ASSEMBLY IN-VITRO OF THIN AND THICK FIBRILS OF COLLAGEN-II FROM RECOMBINANT PROCOLLAGEN-II - THE MONOMERS IN THE TIPS OF THICK FIBRILS HAVETHE OPPOSITE ORIENTATION FROM MONOMERS IN THE GROWING TIPS OF COLLAGEN-I FIBRILS

Human type II procollagen was prepared in a recombinant system and cle aved to pC-collagen II by procollagen N-proteinase. The pC-collagen II was then used as a substrate to generate collagen II fibrils by cleav age with procollagen C-proteinase at 37 degrees C. Electron microscopy of the fibrils demonstrated that, at the early stages of fibril assem bly, very thin fibrils were formed, As the system approached equilibri um over 7-12 h, however, the thin fibrils were largely but not complet ely replaced by thick fibrils that had diameters of about 240 nm and a distinct D-period banding pattern, One typical fibril was photographe d and analyzed in its entirety, The fibril was 776 D-periods (52 mu m) long, It had a central shaft with a uniform diameter that was about 5 16 D-periods long and two tips of about 100 D-periods each, Most of th e central shaft had a symmetrical banding pattern flanked by two trans ition regions of about 30 D-periods each, Measurements by scanning tra nsmission electron microscopy demonstrated that the mass per unit leng th from the tips to the shafts increased linearly over approximately 1 00 D-periods from the fibril end, The linear increase in mass per unit length was consistent with previous observations for collagen I fibri ls and established that the tips of collagen II also had a near parabo loidal shape, However, the orientation of monomers in the tips differe d 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 we re present at early stages of assembly and at equilibrium were compara ble to the collagen II fibrils seen in embryonic tissues and probably represented intermediates on the pathway of thick fibrils formation, T he 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 i n the structural information contained in collagen I and collagen II m onomers.