Abstract
A recently developed recombinant system for synthesis of human procollagen II by stably transfected host cells was used to prepare adequate amounts of protein to study the self-assembly of collagen II into fibrils. The procollagen II was cleaved to pCcollagen II by procollagen N-proteinase (EC 3.4.24.14), the pCcollagen II was chromatographically purified, and the pCcollagen II was then used as a substrate to generate collagen II fibrils by cleavage with procollagen C-proteinase. The kinetics for assembly of collagen II fibrils were similar to those observed previously for the self-assembly of collagen I in that a distinct lag phase was observed followed by a sigmoidal propagation phase. However, under the same experimental conditions, the lag time for assembly 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 fibrils. The relatively long lag time for the assembly of collagen II into fibrils made it possible to demonstrate that most of the conversion of pCcollagen II to collagen II occurred in the solution phase. The critical concentration at 37 degrees C for collagen II was about 50-fold greater than the critical concentration for collagen I. The Gibbs free 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 change for collagen I and about the same as the free energy change for the 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 demonstrated, therefore, that the structural information of the monomer is sufficient to explain the characteristically small diameters and arcade-like geometry of collagen II fibrils found in cartilage and other tissues.
Highlights
From the Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
The kinetics for assembly of collagen I1 fibrils were similar to those observed previouslyfor the self-assemblyof collagen I in that a distinct lag phase was observed followed by a sigmoidal propagation phase
The results demonstrated, that the structural information of the monomer is sufficient to explain the characteristically small diameters and arcade-like geometry of collagen I1 fibrils found in cartilage and other collagen I in the lengthof the critical concentration ogy of the fibrils
Summary
Conditions excepthat themedium usedfor the third24-h period did not Cleavage of Recombinant Procollagen II with Procollagen N - contain I4C-labeled amino acids. 14C-Labeledprocollagen I by separation of the protein by polyacrylamide gel electrophoresis and staining of the gels with Coomassie Blue (30) It had a normal structure as assayed by dideoxynucleotide sequencing of the mRNA-derived cDNA (46). Of Collagen ZZ into Fibrils-To examine fibril assembly of collagen 11,pccollagen I1 was incubated with procollagen C-proteinase (45) ina reaction that contained 150 pg/ml substrate and 37 or 75 unitdm[1] enzyme. To initiate fibril rated by centrifugation, and the supernatant and pellet fracformation, pCcollagen and C-proteinase were mixed a t 4 "C in a 250-pl tions were analyzed by polyacrylamide gel electrophoresis in polypropylene tube ina total tion of 150 pg/ml pccollagen volume of 20 pl and either 37 to or g7i5veuanfiitndaml lcoCn-cpernottreai-naseS.DS (Fig. The tube was capped and placed in a water bath at the temperatures collagen I1 was completely cleaved to collagen I1 by 4 h (Fig. 2).
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