J. Biol. Chem., Vol. 265, Issue 15, 8415-8419, 05, 1990
Specific inactivation of prolyl 4-hydroxylase and inhibition of collagen synthesis by oxaproline-containing peptides in cultured human skin fibroblasts
K Karvonen, L Ala-Kokko, T Pihlajaniemi, T Helaakoski, S Henke, V Gunzler, KI Kivirikko and ER Savolainen
Collagen Research Unit, University of Oulu, Finland.
The crucial role of collagen in fibrotic disorders has prompted attempts to
develop drugs that inhibit collagen accumulation. Peptides containing the
unphysiological amino acid 5-oxaproline (Opr) have recently been found to
act as specific syncatalytic inactivators of pure prolyl 4-hydroxylase, the
enzyme that catalyzes the formation of 4- hydroxyproline in collagens. The
present study indicates that oxaproline-containing peptides
benzyloxycarbonyl-Phe-Opr-Gly-benzyl ester (I) and
benzyloxycarbonyl-Phe-Opr-Gly-ethyl ester (II) inactivate prolyl
4-hydroxylase in cultured human skin fibroblasts, peptide I being about
twice as potent as peptide II. Inactivation by 50% was observed after
culturing with about 20-40 microM concentrations of peptide I for 48 h. The
inactivation appears to be specific, as no changes were found in the
activities of two other intracellular enzymes of collagen synthesis, lysyl
hydroxylase and galactosylhydroxylysyl glucosyltransferase. Synthesis of
4-hydroxyproline by the cells was markedly decreased, and
4-hydroxyproline-deficient procollagen accumulated intracellularly, whereas
no changes were found in the incorporation of [14C]leucine into protein
after culturing of the cells with a 30 microM concentration of peptide I
for 48 h. No changes were seen in the viability of the cells or the release
of lactate dehydrogenase from them into the culture medium. No significant
changes were found in the steady-state levels of the mRNAs for the
pro-alpha 1 chains of type I and type III procollagens or for the alpha and
beta subunits of prolyl 4-hyroxylase or fibronectin after culturing with 75
microM peptide I for 48 h. The data indicate that inactivation of cellular
prolyl 4-hydroxylase has marked effects on cellular 4- hydroxyproline
formation and collagen secretion but no effects on the steady-state levels
of mRNAs for type I and III procollagens or the two types of subunit of
prolyl 4-hydroxylase.
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