BONE-MATRIX PROTEINS - ISOLATION AND CHARACTERIZATION OF A NOVEL CELL-BINDING KERATAN SULFATE PROTEOGLYCAN (OSTEOADHERIN) FROM BOVINE BONE

A small cell-binding proteoglycan for which we propose the name osteoa dherin was extracted from bovine bone with guanidine hydrochloride-con taining EDTA. It was purified to homogeneity using a combination of io n-exchange chromatography, hydroxyapatite chromatography, and gel filt ration. The M-r of the proteoglycan was 85,000 as determined by SDS-PA GE. The protein is rich in aspartic acid, glutamic acid, and leucine. Two internal octapeptides from the proteoglycan contained the sequence s Glu-Ile-Asn-Leu-Ser-His-Asn-Lys and Arg-Asp-Leu-Tyr-Phe-Asn-Lys-Ile. These sequences are not previously described, and support the notion that osteoadherin belongs to the family of leucine-rich repeat protein s. A monospecific antiserum was raised in rabbits. An enzyme-linked im munosorbent assay was developed, and showed the osteoadherin content o f bone extracts to be 0.4 mg/g of tissue wet weight, whereas none was found in extracts of various other bovine tissues. Metabolic labeling of primary bovine osteoblasts followed by immunoprecipitation showed t he cells to synthesize and secrete the proteoglycan. Digesting the imm unoprecipitated osteoadherin with N-glycosidase reduced its apparent s ize to 47 kD, thus showing the presence of several N-linked oligosacch arides. Digestion with keratanase indicated some of the oligosaccharid es to be extended to keratan sulfate chains. In immunohistochemical st udies of the bovine fetal rib growth plate, osteoadherin was exclusive ly identified in the primary bone spongiosa. Osteoadherin binds to hyd roxyapatite. A potential function of this proteoglycan is to bind cell s, since we showed it to be as efficient as fibronectin in promoting o steoblast attachment in vitro. The binding appears to be mediated by t he integrin alpha(v) beta(3), since this was the only integrin isolate d by osteoadherin affinity chromatography of surface-iodinated osteobl ast extracts.