Raj B. Parekh

A COMPARATIVE-ANALYSIS OF DISEASE-ASSOCIATED CHANGES IN THE GALACTOSYLATION OF SERUM IGG

Serum IgG from patients with both adultand juvenile-onset rheumatoid arthritis when compared to age-matched controls has an increased prevalence of N-linked oligosaccharides whose outer arms lack galactose [G(O)] and terminate in Nacetylglucosamine. The reduction in galactosylation was found in lupus patients complicated by Sjogren’s syndrome but not in 85 patients with one of nine other rheumatological disorders, namely SLE, Sjogren’s syndrome (primary), myositis, scleroderma, osteoarthritis, psoriatic arthropathy, ankylosing spondylitis, postYersinia arthropathy, and gout. Further, this reduction in galactosylation was not simply concomitant with an acute or chronic inflammatory process, as shown by an analysis of the N-glycosylation of serum IgG from 84 patients with one or more than 13 different relevant disorders, namely klebsiella, leprosy, tuberculosis, rubella, parvovirus, mumps, glandular fever, AIDS, sarcoidosis, ulcerative colitis, Crohn’s multiple sclerosis, and Waldenstrom’s macroglobulinaemia. Of these, only serum IgG from patients with tuberculosis and Crohn’s disease had elevated G(0) values. Given the evidence for a major role of IgG autosensitization in the pathogenesis of rheumatoid arthritis, it is striking that this structural change in IgG carbohydrate, which may facilitate the self-association of IgG rheumatoid factors and contribute to autoantigenicity, should be restricted to such a small number of diseases. Human serum IgG is a glycoprotein [ 1, 21 carrying on average 2.8 N-linked oligosaccharides. Of these 2.0 are invariably located in the Fc (at the conserved Nglycosylation site of Asn 297), and additional ones in the variable region of the light and heavy chains, with a frequency and position dependent on the occurrence of the

THE ROLE OF IGG GLYCOFORMS IN THE PATHOGENESIS OF RHEUMATOID-ARTHRITIS

ConclusionsIn conclusion, there is a shift in the population of IgG glycoforms towards those with a higher content of agalactosyl biantennary N-linked oligosaccharides in active rheumatoid arthritis (both juvenile and adult), tuberculosis, and Crohns disease, but not in a variety of other rheumatological, inflammatory, or infectious conditions. This shift may contribute to disease pathogenesis both through immune-complex formation and through disturbance of a cellular network directed against the non-reducing terminal G1cNAc epitope. The precise pathology would in each case be modulated by the anatomical site(s) of production of such IgG, and also by the precise mechanism inducing this change in IgG glycosylation. Important amongst such mechanisms may be cross-reactivity between environmental and endogenous carbohydrate epitopes. It will be interesting to see if future research supports the idea that groups of diseases (e. g., rheumatoid arthritis, tuberculosis, Crohns) are indeed related by a common aetiopathogenesis [i. e., G(0)].

N-glycosylation and the production of recombinant glycoproteins

Abstract Many polypeptides of therapeutic and diagnostic interest that are being produced in recombinant form for in-vivo administration carry, in their native form, N -linked oligosaccharides. In this article we review certain biosynthetic aspects and functional consequences of N -glycosylation that are relevant to the production and administration of recombinant glycoproteins. In so doing, we hope to increase awareness of the biological significance of N -linked oligosaccharides, and to emphasize the need to consider the N -glycosylation of a recombinant glycoprotein prior to therapeutic administration.

Oligosaccharide sequencing based on exo- and endoglycosidase digestion and liquid chromatographic analysis of the products.

Exo- and endoglycosidases are used to sequence oligosaccharides and give valuable information on the monosaccharide sequence, together with the anomericity, the stereochemistry, and in some cases, the substitution pattern of the monosaccharides. Both sequential and parallel methods of oligosaccharide sequencing are discussed.

Human T cell IgD receptors react with O-glycans on both human IgD and IgA1.

Previous studies on murine T cell IgD‐R have shown that these receptors recognize N‐glycans of murine IgD, and not of other Ig isotypes. We have now studied the specificity of IgD‐R on human T cells. Human IgD digested with proteinase K to fragments of < 5 kDa inhibit the ability of T cells to form rosettes with IgD‐coated ox erythrocytes. The same amount of digested IgG does not. We tested all the human Ig isotypes: IgG1, −2, −3, −4, IgA2, IgE and IgM fail to inhibit significantly at 20 μg/assay. However, IgA1 is as effective as IgD itself, showing approximately 60 % and 80 % inhibition at 5 μg and 10 μg/assay. Human IgA1 and IgD both contain Gal‐1 → 3‐GalNac‐rich O‐linked glycans, and on this basis are both bound to ricin and jacalin. The O‐linked glycans may therefore also represent the common moiety binding to IgD‐R. Disaccharides Gal‐1 → 3‐GalNac, and Gal‐1 → 4‐Glc at 10 μg/assay blocked IgD rosetting while Gal‐1 → 6‐Glc did not. We conclude that the human IgD‐R is a lectin, differing from the murine IgD‐R in that it has both IgA1 and IgD as ligands.

Lectin-Carbohydrate Interactions in Disease

Two disease associated lectin-carbohydrate interactions have been studied. (1) A T-cell surface lectin which binds IgA1 and IgD is expressed on CD4+ and CD8+ T-lymphocytes in a number of diseases including systemic lupus erythematosus, rheumatoid arthritis (RA), Behcet’s disease and IgA nephropathy. We have demonstrated that calcium independent binding to this receptor is mediated by the O-linked disaccharide Galβ3GalNAc which is associated with the hinge regions of both IgAl and IgD. (2) In rheumatoid arthritis the proportion of IgGO glycoform populations lacking terminal galactose increases. We have Open image in new window Figure 1 Inhibition studies of the binding of sIgA to T-cells. a) Panel A; Inhibition of binding by the normal serum immunoglobulins IgG, IgM, IgA2, IgD and IgA1 and IgA1 desialylated with neuraminidase. IC 50 values were ≫10-5,≫10-5, >10-5, 1×10-5, 0.1×10-5 and 0.07×10-5 M respectively. b) Panel B; Inhibition by the oligosaccharide library recovered after 65°C hydrazinolysis of IgA1.Inhibition by the desialylated library is shown alongside standards of GalNAc and GlcNAc. IC 50 values were 0.6×10-5,0.3×10-5, 6×10-5 and >>10×10-5 M respectively. c) Panel C; Inhibition by the individual sugars associated with the IgA hingeregion: the neutral sugars GalNAc and Galβ3Gal- NAc, and the sialylated sugars NeuNAc2α3(6)Galβ3GalNAc and NeuN-Ac2α3(6)Galβ3[NeuNAc2α6] GalNAc. IC 50 values were 6×10-5, 1×10-5, 1.1×10-5and 6×10-5 M respectively. shown that terminal GlcNAc residues on oligosaccharides in the Fc region of IgG0 can bind to the C-type lectin, serum mannose binding protein, and thus activate the classical complement pathway. This provides a mechanism of activation of the complement system not available to the other classes of IgG glycoforms.