Self-assembly of collagen I from a proband homozygous for a mutation that substituted serine for glycine at position 661 in the alpha 2(I) chain. Possible relationship between the effects of mutations on critical concentration and the severity of the phenotype.
Procollagen I was isolated from cultured skin fibroblasts from a proband who was homozygous for a mutation in the COL1A2 gene that substituted a serine codon for a glycine codon at position 661 of the alpha 2(I) chain. The procollagen I was cleaved to pCcollagen I by procollagen N-proteinase and the pCcollagen I was used as a substrate for assay of self-assembly of collagen I into fibrils. The mutated pCcollagen I was cleaved to collagen I by procollagen C-proteinase at the same rate as control pCcollagen I. However, self-assembly of the mutated collagen I had a lag period that was 15-fold greater than the lag period observed with normal collagen I under the same conditions. Also, self-assembly of the mutated collagen I had a propagation rate of about one-fourth of the propagation rate of normal collagen I. In addition, the critical concentration for fibril assembly was slightly increased. Rotary shadowing electron microscopy of the mutated procollagen I did not reveal any increased flexibility of the triple helix as was seen previously with two mutated procollagens I in which there were substitutions of cysteine for glycine residues in the alpha 1(I) chain (Vogel, B. E., Doelz, R., Kadler, K. E., Hojima, Y., Engel, J., and Prockop, D. J. (1988) J. Biol. Chem. 263, 19249-19255; Lightfoot, S. J., Holmes, D. F., Brass, A., Grant, M. E., Byers, P. H., and Kadler, K. E. (1992) J. Biol. Chem. 267, 25521-25528). However, morphometric analysis by dark-field light microscopy and electron microscopy showed that the fibrils formed from the mutated collagen I appeared thicker in diameter than the fibrils formed from the normal collagen I. Comparison of the results with similar data on four mutated procollagens previously studied raised the possibility that mutations which markedly increase the critical concentration of fibril assembly produce more severe phenotypes than mutations which change other parameters of fibril assembly.
Romanic
A M
AM
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Spotila
L D
LD
Adachi
E
E
Engel
J
J
Hojima
Y
Y
Prockop
D J
DJ
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005998:Glycine; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D010641:Phenotype; D017354:Point Mutation; D011347:Procollagen / Q000235:genetics* / Q000648:ultrastructure; D011348:Procollagen N-Endopeptidase / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012694:Serine; D012867:Skin / Q000378:metabolism*
D046911:Macromolecular Substances; D011347:Procollagen; D012694:Serine; D003094:Collagen; D011348:Procollagen N-Endopeptidase; D005998:Glycine