Polyreactive Binding of Antibodies Generated by Polyclonal B Cell Activation. I. Polyreactivity Could be Caused by Differential Glycosylation of Immunoglobulins

Abstract

Fernández C, Alarcón-Riquelme ME, Abedi-Valugerdi M, Sverremark E, Cortes V. Polyreactive Binding of Antibodies Generated by Polyclonal B Cell Activation. I. Polyreactivity Could be Caused by Differential Glycosylation of Immunoglobulins. Scand J Immunol 1997;45:231-239 The aim of this study was to gain knowledge of the pre-immune repertoire with reactivity directed to the carbohydrate antigen dextran B512 (Dx). Polyclonal activation of spleen cells in mice has been estimated to be a method of revealing the available repertoire. Hybridoma cell lines derived from lipopolysaccharide-(LPS)stimulated C57BL/6 spleen cells were screened for reactivity with Dx and with the non-related protein bovine serum albumin (BSA). Despite the lack of structural similarity between Dx and BSA we observed that nearly all of the Dx positive monoclonal antibodies (MoAbs) cross-reacted with BSA. The Dx and BSA crossreactive MoAbs were also found to bind to several foreign-and auto-antigens, and therefore we concluded that these MoAbs fulfilled the criteria of polyreactivity. The Dx and BSA cross-reactive antibodies were produced in an apparently random fashion as judged by the use of k and l light chains and the use of V H J558 subfamily genes. There are two possible explanations for this type of polyreactivity: (1) LPS induces a randomly generated polyreactive and a randomly generated specific immunoglobulin (Ig) repertoire; (2) polyreactivity can be the result of post-translational modifications. Since immunoglobulins are glycoproteins we considered the possibility that post-translational modifications such as glycosylation could be responsible for the generation of the polyreactive pool. Dextran-specific and Dx/BSA cross-reactive MoAbs showed different degrees of sensitivity to inhibition of glycosylation performed by treatment with tunicamycin (Tm), an inhibitor of the formation of N-glycosidic linkages. Other polyreactive, connective and specific MoAbs were also tested. The authors found that Tm treatment had a more profound effect in reducing the binding capacity of the polyreactive antibodies (Abs), suggesting that the polyreactive Abs may be more dependent than the specific Abs on the carbohydrate content of the molecule for binding to the antigens. The authors propose that lymphocytes may use differential glycosylation as a means to generate polyreactive or monospecific Abs.

Polyreactive Binding of Antibodies Generated by Polyclonal B Cell Activation. II. Crossreactive and Monospecific Antibodies can be Generated from an Identical Ig Rearrangement by Differential Glycosylation

“…Binding to TNP was not influenced by Tm treatment while binding to other antigens was considerably reduced (Table 4 and Fig. 4) which is in accordance with our data in non-transgenic animals [6]. It is important to point out that differences in the stability of glycosylated and naked Igs and other trivial explanations can be excluded.…”

“…Per cent binding reduction was calculated by comparing equivalent points in the loglinear region of the binding curves. All these procedures have been described in detail elsewhere [6].…”

“…To assess the contribution of glycosylation in binding, we treated mono-and TNP/Dx/BSA-crossreactive clones with the inhibitor of glycosylation Tm [18,19,6]. Binding to TNP was not influenced by Tm treatment while binding to other antigens was considerably reduced (Table 4 and Fig.…”

“…Per cent binding reduction was calculated by comparing equivalent points in the loglinear region of the binding curves. All these procedures have been described in detail elsewhere [6].Quantification of carbohydrate (CHO) content of lgs. The CHO content of polyreactive and monospecific MoAbs was evaluated by the digoxigenin (DIG) glycan-based method using a kit purchased from Boehringer Mannheim.…”

Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma

“…Binding to elements in the site might be related to the polyreactive behavior observed for immunoglobulins expressed by FL cells, 54 which, for antibody molecules, apparently can be influenced by glycosylation. 55,56 However, polyreactivity cannot be the only explanation because some myeloma proteins also exhibit this characteristic. 57 The location of the oligosaccharide in the V-region appears critical, possibly because, in contrast to the oligosaccharides in the constant region, 14 the chains exposed in the V-region may be more available to the glycosyl transferases and therefore may be fully glycosylated.…”