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 procollagen I in detail, the binding characteristics of colligin purified from L6 myoblasts have been studied. A total of 3 mol were found to bind/mol of procollagen I, with a Kd of about 25 nM. Both pure and separated pro alpha 1(I) and procollagen alpha 2 (I) chains were able to compete 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 find if the binding activity of colligin was altered during purification, an assay to measure colligin binding to procollagen in crude myoblast cell extracts was developed. This procedure gave the same binding parameters 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 phosphorylation of colligin were required for the binding of colligin to procollagen I and to obtain enough colligin for further studies, recombinant protein was produced in Escherichia coli. An immunoaffinity purification scheme was used to get virtually pure protein in milligram yields. Comparison of the recombinant colligin with that isolated from L6 myoblasts showed that both types existed in solution as monomers and dimers. In addition, both types of colligins showed identical properties with regard to their binding to procollagen I and the isolated pro alpha 1(I) and pro alpha 2(I) chains. Post-translational modifications of colligin were thus not essential for binding to procollagen I.
Jain
N
N
Department of Biochemistry, University of Western Ontario, London, Canada.
Brickenden
A
A
Lorimer
I
I
Ball
E H
EH
Sanwal
B D
BD
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D003094:Collagen / Q000378:metabolism*; D015342:DNA Probes; D004926:Escherichia coli; D006023:Glycoproteins; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D011347:Procollagen / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D002352:Carrier Proteins; D015342:DNA Probes; D006023:Glycoproteins; D011347:Procollagen; D011994:Recombinant Proteins; C046289:colligin; D003094:Collagen