title: Implications of hemolin glycosylation and Ca2+-binding on homophilic and cellular interactions.
abstract: 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.
authors.affiliation: Department of Genetics, Stockholm University, Sweden.
authors.forename: R
authors.initials: R
authors.lastname: Bettencourt
authors.forename: H
authors.initials: H
authors.lastname: Gunne
authors.forename: L
authors.initials: L
authors.lastname: Gastinel
authors.forename: H
authors.initials: H
authors.lastname: Steiner
authors.forename: I
authors.initials: I
authors.lastname: Faye
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002449:Cell Aggregation / Q000187:drug effects / Q000502:physiology; D017931:DNA Primers / Q000235:genetics; D006026:Glycoside Hydrolases; D006031:Glycosylation; D006434:Hemocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007136:Immunoglobulins; D066298:In Vitro Techniques; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D037102:Lectins / Q000378:metabolism; D008863:Microspheres; D009036:Moths / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D017931:DNA Primers; D007136:Immunoglobulins; D019476:Insect Proteins; D037102:Lectins; D011506:Proteins; D011994:Recombinant Proteins; C066846:hemolin; D006026:Glycoside Hydrolases; D002118:Calcium