From the The Glycobiology Institute, Department of
Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU,
United Kingdom, the ¶ Department of Molecular and Cellular
Pathology, Ninewells Hospital & Medical School, University of Dundee,
Dundee DD1 9SY, United Kingdom, the
Department of Medical Microbiology and
Immunology, University Aarhus, Bartholin Building, DK-8000 Aarhus C,
Denmark, §§ CNRS-CERMAV, Domaine Universitaire
601, rue de la Chimie, BP 53-38041 Grenoble Cedex 9, France, and the
** Medical Research Council Immunochemistry Unit, Department of
Biochemistry, Oxford University, South Parks Road,
Oxford OX1 3QU, United Kingdom
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ABSTRACT |
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The human serum immunoglobulins IgG
and IgA1 are produced in bone marrow and both interact with specific
cellular receptors that mediate biological events. In contrast to IgA1,
the glycosylation of IgG has been well characterized, and its
interaction with various Fc receptors (FcRs) has been well studied.
In this paper, we have analyzed the glycosylation of IgA1 and IgA1 Fab
and Fc as well as three recombinant IgA1 molecules, including two
N-glycosylation mutants. Amino acid sequencing data of the
IgA1 Fc O-glycosylated hinge region indicated that
O-glycans are located at Thr228,
Ser230, and Ser232, while O-glycan
sites at Thr225 and Thr236 are partially
occupied. Over 90% of the N-glycans in IgA1 were sialylated, in contrast to IgG, where <10% contain sialic acid. This
paper contains the first report of Fab glycosylation in IgA1, and (in
contrast to IgG Fab, which contains only N-linked glycans) both N- and O-linked oligosaccharides were
identified. Analysis of the N-glycans attached to
recombinant IgA1 indicated that the C
2 N-glycosylation
site contained mostly biantennary glycans, while the tailpiece site,
absent in IgG, contained mostly triantennary structures. Further
analysis of these data suggested that processing at one Fc
N-glycosylation site affects the other. Neutrophil Fc
R binding studies, using recombinant IgA1, indicated that neither the
tailpiece region nor the N-glycans in the C
2 domain
contribute to IgA1-neutrophil Fc
R binding. This contrasts with IgG,
where removal of the Fc N-glycans reduces binding to the
Fc
R. The primary sequence and disulfide bond pattern of IgA1,
together with the crystal structures of IgG1 Fc and mouse IgA Fab and
the glycan sequencing data, were used to generate a molecular model of
IgA1. As a consequence of both the primary sequence and S-S bond
pattern, the N-glycans in IgA1 Fc are not confined
within the inter-
-chain space. The accessibility of the C
2
N-glycans provides an explanation for the increased
sialylation and galactosylation of IgA1 Fc over that of IgG Fc
N-glycans, which are confined in the space between the two
C
2 domains. This also suggests why in contrast to IgG Fc, the IgA1
N-glycans are not undergalactosylated in rheumatoid arthritis.
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INTRODUCTION |
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Serum IgA, produced in bone marrow, consists predominantly of
monomeric IgA1 (1). IgA interacts with specific Fc receptors (Fc
R)1 present on
granulocytes, monocytes, and macrophages, which can mediate
phagocytosis, superoxide generation, enzyme release, and the clearance
of immune complexes (2). IgA antibody molecules lack the C1q binding
site present on IgG (3) and do not activate the classical complement
pathway (4, 5). The activation of the alternative complement pathway by
IgA remains controversial. A study avoiding artificial aggregation and
chemical modification has shown that chimeric IgA1 bound to C3, but no
C3b was generated (6). In contrast, using chimeric IgA2, binding to C3
leads to C3b formation and the terminal complement complex (7). This is
consistent with the reported anti-inflammatory activity of IgA1
antibodies (8-10).
Human IgA1 contains two conserved N-glycosylation sites in
each -chain (Asn263 and Asn459), while the
IgA2 subclass contains an additional two (IgA2m(1)) or three (IgA2m(2))
conserved N-glycans. In addition, Putnam and colleagues
identified an N-glycosylation acceptor sequon in the variable region of the Bur myeloma
-chain of IgA1 (11), which was
not present in either the IgA2 subclass (12) or IgA1 isolated from
other myeloma patients (13). The N-glycans of myeloma IgA1 have been reported as biantennary complex-type structures with sialic
acids attached exclusively in the
2-6 linkage (13). The first
detailed structural analysis of the glycans of normal serum IgA1 was
published by Field et al. (14). However, as with other
studies (15), the analysis was carried out on the desialylated glycan
pool from intact IgA1 and sialylated glycans were not directly analyzed. The N-glycans identified by Field et
al. (14) were predominantly of the biantennary complex type
with a core Fuc, bisecting GlcNAc, or both, and were generally similar
to those reported for myeloma IgA1. In addition, approximately
13.6% of tri- and tetraantennary structures were reported.
IgA1 contains a proline-rich hinge sequence between the Fab and Fc
regions of the glycoprotein (Fig. 1). Within this sequence, there are
nine potential O-glycosylation sites/-chain (18 sites/molecule). The occupancy of these sites in normal serum IgA1 is
not known. In myeloma serum IgA1, the sites of
O-glycosylation were located at the five serine residues
(16). In contrast, both serine and threonine residues are
O-glycosylated in the hinge region of serum IgD (17) and
secretory IgA1 (18), although in the latter case no experimental data
was presented. These differences in O-glycan site occupancy
as well as the finding that the O-glycans of secretory IgA1
are larger and more heterogeneous than in myeloma IgA1 (18, 19) may
reflect tissue-specific glycosylation.
IgA N-glycosylation is required for the intracellular stability and secretion of mouse IgA (20, 21). However, recent studies have shown that this is not the case for either human IgA1 (6) or human IgA2 (7). The IgA1 study also indicated a role for N-glycosylation in antigen binding, suggesting that the secretion of glycosidases by bacteria may aid their survival in the host. In addition, certain species of bacteria may avoid an immune response by secreting IgA1-specific proteases. These proteases cleave the highly stable O-glycosylated hinge region of IgA1, thereby separating the two monomeric antigen-binding regions from the effector region of the antibody. These IgA1 protease-producing bacteria include the three leading agents of bacterial meningitis as well as other clinically important bacterial infections of the genitourinary and respiratory tracts (reviewed in Ref. 22). Interestingly, the O-glycosylation status of the immunoglobulin is known to affect the activity of some of the proteases (23). IgA1 glycans have been implicated in the pathology of a number of diseases including IgA nephropathy (24-27).
In this study of pooled serum IgA1, we have (a) determined
the sialylation of the N- and O-linked
oligosaccharides; (b) defined the sites of
O-glycosylation in the proline-rich hinge region of IgA1;
(c) provided the first report that the Fab region of IgA1 is
glycosylated, identifying both N- and O-glycans;
(d) compared the N-glycosylation of wild-type
recombinant IgA1 and N-glycan deletion mutants to determine
whether occupancy of one Fc N-glycosylation site affects
processing at the other; and (e) explored the location and
orientation of the IgA1 N-glycans in a molecular model of IgA1, constructed from the crystal structures of human IgG1 Fc fragment
(28), mouse Fab fragment (29), and the analytical data obtained in this
study. This model suggested some possible explanations for the
differences in the composition of the glycan populations in IgA1 and
IgG and in their structural and functional roles. In IgG, the conserved
Fc N-glycans are situated in the interstitial region between
the CH2 domains and, as a consequence, are incompletely galactosylated
and sialylated. Binding to C1q is reduced upon degalactosylation (30)
or deglycosylation (31), and, although the glycans are not required for
binding to protein A (32), they are necessary to maintain the
conformation of the Fc region to allow binding to FcR (30).
Therefore, we tested the possibility that IgA1 Fc N-glycans
may play a similar structural role to those in IgG Fc by comparing the
binding of carbohydrate-deficient IgA1 mutants to the neutrophil Fc
R
with that of wild-type recombinant IgA1.
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EXPERIMENTAL PROCEDURES |
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Materials--
Goat F(ab)2 anti-human IgA
fluorescein isothiocyanate conjugate was purchased from Caltag
(Bradsure Biologicals, UK). ProSorb cartridges were obtained from
Perkin-Elmer Ltd. (Applied Biosystems Division, Warrington, UK). Glycan
sequencing HPLC columns (GlycoSep-C and GlycoSep-N) were purchased from
Oxford GlycoSciences Ltd. Enzymes were obtained as follows: PNGase F
from New England Biolabs (Hitchin, UK); Arthrobacter
ureafaciens neuraminidase, almond meal
-fucosidase, Newcastle
disease virus Hitchner B1 strain neuraminidase, and bovine testes
-galactosidase from Oxford GlycoSciences Ltd.; jack bean
N-acetyl
-hexosaminidase, Charonia lampas
-fucosidase, and jack bean
-mannosidase from Oxford Glycobiology
Institute.
Purification of Monomeric Serum IgA1-- Pooled human serum IgA1 was prepared as previously reported (14).
Digestion of IgA1 with Clostridium ramosum IgA Protease-- Affinity-purified IgA1 (2 mg/ml in phosphate-buffered saline, pH 7.4) was incubated for 36 h at 37 °C with the extracellular bacterial protease secreted by C. ramosum (33). The Fc and Fab fragments (Fig. 1) were purified by gel filtration (GF)-HPLC (TSK SW3000GW; 21.5 × 600 mm; 0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2). Fractions containing Fc and Fab were identified by molecular weight (SDS-PAGE), pooled appropriately, and purified to homogeneity by repetitive GF-HPLC. The purity of the isolated fragments was confirmed by reducing SDS-PAGE (12.5%), GF-HPLC, and N-terminal sequencing.
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Peptide:N-Glycosidase F Digestion of IgA1 Fc--
The purified
Fc fragment (15 µg) was denatured by boiling for 10 min in 4 µl of
50 mM sodium phosphate (pH 7.5) containing 0.5% SDS and
1% -mercaptoethanol. After cooling, 2 µl of 5% Nonidet P-40 was
added followed by the 6 µl of PNGase F (3000 units), and the mixture
was incubated overnight at 37 °C. Samples were analyzed by
SDS-PAGE.
N-terminal Amino Acid Sequencing-- All samples were analyzed on an Applied Biosystems (ABI) 470A protein sequencer with on-line phenylthiohydantoin analyzer (Perkin-Elmer). The Fc fragment was analyzed using an elevated temperature (48 °C) to facilitate quantitative cleavage of the prolyl residues from the peptide chain during Edman reaction. Samples were transferred to polyvinylidene difluoride membrane prior to sequencing using ProSorb cartridges (Perkin-Elmer).
Expression and Purification of Recombinant Human IgA1
Molecules--
A wild-type plasmid vector based on pEE6.hCMV (34) and
containing the human 1 gene downstream of a mouse Vnp domain has been described previously (4). Construction of an expression vector for
the IgA1 mutant Pterm455, in which the entire tailpiece was deleted,
involved replacing the first tailpiece residue (proline) with a stop
codon (35). To generate the Asn263 to Ala263
mutant (N263A, lacking the Asn263 N-glycan
acceptor site) the wild-type IgA1 expression vector was used as a
template for polymerase chain reaction-based mutagenesis. Mutagenesis
was performed using a 3
-primer annealing to a unique restriction site
(SalI; as described by Atkin et al. (35)) and a
5
-mismatch primer
(5
-CTGCACCGACCGGCCCTCGAGACCTGCTCTTAGGTTCAGAAGCGGCCCTCACGTGCACACTGACC-3
) annealed across a unique restriction site (XhoI) and in
which the codon AAC (coding for Asn263) was replaced with
GCC (coding for Ala). Following digestion with XhoI and
SalI, polymerase chain reaction products were ligated into
the XhoI/SalI-digested
1 expression vector,
replacing the wild-type sequence in this region. Restriction analysis
confirmed that the insert was correctly oriented. DNA sequencing (36) confirmed that the predicted mutation had been incorporated and that no
misincorporations had occurred during polymerase chain reaction
amplification.
Oligosaccharide Release and Labeling-- N-Glycans were released from the purified glycoproteins (IgA1, 1065 µg; Fab fragment, 610 µg; Fc fragment, 280 µg; recombinant IgA1 wild type, 120 µg; N263A, 84 µg; and Pterm455, 200 µg) by hydrazinolysis at 95 °C using a GlycoPrep1000 (Oxford GlycoSciences) optimized for maximum recovery (~85%) of glycans (37).
IgA1, Fab, and Fc were cryogenically dried over activated charcoal atAnalysis of Oligosaccharides by Weak Anion Exchange (WAX) Chromatography-- High performance anion exchange chromatography was carried out using a GlycoSep-C column (Oxford GlycoSciences Ltd.) according to the conditions described (39). The column was calibrated by the analysis of 2-AB-labeled sialylated glycans from bovine fetuin.
Analysis of Neutral and Acidic Oligosaccharides by Normal Phase Chromatography (NP-HPLC)-- Normal phase HPLC was carried out using the 50 mM ammonium formate, pH 4.4, buffer system on a GlycoSep-N column over 180 min, as described previously (40). The column was calibrated using 2-AB-labeled dextran hydrolysate, the elution positions of which were used to obtain the NP-HPLC glucose unit (gu) values for each glycan.
Exoglycosidase Digestion Conditions-- Enzyme digests were performed at 37 °C for 16-24 h in 100 mM citrate/phosphate buffer, pH 4.5, 0.2 mM zinc acetate, 0.15 M sodium chloride in 10-µl volumes. The enzymes were used either together in various combinations (termed an enzyme array) or alone, as specified in the appropriate figure legend. After incubation, proteins were removed by filtration through nitrocellulose (Pro-Spin, Radleys, UK) as described (41). An aliquot of the aqueous sample (~25 pmol) was mixed with acetonitrile in the ratio 30:70 and applied to the NP-HPLC column. Conditions for the digestions are as described previously (42). The cleavage positions for the exoglycosidases are shown in Fig. 2b.
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Interaction of Recombinant IgA with Neutrophil Fc
Receptors--
Rosette assays were performed (43) using
NIP-derivatized RBC sensitized with wild-type or mutant IgA1. Coating
levels for each antibody were found to be equivalent by reactivity with
goat F(ab
)2 anti-human IgA fluorescein isothiocyanate
conjugate as assessed by flow cytometry. Rosetting of sensitized RBC to
neutrophils was performed in V-bottomed microtiter plates. An effector
cell with three or more RBC attached was scored as a rosette.
Molecular Modeling-- Molecular modeling was carried out on a Silicon Graphics Indigo II computer using the Insight 95 and Discover software package (Biosys Tech, Inc., San Diego, CA). Initially, the Fab and Fc structures were modeled separately, linked to give a model of the entire antibody, and then minimized. The model of the human IgA1 Fab was based on the mouse IgA Fab crystal structure (29), while the human IgA1 Fc model was based on the human IgG1 Fc (28). Protein crystal coordinates were obtained from the Brookhaven data base (44). Both the sequence alignment (45) and inspection of the protein tertiary structure determined the location of sequence insertions and deletions. In all cases, insertions and deletions could be made in loops or linker regions between the domains; these regions were reconstructed using structural homology searches. The structure of the proline-rich hinge region (Val222 to Pro240) was modeled as an extended peptide sequence with all proline residues in the trans conformation (46). The tailpiece (residues 454-472) was also modeled as an extended peptide.
The modeling procedure required (a) construction of the alignment data sets, including positions of disulfide bonds (47-49), amino acid changes, and insertions/deletions (45); (b) fitting the amino acid changes to the IgG1 Fc structure and the mouse IgA Fab structure; (c) reconstruction of loop regions where insertions or deletions were required using structural homology searches; (d) altering the quaternary structure of the CH2 domain to conform to the disulfide pattern reported; and (e) introduction of the hinge and tailpiece polypeptides. After each step, the resulting model was optimized by energy minimization. Initial unfavorable nonbonded contacts that resulted in energy conflicts were adjusted by altering the side chain torsion angles. Subsequent optimization of nonbonded and bond geometries were carried out using the steepest descent energy minimization (minimum of 500 steps/cycle) with the AMBER force field parameters (50). All minimizations were carried out in vacuo, using a dielectric constant of 80. The final model of the IgA1 Fc fragment was obtained by firstly attaching the N- and O-linked oligosaccharides as preminimized intact units to the IgA1 Fc structure and optimizing the model by energy minimization. This was followed by attaching the Fab fragment to each heavy chain after which the model was again optimized by energy minimization. The structures for the individual domains resulting from the insertions, deletions, and point replacements were verified by examination of the amino acid distribution within the individual domains. In all cases, hydrophobic residues were found in the core of the domains or in the domain interfaces (VH:VL, C ![]() |
RESULTS |
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Purification of Monomeric IgA1, Fab, and Fc
Affinity-purified IgA1 was fractionated by GF-HPLC to remove polymeric immunoglobulins and contaminating C1 esterase. The final yield was 0.6 mg/ml serum. The IgA1 was >98% pure as determined by SDS-PAGE, immunoprecipitation, and N-terminal sequencing. The preparation gave two bands on reducing SDS-PAGE at 58 kDa (heavy chain) and 25 kDa (light chain; Fig. 3a, lane 1). Immunoprecipitation confirmed the presence of IgA1, while other immunoglobulins (IgG, IgM, and IgA2) were not detected. No N-terminal sequence was detected apart from the sequence for IgA.
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Exhaustive proteolysis of affinity-purified IgA1 by C. ramosum protease released the expected Fc- and Fd-light chain heterodimer (Fab) fragments. The digestion products were purified to homogeneity and shown to be >98% pure by N-terminal sequencing and reducing SDS-PAGE (Fig. 3a, lanes 2 and 3, respectively). On SDS-PAGE, the Fab migrated as a broad band at 28 kDa, while the Fc separated into two discrete bands migrating at 35 and 38 kDa (Fig. 3a, lane 5), and unglycosylated IgA1 Fc was not detected. PNGase F digestion of the IgA1 Fc yielded a single band on SDS-PAGE. This band migrated at a molecular mass (33 kDa) equivalent to the nonglycosylated IgA1 Fc, indicating that the Fc doublet was due to variable N-glycan site occupancy (Fig. 3a, lane 4).
Generation of Recombinant IgA1 Molecules
Following transfection of mutant heavy chain expression vectors
into a light chain-producing CHO cell line, stable cell lines were
established, and the secreted antibodies were purified by hapten
affinity purification. Fig. 3b shows an SDS-PAGE gel of the
Pterm455 mutant, in which the tailpiece has been deleted. Other
antibodies displayed similar levels of purity. Under nonreducing conditions (lane 2), two major bands of 170 and 80-90 kDa
were obtained for the Pterm455 mutant. These two bands have been shown previously to represent monomers (H2L2) and half-molecules (HL), respectively (35), suggesting that the tailpiece may play some role in
bringing together the two -chains in IgA assembly. There are minor
bands above 250 kDa and at 60 and 25 kDa, which may result from
aggregated IgA, heavy chains, and light chains, respectively, since
under reducing conditions (lane 1), only heavy and light chains were detected.
Identification of IgA1 Hinge O-Glycosylation Sites
The Fc fragment from IgA1 was analyzed by N-terminal amino acid sequencing using an increased temperature sequencer cycle to maximize the release of proline residues. This enabled the identification of sites of O-glycosylation by comparison of the yield of hydroxyamino acids relative to expected yields of nonglycosylated hydroxyamino acid residues. Under the sequencing conditions used, the expected yields of unmodified serine and threonine, relative to other amino acids, were 30-40% and 70-80%, respectively. During sequencing cycles for Thr228, Ser230, and Ser232, no hydroxyamino acids were identified. Thr225 and Thr236 produced depressed signals (30 and 60% relative to the expected signal for threonine), while hydroxyamino acids at other positions gave the expected yield of an unmodified residue. Depressed yields of hydroxyamino acids are consistent with O-glycosylation, since glycosylated anilinothiozoline-amino acids are not extracted from the sequencer reaction chamber under the conditions used (51-53).
Strategy for Glycan Analysis
The overall scheme of intact IgA, Fab, and Fc glycan analysis is shown in Fig. 2a and described below.
A. Analysis of Pooled Human Serum IgA1, Fab, and Fc N-Linked Oligosaccharides by WAX-- The 2-AB-labeled glycan pool from serum IgA1 was separated according to charge by WAX chromatography (Fig. 4a). Five populations were assigned by comparison with an analysis of standard sugars from fetuin resolved on the same HPLC system. Fractions were pooled according to charge and analyzed by NP-HPLC (see step B).
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B. Analysis of Neutral, Mono-, Di-, and Trisialylated Glycans from
Human Serum IgA1, Fab, and Fc by NP-HPLC--
NP-HPLC analysis of the
neutral WAX fraction (Fig. 4b) identified an oligomannose
series representing less than 0.1% of the total N-glycan
pool, and other neutral structures accounted for 1.8% of the total
N-glycan population. All sialylated structures eluted as
neutral glycans after digestion with A. ureafaciens neuraminidase, indicating that the charge was entirely due to sialic
acid. Newcastle disease virus neuraminidase was used to investigate the
linkage position of sialic acid to the glycan structures, since this
exoglycosidase removes 2-3- but not
2-6-linked sialic acid
residues. NP-HPLC analysis of each sialylated WAX fraction from IgA1
(Fig. 4, c-e), Fab (Fig. 4, f-h), and Fc (Fig. 4, i-k) indicated that the mono- and disialylated
N-glycans contained only
2-6-linked sialic acids, while
trisialylated oligosaccharides contained both
2-6- and
2-3-linked sialic acids.
C. Analysis of the Total N-Glycan Pools from Human Serum IgA1, Fab, and Fc by NP-HPLC-- The heterogeneous mixtures of neutral and sialylated N-linked oligosaccharides from IgA1, Fc, and Fab were analyzed by NP-HPLC (Fig. 5, a-c) and resolved into at least 20 peaks. Each peak was assigned a NP-HPLC gu value by comparison with the elution position of a standard 2-AB-labeled dextran hydrolysate mixture (shown at the top of each NP-HPLC panel). Individual peaks were then assigned preliminary structures from their gu values using the positions of standard glycans and predetermined incremental values for monosaccharide residues (Ref. 40; Table I). These predicted structures were confirmed as follows: (a) sialylation status was confirmed by WAX chromatography followed by NP-HPLC (see step B), and (b) analysis of digestions with exoglycosidase enzymes confirmed the bi- and triantennary complex glycans and the oligosaccharides containing core and/or outer arm fucose (see step D).
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D. Simultaneous Sequencing of the Total Glycan Pool Using Exoglycosidase Arrays-- The preliminary assignment of structures to the N-glycans from human serum IgA1 was made using the HPLC gu values as described (40-42). The digestion of aliquots of both individual WAX fractions (data not shown) or the total glycan pool of each sample confirmed these preliminary assignments. Fig. 6 shows the digestion of the total glycan pool from IgA1 with different exoglycosidase arrays monitored by NP-HPLC. The assignment of each of the main peaks in the IgA1 glycan pool was confirmed by following its predicted elution position through each of the enzyme digestions (Table II) as described (42). Predicted elution positions were based upon predetermined incremental values for the monosaccharide additions to standard glycan cores (40) and the known specificity of the enzymes (Fig. 2b).
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The Analysis of the O-Linked Glycans from Human Serum IgA1, Fab, and Fc by NP-HPLC
The O-glycans from intact serum IgA1, Fab, and Fc were
released by hydrazinolysis, under conditions that optimized the release and recovery of intact O-glycans, and analyzed by NP-HPLC
(Fig. 7, a-c). The data were
generally consistent with previous reports (14, 26, 54, 55). Table
III shows the relative proportions of the glycan species. The most abundant was the core 1 monosialylated T antigen (NeuNAc2-3Gal
1-3GalNAc) confirmed by
exoglycosidase digestion and co-elution with authentic
Gal
1-3GalNAc. Larger neutral and sialylated tetrasaccharide
structures (Gal
1-4GlcNAc
1-6Gal
1-3GalNAc) were also
identified. Degradation of O-glycans by
-elimination (peeling) during hydrazinolysis gives rise to
NeuNAc
2-3/6Gal and GalNAc. Quantitation of these structures
during NP-HPLC analysis indicated that peeled products represented less
than 2% of the total glycans in IgA and Fab and less than 10% in Fc.
The O-glycans released from the Fc (Fig. 7b,
Table III) were identical to those released from IgA1. Although the
GalNAc neoantigen was detected, the abundance of this monosaccharide
was comparable with that of the
-elimination degradation product,
NeuNAc
2-3/6Gal, suggesting that GalNAc was an artifact of
O-glycan release and not a naturally occurring
O-glycan. The same range of O-glycans was
identified on the Fab (Fig. 7c, Table III).
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The Analysis of the Glycans from Recombinant IgA1 and Two Carbohydrate-deficient Mutants
The glycosylation of the two conserved N-glycosylation
sites (Asn263 and Asn455) was probed by
comparing the glycosylation of carbohydrate-deficient mutant IgA1
molecules (N263A, Pterm455) with that of wild-type recombinant IgA1
(Fig. 8). Panel a shows the
NP-HPLC desialylated N-glycan profile for the wild-type
antibody. 37% of the glycans consist of the triantennary
oligosaccharide, A3G3 (Fig. 8a, peak 25; Table
IV). The deletion of the C2 domain
N-glycosylation site (N263A, Fig. 8b) gave rise
to an increase in A3G3 to 74% of the total N-glycan pool.
In contrast, the Pterm455 mutant (Fig. 8c), where the whole
tailpiece polypeptide was deleted including the N-glycan
site at Asn455, gave a glycosylation profile dominated by
the biantennary structure, A2G2 (82.9%, peak 22). All
glycan assignments were confirmed by the sequencing strategy described
above. The data suggest that the C
2 glycosylation site contains less
processed N-glycans than the tailpiece site.
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Recombinant IgA1 and the Carbohydrate-deficient Mutants Interact
with Neutrophil FcR
Wild-type recombinant IgA1 has been shown previously to bind to
the myeloid FcR with similar affinity to that of serum IgA1 (4). In
this study, the IgA1 mutant N263A, which lacks the C
2
N-glycan, showed a similar capacity to bind the Fc
R on
neutrophils as wild-type IgA1 (Fig. 9).
Both wild-type and mutant N263A antibodies have similar affinities for
the receptor, as determined by their comparable percentage rosette
formation at different concentrations of coating antibody. Rosetting
analysis suggests that the other IgA1 mutant, Pterm455, also interacts
with the receptor with an affinity similar to that of wild-type
IgA1.2
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DISCUSSION |
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Occupancy of IgA1 N-Glycosylation Sites-- Although IgA1 is more resistant than IgG or IgM to cleavage by common proteolytic enzymes, it is susceptible to specific proteases secreted by certain pathogenic bacteria (reviewed in Ref. 22). Here IgA1 was cleaved with the C. ramosum protease to generate two Fab fragments and the Fc (Fig. 1). The cleavage site, between Pro221 and Val222, allowed the entire O-glycosylated hinge region to be associated with the Fc. The Fc fragment was homogeneous by N-terminal sequencing, although it migrated as a doublet on reducing SDS-PAGE. PNGase F digestion of the Fc doublet yielded a single band with a lower molecular weight than either of the native bands. This suggested that the upper band contained a single Fc heavy chain in which both N-glycan sites are occupied, while the lower band contained Fc heavy chain with only one N-glycan site occupied. These data are consistent with preliminary NMR studies of pooled serum human IgA1 Fc.3 Total occupancy of the N-glycan sites is necessary for the intracellular stability and secretion of murine IgA (20, 21). However, in this study no significantly reduced expression levels were noted for either of the recombinant N-glycan mutants expressed in CHO-K1 cells compared with wild-type IgA (data not shown). These observations are consistent with the secretion of both IgA1 N-glycosylation mutants (6) and of IgA2 in the presence of tunicamycin (7).
Analysis of IgA1 N-Glycans--
Recently developed technology (40)
has enabled this first direct analysis of the sialylated glycans of
human serum IgA1, which make up over 98% of the total
N-glycan population. The mono- and disialylated
N-glycans contained only 2-6-linked sialic acids, while
trisialylated oligosaccharides contained one
2-6- and two
2-3-linked sialic acids (Table I). Interestingly, although A2G2BS2 represented less than 0.2% of the N-glycan pool, the
monosialylated structure A2G2BS(6) was relatively abundant (8.3%),
suggesting that the sialyltransferase preferentially sialylates one arm
of the biantennary glycan. In common with studies of monomeric serum IgA1 from myeloma (13, 15) and normal sera (14), small amounts (~0.1%) of oligomannose structures were detected in this study of
normal pooled human serum IgA1. Increased proportions of oligomannose structures have been noted in human myeloma serum from both polymeric (14%) and monomeric IgA1 (7%; Ref. 15) and have been proposed to act
as ligands for the mannose-specific lectin found on type I fimbriae of
Enterobacteriaceae (56). In pentameric IgM, the predominance
of oligomannose glycans attached to the tailpiece (57) is consistent
with steric hindrance of the region following polymerization of
monomeric IgM in the endoplasmic reticulum (58). Similarly, the
presence of J-chain in polymeric IgA, compared with monomeric IgA, may
be expected to result in increased proportions of oligomannose glycans
at Asn459 in the tailpiece. The proportion of bisected
structures in pooled serum IgA1 (24%) was higher than that in IgG
(14%; Ref. 59) although lower than that previously reported for IgA1
isolated from individuals (40%; Ref. 14). This suggests that the
proportion of bisected oligosaccharides may differ significantly
between individuals.
Occupancy of Potential O-Glycosylation Sites in the IgA1 Hinge Region-- At particular cycles in the amino acid sequencing, signal depression (Thr225 and Thr236) or absence (Thr228, Ser230, Ser232) was noted for some serine and threonine residues. Other than O-glycosylation, no other co- or post-translational modifications (phosphorylation or acetylation) have been reported on the IgA1 hinge. Therefore, these data indicate that the amino acids at these positions were glycosylated. These data are consistent with jacalin precipitation studies of IgA1 fragments (60), which suggested that jacalin interacts with the Ser230 and/or Ser232 and not Ser238 and Ser240. Variable site occupancy at Thr225 and Thr236, indicated by signal depression rather than absence, suggests that IgA1 exists as an array of glycoforms in which different potential O-glycan sites in the hinge region are occupied. In addition, each site may contain a range of oligosaccharides. This complex range of glycosylated variants may also exist in IgD, where variable site occupancy has been reported (61, 62). The finding that certain myeloma serum IgA1 proteins are glycosylated at all five serine residues (16) may be the result of an up-regulation of N-acetyl-galactosaminyltransferase or a modification in its specificity. Indeed, changes in O-glycosylation are associated with many carcinomas through either altered site occupancy (63, 64) or altered processing (65, 66). This may be significant in diseases such as IgA nephropathy, where altered O-glycosylation has been observed (24, 25, 27). In a preliminary study of serum IgA1 Fc isolated from IgA nephropathy patients, the recovery of O-glycans was significantly reduced, suggesting underoccupancy of O-glycosylation sites in the hinge region (41). The O-glycosylation of both serine and threonine residues found in this study is significant, since this indicates that all IgA1 specific proteases of the serine and metallo types cleave immediately adjacent to carbohydrate side chains, in contrast to the cysteine proteases of Prevotella species.
IgA1 Fc O-Glycans-- The O-glycan pool from IgA1 Fc containing the entire hinge region peptide was analyzed. The sialylated and neutral glycans identified by NP-HPLC were consistent with earlier reports of the analysis of the desialylated glycan pool (14, 16, 26, 54, 55) and confirmed previous findings from this laboratory (14), which indicated that serum IgA1 does not carry the GalNAc neoantigen. The limited repertoire of O-glycan structures attached to serum IgA1, compared with the plasma cells producing IgA1 for mucosal secretion (18, 19), indicates tissue-specific O-glycan processing and may reflect the different environments in which these molecules are required to function.
Comparison of the N- and O-Glycosylation of IgA1 Fab and
Fc--
This study contains the first report of Fab glycosylation in
human serum IgA1. The NP-HPLC glycan profile of IgA1 Fc did not account
for the heterogeneity in the glycan profile of intact IgA1 (Fig. 5),
consistent with Fab glycosylation. The glycan profile of the Fab
revealed the glycan structures identified in IgA1 that were absent or
reduced in the Fc. 30% of Fab fragments were glycosylated, suggesting
that Fab glycosylation is as common in human serum IgA1 as in human
serum IgG, where approximately 25% of Fab regions contain sugars (67).
Equivalent levels of Fab glycosylation of IgA and IgG may reflect
similar variable region utilization. Previous studies of myeloma IgA1
-chain identified an N-glycosylation acceptor sequon in
the variable region (11). However, this sequon was not identified in
either the IgA2 subclass (12) or in myeloma IgA1 purified from a
different patient (13).
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Glycosylation of Recombinant IgA1 Mutants--
Triantennary
structures with bisected and fucosylated glycans were predominant in
the wild-type anti-NIP recombinant IgA1. In serum IgA1, the major
structures were of the biantennary type. These differences may reflect
cell-specific glycosylation, highlighting the differences in the
glycosylation pathways between human lymphoid cells and the CHO cell
culture. Cell culture conditions and the particular host cell are known
to influence protein glycosylation (78). However, comparison of the
oligosaccharides of recombinant proteins produced by a variety of
expression systems has established CHO as one of the most suitable
(79). Glycosylation mutants generated by site-directed mutagenesis of
recombinant IgA1 were analyzed by comparative NP-HPLC of the released
glycans. The glycan profile for the N263A mutant, in which the
N-glycosylation site at Asn263 was deleted,
showed that the Asn459 N-glycosylation site
contained mostly triantennary oligosaccharides (Fig. 8, Table IV).
Glycan analysis of the Pterm455 mutant, in which the whole tailpiece
was deleted including the Asn459 N-glycosylation
site, showed that 83% of the structures were A2G2. This suggested that
the C2 N-glycosylation site contains less processed
N-glycans than the tailpiece N-glycosylation site, and this
may reflect the greater accessibility of the tailpiece glycans to the
glycosyltransferases. The summation of the two N-glycan profiles for the mutants did not result in a profile comparable with
that of the wild-type, suggesting that processing at one Fc
N-glycosylation site affects another.
Functional Analysis of Glycosylation Mutants--
The CH2
N-glycan in CHO-K1-derived IgA1 does not influence the
interaction with the neutrophil FcR, since mutant N263A mediated rosette formation to the same extent as wild-type IgA1. This is in
contrast to previous findings, where the mutation of Asn263
to Glu in IgA expressed in insect cells produced an antibody incapable
of mediating rosette formation (80). The differences may arise from the
different expression systems used, particularly since insect cells tend
to attach unusually large oligosaccharides, which may affect the
suitability of the insect cell-derived wild-type antibody for
comparative purposes. A detailed carbohydrate analysis of the
recombinant antibodies was not presented in this earlier study.
Further, although an enzyme-linked immunosorbent assay was used to
assess antibody concentration prior to coating RBC, it is possible that
problems with mutant assembly, undetectable by enzyme-linked
immunosorbent assay, prevented coating levels comparable to that of
wild type. We have controlled for this possibility in our study by
assessing reactivity with an anti-IgA antibody by
fluorescence-activated cell sorting analysis after erythrocyte coating.
Molecular Modeling of IgA1--
A molecular model of an IgA1
glycoform was generated from the crystal structures of related Ig
fragments and the glycan sequencing data (Fig. 10b; see
"Experimental Procedures"). Displacement of the CH2 domains was
required to accommodate the disulfide bond pattern reported in IgA1
(47-49). This leads to a reduced interstitial region and steric
crowding around the N-terminal region of the hinge compared with IgG1
(28). The disulfide pattern also restricts the movement of the
C-terminal region of the hinge and N-terminal region of the C2
domains. The orientation and surface location of Cys311 in
the CH2 domain does not allow the formation of intra- or
inter-
-chain S-S bonds, since these would require disruption of the
tertiary structure of the CH2 Ig domain. However, this surface location of Cys311 is consistent with the reported interaction of
this residue with secretory component in polymeric IgA (49, 86). The
IgA1 hinge region was modeled as an extended structure, based on
findings that proline-rich sequences (87) and heavily
O-glycosylated mucin peptides (88, 89) adopt extended
conformations in solution. As a consequence of the high proline content
of the hinge, the hydroxy-side chains extend from the protein backbone
at different angles and provide for a continuous coat of glycan along
the length of the exposed surface (Fig. 11). The amino acid sequencing
data, which indicated that Ser238 and Ser240
are unglycosylated, is consistent with the steric crowding in this
region of the hinge observed in the molecular model. As a result of the
elongated IgA1 hinge region, each heavy chain diverges from the
inter-
-chain disulfide bridge across Cys241, allowing
the Fab regions greater conformational freedom relative to the
corresponding domains in IgG1.
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ACKNOWLEDGEMENTS |
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We thank Anand Mehta and Nicole Zitzmann for critically reading the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed.
Supported by the Wellcome Trust and the Scottish Office Home
and Health Department (K/MRS/50/C2169).
1
The abbreviations used are: FcR, receptor for
the Fc region of IgA; Fc
R, receptor for the Fc region of IgG; CHO,
Chinese hamster ovary; GF, gel filtration; HPLC, high performance
liquid chromatography; NIP, 3-nitro-4-hydroxy-5-iodophenylacetate;
NP-HPLC, normal phase high performance liquid chromatography; WAX, weak anionic exchange; 2-AB, 2-aminobenzamide; gu, glucose unit; PNGase F,
peptide N-glycosidase F. The nomenclature for describing
oligosaccharide structures is as follows: An (where
n = 1, 2, 3, or 4) indicates the number of antennae
linked to the trimannosyl core; Gn (where n = 0-4) indicates the number of terminal galactose residues in the
structure; F, core fucose; Fo, outer arm fucose attached
1-3 to
GlcNAc; B, bisecting GlcNAc; S, sialic acid (indicated within brackets
are the specific linkages (6 or 3), and subscript numbers after these
parentheses indicate the number of these residues in the specific
linkage; M or Man, mannose; GalNAc, reducing terminal N-acetyl galactosamine of mucin-like O-linked
glycans.
2 R. J. Pleass, C. M. Anderson, J. I. Dunlop, and J. M. Woof, manuscript in preparation.
3 M. Wormald and Y.-L. Pao, personal communication.
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REFERENCES |
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