Inositol polyphosphate 1-phosphatase (INPP1) is a prototype member of metal-dependent/lithium-inhibited phosphomonoesterase protein family defined by a conserved three-dimensional core structure. Enzymes within this family function in distinct pathways including inositide signaling, gluconeogenesis, and sulfur assimilation. Using structural and biochemical studies, we report the effect of substrate and lithium on a network of metal binding sites within the catalytic center of INPP1. We find that lithium preferentially occupies a key site involved in metal-activation only when substrate or product is added. Mutation of a conserved residue that selectively coordinates the putative lithium-binding site results in a dramatic 100-fold reduction in the inhibitory constant as compared with wild-type. Furthermore, we report the INPP1/inositol 1,4-bisphosphate complex which illuminates key features of the enzyme active site. Our results provide insights into a structural basis for uncompetitive lithium inhibition and substrate recognition and define a sequence motif for metal binding within this family of regulatory phosphatases. Among the numerous lipid and soluble IP messengers, inositol 1,4,5-trisphosphate  include structural studies of the archael IMPA1/FBP1s implicating conformational changes associated in a loop region responsible for forming metal site 3 , a potent competitive inhibitor with respect to magnesium, was used in the presence of Li+ as one of the heavy atom derivatives and provides two key observations. The gadolinium ions found in the INPP1/Gd3+/Li+ structure bound predominantly to two sites in the INPP1 active site. Compared with the native INPP1/Mg2+/Li+ structure, the first gadolinium site (GD1) was well correlated with the position of the magnesium metal site 1 (MG1). The second gadolinium, however, was found 3.7 Å from the magnesium in site 2 (MG2) and was coordinated by a different set of protein and (presumably) solvent interactions. Given the anomalous differences of Gd3+, the identity of the metal in sites GD1 and the previously unseen metal site GD3 is unequivocal. Second, based upon difference Fourier analysis, the gadolinium metal density peaks were unequally occupied. Magnesium in the native structure had densities peaks of 4.4σ and 3.3σ in metal sites MG1 and MG2, respectively, giving a relative occupancy of 1.3. The gadolinium derivative showed a strong density peak (34.9σ) at GD1 and a relatively much weaker density peak (7.9σ) at GD3, giving a relative occupancy of the sites of 4.4 (A).We initiated a series of crystallographic studies building upon the original structure determination of INPP1 by multiple isomorphous replacement . In thess of 4.4 A.Figure + concentrations . The Fo-Fc difference density gave peaks of 18.0σ and 10.5σ in metal sites GD1 and GD3, respectively, with a relative occupancy of 1.7. Similarly, the anomalous difference density showed peaks of 13.7σ and 5.7σ in metal sites GD1 and GD3, respectively, giving a relative occupancy of 2.4 (C). In the absence of substrate, both the Gd3+ and Gd3+/Li+ structures had gadolinium atoms that were poorly ordered in metal site GD3, refined to only half occupancy and had B-factors more than two-fold higher than the global average B-factor. This indicates in the absence of substrate or product, the addition of Li+ did not significantly affect the ratio of Gd3+ at sites GD1 and GD3, with metal binding strongly preferred at site GD1.The crystals of INPP1 used for the initial structure determinations were grown in the presence of very high Litrations , 31. Thuccupancy B. The Foy of 2.4 C. In the2+ having a Hill co-efficient of 1.9 P3 substrate and 2 mM inhibitory Gd3+. Although we could not confidently model the substrate in the resultant structure, metal sites GD1 and GD3 became equivalently occupied (D). Difference Fourier analysis showed densities peaks of 20.5σ and 20.0σ in metal sites GD1 and GD3, respectively, giving a relative occupancy of 1.0. Consistent with the Fo-Fc differences, anomalous differences peaks of 15.4σ and 14.0σ were calculated, giving a relative occupancy 1.1 (E). Whereas in the absence of substrate, the gadolinium in metal site GD3 were found at half occupancy and had B-factors greater than twice the average, and in the presence of substrate, the gadolinium in site metal site GD3 was well ordered, refined to full occupancy and had B-factors well below the global average.The activity of INPP1 is cooperative with Mgt of 1.9 , supportoccupied D. Differancy 1.1 E. Wherea3+ metal binding in INPP1. These data are consistent with an uncompetitive pattern of Li+ inhibition that predicts that substrate or product must be bound in order for Li+ inhibition to occur. We therefore tested the effect lithium addition in the presence of substrate. INPP1 was co-crystallized in the presence of 10X molar excess of InsP3 and 2 mM Gd3+, but also 200 mM Li+. As seen in Figure 2F, in the presence of substrate, no electron density peak was observed for Gd3+ at metal site GD3 with little change in density observed at metal site GD1. A 15.4σ difference density peak was found for metal site GD1, with no detectable peak for metal site GD3 indicating lithium displaces metal at site GD3 and that binding requires the presence of substrate. Interestingly, a 4.9σ difference density peak was found for gadolinium binding at GD2, the equivalent of MG2, indicating poorly ordered Gd3+ in that site (F). This provides further evidence Li+ does not displace metals at either sites GD1 or GD2.In the absence of substrate, the addition of lithium did not eliminate Gdhat site F. This p2+ in metals sites MG1 and MG2, are essentially identical. In conjunction with the four INPP1-Gd3+ complexes presented here, at least three metal binding sites are seen in the active site of INPP1. Metal site MG1/GD1 has been observed in all structures, metal site MG2/GD2 has been observed in the Mg2+/Li+ and Gd3+/Li+/InsP3 structures, and the inhibitory metal site GD3 has been observed in the Gd3+, Gd3+/Li+, Gd3+/InsP3 structures.The native INPP1 structure was re-refined and agrees well with the original structure with an all-atom root mean square deviation of 0.25 Å. Importantly, the structural core and active site, including MgA) (A). The GD3/coordinating water site is also in close proximity to a lysine residue in IMPA1 that when mutated dramatically reduces lithium sensitivity and renders the enzyme resistant to Mg2+ inhibition at high concentration . T. TA) (21A) A. The GDntration , furtherB) (B). Notably, this site is distinct from the Li/GD3 binding site observed in the INPP1 structures, and its position away from putative water nucleophile may explain the inhibitory effect these metals exert.An overlay of the INPP-metal complexes with structurally related lithium-inhibited phosphomonoesterase proteins with one or more active site metals reveals that their active site metals cluster around defined sites B 11, 12, 12B (11B) , 35, 36.B) B. Notabl+ inhibition upon the wild-type and mutant enzymes. Mutant INPP1D54A exhibited a 4300-fold inactivation relative to the wild-type enzyme with a Vmax of 13.9 nmol/min/mg compared with 59.7 μmol/min/mg for the wild-type enzyme (A)  P3 by the D54A mutant and wild-type enzyme. As seen in Figure 4B, the x-intercept of this plot P3])) gives a Ki value of 107 mM for the D54A mutant. This represents a >100-fold decrease in Li+ sensitivity for the D54A mutant compared with the wild-type INPP1 (C). The results from the kinetic studies corroborate our structural studies that the site of Li+ inhibition in INPP1 is located at metal site GD3.Based on our crystallographic studies, we found that the lithium-sensitive, inhibitory metal binding site was coordinated by the carboxylates of two conserved residues, D54 and E80. Residue D54 was mutated to alanine, and we kinetically examined the effect of Lie enzyme A 31). W. W+ inhi enzymes A (31). Ape INPP1 C. The re3+ and Gd3+/Li+ structures, the sulfates originated presumably as the Gd3+ counter-ion from the Gd2(SO4)3 used in the crystallization conditions. In the Gd3+/InsP3 and Gd3+/Li+/InsP3 structures, the ion could either be a phosphate from the InsP3 substrate molecule or a sulfate from the Gd2(SO4)3 in the crystallization solutions. We were unable to distinguish these possibilities. However, as seen in Figure 3A, a sulfate (or phosphate) in the INPP1 structures correlates well with the 1-position phosphate of Ins(1)P1 observed in the IMPA1/Ins(1)P1 complex P3 substrate, we hypothesize the sulfates and/or phosphates seen in the INPP1 structures represent the 1- and 4-PO4 positions that would be found with ordered substrate or reaction product.Two sulfate and/or phosphate ions were found in the active sites of the INPP1 structures. In the Gd complex , and theD54A in the presence of calcium. One calcium ion at metal site 1 is found in the INPP1D54A/Ca2+ structure, alongside two sulfate ions similar to those found in Gd3+ structures. The INPP1D54A/Ca2+ structure reveals that D54A mutation does not cause obvious conformational changes, exhibiting an root mean square deviation of 0.6 Å between main-chains of the mutant and the wild-type proteinWe next attempted to directly examine substrate binding by INPP1. As mutation of D54 results in loss of INPP1 activity without significantly changing the substrate affinity, we hypothesize that the D54A mutant can be utilized to trap the substrate in INPP1. To rule out the possibility of unanticipated conformational changes, we first determined the structure of INPP1D54A/Ca2+ crystals with the InsP2 substrate. Indeed, the D54A mutant led to structure determination of the substrate bound INPP1D54A/Ca2/InsP2 complex. Two calcium ions (CA1 and CA2) are found at metal sites 1 and 2. We did not observe any metal binding at MG3/GD3 in the D54A mutant. The mFo-DFc omit map of the InsP2 substrate exhibits continuous electron density that agrees with the shape of InsP2 in the active site of the enzyme P1 structure P2 is reinforced by interactions between the inositol ring and surrounding residues. Together, the INPP1D54A/Ca2/InsP2 complex structure reveals the molecular basis for substrate recognition by INPP1.We next soaked the INPP1e enzyme . Substratructure . The 4-P2 substrate binding. Our systematic approach provides snapshots of how substrate and/or product influence metal binding as well as outlining critical residues that mediate the effects. Overall, we summarize our model using the schematic (A). INPP1 catalyzes the metal-dependent hydrolysis of the phosphomonoester bond from the 1 position phosphate of InsP2 or InsP3 to yield inositol 4-phosphate and inositol 3,4-bisphosphate, respectively  the role of a conserved sequence motif responsible for lithium’s effect; (2) lithium’s uncompetitive pattern of inhibition; and (3) InsPchematic A. INPP1 ectively , 37, 38.ectively , 22, 23,3+, an electron dense lanthanide, as a proxy for catalytically required Mg2+. Despite gadolinium’s coordination ligand number of 6 to 9, as compared with 4 to 6 for magnesium, we observed that the positioning of three Gd3+ sites identified are similar to those observed or proposed for Mg2+. In the absence of substrate, co-crystallization of INPP1 with Gd3+, a competitive inhibitor of Mg2+, resulted in preferred metal binding to site GD1 to GD3. Addition of Li+ in the absence of substrate did not significantly affect the ratio of Gd3+ at these sites. Co-crystallization with Gd3+ but in the presence of substrate P3) resulted in equivalent occupancies of metal sites GD1 and GD3 consistent with the hypothesis that substrate binding alters affinity at metal site GD3 by contributing an additional coordinating ligand through phosphate oxygen. The observation occupancy at a second/third site is also consistent with previous kinetic studies which demonstrated metal binding in INPP1 is cooperative with a Hill co-efficient of 1.9 which predicts if metal site 1 is predominantly bound in the absence of substrate, that an additional two metals are bound cooperatively upon substrate binding P3 and Li+ resulted in a stunning complete loss of electron density at metal site GD3 (no observable density even at below 1 sigma) with little change in density at site GD1 (remained above 20 sigma). Because we did not observe significant changes in the side-chain geometry for residues that coordinate the metal binding sites, we infer the parsimonious explanation is lithium replaces gadolinium at metal site 3. A partially ordered Gd3+ was found in metal site GD2 in the substrate-bound, lithium-treated structure and importantly excludes this metal site as the lithium-binding site. This hypothesis is supported by our kinetic analysis of the INPP1D54A mutant protein which demonstrated a 100-fold reduction in the potency of lithium inhibition (Ki went from 1 mM to over 100 mM with no significant change in Km).The uncompetitive pattern inhibition of INPP1 by LiB). It is important to note, studies of IMPA1 indicate potential differences for the precise site at which lithium acts among family members (B) . T. TB). Itbers (B) , 27, ourhibition , 29. Furtrations . Becausevia Baculovirus infection and grown until enzyme levels were >25 mg/l of medium. Over-expressed protein was concentrated, dialyzed, and sequentially passed over diethylaminoethyl cellulose and phenyl HPLC columns and was purified to homogeneity as judged by SDS-PAGE analysis. The INPP1D54A mutant used for the kinetic and structural studies was over-expressed and purified to homogeneity as described for wild type.Recombinant INPP1 was prepared as previously described . Briefly3+, a competitive inhibitor of Mg2+, has a useful anomalous absorption edge at the CuKα wavelength and therefore was used in the co-crystallization or soaking conditions. Four separate conditions were used to produce crystals in the course of these studies. In each case, purified recombinant bovine INPP1 from pooled Phenyl HPLC fractions was concentrated in a Centricon-30 concentrator, then exchanged with buffer 50 mM Tris HCl (pH 6.5) five times . For crystallization, 3 μl of 13 mg/ml INPP1 was added to 3 μl of reservoir solution: (1) -Mg2+/-Li+/-InsP3/+Gd3+ (6WRO): Reservoir solution consisted of 10% PEG 8000, 80 mM Bis-Tris, pH 5.25, 2 mM Gd2(SO4)3; (2) +Mg2+/+Li+/-InsP3/soak Gd3+ (6WRR): Reservoir solution consisted of 13% PEG8000, 200 mM Li2SO4, and 100 mM Tris, pH 6.3. Upon reaching maximum size after ∼5 days, a Gd3+ heavy atom derivative was prepared by adding 0.5 μl of 17.5 mM Gd2(SO4)3 in reservoir solution to 3 μl hanging drops  containing native crystals for 48 h; (3) -Mg2+/-Li+/+InsP3/+Gd3+ (6WRY): Reservoir solution consisted of 10% PEG 8000, 80 mM Bis-Tris, pH 5.25, 2 mM Gd2(SO4)3, and 1 mM InsP3; and (4) -Mg2+/+Li+/+InsP3/+Gd3+ (6X25): Reservoir solution consisted of 10% PEG 8000, 80 mM Bis-Tris, pH 5.25, 2 mM Gd2(SO4)3, 1 mM InsP3, and 200 mM Li2SO4.INPP1 tetragonal-shaped crystals amenable to diffraction studies were grown by the hanging drop vapor diffusion method on silanized glass covers slips at 20 °C and grew with dimensions routinely exceeding 0.1 × 0.1 × 0.5 mm. GdD54A was concentrated in a Centricon-30 concentrator, then exchanged with buffer 50 mM Tris HCl (pH 6.5) five times . Enzyme concentration was determined using the Bio-Rad protein dye assay. INPP1D54A yielded crystals using PEG6K as a precipitant. Briefly, 5 to 10 mg/ml INPP1D54A in 50 mM Tris HCl (pH 6.5) was mixed with an equal volume of precipitant A—0.1 M Tris HCl pH 5.5, 40 to 43% saturated (NH4)2SO4, 2 mM Gd2(SO4)3—or precipitant B—0.1 M sodium citrate pH 4.3, 16 to 18% PEG6K, 5 mM CaCl2—and subjected to hanging drop vapor diffusion trials at 17 °C. Crystals typically appeared after 3 to 4 days and stopped growing after 1 week. Enzyme substrate complexes were formed in situ by soaking mutant crystals which had been washed 3 times in precipitant A or B solution in 50 to 200 μM of substrate InsP2 for 1 to 3 h.Purified recombinant bovine INPP1h,k,l to h, -k, -l to keep indexing consistent. This problem arises from the physical orientation along the C axis of crystals with P4 point groups. Inverse beam experiments were used to collect anomalous data by complementing ϕ,χ orientations with ϕ + 180°, -χ orientations. Data were reduced, merged, and scaled using the area detector software  at 4 °C to prevent crystal decay and were processed using DENZO and SCALEPACK  was solved and refined before the use of Rata sets . We therve INPP1  was convh Phaser  using th in Coot  using di in Coot , includi in Coot .3+, Gd3+/Li+, Gd3+/InsP3, and Gd3+/Li+/InsP3 were solved by direct Fourier transform in Refmac P3, and Gd3+/Li+/InsP3 data. Anomalous difference maps for the Gd3+ and Gd3+/InsP3 data were calculated in fast fourier transfaorm . Data collection and refinement statistics are given in The structures of INPP1 co-crystallized or soaked in the presence of Gdn Refmac  using phn Refmac  using din Refmac , includiansfaorm  using phlProbity . MoleculD54A/Ca2+ and INPP1D54A/Ca2+/InsP2 were solved by direct Fourier transform in Refmac P2 structure, programs from the CCP4 suite P2 was initially modeled to the positive density using Turbo/Frodo program. Model building was completed with iterative rounds of building in Coot P2 was carried out in both reciprocal space and real space using Phenix  InsP3, ∼2000 cpm of 3H-InsP3, 50 mM HEPES, pH 7.5, 1 mM EGTA, 3 mM MgCl2, and 0 to 100 mM LiCl  were incubated at 37 °C for 15 min, then diluted with 0.5 ml of 0.35 M NH4COO, 0.01 M COOH (IP3 formate) to stop the reactions. Stopped reaction mixtures were divided into two fractions and passed over 200 μl AG 1-X8  anion exchange columns (Bio-Rad). The columns were each washed with 3 ml IP3 formate and the eluate P2 product) collected into scintillation vials containing 12 ml of Tru-Count high salt capacity scintillation fluid (Tru-Lab). Vials were counted in a Packard 1600 TR liquid scintillation analyzer. Percent hydrolysis of the substrate was 20 to 40% for each of the reactions. Nonradioactive InsP3 was purchased from Matreya, and 3H-InsP3 was prepared by digesting 1 μCi of 3H-InsP4 with ∼100 ng of type I 5-phosphatase for 1 h at 37 °C, stopping the reaction at 100 °C for 10 min, then centrifuging the mixture to clear the 5-phosphatase. An aliquot of the supernatant was run on an anion exchange HPLC column to confirm complete digestion of the substrate. The Vmax and Km values for the D54A INPP1 mutant were determined by similar kinetic studies using 0 to 50 μM of InsP3 in the absence of Li+.Limax and Km values for the D54A mutant were determined by fitting kinetic data by nonlinear regression to the one site binding hyperbola equation Y = Vmax∗X/(Kd + X) with an r2 value = 0.99 and the kinetic parameters determined. Data for Li+ inhibition for the D54A mutant and wild-type enzyme were fit by linear regression with r2 values of 0.95 and 0.99, respectively, to a plot of 1/v versus [Li+], for the hydrolysis of Ins-1,3,4-P3. The Ki value was determined according to the following equation: X int = −Ki . All enzyme activities are the averages of three independent measurements.All kinetic data were evaluated using the program Prism . V2+/Li+ (1INP), INPP1/Gd3+ (6WRO), INPP1/Gd3+/Li+ (6WRR), INPP1/Gd3+/InsP3 (6WRY), and INPP1/Gd3+/Li+/InsP3 (6X25); INPP1D54A/Ca2+ (7KIO); INPP1D54A/Ca2+/IP2 (7KIR)Most data are provided in the manuscript; however, diffraction data and coordinates have been deposited in the Protein Data Bank for following INPP1 complexes: INPP1/MgThe authors declare that they have no conflicts of interest with the contents of this article.


Protein N-myristoylation of Src-family kinases (SFKs) is a critical co-translational modification to anchor the enzymes in the plasma membrane. Phosphorylation of SFKs is also an essential modification for regulating their enzymatic activities. In this study, we used Phos-tag SDS-PAGE to investigate N-myristoylation-dependent phosphorylation of SFKs and their non-N-myristoylated G2A mutants. The serine-13 residue of Lyn (Lyn-S13) was shown to be N-myristoylation-dependently phosphorylated. Although there have been more than 40 reports of mass spectrometric studies on phosphorylation at Lyn-S13, the kinase responsible remained unclear. We succeeded in identifying casein kinase 1γ (CK1γ) as the kinase responsible for phosphorylation of Lyn-S13. In HEK293 cells co-expressing Lyn and CK1γ, the phosphorylation level of Lyn-S13 increased significantly. CK1γ is unique among the CK1 family  in carrying an S-palmitoylation site for membrane binding. Co-expression with the non-S-palmitoylated CK1γ mutant, which localized in the cytosol, gave no increase in the phosphorylation level at Lyn-S13. In HEK293 cells expressing the non-S-palmitoylated Lyn-C3A mutant, on the other hand, the Lyn-C3A mutant was phosphorylated at Lyn-S13, and the mutant remained at the Golgi. These results showed that S-palmitoylated CK1γ can phosphorylate S13 of N-myristoylated Lyn at the Golgi during intracellular protein traffic. Human SFKs include nine members , all of which play crucial roles in a wide variety of cellular functions, such as proliferation, survival, migration, growth, or cytokine stimulation. The eight members other than Frk contain an N-terminal Met-Gly motif  are nonreceptor tyrosine kinases that act as key mediators of intracellular signal transduction12. The C-terminal tyrosine-508 residue (Tyr-508) in Lyn or the corresponding conserved tyrosine residue in each of the other members of the SFK family is the primary site for regulating tyrosine kinase activity by reversible phosphorylation. Phosphorylation at the tyrosine residue induces a closed conformation that is enzymatically inactive. Dephosphorylation of the residue results in an open conformation of the SFK; this is followed by autophosphorylation at another tyrosine residue in the kinase domain to produce the active form. In the case of Lyn, a loss-of-phosphorylation mutation at Tyr-508 (Lyn-Y508F) leads to autophosphorylation of Tyr-397 in the kinase domain and induces constitutive kinase activation. It has also been reported that serine phosphorylation in Src, Fyn, and Lck is an important modification for controlling the activities of these enzymes15. Furthermore, mass spectrometry (MS)-based phosphoproteomic techniques have permitted the identification of many sites for the phosphorylation reactions of serine, threonine, and tyrosine residues in SFKs  and non-N-myristoylated G2A mutants of the N-myristoylated model proteins formin-like 2 and formin-like 323, expressed in HEK293 cells. Differences in the banding patterns in Phos-tag SDS-PAGE between the WT and G2A-mutant proteins readily revealed the presence of specific N-myristoylation-dependent phosphorylation sites in the model proteins. Subsequent Phos-tag SDS-PAGE analyses by using Ala-scanning mutants (loss-of-phosphorylation mutants), in which putative phosphorylation sites, as registered in an online database of phosphorylation sites , were replaced with alanine24, permitted the identification of N-myristoylation-dependent phosphorylation sites in the target proteins. The use of Phos-tag SDS-PAGE, in conjunction with existing information on locations of phosphorylation sites (phosphoproteomic data deposited in the database), permitted the identification of specific N-myristoylation-dependent phosphorylation sites; the cellular physiological role of crosstalk between protein N-myristoylation and phosphorylation/dephosphorylation could then be studied.We recently established an original strategy for identifying N-myristoylation-dependent phosphorylation of cellular proteinsIn the present study, we used Phos-tag SDS-PAGE to investigate a novel N-myristoylation-dependent phosphorylation site in SFKs. As a result, we found that Lyn was N-myristoylation-dependently phosphorylated at the Ser-13 residue (Lyn-S13). According to the PhosphoSitePlus database, Lyn-S13 has been identified as a putative phosphorylation site in over 40 reported studies that used MS-based phosphoproteomic methods, whereas there are no previous reports on the use of biochemical site-specific methods, such as amino acid sequencing, site-directed mutagenesis, or site-specific antibody studies , conventional SDS-PAGE and Phos-tag SDS-PAGE were performed by using C-terminally FLAG-tagged SFKs; the corresponding C-terminally FLAG-tagged G2A mutants were used as their non-N-myristoylated counterparts. The phosphorylation status of each WT SFK was compared with that of its G2A mutant. We first analyzed the eight SFKs, transiently expressed in HEK293 cells, by conventional SDS-PAGE followed by western blotting with anti-FLAG antibody , and it was confirmed that the band indicated by an open arrowhead corresponded to the nonphosphorylated form, and that the multiple upshifted bands corresponded to the phosphorylated forms.To identify the N-myristoylation-dependent phosphorylation site of Lyn, we used existing information on the location of phosphorylation sites recorded in the online database PhosphoSitePlus, as described previously5. Samples for Phos-tag SDS-PAGE analysis were prepared as expressed proteins in HEK293 cells in the absence or presence of bengamide B. Figure . This showed that N-myristoylation of Lyn-G2 is essential for phosphorylation to occur at Lyn-S13.To verify pharmacologically whether phosphorylation at Lyn-S13 requires N-myristoylation, we next examined the effect of the methionine aminopeptidase inhibitor bengamide B on the phosphorylation reaction of Lyn WT. It has been reported that bengamide inhibits the removal of the initiating methionine of an SFK protein and significantly decreases the N-myristoylation level of the target27. To determine whether myristoylation of Lyn-G2 is necessary for phosphorylation of Lyn-S13 in the cell-free system, we conducted Phos-tag SDS-PAGE using Lyn WT and its G2A mutant with a reference sample of the S13A mutant, all prepared by cell-free protein synthesis  of a synthesized protein of interest28. Newly synthesized N-myristoylated Lyn initially enters the Golgi system, where palmitoylation probably occurs, providing entry into the membrane secretory transport pathway en route to the plasma membrane. To examine whether an intracellular transport system through the Golgi is required for phosphorylation of Lyn-S13, we used immunofluorescence microscopy to study the cellular localization of Lyn WT and its G2A and S13A mutants expressed in HEK293 cells  expressed in HEK293 cells ], U0126 and PD98059 [for mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)], ML7 [for myosin light-chain kinase (MLCK)], IC261 [for casein kinase 1 (CK1)], and staurosporine (for broad-range protein kinases) were added to reaction mixtures of the cell-free system at concentrations of up to 500 µM, and the phosphorylation states of the resulting Lyn WT protein were analyzed by Phos-tag SDS-PAGE. None of the inhibitors apart from staurosporine produced a significant change in the banding pattern  and CX4945 (for CK2). Treatment with D4476 dose-dependently inhibited the generation of the Ser-13-phosphorylated species, which disappeared completely at a concentration of 125 µM, whereas the lowest upshifted band still remained in the presence of higher concentrations of CX4945. It has been reported that D4476 is much more potent and specific than IC261 and that it is useful for identifying physiological substrates of CK130. Thus, our inhibitor-screening study demonstrated that phosphorylation of Lyn-S13 was markedly reduced by the CK1 inhibitor D4476, suggesting that the kinase responsible for the phosphorylation reaction at Ser-13 in the cell-free system might be CK1. We also performed in vivo inhibition profiling by using the same inhibitor, D4476, in HEK293 cells expressing Lyn WT protein, but effective inhibition of phosphorylation of Lyn-S13 was not observed. The action of the inhibitor might be affected by several other factors under in vivo conditions (data not shown).Our next aim was to identify the kinase responsible for phosphorylation of Lyn-S13. We therefore tested the effects of various kinase inhibitors on phosphorylation of Lyn-S13 by using the TnT T7 Insect Cell Extract Protein Expression System in conjunction with Phos-tag SDS-PAGE as our preliminary experiments to search for a candidate kinase responsible for phosphorylation of Lyn-S13. In the first inhibitory screening, inhibitors H-89 and KT5720 [for cAMP-dependent protein kinase (PKA)], Ro 31-8220 [for protein kinase C (PKC)], KN-93 . To permit the adjustment of the amount of each FLAG-tagged protein loaded onto the Phos-tag SDS-PAGE gel, the expression levels of FLAG-tagged Lyn or its mutant proteins contained in the lysate samples were determined by conventional SDS-PAGE followed by western blotting with anti-FLAG antibody. The lysate samples were analyzed by Phos-tag SDS-PAGE followed by western blotting. For analyses by immunofluorescence microscopy, 1 × 105 cells/500 µL were plated onto a collagen-coated cover glass laid on a 24-well plate for 6 h and then transfected with each of the expression vectors (10 μg) by using ScreenFect A plus reagents and subsequently incubated for 20 h.HEK293 cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% v/v fetal bovine serum, 100 units/mL of penicillin, and 100 μg/mL of streptomycin in a humidified atmosphere of 5% CO21. Phos-tag Acrylamide AAL-107 (20 μM) and two equivalents of ZnCl2 (40 μM) were added to the separating gel before polymerization. An acrylamide stock solution was prepared containing a 39:1 acrylamide–Bis mixture. The running buffer consisted of 0.10 M Tris and 0.10 M 3-(N-morpholino)propanesulfonic acid (MOPS) containing 0.10% w/v SDS and 5.0 mM NaHSO3, the latter being added immediately before use. Electrophoresis was performed at 30 mA/gel until the bromophenol blue dye reached the bottom of the separating gel. Subsequently, western blotting was performed by the wet-tank method, as described previously44.Phos-tag SDS-PAGE was performed by using a 1-mm-thick, 9-cm-wide, and 9-cm-long gel on a mini-type PAGE apparatus . We used a separating gel (6.3 mL) consisting of 7% w/v polyacrylamide and 357 mM 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol hydrochloride  and a stacking gel (1.8 mL) consisting of 4% w/v polyacrylamide and 357 mM Bis–Tris–HCl buffer (pH 6.8) as a neutral phosphate-affinity SDS-PAGE systemThe transfected cells were stained with Hoechst 33342, washed with a phosphate-buffered saline (PBS), fixed with 4% v/v paraformaldehyde in PBS for 15 min, permeabilized with 0.1% v/v Triton X-100 in PBS for 10 min at room temperature, and finally washed with 1.0% w/v bovine serum albumin in a Tris-buffered saline containing Tween 20 (TBS-T). The permeabilized cells were incubated with specific antibodies in TBS-T for 1 h at room temperature. After washing with TBS-T, the cells were incubated with the fluoro-labeled secondary antibodies for 1 h at room temperature. The cells were then washed with TBS-T and mounted by using ProLong Gold Antifade reagents. The stained cells were observed by using a confocal laser microscope .cDNA encoding Lyn was subcloned into pF25A ICE T7 Flexi Vector, and each plasmid was prepared by using QIAprep Spin Miniprep Kit. In vitro protein synthesis was performed by using the TnT T7 Insect Cell Extract Protein Expression System. A mixture composed of 4.0 µL of the TNT T7 Insect Cell Extract Master Mix and 1.0 µL of each plasmid (0.25 µg) was incubated at 30 °C for 4 h. The sample-loading buffer for SDS-PAGE was then added. For kinase inhibitor assays, 4.0 µL of the TNT T7 Insect Cell Extract Master Mix, 1.0 µL of each plasmid (0.25 µg), and 0.5 µL of inhibitor solution were incubated.Supplementary information.


The PfRh5-Basigin ligand–receptor interaction is an essential step in the merozoite invasion process and represents an attractive vaccine target. To reveal genotype–phenotype associations between naturally occurring allelic variants of PfRh5 and invasion inhibition, we performed ex vivo invasion inhibition assays with monoclonal antibodies targeting basigin coupled with PfRh5 next-generation amplicon sequencing. We found dose-dependent inhibition of invasion across all isolates tested, and no statistically significant difference in invasion inhibition for any single nucleotide polymorphisms. This study demonstrates that PfRh5 remains highly conserved and functionally essential, even in a highly endemic setting, supporting continued development as a strain-transcendent malaria vaccine target. Plasmodium falciparum1. The need for a highly effective second generation malaria vaccine that induces cross-strain protection remains as important as ever. One challenge in the creation of a highly effective vaccine is the extensive genetic diversity of P. falciparum. P. falciparum invasion of human erythrocytes is an essential stage of the life cycle and represents a stage amenable to targeting by both therapeutics and vaccines. Due to the essential nature of invasion for parasite survival, the merozoite form of P. falciparum uses an expanded repertoire of invasion ligands, which can be both variantly expressed and polymorphic, facilitating immune evasion and alternative receptor utilization2. While most of the invasion ligands involved in tight junction formation are dispensable and variant, Plasmodium falciparum reticulocyte binding protein homologous 5 (PfRh5) has been shown to be essential3. Antibodies targeting the PfRh5 receptor, basigin (BSG), are potently inhibitory across all laboratory adapted isolates from diverse geographic origins and a collection of short-term adapted P. falciparum field isolates3 and anti-PfRh5 antibodies have also been shown to be braodly neutralizing4. Vaccine-induced anti-PfRh5 antibodies from Aotus monkeys have been shown to be protective in a strain-transcendent manner in in vitro growth inhibition assays (GIA) and when naturally challenged with heterologous parasite genotypes6. Additionally, vaccine-induced antibodies in humans have been shown to sufficiently inhibit parasite growth across heterologous parasite genotypes in GIA assays8. A PfRh5 vaccine is currently in Phase I/IIa clinical trials.Despite tremendous strides in malaria control, progress has plateaued and malaria is responsible for over 405,000 deaths and 228 million cases of disease globally each year, with the large majority of these cases and deaths being caused by the species www.malariagen.net/pf3k)10. Of these, only five have been found at frequencies of 10% or more in any given population globally11. Antibodies raised against PfRh5 from the 3D7 reference strain have been shown to inhibit invasion across genetic variants in growth inhibition assays4. However, invasion assays with diverse field isolates are needed to discover novel SNPs and assess whether these genetic variations produce significant phenotypic impacts that significantly alter the invasion mechanism ex vivo. In the case of PfRh5, GIA assays have been shown to be a true mechanistic correlate of protection and so these assays, performed ex vivo with naturally occurring isolate populations, will be incredibly informative as to the degree of genotypic and phenotypic variation in the PfRh5-BSG pathway12. In this study, we investigate genotype–phenotype association studies ex vivo in a highly endemic region of Senegal to determine whether specific polymorphisms can influence the inhibition of monoclonal antibodies (mAbs) targeting the PfRh5-BSG invasion pathway.While essential to erythrocyte invasion, PfRh5 is also highly conserved, with 16 nonsynonymous single nucleotide polymorphisms (SNPs) having been described in published data, and 35 in unpublished data from the Pf3k project  within 96 h of starting the assay. Of these 31 harvested assays, 17 showed successful re-invasion with a PMR greater than or equal to 1. These 17 assays were analyzed and inhibition data is presented here 10, K429N has only been described once before, and also in Senegal13. A Mann-Whitney U Test found no statistically significant difference between the degree of invasion inhibition with anti-BSG mAb and the presence or absence of the C203Y SNP, for any concentration of antibody  was performed on the 17 samples from the successful ex vivo assays. SNPs were called using a 25% “dominant” cut-off, reflecting a frequency likely to contribute to a potential measurable phenotypic difference. SNPs were found in 11/17 (64.7%) of the samples  would be greater than 10%, and in practice, 20% or more. We have a sufficient sample size to detect a difference in inhibition of 20–23% with 80% power and an alpha level of 0.05. We also calculated Cohen’s d which is a measure of effect size, and our d values for 0.1 We next sought to determine whether some other measure of invasion efficiency or overall genetic diversity was associated with invasion inhibition. Using both linear regression and correlation coefficients on all data, as well as Mann-Whitney U Test to compare the extreme top and bottom quartiles of inhibition Fig. C,D, we fFurther, by applying a more lenient “discovery” SNP calling threshold of 1% (variant coverage of at least 50 reads), we identified additional SNPs at low frequency in 2 additional samples (13/17 (76.5%), and the overall number of detectable SNPs per sample increased Fig. . This fiP. falciparum exhibit similar levels of invasion inhibition as lab and culture-adapted isolates when presented with varying levels of anti-BSG monoclonal antibody, supporting the hypothesis that the PfRh5-BSG invasion pathway is essential in field parasite populations and that there is minimal phenotypic variation in utilization of this pathway. The inhibitory monoclonal antibody used in this study (MEM-M6/6) binds to the membrane proximal Ig domain D2 of basigin. We have shown the presence of the C203Y SNP, a SNP which is present at the PfRh5-BSG interface and is predicted to interact with the D2 of basigin14, does not have a significant impact on the parasites’ dependence on the PfRh5-BSG invasion pathway, as measured by inhibition with MEM-M6/6, in these ex vivo associative studies. While PfRh5 interacts with other parasite proteins as a part of a larger invasion complex, including PfRipr, PfCyRPA, and P11315, PfRh5 is the only member of this complex of proteins that binds to basigin directly15. However, recent studies have found that S197Y, which is also present at the PfRh5-BSG interface, contributes to immune evasion for a neutralizing monoclonal antibodies directed against PfRh57. Our identification of the K429N SNP in Kédougou, previously described in Thiès, Senegal13, highlights potential region and even country-specific selective pressures that may arise in critical Plasmodium antigens and emphasizes the importance of conducting genotype–phenotype association studies in the field. The novel low frequency SNPs identified in this study will be of interest to pursue in terms of their function. Further studies combining genome editing to test the role of polymorphisms in an isogenic background would allow for precise investigation of whether genotypes observed in the field can alter inhibition with antibodies targeting the receptor–ligand interface. These results show PfRh5 phenotypic function and subsequent immune function are not impacted by diversity and the PfRh5-BSG invasion pathway remains essential in field samples. Previous work had shown similar results, but in lab strains and culture-adapted samples3. Here, we add the substantial evidence from ex vivo field samples from a highly endemic site, known to contain high levels of genetic diversity, exhibiting similar behavior. By demonstrating similar behaviors in field isolates that were observed in lab isolates, we add to the growing body of evidence that PfRh5 is an important, and functionally conserved target for a malaria vaccine.This study shows that field isolates of This study has some limitations. First, the sample size of successful invasion assays is small (n = 17) for a genotype–phenotype association study. However, we have performed power calculations that indicate that we are adequately powered to detect a biologically relevant effect size, a difference in invasion inhibition of 20–23% or greater. An additional limitation of the study is that these ex vivo assays measure the phenotypic behavior of parasite populations within each patient (isolates), not individual parasite clones (strains). However, polyclonal infections are common in highly endemic regions, such as Kédougou and so this limitation may also be viewed as a strength in reflecting the reality of the field. Demonstrating that anti-BSG antibodies can significantly inhibit invasion across a genetically diverse parasite population provides a realistic idea of the potential success of a neutralizing antibodies from a PfRh5 vaccine in patients living in endemic regions.11. Here, all variants had very similar biophysical binding parameters (Kd)11, indicating that these specific SNPs in PfRh5 do not show a dramatic difference in binding kinetics to basigin in vitro. These findings are in contrast to those evaluating the role of a rare basigin variant—E92K—the genetic determinant of the Ok3. The basigin E92K variant had a two-fold lower affinity for PfRh5, due to slower association and a faster dissociation rate, and impacted Plasmodium invasion3. Our experimental future directions are to quantitatively test binding kinetics of each of the PfRh5 variants identified in this study. Our associative experiments in ex vivo isolates are important for identifying associations between invasion pathway and immune evasion; however, to conclusively show that any given SNP does not impact receptor binding or immune evasion, ideally each SNP would be introduced into an isogenic background using genome editing approaches (such as with CRISPR-Cas9), thereby specifically confirming the associations observed in the field. Taken together, our findings support the potential for strain-transcendent immunity generated in response to antibodies induced by the PfRh5 vaccine as thus far we have yet to identify any genotypes that escape potent neutralizing antibodies.An area for future directions is to understand at a deeper, more mechanistic level the contribution of specific SNPs to receptor binding and immune evasion, both the dominant SNPs as well as emerging novel SNPs found at low frequencies. In vitro protein–protein interaction assays have been previously performed for a subset of these dominant SNPs which are prevalent globally. Surface plasmon resonance (SPR) was performed with recombinant basigin as well as wild-type PfRh5 (from 3D7) and variants from 7G8 (C203Y) and GB4 (C203Y + I407V) to determine the binding kinetics and affinitiesP. falciparum on a Pf-specifc HRP2/3 rapid diagnostic test (RDT), a study clinical staffer assessed eligibility and obtained informed consent. A venous blood sample of 5ml in a EDTA vacutainer was obtained from consenting, enrolled patients and transported at room temperature from the clinic to the field lab for processing; no more than 6 h between draw and processing. Thin and thick blood smears were made for each sample to confirm mono-infection with P. falciparum by microscopy. Patient demographics and clinical parameters are described in Table This study was conducted with ethical approval from the National Ethics Committee of Senegal (CNERS) and the Institutional Review Board of the Yale School of Public Health. All research was performed in accordance with relevant guidelines and regulations, and informed consent was obtained from all participants and/or their legal guardians. Samples used in this study were collected as part of ongoing surveillance conducted by Institut Pasteur de Dakar investigating causes of febrile illness. Patients were recruited from five clinics in Kédougou, Senegal. Eligibility criteria for the main study was the presence of a fever  and/or a fever in the past 24 h. If a patient tested positive for 3. All antibodies used in the study were azide-free. The anti-BSG monolonal antibodies used in this study have been shown to specifically interrupt the interaction between PfRh5 and basigin in vitro by surface plasmon resonance (SPR)16. Additionally, wells were plated to allow for microscopic evaluation of assay progression and morphologic staging. Assay plates were cultured in a candle jar culture in a 37 degree C incubator. Assays were harvested when the smear-only control wells showed that at least 95% rings (no more than 5% schizonts remained) for 100 parasitized cells counted. If this criterion was not met, the culture was placed back into the incubator and smears were periodically made until it was ready to harvest. If a sample had not progressed past the schizont stage 96 h after starting the assay, it was discarded as it did not meet the criteria for a successful assay. Upon successful re-invasion, microscopy slides were made, stained with freshly diluted 10% Giemsa (Sigma-Aldrich) and counting was performed blinded. The parasitemia from the RPMI only wells were counted and averaged and these parasitemias were used to determine parasite multiplication rate (PMR). PMR is calculated as: Final parasitemia /Initial parasitemia . Parasitemia from the duplicate test wells were averaged and invasion inhibition, relative to isotype controls, was calculated as follows: 100- [(Average Percent invasion in anti-BSG MEM-M6/6 antibody wells)/(Average parasitemia in IgG1 isotype control wells) * 100]. Of the n = 31 ex vivo invasion assays that had re-invaded and were harvested, the RPMI control wells were first counted by microscopy to determine if they reached a parasite multiplication rate (PMR) of at least 1; a quality control criteria for successful reinvasion. Seventeen of the 31 harvested assays showed a PMR of at least 1, and the inhibition data for these 17 assays are shown here.Initial parasitemia was calculated by thin smear microscopy for each sample. Samples were washed twice with unsupplemented RPMI media and resuspended in supplemented media. All cultures were plated with a starting parasitemia between 0.25%–1.0%, at 4% hematocrit with supplemented RPMI media. Samples with parasitemia greater than 1% were diluted to 1% parasitemia with uninfected O+ erythrocytes. Three concentrations of monoclonal anti-BSG MEM-M6/6 antibody  (10 13 and high fidelity polymerase. Size and purity of the PCR amplicon was confirmed on an agarose gel prior to Next Generation Sequencing (NGS). PCR amplicons for PfRh5 exon 2 were bead-purified (Omega), quantified by Qubit and Bioanalyzer, and normalized by KAPA qPCR and bioanalyzer. Library prep was performed using unique dual indexes (UDIs), samples were multiplexed, pooled, and sequenced with a NovaSeq with targeted coverage of 500,000 reads per sample at the Yale Center for Genome Analysis. Geneious Prime Software version 2020.0.5 (https://www.geneious.com) was used to analyze the sequencing data. Paired-end reads were trimmed with BBDuk plugin ; trimmed sequences were mapped to the PfRh5 reference sequence from 3D7 (PF3D7 0424100). For dominant SNPs, we used a threshold of 25% of reads given minimum read coverage of 5000 reads. For SNP discovery, especially given a high complexity of infection among the samples, SNPs were called given a minimum frequency of 1% of all reads and coverage of minimally 5000 reads. Specific criteria for SNP calling were based on17 and were as follows: minimum quality score of Q35, found in both forward and reverse reads from each sequencing lane, minimum variant frequency of 25% (dominant) or 1% (discovery) and minimum coverage of 5000 reads, maximum variant P value of 0.0001, and minimum strand bias P value of 0.0005 when exceeding 65% strand bias. Final SNPs were independently validated by two individual researchers (A.J.M and A.K.B) and were further confirmed by visual inspection of individual mapped reads.DNA was extracted from dried blood spots (DBS) using QIAmp DNA Blood Mini Kit according to manufacturer’s instructions. PfRh5 exon 2 was PCR amplified using previously described primers18. Specifically, the precision for replicates of 3D7 was calculated as 7.9% and precision for replicates of FVO is was 6.9%18. Given this, a meaningful difference in invasion inhibition  would be greater than 10%, and in practice, 20% or more. This study has 80% power to detect a difference in invasion inhibition of 20–23% or greater between two groups, with an alpha level of 0.05.GraphPad Prism 8.2.4 was used for all statistical analysis. We performed power calculations to determine whether the sample sizes in this study are enough to detect a biologically relevant effect. Based on previous precision estimates for GIA assays with multiple strains, an inhibition value within 10% would be within the limit of precision for biological replicate of a single genotypeA Mann-Whitney U Test was performed to determine if mean inhibition was significantly different for isolates with mutation or without. Statistical associations between antibody-mediated invasion inhibition and PMR, parasitemia or number of SNPs were assessed using a linear regression model and Spearman nonparametric rank correlation coefficients (data not shown).Supplementary Information.


Streptococcus species and in Staphylococcus aureus. Fifty-three are integrated into the origin of transfer (oriT) of a host integrative conjugative element (ICE), encoding a MobT relaxase and belonging to three distant families: ICESt3, Tn916, and ICE6013. The others are integrated into an unrelated IME or in chromosomal sites. After labeling by an antibiotic resistance gene, the conjugative transfer of one of these IMEs (named IME_oriTs) and its host ICE was measured. Although the IME is integrated in an ICE, it does not transfer as a part of the host ICE (no cis-mobilization). The IME excises and transfers separately from the ICE (without impacting its transfer rate) using its own relaxase, distantly related to all known MobT relaxases, and integrates in the oriT of the ICE after transfer. Overall, IME_oriTs use MobT-encoding ICEs both as hosts and as helpers for conjugative transfer. As half of them carry lsa(C), they actively participate in the dissemination of lincosamide–streptogramin A–pleuromutilin resistance among Firmicutes.Integrative mobilizable elements (IMEs) are widespread but very poorly studied integrated elements that can excise and hijack the transfer apparatus of co-resident conjugative elements to promote their own spreading. Sixty-four putative IMEs, harboring closely related mobilization and recombination modules, were found in 14  The relaxase binds covalently to DNA and, by interacting with a so-called coupling protein (CP), brings one of the strands of DNA to an active transport system encoded by the conjugative element. This system of mating pair formation (MPF) is a macromolecular protein complex belonging to the type IV secretion systems (T4SS) [916 superfamily), which includes distantly related families such as Tn916, ICESt3, and ICE6013 families. All MobM-encoding plasmids and all MobT-encoding ICEs encode TcpA CPs that are unrelated to VirD4 [FATA [Integrative conjugative elements (ICEs) are mobile genetic elements (MGEs) integrated in bacterial chromosomes (or plasmids) that are able to excise as a circular form and transfer autonomously by conjugation  . The rels (T4SS) ,6,7. It s (T4SS) ,8. The ms (T4SS) . The knoD4 [FATA .oriT that is sufficiently similar to that of their helper conjugative element to be recognized by its relaxase. Almost all IMEs from Firmicutes characterized until now encode their own relaxase that always belongs to the canonical families also found in Proteobacteria. However, most of the 144 putative IMEs revealed by the analysis of 124 genomes of streptococci encode putative relaxases belonging to five families of RCR initiators of viruses or plasmids [in trans by conjugative elements. However, their integration in ICEs suggests that they could also be transferred as a part of the ICE , are able to transfer by conjugation although they do not encode the MPF. All of the very few exhaustive searches of IMEs and ICEs in genomes revealed more IMEs than ICEs ,12,13,14plasmids . Some oforiT of ICEs belonging to the Tn916 superfamily  [Streptococcus agalactiae, in two strains of Streptococcus mutans, in one strain of Streptococcus mitis, and in one strain of unidentified Streptococcus species. Three of them carry a lsa(C) gene, conferring cross-resistance to lincosamides, streptogramin A, and pleuromutilins in S. agalactiae [oriT, which is conserved in diverse and broadly distributed ICEs from Firmicutes, provides a safe and widespread site of integration. Douarre et al. [oriT family by examining 531 genomes of S. agalactiae. They performed conjugation experiments in order to study the transferability of two IMEs and of their cargo lsa(C). Despite repetitive experiments, only one transconjugant was obtained. Characterization of this transconjugant indicated that the IME_oriT was present in two copies . Transfer of the ICE hosting IME_oriT  in the donor strain was not observed [in trans, thus enabling its co-transfer with the ICE? Does the use of the ICE T4SS by the IME interfere with ICE transfer? Overall, is this IME a harmful pirate or a parasite of the ICE that hampers ICE transfer or a gentle hitchhiker that pacifically co-exists with the ICE?We previously identified a family of putative IMEs that are integrated inside the E_oriTs) . Such IMe et al.  later idobserved . This raobserved )? Is theoriT of ICEs revealed 64 elements, not only in many species of streptococci but also in one strain of Staphylococcus aureus. In order to answer the questions raised above, we studied excision and transfer by conjugation of an IME_oriT. For these purposes, we made genetic constructs to create adequate donor and recipient strains that were then used in various conjugation experiments. These mating assays demonstrated that this IME_oriT can be mobilized in trans but not in cis by the ICE, and does not interfere with ICE transfer.In this study, an exhaustive search of this family of IMEs that target SagLMG15084_oriT described previously [oriTs were also identified using a previously described method [Streptococcus salivarius sequenced in our laboratory  and the name of the bacterial strain that hosts the IME. Denominations also include the name of the target sequence (oriT or oriT* if the IME is integrated in a secondary site). A multiple alignment of the IME sequences was made using Clustal Omega 1.2.2 in order to construct a full distance matrix. This enabled the selection of representative sequences for further analyses. Based on the distance matrix obtained for IME sequences, sequences were aligned two-by-two using BlastN (with default parameters). These comparison files were then used to draw alignment graphs using Artemis Comparison Tool (ACT) provided by the Sanger Centre [The relaxase (GenBank ID: EPW57073.1) and integrase (GenBank ID: EPW57075.1) of IME_d method  on 21 geng paper ) and werr Centre . Manual https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).Cargo genes of the putative IMEs were analyzed using the Conserved Domain search tool on NCBI website  proteins encoded by the putative IMEs were extracted using Geneious Prime  and aligned using ClustalW. Phylogenetic trees were built with MEGA7  using thThe bacterial strains used in this study are indicated in S. salivarius and S. thermophilus strains were grown in M17 broth supplemented with 0.5% lactose (LM17) at 37 or 42 °C, respectively, without shaking. Solid cultures were made in oxygen-free environment induced by GasPak utilization . When required, cultures were supplemented with the following antibiotics: chloramphenicol , erythromycin 5 µg·mL−1, spectinomycin , and kanamycin .S. salivarius L25 as described previously [SsalF1-4_fda in S. salivarius F1-4 and ICESt3 of S. thermophilus in LMG18311) was done by insertion of a chloramphenicol resistance cassette as described previously [SsalL25_oriT and 1-kb-upstream and downstream flanking regions were PCR-amplified before natural transformation of S. thermophilus LMG18311(ICESt3) as described by Gardan et al. [SsalL25_oriTΔrel to avoid introducing an antibiotic resistance gene and polar effect, was similar to the strategy used previously [Overlap PCR was used to insert an antibiotic resistance gene in integrative elements of eviously . Deletioeviously . IME_Ssaeviously . SequencattI and attB were sequenced .Excision tests were done as described previously . SequencS. salivarius and S. thermophilus strains were made as described previously [t-test. A p-value < 0.05 was considered as significant.Mating experiments using eviously ,24. All oriT of an ICE that were previously described [SagLMG15084_oriT to search for homologs in bacterial genomes available on the NCBI website and in 21 genomes of S. salivarius sequenced in our laboratory [SagLMG15084_oriT were found in 14 different Streptococcus species and in one strain of S. aureus. Analysis of these putative relaxases indicate that they all exhibit a PF2486 domain characteristic of Rep_trans_proteins. This family includes the MobT relaxases of ICEs belonging to the Tn916 superfamily  and the RCR initiators involved in the maintenance of many small plasmids of Firmicutes , the integrase gene is truncated (pseudogene). Thus, these elements were considered as defective and were not included in SagGB00984_oriT in 10 other strains of S. agalactiae, one strain of Streptococcus cristatus (98% of nucleotide identity) and one strain of S. mitis (99% of nucleotide identity); (ii) IME_SagUCN70 carrying lsa(C) found in another strain of S. agalactiae, four strains of S. salivarius, four strains of Streptococcus parasanguinis, two strains of S. pseudopneumoniae, and two strains of Streptococcus sp. (98 to 99% of nucleotide identity), and (iii) IME_SaurER01570.3_oriT carrying lsa(C) found in one strain of S. aureus, one strain of S. oralis, and two strains of Streptococcus sp. (100% of nucleotide identity).All the IMEs integrated in an escribed ,17 encodboratory . Sixty-nof pC221 ). Even tof pC221 ,26. Sequn = 53), integration of the IME occurred upstream the gene encoding the MobT relaxase: in Tn916-related ICEs , in ICESt3-related ICEs , and in one ICE related to ICE6013  of the ICE [nic sequence demonstrated for ICEBs1 by Lee and Grossman [oriT was also detected inside an unrelated IME (integrated in rpmG) between the gene encoding a putative relaxase  and the gene encoding its putative TcpA CP  or different integration sites  do not group together on the phylogenetic tree (In most of the cases (nt sites ) in S. a. aureus . For ICE the ICE ,28. As dGrossman ). Intere TcpA CP . In addi TcpA CP . In the tic tree . ElementoriTs, except two), all the IME_oriTs detected by the in silico analysis carry diverse cargo genes  of the detected IMEs carry a lsa(C) resistance gene that is known to confer cross-resistance to lincosamides, streptogramin A, and pleuromutilins in S. agalactiae [S. agalactiae a variant of Lsa(C) that shares 93% of amino acid identity with Lsa(C) found initially in S. agalactiae strain UCN70 [S. agalactiae . A second variant of Lsa(C) showing 96% of amino acid identity with Lsa(C) of S. agalactiae strain UCN70 was discovered in two strains of S. pseudopneumoniae  was found on the IMEs hosted by S. agalactiae LMG15084, 18RS21, and NCTC11079.Several encode proteins that could confer resistance against antibiotics, bacteriocins, or heavy metals . In partalactiae . Douarrealactiae  describein UCN70 . This varistatus . These Ieumoniae . In addioriTs also convey genes that are not involved in resistance: a putative alkylation repair protein encoded by an IME found in S. anginosus (strain NCTC11064), a putative regulator , a two-component system (in strain A12), and a protein with a domain found in chromosomal segregation proteins   . S. salivarius strains, an IME_oriT is integrated into oriT of an ICE of the ICESt3 family, which is integrated in the 3′ end of fda. In S. salivarius L45, another IME_oriT is integrated in oriT of an ICESt3-related ICE integrated in the 3′ end of rpsI. Excision of these three IMEs and of their host ICEs was tested by PCR for wild type strains. All the six integrative elements (IMEs and ICEs) were found to excise  were labeled by kanamycin and spectinomycin resistance genes, respectively, to study their transfer by conjugation. The recipient strain used in the mating pair was S. salivarius strain F1-4 that hosts an ICE of the ICESt3 family integrated in fda. To immobilize this ICE in the recipient strain, the integrase gene of this ICE was deleted and replaced by a chloramphenicol resistance gene  to a S. thermophilus strain (LMG18311) that proved proficient for ICE transfer [St3 (labeled by a chloramphenicol resistance gene) [St3::IME_SsalL25_oriT)) as donor in the mating pairs. To increase the transfer frequency, we used S. thermophilus LMG18311ΔepsE mutant, as recipient strain [St3::IME_SsalL25_oriT) (donor)/LMG18311ΔepsE (recipient), the frequencies of transfer of the ICE and of the IME were 3.3 × 10−4 ± 1.3 × 10−4 and 3.1 × 10−6 ± 1.5 × 10−6 TC/donor cell, respectively  the frequency of TC obtention was 3-fold higher (9.3 × 10−6 ± 6.5 × 10−6 TC/donor cell)  . ICE andt strain . Using tectively . IME_Ssaor cell) .SsalL25_oriT can be mobilized in trans, other strains were constructed. The integrase gene of the ICE hosted by strain LMG18311(ICESt3) was deleted, resulting in LMG18311(ICESt3Δint). Excision and conjugative transfer of this element were not detected. IME_SsalL25_oriT was transferred (by conjugation) in this strain to obtain LMG18311. Then, epsE was also deleted in the LMG18311(ICESt3Δint) strain to obtain LMG18311ΔepsE(ICESt3Δint) strain. In conjugation experiments using the mating pair LMG18311 as donor and LMG18311ΔepsE(ICESt3Δint) as recipient, transconjugants were obtained at a frequency of 4.8 × 10−6 ± 4.0 × 10−6 TC/donor cell  (donor)/LMG18311ΔepsE (recipient without integration site for the IME) gave no transconjugant, indicating that the IME_ SsalL25_oriT integrates efficiently only in an oriT  and conjugation experiments were performed using this strain ) as donor and the LMG18311ΔepsE (ICESt3Δint) strain as recipient. No transfer of the IME was observed using this mating pair  of IME_oriT could be detected.The IME_f ICESt3  and couling pair . Thus, tobserved . TherefoSsalL25_oriT was transferred by conjugation into a strain deleted for the relaxase gene of ICESt3 to obtain LMG18311 used as donor in conjugation experiments with LMG18311ΔepsE strain as recipient. This ICE was still able to excise (data not shown) but no transconjugant was obtained . Freque similar . This in similar .int mutant of ICESt3) does not have any impact on the IME transfer rate in the tested laboratory conditions  belong to a larger family of proteins, Rep_trans, that are classically considered as RCR initiators mainly involved in plasmid maintenance [, which do not encode any MPF protein, encode Rep_trans “RCR initiators” that are very distantly related to genuine MobTs from ICEs [oriTs) are actually relaxases involved in their conjugative transfer; and (ii) these elements are IMEs that use the MPFFA and TcpA of helper ICEs to promote their own transfer. This suggests that the tridimensional structures of the proteins are conserved despite a low conservation of the primary sequence, as previously described for the Rep proteins RepSTK1 of Geobacillus stearothermophilus and staphylococcal Rep proteins [Bs1) has been described for plasmids that encode RCR Initiators of the Rep_1 family [916 and ICEBs1 also catalyze the initiation of RCR needed for the maintenance of the excised ICEs [MobT relaxases from ICEs of the Tnntenance ,31. Nonerom ICEs ,13,14,15proteins . This wo1 family . In addised ICEs ,33. As ssed ICEs .oriT family [oriT. These 64 elements are distributed in 14 streptococcal species and in S. aureus. Almost identical elements were found in distant species, indicating a very recent horizontal transfer of these elements. These elements target the oriT of ICEs belonging to three distantly related families  of the Tn916 superfamily that all exhibit a MobT relaxase. More surprisingly, one IME_oriT was found inside an IME integrated in rpmG. It is worth mentioning that this IME also encodes a relaxase belonging to the enlarged MobT family. Therefore, this suggests that the IME_oriT recognizes specifically the oriT associated to MobT relaxases and more precisely a very short conserved sequence that includes the site that is nicked by the relaxase of a very large array of integrated elements encoding very distant relaxases. However, some of IME_oriT elements were also found integrated in other chromosomal sites (neither in ICEs nor in IMEs) even though they do harbor an integrase closely related to the one of the IMEs integrated in oriT. These sites could be secondary ones that present some similarities with the site targeted by the integrase. We previously described twelve members of this IME_T family . In thisoriT family. Half of these elements carry lsa(C) that confers cross-resistance to lincosamides, streptogramin A, and pleuromutilins. Thus, these IMEs play a major role in the dissemination of this particular cross-resistance. Interestingly, one of these IMEs carrying lsa(C) is integrated in an ICE6013. This is only the second description of an ICE6013 carrying an antibiotic resistance gene. The first description is an ICE6013 carrying a penicillin resistance gene through insertion of Tn552 but, in this case, the transposon disrupts the DDE transposase gene of the ICE and thus impairs its excision and transfer [Streptococcus suis [merA that encodes a mercuric reductase conferring mercury resistance. This gene is frequently found on MGEs, in particular, in oral bacteria belonging to the Firmicutes group [Diverse cargo genes were found on the IMEs of the IME_transfer . In the cus suis . IMEs haes group  due to tes group . Bacteries group . This faes group . oriT failed to find any mobilization in cis by its host ICE. This suggests that disruption of the origin of transfer of the ICE by integration of the IME impairs ICE transfer and that prior excision of the IME is required to visualize ICE transfer. Furthermore, conjugative transfer of the IME by mobilization in trans was observed, albeit at a 100-fold lower frequency than that of ICE. This transfer does not impair the ICE transfer rate. This indicates that IME_oriT benefits from the DNA transport system encoded by their helper ICE but does not impair ICE transfer. Similar interactions could also occur between the many IMEs that integrate inside the genes encoding conserved or essential proteins (such as VirD4 CP) of ICEs of another superfamily widespread in Firmicutes (Tn5252 superfamily) and their ICE host [Salmonella [oriT studied here, these IMEs are harmful pirates of conjugative IncA/C plasmids that await the arrival of the conjugative plasmid and then are probably transferred to all the cells harboring the plasmids and then eliminate these plasmids [Although the elements are integrated in ICEs, the experimental characterization of the mobility of an IME_ICE host ,12. The lmonella ,40,41,42oriTs are the first elements that are clearly shown to use a conjugative element both as a host for their integration and as helper supplying their transfer engine (CP and MPF). Some IMEs, like SGI1, can act as harmful pirates of their host/helper but some (such as IME_oriTs) can also be harmless hitchhikers of the ICEs. Other unrelated IMEs in Firmicutes may have similar capacities. Further studies are needed in order to decipher the mechanistic aspects of their mobilization.In conclusion, IMEs that integrate inside the origin of transfer of both ICEs and IMEs are widespread in streptococci and are also found in other Firmicutes. They vehicle cargo genes, particularly antibiotic resistance genes, and can thus participate in the dissemination of these threatening genes. They can also offer other helpful traits to their bacterial host , which help them persist in bacterial communities. The IME_


Lupinus albus seeds, but its function is still unknown. It shares high structural similarity with xyloglucan-specific endo-glucanase inhibitor proteins (XEGIPs) and, to a lesser extent, with Triticum aestivum endoxylanase inhibitors (TAXI-I), active against fungal glycoside hydrolases GH12 and GH11, respectively. However, γC lacks both these inhibitory activities. Since β-galactomannans are major components of the cell walls of endosperm in several legume plants, we tested the inhibitory activity of γC against a GH2 β-mannosidase (EC 3.2.1.25). γC was actually able to inhibit the enzyme, and this effect was enhanced by the presence of zinc ions. The stoichiometry of the γC/enzyme interaction was 1:1, and the calculated Ki was 1.55 μM. To obtain further insights into the interaction between γC and β-mannosidase, an in silico structural bioinformatic approach was followed, including some docking analyses. By and large, this work describes experimental findings that highlight new scenarios for understanding the natural role of γC. Although structural predictions can leave space for speculative interpretations, the full complexity of the data reported in this work allows one to hypothesize mechanisms of action for the basis of inhibition. At least two mechanisms seem plausible, both involving lupin-γC-peculiar structures.γ-conglutin (γC) is a major protein of  Glycoside hydrolases (GHs) are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides . The GHsSome pathogenic microorganisms are able to secrete GHs to penetrate plant cell walls . As a reLupinus albus /[GH2] was 0.99. The resulting Ki was 1.55 ± 0.08 μM, assuming a competitive mechanism of action of the inhibitor. This inhibition mechanism seems the most plausible, given the mechanism of action of the other GH inhibitors /[enzyme] ratio.r et al. , using tr et al. . The inprding to ; inhibitThe stoichiometry of γC/enzyme binding was determined according to , tracingL. albus γC (UniprotKB: Q9FSH9I) and β-mannosidase (UniprotKB: Q9XCV4) sequences were retrieved from the UniProtKB/Swiss-Prot database (www.expasy.org).The L. albus γC was created using the Swiss Model homology modelling pipeline [https://swissmodel.expasy.org.The PDB model structure of pipeline , a tool The UCSF Chimera software was used for molecular graphics, and surfaces were created with the MSMS package .L. albus γC model and β-mannosidase with the best resolution in the PDB repository, namely, the enzyme from T. harzianum ThMan2A (PDB: 4CVU) [Docking analyses were performed with the B: 4CVU) . These eB: 4CVU) , ClusProB: 4CVU) , PRISM 2B: 4CVU) ,67, GRAMB: 4CVU) , HDOCK SB: 4CVU)  and FRODB: 4CVU) . All theB: 4CVU) .2+ binding sites of γC. This tool [The Metal Ion-Binding site prediction and docking server (MIB) was used to analyze the potential Znhis tool  takes adhis tool ,74.Glycosylation was added to the γC model using the Glyprot software , availabt-tests. p values < 0.05 were considered to be statistically significant.All determinations were carried out in triplicate. Data reported in the histograms are expressed as the means ± S.E. Data were analyzed by L. albus γC, an effect sought for a long time in vain. Considering its homology with XEGIPs, attention has been previously focused on investigating its activity towards those enzymes targeted by XEGIPs themselves. However, switching the target towards enzymes potentially more relevant to legumes’ cell wall attacks, namely, enzymes acting on β-galactomannan degradation, enabled us to obtain first insights into possible γC involvement in cellular responses to pathogens, strengthening the possibility that γC is a multi-functional protein. From an evolutionary point of view, the production and accumulation in seeds of huge amounts of a particular protein that would be needed only if certain microbes attacked, is unfavorable. Normally, antimicrobial compounds are conditionally expressed [In this work, we first describe an inhibitory activity of xpressed . Thus, axpressed  and consxpressed .2+ in enhancing the inhibitory activity of γC deserves further research.In silico predictions showed that the interaction of γC with GH2 β-mannosidase can occur in proximity to the active site and allowed us to propose that the potential inhibitory activity could be mediated by structures that are peculiar to γC, given the lack of XEGIPs’ IL1 and IL2 characteristics. These findings are consistent with the experimental activities since a stoichiometry of 1:1 can be hypothesized from the titration curve of GH2 β-mannosidase with γC. Interestingly, this inhibitory activity described is mediated by the presence of a metal ion. This finding raises new questions on the inhibitory mechanisms of GHIPs, in particular, those that, to date, have been identified as homologs but have failed to demonstrate inhibition, for example, soybean Bg7S. The role of ZnBy and large, this work describes experimental findings that highlight interesting new scenarios for understanding the natural role of γC. Although structural predictions can leave space for speculative interpretations, the full set of data reported in this work allows one to hypothesize possible and allows one to hypothesize possible mechanisms of action for the basis of inhibition. As a matter of fact, at least two mechanisms seem plausible, both involving elements that are peculiar to the lupin γC structure.


The human plasma site-specific glycan map described herein has utility in applications ranging from glycan biomarker research and discovery to the development of novel glycan-altering interventions.Alterations in the human glycome have been associated with cancer and autoimmunity. Thus, constructing a site-specific map of the human glycome for biomarker research and discovery has been a highly sought-after objective. However, due to analytical barriers, comprehensive site-specific glycoprofiling is difficult to perform. To develop a platform to detect easily quantifiable, site-specific, disease-associated glycan alterations for clinical applications, we have adapted the multiple reaction monitoring mass spectrometry method for use in glycan biomarker research. The adaptations allow for highly precise site-specific glycan monitoring with minimum sample prep. Using this technique, we successfully mapped out the relative abundances of the most common 159 glycopeptides in the plasma of 97 healthy volunteers. This plasma glycome map revealed 796 significant (FDR < 0.05) site-specific inter-protein and intra-protein glycan associations, of which the vast majority were previously unknown. Since age and gender are relevant covariants in biomarker research, these variables were also characterized. 13 glycopeptides were found to be associated with gender and 41 to be associated with age. Using just five age-associated glycopeptides, a highly accurate age prediction model was constructed and validated (r Traditionally, proteins are considered the end-product of the information stored in a cell’s genome. However, in order to function appropriately, many proteins require post-translational modifications, and these are commonly glycans. As modifiers, glycans can function as protein “on and off” switches or as “analog regulators” to fine-tune protein function2. The process that synthesizes and enzymatically attaches glycans to organic molecules is called glycosylation and it can produce thousands of unique glycan structures by linking together a finite set of sugar monomers3. However, unlike DNA, RNA and protein synthesis, there is no template to guide the production of glycans. The process is thus immensely complex and impossible to predict from gene expression profiles alone. In fact, when one considers the massive 3-dimensional structural diversity of glycans combined with their variation in attachment sites, the complexity of the glycome parallels that of the genome2.Glycans (oligosaccharides) are one of the four fundamental molecules that make up all living systems2, the National Research Council of the U.S. National Academies highlighted the importance of developing a site-specific map of the serum glycome, which would aid in the development of glycans as biomarkers of human diseases. One reason for the excitement around the use of glycans as disease-specific biomarkers is that glycosylation is a process influenced by a variety of factors including: the type of cell and its activation state; environmental factors, such as the presence of available metabolites; the age of the cell, as glycan moieties can be lost over time; and inflammatory mediators, such as cytokines and chemokines. All these factors can be altered in the setting of human diseases, making the glycome an expression of the overall health status of an individual. Furthermore, it has been hypothesized that glycans not only become altered in the setting of human disease but that they actually play a major role in the etiology of all human diseases2. It is therefore not surprising that alterations in the glycome have already been linked to a variety of human diseases, especially cancer and autoimmunity16. Most of these prior studies used labor-intensive methodologies to characterize glycans released from purified proteins and perhaps for this reason, detailed analyses have only been conducted on a relatively small number of patients. Lower resolution techniques, which yield limited structural information or no site-specific information, have been used to characterize larger patient cohorts, but such analyses are not ideally suited for biomarker discovery research. As a result, the sensitivity and specificity of site-specific glycosylations as disease-specific multi-analyte classifiers of autoimmunity is currently unknown.As part of their glycoscience “Roadmap”2. In this regard, glycoscience is similar to where the field of genetics was during the initial stages of the human genome project2. Mass spectrometry (MS)-based technologies remain very appealing for glycan biomarker research because glycans are ionizable molecules. Also, the potential to accurately profile and quantitate thousands of glycan structures from a relatively small amount of starting material (e.g. 2 µl of serum) makes glycans superior to other molecules traditionally used as biomarkers of human diseases. For example, a site-specific glycoprofiling method could theoretically increase the accuracy of a serum protein biomarker by subdividing it into its different glycoforms.In comparison to the advances made in the fields of genomics and proteomics, glycoscience remains relatively understudied, which is due to a lack of the analytical tools needed to drive the field forward17. Although MRM MS is mainly used in the fields of metabolomics and proteomics21, its high sensitivity and linear response over a wide dynamic range makes it especially suited for glycan detection22. Herein, we employ MRM MS to construct a detailed site-specific structural map of the human plasma glycome of healthy individuals and to characterize the glycans’ inter- and intra-molecular correlations. Glycan alterations associated with age and gender (common covariants in biomarker research and discovery) were also identified and multi-analyte classifiers capable of predicting age were constructed and validated.With the goal of deploying glycan biomarkers clinically, we have developed Multiple Reaction Monitoring (MRM) to site-specifically characterize the human glycome in a rapid and reproducible fashion23 (Table With knowledge of the retention times and collision induced dissociation (CID) behavior of the most abundant serum glycoforms23 Table , we char23 Table  and S2 w23 Table  and S2 w23 Table  and S3 a23 Table .Figure 1Having calculated the relative contribution of each glycopeptide that make up the bulk of the plasma glycome Fig. , we nextP < 2E−16)    Fig.  at site 16) Fig.  positiveP < 2E−16)   with one another at site 176 of alpha 2-HS glycoprotein (A2HSG)  with 5402 at A2MG position 1424 (06) Fig.  which poP < 2E−16) between glycan 5412 at position 70 of Alpha-1 Antitrypsin (A1AT) with glycan 5412 at position 630 of transferrin (TF)  with one another]. This was especially true for the different immunoglobulin subclasses. For example, the abundance of glycan modifiers on IgG1 correlated with their identical counterparts on IgG2  Fig. . This fiP = 0.006) but had a strong highly significant negative correlation (PPMCC -0.81) with the serum concentration of A1AT when present at A1AT site 271 (P < 2E−16)   with A1AT’s serum concentration when present at site A1AT site 70 (16) Fig.  occurrin28. Thus, we sought to characterize the site-specific glycan alterations that could be contributed to the age and gender effect   Fig. A. Thus, 30. Thus, characterizing gender-specific alterations in glycosylation is an important step in developing glycans as biomarkers of human disease. Figure Many biological processes are altered by gender and ultimately this leads to differences in disease frequencies and treatment outcomes2 = 0.62 ± 0.12, fivefold CV)  Table .Figure 4P < 2E−16 and P = 8.4E−06, respectively) with no evidence  of any substantial residual heterogeneity (i.e. there was no remaining variability in effect sizes that was unexplained)  Fig. .2 = 0.67 ± 0.05, fivefold CV)  and 1 serum protein (IgG3). This model was also highly accurate in its ability to predict age  Fig. . AdditioHere we present a detailed site-specific map of the human serum glycome, which reveals many novel features of glycosylation. In some cases, glycosylation varied with protein abundance, such that the probability of a particular site-specific glycosylation occurring became rare as the serum concentration of the protein increased Fig. F. We canOther interesting phenomena that came to light from our study include the observed correlations of site-specific glycosylations across different proteins. This was especially true for IgG1 and IgG2 glycosylations Fig. . However32. Specifically, the prior methods required purification of IgG and enzymatic release of the modifying glycans. Since IgG has multiple sites of glycosylation, analyzing released glycans does not provide site-specific information about the glycans present at the important Ig Fc CH2-84.4 glycosylation site. In contrast, our MRM MS method is site-specific and requires no protein purification. Thus, our glycan mapping results differ significantly from those previously reported32. Furthermore, some amount of glycan structural information is inevitably lost or altered during the ionization process. Thus, different MS ionization and analysis methods will yield different efficiencies of detection for different glycan structures. Thus, our goal was not to definitively determine the hierarchy of occupancy of a particular glycosylation site. Rather, we set out to develop a highly precise method of site-specific glycan detection  the MRM approach employed here eliminates the need for additional protein purification or chemical processing, which allowed for large patient cohorts to be rapidly characterized; (3) the analysis was precise, rapid, and automated for high throughput; (4) it required only 2 µl of serum or plasma and little sample preparation, current techniques require over an mL of blood to quantitate Ig levels; and (5) in addition to total protein quantification (including IgG subtype quantification), the technique provided the relative abundance of each glycopeptide, making it more suitable for biomarker research and discovery. For these reasons, the development of this approach as a clinical diagnostic tool is very appealing, especially when compared to its more labor-intensive alternatives32. Since our method is rapidly evolving, with more site-specific glycosylations being incorporated every month, we anticipate that in the near future glycan analysis will become integral to the diagnosis and management of human diseases, especially diseases of the immune system and cancer.Our study is unique for a variety of reasons: (1) glycan quantification was site-specific across multiple serum proteins including different Ig classes and subclasses, previous studies typically focus on characterizing released glycans or glycoprofiled only a few serum proteinsThe objective of this study was to identify the relative abundance of site-specific glycosylations of the most abundant plasma proteins and then to use this information to better understand patterns of glycosylation in humans, with the ultimate goal of developing a robust glycan biomarker discovery platform. Healthy individuals were recruited from the University of California (UC) Davis Medical Center. The University of California, Davis Institutional Review Board (Committee B) approved this study. Research was performed in accordance to relevant guidelines and regulations. All participants provided their written informed consent.35. To allow for absolute quantification, 100 µg of each monitored protein  was digested according to the same protocol and a dilution series was made prior to sample injection.For each individual enrolled, plasma was separated from whole blood using a Ficoll gradient. From each plasma preparation, a 2 µL aliquot was reduced, alkylated, and then subjected to trypsin digestion at 37 °C36. Briefly, an Agilent Eclipse plus C18  coupled with an Agilent Eclipse plus C18 pre-column  was used for UPLC separation. 1.0 µL of the digested plasma samples was injected and analyzed using a 25-min binary gradient consisting of solvent A of 3% acetonitrile, 0.1% formic acid, solvent B of 90% acetonitrile, 0.1% formic acid in nano-pure water (v/v) at a flow rate of 0.5 mL/min.The neat enzymatically-prepared samples containing both peptides and glycopeptides were then directly analyzed without further hands-on sample cleanup or dilution using an Agilent 1290 infinity liquid chromatography (LC) system coupled to an Agilent 6490 triple quadrupole (QqQ) mass spectrometer , as previously described36. The specific method used herein has been highly validated and the monitored transitions have been described in detail36. Results were integrated using Agilent MassHunter Quantitative Analysis B.5.0 software. Protein concentrations were determined based on calibration curves and glycopeptide relative responses were calculated using the area under the curves of the glycopeptide and a non-glycosylated reference peptide from the same protein.The MRM MS method used for this study requires predetermined knowledge of the peptide or glycopeptide’s LC retention time and its collision induced dissociation (CID) behavior, which we have previously determined for all the non-glycosylated peptides and glycopeptides used in this study Fig. 17,35,36.37. For each analyte, skewedness was calculated, and data was log transformed when necessary to remove excessive skewness. Outliers were identified using R package “extreamvalues”38, and when present, were winsorized from the analysis, so that the outliers were set equal to the nearest non-outlier value. Analytes could be detected in all samples; thus, there was no need for imputation of missing data. ANCOVA and linear regression assumptions about the normality of residuals were examined by use of the Shapiro–Wilk test. Colinearity of variables in the multivariate models was examined by calculating variance inflation factor (excessive if > 2.5) with R package “car”39. Nonlinear relationships between the analytes and the outcome were evaluated with R package “mfp” using a multiple fractional polynomial method40. Variable selection in the multiple linear regressions analyses was performed by forward stepwise exhaustive search using “leaps” R package41. The algorithm searched the best models of all sizes up to the specified maximum number variables. To identify the best number of variables, each model’s performance was estimated by the leave-one-out cross validation method using “caret”42 R package and the number with minimum root-mean-square error (RMSE) was selected. Logistic regression models were fitted using Firth's bias reduction method with the R package “logistf”43. This package was also used for automated variable selection based on penalized likelihood ratio tests. Model performance estimated by fivefold cross-validation was calculated using R package "HandTill2001"44. Meta-analyses were conducted to assess findings across the multiple datasets using R package “metafor”45. A weighted random-effects model was used to estimate a summary effect size. Restricted maximum-likelihood estimator was selected to estimate between-study variance. Weighted estimation with inverse-variance weights was used to fit the model. To present the correlations between all analytes simultaneously, the dimensionality reduction algorithm “t-distributed stochastic neighbor embedding” (t-sne) was used, implemented in the R package “Rtsne”46.All statistical analysis were done using R softwareSupplementary Information 1.Supplementary Information 2.Supplementary Dataset.