Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N-glycans with Gal(beta1-6)Man-motifs.

Keyhole limpet (Megathura crenulata) hemocyanin (KLH), an extracellular respiratory protein, is widely used as hapten carrier and immune stimulant. Although it is generally accepted that the sugar constituents of this glycoprotein are likely to be implicated in the antigenicity and biomedical properties of KLH, knowledge of its carbohydrate structure is still limited. Therefore, we have investigated the N-linked oligosaccharides of KLH. Glycan chains were enzymatically liberated from tryptic glycopeptides, pyridylaminated and separated by two-dimensional HPLC. Only neutral oligosaccharides were obtained and characterized by carbohydrate constituent and methylation analyses, MALDI-TOF-MS, ESI-ion trap-MS and sequential exoglycosidase digestion. The results revealed that KLH is carrying high mannose-type glycans and truncated sugar chains derived thereof. As a characteristic feature, a number of the studied N-glycans contained a Gal(beta1-6)Man-unit which has not been found in glycoprotein-N-glycans so far. Hence, our studies demonstrate that this marine mollusk glycoprotein is characterized by a unique oligosaccharide pattern comprising, in part, novel structural elements.