G76E substitution in type I collagen is the first nonlethal glutamic acid substitution in the alpha1(I) chain and alters folding of the N-terminal end of the helix.
The majority of osteogenesis imperfecta (OI) is caused by substitutions for glycine residues in the two alpha chains of type I collagen. Since only 4% of possible nucleotide changes in type I collagen glycine codons would result in a glutamic acid substitution, these are predicted to be infrequent. Only one glutamic acid substitution in type I collagen has been fully reported. We describe here the clinical, biochemical, and molecular characterization of a girl with severe type III OI caused by a G76E substitution in COL1A1. This is the first delineation of a glutamic acid substitution in the alpha1(I) chain causing nonlethal osteogenesis imperfecta. The proband's fibroblast type I collagen chains and cyanogen bromide peptides were electrophoretically normal, while osteoblast collagen was slightly overmodified. This suggested a mutation near the N-terminal end of the collagen helix. A mismatch was detected by RNA:DNA hybrid analysis in cDNA coding for 106 amino acids at the N-terminal end of the helical region. Subclones of both alleles were sequenced and revealed a G --> A (c.761G > A) mutation causing an alpha1(I) G76E substitution in one allele. The presence of the mutation in the proband's leukocyte gDNA, and its absence in parental gDNA, was confirmed by Tsp509I digestion. The glutamic acid substitution alters the folding of the mutant collagen helices. Pericellular processing of type I collagen by the proband's fibroblasts yielded an earlier appearance of the pC-alpha1(I) form and of mature alpha chains as compared to control cell processing. Also, the presence of the glutamic acid substitution apparently exposes the adjacent Arg75 residue in the alpha1 chain. Trypsin digestion of proband fibroblast collagen resulted in shortened alpha1 chains, as confirmed by CNBr analysis. In addition, the Tm for mutant helices from fibroblasts and osteoblasts was decreased 2-4 degrees C versus controls, demonstrating a decrease in helix stability. These findings increase our understanding of the disruptive effect of glutamic acid substitutions in collagen.
Cabral
W A
WA
Section on Connective Tissue Disorders, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
Chernoff
E J
EJ
Marini
J C
JC
D000293:Adolescent; D019943:Amino Acid Substitution; D001842:Bone and Bones / Q000000981:diagnostic imaging; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D018698:Glutamic Acid / Q000235:genetics* / Q000502:physiology; D005998:Glycine / Q000235:genetics* / Q000502:physiology; D006358:Hot Temperature; D006801:Humans; D010006:Osteoblasts / Q000201:enzymology; D010013:Osteogenesis Imperfecta / Q000235:genetics*; D010434:Pepsin A / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000502:physiology*; D012313:RNA / Q000032:analysis; D011859:Radiography; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D018698:Glutamic Acid; D012313:RNA; D003094:Collagen; D010434:Pepsin A; D005998:Glycine