ROLE OF THE PRO-ALPHA-2(I) COOH-TERMINAL REGION IN ASSEMBLY OF TYPE-ICOLLAGEN - DISRUPTION OF 2 INTRAMOLECULAR DISULFIDE BONDS IN PRO-ALPHA-2(I) BLOCKS ASSEMBLY OF TYPE-I COLLAGEN

Collagen biosynthesis is a complex process that begins with the associ ation of three procollagen chains. A series of conserved intra- and in terchain disulfide bonds in the carboxyl-terminal region of the procol lagen chains, or C-propeptide, has been hypothesized to play an import ant role in the nucleation and alignment of the chains. We tested this hypothesis by analyzing the ability of normal and cysteine-mutated pr o-alpha 2(I) chains to assemble into type I collagen heterotrimers whe n expressed in a cell line (D2) that produces only endogenous pro-alph a 1(I). Pro-alpha 2(I) chains containing single or double cysteine mut ations that disrupted individual intra- or interchain disulfide bonds were able to form pepsin resistant type I collagen with pro-alpha 1(I) , indicating that individual disulfide bonds were not critical for ass embly of the pro-alpha 2(I) chain with pro-alpha 1(I). Pro-alpha 2(I) chains containing a triple cysteine mutation that disrupted both intra chain disulfide bonds were not able to form pepsin resistant type I co llagen with pro-alpha 1(I). Therefore, disruption of both pro-alpha 2( I) intrachain disulfide bonds prevented the production and secretion o f type I collagen heterotrimers. Although none of the individual disul fide bonds is essential for assembly of the procollagen chains, the pr esence of at least one intrachain disulfide bond may be necessary as a structural requirement for chain association or to stabilize the prot ein to prevent intracellular degradation. J.Cell. Biochem. 71 :233-242 , 1998. (C) 1998 Wiley-Liss, Inc.