Complex-type Biantennary N-glycans Research Articles

Characterization of novel endo-β-N-acetylglucosaminidases from intestinal Barnesiella intestinihominis that hydrolyze multi-branched complex-type N-glycans

Endo-β-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze N-linked glycans. Many ENGases have been characterized, but few have been identified with hydrolytic activity towards multi-branched complex-type N-glycans. In this study, three candidate ENGases were identified from Barnesiella intestinihominis based on database searches and phylogenetic analysis. A domain search identified the NxE motif in all three candidates, suggesting that they were members of glycosyl hydrolase family 85 (GH85). The three candidate ENGases, named Endo-BIN1, Endo-BIN2, Endo-BIN3, were expressed in Escherichia coli cells, and their hydrolytic activity towards N-glycans and glycoproteins were measured by HPLC analysis and SDS-PAGE analysis. All ENGases showed hydrolytic activity towards glycoproteins, but only Endo-BIN2 and Endo-BIN3 showed hydrolytic activity towards pyridylaminated N-glycans. Optimum pH of Endo-BIN1 Endo-BIN2, End-BIN3 were pH 6.5, 4.0, and 7.0, respectively. We measured substrate specificities of Endo-BIN2 and Endo-BIN3 towards pyridylaminated N-glycans, and found that the two Endo-BIN enzymes showed similar substrate specificity, preferring bi-antennary complex-type N-glycans with galactose or α2,6-linked sialic acid residues at the non-reducing ends. Endo-BIN2 and Endo-BIN3 were also able to hydrolyze multi-branched complex-type N-glycans. SDS-PAGE analysis revealed that all Endo-BIN enzymes were capable of releasing complex-type N-glycans from glycoproteins such as rituximab, transferrin, and fetuin. We expect that B. intestinihominis possesses ENGases to facilitate the utilization of complex-type N-glycans from host cells. These findings will have applications in N-glycan remodeling of glycoproteins and the development of pharmaceuticals.

Enabling Chemoenzymatic Strategies and Enzymes for Synthesizing Sialyl Glycans and Sialyl Glycoconjugates.

Sialic acids are fascinating negatively charged nine-carbon monosaccharides. Sialic acid-containing glycans and glycoconjugates are structurally diverse, functionally important, and synthetically challenging molecules. We have developed highly efficient chemoenzymatic strategies that combine the power of chemical synthesis and enzyme catalysis to make sialic acids, sialyl glycans, sialyl glycoconjugates, and their derivatives more accessible, enabling the efforts to explore their functions and applications. The Account starts with a brief description of the structural diversity and the functional importance of naturally occurring sialic acids and sialosides. The development of one-pot multienzyme (OPME) chemoenzymatic sialylation strategies is then introduced, highlighting its advantages in synthesizing structurally diverse sialosides with a sialyltransferase donor substrate engineering tactic. With the strategy, systematic access to sialosides containing different sialic acid forms with modifications at C3/4/5/7/8/9, various internal glycans, and diverse sialyl linkages is now possible. Also briefly described is the combination of the OPME sialylation strategy with bacterial sialidases for synthesizing sialidase inhibitors. With the goal of simplifying the product purification process for enzymatic glycosylation reactions, glycosphingolipids that contain a naturally existing hydrophobic tag are attractive targets for chemoenzymatic total synthesis. A user-friendly highly efficient chemoenzymatic strategy is developed which involves three main processes, including chemical synthesis of lactosyl sphingosine as a water-soluble hydrophobic tag-containing intermediate, OPME enzymatic extension of its glycan component with a single C18-cartridge purification of the product, followed by a facile chemical acylation reaction. The strategy allows the introduction of different sialic acid forms and diverse fatty acyl chains into the products. Gram-scale synthesis has been demonstrated. OPME sialylation has also been demonstrated for the chemoenzymatic synthesis of sialyl glycopeptides and in vitro enzymatic N-glycan processing for the formation of glycoproteins with disialylated biantennary complex-type N-glycans. For synthesizing human milk oligosaccharides (HMOs) which are glycans with a free reducing end, acceptor substrate engineering and process engineering strategies are developed, which involve the design of a hydrophobic tag that can be easily installed into the acceptor substrate to allow facile purification of the product from enzymatic reactions and can be conveniently removed in the final step to produce target molecules. The process engineering involves heat-inactivation of enzymes in the intermediate steps in multistep OPME reactions for the production of long-chain sialoside targets in a single reaction pot and with a single C18-cartridge purification process. In addition, a chemoenzymatic synthon strategy has been developed. It involves the design of a derivative of the sialyltransferase donor substrate precursor, which is tolerated by enzymes in OPME reactions, introduced to enzymatic products, and then chemically converted to the desired target structures in the final step. The chemoenzymatic synthon approach has been used together with the acceptor substrate engineering method in the synthesis of complex bacterial glycans containing sialic acids, legionaminic acids, and derivatives. The biocatalysts characterized and their engineered mutants developed by the Chen group are described, with highlights on synthetically useful enzymes. We anticipate further development of chemoenzymatic strategies and biocatalysts to enable exploration of the sialic acid space.

Site-selective sulfation of N-glycans by human GlcNAc-6-O-sulfotransferase 1 (CHST2) and chemoenzymatic synthesis of sulfated antibody glycoforms

Sulfation is a common modification of glycans and glycoproteins. Sulfated N-glycans have been identified in various glycoproteins and implicated for biological functions, but in vitro synthesis of structurally well-defined full length sulfated N-glycans remains to be described. We report here the first in vitro enzymatic sulfation of biantennary complex type N-glycans by recombinant human CHST2 (GlcNAc-6-O-sulfotransferase 1, GlcNAc6ST-1). We found that the sulfotransferase showed high antennary preference and could selectively sulfate the GlcNAc moiety located on the Manα1,3Man arm of the biantennary N-glycan. The glycan chain was further elongated by bacterial β1,4 galactosyltransferase from Neiserria meningitidis and human β1,4 galactosyltransferase IV（B4GALT4）, which led to the formation of different sulfated N-glycans. Using rituximab as a model IgG antibody, we further demonstrated that the sulfated N-glycans could be efficiently transferred to an intact antibody by using a chemoenzymatic Fc glycan remodeling method, providing homogeneous sulfated glycoforms of antibodies. Preliminary binding analysis indicated that sulfation did not affect the apparent affinity of the antibody for FcγIIIa receptor.

Identification of N-glycan positional isomers by combining IMS and vibrational fingerprinting of structurally determinant CID fragments.

While glycans are present on the surface of cells in all living organisms and play key roles in most biological processes, their isomeric complexity makes their structural characterization challenging. Of particular importance are positional isomers, for which analytical standards are difficult to obtain. We combine ultrahigh-resolution ion-mobility spectrometry with collision-induced dissociation and cryogenic infrared spectroscopy to determine the structure of N-glycan positional isomers. This approach is based on first separating the parent molecules by SLIM-based IMS, producing diagnostic fragments specific to each positional isomer, separating the fragments by IMS, and identifying them by comparing their IR fingerprints to a previously recorded spectral database. We demonstrate this strategy using a bottom-up scheme to identify the positional isomers of the N-linked glycan G0-N, in which a terminal N-acetylglucosamine (GlcNAc) is attached to either the α-3 or α-6 branch of the common N-glycan pentasaccharide core. We then use IR fingerprints of these newly identified isomers to identify the positional isomers of G1 and G1F, which are biantennary complex-type N-glycans with a terminal galactose attached to either the α-3 or α-6 branch, and in the case of G1F a fucose attached to the reducing-end GlcNAc. Starting with just a few analytical standards, this fragment-based spectroscopy method allows us to develop a database which we can use to identify positional isomers. The generalization of this approach would greatly facilitate glycan analysis.

Preparation and biological activities of anti-HER2 monoclonal antibodies with multibranched complex-type N-glycans.

Immunoglobulin G (IgG) has a conserved N-glycosylation site at Asn297 in the fragment crystallizable (Fc) region. Previous studies have shown that N-glycosylation of this site is a critical mediator of the antibody's effector functions, such as antibody-dependent cellular cytotoxicity. While the N-glycan structures attached to the IgG-Fc region are generally heterogenous, IgGs engineered to be homogenously glycosylated with functional N-glycans may improve the efficacy of antibodies. The major glycoforms of the N-glycans on the IgG-Fc region are bi-antennary complex-type N-glycans, while multibranched complex-type N-glycans are not typically found. However, IgGs with tri-antennary complex-type N-glycans have been generated using the N-glycan remodeling technique, suggesting that more branched N-glycans might be artificially attached. At present, little is known about the properties of these IgGs. In this study, IgGs with multibranched N-glycans on the Fc region were prepared by using a combination of the glycosynthase/oxazoline substrate-based N-glycan remodeling technique and successive reactions with glycosyltransferases. Among the IgGs produced by these methods, the largest N-glycan attached was a bisecting N-acetylglucosamine containing a sialylated penta-antennary structure. Concerning the Fc-mediated effector functions, the majority of IgGs with tri- and tetra-antennary N-glycans on their Fc region showed properties similar to IgGs with ordinary bi-antennary N-glycans.

Site-specific N-glycan Analysis of Antibody-binding Fc γ Receptors from Primary Human Monocytes

FcγRIIIa (CD16a) and FcγRIIa (CD32a) on monocytes are essential for proper effector functions including antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Indeed, therapeutic monoclonal antibodies (mAbs) that bind FcγRs with greater affinity exhibit greater efficacy. Furthermore, post-translational modification impacts antibody binding affinity, most notably the composition of the asparagine(N)-linked glycan at N162 of CD16a. CD16a is widely recognized as the key receptor for the monocyte response, however the post-translational modifications of CD16a from endogenous monocytes are not described. Here we isolated monocytes from individual donors and characterized the composition of CD16a and CD32a N-glycans from all modified sites. The composition of CD16a N-glycans varied by glycosylation site and donor. CD16a displayed primarily complex-type biantennary N-glycans at N162, however some individuals expressed CD16a V158 with ∼20% hybrid and oligomannose types which increased affinity for IgG1 Fc according to surface plasmon resonance binding analyses. The CD16a N45-glycans contain markedly less processing than other sites with >75% hybrid and oligomannose forms. N38 and N74 of CD16a both contain highly processed complex-type N-glycans with N-acetyllactosamine repeats and complex-type biantennary N-glycans dominate at N169. The composition of CD16a N-glycans isolated from monocytes included a higher proportion of oligomannose-type N-glycans at N45 and less sialylation plus greater branch fucosylation than we observed in a recent analysis of NK cell CD16a. The additional analysis of CD32a from monocytes revealed different features than observed for CD16a including the presence of a predominantly biantennary complex-type N-glycans with two sialic acids at both sites (N64 and N145).

Glycoanalysis of the placental membrane glycoproteins throughout placental development

Structural changes of glycans are observed in different (patho)physiological conditions. Human placental membrane (glyco)proteins were isolated from the first and third trimester placentas of mothers at different ages. By using lectin microarray, we demonstrated that the placental membrane N-glycome contains several N-glycan groups: high mannose, asialylated and sialylated biantennary moieties, bisected, core fucosylated, fucosylated at other positions (bearing terminal and/or antennary Fuc), α2-6 and α2-3 sialylated structures. Employing MALDI-TOF MS enabled identification of over sixty different N-glycan structures in all samples, with 17 moieties exceeding the relative abundance of 2%. The major MS peaks originated from: 1) biantennary complex type N-glycan with a bisecting GlcNAc residue and 2) a core Fuc paucimannosidic and high mannose type structures M3-M9. Age of mothers and the stage of placental development affected N-glycome. The work presented in this article is the first comprehensive mass spectrometric study of the N-glycome of human placental membrane proteins. Our results may be seen as the baseline which can serve for future MALDI MS profiling of the placental membrane N-glycome in different pathophysiological conditions.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling.

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise α-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial β-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Revisiting the substrate specificity of mammalian α1,6-fucosyltransferase reveals that it catalyzes core fucosylation of N-glycans lacking α1,3-arm GlcNAc

The mammalian α1,6-fucosyltransferase (FUT8) catalyzes the core fucosylation of N-glycans in the biosynthesis of glycoproteins. Previously, intensive in vitro studies with crude extract or purified enzyme concluded that the attachment of a GlcNAc on the α1,3 mannose arm of N-glycan is essential for FUT8-catalyzed core fucosylation. In contrast, we have recently shown that expression of erythropoietin in a GnTI knock-out, FUT8-overexpressing cell line results in the production of fully core-fucosylated glycoforms of the oligomannose substrate Man5GlcNAc2, suggesting that FUT8 can catalyze core fucosylation of N-glycans lacking an α1,3-arm GlcNAc in cells. Here, we revisited the substrate specificity of FUT8 by examining its in vitro activity toward an array of selected N-glycans, glycopeptides, and glycoproteins. Consistent with previous studies, we found that free N-glycans lacking an unmasked α1,3-arm GlcNAc moiety are not FUT8 substrates. However, Man5GlcNAc2 glycan could be efficiently core-fucosylated by FUT8 in an appropriate protein/peptide context, such as with the erythropoietin protein, a V3 polypeptide derived from HIV-1 gp120, or a simple 9-fluorenylmethyl chloroformate-protected Asn moiety. Interestingly, when placed in the V3 polypeptide context, a mature bi-antennary complex-type N-glycan also could be core-fucosylated by FUT8, albeit at much lower efficiency than the Man5GlcNAc2 peptide. This study represents the first report of in vitro FUT8-catalyzed core fucosylation of N-glycans lacking the α1,3-arm GlcNAc moiety. Our results suggest that an appropriate polypeptide context or other adequate structural elements in the acceptor substrate could facilitate the core fucosylation by FUT8.

Exploring Site-Specific N-Glycosylation of HEK293 and Plant-Produced Human IgA Isotypes.

The full potential of recombinant Immunoglobulin A as therapeutic antibody is not fully explored, owing to the fact that structure–function relationships of these extensively glycosylated proteins are not well understood. Here monomeric IgA1, IgA2m(1), and IgA2m(2) variants of the anti-HER2 antibody (IgG1) trastuzumab were expressed in glyco-engineered Nicotiana benthamiana plants and in human HEK293-6E cells. All three IgA isotypes were purified and subjected to biophysical and biochemical characterization. While no differences in assembly, antigen binding, and glycosylation occupancy were observed, both systems vary tremendously in terms of glycan structures and heterogeneity of glycosylation. Mass-spectrometric analysis of site-specific glycosylation revealed that plant-produced IgAs carry mainly complex-type biantennary N-glycans. HEK293-6E-produced IgAs, on the contrary, showed very heterogeneous N-glycans with high levels of sialylation, core-fucose, and the presence of branched structures. The site-specific analysis revealed major differences between the individual N-glycosylation sites of each IgA subtype. Moreover, the proline-rich hinge region from HEK293-6E cell-derived IgA1 was occupied with mucin-type O-glycans, whereas IgA1 from N. benthamiana displayed numerous plant-specific modifications. Interestingly, a shift in unfolding of the CH2 domain of plant-produced IgA toward lower temperatures can be observed with differential scanning calorimetry, suggesting that distinct glycoforms affect the thermal stability of IgAs.

The Emerging Importance of IgG Fab Glycosylation in Immunity.

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity.

Exploring N-Glycan Conformers: Assessment of Enhanced Sampling Algorithms

N-glycans (oligosaccharides linked to proteins via the side chain of asparagine) play an important role in protein folding control, cell adhesion, organism immune response, etc. The functional versatility comes from structural variety and the inherit flexibility of glycosidic linkages. Each N-glycan has several co-existing conformers posing a challenge for experimental characterisation and computational modelling. In this work, we assess the enhanced sampling capabilities of the replica-exchange MD (REMD) and metadynamics (MTD). While REMD has been used in the previous studies of N-glycans, it is the first application of MTD, as we know. Our proposed approach is based on the multi-replica MTD, where some degrees of freedom are biased explicit by the MTD procedure and the others implicitly by using replicas at higher temperatures. In addition, we apply the replica state exchange MTD (RSE-MTD), our proposed enhancement to increase replica exchange efficiency. Finally, the methods have been tested on Bi9, a biantennary complex-type N-glycan. We demonstrate that REMD, following a protocol from the previous studies, fails to provide consistent results, in contrast, our MTD approach enhances sampling along the critical collective variables and ensures converged results. The estimated conformer populations of Bi9 are in agreement with the experimental results obtained from the NMR pseudo-contact shift (PCS) measurements.

Expression of natural human β1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans.

β1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human β1,4-galactosyltransferase 1 (GalT). Here we show that these differences are associated with differences at its N-terminus: the natural short variant of human GalT results in hybrid type N-glycans, whereas the long form generates bi-antennary complex type N-glycans. Furthermore, expression of non-mammalian, chicken and zebrafish GalT homologues with N-termini resembling the short human GalT N-terminus also induce hybrid type N-glycans. Providing both non-mammalian GalTs with a 13 amino acid N-terminal extension that distinguishes the two naturally occurring forms of human GalT, acted to increase the levels of bi-antennary galactosylated N-glycans when expressed in tobacco leaves. Replacement of the cytosolic tail and transmembrane domain of chicken and zebrafish GalTs with the corresponding region of rat α2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further.

A simplified procedure for gram-scale production of sialylglycopeptide (SGP) from egg yolks and subsequent semi-synthesis of Man3GlcNAc oxazoline

Heterogeneity of glycan structures in native glycoconjugates always hampers precise studies on carbohydrate-involved biological functions. To construct homogeneous glycoconjugates from natural resource of homogeneous glycans is therefore a practical approach to solve this problem. We report here an optimized procedure for gram-scale production of sialylglycopeptide (SGP) containing a disialyl biantennary complex-type N-glycan from egg yolks. Our new procedure simplified the extraction process by treating the egg yolk powder with 40% acetone, avoiding massive emulsification, high-speed centrifugation, and sophisticated chromatography in reported methods. Subsequent semi-synthesis of the N-glycan core Man3GlcNAc oxazoline from SGP was accomplished for the first-time via glyco-trimming and successive oxazoline formation. This efficient semi-synthesis provides an alternative to the pure chemical approach that involves multi-step total synthesis and facilitates the application of endo-glycosidase-enabled chemoenzymatic synthesis of various homogeneous glycoconjugates.

A1.45 Hyperglycosylation of ACPA-IGG variable domains modulates reactivity to citrullinated antigens

Background and ObjectivesAutoantibodies specific for citrullinated antigens are highly relevant diagnostic and prognostic biomarkers in rheumatoid arthritis and have been considered to be involved in disease pathogenesis. Previous studies have...

Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-LeX identified in a previous glycan microarray screen.

Human gastric TFF2 peptide contains an N-linked fucosylated N,N'-diacetyllactosediamine (LacdiNAc) oligosaccharide

In the human stomach, the peptide trefoil factor family 2 (TFF2) is secreted together with the mucin MUC6 by mucous neck cells (MNCs) and antral gland cells. TFF2 is strongly associated with the gastric mucus and promotes gastric restitution. Here, TFF2 was purified from the human corpus and antrum, respectively, by size-exclusion chromatography, and the N-linked glycan structure at N-15 of the mature peptide was determined. As a hallmark, the unusual monofucosylated N,N'-diacetylhexosediamine (tentatively assigned as GalNAcβ1→4GlcNAc, LacdiNAc) modification was detected as the terminal structure of a bi-antennary complex type N-glycan exhibiting also core fucosylation. Replicate analyses did not show microheterogeneities in the fraction of peptide-N-glycosidase F cleaved and permethylated N-glycans when analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). On the glycopeptide level, a minor glycan microheterogeneity was evident in liquid chromatography-electrospray ionization (ESI)-MS, demonstrating the presence of underfucosylated species. The tryptic TFF2 N-glycopeptide p34-39 (LSPHNR N-glycosylated with Fuc3Hex3HexNAc6) was identified by both ESI-tandem mass spectrometry and MALDI-post-source decay analysis. Lectin analyses with the Wisteria floribunda agglutinin indicated the potential presence of LacdiNAc terminating glycans and revealed minor differences between TFF2 from fundic units, i.e. MNCs, and antral units, i.e. antral gland cells. Strikingly, on the level of the primary structure, there was no indication that the formation of the proposed LacdiNAc structure is cis-controlled by a peptidic determinant related to the published sequences.

Effect of Bisecting GlcNAc and Core Fucosylation on Conformational Properties of Biantennary Complex-Type N-Glycans in Solution

The introduction of bisecting GlcNAc and core fucosylation in N-glycans is essential for fine functional regulation of glycoproteins. In this paper, the effect of these modifications on the conformational properties of N-glycans is examined at the atomic level by performing replica-exchange molecular dynamics (REMD) simulations. We simulate four biantennary complex-type N-glycans, namely, unmodified, two single-substituted with either bisecting GlcNAc or core fucose, and disubstituted forms. By using REMD as an enhanced sampling technique, five distinct conformers in solution, each of which is characterized by its local orientation of the Manα1-6Man glycosidic linkage, are observed for all four N-glycans. The chemical modifications significantly change their conformational equilibria. The number of major conformers is reduced from five to two and from five to four upon the introduction of bisecting GlcNAc and core fucosylation, respectively. The population change is attributed to specific inter-residue hydrogen bonds, including water-mediated ones. The experimental NMR data, including nuclear Overhauser enhancement and scalar J-coupling constants, are well reproduced taking the multiple conformers into account. Our structural model supports the concept of "conformer selection", which emphasizes the conformational flexibility of N-glycans in protein-glycan interactions.

An N-glycosylation Analysis of Human Alpha-2-Macroglobulin Using an Integrated Approach

Assignment of glycosylation sites and site microheterogeneity is of both biological and clinical significance. Herein, the detailed N-glycosylation pattern of human serum alpha-2-macroglobulin was studied using an integrative approach, including permethylation of N-glycans, collision induced dissociation (CID) and electron transfer dissociation (ETD) of chymotryptic N-glycopeptides, and partial deglycosylation of chymotryptic N-glycopeptides with endo-β-N-acetylglucosaminidase F3 (Endo F3). Three N-glycosylation sites were found to be occupied by four biantennary complex type N-glycans using N-glycan analysis and the ETD/CID method. Endo F3 assisted mass spectrometric analysis yielded five N-glycosylation sites with and without core fucosylation. In total, six out of eight potential N-glycosylation sites were identified using this approach. This integrative approach was performed using only 10 μL of human serum for both N-glycosylation site assignment and site microheterogeneity determination.

1PT212 バイセクティングGlcNAcとフコースの導入が水中N型糖鎖の構造多様性に及ぼす影響(日本生物物理学会第50回年会(2012年度))

1PT212 バイセクティングGlcNAcとフコースの導入が水中N型糖鎖の構造多様性に及ぼす影響(日本生物物理学会第50回年会(2012年度))