SELF-ASSEMBLY INTO FIBRILS OF COLLAGEN-II BY ENZYMATIC CLEAVAGE OF RECOMBINANT PROCOLLAGEN-II - LAG PERIOD, CRITICAL CONCENTRATION, AND MORPHOLOGY OF FIBRILS DIFFER FROM COLLAGEN-I

A recently developed recombinant system for synthesis of human procoll agen II by stably transfected host cells was used to prepare adequate amounts of protein to study the self-assembly of collagen II into fibr ils. The procollagen II was cleaved to pCcollagen II by procollagen N- proteinase (EC 3.4.24.14), the pCcollagen II was chromatographically p urified, and the pCcollagen Il was then used as a substrate to generat e collagen II fibrils by cleavage with procollagen C-proteinase. The k inetics for assembly of collagen II fibrils were similar to those obse rved previously for the self-assembly of collagen I in that a distinct lag phase was observed followed by a sigmoidal propagation phase. How ever, under the same experimental conditions, the lag time for assembl y of collagen II fibrils was 5-6-fold longer, and the propagation rate for collagen II fibrils was about 30-fold lower than for collagen I f ibrils. The relatively long lag time for the assembly of collagen II i nto fibrils made it possible to demonstrate that most of the conversio n of pCcollagen II to collagen II occurred in the solution phase. The critical concentration at 37-degrees-C for collagen II was about 50-fo ld greater than the critical concentration for collagen I. The Gibbs f ree energy change for the assembly of collagen II into fibrils was -40 kJ/mol, a value that was about 14 kJ/mol less than the free energy ch ange for collagen I and about the same as the free energy change for t he homotrimer of collagen I. Dark-field light microscopy and negative- staining electron microscopy demonstrated that the collagen II fibrils were thin and formed network-like structures. The results demonstrate d, therefore, that the structural information of the monomer is suffic ient to explain the characteristically small diameters and arcade-like geometry of collagen II fibrils found in cartilage and other tissues.