Anomalous cysteine in type I collagen. Localisation by chemical cleavage of the protein using 2-nitro-5-thiocyanobenzoic acid and by mismatch analysis of cDNA heteroduplexes.
A method is presented for the localisation of an anomalous cysteine inside the triple helical domain of type I collagen from a patient affected with Osteogenesis Imperfecta. The chemical cleavage used relies on the specificity and reactivity of the thiol side chain versus 2-nitro-5-thiocyanobenzoic acid, to yield cyanocysteine; in mild alkaline conditions this derivative will undergo the breakdown of its N-side peptide bond. This method could allow a more precise localisation of anomalous cysteine in both type I collagen alpha chains, alpha 1(I) and alpha 2(I), compared to previous analytical methods on CNBr peptides. For the mutant alpha 1(I) chains from a patient affected by Osteogenesis Imperfecta, we found a location of cysteine in the peptide alpha 1(I)CB8, between amino acids 170-200. Biochemical localisation was confirmed by a chemical cleavage method for mismatched cytosines on heteroduplexes obtained after denaturation and annealing of a 233 bp cDNA fragment amplified by PCR from the heterozygote patient.
Tenni
R
R
Dipartimento di Biochimica, University of Pavia, Italy.
Rossi
A
A
Valli
M
M
Mottes
M
M
Pignatti
P F
PF
Cetta
G
G
D001483:Base Sequence; D003094:Collagen / Q000032:analysis* / Q000235:genetics; D003545:Cysteine / Q000032:analysis*; D004247:DNA; D005347:Fibroblasts; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010013:Osteogenesis Imperfecta / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012867:Skin / Q000032:analysis*; D013861:Thiocyanates
D013861:Thiocyanates; D003094:Collagen; D004247:DNA; D003545:Cysteine; C008658:2-nitro-5-thiocyanobenzoic acid