Formation of collagen fibrils by enzymic cleavage of precursors of type I collagen in vitro

Abstract

Two systems were used to generate collagen fibrils in vitro by enzymic cleavage of intermediates in the conversion of procollagen to collagen. In one system fibrils were generated by using procollagen NH2-terminal proteinase to cleave pNcollagen, the intermediate which contains the NH2-terminal but not the COOH-terminal propeptides found in procollagen. When pNcollagen was incubated with procollagen NH2-terminal proteinase, the NH2-terminal propeptides were enzymically cleaved from the protein, and there was an increase in the turbidity of the solution over and above the turbidity observed with pNcollagen alone. Electron microscope examination of the samples demonstrated that the increase in turbidity was associated with the assembly of collagen fibrils. The fibrils had a mean diameter of 104 nm +/- 51.7 S.D. or about the same as fibrils formed from pNcollagen alone. However, the fibrils formed by enzymic cleavage of pNcollagen had a more distinct gap-overlap pattern and they appeared to be more tightly packed than fibrils of pNcollagen. Varying the concentration of enzyme varied both the rate of enzymic cleavage of the pNcollagen and the rate of fibril assembly, but there was no consistent effect on the diameter or morphology of the fibrils. In the second system, fibrils were generated with a recently described procedure (Miyahara, M., Njieha, F. K., and Prockop, D. J. (1982) J. Biol. Chem. 257, 8442-8448) in which procollagen COOH-terminal proteinase is used to cleave pCcollagen, the intermediate containing the COOH-terminal but not the NH2-terminal propeptides found in procollagen. When incubated with procollagen COOH-terminal proteinase, the COOH-terminal propeptides were cleaved and collagen fibrils assembled. The collagen fibrils were unusually thick with a mean diameter of 1184 nm +/- 291 S.D. The large diameters of the fibrils made it possible to demonstrate by scanning electron microscopy that each fibril was comprised of a bundle of subfibrils packed into a right-handed helix. The fibrils frequently had branch points which appeared to consist of subfibrils which separated from the main axis of the structure. Also, the surface of the fibrils was scalloped at 270- to 300-nm intervals, suggesting that some of the collagen molecules on the surface were in a 4D staggered array. The results suggested the hypothesis that the order in which the NH2-terminal and COOH-terminal propeptides are cleaved in the conversion of procollagen to collagen may provide a mechanism for controlling the diameter, or both the diameter and morphology, of collagen fibrils.