved , 27. Likn solved , 29 and n solved , 29. In n solved . In addia, b, b’, and a’ domains . Th. Tha, b,ce motif  and S2. tetramer , 33, 34.tetramer . Also kntetramer . The α-stetramer .2β2 C-P4H-I tetramer, the CAT domains of the two α-subunits point away from the α2-dimer interface  and are capped by the β/PDI subunit, positioned at both ends of the elongated tetramer, suggesting that the β/PDI subunit interacts solely with the CAT domain . Th. Th2β2 CT domain .2β2 heterotetramer, which is also discussed.Here, we report on the characterization of two truncated human C-P4H-II variants in which the α-subunit is lacking either the N-terminal dimerization domain (C-P4H-II-Δ140) or the dimerization domain and the PSB domain (C-P4H-II-Δ281). These truncated variants of C-P4H-II are stable and soluble heterodimer complexes. The C-P4H-II-Δ281 complex could be crystallized, and its crystal structure reveals the structure of the CAT domain as well as its interactions with the β/PDI subunit. This crystal structure, together with the AlphaFold2 (DeepMind company and EMBL-EBI)  model ofEscherichia coli Origami strain (E. coli CyDisCo expression system (A) show that wildtype C-P4H-II and its two truncated variants have the expected secondary structure properties. The CD melting curves of the truncated complexes (B) show Tm values of 50.2 and 53.2 °C for, respectively, C-P4H-II-Δ140 and C-P4H-II-Δ281, slightly higher than the Tm of mature C-P4H-II (Tm = 46.5 °C)  , which c46.5 °C) . Subsequa, b’, and a’ domains of human PDI, as described in the The C-P4H-II-Δ281 complex was crystallized in space group R3. A complete dataset was collected at the P14 beamline  at 3.8 Å resolution . The strThe CAT domain is folded into the typical DSBH-fold , as alsoN-ethylmaleimide (NEM)-labeled sample, in agreement with the presence of a disulfide bridge between Cys294 and Cys504  and Cys504, located in β-strand βVII of the DSBH-fold . The latd Cys504  and S3. d Cys504  for the d Cys504 , 47, 48.d Cys504  and in fd Cys504  and Cr-Pd Cys504  but not d Cys504 , 43.a’, b’, and a domains of PDI, whereas there are no interactions between the CAT domain and the b domain (a’ domain and around Ile324 (α14-helix) with the b’ domain. A third main interaction region is near Cys529 (near the C terminus of the CAT domain), which interacts with the a domain of the β/PDI subunit  include disulfide bridges with Cys397 and Cys53 (of PDI), respectively, which are the proximal cysteines  of the C domains , and S7. domains . The pre domains . This swminantly .Figure 4a’ domain) and Tyr99 (of the a domain), respectively, which are possible because of the conserved cis-(Phe440-Pro441) and cis-(Tyr99-Pro100) peptide bond (a’ and a domains are complemented with interactions of the β2-region (near Glu330) and the βVI–βVII loop region (near Ser492) of the CAT domain with the CGHC loop of the a’ and a domains of PDI, respectively (The sequences around the Cys311 (β1 β-strand of the CAT domain) and Cys529 (C terminus of CAT domain) that interact with the PDI domains are very different  and S10,ide bond  and S7. ectively . Cys311 ectively , and theectively , 48 thatIt is possible that the intersubunit disulfide bridges formed between CAT and PDI are transient disulfide bonds, which are easily converted into intrasubunit disulfide bridges (between the cysteines of the CGHC-cysteines) by nucleophilic attack of the distal cysteine of PDI, as suggested by other studies , 49. Thia and a’ domains (A), and this geometry is within acceptable ranges of disulfide bridges when compared with the geometry of disulfide bridges in other proteins (a domain of ERp57 and tapasin (B). The PDI geometry is also conserved in the structure of the MTP complex when considering the interface of the a’ domain of PDI and the MTP α-subunit (C). In the ERp57–tapasin complex  with the structure of the oxidized a’ domain of the uncomplexed PDI (with a disulfide bond between Cys397 and Cys400) shows no structural differences between these two states. Clearly, the various oxidation states of the PDI domains can be accommodated with minor structural changes.The geometries of the intersubunit disulfide bridges formed between Cys311(CAT) and Cys397(PDI) and between Cys529(CAT) and Cys53(PDI) are very similar, as shown when superimposing the  domains A, and thproteins . This ge tapasin B. The PD-subunit C. In theb’ domain. Various studies on the interactions of PDI with peptides have highlighted the importance of the b’ hydrophobic pocket for the PDI peptide interactions . There is no further sequence or structural similarity in the regions of the α-subunits of the C-P4H-II-Δ281 and MTP complexes, which interact with the b’ domain, but nevertheless, there are no conformational differences between the structural elements of the b’ domain that form the binding pocket in these two complexes. This is consistent with the required promiscuity of substrate binding that this pocket must have as part of PDIs role in oxidative folding. The structure of the C-P4H-II-Δ281 complex suggests that the a’ and b’ interfaces contribute much to the stability of the complex, which is consistent with previous mutational studies, which have shown that the a’ and b’ domains are most important for the assembly of the mature complex  ion, which is consistent with the presence of EDTA in the protein buffer of the crystallization experiment. The main chain and side chains of the fully conserved His-X-Asp----His motif  residue superimp5 substrate peptide, by which the peptide binds in a tunnel, so that residue Pro5 of this peptide points toward the catalytic metal ion (3HQR)  and the βII-βIII loop (residues His148 to Gly159 in Cr-P4H) sequester its (Ser-Pro)etal ion . It can n (3HQR) , the hain (3HQR) . Large sn (3HQR) . For exan (3HQR)  41). In. In5 subn (3HQR) , whereasn (3HQR) .B) 5 peptide that is hydroxylated in the catalytic cycle. This tyrosine, which is Tyr427 in C-P4H-II, is highly conserved in P4H sequences . The notion that the conformation of the βI-βII region of the C-P4H-II-Δ281 complex is different from the conformation competent for catalysis is consistent with the observation that the C-P4H-II-Δ281 complex has low catalytic activity , showing that the flipped-out conformation, as observed in the Cr-P4H structure, is not possible in the structure of the assembly that is captured in this crystal form of the C-P4H-II-Δ281 complex.Previous structural studies of Cr-P4H have shown that conformational flexibility of the βI-βII catalytic loop  , 27 is iequences , and indactivity . The fli2-dimer, using the sequence of the mature α-subunit, starting at Glu22  is required for exchanging the succinate with 2-oxoglutarate after which the next reaction cycle can start. The notion that the procollagen substrate remains bound at the nearby PSB domain at the end of the catalytic cycle, and is not released in bulk solvent, is in line with the observed processivity properties of this enzyme  and C-P4H-II (gene name: P4HA2_HUMAN). These studies were done with the P13674-1 (UniProt) isoform-1 splice variant of C-P4H-I (in which exon 10 is expressed)  and withE. coli expression strain. In the protocol, on day 2, a single bacterial colony was first inoculated in Luria–Bertani medium containing 100 μg/ml ampicillin and grown overnight at 37 °C in a shaker. The overnight culture was diluted on day 3 (100-fold) in 1 l of Terrific broth medium supplemented with 100 μg/ml ampicillin and incubated at 30 °C with vigorous shaking (250 rpm) until the absorbance reached 0.6 to 0.7 at 600 nm. Then the culture was cooled to the induction temperature (20 °C), and the expression was induced by adding 0.5 mM IPTG and continued to grow overnight (∼17 h) at 20 °C (250 rpm). After expression, on day 4, the cells were harvested by centrifugation  and frozen with liquid nitrogen and stored at −70 °C for later use.The expression of the recombinant proteins was initiated by transforming the CyDisCo plasmid, which encodes also the α-subunit, the β/PDI subunit, and Erv1, into the K12 The protein purification was carried out at 4 °C. The frozen pellets, containing the recombinant proteins with the N-terminal 6× His tag in the α-subunit, were suspended in lysis buffer containing 50 mM Tris, pH 7.8, 100 mM NaCl, and 100 mM glycine, supplemented with SIGMAFAST protease inhibitor cocktail tablets (one tablet per 100 ml). The cell pellet was sonicated at 36% amplitude for 7 min with 2 s ON, 4 s OFF pulse on ice bath, and the supernatant was loaded onto a Talon immobilized metal-ion affinity chromatography column, washed with the lysis buffer, and eluted with the same buffer containing also 250 mM imidazole. The protein was then passed through a PD10 column pre-equilibrated with a buffer containing 10 mM Tris, pH 7.8, 50 mM NaCl, and 100 mM glycine. After this, the sample was subjected to anion exchange chromatography  connected to BioLogic DuoFlow purification device  with the same buffer and eluted by using an NaCl gradient from 50 to 1000 mM. All fractions containing the protein  were then pooled and concentrated with 50 kDa molecular weight cutoff Amicon Ultracentrifugal filters (Merck Millipore) before loading them on to a Superdex 200 10/300 GL column  pre-equilibrated with SEC buffer containing 50 mM Tris, pH 7.8, 50 mM NaCl, and 100 mM glycine. All fractions containing the protein were pooled and concentrated to about 3 to 10 mg/ml. Mostly, samples were frozen with liquid nitrogen in 100 μl aliquots (in SEC buffer) before being used for the subsequent experiments. The sample quality was monitored by SDS-PAGE analysis.Peptide MS fingerprints and selected MSMS spectra were used to confirm the presence of the protein loaded on the SDS-PAGE gels after the purifications. Bands were excised, alkylated (DTT/iodoacetamide), and trypsinized using standard procedures. Peptides were eluted from the gel and measured on an UltrafleXtreme Maldi-Tof mass spectrometer (Bruker). Raw data were processed with BioTools and subjected to database search using Mascot (Matrix Science).The SEC-MALS analysis of the purified proteins (in SEC buffer) was carried out using a miniDAWN detector from Wyatt Technologies in on-line mode (connected to the Shimadzu HPLC system). Before entering the MALS detector, the sample flows through an Optilab refractive index detector from Wyatt Technologies. This is used to measure the concentration of the protein sample. Molecular mass and polydispersity measurements were carried out using the ASTRA software from Wyatt Technologies. About 30 to 50 μl of the purified (and frozen-thawed) sample was filtered and loaded with flow rate 500 μl/min onto the Superdex 200 Increase 10/300 GL column pre-equilibrated with SEC buffer (0.1 μm filtered and degassed).Tm calculations as the proteins started to aggregate. Data analysis was carried out using the Chirascan Pro-Data Viewer (Applied Photophysics) and CDNN . The direct CD measurements  were converted into mean residue molar ellipticity ([θ]MR) by Pro-Data Viewer. The Tm was calculated with the Global 3 software (Applied Photophysics) using a one-transition model and using the same wavelength range as aforementioned.Far-UV CD spectra of the purified proteins were recorded using a Chirascan CD spectrophotometer (Applied Photophysics Ltd) in a cuvette with 1 mm path length, using a wavelength range of 190 to 280 nm. The purified protein samples (in SEC buffer) were diluted to 0.1 mg/ml using Milli-Q water, and the final protein concentrations were determined from the absorbance values at 214 nm. The CD data were acquired every 1 nm with 0.5 s as an integration time and repeated three times with baseline correction. The samples were then heated from 22 to 94 °C using a Peltier temperature controller at a rate of 1 °C/min for obtaining a CD melting curve, recording at each temperature the CD spectrum using the same wavelength range as mentioned previously. The data collected between 72 and 94 °C were not used for the 14C]glutarate (PerkinElmer)  liquid scintillation counter. The results were calculated as disintegrations per minute per enzyme active site, taking into account that the number of C-P4H active sites is two per mature C-P4H-II tetramer and one per heterodimer for the truncated variants.Two assays were used to characterize the enzymatic properties of C-P4H-II and its truncated variants. First, the catalytic activity of the purified enzymes was measured by an indirect activity assay based on the hydroxylation-coupled decarboxylation of 2-oxo[1-inElmer) . About 1ct assay , based om/z range of 387 to 5000. The mass spectra were further internally recalibrated using monoisotopic masses of the selected peptides, whose sequences were confirmed by MS/MS (collision-induced dissociation) measurements. The peptide monoisotopic masses were obtained by using a built-in SNAP2 peak picking algorithm and were then searched against the C-P4H-II sequence with GPMAW 9.2 software (Lighthouse Data) and ProteinProspector (https://prospector.ucsf.edu/prospector/mshome). Because of the broad cleavage specificity of pepsin, the peptide mapping was performed as follows. The peptides with a single hit were assigned first, and the observed cleavage sites of these peptides were then used to assign the other peptides with multiple possible locations. No restrictions were applied to the digestion sites, that is, nonspecific cleavage was used, and only the peptides with less than 10 ppm mass error were considered. All possible disulfide linkages were initially considered in the search of the peptides including intersubunit and intrasubunit ones by searching α- and β-subunits simultaneously. The search was repeated by limiting the disulfide pairing to those observed in the crystal structure. The identified peptides as well as the sequence coverages in each of the three experiments are listed in The MS analyses were performed with native (untreated), DTT-reduced, and NEM-labeled samples of the mature C-P4H-II (in SEC buffer). The treatment with DTT reduces disulfide bridges, and the treatment with NEM will modify free SH groups. First, the protein solution was buffer-exchanged to 100 mM ammonium acetate (pH 6.8) by using a PD-10 column (Cytiva). In the next step, three 50-μl aliquots were taken from the C-P4H-II sample (3.5 μM), the first being the native sample, the second reduced with DTT , and the third treated with NEM . In the third step, each sample was diluted  with ammonium acetate (pH 2.6) (to reduce pH to around 4.1 to facilitate on-line pepsin digestion and to prevent cysteine oxidation). The on-line pepsin digestion tubing was prepared as follows : a 50-μlExtensive crystallization screening was performed with the C-P4H-II-Δ281 complex. After SEC, the protein (in SEC buffer) was concentrated to 11 mg/ml by using 50 K Amicon Ultra-15 Centrifugal Filter concentrator (Merck). The protein was subjected to extensive crystallization screening by using the equipment of the BCO, University of Oulu Structural Biology core facility. Initial screening for crystallization was carried out by the sitting drop vapor diffusion method using in-house screens in IQ 96-well triple sitting drop plates (TTP Labtech) at two different temperatures (22 and 4 °C). The protein and precipitant solutions were mixed together in three volume ratios  in a final volume of 300 nl with the help of the Mosquito LCP nanodispenser (TTP Labtech). The plates were imaged using Formulatrix Rock Imagers RI54 (at 22 °C) and RI27 (at 4 °C). The crystallization results were monitored with the IceBear software expert system .The first, poorly diffracting, crystals of the CP4H-II-Δ281 complex were obtained at 4 °C using a well solution of 0.1 M MES, pH 6.0, containing 20% (w/v) PEG4000, 0.2 M lithium sulfate  at a volume ratio of 1:2 for the protein and well solutions, respectively. In subsequent purifications, the SEC buffer of the last step of the purification protocol was supplemented with 1 mM EDTA, and in further crystallization experiments, several additive screens were tested to improve the crystal quality. The best crystals were eventually obtained at 4 °C by mixing equal volumes of a protein solution of 16.5 mg/ml in 50 mM Tris–HCl, pH 7.8 (at 4 °C), 50 mM NaCl, 100 mM glycine, 1 mM EDTA, with a well solution of 0.1 M MES, pH 6.0 (at 4 °C), 20% (w/v) PEG4000, 0.2 M lithium sulfate, and 3% methanol. The initial crystal testing was done in the BCO Structural Biology core facility/Biocenter Finland national data collection core facility, using the in-house Microstar X8 Proteum X-ray generator (Bruker). Crystals were cryoprotected, before cryocooling by immersing in liquid nitrogen, by quick transfer (few seconds of equilibration) in a cryobuffer . All the crystal fishing and cryocooling protocols were performed in the cold room at 4 °C. Many crystals were tested with the home source X-ray generator, and the best crystals (showing diffraction between 6 Å and 9 Å resolution) were stored and shipped to synchrotrons . The best dataset (3.8 Å resolution) was collected at the beamline P14, PETRA III (DESY), EMBL Hamburg. The X-ray images were processed by XDS , and the2JIG)  domains of the β/PDI subunit were positioned using Phaser. The a and a’ domains were found by using as search models the structures of these domains, as present in the reduced (“open”) form of human PDI (PDB entry: 4EL1)  was superposed onto the already positioned a and b' domains using Coot    was used2JIG) . Such a y: 4EL1) , and thery: 6I7S ). The PDry: 6I7S , Refmac5ry: 6I7S , and Lowry: 6I7S  gave Rwoing Coot , and subLimited) , and modLimited) . In thisAt this stage, the predicted models of the AlphaFold2 , 69 and Rwork/Rfree in the last BUSTER refinement run before and in the first BUSTER refinement run after the Refmac5 series were 27.8%/30.4% and 26.0%/28.0%, respectively. The slow progress of the refinement is possibly associated with the inaccuracy of the initial positioning of the domains of the less ordered β/PDI subunits and smaller gradients for their atoms during refinement .The final structure is the result of three additional series of refinement with BUSTER, Refmac5, and again BUSTER. Model correction in Coot was done using BUSTER maps, whereas the intermediate Refmac5 series helped pushing optimization forward. B-factor (5–7 “big” cycles or 20 cycles). In both protocols, the relative weight between X-ray and geometry terms was estimated by the respective program. B-factors of all atoms were reset to 50 Å2 prior to each refinement run. In the TLS refinement cycles, the entire CAT domains and individual domains of the β/PDI subunits were assigned as TLS groups . TLS parameters were recalculated from scratch in each run. The AlphaFold2 models of the CAT domain and the β/PDI subunit were generated using AlphaFold2, as implemented at the EBI (The refinement protocols for BUSTER and Refmac5 were similar and included TLS refinement (one “big” cycle in case of BUSTER or 10 cycles in case of Refmac5) followed by restrained refinement of atomic coordinates and the overall  the EBI , and the the EBI . In case the EBI , and weiRwork = 24.4%, Rfree = 27.8%, as calculated by BUSTER) are listed in a’ and b’ domains of both β/PDI subunits are nicely defined by the electron density map . Inventor: L. W. R.A patent for the production system used to make the protein for structural studies using sulfhydryl oxidases in the cytoplasm of J. M. owns equity in FibroGen, Inc, which develops HIF-P4H inhibitors as potential therapeutics. This company supports research in the J. M. group.All the other authors declare that they have no conflicts of interest with the contents of this article.


PIK3CA, encoding p110α, is frequently mutated in human cancers. Most PIK3CA mutations are clustered in the helical domain or the kinase domain. Here, we report that p85β disassociates from p110α helical domain mutant protein and translocates into the nucleus through a nuclear localization sequence (NLS). Nuclear p85β recruits deubiquitinase USP7 to stabilize EZH1 and EZH2 and enhances H3K27 trimethylation. Knockout of p85β or p85β NLS mutant reduces the growth of tumors harboring a PIK3CA helical domain mutation. Our studies illuminate a novel mechanism by which PIK3CA helical domain mutations exert their oncogenic function. Finally, a combination of Alpelisib, a p110α-specific inhibitor, and an EZH inhibitor, Tazemetostat, induces regression of xenograft tumors harboring a PIK3CA helical domain mutation, but not tumors with either a WT PIK3CA or a PIK3CA kinase domain mutation, suggesting that the drug combination could be an effective therapeutic approach for PIK3CA helical domain mutant tumors.PI3Ks consist of p110 catalytic subunits and p85 regulatory subunits.  The mechanisms behind the oncogenic role of the PIK3CA helical domain mutant is poorly understood. Here, the authors show that its oncogenic function depends on the release of p85β from mutated p110α, its translocation to the nucleus and the consequent increased activity of EZH proteins. PIK3CA, which encodes the p110α catalytic subunit of PI3 kinase, is one of the most frequently mutated oncogenes in human cancers3. Recently, the FDA approved the combination of p110α-specific inhibitor Alpelisib and the estrogen receptor antagonist Fulvestrant to treat PIK3CA-mutant breast cancer patients whose tumors are hormone receptor (HR)-positive and HER2-negative4. This approval highlights mutant PIK3CA (encoding p110α protein) as a critical cancer drug target and the importance of further understanding the molecular mechanisms by which PIK3CA mutations drive tumorigenesis. PI3Ks are heterodimers consisting of a p110 catalytic subunit and a p85 regulatory subunit5. Normally, the p85 subunits bind and stabilize the p110 subunit and inhibit its enzymatic activity6. Upon growth factor stimulation, the SH2 domains of p85 bind to the phospho-tyrosine residues on receptor protein kinases or adaptor proteins such as insulin receptor substrate 1 (IRS1), which activates PI3K and catalyzes the conversion of phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3)5. PIP3 recruits pleckstrin homology domain-containing proteins, including PDK1 and AKTs, to the cell membrane to activate signaling pathways5.PIK3CA mutations are clustered in two hotspots in p110α protein: the E542, E545, and Q546 residues in the helical domain and the H1047 residue in the kinase domain7. Accumulating evidence suggests that the helical domain mutations and kinase domain mutations exert their oncogenic functions through distinct molecular mechanisms. p110α helical domain mutations alleviate the inhibitory interaction between p110α and the N-terminal SH2 domain (nSH2) of p85α and β, and facilitate direct interaction of p110α with IRS112. The E545K helical domain mutation in p110α does not increase lipid kinase activity in response to phosphorylation of receptor tyrosine kinase or adaptor proteins12. Moreover, the p110α E545K helical domain mutant requires the Ras-binding domain to transform chicken embryonic fibroblasts13, whereas the H1047R kinase domain mutant requires the p85-binding domain. Phenotypically, breast cancer cells expressing a PIK3CA helical domain mutation showed more severe metastatic phenotypes than that of wild-type or PIK3CA H1047R mutation in an isogenic background14. Furthermore, patients with a PIK3CA H1047R mutation have a better response to PI3K/AKT/mTOR inhibitors than patients with a PIK3CA helical domain mutation in early phase clinical trials16.Most tumor-derived PIK3R1) and p85β (encoded by PIK3R2) are ubiquitously expressed, but they seem to play opposite roles in tumorigenesis17. Loss-of-function mutations of p85α frequently occur in endometrial and brain cancers19, suggesting that it normally functions as a tumor suppressor21. In contrast, p85β often is overexpressed in diverse cancers and depletion of p85β impairs tumor formation in vivo and in vitro26, suggesting that it plays an oncogenic role in tumorigenesis. Intriguingly, several studies have demonstrated that p85 proteins can regulate biological processes through nuclear translocation31. It has been shown that p85α and p85β interact with nuclear Bromodomain-containing protein 7 (BRD7) and X-box binding protein 1 (XBP1) to regulate glucose homeostasis31. However, it remains unknown whether nuclear p85 proteins play an important role in tumorigenesis.The regulatory subunits of PI3K p85α . Moreover, we demonstrate that a combination of Alpelisib and an EZH inhibitor, Tazemetostat, induces regression of xenograft tumors harboring a 12. To gain insights into how the p110α helical domain mutations impact PI3K complex formation, we immunoprecipitated either wild-type (WT) p110α or p110α E545K mutant protein in cell lines with the WT or mutant endogenous p110α proteins tagged with 3 × FLAG , GSK2816126 , a combination of BYL719 and EPZ-6438, or a combination of BYL719 and GSK2816126.Alpelisib (BYL719) and Tazematostat (EPZ-6438) were dissolved in 0.5% carboxymethylcellulose sodium salt (CMC). GSK2816126 was dissolved in 20% Captisol. Once tumor sizes reached 100–150 mmKR-AA mutant cell using standard procedures. Briefly, total RNAs were extracted using TRIzol reagents, and Illumina high-throughput sequencing libraries were constructed and sequenced according to the manufacturer’s instructions. The raw paired-end reads were trimmed and quality controlled by SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) with default parameters. To identify differential expression genes (DEGs) between the two samples, the expression level of each transcript was calculated according to the fragments per kilobase of exon per million mapped reads (FPKM) method. Differential expression of genes  were analyzed and selected for subsequent analysis.High-Throughput RNA sequencing was performed in DLD1 parental cell and DLD1 p85β NLSKR-AA mutant cells were sonicated and immunoprecipitated with an anti-H3K27me3 antibody. Purified ChIP DNA was verified on an agarose gel to ensure proper fragmentation for Illumina library construction. Next-gene sequencing was performed using an Illumina Hi-Seq 2000 machine. Raw reads  were aligned to human reference genome hg19 using bowtie (v1.1.0), allowing up to one mismatch. Peak calling was performed using model-based analysis of ChIP–seq  with a cutoff of P ≤ 1 × 10−8. Promoter regions were defined as regions 3 kb upstream to 3 kb downstream of the TSS. The H3K27me3 occupancies at promoter regions were normalized as reads per kilobase per million reads (RPKMs). Fold changes and significance of H3K27me3 differences between conditions were determined using an MA-plot-based method with a random sampling model, which was implemented in the R package DEGseq. Enriched regions were determined by the HOMER program  with an FDR value cutoff of 0.05. ChIP-seq density heatmaps and histograms were generated using ngs.plot. The validation experiment was performed using ChIP-PCR with primers listed in Table Chromatin Immunoprecipitation (ChIP) was performed using the SimpleChIP Enzymatic Chromatin IP Kit  following the manufacturer’s instructions. Chromatin from DLD1 and DLD1 p85β NLS51. Briefly, paraffin-embedded mouse and human tissues were deparaffinized in xylene and antigen retrieved by boiling the sample for 20 min. Samples were incubated with primary antibodies at 4 °C overnight. The sections were stained with secondary antibodies for 30 min at room temperature and then stained with an EnVision-HRP kit (Dako).Immunohistochemistry was performed as described previouslyThe dataset files of colorectal cancer (COAD), bladder carcinoma (BLCA), endometrial carcinoma (UCEC), and breast cancer (BRCA) were downloaded from the TCGA website. The files include the RNA-seq files providing normalized FPKM values, the somatic mutations, and the 5-year survival of each patient.For gene expression analysis, FPKM values of indicated genes from tumor samples and corresponding normal tissue samples, if available, were plotted. The statistical significance difference of tumor versus non-tumor was calculated using the student t-test.PIK3R2 expression with 5-year survival, patients were divided into three groups according to their PIK3CA mutation status: helical domain mutation group , non-helical domain mutation group (Patients with PIK3CA mutation at H1047 and other sites) and wild type group (Patients with wild-type PIK3CA), and then patients were further divided into PIK3R2 high and PIK3R2 low according to the median expression of PIK3R2 in each group. Due to the limitation of the patient number of helical domain mutation groups in individual tumor type, PIK3R2 high and PIK3R2 low in each group of four tumor types were combined to assess the relevance of PIK3R2 expression and 5-year survival of all patients. Kaplan-Meier analysis of 5-year survival was performed with a Cox proportional hazards model.To analyze the association of t-test to compare the means between the two groups, assuming unequal variances. For xenograft growth, we carried out ANOVA for repeated measurements to test whether there is an overall difference in the tumor sizes by testing group differences as well as whether there was a difference in the development of tumor sizes over time between the two groups by testing the interaction between time and group. The combinatorial effect of drug treatment in vivo was evaluated by combination ratio using the fractional product method. FTV  = mean of final tumor volume of treated group/mean of final tumor volume of the control group. Expected FTV = (FTV of agent1) * (FTV of agent2). Observed FTV = mean final tumor volume of combinatorial treatment/means final tumor volume of the control group. Combination ratio = expected FTV/observed FTV. Combination ration > 1, = 1, or < 1, was defined as synergy, additive effect, or antagonism, respectively.GraphPad Prism software was used to create the graphs. Data are plotted as mean ± SEM. We applied the two-sided Further information on research design is available in the Supplementary materialsPeer Review FileVideo 1Video 2Video 3Reporting Summary


Gossypium hirsutum was studied using sequence and structure bioinformatic tools. The encoded protein (UniProt A0A1U8MGX4) was predicted to localize to the nucleus, was found to retain the B3 transcription factor domain with conserved DNA-binding residues and to most closely cluster with REM subfamily members of B3-domain containing proteins.A gene of unknown function, Gohir.A02G044702.1, identified in IntroductionGossypium hirsutum (L. accession Texas Marker-1 (TM-1) version 2.0 and annotation version 2.1) , the gene, LOC107936594 (CottonGen: Gohir.A02G044702_UTX-TM1_v2.1), was identified to encode a conserved protein of unknown function labeled 'B3 domain-containing protein At1g05920-like' . Here we present evidence that supports this protein, referred to here as GhB3-A0A1U8MGX4, is part of the B3 domain-containing family, likely within the highly diverse REM subfamily, and contains the sequence and structure features required for nuclear-localization and DNA-binding to function as a transcription factor (TF). Members of the B3 superfamily all contain a B3 domain, a ~100 amino acid DNA-binding domain specific to plants and found in even unicellular green algae . It is prevalent in flowering plants with around 100 B3-containing proteins identified in Arabidopsis . This superfamily is involved in regulating many key processes, including stress and hormone responses , embryogenesis  and development . The superfamily has been classified phylogenetically into four subfamilies: LAV (LEC/ABI/VAL), RAV (Related to ABI3/VP1), ARF (Auxin Response Factor) and REM (Reproductive Meristem), which differ in their DNA recognition sequence .In a recent sequencing of the genome of upland cotton,Sequence FeaturesFigure 1A). ConSurf  was used to calculate the evolutionary conservation of each residue and the most highly conserved residues were found within the DUF 313 domain region (amino acids 120-213), these residues are indicated in the multi-sequence alignment by asterisks . The full ConSurf results are available as Extended Data.InterPro webserver identified the 241–amino acid GhB3-A0A1U8MGX4 protein as a member of the ‘DNA-binding pseudobarrel domain’ superfamily (IPR015300) and the ‘B3 domain-containing protein At2g31720-like’ family (IPR005508) with a domain of unknown function DUF 313 (PF03754). This plant-specific family of unknown function includes At2g31720, Auxin Response Factor 70 , a protein linked to stress response through yeast one-hybrid screening of TF-promoter interactions . Sequence analysis of GhB3-A0A1U8MGX4 by subcellular localization programs BUSCA and LOCALIZER predicted a nuclear localization sequence (NLS), KRKR, in the N-terminal region, in agreement with the function as a transcription factor. A domain architecture was created to visualize these sequence features . The cotton paralogs separate into two distinct clusters with the most closely aligned Arabidopsis sequences to GhB3-A0A1U8MGX4  being proteins of unknown function but listed as belonging to the REM subfamily of B3 domain-containing proteins .Of the 40 plant and 10 non-plant genomes used by the PhyloGenes webserver, homologs of GhB3-A0A1U8MGX4 were found in 25 species, confined to magnoliophyta (angiosperms), which agrees with the species listed by pfam for DUF 313 domain (PF03754) containing proteins. Arabidopsis ARF70 (Q8RV83) is a listed homolog but none of the other proteins have been functionally characterized or named. This indicates that these proteins belong to a set of B3 domain-containing proteins distinct from any currently characterized member of this family. PhyloGenes identified 14 cotton paralogs and 23 Arabidopsis orthologs of GhB3-A0A1U8MGX4. ClustalOmega was used to create a multi-sequence alignment (available as Extended Data) and a phylogenetic tree (Neighbor-joining) of these sequences . The model showed a high level of confidence across amino acids 111-241, which includes the entirety of the DUF 313 domain region. This domain showed a seven-stranded, beta-sheet open barrel flanked by two short alpha helices characteristic of B3 DNA binding domains . The lower confidence N-terminal 110 amino acids were deleted from the structure for remaining structure analyses.The AlphaFold tool in UCSF ChimeraX  was used to predict a structure model (available as Extended Data) for the GhB3-A0A1U8MGX4 protein . The multi-sequence alignment (MSA) shows the domain region where the greatest similarity was seen. The GhB3-A0A1U8MGX4 sequence retains many of the residues conserved across subfamilies  along with a number of residues identified as involved in DNA binding in other homologs (yellow boxes) which ConSurf also identified as evolutionarily conserved (asterisks).A structure analog search using the DALI server  identified structures of B3 domain-containing proteins from each of the four subfamilies  that closely matched the GhB3-A0A1U8MGX4 model  even though sequence similarity was low (10-27%). Two structures were chosen from each subfamily and their sequences were aligned with GhB3-A0A1U8MGX4 using ClustalOmega and ESPript3 to create a multi-sequence alignment . Subfamilies have been reported to have characteristic motifs within the DNA-interacting C-arm loop  . These motifs can be seen for each subfamily within the MSA but it is interesting that the GhB3-A0A1U8MGX4 C-arm loop sequence doesn’t match any of these motifs. Differences in this region have been attributed to DNA-binding specificity so this could indicate that GhB3-A0A1U8MGX4 binds to a distinct DNA sequence. This is still in agreement with belonging to the REM subfamily as it is known to be the largest and most diverse subfamily with highly diverged DNA binding .Figure 1E). There is good alignment (r.m.s.d. of 1.014 angstroms over 53 of 96 pruned atom pairs) with DNA-interacting N- and C-arm loops associating with the major groove. A characteristic feature of B3 DNA-binding domains is a large positively charged area around the DNA binding site . The GhB3-A0A1U8MGX4 model contains a similar distinct and large area of positively charged residues in the modeled binding site. Mapping of ConSurf conservation scores onto the structure indicates that these residues are also highly conserved .DNA was modeled into the predicted binding site using a structure overlay between the GhB3-A0A1U8MGX4 model and a LEC2-DNA structure (PDB 6J9C) using UCSF ChimeraX MatchMaker (ConclusionEvidence from the sequence, homology and structure analyses support GhB3-A0A1U8MGX4 being a B3 domain-containing protein within the REM subfamily, with a retained DNA-binding site. The nuclear subcellular localization and conserved and charged binding site features indicate a DNA binding function such as a transcription factor, similar to other members of this family, although potentially with a unique DNA recognition sequence. With a distinct binding motif and largely uncharacterized homologs, this protein could belong to a new subset of REM proteins involved in regulating a distinct plant process, likely specific to angiosperm species.10.22002/D1.20239Description: ConSurf sequence conservation results for A0A1U8MGX4 . Resource Type: Dataset. DOI:10.22002/D1.20240Description: AlphaFold modeled structure of A0A1U8MGX4. Resource Type: Model. DOI:10.22002/D1.20241Description: ClustalOmega MSA of PhyloGenes phylogenetic tree. Resource Type: Dataset. DOI:


Extracellular vesicles (EVs) are known to have a significant role in the central nervous system (CNS) and neurodegenerative disease.PubMed, Scopus, ISI Web of Science, EMBASE, and Google Scholar were used to identify published articles about EV modifications (2012 to Feb 2022).In total, 1,435 published papers were identified among the searched articles, with 1,128 non-duplicate publications being identified. Following the screening of titles and abstracts, 214 publications were excluded; following the full-text screening of 93 published articles, another 33 publications were excluded. The remaining 60 studies were considered. The kappa statistic of 0.868 indicated that the raters were highly reliable. Furthermore, the inter-reliability and intra-reliability coefficients were found to be 0.931 and 0.908, respectively, indicating strong reliability and consistency between the eligible studies identified by the raters. A total of 27 relevant studies demonstrated the role of EVs as therapeutic and diagnostic biomarkers in neurodegenerative diseases. Of note, 19 and 14 studies, respectively, found EVs to be pioneering in diagnostic and therapeutic roles.EVs play an important role in the central nervous system (CNS), aiding in cell-to-cell communication and serving as a diagnostic marker and therapeutic target in a variety of neurodegenerative diseases. EVs are the home of several proteins [including-synuclein (-syn) and tau proteins], lipids, and genetic materials such as DNA and RNA. The presence of novel miRNAs in EVs suggests biomarkers for the diagnosis and screening of neurodegenerative disorders. Furthermore, EVs play an important role in the pathogenesis of such disorders. This systematic review discussed the current state of EVs’ role in neurological diseases, as well as some preclinical studies on the therapeutic and diagnostic potential of EVs. Extracellular vesicles (EVs) are defined as naturally releasing lipid bilayer delimited vesicles from a cell that cannot replicate. It, which is secreted by the cells, mediates conserved intercellular communications through the content present in their lipid bilayer . The ISEvia the endosomal sorting complex required for transport (ESCRT) dependent and independent pathway. The ESCRT-dependent sorting of cargo involves sequential interaction of ESCRT subunits I, II, and III followed by Vps4 protein for initiating the budding and detachment process from the ESCRT III complex  recommendations .By searching PubMed, Scopus, ISI Web of Science, EMBASE, and Google Scholar for all articles on EV modifications, published articles were chosen (2012 to Feb 2022). Searches were conducted using the following keywords: “extracellular vesicles” [Medical Subject Headings (MeSH)] OR “exosomes” [Medical Subject Headings (MeSH)] OR “Neurodegenerative disease” (MeSH) OR “therapeutics” (MeSH) OR “diagnostic biomarker” (MeSH). Two independent investigators reviewed the titles, aims, and abstracts of published articles to determine eligibility. Full-length articles were evaluated by the same investigators, and inclusion and exclusion criteria were applied to each article. Furthermore, the same researchers screened the references of the initial eligible articles to identify all eligible articles for inclusion in the final list. We calculated the overall risk biasness and inter-reliability agreement.The quality of the systematic reviews implicated in the present systematic review was evaluated using the GRADE system .In brief, the GRADE scoring system has four levels of evidence including very low, low, moderate, and high. All eligible studies included in this systematic review were assessed for the following characteristics: imprecision, inconsistency, risk of bias, indirectness, and biasness of publication. Publication bias referred to the application of EVs in the extracted data of reviewed papers and the highlights drawn from the results of such studies because studies without references and publications are not available. The validity of the eligible studies was independently assessed by the two reviewers using a standard checklist. The index of inter-and intra-rater agreement, calculated using the kappa statistic, was used to evaluate the items relevant to the review.For this study, research papers screened during the literature search met the following inclusion criteria: this study included the following topics: (1) EVs; (2) neurodegenerative diseases; (3) therapeutic and diagnostic biomarkers; (4) exosomes; and (5) a published original article with all full-text literature and properly cited references. The following studies were excluded: (1) insufficient reported data with uncited references; (2) published conference proceedings; (3) published review articles, letters, or text written in a language other than English; or (4) repetition of previously published articles.The names of the authors, the year of publication, the country of origin, and the mode of EV application (therapeutic or diagnostic biomarker) were extracted. The majority of articles decipher the role of EVs in neurodegenerative diseases, either as a therapeutic/diagnostic biomarker or both.A total of 1,435 articles were screened, and 1,128 non-duplicate publications were found. Following the screening of titles and abstracts, 214 publications were excluded; following the full-text screening of 93 published articles, another 33 publications were excluded. As shown in the PRISMA flow diagram, the remaining 60 studies were included in this systematic review .Study characteristics of the 27 eligible studies showed that most authors demonstrated the role of EVs as therapeutic and diagnostic biomarkers in neurodegenerative diseases as shown in The systematic review of the published articles was found consistent in validity appraisal among the two raters, as assessed using a kappa statistic of 0.868. Inter-reliability coefficient and intra-reliability coefficient showed strong agreement between the reviewer’s decisions as shown in In the present systematic review, the role of EVs in neurodegenerative diseases was evaluated. Altogether, the findings suggested that EVs can be used both as diagnostic biomarkers and therapeutics in neurodegenerative diseases. EVs are nano-sized lipid bilayer vesicles sized between 30 and 1,000 nm that are released by several cell types including healthy and diseased cells. These secreted EVs play an important role in intercellular communication mediated through their biological content . Previous ample evidence suggested that EVs are the carrier of mRNAs, miRNAs, long non-coding RNAs, and proteins that are associated with the neurodegenerative changes in the body, which may be exchanged between cells of the target. Bestowing this reason, EVs attract special space for acting both as diagnostic markers and therapeutics in neurodegenerative diseases. The majority of studies observed the role of EVs in both diagnostics and therapeutics . NotablyOne of the previously published studies performed on cell culture showed the presence of proteins susceptible to aggregation, which was proven a classical hallmark in neurodegenerative diseases . A diseaNonetheless, EVs are considered to be an ideal reservoir for specific biomarkers of neurodegenerative changes. EVs are released by all cells including the CSF, blood, and urine, which will be a beneficial approach for the detection of the brain and neural cell-related changes. EVs are resistant to enzymatic degradation and thereby provide stability during the processing and isolation of EV biomarkers. Therefore, EVs explore the potential of liquid biopsy for the investigation of neurodegenerative diseases.A previously published study observed that EVs of neurodegenerative diseases exhibit the presence of pathogenic proteins including α-synuclein (α-syn) and tau proteins, which serve as diagnostic biomarkers and therapeutic targets in such diseases . Over thExtracellular vesicles possess the ability to cross the blood-brain barrier (BBB) and have the potential to efficiently deliver therapeutic molecules into the cells providing an attractive opportunity for delivering mRNAs, miRNAs, and drugs to show a protective effect in neurodegenerative diseases. A previously published study showed that astrocyte-derived EVs (AEVs) exhibit neuroprotection in ischemic stroke conditions . AEVs shExtracellular vesicles are known to play a dual role in diagnostics as well as therapeutics in neurodegenerative diseases. In one of the previously published studies, it was demonstrated that intraperitoneal injection of EVs derived from umbilical cord MSCs showed improved cognitive outcomes through the decrease in neurological severity scores and improved reflex and sensation mediated by an HDAC1-Dependent EGR2/Bcl2 Axis . SimilarExtracellular vesicles exhibit promising therapeutic and diagnostic uses in biomaterials. These tailored nano-vesicles can be loaded with desired biomolecules, such as proteins, lipids, nucleic acids, and drugs, using different modification approaches to obtain functionalized EVs. These functionalized EVs can be exploited in the treatment of various diseases, including neurodegenerative diseases. In the recent decade, there has been an increasing trend of research into exosomes, which has expanded into neurodegenerative diseases. The body of evidence demonstrates that exosomes play an important part in communication in the brain. On understanding the physical nature of exosomes, it may be possible to manipulate their contents to deliver therapeutic factors to delay the onset of neurodegeneration. Bringing the field of immunology, neurology, and oncology along with infectious diseases could help in exploiting all the aspects of EVs for giving an understanding pathogenesis and management of human diseases. Combining immunology, neurology, oncology, and infectious diseases could aid in the exploitation of all aspects of EVs for understanding the pathogenesis and management of human diseases.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.AR, G-BJ, MS, RG, and SA: conceptualization. AR and G-BJ: methodology. AR: software, investigation, and writing—original draft preparation. G-BJ, KG, SA, MS, RG, and SK: validation. G-BJ, SA, MS, RG, and SK: formal analysis. SK: resources and project administration. AR and KG: data curation. KG, RG, SA, MS, and G-BJ: writing—review and editing. SA, MS, RG, and SK: visualization. SA, MS, RG, G-BJ, and SK: supervision. All authors contributed to the article and approved the submitted version.


Geobacillus 12AMOR1, and the first one representinga non-permuted GH70 enzyme, reveals high structural similarity inthe core domains with most GtfBs, featuring a similar tunneled activesite. We propose that GtfC (and related GtfD) enzymes evolved fromstarch-degrading α-amylases from GH13 by acquiring α-1,6transglycosylation capabilities, before the events that resulted incircular permutation of the catalytic domain observed in other GH70enzymes . AlphaFold modelingand sequence alignments suggest that the GbGtfC structure representsthe GtfC subfamily, although it has a so far unique alternating α-1,4/α-1,6product specificity, likely determined by residues near acceptor bindingsubsites +1/+2.GtfC-type 4,6-α-glucanotransferase(α-GT) enzymes fromGlycoside Hydrolase Family 70 (GH70) are of interest for the modificationof starch into low-glycemic index food ingredients. Compared to therelated GH70 GtfB-type α-GTs, found exclusively in lactic acidbacteria (LAB), GtfCs occur in non-LAB, share low sequence identity,lack circular permutation of the catalytic domain, and feature a single-segmentauxiliary domain IV and auxiliary C-terminal domains. Despite thesedifferences, the first crystal structure of a GtfC, GbGtfC-ΔCfrom  More recently, however,enzymes with 4,6-α-GT reaction specificity were also characterizedin non-LAB species, sharing low sequence similarity with GtfB enzymes(<30%); these were designated the GtfC subfamily.18 Of the 30 putative GtfC enzymes found in public databasesby 2018,2 four have been biochemicallycharacterized;22 among them are enzymes from thermophilic bacteria, increasing thepotential of these enzymes in an industrial setting, as they wereable to convert starch into linear isomalto-/maltooligosaccharidesat high temperatures (60–68 °C).22 For example, adding the Geobacillus 12AMOR1 GtfC(GbGtfC) enzyme during bread baking showed antistaling effects. Inaddition to GtfCs, a few 4,6-α-GT enzymes with even lower sequencesimilarity were identified in (plant-associated) bacteria. The characterizedenzymes in this group synthesized reuteran-like branched α-glucansinstead of linear products,24 thus defining anotherGH70 4,6-α-GT subfamily (GtfD).Starch is a major energy-providingingredient in many of our foods;it is digested by starch-degrading human enzymes in the gastrointestinaltract. The action of these enzymes, such as α-amylases and glucosidases,may result in an undesirably rapid release of glucose in the blood,increasing the risk of cardiovascular diseases in the long term.26 and proposed a reaction scheme involving sliding of intermediateproducts through the binding groove. We then hypothesized that thesubstrate and product specificity of different GtfB-type 4,6-α-GTsis related to the accessibility of the active site binding groove,which is defined by two loops (A1 and B). A (phylogenetic) surveysuggested that about 80% of enzymes in the GtfB subfamily featurea tunneled binding groove,26 while theremaining ones are (much) more open, allowing for the processing ofbranched substrates and products. The so far characterized GtfC-type4,6-α-GTs generated linear products, although it has to be notedthat the tested substrates were also largely linear. To date, no GtfCprotein 3D structures have been reported; given the low sequence identitywith GtfB-type 4,6-α-GTs (<30%) the question is whether GtfCsfeature a tunnel or not and if a similar diversity with regard toactive site openness exists. Interestingly, the GtfC from Geobacillus 12AMOR1 (GbGtfC) was found to have a uniqueproduct specificity.22 With a limited hydrolyticactivity, GbGtfC releases mainly maltose instead of glucose from amyloseV or maltoheptaose substrate, synthesizing a main product containingalternating α-1,4/α-1,6 linkages instead of consecutiveα-1,6 linkages. This suggests that GbGtfC exclusively transfersmaltosyl units instead of glucosyl units, but the structural detailsthat confer this property remain to be uncovered.The transglycosylationreaction catalyzed by GH70 4,6-α-GTsinvolves three catalytic residues (two Asp and one Glu) and has beendescribed by two half-reactions, each involving an oxocarbenium-iontype transition state, stabilized by an Asp residue. The first half-reactionis α-1,4 specific cleavage of the substrate and results in acovalent enzyme-glycosyl intermediate, which is transferred with α-1,6specificity to an acceptor substrate in the second half-reaction,leading to isomalto-/maltooligosaccharide (IMMO/IMMP) products containingα-1,6 linked units at the non-reducing end. Previously, we structurallycharacterized 4,6-α-GT enzymes of the GtfB subfamily8-barrel in the catalytic domain A, as is the case in GH13α-amylases belonging to the same clan GH-H.28 Despite this absence of permutation, allseven conserved sequence regions I–VII found in GH-H enzymeswere predicted to be present.18 Second,GtfCs were predicted to lack domain V and to have a single-segmentdomain IV. This domain IV was proposed to have been inserted intodomain B of an ancestor α-amylase of the GH13_5 subfamily, whichmainly originate from bacteria and also act on starch-like substrates,but lack this domain IV.28 Finally, some GtfC-type enzymeswere predicted to feature additional C-terminal domains of the bacterialIg (type 2) fold.18 Phylogenetic analysis and predicteddomain organization lead to the hypothesis that the GtfC subfamilyrepresents an intermediate in a linear evolutionary pathway betweenGH13_5 α-amylases and GtfB-type 4,6-α-GTs.18 Yet, since no GtfC 3D structures have been reported, it is stillunknown whether GtfCs resemble more the α-amylases or the GtfBsstructurally.Importantly,the GtfC-type 4,6-α-GTs differ from their GtfB-typerelatives (and GH70 glucansucrases) regarding domain organization.First, GtfCs lack the circular permutation of the (β/α)Geobacillus 12AMOR1 (GbGtfC),revealing the 3D structure of the core domains A, B, C, and the single-segmentdomain IV. Despite the absence of circular permutation, GbGtfC featuresa tunneled active site architecture that closely resembles the majorityof GtfB-type 4,6-α-GTs. The obtained structure of the GbGtfC-ΔCenzyme (at 2.25 Å resolution), together with docking experimentsdepicting donor and acceptor reactions, allowed us to pinpoint theresidues in the active site that likely contribute to its unique “alternating”specificity. AlphaFold modeling confirmed that GbGtfC features twoC-terminal domains of the Ig (type 2) fold that are absent in thecrystallized construct. Finally, we show that the GbGtfC 3D structurerepresents the GtfC α-GT subfamily as currently known, suggestingthat the structural changes necessary to acquire the α-1,6 starch-transglycosylatingspecificity of GH70 α-GTs from starch-degrading GH13 α-amylasestook place before domain permutation events.Here, we report the first crystal structure ofa GtfC-type enzyme,the 4,6-α-GT from Geobacillus 12AMOR1 GtfC and a 20-residue N-terminalHis-tag, have been described before.22 Briefly,the pET15b vector carrying the gtfC construct wasoverexpressed in E. coli BL21 (DE3)cultures grown at 37 °C; harvested cells were resuspended andbroken by sonication; cell-free extract (CFE) was stored at 4 °C.The GbGtfC-ΔC protein in the CFE was captured by immobilizedmetal affinity chromatography (IMAC) on a Ni-Sepharose column  using an elution buffer containing 20 mM Tris-HCl,pH 8.0, 100 mM NaCl, and 350 mM imidazole. Fractions with the highestabsorbance at 280 nm were pooled and concentrated using a VivaSpin4 (molecular weight cutoff 10 kDa) at 4000g. Thefinal purification step was done via size exclusionchromatography on an Äkta Micro system equipped with a Superdex200 Increase 10/300 column  at 12 °C.The elution buffer contained 20 mM MES-NaOH, pH 6.1, 100 mM NaCl,and 1 mM CaCl2. The center fractions of the peak elutingat 13.3–14.8 mL were pooled  and concentrated as described above to obtain the final GbGtfC-ΔCprotein sample suitable for crystallization. Protein concentrationswere determined by measuring the absorbance at 280 nm using a NanoDropOne spectrophotometer .The cloning and expressionof the GbGtfC-ΔC construct, containing residues 33–738of 2. The reservoirsolution contained 1.07–1.14 M (NH4)2SO4, 0.1 M MES-NaOH, pH 6.5, and 0.4 M Na3citrate,and hanging drops were prepared by mixing 1.5 μL of proteinsolution and 1.5 μL of reservoir solution. Prior to data collection,crystals were briefly transferred to 1.25 M (NH4)2SO4, 0.05 M MES-NaOH, pH 6.5, 0.2 M Na3citrate,and 30% (v/v) glycerol and flash-cooled in liquid nitrogen. X-raydiffraction data were collected at beamline I03 of the Diamond LightSource (UK) and processed using XDS;29 statisticsare given in Crystals of GbGtfC-ΔCwere grown at 20 °C using a 10.0 mg/mL protein solution, 20 mMMES-NaOH, pH 6.1, 100 mM NaCl, and 1 mM CaCl30 a template modelwas generated by the one-to-one protocol of Phyre31 based on the highest scoring structure from a Phyre search,the crystal structure of the α-amylase from Halothermothrixorenii (PDB: 3BC9).32 The asymmetric unitof the I4122 cell contains one proteinmolecule. Refinement and model building was carried out using Refmac33 and COOT;34 groupsfor TLS refinement were determined using Phenix35 and were edited manually to include domain IV as a separateTLS group. The B-factor distribution showed a large range of values,with relatively high values for domains C and IV . Some stretches of residues in domain IV lackedgood electron density, especially residues 271–282, which werelater modeled guided by an AlphaFold generated model.37The crystalstructure of GbGtfC-ΔC was determined by the molecular replacementmethod using PHASER;38 was used to define secondary structure. Atomic coordinates and structurefactors have been deposited at the Protein Data Bank with entry 7ZC0. PDBeFold39 was used to analyze structural similarities,with a lowest acceptable match threshold of 70% or 40%.The final refinement statistics and model quality are listedin 37 The modelwith the highest overall pLDDT (per-residue confidence) score wasused for comparison with the crystal structure.The fullsequence of GbGtfC  wassubjected to AlphaFold modeling.Table S1), from Heyndrickxia sporothermodurans (902 residues), Weizmannia coagulans DSM1 (954residues), Exiguobacterium sibiricum 255-15 (893residues), and Exiguobacterium acetylicum (892 residues).Additionally,AlphaFold models were calculated for 4 other GtfC-type enzymes , yielding a final maltooctaose(G8) model. The corresponding residues from four other GtfC enzymes, as well as a GtfB-type 4,6-α-GT from L. reuteri 121 (Q5SBM0), were selected for a sequence alignment with ESPript3.0.40We used the nativecrystal structure to map the substrate binding groove of GbGtfC-ΔC;an initial model was obtained by superposition with maltoheptaose(G7) bound to subsites +2 to −5 of Lr121 GtfB41 and AutoDock Tools (version 1.5.6)42 anddocked in the crystal structures of GbGtfC (this study) and Lr121GtfB25 using Vina-Carb,43 representing scenarios for the donor reaction or for theacceptor reaction with a covalent glucosyl-enzyme intermediate atthe catalytic nucleophile D413. All docking results were visuallyinspected in PyMOL, judged by hydrogen-bond interactions with catalyticresidues, and then grouped by visual similarity. Details of the dockingprocedures and interpretation of the results are given in the Supporting Information.Mixed isomalto-maltooligosaccharides(DP1–6) were setup using SWEET2Geobacillus 12AMOR1 GtfC (Genbank AKM18207.1). Using thefull sequences of the resulting hits, multiple sequence alignmentswere performed with MUSCLE44 and inspectedwithin JalView 2;45 sequences lacking significantparts of the GH70 core (containing the conserved sequence regions(motifs) I–VII) were deleted. This initial alignment was extendedby three extra sets of sequences representing biochemically characterizedbacterial enzymes: (a) eight canonical α-amylases from GH13subfamily 5 (GH13_5); (b) five GH70 glucansucrase sequences; and (c)six GH70 GtfB sequences.26 The sequencesused for the final alignment are shown in Table S2. Residues constituting three important loops in GH70 GtfB-type4,6-α-GTs were identified on the basis of previously determinedstructures:26 loop B in domain B and loopsA1 and A2 in domain A . A phylogenetic tree was constructedin MEGA X46 using the Maximum Likelihoodmethod; the tree with the highest log likelihood was used. Initialtree(s) for the heuristic search were obtained automatically by applyingNeighbor-Join and BioNJ algorithms to a matrix of pairwise distancesestimated using a JTT model and then selecting the topology with superiorlog likelihood value. Branch lengths were measured in the number ofsubstitutions per site. All positions with less than 95% site coveragewere eliminated, i.e., fewer than 5% alignment gaps, missing data,and ambiguous bases were allowed at any position . There was a total of 512 positions in the final data set.The bootstrap consensus tree was inferred from 1000 bootstrap replicates.47A BLASTp search with defaultparameters was performed  with the sequence of 26 this subset contained233 sequences with long loops A1 and B ,likely featuring a tunneled binding groove.For a comparison between acceptor subsiteresidues in GtfC- and GtfB-type 4,6-α-GTs, the 63 putative GtfCsequences were aligned with a subset of the 283 putative GtfB sequencesfrom Pijning et al.;8 barrelalso found in other GH70 enzymes, but, like in GH13 α-amylases,it starts with strand β1 and is interrupted after helix α3by a long insertion, forming domain B, as well as the auxiliary domainIV, which is absent in GH13 enzymes. Despite being non-permuted, theoverall topology of domain A is very similar to that of other GH70structures . Domain B (residues145–222 and 333–386) has the central twisted five-strandedantiparallel β-sheet also observed in other GH70 structuresbut is more compact, mainly due to shorter connections between theβ-strands. For example, the connection between strands β2and β3 (residues 191–210) is about 30 residues shorterthan it is in Lr121 GtfB and lacks two α-helices, while theloop connecting strands β4 and β5 (residues 357–380)is about nine residues shorter. The connection between strands β3and β4 is “extended” by the insertion of about110 residues that constitute domain IV (residues 223–332).Finally, domain C (residues 631–736) displays a similar Greekkey topology as in other GH70 and GH13 structures, albeit some loopsthat connect the β-strands are either shorter or longer.We determinedthe crystal structureof GbGtfC-ΔC at a resolution of 2.25 Å from crystals containingone protomer in the asymmetric unit 1a consis26 Yet, PDBeFold analysis of thecore domains  of the GbGtfC-ΔC crystal structurerevealed that the closest structural homologues are α-amylasesfrom Alicyclobacillus sp. (PDB: 6GXV)48 and Geobacillus stearothermophilus (PDB: 4UZU)49 with Q-scores of 0.46/0.44 and root-mean-square deviations(RMSD) of 1.95/1.88 Å, respectively. Both these α-amylasesbelong to subfamily GH13_5, confirming structurally the previous observationthat this is the α-amylase subfamily to which GH70 enzymes areevolutionary closest.25 Only after includingdomain IV to the PDBeFold search, structural homologues of GH70 enzymeswere detected, the closest one being the 4,6-α-GT GtfB-ΔNΔVfrom L. reuteri 121 25 with a lower Q-score (0.24) than the α-amylases butalso a somewhat lower RMSD value (1.72 Å).Despite the low sequencesimilarity, the GbGtfC-ΔC core structureclosely resembles that of GtfB-type 4,6-α-GTs.The GbGtfC-ΔC3D structure confirms the earlier notion thatat the domain level it represents an intermediate between GH13 α-amylasesand GH70 GtfB-type α-GTs; regarding the structural details ofthe core domains, and especially the active site region, it is clearlysimilar to the GH70 GtfB-type α-GTs and more distant from theGH13 α-amylases.via two loops that lie adjacent to each other. The crystallographicB-factors for domain IV are on average higher than for other domains, indicating that this domainmay be flexible due to a hinged connection with domain B. Some ofits residues hardly showed electron density , but since domain IV superimposed almost perfectly withthat of the AlphaFold model 25 and Limosilactobacillus reuteri NCC2613(Lr2613) GtfB ;this domain thus can be considered a previously unobserved fold. Still,a manual inspection showed that parts of domain IV can be superimposedwith that of other GH70 structures  2a, takin25 Alignmentof these loops  andB (residues 338–352) near the binding groove results in a tunnel-likearchitecture that encompasses donor subsites −2 and −32b, similse loops 3 reveals51 We therefore compared the 3Dstructures of GbGtfC (this study) and Lr121 GtfB25 and used them to perform molecular docking with donor andacceptor substrates. The active site of GbGtfC seems more constrainedaround subsites +1/+2 than in Lr121 GtfB ; moreover, while the residues surrounding donor subsitesare largely conserved, GbGtfC differs at four positions near acceptorbinding subsites of the products synthesized from amylose.25 The third and fourth non-conserved positions, T346 and V348 fromloop B, locate at the opposite side of the subsite +1 sugar unit;they are replaced by S918 and T920 in Lr121 GtfB. Together, whilethe four positions are largely conserved in a subset of 233 GtfBsthat likely feature a tunnel , the 63 putative GtfCs have a different and less conserved set.Notably, GbGtfC is unique among GtfCs with Y375 replacing D or N orK and the T346/V348 pair replacing mostly S/I or S/S. This suggeststhat Y375, T346, and V348 of GbGtfC contribute to its unique productspecificity. Supporting evidence comes from a recent study with H. sporothermodurans GtfC52 postulatingthat mutation of the corresponding S345/I347 to T/V resulted in productswith alternating α-1,6/α-1,4 linkages rather than consecutiveα-1,6 linkages.Given the observed structural similarityin the binding groove between GbGtfC and Lr121 GtfB, it is intriguingthat Lr121 GtfB (and other GtfBs) synthesize products with consecutiveα-1,6 linkages, whereas GbGtfC forms alternating α-1,4/α-1,6linkages. In fact, GbGtfC so far is the only biochemically characterizedGtfC-type 4,6-α-GT displaying this specificity; understandingthis unique property is important regarding its application in starchmodification.121 GtfB S5a; more22 However,the synthesis of α-glucans by 4,6-α-GTs proceeds throughmany cleavage and transfer steps. To understand how the final productspectrum is obtained would require a systematic analysis of everypossible reaction for each possible donor or acceptor substrate. Indeed,our docking experiments suggested that the situation is more complicatedthan can be explained by a single transglycosylation preference. Nevertheless,the docking experiments with GbGtfC and Lr121 GtfB  did allow us to derivesome principles that agree with the experimentally observed end productsof either enzyme.25 First, a general and rather unexpectedobservation was that, for both enzymes, donor and acceptor reactionsdo not seem to be restricted to α-1,4-resp. α-1,6-specificity,but also can occur with α-1,6- resp. α-1,4-specificity.Yet, α-1,6 transglycosylations become dominant over α-1,4transglycosylations, because (intermediate) products of the lattercan easily “react back” because the glucosyl moietyin subsite +1 hardly requires a change in conformation to act in asubsequent donor reaction . Theresult is that, with GbGtfC, intermediate products with NR maltosylends “survive”, and these are easily elongated by α-1,6-transglycosylation,favoring the formation of alternating glucan products. The experimentallyobserved maltose in the reaction pool of GbGtfC22 likely results from a more efficient α-1,4-transglycosylationof glucose than in Lr121 GtfB. Finally, the docking results suggestthat the described differences between GbGtfC and Lr121 GtfB relateto interactions of donor/acceptor substrates in subsites +1 and +2with the non-conserved residues described above (Table S3), further supporting the role of these residues indetermining the unique product specificity of GbGtfC.It was proposed earlier that α-1,4/α-1,6alternatingend products of GbGtfC can be explained by an α-1,6 transglycosylationpreference for maltosyl rather than glucosyl moieties, supported bythe accumulation of maltose and hardly any glucose upon incubationof the enzyme with amylose V.Figure S6a); omittingthese residues improved the average pLDDT to 94.8 (Table S1), indicating a highly reliable model. The AlphaFoldmodel superposed well with the crystal structure (RMSD = 0.79 Åfor 591 Cα atoms), even for most of the loop regions (The average per-residue confidence score (pLDDT) of the highestranked AlphaFold model of GbGtfC was 92.6. The N-terminal 32 residuesof GbGtfC correspond to the signal peptide and expectedly showed significantlylower pLDDT scores S6a; omit22 they form two bacterial Ig-liketype 2 domains (Ig2), which connect to domain C via a short loop (residues 734–738)  indicatereliable modeling of their fold, the relative orientation of thesedomains is modeled with less confidence, especially regarding theC-terminal Ig2 domain. Domain Ig2a (residues 739–823) and domainIg2b (residues 824–903) share low sequence identity (26.2%)but have the same immunoglobulin fold; they can be superimposed givingan RMSD of 0.74 Å. Both domains contain nine β-strandsand form two opposing, mostly antiparallel β-sheets  6a. Altho734–738) S6a indicGeobacillus species, and the closest homologues in termsof sequence (from H. sporothermodurans) show 76.3%sequence identity. Some of its residues near the binding groove aredifferent from most GtfC sequences (see above). This raised the questionhow representative the GbGtfC 3D structure is for the GtfC subfamilyof 4,6-α-GTs. We therefore constructed AlphaFold models of fourother GtfC-type GTs (Table S1), three ofwhich were characterized as 4,6-α-GTs synthesizing linear isomalto/maltooligosaccharideswith consecutive α-1,6 linkages. The AlphaFold models showedcomparable pLDDT scores and very similar folds , reflected in low RMSD values of 0.54–0.72Å upon Cα superposition with GbGtfC. Notably, the highstructural conservation includes not only the core domains A, B, andC but also domain IV. Near the active site region, loops A1, A2, andB have somewhat lower pLDDT scores (not shown). Although there areslight differences in position with differences up to 3.8 Å (inthe tip of loop A1), these loops have the same architecture as inGbGtfC and form a tunnel at the donor side of the binding groove . We thus suggest that, although Geobacillus 12AMOR1 GbGtfC has some unique features nearthe active site, the 3D structure of the core domains of this enzymerepresents the whole GtfC subfamily, at least for the 63 sequencesfound so far.The BLASTp resultsindicate that on a residue level GbGtfC is ratherunique among GtfC subfamily enzymes: it is the only enzyme from a H.sporothermodurans, E. acetylicum, and E. sibiricum 255-15 feature two Ig2 domains; for the latter,this was already predicted in an earlier study.18 Ig2 domains occur in various bacterial and phage surfaceproteins and have been proposed to play a role in cell surface adhesionor carbohydrate binding.53 For GtfCs, thisremains to be investigated, but since these enzymes are extracellularand process carbohydrates, such functions seem to be possible. Onthe other hand, the predicted structure of the W. coagulans DSM1 GtfC features three C-terminal SRC Homology 3 (SH3) domains of about 60 residues each;SH3 domains are thought to mediate protein–protein interactions.54 Variations in the length of the C-terminal partsof the GtfC sequences found by the BLASTp search (see below) suggeststhat the type and the number of copies of the C-terminal domains couldbe related to the bacterial species and its specific natural environment.Like GbGtfC, the C-terminal domains of the GtfCfrom Geobacillus 12AMOR1 GtfC (GbGtfC)sequence yielded a total of 102 putative non-permuted bacterial sequencescontaining the four conserved GH70 motifs in the order I–II–III–IV(Table S2). All sequences originate fromnon-LAB species, but based on their sequence alignment they couldbe divided in two groups. The first group contains 63 hits, more thandouble the number of sequences identified in 20182 and shows sequence identities of 52.9–76.3% withGbGtfC. The enzymes within this group originate mainly from Gram-positivesoil or marine bacteria such as Weizmannia coagulans or Exiguobacterium species; for example, the earliercharacterized GtfCs from Exiguobacterium sibiricum 255-1518 and Weizmannia coagulans DSM120 belong to this group. Most sequenceshave a length of around 900 residues and share high sequence similarity,suggesting that they are GtfC-type α-glucanotransferases constitutinga similar domain organization with the three core domains , an inserted domain IV, and extra C-terminal domains. The secondgroup, containing the remaining 39 sequences, showed lower overallsequence identities (40.4–49.9% to GbGtfC) and originate mostlyfrom Gram-negative bacteria such as Azotobacter chroococcum (a plant-associated nitrogen-fixing species) or Burkholderia . Including the previously characterizedenzymes from Azotobacter chroococcum NCIMB 800324 and from the Gram-positive Paenibacillusbeijingensis DSM 24997,23 thisgroup represents putative GtfD-type α-glucanotransferases. Ingeneral, the sequences in this group are shorter at the C-terminalend, suggesting that they do not feature Ig-like domains.A BLASTpsearch with the Table S2). The Geobacillus 12AMOR1 GtfC sequenceis rather unique in these regions (note that it is the only Geobacillus entry found). Nevertheless, the alignment stronglysuggests that all 63 putative GtfC-type α-GTs found so far featurea tunneled binding groove, prefer mostly linear starch substrates,and synthesize linear α-glucan products; this is also supportedby the AlphaFold models of selected GtfCs (see above). Whether GbGtfCis the only GtfC synthesizing products with alternating α-1,4/α-1,6linkages remains to be investigated. A detailed biochemical characterizationof more GtfC-type GTs and their products is needed to confirm this.Amore detailed analysis of the sequence alignment of the GH70 motifs,loops A1, A2, B, and the 370s loop within the GtfC group revealedthat they are highly conserved regarding residue type  are apparent (Table S2) likely related to their reaction specificity involvingmore branched substrates and products.24 On the otherhand, in LAB species, permutation did take place (via gene duplication)  7; a late56 in GbGtfC (and other GH70enzymes), domain IV structurally does not resemble a CBM domain anddid not reveal carbohydrate binding sites. Finally, the phylogenetictree shows that within the GtfC clade, the Geobacillus 12AMOR1 GtfC is in a rather unique position, perhaps related tothe observed differences in residues surrounding the binding grooveas described above.Notably, despite the absence of permutation anddespite a differentdomain composition, the GtfC- and GtfD-clades are phylogeneticallycloser to other GH70 enzymes  than they are to the GH13_5 α-amylases.The GbGtfC-ΔC crystal structure (as well as the AlphaFold modelsof other GtfC enzymes) clearly confirms this, showing the high structuralsimilarity with GtfB enzymes, but differing from the GH13_5 α-amylases,which have a more open active site groove and lack certain structuralelements in the core domains . Thus,the high structural similarity between GtfC and GtfB-type α-GTsshows that the gene duplication step occurring in LAB did not leadto large structural changes in the core domains, consistent with theirshared substrate and reaction specificity . Thisalso suggests that the changes that were necessary to acquire α-1,6transglycosylation specificity, as well as the insertion of domainIV, took place before the division between LAB and non-LAB (bifurcationpoint I), likely in bacterial α-amylase enzymes and leadingto an ancestor α-GT enzyme 8. The ro


In this study, native lotus seed flour (N-LSF) was modified by different methods, namely, partial gelatinization (PG), heat–moisture treatment (HMT), or pullulanase treatment (EP). Their composition, functional properties, starch composition, and estimated glycemic index (eGI) were compared. PG contained similar protein, soluble dietary fiber, and insoluble dietary fiber contents to N-LSF, while those of HMT and EP differed from their native form. PG increased rapid digestible starch (RDS) but decreased resistant starch (RS); while HMT and EP increased amylose and RS contents to 34.57–39.23% and 86.99–92.52% total starch, respectively. Such differences led to the different pasting properties of the modified flours rather than PG, which was comparable to the native flour. HMT had limited pasting properties, while EP gave the highest viscosities upon pasting. The eGI of all samples could be classified as low (<50), except that of PG, which was in the medium range (60). It was plausible that lotus seed flour modified either with HMT or EP could be used as carbohydrate source for diabetes patients or health-conscious people. Diabetes mellitus (DM) is one of the metabolic diseases characterized by high fasting blood glucose levels, of which the prevalence and incidence are currently increasing glob-ally. It has been reported in 2021 that 537 million adults around the world are living with DM, and the number estimated in 2030 will rise to more than 643 million [Nelumbo nucifera), is a rich source of starch, which provides carbohydrates as a nutrient and energy source for the human body. Many studies have reported that total starch content in lotus seed can reach over 60% on a dry basis [Bifidobacterium adolescentis growth and produce short chain fatty acids when tested in the simulated conditions of the human digestive system. Thus, lotus seed can be used as a prebiotic. In addition, lotus seed starch possessed low swelling power, and consequently high pasting and gelatinization temperatures, though the gelatinized granules are highly susceptible to enzymatic digestion. It also showed higher gel syneresis and higher ratio setback to peak viscosity during pasting, suggesting a stronger tendency for short-term retrogradation [Lotus seed, particularly of sacred lotus  were purchased in four batches from a local farm located in Nakhon Sawan province, Thailand during July 2020 to September 2020. Once received, the seeds were sampled for moisture analysis, vacuum-packed in polyethylene plastic bags and stored at room temperature until being used for flour preparation. Moisture content of the lotus seeds ranged from 6.5–7% w/w. Prior to flour preparation, lotus seeds were soaked in deionized (DI) water  overnight, peeled, removed the embryos by hands, and dried in a hot air oven at 40 °C for 16–18 h to obtain dried lotus seeds with the final moisture content of 7–8% w/w. Native lotus seed flour (N-LSF) was prepared by grinding the dried seeds using a grinder mill machine, followed by sieving through a 100-mesh sieve. The flour was packed in polyethylene plastic bags and kept at room temperature until being used. The production yield of N-LSF from dried lotus seeds was 80–82%.Sun-dried seeds of sacred lotus  and kept under refrigeration at 4 °C for 12 h. They were parboiled at 75 °C for 10 min to achieve partial gelatinization of starch. Then, the parboiled seeds were dried at 60 °C in a hot air oven until the moisture content was 10–12% w/w. After drying, the seeds were ground and sieved through 100-mesh sieve to obtain partially gelatinized lotus seed flour (PG). The samples were packed in polyethylene plastic bags and kept in a desiccator. The yield of production of PG from dried lotus seeds was up to 80%.Dried lotus seeds were soaked in DI water  was prepared by slightly modifying the method of Whantongkhum and Suwannaporn . DI watew/v) of N-LSF in DI water was prepared. The pH of the slurry was adjusted to 4.5 with 0.1 M sodium acetate buffer solution pH 4.5. Pullulanase  was added to the slurry at an enzyme-to-substrate ratio of 20 NPUN/g dry flour. Enzymatic treatment was performed at 55 °C for 6 h in a shaking water bath. The treated slurry was then heated at 70 °C for 10 min to stop the enzyme reaction. Sample was centrifuged at 4600× g for 10 min, and the precipitate was washed twice with DI water. The precipitate was collected and dried at 45 °C in a hot air oven until the final moisture content reached 10–12% w/w to obtain the enzymatically modified flour (EP). The recovery of EP from N-LSF was about 70%.Enzymatic modification of lotus seed flour was performed using pullulanase according to the method of Wattananapakasem et al.  with sliChemical composition of native and modified lotus seed flours was determined according to the AOAC Official Methods . Moistur2 + 2 g KI in DI water, 800 μL). A final volume was then made up to 12 mL with DI water. The sample was immediately mixed and chilled in the refrigerator for 20 min before test. Amylose content was determined using 96-well UV visible microplate reader spectrophotometer  at the wavelength of 620 nm. The absorbance values were converted to percent of amylose by standard curve of amylose from potato .Amylose content was determined using the iodine absorption method  with sliRapid digestible starch (RDS) and slow digestible starch (SDS) contents were analyzed based on in vitro starch digestibility procedure . Digestag for 10 min. The residue as washed with 10 mL of DI water and recentrifuged at the same condition. Then, 3 mL of DI water and 3 mL of 4 M KOH was added and the mixture was incubated at room temperature for 30 min. After that, 5.5 mL of 2 M HCl, 3 mL of 0.4 M sodium acetate buffer pH 4.75 and 80 μL (3.26 U) of amyloglucosidase in 0.4 M sodium acetate buffer pH 4.75 were added, followed by vortexing. The mixture was incubated at 60 °C for 45 min in a shaking water bath. Next, it was centrifuged at 4600× g for 10 min to collect the supernatant. The residue was washed with 10 mL of DI water and centrifuged again. The supernatant from the first and second extractions were combined for the measurement of glucose content using glucose oxidase–peroxidase kit GOPOD reagent enzymes .Resistant starch (RS) content was analyzed based on an in vitro starch digestibility procedure  with slig for 10 min. The supernatant was carefully pulled out using a syringe, dried for 24 h at 105 °C and weighed. The precipitates were immediately weighed. Solubility and swelling power were calculated according to the following equations:Solubility and swelling power and of samples were determined according to method of . Flour s® v.2.2, Newport Scientific).Pasting properties of samples were investigated according to the AACC method 76–21.01  using a g for 10 min to separate precipitate for RS analysis. The supernatant was further added with 0.4 M sodium–acetate buffer  and amyloglucosidase from Aspergillus niger . Samples were incubated for 45 min at 60 °C. Glucose concentration in sample was then measured using glucose oxidase–peroxidase kit GOPOD reagent enzymes (Megazyme). Rate of starch digestion was expressed as a percentage of glucose at different times . The hydrolysis index (HI) was initially determined using area under the curve from GraphPad Prism 7 software  and using white bread as a reference food. From the linear relationship between hydrolysis index and glycemic index, estimated glycemic index (eGI) was calculated according to the equation:The rate of in vitro starch hydrolysis was analyzed according to the method recommended by . BrieflyIn addition, glycemic load (GL) of the flour samples was determined from eGI and the amount of available carbohydrate in a common serving, i.e., 30 g (1/4 cup). Available carbohydrate was calculated by subtracting total dietary fibers from total carbohydrates. GL was calculated using the following equation:p < 0.05).Statistical analyses were performed using the IBM SPSS Statistic 19 . All results were reported as mean and standard deviation of three replicates, except that chemical composition was reported as mean of duplicate analysis. Differences between lotus seed flour and its modified samples were determined using Analysis of Variance with Tukey’s post hoc test . Protein content of N-LSF was 26.79% on a dry basis, which was the highest among other samples. Carbohydrate was the major composition of N-LSF, accounting for 67%, while fat and ash contents were only about 2 and 4%, respectively, on a dry basis. Partial gelatinization and pullulanase treatment caused the lotus seed flour to have slightly less protein than the native flour, while heat–moisture treatment decreased the protein content in lotus seed flour to about 12% dry basis. The preparation of HMT involved soaking of the flour, which probably led to the loss in soluble protein by leaching. Denaturation of protein during the heat moisture treatment may also have contributed to the decrease in protein content of HMT. This result is in agreement with work of Jia et al. [Moisture content of native lotus seed flour was 7.92% .48% w/w . All lota et al.  that theIt is noticeable that protein content of N-LSF was significantly higher than other conventional flours, such as rice (9%), corn (8%), wheat (15%), and potato (9%) ,38. MoreFat content of PG and HMT was similar to that of native flour (about 2% dry wt) but EP contained slightly lower fat than the others. This was because all modification methods did not involve any hydrophobic solvent that could lead to the change in fat content. Similar ash content of 4.3% was also observed for N-LSF and PG, while HMT and EP contained less ash at 2.33 and 1.06%, respectively. It was plausible that the decrease in ash content of HMT and EP was caused by leaching of minerals into water during those modification processes. The total carbohydrates content of HMT and EP was higher than N-LSF and PG. This finding could be explained by the lower protein and ash contents of HMT, and the lower fat and ash content of EP, when compared with N-LSF and PG.N-LSF contained 8.36 and 3.59% of soluble and insoluble dietary fiber, respectively, on a dry basis. Slightly lower amounts of both types of dietary fibers were found in PG. Significant alteration of dietary fiber content was observed in HMT and EP. Heat–moisture treatment gave lotus seed flour with lower soluble fiber but higher insoluble fiber content. An opposite trend was observed for EP, of which most of the dietary fiber was soluble fiber and there was only <0.10 g of insoluble fiber in 100 g dry wt. Soluble components including soluble dietary fiber might have lost upon the preparation steps of HMT. In addition, it was possible that heat–moisture treatment led to the denaturation of protein in the lotus seed flour; thus, the denatured protein could form a layer on the surface of starch granules. Consequently, the protein-coated starch granules were inaccessible to the enzymes used for dietary fiber analysis and became less soluble in water. Dupuis et al.  demonstrBased on the colorimetric iodine method, the percentage of amylose in N-LSF was 24.30% on a dry basis . It has The enzymatic digestibility of native and modified lotus seed flours was investigated. RDS, SDS, and RS, which were classified consecutively by reaction times, represented the three different starch materials found in lotus seed flour samples . N-LSF cSolubility, also known as water-soluble index, of native and modified lotus seed flours were studied at 55, 65, 75, 85, and 95 °C, and the results are shown in The swelling power, which indicates the amount of water that can penetrate into and be adsorbed by the flour, of different lotus seed flour samples is presented in Although swelling power is known to be harmonious with solubility ,18, therPasting properties are associated with the rigidity or swelling potential of starch granules and the leaching of amylose molecules. The results showed that all pasting parameters of N-LSF were relatively closed to those of native lotus seed starch in the literature . DiffereThe pasting properties, particularly peak viscosity, peak time and pasting temperature, of native and modified lotus seed flours correlated with their swelling power . Flours For EP, the short linear amylose chains, which were abundant in EP , might lIn this study, starch hydrolysis was carried out in vitro to simulate the in vivo digestive characteristics of lotus seed flours and to estimate their glycemic indices. As shown in Starch modification based on partial gelatinization, heat–moisture treatment, and pullulanase enzyme treatment significantly altered the composition and properties of lotus seed flour to different extents. Partial gelatinization did not affect the chemical composition and pasting properties of the flour but largely increased the digestibility of starch. Heat–moisture treatment resulted in the modified lotus seed flour with less protein but more amylose, insoluble fiber, and RS, which restricted its swelling and pasting behavior while maintaining the low GI value of the native flour. Treatment with pullulanase altered the properties of lotus seed flour in a similar manner to heat–moisture treatment, but the enzyme-modified flour exhibited more rapidly pasting and much larger changes in viscosity during pasting cycle than both the native and other modified flours. Based on the obtained results, heat–moisture treatment could be a potential modification method for producing low glycemic lotus seed flour that could be applied in food product without affecting the consistency of food. On the other hand, pullulanase treatment could improve the thickening effect of lotus seed flour while maintaining the low glycemic value.