Role of the pro-alpha2(I) COOH-terminal region in assembly of type I collagen: disruption of two intramolecular disulfide bonds in pro-alpha2(I) blocks assembly of type I collagen.
Collagen biosynthesis is a complex process that begins with the association of three procollagen chains. A series of conserved intra- and interchain disulfide bonds in the carboxyl-terminal region of the procollagen chains, or C-propeptide, has been hypothesized to play an important role in the nucleation and alignment of the chains. We tested this hypothesis by analyzing the ability of normal and cysteine-mutated pro-alpha2(I) chains to assemble into type I collagen heterotrimers when expressed in a cell line (D2) that produces only endogenous pro-alpha1(1). Pro-alpha2(I) chains containing single or double cysteine mutations that disrupted individual intra- or interchain disulfide bonds were able to form pepsin resistant type I collagen with pro-alpha1(I), indicating that individual disulfide bonds were not critical for assembly of the pro-alpha2(I) chain with pro-alpha1(I). Pro-alpha2(I) chains containing a triple cysteine mutation that disrupted both intrachain disulfide bonds were not able to form pepsin resistant type I collagen with pro-alpha1(I). Therefore, disruption of both pro-alpha2(I) intrachain disulfide bonds prevented the production and secretion of type I collagen heterotrimers. Although none of the individual disulfide bonds is essential for assembly of the procollagen chains, the presence of at least one intrachain disulfide bond may be necessary as a structural requirement for chain association or to stabilize the protein to prevent intracellular degradation.
Doyle
S A
SA
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118, USA.
Smith
B D
BD
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003094:Collagen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D017931:DNA Primers; D004220:Disulfides; D011498:Protein Precursors; D003094:Collagen; D003545:Cysteine; D000409:Alanine