Role Of Colligin In Collagen Biosynthesis

Colligin, a glycoprotein of molecular mass 46 kDa, is localized in the endoplasmic reticulum of diverse kinds of cells which produce collagen I. It binds collagen type I and type IV. As a step towards defining the role of colligin in the cell, the binding characteristics were studied in detail. Two different binding assays were developed. These studies showed that three moles of colligin bind per mole of procollagen with a K{dollar}\sb{lcub}\rm d{rcub}{dollar} of about 25 nM. In vivo cross-linking experiments revealed that colligin also binds to procollagen in the ER along with two other ER resident proteins, PDI and GRP78.;In order to understand the role of colligin in differentiation, the rate of procollagen biosynthesis in L6 myoblasts was compared with that of differentiation-defective C8 mutant cells, where the level of colligin is lower. Pulse-chase experiments showed that the rate of degradation of pro{dollar}\alpha\sb2{dollar}(I) chains is higher in C8. Further, the lower rate of hydroxylation and secretion of procollagen I and its less stable triple-helical structure in C8 suggests that colligin probably plays an important role in procollagen biosynthesis.;A considerable amount of evidence suggests that the ER has specific proteases. To specifically examine procollagen degradation, pulse labelled procollagen was quantitated in cells treated with brefeldin A. Procollagen was degraded by ER resident proteases under these conditions. The in vivo degradation of procollagen was inhibited by TLCK and TPCK; inhibitors of serine proteases. It was also found that procollagen is degraded in vitro in microsomal preparations and colligin protected it from this degradation. Another possible function of colligin may thus be to protect procollagen chains from degradation in the ER.;A regulatory linkage between the biosynthesis of colligin and procollagen was examined under a variety of conditions, notably in the presence of steroid hormones and growth factors. These studies establish that colligin and procollagen are regulated in parallel, suggesting an important functional linkage.;In efforts to understand a possible role of colligin in procollagen secretion, a line of lung fibroblasts having defective procollagen chains and negligible levels of colligin were transfected with colligin cDNA. These studies showed that colligin helps in the solubilization of defective pro {dollar}\alpha\sb1{dollar}(I) chain aggregates in the ER and also prevents secretion of these mutant chains.;To examine whether glycosylation and phosphorylation of colligin are required for binding, and to obtain enough colligin for further studies, a method for overproducing colligin in E. coli was developed. Recombinant colligin with molecular mass of 41 kDa was purified almost to homogeneity from the bacterial system. Comparison of the recombinant colligin with that isolated from myoblasts showed that the post-translational modifications of colligin are not essential for binding. Both types of colligin showed identical properties with regard to their binding properties to procollagen and their ability to protect procollagen from microsomal degradation.;The work presented in this report showed that colligin is a multifunctional protein involved in collagen biosynthesis in the ER. It is very likely that it acts as a \u27specific chaperone\u27 in order to protect unassembled procollagen chains from degradation and also help in the oligomerization and secretion of these chains