Procollagen polypeptides containing cis-4-hydroxy- l-proline are overglycosylated and secreted as nonhelical pro-γ-chains

Abstract

Incorporation of a proline analog into collagen polypeptides was studied by incubating matrix-free tendon cells from 17-day-old chick embryos with cis-4-hydroxy- l-proline. Velocity sedimentation of intracellular polypeptides provided further evidence that incorporation of the analog into protein prevented the pro-α- and pro-γ-chains of procollagen from folding into a stable triple-helical conformation. The size of the newly synthesized intracellular and extracellular protein was examined under conditions which prevented proteolysis during processing of the samples. In contrast to previous observations, the results demonstrated that there was little if any intracellular degradation of nonhelical pro-α-and pro-γ-chains containing the proline analog, and a fraction of the nonhelical pro-γ-chains was secreted into the medium without extensive degradation. In further studies, the cells were incubated with 14C lysine, and the synthesis of glycosylated hydroxylysyl residues was measured in control cells and in cells incubated with cis-4-hydroxy- l-proline. The results demonstrated that the content of glycosylated hydroxylysyl residues in nonhelical pro-γ-chains containing cis-4-hydroxy- l-proline was increased twofold as compared to the triple-helical procollagen in control cells. The results suggested that under control conditions folding into the triplehelical conformation limits the extent of glycosylation of collagen. If folding is prevented or delayed, procollagen polypeptides are more extensively glycosylated.