N-linked Glycan Sites Research Articles

Modification of N-Linked Glycan Sites in Viral Glycoproteins.

Post-translational modification of proteins by the addition of sugar chains, or glycans, is a functionally important hallmark of proteins trafficked through the secretory system. These proteins are termed glycoproteins. Glycans are known to be important for initiating signaling through binding of cell surface receptors, facilitating protein folding, and maintaining protein stability. For pathogens, glycans can also mask vulnerable protein regions from neutralizing antibodies. Thus, there is a need to develop methods to decipher the role of specific glycans attached to proteins in order to understand their biological role. Here, we describe established methods for identifying glycosylated residues and understanding their role in protein synthesis and function using viral glycoproteins as a model.

Elucidating the Structural Dynamics of Integrin α IIbβ 3 from Native Platelet Membranes By Cryo-EM Coupled with Build and Retrieve Data Processing Methodology

Background: Platelets play a critical role in physiological processes by sensing the extracellular environment through their membrane proteins. Changing the quantity or quality of these proteins can profoundly affect platelet physiology and impact health. The native membrane environment provides essential additional regulatory cues that impact the protein structure and mechanism of action. Single-particle cryogenic electron microscopy (cryo-EM) has transformed structural biology by allowing high-resolution structures of membrane proteins and large macromolecules to be solved from highly purified, homogeneous samples. Our recent breakthroughs in data processing make it feasible to obtain high-resolution protein structures from crude preparations in their native environments by integrating cryo-EM with the novel “Build and Retrieve” (BaR) data processing methodology. By performing in silico purification, image sorting, and model building from large heterogenous datasets, this iterative bottom-up methodology opens new avenues for an in-depth systems biology approach with structural biology that will uncover how the native environment, including lipid and metal binding, post-translational modifications, and co-factor associations regulate platelet function at the molecular level. Method: Whole blood was collected, and the platelet membrane proteins were solubilized by 1% n-Dodecyl-β-D-Maltoside (DDM) and incorporated into lipidic nanodisc MSP1E3D1 directly from the endogenous source. After being enriched by size exclusion chromatography, the mixture of proteins at ~200 kDa was directly applied on cryo-EM grids. Data were collected on Titan Krios G3i cryogenic transmission electron microscope equipped with a BioQuantum K3 camera. All data processing was done in cryoSPARC using the BaR protocol. The near-atomic-resolution cryo-EM maps were used for protein identification using the program phenix.sequence_from_map in the PHENIX suite. The final protein models were built by Coot and refined by PHENIX. Results: We have used using cryo-EM followed by the BaR method to solve the first unmodified integrin α IIbβ 3 structure directly from resting human platelet membranes in its inactivated and intermediate states at 2.76Å and 2.49Å, respectively. From these two structures, we observed the ion binding sites at the β1-domain and β-propeller domain in agreement with previous “classical” α IIbβ 3 structural studies using a purified sample. More importantly, we were able to assign endogenous N-linked glycan sites, which reflected how glycosylation naturally regulates α IIbβ 3 dynamic and function on resting human platelets. Finally, we also solved a novel third unique dimer conformation of α IIbβ 3 at 2.61Å formed by two intermediate-states of α IIbβ 3. This may indicate a previously unknown self-regulatory mechanism of α IIbβ 3 in its native environment. Conclusion: In conclusion, our data show the power of using cryo-EM with the BaR method to uncover novel structures directly from natural sources to identify unrecognized regulation mechanisms for proteins without artifacts due to purification processes. A distinct advantage of the BaR method is that it is an unbiased approach for solving multiple structures from raw samples, which highlights the potential of building a protein structural atlas from platelets. These data have the potential to enrich our understanding of platelet signaling circuitry and foster the development of higher-quality diagnoses and treatments for patients with platelet disorders.

Determining Site-Specific Glycan Profiles of Recombinant SARS-CoV-2 Spike Proteins from Multiple Sources.

Glycopeptide Abundance Distribution Spectra (GADS) were recently introduced as a means of representing, storing, and comparing glycan profiles of intact glycopeptides. Here, using that representation, an extensive analysis is made of multiple commercial sources of the recombinant SARS-CoV-2 spike protein, each containing 22 N-linked glycan sites (sequons). Multiple proteases are used along with variable energy fragmentation followed by ion trap confirmation. This enables a detailed examination of the reproducibility of the method across multiple types of variability. These results show that GADS are consistent between replicates and laboratories for sufficiently abundant glycopeptides. Derived GADS enable the examination and comparison of the glycan profiles between commercial sources of the spike protein. Multiple distinct glycopeptide distributions, generated by multiple proteases, confirm these profiles. Comparisons of GADS derived from 11 sources of recombinant spike protein reveal that sources for which protein expression methods were the same produced near-identical glycan profiles, thereby demonstrating the ability of this method to measure GADS of sufficient reliability to distinguish different glycoform distributions between commercial vendors and potentially to reliably determine and compare differences in glycosylation for any glycoprotein under different conditions of production. All mass spectrometry data files have been deposited in the MassIVE repository under the identifier MSV000091776.

Uncovering the Role of N-Glycan Occupancy on the Cooperative Assembly of Spike and Angiotensin Converting Enzyme 2 Complexes: Insights from Glycoengineering and Native Mass Spectrometry.

Interactions between the SARS-CoV-2 Spike protein and ACE2 are one of the most scrutinized reactions of our time. Yet, questions remain as to the impact of glycans on mediating ACE2 dimerization and downstream interactions with Spike. Here, we address these unanswered questions by combining a glycoengineering strategy with high-resolution native mass spectrometry (MS) to investigate the impact of N-glycan occupancy on the assembly of multiple Spike-ACE2 complexes. We confirmed that intact Spike trimers have all 66 N-linked sites occupied. For monomeric ACE2, all seven N-linked glycan sites are occupied to various degrees; six sites have >90% occupancy, while the seventh site (Asn690) is only partially occupied (∼30%). By resolving the glycoforms on ACE2, we deciphered the influence of each N-glycan on ACE2 dimerization. Unexpectedly, we found that Asn432 plays a role in mediating dimerization, a result confirmed by site-directed mutagenesis. We also found that glycosylated dimeric ACE2 and Spike trimers form complexes with multiple stoichiometries (Spike-ACE2 and Spike2-ACE2) with dissociation constants (Kds) of ∼500 and <100 nM, respectively. Comparing these values indicates that positive cooperativity may drive ACE2 dimers to complex with multiple Spike trimers. Overall, our results show that occupancy has a key regulatory role in mediating interactions between ACE2 dimers and Spike trimers. More generally, since soluble ACE2 (sACE2) retains an intact SARS-CoV-2 interaction site, the importance of glycosylation in ACE2 dimerization and the propensity for Spike and ACE2 to assemble into higher oligomers are molecular details important for developing strategies for neutralizing the virus.

Variations within the Glycan Shield of SARS-CoV-2 Impact Viral Spike Dynamics

The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity, whether arisen naturally or through vaccination. Understanding the structure of the viral spike assists in determining the impact of mutations on the antigenic surface. One class of mutation impacts glycosylation attachment sites, which have the capacity to influence the antigenic structure beyond the immediate site of attachment. Here, we compare the site-specific glycosylation of recombinant viral spike mimetics of B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), B.1.1.529 (Omicron). The P.1 strain exhibits two additional N-linked glycan sites compared to the other variants analyzed and we investigate the impact of these glycans by molecular dynamics. The acquired N188 site is shown to exhibit very limited glycan maturation, consistent with limited enzyme accessibility. Structural modeling and molecular dynamics reveal that N188 is located within a cavity by the receptor binding domain, which influences the dynamics of these attachment domains. These observations suggest a mechanism whereby mutations affecting viral glycosylation sites have a structural impact across the protein surface.

N-Linked Glycan Sites on the Influenza A Virus Neuraminidase Head Domain Are Required for Efficient Viral Incorporation and Replication.

N-linked glycans commonly contribute to secretory protein folding, sorting, and signaling. For enveloped viruses, such as the influenza A virus (IAV), large N-linked glycans can also be added to prevent access to epitopes on the surface antigens hemagglutinin (HA or H) and neuraminidase (NA or N). Sequence analysis showed that in the NA head domain of H1N1 IAVs, three N-linked glycosylation sites are conserved and that a fourth site is conserved in H3N2 IAVs. Variable sites are almost exclusive to H1N1 IAVs of human origin, where the number of head glycosylation sites first increased over time and then decreased with and after the introduction of the 2009 pandemic H1N1 IAV of Eurasian swine origin. In contrast, variable sites exist in H3N2 IAVs of human and swine origin, where the number of head glycosylation sites has mainly increased over time. Analysis of IAVs carrying N1 and N2 mutants demonstrated that the N-linked glycosylation sites on the NA head domain are required for efficient virion incorporation and replication in cells and eggs. It also revealed that N1 stability is more affected by the head domain glycans, suggesting N2 is more amenable to glycan additions. Together, these results indicate that in addition to antigenicity, N-linked glycosylation sites can alter NA enzymatic stability and the NA amount in virions.IMPORTANCE N-linked glycans are transferred to secretory proteins upon entry into the endoplasmic reticulum lumen. In addition to promoting secretory protein maturation, enveloped viruses also utilize these large oligosaccharide structures to prevent access to surface antigen epitopes. Sequence analyses of the influenza A virus (IAV) surface antigen neuraminidase (NA or N) showed that the conservation of N-linked glycosylation sites on the NA enzymatic head domain differs by IAV subtype (H1N1 versus H3N2) and species of origin, with human-derived IAVs possessing the most variability. Experimental analyses verified that the N-linked glycosylation sites on the NA head domain contribute to virion incorporation and replication. It also revealed that the head domain glycans affect N1 stability more than N2, suggesting N2 is more accommodating to glycan additions. These results demonstrate that in addition to antigenicity, changes in N-linked glycosylation sites can alter other properties of viral surface antigens and virions.

ADAMTS‐13 glycans and conformation‐dependent activity

Background ADAMTS-13 activity can be regulated by its conformation, whereby interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS-13 in a closed conformation. ADAMTS-13 contains 10 N-linked glycans, with four sites present in theTSP2 through to CUB domains that may contribute to its conformation. Objectives/Methods We hypothesized that glycosylation contributes to ADAMTS-13 conformation and function. The proteolytic activity of glycan-modified ADAMTS-13 was assessed under static and shear stress conditions. Results Enzymatic removal of terminal silaic acid or entire N-linked glycan chains decreased activity against FRETS-VWF73 at pH 7.4 and against full-length von Willebrand factor (VWF) under shear stress. Using truncated ADAMTS-13, we demonstrated that this was attributable to loss of sialic acid from the glycans in the metalloprotease domain and an effect of N-linked glycosylation in the TSP2 through to CUB domains. Mutation of the N-linked glycan sites in the MDTCS domains reduced or abolished protein expression. However, the N707Q, N828Q, N1235Q and N1354Q (TSP2, TSP4, CUB1, and CUB2 domains, respectively) variants were expressed normally. Interestingly, the N707Q and N828Q variants showed reduced activity against FRETS-VWF73, but normal activity under flow conditions. In contrast, the N1235Q and N1354Q variants had enhanced activity against FRETS-VWF73 and VWF under shear stress. Immunoprecipitation experiments confirmed that loss of N-linked glycans in the CUB domains significantly reduced the interaction with the spacer domain and enhanced binding to the 6A6 anti-ADAMTS-13 antibody, which recognizes a cryptic epitope in the metalloprotease domain. Conclusions Together, these data demonstrate that the N-linked glycans of ADAMTS-13 play a crucial role in regulating ADAMTS-13 activity.

Glycan-independent binding and internalization of human IgM to FCMR, its cognate cellular receptor

IgM is the first antibody to be produced in immune responses and plays an important role in the neutralization of bacteria and viruses. Human IgM is heavily glycosylated, featuring five N-linked glycan sites on the μ chain and one on the J-chain. Glycosylation of IgG is known to modulate the effector functions of Fcγ receptors. In contrast, little is known about the effect of glycosylation on IgM binding to the human Fcμ receptor (hFCMR). In this study, we identify the Cμ4 domain of IgM as the target of hFCMR, and show that binding and internalization of IgM by hFCMR is glycan-independent. We generated a homology-based structure for hFCMR and used molecular dynamic simulations to show how this interaction with IgM may occur. Finally, we reveal an inhibitory function for IgM in the proliferation of T cells.

Removal of N-Linked Glycosylations at Acidic pH by PNGase A Facilitates Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of N-Linked Glycoproteins.

Protein glycosylation is the most frequent post-translational modification and is present on more than 50% of eukaryotic proteins. Glycosylation covers a wide subset of modifications involving many types of complex oligosaccharide structures, making structural analysis of glycoproteins and their glycans challenging for most analytical techniques. Hydrogen/deuterium exchange monitored by mass spectrometry is a sensitive technique for investigation of protein conformational dynamics of complex heterogeneous proteins in solution. N-linked glycoproteins however pose a challenge for HDX-MS. HDX information can typically not be obtained from regions of the glycoprotein that contain the actual N-linked glycan as glycan heterogeneity combined with pepsin digestion yields a large diversity of peptic N-glycosylated peptides that can be difficult to detect. Here, we present a novel HDX-MS workflow for analysis of the conformational dynamics of N-linked glycoproteins that utilizes the enzyme PNGase A for deglycosylation of labeled peptic N-linked glycopeptides at HDX quench conditions, i.e., acidic pH and low temperature. PNGase A-based deglycosylation is thus performed after labeling (post-HDX) and the utility of this approach is demonstrated during analysis of the monoclonal antibody Trastuzumab for which it has been shown that the native conformational dynamics is dependent on the N-linked glycan. In summary, the HDX-MS workflow with integrated PNGase A deglycosylation enables analysis of the native HDX of protein regions containing N-linked glycan sites and should thus significantly improve our ability to study the conformational properties of glycoproteins.

N-Linked Glycans within the A1A2A3 Domains of VWF Play a Critical Role in Modulating Macrophage-Mediated Clearance

Enhanced plasma clearance of von Willebrand factor (VWF) plays an important role in the etiology of both type 1 and type 2 VWD. Nevertheless, although significant progress has been achieved in understanding the structure and functional properties of VWF, the mechanism(s) responsible for modulating VWF clearance from the plasma remain poorly understood. Accumulating recent data suggests that hepatic and splenic macrophages play key roles in modulating VWF clearance. A number of putative macrophage receptors for VWF have been also been described, including LRP1, β2-integrins and Siglec-5. In addition, it is well recognised that variation in VWF glycan expression significantly influences its clearance rate. In particular, terminal ABO(H) blood group determinants which are predominantly expressed on the N-linked glycans of human VWF significantly modulate its rate of clearance. Critically however, the molecular mechanisms through which specific macrophage receptors interact with particular regions of the complex VWF glycoprotein have not been defined.To investigate the role of VWF glycans and specific VWF domains in regulating VWF clearance, we expressed and purified a series of recombinant VWF variants and truncations with/without specific glycan sites. In addition, VWF glycosylation was modified using specific exoglycosidase digestions. Subsequently, recombinant VWF variants and glycoforms thereof were injected into VWF-/-mice, and plasma VWF clearance rates determined by ELISA. VWF-macrophage interactions were also quantified in vitro using phorbol ester-differentiated monocytic THP-1 cells, and primary human monocytes, in a High Content Analysis Imaging system.In keeping with previous reports, we observed that clearance of a truncated VWFA1A2A3 fragment in VWF-/-mice was very similar to that of full-length wild type (WT-) VWF (VWFA1A2A3; t1/2 = 6.3 min versus rWT-VWF; t1/2 = 7.9 min). Furthermore, chemical depletion of macrophages using clodronate liposomes administration significantly inhibited A1A2A3 clearance in vivo (1.7-fold at 10 min time point) to a similar extent to that observed with full length VWF. In vitro binding experiments confirmed that A1A2A3 bound to differentiated THP-1 cells in a dose- and time- dependent manner. Interestingly, this binding was significantly enhanced in the presence of ristocetin. Cumulatively, these data demonstrate that the A1A2A3 domains of VWF contain a critical receptor-binding site for macrophage-mediated clearance.Interestingly, we observed that the half-life of infused human plasma-derived VWF and recombinant VWF expressed in HEK293T cells in VWF-/- mice were significantly different. Furthermore, treatment with PNGase F to completely remove N-linked glycan structures markedly enhanced the clearance of full length VWF (t1/2 2.1 min; p<0.05). Collectively, these findings highlight the essential roles played by N-glycans in regulating VWF survival. Two N-linked glycan sites are located within A1A2A3 at N1515 and N1574 respectively. Importantly, we found that PNGase digestion of A1A2A3 resulted in markedly enhanced macrophage binding in vitro. Consequently we hypothesized that the two N-glycans located within the A2 domain might be important in regulating VWF clearance by macrophages. Targeted disruption of these individual N-glycan sites by site-directed mutagenesis (A1A2A3-N1515Q and A1A2A3-N1574Q respectively) resulted in significantly enhanced macrophage binding in vitro compared to wild type A1A2A3. Furthermore, following tail vein infusion in VWF-/-mice, full length VWFN1515Q and VWFN1574Q both demonstrated markedly reduced half-lives compared to wild type VWF (VWFN1515Q; t1/2 = 3.7 min, VWFN1574Q; t1/2 = 5.5 min). Finally, introduction of the N1515Q point mutation into truncated A1A2A3 also served to significantly enhance plasma clearance, (A1A2A3N1515Q-VWF; t1/2 = 3.1 min versus A1A2A3-VWF; t1/2 = 6.3 min).In conclusion, our novel data identify a crucial role of the VWF A domains in regulating macrophage-mediated VWF clearance. In addition, we further demonstrate that the N-linked glycans structures located at N1515 and N1574 within the A2 domain play specific roles in protecting VWF against in vivo clearance by macrophages. Given the important role played by enhanced VWF clearance in the etiology of type I VWD, these findings are of direct clinical importance. DisclosuresNo relevant conflicts of interest to declare.

Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment

Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs.

Comprehensive Functional Analysis of N-Linked Glycans on Ebola Virus GP1

ABSTRACTEbola virus (EBOV) entry requires the virion surface-associated glycoprotein (GP) that is composed of a trimer of heterodimers (GP1/GP2). The GP1 subunit contains two heavily glycosylated domains, the glycan cap and the mucin-like domain (MLD). The glycan cap contains only N-linked glycans, whereas the MLD contains both N- and O-linked glycans. Site-directed mutagenesis was performed on EBOV GP1 to systematically disrupt N-linked glycan sites to gain an understanding of their role in GP structure and function. All 15 N-glycosylation sites of EBOV GP1 could be removed without compromising the expression of GP. The loss of these 15 glycosylation sites significantly enhanced pseudovirion transduction in Vero cells, which correlated with an increase in protease sensitivity. Interestingly, exposing the receptor-binding domain (RBD) by removing the glycan shield did not allow interaction with the endosomal receptor, NPC1, indicating that the glycan cap/MLD domains mask RBD residues required for binding. The effects of the loss of GP1 N-linked glycans on Ca2+-dependent (C-type) lectin (CLEC)-dependent transduction were complex, and the effect was unique for each of the CLECs tested. Surprisingly, EBOV entry into murine peritoneal macrophages was independent of GP1 N-glycans, suggesting that CLEC-GP1 N-glycan interactions are not required for entry into this important primary cell. Finally, the removal of all GP1 N-glycans outside the MLD enhanced antiserum and antibody sensitivity. In total, our results provide evidence that the conserved N-linked glycans on the EBOV GP1 core protect GP from antibody neutralization despite the negative impact the glycans have on viral entry efficiency.

The N Domain of Human Angiotensin-I-converting Enzyme: THE ROLE OF N-GLYCOSYLATION AND THE CRYSTAL STRUCTURE IN COMPLEX WITH AN N DOMAIN-SPECIFIC PHOSPHINIC INHIBITOR, RXP407

Angiotensin-I-converting enzyme (ACE) plays a critical role in the regulation of blood pressure through its central role in the renin-angiotensin and kallikrein-kinin systems. ACE contains two domains, the N and C domains, both of which are heavily glycosylated. Structural studies of ACE have been fraught with severe difficulties because of surface glycosylation of the protein. In order to investigate the role of glycosylation in the N domain and to create suitable forms for crystallization, we have investigated the importance of the 10 potential N-linked glycan sites using enzymatic deglycosylation, limited proteolysis, and mass spectrometry. A number of glycosylation mutants were generated via site-directed mutagenesis, expressed in CHO cells, and analyzed for enzymatic activity and thermal stability. At least eight of 10 of the potential glycan sites are glycosylated; three C-terminal sites were sufficient for expression of active N domain, whereas two N-terminal sites are important for its thermal stability. The minimally glycosylated Ndom389 construct was highly suitable for crystallization studies. The structure in the presence of an N domain-selective phosphinic inhibitor RXP407 was determined to 2.0 Å resolution. The Ndom389 structure revealed a hinge region that may contribute to the breathing motion proposed for substrate binding.

Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein.

The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008), Nature (London), 454, 177-182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glycoprotein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B-value-sharpened electron-density maps and the proper sequence register was confirmed by building alternate sequences using N-linked glycan sites as anchors and secondary-structural predictions.

N-Glycans and the N Terminus of Protein C Inhibitor Affect the Cofactor-enhanced Rates of Thrombin Inhibition

Protein C inhibitor (PCI) is a serine protease inhibitor, displaying broad protease specificity, found in blood and other tissues. In blood, it is capable of inhibiting both procoagulant and anticoagulant proteases. Mechanisms that provide specificity to PCI remain largely unrevealed. In this study we have for the first time provided a full explanation for the marked size heterogeneity of blood-derived PCI and identified functional differences between naturally occurring PCI variants. The heterogeneity was caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of a Delta6-N-cleaved form. Bi-, tri-, and tetra-antennary complex N-glycans were identified. Fucose residues were identified both on the core GlcNAc and as parts of sialyl-Le(a/x) epitopes. Moreover, a glycan with a composition that implied a di-sialyl antenna was observed. PCI was N-glycosylated at all three potential N-glycosylation sites, Asn-230, Asn-243, and Asn-319, but a small fraction of PCI lacked the N-glycan at Asn-243. The overall removal of N-glycans affected the maximal heparin- and thrombomodulin-enhanced rates of thrombin inhibition differently in different solution conditions. In contrast, the Delta6-N-region increased both the heparin- and the thrombomodulin-enhanced rates of thrombin inhibition at all conditions examined. These results thus demonstrate that the N-linked glycans and the N-terminal region of blood-derived PCI in different ways affect the cofactor-enhanced rates of thrombin inhibition and provide information on the mechanisms by which this may be achieved. The findings are medically important, in view of the documented association of PCI with atherosclerotic plaques and the promising effect of PCI on reducing hypercoagulability states.

N-linked glycosylation of VWF modulates its interaction with ADAMTS13

AbstractWe examined the role of N-linked glycan structures of VWF on its interaction with ADAMTS13. PNGase F digestion followed by lectin analysis demonstrated that more than 90% of VWF N-linked glycan chains could be removed from the molecule (PNG-VWF) without disruption of its multimeric structure or its ability to bind to collagen. PNG-VWF had an approximately 4-fold increased affinity for ADAMTS13 compared with control VWF. PNG-VWF was cleaved by ADAMTS13 faster than control VWF and was also proteolysed in the absence of urea. Occupancy of the N-linked glycan sites at N1515 and N1574 and their presentation of ABO(H) blood group sugars were confirmed with an isolated tryptic fragment. Recombinant VWF was mutated to prevent glycosylation at these sites. Mutation of N1515 did not alter ADAMTS13 binding or increase rate of ADAMTS13 proteolysis. Mutation of N1574 increased the susceptibility of VWF to ADAMTS13 proteolysis and allowed cleavage in the absence of urea. Mutation of N1574 in the isolated recombinant VWF-A2 domain also increased binding and ADAMTS13 proteolysis. These data demonstrate that the N-linked glycans of VWF have a modulatory effect on the interaction with ADAMTS13. At least part of this effect is conformational, but steric hindrance may also be important.

N-Linked Oligosaccharides Direct the Differential Assembly and Secretion of Inhibin α- and βA-Subunit Dimers

The biosynthetic pathway governing inhibin heterodimer (alpha/beta) and activin homodimer (beta/beta) assembly and secretion from ovarian granulosa cells is not fully understood. Here, we examined the role of inhibin subunit glycosylation in the assembly and secretion of mature inhibin A and activin A. Inhibition of subunit glycosylation by tunicamycin treatment of alpha- and beta(A)-expressing CHO cell lines reduced inhibin but not activin secretion. Dimeric inhibin A is preferentially secreted from parental isogenic wild-type (wt) cell lines (alpha(wt)beta(wt)). Mutation of a single glycosylation site at asparagine 268 (alpha(Delta268)beta(wt)) reduces inhibin secretion by 78% and permits beta/beta assembly and secretion. Conversely, gain of a glycosylation (GOG) site in the analogous region of the beta(A)-subunit (alpha(wt)beta(GOG327)) enhances inhibin A secretion. The present study demonstrates that N-linked glycan sites direct heterodimer vs. homodimer assembly, and prevention of glycosylation abrogates inhibin secretion. These data support a definitive role for site-specific N-glycosylation in governing inhibin/activin dimer assembly and secretion.

The Effect of Von Willebrand Factor Glycans on the Interaction with ADAMTS13.

Von Willebrand factor (VWF) is a large plasma glycoprotein that mediates platelet tethering at sites of vascular injury. This function is dependent upon its multimeric size, which is modulated in plasma through proteolysis by ADAMTS13. Terminal ABO blood group sugars presented on the N-linked glycans of VWF alter the susceptibility of VWF to cleavage by ADAMTS13. Two predicted N-linked glycan sites (N1515 & N1574) are in close proximity to the ADAMTS13 cleavage site (Y1605-M1606). However, is it not known whether these predicted sites are occupied, or indeed, present the ABO sugars. In this study, purified plasma VWF was digested with trypsin, and the resulting fragments separated by ion-exchange chromatography. A 55kDa fragment observed under reducing SDS-PAGE was identified as VWF residues 1493–1849 by mass spectrocosopy and N-terminal sequencing. This fragment encompassed the predicted N-linked glycan sites at N1515 & N1574. Incomplete PNGase F digestion produced three protein bands, indicating that both glycosylation sites were occupied. Furthermore, analysis with blood group specific lectins demonstrated presentation of the ABO sugars on these glycan chains. This data suggests that the N1515 and N1574 glycosylation sites and subsequent ABO(H) sugar modification may influence VWF proteolysis. To further elucidate the role of other VWF glycan structures on susceptibility to ADAMTS13 cleavage, purified VWF was treated with neuraminidase to remove terminal sialic acid residues, producing asialo VWF (As-VWF), and PNGase F to remove whole N-linked glycan chains. (Nless-VWF). Lectin analysis demonstrated removal of >95% of terminal sialic residues and >75% whole N-linked glycan chains. Both AsVWF and Nless-VWF bound to type III collagen with similar affinity to wild type VWF (KD 1.9nM, 1.6nM, 1.4nM respectively) and demonstrated similar multimeric composition. Interestingly, As-VWF had decreased susceptibility to ADAMTS13 cleavage, but bound ADAMTS13 with comparable affinity to wild type VWF (KD 11nM & 12nM respectively). Nless-VWF was cleaved faster by ADAMTS13 and bound ADAMTS13 with ~ 4-fold increased affinity (KD 3.4nM).This data demonstrates that the glycan moiety of VWF plays an important role in mediating the interaction with ADAMTS13.

Investigation of cationic peanut peroxidase glycans by electrospray ionization mass spectrometry

Cationic peanut peroxidase (CP) was isolated from peanut ( Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by 1H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry.