HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Arnold, J. N. Articles by Rudd, P. M. Search for Related Content PubMed PubMed Citation Articles by Arnold, J. N. Articles by Rudd, P. M.

The Glycosylation of Human Serum IgD and IgE and the Accessibility of Identified Oligomannose Structures for Interaction with Mannan-Binding Lectin¹

James N. Arnold^2,*, Catherine M. Radcliffe, Mark R. Wormald, Louise Royle, David J. Harvey, Max Crispin, Raymond A. Dwek, Robert B. Sim^* and Pauline M. Rudd

^* Medical Research Council (MRC) Immunochemistry Unit and Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn³⁵⁴ are inaccessible because the complex glycans at Asn⁴⁴⁵ block access to the site. On IgE, the additional C_H2 hinge domain blocks access to the oligomannose glycans at Asn³⁹⁴ on one H chain by adopting an asymmetrically bent conformation. IgE contains 8.3% Man₅GlcNAc₂ glycans, which are the trimmed products of the Glc₃Man₉GlcNAc₂ oligomannose precursor. The presence of these structures suggests that the C_H2 domain flips between two bent quaternary conformations so that the oligomannose glycans on each chain become accessible for limited trimming to Man₅GlcNAc₂ during glycan biosynthesis. This is the first study of the glycosylation of human serum IgD and IgE from nonmyeloma proteins.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
There are five classes of Ig in humans, IgG, IgM, IgA, IgD, and IgE. All are glycoproteins and therefore their populations may contain glycoforms which can be recognized by the lectin-like recognition proteins of the innate immune system, such as mannan-binding lectin (MBL),³ macrophage mannose receptor (mMR), and the surfactant proteins SP-A and SP-D. Much is already known about the glycosylation of IgG (1) and IgA (2, 3). IgG contains a conserved N-glycosylation site in the C_H2 domain (Fig. 1) at Asn²⁹⁷ in the Fc region. The biantennary complex glycans at this site have been shown to be variable (4). One set of glycoforms, referred to as IgG-G2, contains glycans at the Asn²⁹⁷ site terminating in a galactose residue on both arms. In IgG-G1 glycoforms, a terminal galactose residue is missing from one arm, exposing a GlcNAc residue. In IgG-G0 glycoforms, neither arm is galactosylated so that a GlcNAc residue is exposed at the terminals of each arm. In normal human serum, 20% of IgG glycans at this glycosylation site terminate in GlcNAc on both arms. The IgG-G0 glycoforms have been shown to bind MBL (5) and also to interact with the mMR (6). MBL has been shown to bind to polymeric forms of serum IgA (7). The interaction of MBL with human polymeric IgM is uncertain. It has been shown that mouse IgM and to a lesser extent, human serum IgM can be purified by affinity chromatography using immobilized rabbit MBL (8), but immobilized polyclonal human IgM does not bind MBL (9). The L chains of the Igs contain no conserved N- or O-linked glycosylation sites.

View larger version (55K): [in this window] [in a new window]   FIGURE 1. Diagrammatic structure of IgG, IgD, and IgE. Diagrammatic representation of IgG, IgD, and IgE showing their variable H chain (VH), constant H chain (CH) domains, and their two L chain domains. The diagram also shows the split into Fab and Fc domains. The structures of IgD and IgE are hypothetical as no crystal structures exist, although the Fc region of IgE has been crystallized (43 ). The asparagines to which N-linked glycans are attached are marked in sites predicted by molecular modeling. The O-linked glycosylation sites of IgD, which are found in the extended hinge region of the CH1 and CH2 domains are shown. It is uncertain whether both Thr131 and Thr132 or only one are glycosylated (27 ). Asn383 on IgE is a potential N-link glycosylation site, but was shown not to be glycosylated in a myeloma IgE (31 ).

An earlier report on glycosylation of a myeloma IgD indicated that the Ig contains oligomannose glycans (23), that are potential binding sites for MBL. The ability of MBL to bind IgD and IgE has not previously been examined. Normal, polyclonal IgD and IgE are difficult to purify from human plasma due to their low abundance (30 µg/ml and <1 µg/ml, respectively). The biological role of IgD in blood is uncertain. In 1972, IgD was found to be membrane-bound as part of the BCR (24) on immature B cells. IgD is secreted into the serum as part of the primary Ab response upon B cell activation. The serum half-life of IgD is short at 2.8 days (25) perhaps because of an extended hinge region between the Fc and Fab which renders IgD susceptible to proteolytic degradation (26). IgD has three N-linked glycosylation sites (Fig. 1) in the Fc region at positions Asn³⁵⁴, Asn⁴⁴⁵, Asn⁴⁹⁶ (27). Studies of a myeloma IgD protein (23) found that Asn³⁵⁴ in the C_H2 domain was occupied by oligomannose structures. These if exposed, could provide a binding site for MBL or macrophage mannose receptor. The other sites contained sugars terminating in galactose and sialic acid (complex glycans). The oligomannose sugars of Asn³⁵⁴ have been shown to play a structural role, as mutagenesis of this site resulted in nonsecretion of the H chain (28). The hinge region of IgD contains multiple O-linked glycosylation sites (Fig. 1), though it has been found that complete inhibition of O-linked glycosylation using benzyl 2-acetamido-2-deoxy--D-galactopyranosidase did not affect assembly and secretion (28).

IgE is the least abundant serum Ab. IgE directed toward allergens leads to the symptoms of allergy through binding to the FcRI found on tissue mast cells and basophils (reviewed by Refs. 29 and 30). This binding leads to the release of proinflammatory mediators such as histamine, causing the symptoms of allergy. There are seven N-linked glycosylation sites in the -chain at Asn¹⁴⁰, Asn¹⁶⁸, Asn²¹⁸, Asn²⁶⁵, Asn³⁷¹, Asn³⁸³, and Asn³⁹⁴ in the constant region of IgE (Fig. 1) (31). Quantitative site analysis of glycans from a myeloma IgE protein identified Asn³⁸³ as an unoccupied site and Asn³⁹⁴ as containing oligomannose structures (31).

We report here the first study of serum (nonmyeloma) IgD and IgE glycosylation. This study confirms the presence of oligomannose glycans and evaluates the accessibility of these N-linked glycans for MBL binding using a combination of molecular modeling and ELISA-style binding assays. It has previously been demonstrated that MBL binds to the IgG-G0 glycoforms and polymeric forms of serum IgA. Serum IgD and IgE have not previously been assessed as targets of MBL binding.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abs and antisera

Purified human IgD from pooled normal serum was supplied by Kent Laboratories (cat. no. 12770; Bellingham, WA) and human IgE from single donor hyperimmune serum was supplied by Biodesign International (A10164H; Saco, ME). IgG known to contain 34% IgG-G0 (data not shown) was obtained from the Glycobiology Institute (Oxford, U.K.). The samples were shown to be pure by SDS-PAGE and direct ELISA using goat anti-human IgD (-chain specific) alkaline phosphatase conjugate (A6406), goat anti-human IgE (-chain specific) alkaline phosphatase conjugate (A3525), and goat anti-human IgG (-chain specific) alkaline phosphatase conjugate (A3187) obtained from Sigma-Aldrich (Poole, U.K.). Rabbit anti-MBL polyclonal antiserum was raised by Charles River Laboratories (Romans, France) against the recombinant head and neck region of MBL (N. K. Lim, MRC Immunochemistry Unit; unpublished work). The antiserum was depleted of anti-mannan Abs by passage on a mannan-agarose resin (M9917; Sigma-Aldrich) run in Dulbecco’s PBS (8.2 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 139 mM NaCl, 3 mM KCl, pH 7.4) obtained from Oxoid (Hampshire, U.K.) made 0.5 mM EDTA.

MBL purification

The purification was based on the method of Tan et al. (32). One liter of citrated human plasma (HDS Supplies, High Wycombe, U.K.) was made 20 mM CaCl₂ and left overnight at 4°C to clot. The clot was filtered through muslin, and the serum made 7% w/v polyethylene glycol (PEG; m.w. 3350, P-3640; Sigma-Aldrich) and left stirring for 2h at 4°C for a precipitate to form. The precipitate was spun down at 11,000 x g for 30 min at 4°C. The supernatant was discarded and the pellet was resuspended and washed in 400 ml of TBS-TCa-PEG (50 mM Tris, 140 mM NaCl, 0.05% Tween 20, 20 mM CaCl₂, 7% PEG, pH 7.8). The pellet was redissolved in 50 ml of HS-TBS-TCa (50 mM Tris, 1M NaCl, 0.05% Tween 20, 20 mM CaCl₂, pH 7.8). These dissolved proteins were then incubated for 2 h at 4°C with 20 ml of mannan-agarose resin (M-9917; Sigma-Aldrich) equilibrated with HS-TBS-TCa, rotating slowly. The resin was then washed with 500 ml of HS-TBS-TCa at 4°C and packed into a 1.5-cm diameter column. The MBL/MASPs were eluted with HS-TBS-TEDTA (50 mM Tris, 1M NaCl, 0.05% Tween 20, 10 mM EDTA, pH 7.8) and fractions collected and pooled. The MBL concentration was calculated using a standardized MBL detection ELISA (as described below). This partially purified MBL, which was used for the binding studies, contained contaminants of MASPs, IgG, and IgM and traces of other proteins. Highly pure MBL was obtained by dialyzing the preparation into gel filtration running buffer (0.1 M sodium acetate, 0.2 M NaCl, 5 mM EDTA, pH 5.0) and running on a Superose 6 gel filtration column. The fractions (1 ml), judged to contain MBL, were analyzed by SDS-PAGE and pooled. This material, which still contained some IgM contamination, was dialyzed into PBS 0.5 mM EDTA and run on a 5-ml anti-human IgM (µ-chain specific) agarose resin (A9935; Sigma-Aldrich). The resulting MBL was pure and quantified by amino acid analysis.

MBL quantification ELISA

ELISA plates (Nunc-Maxisorp; Roskilde, Denmark) were coated with 100 µl of 50 µg/ml mannan (M-7504; Sigma-Aldrich) in 0.1 M NaHCO₃, pH 9.5. After each step the wells were washed three times at room temperature (RT) with 200 µl of wash buffer (10 mM HEPES, 5 mM CaCl₂, 1 M NaCl, 0.1% Tween 20, pH 7.4). The wells were blocked with 400 µl of PBS, 0.1% Tween 20 (PBST) for 2 h at RT and then washed and incubated for 1 h at RT with 1/100–1/10,000 (v/v) dilutions of the purified MBL in 10 mM HEPES, 1 M NaCl, 5 mM CaCl₂, pH 7.4. Negative controls were diluted in 10 mM HEPES, 1 M NaCl, 5 mM EDTA, pH 7.4, to prevent MBL binding. The ELISA was standardized using the highly purified MBL. The wells were washed and then incubated for 1 h at RT with 100 µl of a 1/250 (v/v) dilution of rabbit anti-MBL antiserum, washed and incubated with 100 µl of a 1/700 (v/v) dilution of goat anti-rabbit IgG (whole molecule) Ab alkaline phosphatase conjugate (A-3812; Sigma-Aldrich). After another washing, 100 µl of 1 mg/ml p-nitrophenyl phosphate substrate in 0.2 M Tris buffer (N-2770; Sigma-Aldrich) was added. The absorbances at 405 nm were recorded after 10 min.

Denaturation of Igs

A total of 20 µg of IgE (in 100 mM Tris, 200 mM NaCl, 0.1% sodium azide, pH 7.5) or IgD (in 10 mM Tris, 200 mM NaCl, 0.05% sodium azide, pH 8) was denatured by mixing 1:1 (v/v) with 0.2 M Tris, 40 mM DTT, 8 M guanidine-HCl, pH 8.2, and incubated for 2 h at 37°C. The samples were then made 42 mM iodoacetamide and incubated at 37°C for a further 15 min. The guanidine was then removed by microdialysis for 20 h against PBS at 4°C.

Assay for MBL binding to targets

ELISA plate (Nunc-Maxisorp) wells were coated with 100 µl of 10 µg/ml IgD, IgE, or IgG, the last of which was used as a positive binding control. BSA (A3912; Sigma-Aldrich) was used as a negative binding control and all were diluted in 0.1 M NaHCO₃, pH 9.5. The wells were incubated at 4°C overnight, then blocked with 400 µl of PBST for 2 h at RT, washed three times with 200 µl of wash buffer (10 mM HEPES, 1 M NaCl, 5 mM CaCl₂, 0.1% Tween 20, pH 7.4), and incubated in triplicate for 1 h at 37°C with 50 ng/well of the purified MBL diluted in 10 mM HEPES, 1 M NaCl, 5 mM CaCl₂, pH 7.4. Other wells were incubated with MBL diluted with 10 mM HEPES, 1 M NaCl, 5 mM EDTA, pH 7.4, as controls for non-calcium-dependent binding. The wells were washed three times with the wash buffer and incubated for 1 h at RT with 100 µl of 1/250 (v/v) anti-MBL polyclonal antiserum in wash buffer. The wells were washed and incubated with 100 µl of a 1/2000 (v/v) dilution of monoclonal anti-rabbit IgG (-chain specific: clone RG-96 alkaline phosphatase conjugate; A-2556, Sigma-Aldrich) in wash buffer, washed again, and 100 µl of an AmpliQ DakoCytomation (Cambridgeshire, U.K.) amplification kit reagent mixture was added and the OD was read at 492 nm after 30 min.

Removal of N-linked glycans for analysis

SDS-PAGE gels were prepared and run according to Küster et al. (33) and in-gel N-linked glycan release was performed as described by Radcliffe et al. (34). IgE (10 µg in 100 mM Tris, 200 mM NaCl, 0.1% sodium azide, pH 7.5) or IgD (10 µg in 10 mM Tris, 200 mM NaCl, 0.05% sodium azide, pH 8) was reduced with 50 mM DTT and incubated for 10 min at 70°C then alkylated with 10 mM iodoacetamide. The sample was then incubated at RT for 30 min in the dark before being loaded onto the gel, and run at 500 V, 25 mA for 1 h. The separated glycoproteins were visualized with Coomassie blue stain and the relevant bands were cut into small pieces and dried using vacuum centrifugation. Three units of peptide N-glycanase F (PNGase, EC 3.5.1.52, 1000 U/ml) diluted in 27 µl of 20 mM NaHCO₃ was added per 10–15 mm³ of gel and incubated for 16 h at 37°C. N-linked glycans were extracted from the gel pieces by collecting the supernatants of sequential gel incubations with 3x 200 µl of water, then 200 µl of acetonitrile, then 200 µl of water, and finally 200 µl of acetonitrile in a sonicating water bath for 30 min at RT. The collected supernatants were concentrated in the vacuum centrifuge to 500 µl, then depleted of any ions using 50 µl of an AG-50 X12 (H⁺ activated) ion-exchange resin, which was removed by filtering through a 0.45-µm LH Millipore filter using a syringe. The N-linked glycans were dried for 2-aminobenzamide (2AB) labeling.

Release of O-glycans by hydrazinolysis

The O-linked glycans were released chemically with anhydrous hydrazine as described by Royle et al. (35).

2AB labeling

Released glycans were labeled by reductive amination with the fluorophore 2AB according to Bigge et al. (36), using a Ludger Tag 2AB glycan labeling kit (Ludger, Oxford, U.K.). Excess 2AB reagent was removed by ascending chromatography on Whatman 3MM paper (Clifton, NJ) in acetonitrile.

Normal phase-HPLC (NP-HPLC) and anion exchange-HPLC

Labeled glycans were separated on NP-HPLC as described by Guile et al. (37). NP-HPLC used a 4.6 x 250-mm TSK amide-80 column (Anachem, Luton, U.K.) with a linear gradient of 20–58% solvent A (50 mM formic acid adjusted to pH 4.4 with ammonium hydroxide) with solvent B (acetonitrile). Fluorescence was measured at 420 nm with excitation at 330 nm. Weak anion exchange HPLC was conducted as described by Zamze et al. (38) using a Vydac 301VHP575 7.5 x 50-mm weak anion exchange column (Hichrom, Berkshire, U.K.). Glycan profiles from NP-HPLC were calibrated against a dextran ladder prepared from hydrolyzed and 2AB-labeled glucose oligomers (37). Glycans were assigned glucose unit (GU) values and glycan structure/composition was predicted by reference to a glycan database using the program PeakTime (E. Hart, R. A. Dwek, P. M. Rudd (Glycobiology Institute), unpublished).

Exoglycosidase digestions

Exoglycosidases were used to confirm the structures of glycans present in the preparations, in conjunction with HPLC (34). Enzymes were used at manufacturers’ recommended concentrations and digests were conducted using 50 mM sodium acetate buffer, pH 5.5, for 16 h at 37°C. Enzymes were supplied by Glyko (Upper Heyford, U.K.); 1–2 U/ml Arthobacter ureafaciens sialidase (ABS; EC3.2.1.18); 3 mU/ml almond meal -fucosidase (AMF; EC 3.2.1.111); 1 U/ml bovine testis -galactosidase (BTG, EC 3.2.1.23); 100 mU/ml jack bean -mannosidase (JBM; EC 3.2.1.24); 120 U/ml Streptococcus pneumonia -hexosaminidase (SPH; EC 3.2.1.30); 40 U/ml -N-acetylglucosaminidase (GuH) (cloned from S. pneumonia, expressed in Escherichia coli, EC 3.2.1.30) (Europa Bioproducts, Cambridge, U.K.); 1 U/ml S. pneumonia sialidase recombinant from Escherichia coli (NAN1, EC 3.2.1.18); 100 U/ml bovine kidney fucosidase (BKF; EC 3.2.1.51); glucosidase II (GlcII) was prepared in the Glycobiology Institute (Oxford, U.K.).

Mass spectrometry

Non-2AB-labeled N-linked glycans were analyzed by MALDI-MS and O-linked glycans were analyzed by liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS)/MS as described earlier (35).

Molecular modeling data

Sequence alignment was performed using Align (39) on the equivalent domains of IgG, IgD, and IgE (Swiss-Prot: P01857, P01880, and P01854, respectively). Molecular modeling was performed on a Silicon Graphics Fuel workstation using InsightII and Discover software (Accelrys, San Diego, CA). Figures were produced using the program Molscript (40). Crystal structures used as the basis for modeling were obtained from the Protein Data Bank (41). Molecular models for the Fc domains of IgG and IgD were based on the crystal structure of IgG Fc (42) and the molecular model for IgE was based on the crystal structure of IgE hinge and Fc domains (43). N- and O-glycan structures were generated using the database of glycosidic linkage conformations (44, 45) and in vacuo energy minimization to relieve unfavorable steric interactions. The Asn-GlcNAc linkage conformations were based on the observed range of crystallographic values (46).

Glycosylated Asn side-chains can adopt three distinct conformations (46), with ₁/₂ values of 60°/180°, 180°/180°, and 300°/180° (₁ = N-C – C – C and ₂ = C – C – C – N), each conformation being reasonably flexible and with easy interconversion between them. In IgD, Asn³⁵⁴ and Asn⁴⁹⁶ can only adopt one of these conformations, whereas two of these conformations are sterically allowed for Asn⁴⁴⁵ (180°/180° and 300°/180°). Thus, IgD was modeled with Asn⁴⁴⁵ on one H chain in the 180°/180° conformation and on the other H chain in the 300°/180° conformation. In IgE, Asn³⁸³ and Asn³⁹⁴ can only adopt one of the side-chain conformations. All three distinct side-chain conformations are sterically allowed for Asn³⁷¹, and so IgE was modeled with Asn³⁷¹ on one H chain in the 180°/180° conformation and on the other H chain in the 300°/180° conformation. For Asn²⁶⁵, none of the three possible side-chain conformations is sterically disallowed due to interactions with the C_H2 domain (the domain to which it is attached). However, for the Asn²⁶⁵ on the H chain closer to the C_H4 domains, the –60°/180° conformation leads to fewer steric interactions between the glycan outer arms and the C_H3/C_H4 domains and so this conformation was used.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
N-linked glycans of IgD

2AB-labeled glycans released from the -chain (Fig. 2) were assigned structures from GU values, shifts with the enzyme digest arrays and MALDI-MS (Fig. 3 and Table I). Sialylated glycan peaks digested with ABS but not with NAN1 indicating that all sialic acid residues were 2,6- (and not 2,3-) linked to galactose. Weak anion exchange HPLC and digests showed the presence of neutral, mono-, and disialylated structures. Oligomannose sugars Man₅-Man₉ (for oligomannose structures see Table IV) were identified as previously described on an IgD myeloma WAH (23). In addition, there was a Man₉Glc₁ structure (Table I) that accounted for 3.3% of the glycan pool. A GlcII digest of Man₈ and Man₉ collected from NP-HPLC revealed the presence of a Man₈Glc₁ structure which accounted for 15% of the GU 9.61 peak (Fig. 4). The sum of the oligomannose structures comprised 37% of the glycan pool, which is compatible with the occupation of one of the three N-linkage glycosylation sites and in agreement with data from the work of Mellis and Baenziger (23), who showed that in a myeloma IgD, oligomannose glycans occupy the Asn³⁵⁴ site. The remaining 63% of glycan structures terminated in galactose or sialic acid. No glycans were detected on the L chain of IgD by NP-HPLC (data not shown).

View larger version (74K): [in this window] [in a new window]   FIGURE 2. SDS-PAGE analysis of reduced and alkylated IgD and IgE. The gels were 10% acrylamide, the molecular mass marker (molecular mass 6,500–205,000) was supplied by Sigma-Aldrich (M4038). The presence of IgD was confirmed using goat anti-human IgD (-chain specific) alkaline phosphatase conjugate. The presence of IgE was confirmed using goat anti-human IgE (-chain specific) alkaline phosphatase conjugate. The H chains appeared as doublets. Bands were excised and analyzed separately but as the glycan profiles were identical the doublets were pooled. A trace of IgG is present in the IgE preparation, identified as IgG H chain from its molecular mass and by ELISA using a goat anti-human IgG (-chain specific) alkaline phosphatase conjugate supplied by Sigma-Aldrich (A3187).

View larger version (42K): [in this window] [in a new window]   FIGURE 3. NP-HPLC exoglycosidase digestion profile of glycan pool from IgD. 2AB-labeled N-linked glycans were digested by exoglycosidases and analyzed by NP-HPLC. Structure abbreviations: all N-glycans have two core N-acetyl-glucosamines (GlcNAcs): Fc, core fucose linked -1–6 to the inner GlcNAc: Man, number of mannose residues on core GlcNAcs: A, number of antennae on the trimannosyl core; A2, bianntenary; B, bisecting GlcNAc linked 1–4 to inner mannose; G, number of galactose residues on antennae; S, number of sialic acids on antennae. The profile shows most notably the presence of oligomannose structures. Percentage areas and GU values are shown in Table I. The exoglycosidases used were: ABS (removes sialic acid), BTG (removes galactose), BKF (removes core fucose), GuH (removes terminal GlcNAc).

View this table: [in this window] [in a new window]   Table I. Pooled normal IgD chain N-linked glycans

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Man8Glc1 glycan identified under the Man9 peak from IgD. The -GlcII (removes glucose) digestion of the GU 9.61 peak fraction analyzed by NP-HPLC results in a Man8 peak, showing that a Man8Glc1 species is present in addition to Man9. Man8Glc1 makes up 15% of the GU 9.61 peak and 2.4% of the overall glycan pool

The O-linked glycans were removed chemically by hydrazinolysis. The 2AB-labeled glycans were run on NP-HPLC before and after digestion with ABS, and ABS + BTG. Peaks were distinguished from background noise and structural assignments were made based on GU values. The profile (Fig. 5) shows the presence of neutral, mono-, and disialylated Core I structures. The HPLC assignments were consistent with results from LC-ESI-MS/MS (Table II).

View larger version (28K): [in this window] [in a new window]   FIGURE 5. NP-HPLC analysis of O-linked glycans from IgD. NP-HPLC of the O-linked glycans of IgD before and after sialidase digestion. Numbers are GU values for the glycan peaks. All other peaks are not glycans. O-linked glycans identified were neutral, mono-, and disialylated Core I structures as shown. The sialylated peak at GU 2.27 is a product of peeling where Core I glycans have broken down in the release procedure and the galactose attached to a sialic acid is 2AB labeled. The structures are represented by: GlcNAc, ; galactose, ; fucose, with a dot inside; sialic acid, ; linkage, solid line; linkage, dotted line; 1–6 linkage, \; 1–4 linkage, —; 1–2 linkage, /.

View this table: [in this window] [in a new window]   Table II. Pooled normal IgD chain O-linked glycans

The structures of 2AB-labeled glycans from the -chain (Fig. 2) were assigned from GU values, shifts with enzyme digestion arrays, and MALDI-MS (Fig. 6 and Table III). Weak anion exchange chromatography separated the neutral, mono-, di-, and confirmed the absence of trisialylated structures. A NAN1 digestion showed no 2,3-linked sialic acid, indicating that all sialic acids were 2,6-linked to galactose as subsequently confirmed by digestion with ABS. Oligomannose structures Man₄-Man₈ comprised 14.2% of the glycan pool, which is compatible with the occupation of one of the potential seven N-linkage sites. The presence of Man₄ is caused by an incomplete digestion during glycan processing of Man₅ to Man₃ by -mannosidase II or -mannosidase III in the golgi. Man₅ was the most abundant oligomannose structure that accounted for 8.3% of the glycan pool. Dorrington and Bennich (31) established that Asn³⁹⁴ was occupied by oligomannose structures in an IgE myeloma. The remaining 85.8% of glycans terminated in galactose or sialic acid. No glycans were detected on the L chain of IgE by NP-HPLC (data not shown).

View larger version (39K): [in this window] [in a new window]   FIGURE 6. NP-HPLC exoglycosidase digestion profile of glycan pool from IgE. NP-HPLC profiles of N-linked IgE glycans, and results of digestion arrays of exoglycosidases (see legend of Fig. 4 for details).

View this table: [in this window] [in a new window]   Table III. IgE chain N-linked glycans (see Table I for details)

The N-linked glycans of IgD and IgE contain many of the same glycan structures (Table I and III). IgD and IgE both contain oligomannose ladders (Man₅-Man₉ and Man₄-Man₈, respectively). Comparing the overall glycan profiles, 97% of the structures present in IgE are also present on IgD. IgD, however, contains a larger number of glycan structures, and only 65% of the structures found in IgD were also present in IgE.

MBL-binding studies

The binding of MBL to the Igs was assessed using ELISA plates coated with IgD, IgE, and IgD and IgE that had been denatured to expose any inaccessible oligomannose glycans present (Fig. 7). IgG was used as a positive binding control and BSA was used as a negative binding control. Assays were done in triplicate with additional EDTA negative controls. There was no significant binding of MBL to native IgD, however, unfolding of the IgD resulted in significant binding to MBL, indicating that the oligomannose structures became exposed upon unfolding. MBL showed low levels of binding to IgE which increased after unfolding, again indicating exposure of oligomannose structures after denaturing.

View larger version (7K): [in this window] [in a new window]   FIGURE 7. MBL binding to native and unfolded Igs. Microtiter plate wells were coated with 1 µg/well of each Ig, and incubated with 50 ng/well MBL. Wells were then incubated with the anti-MBL antiserum and then the monoclonal anti-rabbit IgG (-chain specific) alkaline phosphatase conjugate, as described in Materials and Methods. The bars show the mean of the triplicate MBL binding assay ±1 SD unit, read at 492 nm. EDTA and BSA negative control readings were subtracted from the results. Results are shown for both native IgD and IgE and the denatured Abs. A positive control of native IgG (determined to contain 34% IgG-G0 glycoform) was used. The absorbances were read after 30 min.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IgD contained a variety of N-linked glycans terminating in galactose and sialic acid, as well as oligomannose structures (Table I). The oligomannose glycans comprised 37.1% of the N-linked glycan pool, consistent with occupation of one of the three N-linked glycosylation sites. Previous studies on a human myeloma IgD have shown that the Asn³⁵⁴ N-linkage site contains exclusively oligomannose glycans (Man₅-Man₉), including the glucosylated Man₉Glc₁, Man₈Glc₁, and Man₇Glc₁ (23). Serum IgD shows the same range of oligomannose glycans and glucosylated oligomannose glycans as the myeloma, with the exception of Man₇Glc₁. Man₉Glc₁ is an endoplasmic reticulum (ER) retention signal for the glycoprotein to which it is attached, to ensure proper quality control and correct folding (47). A Man₉Glc₃ structure becomes attached to the Asn of the N-link site cotranslationally in the ER. Man₉Glc₃ is digested to Man₉Glc₁ by ER glucosidase I and II. Monoglucosylated glycans on secreted proteins are discussed in more detail by Crispin et al. (48).

The complex glycans on myeloma IgD previously identified by Mellis and Baenziger (23) are very similar to those that have been identified in this study of normal serum IgD. The biggest difference was in the quantity of sialylated structures. In normal serum IgD, 45% of the total glycan pool was made up of sialylated structures, both mono- (24%) and disialylated (21%). This is a higher overall sialylation level than reported for the myeloma IgD where Mellis and Baenziger (23) reported that sialylated structures were present at Asn⁴⁴⁵, and at this site only 50% of the glycans were monosialylated (of the overall glycan pool 16% of the glycans were sialylated).

The O-linked glycans released from IgD are present in the hinge region (Fig. 1) (49). Neutral, mono-, and disialylated Core I structures were identified (Table II). This finding is consistent with those of Mellis and Baenziger (49) who report the same Core I glycans present on a myeloma IgD. Our study also identified the product of peeling which is a sialylated galactose from the breakdown of Core I structures from the release and extraction procedure (Fig. 5) that becomes 2AB labeled. The Core I structures attached to IgD all terminate in galactose or sialic acid and these sugars are not potential ligands for MBL.

Serum IgE glycosylation

Analysis of serum IgE N-linked glycans revealed the presence of oligomannose sugars Man₄-Man₈ that composed 14.2% of the glycan pool (Table III). Of the seven potential N-linked glycosylation sites per H chain of IgE, only Asn³⁹⁴ contained solely oligomannose glycans in a myeloma IgE (50, 31). We report here that oligomannose glycans account for one-seventh of the glycan pool. It has been previously found by Dorrington and Bennich (31) that Asn³⁸³ in the C_H3 domain is an unoccupied site in a myeloma IgE. Modeling to identify the positioning of this site showed that Asn³⁸³ is on the surface of the quaternary structure and therefore completely accessible for glycan processing. As the oligomannose structures identified accounted for one-seventh of the glycan pool and not one-sixth, these findings suggest that all seven N-linked sites are occupied in the normal serum IgE. Modeling of glycans onto this site also showed that glycosylation would not hinder or block the docking of the FcRI from the crystal structure of Garman et al. (51).

Both mono- and disialylated glycans have been shown to be present on a myeloma IgE (50), and were assigned to the N-linkage sites Asn²¹⁸ and Asn²⁶⁵ (31). Here, we report the presence of 39% of monosialylated and 36% disialylated glycans on serum IgE, indicating that other N-linked sites must also be occupied by sialylated glycans. No O-linked glycans have been described in the conserved regions on IgE.

The conserved Ig Fc glycosylation site

IgG has a single glycosylation site at Asn²⁹⁷ in the C_H2 region of the Fc domain. This glycosylation site is localized on a -turn at the top of the Fc domain, near the hinge and the glycans are situated in the space between the two C_H2 domains (42). Complete removal of this glycan alters both the stability of the Fc domain and its ability to bind FcRs (reviewed by Ref.52). The sequence of the glycan at Asn²⁹⁷ has also been shown to modulate receptor binding (53).

Sequence alignment between IgG, IgD, and IgE indicates that the IgG Asn²⁹⁷ site is completely conserved in all three, corresponding to site Asn³⁵⁴ in IgD and Asn³⁹⁴ in IgE. None of the other glycosylation sites are conserved between IgD and IgE suggesting that this Asn-common glycan may have a conserved role in the folding, assembly, or function of Ig Fc domains.

Glycan processing at the conserved Ig Fc glycosylation site

In IgG, the Asn-common glycans are complex and thus accessible to ER and golgi glycan-processing enzymes (54, 55). It has been shown that the glycans IgG-G1 and -G2 that collectively make up 80% of normal human serum IgG glycoforms have restricted motion because the terminal galactose residues interact with the peptide surface, holding them in place. The IgG-G0 glycans are free to move as their glycans are missing the terminal galactose residues. As a result the glycans are more accessible at this stage for processing (5), this explains why most glycans get terminal galactoses attached to the 6-arm GlcNAc. IgD has the same domain structure as IgG; however the glycans found at this site are virtually unprocessed oligomannose or glucosylated oligomannose glycans, indicating low accessibility to ER and golgi glycan processing enzymes. Modeling of IgD (Fig. 8) showed that access to glycans at Asn³⁵⁴ may be blocked by the glycans attached to Asn⁴⁴⁵. This steric hindrance to access by the ER and golgi glycosidases and by glycosyltransferases to the glycans of Asn³⁵⁴, may thus prevent further processing of the Asn³⁵⁴ glycans once the protein quaternary structure has formed. Precedents within the glycosylation of CD4 have also shown the environment around a glycosylation site and the attached glycan to be important to site-specific processing (56).

View larger version (42K): [in this window] [in a new window]   FIGURE 8. Molecular model of IgD Fc. A model of IgD Fc (CH2 and CH3 domains) based on the crystal structure of IgG Fc (see Materials and Methods for details) showing (a) front view and (b) side view (rotated by 90° clockwise about the y-axis relative to a). The peptide backbone and glycosylated Asn residues are shown in gray. An oligomannose glycan (Man9) is attached to Asn354 on both H chains (blue). Complex biantennary glycans, with core fucose, bisecting GlcNAc and sialic acid (FcA2BG2S2) are attached to Asn445 (red) and Asn496 (green) on both H chains. The Asn445 side chains on the two H chains have been modeled in the 180°/180° (left side of b) and the 300°/180° (right side of a) conformations. The Asn445 side chains are expected to be flexible so the glycans attached to these residues will move between the two positions.

View larger version (41K): [in this window] [in a new window]   FIGURE 9. Molecular model of IgE Fc and hinge domain. A model of IgE Fc (CH2, CH3, and CH4 domains) based on the crystal structure of IgE Fc (see Materials and Methods for details) showing (a) front view, with the CH2 domains behind the CH3 domains, and (b) side view (rotated by 90° clockwise about the y-axis relative to a), with the CH2 domains to the right. The peptide backbone and glycosylated Asn residues are shown in gray. An oligomannose glycan (Man9) is attached to Asn394 on both H chains (blue). Complex biantennary glycans, with core fucose, bisecting GlcNAc and sialic acid (FcA2BG2S2) are attached to Asn265 (light blue), Asn371 (red) and Asn383 (green) on both H chains. The Asn371 side chains on the two H chains have been modeled in the 300°/180° (left of a) and the 180°/180° (right side of a) conformations. The Asn265 and Asn371 side chains are expected to be flexible.

The pattern of MBL binding to folded proteins (Fig. 7) mirrors almost exactly the accessibility of the glycans to processing. Although IgD and IgE have glycans to which MBL binds (Table IV), native IgD shows no interaction with MBL and native IgE only weak binding, ruling out these as potential binding targets and inducers of the activation of the lectin pathway of complement. There are previous reports of glycoproteins that contain oligomannose glycans that are inaccessible for MBL binding. Solis et al. (57) reported that the oligomannose structures present on complement component C3 and RNase B were negative for MBL binding in the native proteins, although the isolated oligomannose structures converted to neoglycolipids (20) did bind MBL. It should be noted that both IgD and IgE both contain <1% of glycans that terminate in GlcNAc. These glycans are potential targets for MBL binding and must occupy a site other than the Asn-common. Although these glycans are likely to be accessible, at the quantities they appear (2% IgD and 1% IgE) they would have no physiological effect. Only one IgD and IgE of eight would carry such a glycan. At these concentrations the abundance would be too low throughout the Ag bound IgD and IgE for MBL to bind with high avidity.

These findings lead to a more general hypothesis that, for serum proteins, the presence of oligomannose glycans on the mature protein indicates a lack of access of the glycan-processing enzymes which will be accompanied by a lack of recognition by MBL. Glycans that are accessible to MBL will also be accessible to the glycan processing enzymes and so be converted to complex glycans in the Golgi. This ensures that self proteins are in general not recognized by lectins such as MBL. This hypothesis holds true for the MBL binding to IgG-G0, where the glycans attached at the Asn-common site are accessible to ER glycan-processing enzymes and as a result are complex glycans. It is only at the point when the terminal galactose is attached to the 6-arm that the glycans become inaccessible as the galactose interacts with the peptide surface (5). It is when these galactoses are missing in the IgG-G0 glycoform that the glycan-protein linkage becomes flexible, and the terminal GlcNAcs are targets for MBL binding.

	Acknowledgments

 
We thank Dr. Tony Merry for help with N-linked glycan analysis and Tony Willis for amino acid analysis of the MBL preparation. The MALDI mass spectrometer was purchased with a grant from the Biotechnology and Biological Sciences Research Council. The LC-ESI mass spectrometer was purchased with a grant from the Wellcome Trust.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This research was supported by the MRC (U.K.).

² Address correspondence and reprint requests to Dr. James N. Arnold, Department of Biochemistry, MRC Immunochemistry Unit, University of Oxford, Oxford OX1 3QU, U.K. E-mail address: james.arnold{at}bioch.ox.ac.uk

³ Abbreviations used in this paper: MBL, mannan-binding lectin; mMR, macrophage mannose receptor; MASP, MBL-associated serine protease; CRD, carbohydrate recognition domain; PEG, polyethylene glycol; RT, room temperature; 2AB, 2-aminobenzamide; NP-HPLC, normal phase HPLC; GU, glucose unit; ABS, Arthobacter ureafaciens sialidase; BTG, bovine testis -galactosidase; GuH, -N-acetyl glucosaminidase; Glc, glucose; GlcII, glucosidase II; ER, endoplasmic reticulum; GlcNAc, N-acetyl glucosamine; BKF, bovine kidney fucosidase; LC-ESI-MS, liquid chromatography/electrospray ionization mass spectrometry; NAN1, Streptococcus pneumonia sialidase; SP, surfactant protein.

Received for publication June 9, 2004. Accepted for publication August 27, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Butler, M., D. Quelhas, A. J. Critchley, H. Carchon, H. F. Hebestreit, R. G. Hibbert, L. Vilarinho, E. Teles, G. Matthijs, E. Schollen, et al 2003. Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis. Glycobiology 13:601.[Abstract/Free Full Text]
2. Royle, L., A. Roos, D. J. Harvey, M. R. Wormald, D. van Gijlswijk-Janssen, R. M. Redwan, I. A. Wilson, M. R. Daha, R. A. Dwek, P. M. Rudd. 2003. Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems. J. Biol. Chem. 278:20140.[Abstract/Free Full Text]
3. Mattu, T. S., R. J. Pleass, A. C. Willis, M. Kilian, M. R. Wormald, A. C. Lellouch, P. M. Rudd, J. M. Woof, R. A. Dwek. 1998. The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fc receptor interactions. J. Biol. Chem. 273:2260.[Abstract/Free Full Text]
4. Parekh, R. B., R. A. Dwek, B. J. Sutton, D. L. Fernandes, A. Leung, D. Stanworth, T. W. Rademacher, T. Mizuochi, T. Taniguchi, K. Matsuta, et al 1985. Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature 316:452.[Medline]
5. Malhotra, R., M. R. Wormald, P. M. Rudd, P. B. Fischer, R. A. Dwek, R. B. Sim. 1995. Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nat. Med. 1:237.[Medline]
6. Dong, X., W. J. Storkus, R. D. Salter. 1999. Binding and uptake of agalactosyl IgG by mannose receptor on macrophages and dendritic cells. J. Immunol. 163:5427.[Abstract/Free Full Text]
7. Roos, A., L. H. Bouwman, D. J. van Gijlswijk-Janssen, M. C. Faber-Krol, G. L. Stahl, M. R. Daha. 2001. Human IgA activates the complement system via the mannan-binding lectin pathway. J. Immunol. 167:2861.[Abstract/Free Full Text]
8. Nevens, J. R., A. K. Mallia, M. W. Wendt, P. K. Smith. 1992. Affinity chromatographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein. J. Chromatogr. 597:247.[Medline]
9. Roos, A., L. H. Bouwman, J. Munoz, T. Zuiverloon, M. C. Faber-Krol, F. C. Fallaux-van den Houten, N. Klar-Mohamad, C. E. Hack, M. G. Tilanus, M. R. Daha. 2003. Functional characterization of the lectin pathway of complement in human serum. Mol. Immunol. 39:655.[Medline]
10. Malhotra, R., J. Lu, U. Holmskov, R. B. Sim. 1994. Collectins, collectin receptors and the lectin pathway of complement activation. Clin. Exp. Immunol. 97:(Suppl. 2):4.
11. Holmskov, U., R. Malhotra, R. B. Sim, J. C. Jensenius. 1994. Collectins: collagenous C-type lectins of the innate immune defense system. Immunol. Today 15:67.[Medline]
12. Valdimarsson, H., M. Stefansson, T. Vikingsdottir, G. J. Arason, C. Koch, S. Thiel, J. C. Jensenius. 1998. Reconstitution of opsonizing activity by infusion of mannan-binding lectin (MBL) to MBL-deficient humans. Scand. J. Immunol. 48:116.[Medline]
13. Petersen, S. V., S. Thiel, J. C. Jensenius. 2001. The mannan-binding lectin pathway of complement activation: biology and disease association. Mol. Immunol. 38:133.[Medline]
14. Hajela, K., M. Kojima, G. Ambrus, K. H. Wong, B. E. Moffatt, J. Ferluga, S. Hajela, P. Gal, R. B. Sim. 2002. The biological functions of MBL-associated serine proteases (MASPs). Immunobiology 205:467.[Medline]
15. Matsushita, M., T. Fujita. 1992. Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease. J. Exp. Med. 176:1497.[Abstract/Free Full Text]
16. Thiel, S., T. Vorup-Jensen, C. M. Stover, W. Schwaeble, S. B. Laursen, K. Poulsen, A. C. Willis, P. Eggleton, S. Hansen, U. Holmskov, et al 1997. A second serine protease associated with mannan-binding lectin that activates complement. Nature 386:506.[Medline]
17. Dahl, M. R., S. Thiel, M. Matsushita, T. Fujita, A. C. Willis, T. Christensen, T. Vorup-Jensen, J. C. Jensenius. 2001. MASP-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway. Immunity 15:127.[Medline]
18. Turner, M. W.. 1996. Mannose-binding lectin: the pluripotent molecule of the innate immune system. Immunol. Today 17:532.[Medline]
19. Weis, W. I., K. Drickamer, W. A. Hendrickson. 1992. Structure of a C-type mannose-binding protein complexed with an oligosaccharide. Nature 360:127.[Medline]
20. Childs, R. A., K. Drickamer, T. Kawasaki, S. Thiel, T. Mizuochi, T. Feizi. 1989. Neoglycolipids as probes of oligosaccharide recognition by recombinant and natural mannose-binding proteins of the rat and man. Biochem. J. 262:131.[Medline]
21. Mizuochi, T., R. W. Loveless, A. M. Lawson, W. Chai, P. J. Lachmann, R. A. Childs, S. Thiel, T. Feizi. 1989. A library of oligosaccharide probes (neoglycolipids) from N-glycosylated proteins reveals that conglutinin binds to certain complex-type as well as high mannose-type oligosaccharide chains. J. Biol. Chem. 264:13834.[Abstract/Free Full Text]
22. Iobst, S. T., M. R. Wormald, W. I. Weis, R. A. Dwek, K. Drickamer. 1994. Binding of sugar ligands to Ca²⁺-dependent animal lectins. I. Analysis of mannose binding by site-directed mutagenesis and NMR. J. Biol. Chem. 269:15505.[Abstract/Free Full Text]
23. Mellis, S. J., J. U. Baenziger. 1983. Structures of the oligosaccharides present at the three asparagine-linked glycosylation sites of human IgD. J. Biol. Chem. 258:11546.[Abstract/Free Full Text]
24. Van Boxel, J. A., W. E. Paul, W. D. Terry, I. Green. 1972. Communications: IgD-bearing human lymphocytes. J. Immunol. 109:648.[Abstract/Free Full Text]
25. Rogentine, G. N., Jr, D. S. Rowe, J. Bradley, T. A. Waldmann, J. L. Fahey. 1966. Metabolism of human immunoglobulin D (IgD). J. Clin. Invest. 45:1467.
26. Griffiths, R. W., G. J. Gleich. 1972. Proteolytic degradation of IgD and its relation to molecular conformation. J. Biol. Chem. 247:4543.[Abstract/Free Full Text]
27. Takahashi, N., D. Tetaert, B. Debuire, L. C. Lin, F. W. Putnam. 1982. Complete amino acid sequence of the heavy chain of human immunoglobulin D. Proc. Natl. Acad. Sci. USA 79:2850.[Abstract/Free Full Text]
28. Gala, F. A., S. L. Morrison. 2002. The role of constant region carbohydrate in the assembly and secretion of human IgD and IgA1. J. Biol. Chem. 277:29005.[Abstract/Free Full Text]
29. Sutton, B. J., H. J. Gould. 1993. The human IgE network. Nature 366:421.[Medline]
30. Gould, H. J., B. J. Sutton, A. J. Beavil, R. L. Beavil, N. McCloskey, H. A. Coker, D. Fear, L. Smurthwaite. 2003. The biology of IgE and the basis of allergic disease. Annu. Rev. Immunol. 21:579.[Medline]
31. Dorrington, K. J., H. H. Bennich. 1978. Structure-function relationships in human immunoglobulin E. Immunol. Rev. 41:3.[Medline]
32. Tan, S. M., M. C. Chung, O. L. Kon, S. Thiel, S. H. Lee, J. Lu. 1996. Improvements on the purification of mannan-binding lectin and demonstration of its Ca²⁺-independent association with a C1s-like serine protease. Biochem. J. 319:(Pt. 2):329.
33. Küster, B., S. F. Wheeler, A. P. Hunter, R. A. Dwek, D. J. Harvey. 1997. Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography. Anal. Biochem. 250:82.[Medline]
34. Radcliffe, C. M., G. Diedrich, D. J. Harvey, R. A. Dwek, P. Cresswell, P. M. Rudd. 2002. Identification of specific glycoforms of major histocompatibility complex class I heavy chains suggests that class I peptide loading is an adaptation of the quality control pathway involving calreticulin and ERp57. J. Biol. Chem. 277:46415.[Abstract/Free Full Text]
35. Royle, L., T. S. Mattu, E. Hart, J. I. Langridge, A. H. Merry, N. Murphy, D. J. Harvey, R. A. Dwek, P. M. Rudd. 2002. An analytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins. Anal. Biochem. 304:70.[Medline]
36. Bigge, J. C., T. P. Patel, J. A. Bruce, P. N. Goulding, S. M. Charles, R. B. Parekh. 1995. Nonselective and efficient fluorescent labeling of glycans using 2-aminobenzamide and anthranilic acid. Anal. Biochem. 230:229.[Medline]
37. Guile, G. R., P. M. Rudd, D. R. Wing, S. B. Prime, R. A. Dwek. 1996. A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem. 240:210.[Medline]
38. Zamze, S., D. J. Harvey, Y. J. Chen, G. R. Guile, R. A. Dwek, D. R. Wing. 1998. Sialylated N-glycans in adult rat brain tissue- a widespread distribution of disialylated antennae in complex and hybrid structures. Eur. J. Biochem. 258:243.[Medline]
39. Pearson, W. R., T. Wood, Z. Zhang, W. Miller. 1997. Comparison of DNA sequences with protein sequences. Genomics 46:24.[Medline]
40. Kraulis, P. J.. 1991. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24:946.
41. Berman, H. M., J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, P. E. Bourne. 2000. The Protein Data Bank. Nucleic Acids Res. 28:235.[Abstract/Free Full Text]
42. Deisenhofer, J.. 1981. Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-Å resolution. Biochemistry 20:2361.[Medline]
43. Wan, T., R. L. Beavil, S. M. Fabiane, A. J. Beavil, M. K. Sohi, M. Keown, R. J. Young, A. J. Henry, R. J. Owens, H. J. Gould, B. J. Sutton. 2002. The crystal structure of IgE Fc reveals an asymmetrically bent conformation. Nat. Immunol. 3:681.[Medline]
44. Petrescu, A. J., S. M. Petrescu, R. A. Dwek, M. R. Wormald. 1999. A statistical analysis of N- and O-glycan linkage conformations from crystallographic data. Glycobiology 9:343.[Abstract/Free Full Text]
45. Wormald, M. R., A. J. Petrescu, Y. L. Pao, A. Glithero, T. Elliott, R. A. Dwek. 2002. Conformational studies of oligosaccharides and glycopeptides: complementarity of NMR, X-ray crystallography, and molecular modelling. Chem. Rev. 102:371.[Medline]
46. Petrescu, A. J., A. L. Milac, S. M. Petrescu, R. A. Dwek, M. R. Wormald. 2004. Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding. Glycobiology 14:103.[Abstract/Free Full Text]
47. Ellgaard, L., M. Molinari, A. Helenius. 1999. Setting the standards: quality control in the secretory pathway. Science 286:1882.[Abstract/Free Full Text]
48. Crispin, M. D. M., G. E. Ritchie, A. J. Critchley, P. B. Morgan, I. A. Wilson, R. A. Dwek, R. B. Sim, P. M. Rudd. Monoglucosylated glycans in the secreted human complement component C3: implications for protein biosynthesis and structure. FEBS Lett. 566:270.
49. Mellis, S. J., J. U. Baenziger. 1983. Structures of the O-glycosidically linked oligosaccharides of human IgD. J. Biol. Chem. 258:11557.[Abstract/Free Full Text]
50. Baenziger, J., S. Kornfeld, S. Kochwa. 1974. Structure of the carbohydrate units of IgE immunoglobulin. I. Over-all composition, glycopeptide isolation, and structure of the high mannose oligosaccharide unit. J. Biol. Chem. 249:1889.[Abstract/Free Full Text]
51. Garman, S. C., B. A. Wurzburg, S. S. Tarchevskaya, J. P. Kinet, T. S. Jardetzky. 2000. Structure of the Fc fragment of human IgE bound to its high-affinity receptor FcRI . Nature 406:259.[Medline]
52. Jefferis, R., J. Lund, J. D. Pound. 1998. IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation. Immunol. Rev. 163:59.[Medline]
53. Mimura, Y., P. Sondermann, R. Ghirlando, J. Lund, S. P. Young, M. Goodall, R. Jefferis. 2001. Role of oligosaccharide residues of IgG1-Fc in FcRIIb binding. J. Biol. Chem. 276:45539.[Abstract/Free Full Text]
54. Wormald, M. R., P. M. Rudd, D. J. Harvey, S. C. Chang, I. G. Scragg, R. A. Dwek. 1997. Variations in oligosaccharide-protein interactions in immunoglobulin G determine the site-specific glycosylation profiles and modulate the dynamic motion of the Fc oligosaccharides. Biochemistry 36:1370.[Medline]
55. Rudd, P. M., R. J. Leatherbarrow, T. W. Rademacher, R. A. Dwek. 1991. Diversification of the IgG molecule by oligosaccharides. Mol. Immunol. 28:1369.[Medline]
56. Ashford, D. A., C. D. Alafi, V. M. Gamble, D. J. Mackay, T. W. Rademacher, P. J. Williams, R. A. Dwek, A. N. Barclay, S. J. Davis, C. Somoza, et al 1993. Site-specific glycosylation of recombinant rat and human soluble CD4 variants expressed in Chinese hamster ovary cells. J. Biol. Chem. 268:3260.[Abstract/Free Full Text]
57. Solis, D., T. Feizi, C. T. Yuen, A. M. Lawson, R. A. Harrison, R. W. Loveless. 1994. Differential recognition by conglutinin and mannan-binding protein of N-glycans presented on neoglycolipids and glycoproteins with special reference to complement glycoprotein C3 and ribonuclease B. J. Biol. Chem. 269:11555.[Abstract/Free Full Text]

This article has been cited by other articles:

				T. A. Bowden, M. Crispin, D. J. Harvey, A. R. Aricescu, J. M. Grimes, E. Y. Jones, and D. I. Stuart Crystal Structure and Carbohydrate Analysis of Nipah Virus Attachment Glycoprotein: a Template for Antiviral and Vaccine Design J. Virol., December 1, 2008; 82(23): 11628 - 11636. [Abstract] [Full Text] [PDF]

				R. M. Anthony, F. Nimmerjahn, D. J. Ashline, V. N. Reinhold, J. C. Paulson, and J. V. Ravetch Recapitulation of IVIG Anti-Inflammatory Activity with a Recombinant IgG Fc Science, April 18, 2008; 320(5874): 373 - 376. [Abstract] [Full Text] [PDF]

				C. M. Radcliffe, J. N. Arnold, D. M. Suter, M. R. Wormald, D. J. Harvey, L. Royle, Y. Mimura, Y. Kimura, R. B. Sim, S. Inoges, et al. Human Follicular Lymphoma Cells Contain Oligomannose Glycans in the Antigen-binding Site of the B-cell Receptor J. Biol. Chem., March 9, 2007; 282(10): 7405 - 7415. [Abstract] [Full Text] [PDF]

				M. van Lith and A. M. Benham The DM{alpha} and DMbeta Chain Cooperate in the Oxidation and Folding of HLA-DM J. Immunol., October 15, 2006; 177(8): 5430 - 5439. [Abstract] [Full Text] [PDF]

				I. Terai, K. Kobayashi, J.-P. Vaerman, and N. Mafune Degalactosylated and/or Denatured IgA, but Not Native IgA in Any Form, Bind to Mannose-Binding Lectin J. Immunol., August 1, 2006; 177(3): 1737 - 1745. [Abstract] [Full Text] [PDF]

				A. C. Smith, J. F. de Wolff, K. Molyneux, J. Feehally, and J. Barratt O-Glycosylation of Serum IgD in IgA Nephropathy J. Am. Soc. Nephrol., April 1, 2006; 17(4): 1192 - 1199. [Abstract] [Full Text] [PDF]

				J. N. Arnold, R. Wallis, A. C. Willis, D. J. Harvey, L. Royle, R. A. Dwek, P. M. Rudd, and R. B. Sim Interaction of Mannan Binding Lectin with {alpha}2 Macroglobulin via Exposed Oligomannose Glycans: A CONSERVED FEATURE OF THE THIOL ESTER PROTEIN FAMILY? J. Biol. Chem., March 17, 2006; 281(11): 6955 - 6963. [Abstract] [Full Text] [PDF]

				J. N. Arnold, M. R. Wormald, D. M. Suter, C. M. Radcliffe, D. J. Harvey, R. A. Dwek, P. M. Rudd, and R. B. Sim Human Serum IgM Glycosylation: IDENTIFICATION OF GLYCOFORMS THAT CAN BIND TO MANNAN-BINDING LECTIN J. Biol. Chem., August 12, 2005; 280(32): 29080 - 29087. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Arnold, J. N. Articles by Rudd, P. M. Search for Related Content PubMed PubMed Citation Articles by Arnold, J. N. Articles by Rudd, P. M.