Clinical Immunology 116 (2005) 3 10 www.elsevier.

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Short Analytical Review

IgA: An immune glycoprotein

Esther M. Yooa,T, Sherie L. Morrisona,b
a

Department of Microbiology, Immunology and Molecular Genetics University of California, 609 Charles E. Young Drive, Los Angeles, CA 90095, USA b The Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA Received 24 February 2005; accepted 15 March 2005 Available online 27 April 2005

Abstract IgA is a glycoprotein containing multiple N-linked carbohydrates as well as O-linked glycans in the case of IgA1. Because of the critical role it plays in providing protection at mucosal surfaces, IgA is an ideal candidate for use as a therapeutic or prophylactic agent. The presence or absence of carbohydrates, as well as their structure, has been found to influence effector functions and binding to specific IgA receptors. In addition, changes in IgA glycosylation are associated with immune pathology. A thorough understanding of the contributions of the glycans to IgA immune protection will aid in the design of clinically suitable antibodies. D 2005 Elsevier Inc. All rights reserved.
Keywords: Immunoglobulin; Glycosylation; Recombinant antibodies; Glycoprotein

IgA IgA is the most abundant class of immunoglobulin synthesized in humans, with 66 mg of IgA/kg of body weight produced daily compared to 34 mg of IgG and 7.9 mg of IgM. Human IgA exists as two isotypes, IgA1 and IgA2, with IgA2 having three allotypes. The most significant difference between the two isotypes of IgA is that IgA1, but not IgA2, possesses a 13 amino acid hinge region containing three to five O-linked carbohydrate moieties (Fig. 1). In IgA1, IgA2m(2) and IgA2(n), but not IgA2m(1), a disulfide bond links the L chain to the H chains [1,2]. Unlike IgG, IgA is a polymeric immunoglobulin with the most abundant species an H2L2 dimer with associated J chain. The presence of multiple binding sites in polymeric IgA provides increased avidity for antigen. Both IgA and IgM, the other polymeric immunoglobulin, possess an 18 amino acid extension of the C terminus (tail-piece), which participates in polymerization through a penultimate Cys residue. In addition to the tail-piece, structural motifs in the constant region domains are critical for polymer assembly and J chain incorporation [3].
T Corresponding author. Fax: +1 310 794 5126. E-mail address: esthery@microbio.ucla.edu (E.M. Yoo). 1521-6616/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2005.03.010

Human serum is 90% IgA1 and only 10% IgA2, while in external secretions, the proportion of IgA2 can be as high as 50%. This characteristic distribution may reflect the fact that plasma cells in the bone marrow are the source of serum IgA and plasma cells in the lamina propria are the source of secreted IgA. Variations in the serum half-life of IgA isotypes could also contribute to these differences. Indeed, recombinant chimeric IgA1 and IgA2 differ in their pharmacokinetic properties [4] with IgA2 rapidly being cleared from the serum of mice by the asialoglycoprotein receptor (ASGP-R) present in the liver.

Glycosylation of serum and secretory IgA There are a variable number of N-linked glycans on IgA depending on both the isotype and allotype. All IgAs contain an N-linked carbohydrate at position 263 in CH2 and at position 459 in the tail-piece extension of CH3. All IgA2s contain additional glycans at position 166 in CH1 and 337 in CH2. IgA2m(2) and IgA2(n) contain a fifth glycan at position 211 in CH1 (Fig. 1). The N-linked glycans can either remain high mannose or can be further processed to complex (Fig. 2A). The degree of processing varies depending on the protein, the position

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Fig. 1. Glycosylation of IgA. IgA1 has N-linked carbohydrate addition sites in CH2 (Asn263) and in CH3 (Asn459) [85] and O-linked carbohydrate in its hinge region. IgA2 lacks O-linked carbohydrate but has additional N-linked sites in CH1 (Asn166) and in CH2 (Asn337). The IgA2m(2) and the IgA2(n) allotypes have a fifth N-linked site in CH1 (Asn211) [85,86].

of the carbohydrate and the expression system. Analysis of pooled monomeric serum IgA1 showed that over 80% of the N-glycans were sialylated biantennary complex oligosaccharides (Fig. 2A) [5]. In mice, Th2 cytokines have been shown to influence the structure of the glycans attached to IgA and altered glycan structures are associated with renal disease in immunized mice [6,7]. The removal of carbohydrate from murine IgA has been found to interfere with its

secretion [8]. However, human IgA1 lacking carbohydrate is not impaired in its ability to be secreted [9]. O-linked glycosylation occurs at Ser and Thr residues within the hinge of IgA1. There is no well-defined motif for the acceptor site other than the proximity of Pro residues (reviewed in [10]), and a single enzyme, UDP-N -acetyl-ad-galactosamine:polypeptide N -acetylgalactosaminyltransferase 2, appears to be responsible for transferring the initial N -acetylgalactosamine (GalNAc) to all of the different sites within the hinge peptide [11]. The O-glycans within the hinge of IgA1 assume many different structures, with the most abundant being monosialylated T antigen (NeuAca23Galh1-3GalNAc) (Fig. 2B). However, larger neutral and sialylated tetrasaccharide structures (Galh1-4GlcNAch16Galh1-3GalNAc) were also present [5]. Secretory IgA (SIgA) contains secretory component (SC) with seven N-glycosylation sites and J chain with one Nglycan. In a recent molecular model of SIgA, the Fab arms form a T shape with SC wrapped around the heavy chains [12]. SC is covalently bound at one end to the Ca2 domain and the other end interacts noncovalently with both the J chain and one of the Ca3 domains of a H chain. N-glycans of SC provide SIgA with bacteria binding sites in addition to those on the four Fab-associated binding domains. SC, through its carbohydrate residues, assures the appropriate in vivo localization of SIgA by anchoring it to the mucus lining of the epithelial surface [13]. It is this improved localization, not the improved stability conferred by the presence of SC, that is responsible for the increased protection provided by SIgA compared to dimeric IgA against Salmonella typhimurium infection. In spite of the superior protection provided by SIgA, it has not been used in any clinical trials to date. One issue is obtaining abundant amounts of SIgA since it is normally the

Fig. 2. Structure of carbohydrate moieties present on IgA. (A) Structure of biantennary complex carbohydrate. The degree of substitution of the Man3GlcNAc2 core is quite variable. Fuc and a bisecting GlcNAc may or may not be present in human IgA. The structure of the sialic acid added depends on the cells in which the immunoglobulin is produced. Human immunoglobulins contain oligosaccharides with NeuAc, whereas mouse Igs contain oligosaccharides with both NeuAc and NeuGc. The bisecting GlcNAc is absent when proteins are produced in murine myelomas and CHO cells. Wild-type CHO cells also appear to lack the glycosyltransferases necessary for generating a2Y6 linked sialic acid and produce sugars with NeuAca2Y3Gal. (B) Structure of the O-linked carbohydrates added to the hinge of IgA1.

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product of two cell types, the plasma cell and the epithelial cell. One solution is in vitro association of recombinant SC with dimeric IgA [14]. The other solution is to synthesize H chain, L chain, SC and J chain in a single cell, as was initially reported using a murine myeloma expression system [15] and then subsequently using Chinese Hamster ovary (CHO) cells [16] (Gurbaxani, unpublished results).

Receptors for IgA pIgR The human polymeric immunoglobulin receptor (pIgR), synthesized by the mucosal and secretory glandular epithelium, consists of an ectoplasmic segment of 589 amino acids, a membrane-spanning domain of 23 amino acids and a 103 amino acid cytoplasmic domain (reviewed in [17]). Newly synthesized pIgR is inserted in the basolateral membrane from which it is endocytosed with or without its ligand, polymeric Ig, and is transported to the apical surface by transcytosis. At the apical surface, an unknown enzyme cleaves the pIgR between the ectoplasmic (SC) and transmembrane domains, releasing the SCIgA complex into external secretions [18]. pIgR specifically binds to polymeric IgA (or IgM) and not to monomeric IgA or IgG, with the presence of J chain required for binding [19]. N-linked glycosylation was not required for IgA1 to associate with the pIgR [9]. Studies in J chain deficient mice indicate that although J chain is necessary for stable association of IgA with SC, it is not necessary for the transport of IgA into external secretions [20]. SIgA in external secretions plays an important role in immune defense at the mucosal surfaces (see above). CD89 CD89 or FcaRI is an IgA-specific receptor expressed on human monocytes, eosinophils, neutrophils and macrophages. No homologous receptor has been found in the mouse. Both monomeric and dimeric IgA bind FcaRI and immune complexes of either monomeric or dimeric IgA can activate phagocytosis. FcaRI and IgA form a 2:1 complex, with one FcaRI binding at each CH2 CH3 interface to residues conserved in all human IgA molecules [2]. In the crystal structure of Fca bound to CD89, the carbohydrate attached at Asn263 is exposed on the surface and approaches of the receptor but does not directly contact it [2]. within 8 A Although alterations in the structure of the carbohydrate can influence binding as oversialylated IgA1 and IgA2 bound less well to FcaRI [21], there is disagreement as to whether this carbohydrate is necessary for CD89 binding [5,22]. TfR Studies have implicated the transferrin receptor (TfR/ CD71) in IgA binding [23]. Although it was initially

reported that TfR bound monomeric better than polymeric IgA1, a more recent publication by the same group revealed that only polymeric IgA1 will bind the TfR on cultured mesangial cells [24]. The binding is inhibited by transferrin [23], and by soluble TfR1 and TfR2 [24]. IgA1 with the hinge of IgA2 failed to bind Daudi cells, suggesting a role for the hinge of IgA1 in recognition. Surprisingly, recombinant IgA1 lacking N-linked carbohydrate sites also failed to bind whereas IgA1 from which the carbohydrates had been removed enzymatically continued to bind although no controls to verify the removal of the carbohydrates were included in the study [24]. ASGP-R The asialoglycoprotein-receptor (ASGP-R) binds oligosaccharides with terminal h-linked N -acetylgalactosamine (GalNAc) or galactose (Gal) and can mediate the rapid clearance of glycoproteins bearing these terminal sugars. Increasing the amount of sialic acid present on the carbohydrate decreases the interaction of IgA1 and IgA2 with the ASGP-R [21]. The ASGP-R is abundantly expressed in the liver and, in the mouse, the liver accounted for more catabolism of monomeric murine IgA than all other tissues combined [25,26]. The liver appears to play a similar role in humans [26,27]. Fca /lR The Fca/A receptor (Fca/AR) is expressed on mature B cells and macrophages, but not on granulocytes, T cells or NK cells. It is also found in the liver, kidney, small and large intestines, testis and placenta although it is unclear if it is expressed on epithelial cells or on infiltrating hematopoietic cells [28,29]. Although the role of this receptor has not been precisely defined, IgM-coated beads can be endocytosed through the receptor. Message for both a membrane and soluble form of Fca/AR has been detected in human mesangial cell (MC) lines following treatment with IL-1 but not with TNF-a [30]. However, Fca/AR does not appear to be the novel Fca receptor that has been identified on MC since that receptor does not bind IgM [31].

Protease sensitivity of IgA The effectiveness of IgA depends on its structural integrity and many bacteria that cause disease by colonizing or infecting human mucosal membranes secrete IgA1 proteases. These enzymes cleave between Pro and Ser or Pro and Thr residues in the hinge region of human IgA1, thereby separating the Fab from the Fc region. Many but not all proteases can cleave a hybrid IgA2/IgA1 with a shortened hinge [32]. Recent studies have shown that both Ca2 and Ca3 are required for the cleavage of the hinge of IgA1 by the protease of Haemophilus influenzae type 1 and

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Neisseria gonorrhoeae type 2 [33]. The absence of N-linked carbohydrate increased the susceptibility to cleavage by a protease from N. gonorrhoeae , but not H. influenzae [33]. IgA1 proteases from different species of Streptococcus all cleave the IgA1 hinge between Pro227 and Thr228 [34]. The removal of only sialic acid residues increases the susceptibility of IgA1 to these streptococcal proteases while a decreased susceptibility to cleavage was observed after extensive deglycosylation of the hinge region [35].

IgA in immune protection IgA plays an important role in providing protection at mucosal surfaces. It is effective in neutralizing bacterial toxins. In fact, polymeric IgA has been found to be more effective than either monomeric IgA or IgG with the same variable regions in neutralizing exotoxins from Clostridium difficile [36]. IgA plays an important role in virus neutralization by inhibiting binding to cell surface receptors. Its binding is also thought to be able to effect changes in the virus that disrupts its structure, thereby preventing either receptor binding or fusion of the virus with the cell membrane. It originally appeared that IgA could act as a Trojan horse, allowing Epstein-Barr virus in complex with IgA to infect epithelial cells using the pIgR; however, more recently, it has been demonstrated that if the cells are polarized, as they would be in vivo, they are not infected [37,38]. IgA prevents bacteria from binding to mucosal surfaces and appears to play a role in inhibiting penetration by commensal bacteria [39]. Antigens present in tissues are eliminated by being bound to dimeric IgA and then transported across the epithelial layer by the pIgR [40]. Recently, dimeric IgA specific for LPS has been shown to play an anti-inflammatory role. Intracellular dimeric IgA interacts with LPS within the apical recycling endosome compartment, reducing NF-?B translocation, and consequently attenuating the proinflammatory response induced by LPS [41].

IgA nephropathya disease associated with altered glycosylation of IgA The diagnostic feature of IgA nephropathy (IgAN) is the presence of IgA1-containing immune complexes in the glomerular mesangium with mononuclear cell infiltration in the kidney correlating with glomerular damage and interstitial tissue injury. Although precisely what is responsible for IgA immune complex deposition remains poorly defined, many studies have found that a deficiency in the processing of the hinge O-glycans is associated with disease. Circulating and glomerular IgA in patients with IgAN showed a decreased presence of carbohydrate in the IgA1 hinge glycopeptides and increased levels of IgA glycoforms with exposed GalNAc (Fig. 2B) [42 45]. The

alterations in glycosylation of the hinge of IgA1 are not associated with alterations in its amino acid sequence [46]. An unresolved issue is how does the alteration in glycosylation relate to disease. One possibility is that the change in carbohydrate structure alters the physiochemical properties of IgA. Indeed, when sialic acid alone or sialic acid and Gal were removed from human IgA1, it was found to accumulate in rat glomeruli and the desialylated/ degalactosylated IgA1 showed an increased tendency to aggregate [47] with the lack of these sugars appearing to make the IgA1 sticky[48]. Receptor binding may also contribute to disease. MC possess receptors whose occupancy by IgA induces mesangial proliferation, phagocytosis of IgA complexes and the release of soluble mediators which can affect cell growth, apoptosis and inflammatory responses [49 51]. At the present time, the identity of the responsible receptor(s) is unknown. Although it does not appear to be CD89 [31,52 54], transgenic mice expressing human CD89 on macrophages/monocytes spontaneously developed massive mesangial IgA deposition, glomerular and interstitial macrophage infiltration, mesangial matrix expansion, hematuria and mild proteinuria [55]. Disease could be induced in RAG/ mice by injection of serum from CD89 transgenic mice and in SCID mice transgenic for CD89 by injection of patient IgA. Although it has been suggested that defective O-glycans in the hinge of IgA1 may directly contribute to glomerular deposition, it is also possible that the immune response to the IgA1 peptide may play a role in pathogenesis through the formation of immune complexes that bind to mesangial cells through the Fc region [52]. Circulating immune complexes isolated from sera of patients with IgAN contain undergalactosylated IgA1 and IgG antibodies specific for GalNAc residues that are present not only in the O-glycans of the hinge region of IgA but also in other non-Ig proteins [56]. A number of microorganisms express GalNAc on their surface and it is possible that the increased levels of antiGalNAc antibodies in IgAN patients result from a preceding infection with GalNAc-expressing microorganisms. Studies have also shown that antibody titers against a synthetic hinge peptide are significantly higher in IgAN patients than in controls. In addition, the reactivity of the serum IgG from the IgAN patients against a monoclonal IgA1 increased as the carbohydrate was successively removed from the IgA1 hinge using neuraminidase, h-galactosidase and a-N -acetylgalactosaminidase [57]. Alterations in glycosylation have also been seen in other diseases. Decreased glycosylation (reduced sialylation and galactosylation) of the IgA1 hinge was found associated with nephritic syndrome following allogeneic bone marrow transplantation [58]. In Henoch Scho nlein purpura, a form of systemic vasculitis characterized by IgA deposition in affected blood vessels, reduction in galactosylation of GalNAc moieties is seen only in those patients who have renal involvement [46,59,60]. In patients with primary

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Sjo grens syndrome, an autoimmune disorder characterized by lymphoid cell infiltration of exocrine organs, in particular lacrimal and salivary glands, monomeric IgA1 contains more sialic acid on the N-linked glycans than found in normals [61].

Recombinant IgA Given its central role in providing protection at mucosal surfaces and its unique combination of properties, it seems certain that therapeutics based on IgA antibodies will take their place along with the IgG-based therapeutic molecules that are assuming an ever-increasing role in the clinic. Given the role of carbohydrate in both the normal and pathologic functions of IgA, special attention must be paid to the carbohydrates attached by different expression systems. Several different expression systems for recombinant IgA production are available, each with its advantages and shortcomings. Although bacteria are often an attractive expression system for the production of recombinant proteins because of the low cost of the media in which they grow, E. coli , the most commonly used expression system, does not add N-linked carbohydrates and hence cannot make antibodies with the whole complement of functional properties. In bacteria, there is also the additional problem of the assembly of the multimeric IgA proteins with multiple intra- and inter-chain disulfide bonds. Yeast can also be grown in simple media and does attach N-linked carbohydrates, but in Saccharomyces cerevisiae , hypermannosylated N-glycan structures with >100 mannose (Man) residues are often present. In Pichia pastoris, hypermannosylation occurs less frequently and Pichia has recently been engineered to attach carbohydrates that are closely related to those attached by mammalian systems [62,63]. Insect cells have also been used for antibody production. Although there appear to be differences among different cell lines, insect cells do not appear to contain all of the enzymes required to produce sialylated complex carbohydrates [64,65]. This may be an important consideration since IgA, in contrast to IgG, can be highly sialylated. IgA1 expressed in cells derived from Spodoptera frugiperda (Sf9) appears to exhibit different functional properties from IgA produced in CHO cells [5,22]. Plants are able to produce proteins with both high Man and complex N-linked glycans having a core with two N acetylglucosamine (GlcNAc) residues. However, this core is substituted by h1Y2linked xylose and a1Y3linked core fucose (Fuc) instead of the a1Y6 linked core Fuc found in mammals. Plants also do not add either Gal or sialic acid to their complex N-glycans. A significant concern is whether the novel glycans attached by plants will be immunogenic. IgA and SIgA have been produced in plants [66 68]. Transgenic animals are attractive expression systems for large-scale production of antibodies. However, there is

species-specific variation in sialic acid structure with N acetylneuraminic acid (NeuAc) and/or N -glycolylneuraminic acid (NeuGc) seen in different animals [69]. There is also species-specific variation in the addition of bisecting GlcNAc, core fucosylation and extent of Gal addition [69]. Murine myeloma and CHO cells have been the most commonly used mammalian expression systems to date for antibody production. However, murine carbohydrates differ from those present in the human. It has been reported that a significant amount of circulating IgG in man specifically interacts with Gala1Y3Galh1Y4GlcNAc, an epitope abundant on glycoconjugates of non-primate mammals, prosimians, and New World monkey but absent from Old World monkeys, apes and man [70]. Some of the carbohydrates introduced in murine expression systems bear this structure, but its presence appears to be quite variable. Although we do not usually find this epitope in the Fc associated carbohydrate of IgG produced in murine myelomas, our recent studies of chimeric IgA produced in murine myelomas has indicated the presence of large quantities of Gala1Y3Galh1Y4GlcNAc on these proteins (Yu et al., in preparation). Given the exposed nature of many of the carbohydrate moieties on IgA, it seems unlikely that murine myelomas would be an appropriate expression system for IgA production. Murine myeloma cells also do not add h1Y4 linked GlcNAc to the Manh1Y4 in the trimannosyl core of N-linked sugars although it is unclear what impact this would have on IgA function [71]. CHO cells appear to lack the glycosyltransferases necessary for generating both the bisecting GlcNAc and a2Y6 linked sialic acid [72] and do not appear to attach the Gala1Y3Galh1Y4GlcNAc epitope. However, we have observed incomplete assembly of IgA2 produced in CHO cells (Gurbaxani et al., submitted for publication). Although recombinant IgA has many properties that make it an attractive therapeutic agent, there are some potential limitations for its use. Selective IgA deficiency (SIgAD) is the most common inherited form of primary immunodeficiency, affecting 1 in 600 people in the Western world [73]. Most people are asymptomatic with no increased incidence of infection. However, there is an increased risk to developing antibody-mediated autoimmune diseases such as myasthenia gravis, insulin-dependent diabetes mellitus, systemic lupus erythematosus, and rheumatoid arthritis (as reviewed in [74]). Common variable immunodeficiency (CVID) is a related syndrome in which patients are deficient in both IgG and IgA. A regulatory defect in B cell differentiation is thought to be responsible for the disease and although the genetic basis of these defects is unknown, both SIgAD and CVID are associated with MHC susceptibility genes [75]. Variable levels of anti-IgA antibodies are detected in 10 40% of adults [76 80] and 60% in children with SIgAD [81]. These antibodies are generally IgGs, although anti-IgA antibodies of IgD, IgE and IgM class have also been described [82 84]. Therefore, it will be critical to screen

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