Isolation of Crosslinked Peptides from Insoluble Human Leiomyoma

Two collageneous crosslinked peptides have been isolated from tryptic digests of sodium cyanoborohydride(NaBH3CN)-reduced insoluble matrix of human leiomyoma. The amino acid composition of both peptides demonstrates that the N-terminal, non-helical region of type III collagen is involved in crosslinking. The monomer (Col1) and dimer (Col1)2 N-terminal, non-helical peptides, consisting of 21 and 42 residues respectively, were found to be cross-linked to a tryptic peptide ('T9′, 9 residues) from the helical region of an adjacent type III molecule, forming the crosslinked peptides Col1 × T9 and (Col1)2× T9. The location of peptide T9 in the C-terminal, helical crosslink region [residues 922–930 of the α1(III)chain] was confirmed by the following observations. a) Its amino acid composition, which was deduced by calculation of the composition difference between the crosslinked peptide (Col1)2× T9 and the non-crosslinked peptide (Col1)2. This was consistent with the corresponding sequence of calf calf α1(III) when methionine is replaced by hydroxyproline. b) Cleavage of the crosslink of Col 1 × T9 by periodate, which released a peptide with the amino acid composition of T9. c) Sequence determination of the crosslinked peptide (Col1)2× T9; since the N-terminus of Col1 is blocked by pyroglutamic acid, sequence determination reveals the N-terminal sequence of T9: The chemical nature of the reduced crosslinks was shown to be different in the two crosslinked peptides. Col1 × T9 contains hydroxylysinonorleucine o5Lys(ω Nle) whereas (Col1)2× T9 contains a trifunctional crosslink, which is different from the trifunctional crosslinks that have been found in type I collagen.