Date of Award
Doctor of Philosophy
A 46 kDa glycoprotein (which had previously been implicated in myoblast differentiation) has been purified to homogeneity from L6 rat skeletal myoblasts. The sequence of 30 N-terminal amino acids has some resemblance to a sequence in myosin light-chain. The oligosaccharide chains of the glycoprotein can be released by treatment with glycosidase H suggesting that gp46 has a high mannose type of glycans. Gp46 is widely distributed and conserved in different cell lines as determined by immunoblotting using a monoclonal anti-gp46 antibody. High levels of gp46 were found in fibroblastic and myogenic, but not in haematopoietic cell lines. A survey of the distribution of gp46 shows that it is present in those cell lines which attach to the substratum and produce collagens.;The results of immunofluorescence show that by far the majority of gp46 is associated with endoplasmic reticulum. Protease treatment of a microsomal fraction revealed that the protein is in the interior of the reticulum.;The characterization of the binding properties of gp46 with the components of extracellular matrix revealed gp46 as a major gelatin/collagen binding protein. The binding of gp46 to collagen and gelatin is disrupted by Arg-Gly-Asp (RGD) containing peptide but not by Arg-Gly-Glu (RGE). These findings suggest that RGD sequence is also used in protein recognition within the cell.;To study the role of procollagen synthesis in the differentiation of myoblasts, a specific inhibitor of collagen synthesis, ethyl-3,4-dihydroxybenzoate (DHB) was utilized. It is shown that DHB reversibly inhibits both morphological and biochemical differentiation of myoblasts, if it is added to the culture medium prior to the cell alignment stage. DHB also decreases markedly the production of gp46.;While investigating the role of oligosaccharide side chains of gp46 in gelatin binding, a 78 kDa gelatin binding protein was discovered which was found to be identical to the glucose regulated protein (grp78). On the basis of selective release of grp78 by ATP, a single step purification of grp78 from rat liver microsomes was developed.;In order to get some clues regarding the function of gp46, wild-type L6, as well as myoblast mutants (C8) were examined for gp46 and collagen biosynthesis. The mutant C8 produces only 30% of the gp46 as compared to wild type. There is a possibility that this mutant secretes significant levels of the homotrimeric form of procollagens instead of the normal heterotrimeric form. These observations are suggestive of a role of gp46 in the assembly of procollagen in the endoplasmic reticulum.
Nandan, Devki, "Characterization And Possible Functions Of A Collagen-binding Protein From Skeletal Myoblasts" (1990). Digitized Theses. 1895.