Karvonen K, Ala-Kokko L, Pihlajaniemi T, Helaakoski T, Henke S, Günzler V, Kivirikko K I, Savolainen E R
Collagen Research Unit, University of Oulu, Finland.
J Biol Chem. 1990 May 25;265(15):8415-9.
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.
胶原蛋白在纤维化疾病中的关键作用促使人们尝试开发抑制胶原蛋白积累的药物。最近发现，含有非生理性氨基酸5-氧代脯氨酸（Opr）的肽可作为纯脯氨酰4-羟化酶的特异性同步催化失活剂，该酶催化胶原蛋白中4-羟脯氨酸的形成。本研究表明，含氧代脯氨酸的肽苄氧羰基-苯丙氨酸-Opr-甘氨酸苄酯（I）和苄氧羰基-苯丙氨酸-Opr-甘氨酸乙酯（II）可使培养的人皮肤成纤维细胞中的脯氨酰4-羟化酶失活，肽I的效力约为肽II的两倍。用约20-40 microM浓度的肽I培养48小时后，观察到50%的失活。这种失活似乎具有特异性，因为胶原蛋白合成的另外两种细胞内酶，赖氨酰羟化酶和半乳糖基羟赖氨酰葡糖基转移酶的活性没有变化。细胞合成4-羟脯氨酸明显减少，细胞内积累了缺乏4-羟脯氨酸的前胶原，而用30 microM浓度的肽I培养细胞48小时后，[14C]亮氨酸掺入蛋白质的情况没有变化。细胞活力或乳酸脱氢酶从细胞释放到培养基中的情况没有变化。用75 microM肽I培养48小时后，I型和III型前胶原的前α1链、脯氨酰4-羟化酶的α和β亚基或纤连蛋白的mRNA稳态水平没有显著变化。数据表明，细胞内脯氨酰4-羟化酶的失活对细胞4-羟脯氨酸的形成和胶原蛋白分泌有显著影响，但对I型和III型前胶原或脯氨酰4-羟化酶的两种亚基的mRNA稳态水平没有影响。
