Implications of hemolin glycosylation and Ca2+-binding on homophilic and cellular interactions.

Insects are useful models for the study of innate immune mechanisms because of their lack of antibodies and receptors involved in adaptive immune response. Nevertheless, hemolin cloned from moths is a soluble and membrane associated Ig-related molecule that is up-regulated during immune response [Lanz-Mendoza, H. & Faye, I. (1999) Dev. Comp. Immunol. 23, 359-374]. The hemolin monomeric form has four, pair-wise, interacting Ig-domains, forming a strongly bent horseshoe structure [Su, X.-D., Gastinel, L.N., Vaughn, D.E., Faye, I., Poon, P. & Bjorkman, P. (1998) Science 281, 991-995]. To elucidate the nature of its homophilic and cellular interactions, the glycosylation and Ca2+-binding properties of hemolin were investigated. We used Hyalophora cecropia hemolin isolated from hemolymph of bacteria-injected pupae, or produced as a recombinant protein in a baculovirus/insect cell system. Both types of hemolin contain N-acetylglucosamine and probably sialic acid, as indicated by peptide:N-glycosidase F and neuraminidase digestion and glycosylation detection by Western-blotting analysis. The N-acetylglucosamine residues on hemolin were confirmed with the use of specific lectins. In addition, hemolin was shown to specifically bind calcium when spotted onto nitrocellulose and treated as for 45Ca2+ autoradiography. Earlier studies demonstrated that hemolin can bind to hemocytes and this was tested for its dependence on calcium and carbohydrates, using hemolin-coated fluorescent microspheres. A greater level of attachment of microspheres occurred in the presence of calcium than if calcium was absent. Furthermore, this binding was inhibited by EGTA and N-acetylglucosamine or N-acetylneuraminic acid, implying that carbohydrates and calcium are crucial factors in homophilic binding and cell-adhesion events mediated by this Ig-superfamily molecule.