Covalent Crosslinking between Molecules of Type I and Type III Collagen

Abstract

Several peptides containing intermolecular crosslinks were isolated from tryptic digests of the insoluble matrix of human leiomyoma and calf aorta. From leiomyoma type 111 collagen, two crosslinked peptides were isolated. The peptide [T,(III)], is a dimer which contains the 73 amino-terminal residues of two, zero-D staggered, crosslinked aI(II1) chains (D being the distance by which molecules or chains are staggered relative to each other). After deblocking with pyroglutamate aminopeptidase, its amino-terminal amino acid sequence was determined to be < Glu-Tyr-Asp-Ser-Tyr-Asp-Val-Xaa-Ser-Gly-Val-Ala-Val-Gly-Gly-Leu-Ala-Gly-Tyr-Hyp-Gly-. The involvement of [TN(II1)l2 in the formation of 4D-staggered intermolecular bonds was demonstrated by sequence analysis of [T,(ITI)], x T(II1). For this three-chained peptide, a double sequence which corresponded to T,(III) and T(III), a nine-residue peptide from the carboxy-terminal helical crosslinking site of type I11 collagen, was found.Crosslinks between type I and type I11 collagens were identified by isolating a peptide T,(III) x T,(I) from leiomyoma. This peptide was characterized, first by digesting it with collagenase giving rise to Col,(III) x Col,(I) and then chymotrypsin giving C(II1) and C(1). C(1) and C(II1) are peptides characteristic for type I and type 111 collagens, respectively. Further, three-component crosslink-containing peptides, from both type I and type I11 collagen, were isolated from aorta and leiomyoma. The peptides Col,(III) x Col,(I) x T(II1) from leiomyoma and Col,(III) x Col,(I) x T(1) from aorta are crosslinked amino-terminal regions of OD-staggered type I and type I11 molecules, joined either to an adjacent 4D-staggered type 111 or type I molecule. Crosslinked peptides involving only type I11 collagen molecules were also isolated from calf aorta [colN(III)]2 and [CoI,(III)], x T(III)].As intramolecular bonds between type 1 and 111 collagens can be excluded the isolation of these crosslinked peptides demonstrated that type I and type I11 collagen molecules are present within the same collagen fibril with OD stagger, bound by intermolecular crosslinks. The significance of these findings with respect to the proposed models for collagen fibres and the physiological properties of the extracellular matrix, are discussed.Covalent crosslinking of collagen molecules within a fibre, which is the final step in the formation of a collagen matrix, has been described for type I, type IT and type I11 collagens (for recent review see [l]). Structural investigations on these mol- Nomenclature of' Crosslinked Peptides. Due to the absence of a systematic nomenclature for crosslinked peptides derived from insoluble collagens, the following system was developed. The type of collagen is denoted by roman numerals in brackets. The enzyme cleavage which produced the peptides is denoted by Col, T or C for bacterial collagenase, trypsin or chymotrypsin, respectively. The subscript N indicates that the peptide contains the amino-terminal nonhelical region of the collagen...

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