Variability in the Structural Requirements for Binding of Human Monoclonal Anti‐Myelin‐Associated Glycoprotein Immunoglobulin M Antibodies and HNK‐1 to Sphingoglycolipid Antigens

A high proportion of patients with neuropathy have immunoglobulin M (IgM) paraproteins that react with carbohydrate determinants on the myelin‐associated glycoprotein (MAG) and two sphingoglycolipids, 3‐sulfoglucuronyl paragloboside (SGPG) and 3‐sulfoglucuronyl lactosaminyl paragloboside. In order to characterize the fine specificities of these human antibodies further, the binding of 10 anti‐MAG paraproteins to several chemically modified derivatives of SGPG was compared with the binding to intact SGPG by both TLC‐overlay and enzyme‐linked immunosorbent assay. The following derivatives were tested: the desulfated lipid, glucuronyl paragloboside (GPG); the methyl ester of GPG (MeGPG); the methyl ester of SGPG, 3‐sulfomethylglucuronyl paragloboside (SMeGPG); and 3‐sulfoglucosyl paragloboside (SGlcPG) produced by reduction of the carboxyl group of the glucuronic acid with sodium borohydride. All 10 IgM paraproteins and the related mouse IgM antibody, HNK‐1, reacted most strongly with intact SGPG, but variations in the reactivity with the derivatives revealed striking differences in the structural requirements for binding between the antibodies. Five distinct patterns of reactivity were observed: (a) three of the human antibodies and HNK‐1 exhibited partial reactivity with the sulfated derivatives, SMeGPG and SGlcPG, but not with GPG or MeGPG, indicating an absolute requirement for the sulfate group; (b) two of the human antibodies reacted only with GPG, demonstrating a requirement for the free carboxyl group on the glucuronic acid; (c) three of the antibodies bound to SMeGPG, SGlcPG, and GPG, but not to MeGPG, suggesting that at least one negative charge was needed for binding; (d) one antibody bound to SMeGPG and GPG, but not to SGlcPG or MeGPG, suggesting that both the carbonyl group and at least one negative charge were required; and (e) another antibody bound to MeGPG, SMeGPG, and SGlcPG, but not to GPG, a pattern that is difficult to explain simply based on the chemical structures. Interestingly, only those antibodies that exhibited reactivity with GPG bound to a third, minor, unidentified glycolipid of normal peripheral nerve that chromatographs faster than SGPG. The results clearly demonstrate heterogeneity in the fine specificities of the anti‐MAG antibodies that may affect their pathogenic properties.