Site-directed mutagenesis of type I collagen: effect on susceptibility to collagenase.
A genetic approach to define the role of collagenase in physiological and pathological bone remodeling is to identify spontaneous mutations in the collagenase gene which alter enzymatic activity. Alternatively it is possible, though site-directed mutagenesis, to alter genes encoding critical amino acid sequences in the collagen substrate, in a manner analogous to the successful development of animal models for osteogenesis imperfecta. We have thus utilized this approach to alter the Col1a1 gene to encode amino acid substitutions in sequences around the known collagenase cleavage site (glycine-isoleucine at positions 775-776) in type I collagen, and transfect these genes into homozygous Mov-13 fibroblasts, in which the endogenous Col1a1 gene is inactive. Nonconservative substitutions of proline for isoleucine at the P1' site and double substitutions of proline for glutamine (P2) and alanine (P2') resulted in type I collagen resistant to hydrolysis by collagenase. Furthermore, in normal fibroblasts transfected with a mutant Col1a1 gene encoding collagenase resistance in which an additional methionine substitution at position 776 provided a marker for the mutant protein, mutant and wild type triple helical molecules were synthesized and secreted as heterotrimers. A single mutant alpha 1(I) chain did not prevent cleavage of the wild type alpha 1(I) chain but it is likely that the uncleaved alpha 1(I) chain would prevent dissociation of the triple helical fragments containing the other cleaved chains. Introduction of these genes into transgenic mice should result in abnormal phenotypes characterized by altered connective tissue remodeling.
Krane
S M
SM
Department of Medicine, Harvard Medical School, Massachusetts General Hospital, Boston 02114.
Jaenisch
R
R
D000595:Amino Acid Sequence; D000818:Animals; D003094:Collagen / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D017364:Collagenases / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010013:Osteogenesis Imperfecta / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity
C000716128:Col1a1 protein, mouse; D000090602:Collagen Type I, alpha 1 Chain; D003094:Collagen; D017364:Collagenases