INTERACTION OF PROCOLLAGEN-I AND OTHER COLLAGENS WITH COLLIGIN

Colligin is a collagen-binding glycoprotein of molecular mass 46000 Da localized to the endoplasmic reticulum (ER) of diverse kinds of cells that produce collagen I. In order to help define its role in collagen biosynthesis and to study the interaction of colligin with procollage n I in detail, the binding characteristics of colligin purified from L 6 myoblasts have been studied. A total of 3 mol were found to bind/mol of procollagen I, with a K-d of about 25 nM. Both pure and separated pro alpha(1)(I) and procollagen alpha(2)(I) chains were able to compet e with procollagen I for binding to colligin. However, colligin binds to pro alpha(2)(I) with higher affinity than to pro alpha(1)(I). To fi nd if the binding activity of colligin was altered during purification , an assay to measure colligin binding to procollagen in crude myoblas t cell extracts was developed. This procedure gave the same binding pa rameters as did the highly purified colligin. Among different collagen types, colligin was found to bind to collagen I and collagen IV, but not to collagen III. In order to examine whether glycosylation or phos phorylation of colligin were required for the binding of colligin to p rocollagen I and to obtain enough colligin for further studies, recomb inant protein was produced in Escherichia coli. An immunoaffinity puri fication scheme was used to get virtually pure protein in milligram yi elds. Comparison of the recombinant colligin with that isolated from L 6 myoblasts showed that both types existed in solution as monomers and dimers. In addition, both types of colligins showed identical propert ies with regard to their binding to procollagen I and the isolated pro alpha(1)(I) and pro alpha(2)(I) chains. Post-translational modificati ons of colligin were thus not essential for binding to procollagen I.