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Volume 271, Number 25, Issue of June 21, 1996 pp. 14864-14869
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Assembly in Vitro of Thin and Thick Fibrils of Collagen II from Recombinant Procollagen II
THE MONOMERS IN THE TIPS OF THICK FIBRILS HAVE THE OPPOSITE ORIENTATION FROM MONOMERS IN THE GROWING TIPS OF COLLAGEN I FIBRILS

(Received for publication, May 4, 1995, and in revised form, February 29, 1996)

Andrzej Fertala , David F. Holmes ^¶ , Karl E. Kadler ^¶ , Aleksander L. Sieron and Darwin J. Prockop

From the  Department of Biochemistry and Molecular Biology Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the ^¶ School of Biological Sciences, Stopford Building 2.205, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom

Human type II procollagen was prepared in a recombinant system and cleaved to pC-collagen II by procollagen N-proteinase. The pC-collagen II was then used as a substrate to generate collagen II fibrils by cleavage with procollagen C-proteinase at 37 °C. Electron microscopy of the fibrils demonstrated that, at the early stages of fibril assembly, very thin fibrils were formed. As the system approached equilibrium over 7-12 h, however, the thin fibrils were largely but not completely replaced by thick fibrils that had diameters of about 240 nm and a distinct D-period banding pattern. One typical fibril was photographed and analyzed in its entirety. The fibril was 776 D-periods (52 µm) long. It had a central shaft with a uniform diameter that was about 516 D-periods long and two tips of about 100 D-periods each. Most of the central shaft had a symmetrical banding pattern flanked by two transition regions of about 30 D-periods each. Measurements by scanning transmission electron microscopy demonstrated that the mass per unit length from the tips to the shafts increased linearly over approximately 100 D-periods from the fibril end. The linear increase in mass per unit length was consistent with previous observations for collagen I fibrils and established that the tips of collagen II also had a near paraboloidal shape. However, the orientation of monomers in the tips differed from the tips of collagen I fibrils in that the C termini instead of the N termini were directed toward the tips. The thin fibrils that were present at early stages of assembly and at equilibrium were comparable to the collagen II fibrils seen in embryonic tissues and probably represented intermediates on the pathway of thick fibrils formation. The results indicated that the molecular events in the self-assembly of collagen II fibrils are apparently similar to those in self-assembly of collagen I fibrils, but that there are also important differences in the structural information contained in collagen I and collagen II monomers.

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