hydroxylysine and Pulmonary-Fibrosis

Rats were intratracheally instilled with bleomycin or with silica (quartz) dust to induce lung fibrosis. Several weeks later, purified collagen chains (or collagen digests) were isolated from the lungs of these animals and from age-matched controls instilled intratracheally with saline solution, and the ratios of hydroxylysine to lysine and of the dysfunctional cross-links DHLNL to HLNL were quantified. Collagen from fibrotic lungs had significantly higher ratios of DHLNL:HLNL than did control lungs, 15.5 +/- 4.8 and 17.1 +/- 4.8 vs. 2.3 +/- 0.5 for the silica-instilled and the bleomycin-instilled animals, respectively. The hydroxylysine:lysine ratio was significantly increased for the alpha 1(I) chain, to a value 170% of that of lung collagen from control animals, and for several of its constituent CNBr peptides. Lung tissue was exhaustively digested with collagenase and specific cross-linked peptides were isolated and characterized. The cross-linked alpha 1(I) x alpha 1(I) peptide linked by the residues 87 x 16C, with a ratio of DHLNL:HLNL of 17:1, demonstrated that the increased hydroxylation of the dysfunctional cross-links in fibrotic lung collagen could be accounted for in part by increased hydroxylation of the lysine residue at position 16C of the C-terminal telopeptide of the collagen alpha 1(I) chain. It proved impossible to locate the corresponding N-terminal cross-linked fragment from alpha 1(I) x alpha 1(I) chains, 9N x 930, possibly due to further reactions of this material to form the material referred to as poly(CB6). Isolated poly (CB6) accounted for more than half of the total alpha 1(I)CB6 peptide expected in lung collagen, and had a hydroxylysine:lysine content 2.8 times greater in bleomycin-treated animals than in their age-matched controls. Evidence was also found for a cross-linked alpha 1(III) x alpha 1(I) peptide linking residue 87 from the alpha 1(III) chain with residue 16C from the alpha 1(I) chain; it also had an increased ratio of DHLNL:HLNL. We conclude that the increased hydroxylation of lysine observed in two different animal models of lung fibrosis occurs preferentially at the N- and C-terminal nonhelical extension peptides of the alpha 1(I) collagen chains, and that this apparent specificity of overhydroxylation of fibrotic collagen may have important structural and pathological consequences.

Topics: Amino Acid Sequence; Animals; Bleomycin; Collagen; Cyanogen Bromide; Hydroxylysine; Lung; Lysine; Male; Molecular Sequence Data; Peptide Mapping; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Silicon Dioxide

Collagen synthesized by tissue minces from lungs of rats administered 1 unit of bleomycin by intratracheal instillation 1 or 2 wk earlier contained relatively more hydroxylysine than did collagen made by lungs from saline-instilled control animals. Most, if not all, of the relative increase in lysine hydroxylation could be localized to the alpha 1 (I) chain of type I collagen. Lung homogenates from bleomycin-treated rats showed increased activity of lysyl hydroxylase (EC 1.14.11.4), the enzyme catalyzing the conversion of collagen-bound lysine to hydroxylysine. Thus, the increased hydroxylation of lysine and of lysine-derived cross-links previously observed in collagen of diseased human lungs and in animal models of lung fibrosis is reflected in an in vitro system.

Topics: Animals; Bleomycin; Collagen; Dose-Response Relationship, Drug; Hydroxylation; Hydroxylysine; Lung; Minoxidil; Mixed Function Oxygenases; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Pulmonary Fibrosis; Rats