ROLE OF N-LINKED GLYCOSYLATION IN HUMAN OSTEONECTIN - EFFECT OF CARBOHYDRATE REMOVAL BY N-GLYCANASE AND SITE-DIRECTED MUTAGENESIS ON STRUCTURE AND BINDING OF TYPE-V COLLAGEN

In this study we demonstrate that the binding region of recombinant tr uncated human bone osteonectin (tHON) for type V collagen resides betw een amino acids 1 and 146. After removal of oligosaccharide chain stru ctures from tHON, bovine bone osteonectin (BBON) and human platelet os teonectin (HPON) by N-glycanase, their ability to bind to type V colla gen is increased, and HPON affinity to collagen V is the same as that of BBON. These data suggest that glycosylation of osteonectin has a di rect or regulatory effect on osteonectin binding to collagen V and tha t the increase in tHON binding upon removal of carbohydrate is the res ult of a loss of a down-regulation site or direct interference of the carbohydrate at the binding site. To determine the specific role of ea ch N-glycosylation site in tHON, Asn(71) and Asn(99) were mutated to G in (N71Q, N99Q) and Thr(73),and Thr(101) mutated to Ala (T73A, T101A) to selectively inhibit oligosaccharide attachment, The binding affinit y of N99Q and T101Q to collagen V is markedly increased over wild-type tHON, whereas N71Q and T73A are the same as wild-type tHON. The doubl ed mutant (N71,99Q) binds identically to collagen V as N99Q and T101A. These data suggest that only the position 99 glycosylation site (Asn( 99)-X-Thr(101)) in tHON is important in the reduction of binding of os teonectin to collagen V. Consistent with the binding data is the obser vation that both the N71Q and T73A mutant proteins migrate on SDS-poly acrylamide gel electrophoresis gels identically to wild-type tHON, sug gesting that there is little or no N-glycosylation of residue 71 in wi ld-type osteonectin.