Involvement of glycans in the immunological cross-reaction between [alpha]-macroglobulin and hemocyanin of the gastropod Helix pomatia

By tandem-crossed immunoelectrophoresis and ELISA experiments an immunological relationship was observed between [alpha]-macroglobulin ([alpha]M) and hemocyanin (Hc) of the terrestrial snail Helix pomatia. Both glycoproteins occur in the hemolymph: [alpha]M (minor component) as a specific proteinase inhibitor, Hc (consisting of three components: [alpha][sub]D-HpH, [alpha][sub]N-HpH and [beta]-HpH) as oxygen transport protein. The cross-reaction was found to be correlated with glycosylation. (i) With [beta]-HpH, which is richer in carbohydrates than [alpha][sub]D-HpH and [alpha][sub]N-HpH, mainly due to a higher 3-O-methyl-d- galactose content, the cross-reaction with Hp[alpha]M was highest. (ii) From the 8 functional units, designated a-h, isolated from [beta]-HpH, two that lack carbohydrates (c and f) were not recognized by antibodies against Hp[alpha]M, while the six glycosylated ones were strongly cross-reacting. The nearly complete loss of the cross-reactivity upon deglycosylation of functional units d and g and the inhibition in competitive ELISA experiments by glycopeptides isolated from both [beta]-HpH and Hp[alpha]M are further evidence that glycans are involved in the immunological relationship between HpH and Hp[alpha]M. Carbohydrate analyses indicated that the glycan structures present on Hp[alpha]M are very similar (or identical) to those found on HpH, suggesting that glycans are common epitopes on both proteins. Especially d-xylose and 3-O-methyl-d-galactose seem to be responsible for the cross-reactivity since the [alpha]- macroglobulin and hemocyanin of the cephalopod Sepia officinalis, which lack these two monosaccharides in their glycan structures, do not immunologically cross-react.