IDENTIFICATION OF PEPTIDE-FRAGMENTS GENERATED BY DIGESTION OF BOVINE AND HUMAN OSTEOCALCIN WITH THE LYSOSOMAL PROTEINASES CATHEPSIN-B, CATHEPSIN-D, CATHEPSIN-L, CATHEPSIN-H, AND CATHEPSIN-S

We have determined the primary cleavage sites in the bone Gla protein (BGP; osteocalcin) for several of the proteases that could act on the protein during bone resorption and turnover, cathepsins B, D, L, H, an d S, The time course of BGP digestion by each cathepsin was first dete rmined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, W e then incubated human and bovine BGP with each cathepsin for a suffic ient time to reduce the level of intact protein by at least 20-fold, i solated the major cleavage peptides, and identified each by N-terminal sequence analysis and by amino acid analysis, Our results show that B GP has relatively few cathepsin-sensitive sites and that these sites a re located at the N and C terminus of the 49-residue protein, Cathepsi ns B, L, H, and S readily cleave BGP at the G(7)-A(8) bond; cathepsin L also cleaves at R(43)-R(44); cathepsin B also cleaves at R(44)-F-45; and cathepsin D cleaves only at A(41)-Y-42. The immunoreactivity of t he major peptides generated by cathepsin cleavage was evaluated using the original radioimmunoassay developed for the detection of BGP in hu man serum, The BGP 8-49 fragment cross-reacts identically with native BGP, while the 8-43 and the 1-44 fragments require 20- to 40-fold high er concentrations to achieve the same level of displacement as the nat ive protein, The 1-41 and 8-41 fragments are unable to significantly d isplace the labeled native BGP tracer at any concentration tested, The se results demonstrate the utility of peptides generated by cathepsin digestion in the mapping of the antigenic epitopes recognized by a giv en BGP immunoassay.