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

By tandem-crossed immunoelectrophoresis and ELISA experiments an immunological relationship was observed between α-macroglobulin (αM) and hemocyanin (Hc) of the terrestrial snail Helix pomatia. Both glycoproteins occur in the hemolymph: αM (minor component) as a specific proteinase inhibitor, Hc (consisting of three components: αD-HpH, αN-HpH and β-HpH) as oxygen transport protein. The cross-reaction was found to be correlated with glycosylation. (i) With β-HpH, which is richer in carbohydrates than αD-HpH and αN-HpH, mainly due to a higher 3-O-methyl-d-galactose content, the cross-reaction with HpαM was highest. (ii) From the 8 functional units, designated a-h, isolated from β-HpH, two that lack carbohydrates (c and f) were not recognized by antibodies against HpαM, 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 β-HpH and HpαM are further evidence that glycans are involved in the immunological relationship between HpH and HpαM. Carbohydrate analyses indicated that the glycan structures present on HpαM 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 α-macroglobulin and hemocyanin of the cephalopod Sepia officinalis, which lack these two monosaccharides in their glycan structures, do not immunologically cross-react.