Sequential cleavage of type I procollagen by procollagen N-proteinase. An intermediate containing an uncleaved Pro.alpha.1(I) chain

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Abstract
The conversion of type I procollagen to type I collagen was studied by cleaving the protein with partially purified type I procollagen N-proteinase from chick embryos. Examination of the reaction products after incubation for varying times at 30.degree. C indicated that, during the initial stages of the reaction, pro.alpha.1(I) and pro.alpha.2(I) chains were cleaved at about the same rate. As a result, all the pro.alpha.2(I) chains were converted to pC.alpha.2(I) chains well before all the pro.alpha.1 chains were cleaved. When the reaction products were examined by gel electrophoresis without reduction of interchain disulfide bonds, a distinct band of an intermediate was detected. The same intermediate was seen when the reaction was carried out at 35, 37 and 40.degree. C. Over 2/3 of the type I procollagen was converted to the intermediate and that this intermediate was then slowly converted to the final product of pCcollagen. The kinetics for the reaction did not fit a simple model for precursor-product relationship among substrate, intermediate and product. Examination of the reaction products with a 2-step gel procedure demonstrated that the intermediate consisted of 3 polypeptide chains in which the N propeptide was cleaved from one pro.alpha.1 chain and one pro.alpha.2(I) chain but the N propeptide was still present on one of the pro.alpha.1(I) chains. In further experiments it was demonstrated that a similar intermediate was seen when a homotrimer of pro.alpha.1(I) chains was partially cleaved by the enzyme. No intermediate was detected when the enzyme was used to cleave type II procollagen, a homotrimer of pro.alpha.1(II) chains. If the partially cleaved intermediate of type I procollagen is generated during fibril assembly in vivo, it may help to determine the structure of the collagen fibrils found in tissues.