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Collagen prolyl 4-hydroxylase isoenzymes I and II have sequence specificity towards different X-Pro-Gly triplets
Matrix Biology ( IF 4.5 ) Pub Date : 2023-12-09 , DOI: 10.1016/j.matbio.2023.12.001
Antti M Salo 1 , Pekka Rappu 2 , M Kristian Koski 1 , Emma Karjalainen 1 , Valerio Izzi 3 , Kati Drushinin 1 , Ilkka Miinalainen 4 , Jarmo Käpylä 2 , Jyrki Heino 2 , Johanna Myllyharju 1
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Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.



中文翻译:


胶原蛋白脯氨酰 4-羟化酶同工酶 I 和 II 对不同的 X-Pro-Gly 三联体具有序列特异性



胶原蛋白生物合成需要对赖氨酸和脯氨酸残基进行多种共翻译和翻译后修饰,以形成结构和功能上具有竞争力的胶原蛋白分子。在重复的-XY-Gly-序列的Y位脯氨酸中形成4-羟基脯氨酸(4Hyp)为三螺旋胶原蛋白分子提供热稳定性。 4Hyp 的形成由由三种同工酶组成的胶原脯氨酰 4-羟化酶 (C-P4H) 家族催化。在这里,我们通过对来自细胞和组织样品的 I 型和 IV 型胶原蛋白进行详细的 4Hyp 分析,确定了两种主要 C-P4H 同工酶在胶原蛋白羟基化中的具体作用。 C-P4H-I 的丢失导致胶原蛋白羟基化不足,其中受影响的脯氨酸分布不均匀,但主要存在于相邻 X 位氨基酸具有带正电或极性不带电侧链的位点。相反,C-P4H-II 的缺失会导致三联体羟基化不足,其中 X 位被带负电的氨基酸谷氨酸或天冬氨酸占据。人们发现这些三联体的羟基化很重要,因为单独失去 C-P4H-II 会导致胶原熔化温度降低并改变胶原原纤维和基底膜的组装。观察到的 C-P4H 同工酶底物特异性差异可以通过底物与活性位点的选择性结合导致 Km 和 Vmax 值的明显差异来解释。此外,我们的结果清楚地表明底物脯氨酸的选择不依赖于胶原类型,但主要决定因素是-X-Pro-Gly-三联体的X位置氨基酸。 尽管我们的数据清楚地显示了 C-P4H-I 和 II 对于正常脯氨酰 4-羟基化和胶原蛋白功能的必要性,但令人惊讶的是,同工酶与各种前胶原的 mRNA 表达并不紧密协调,这表明存在额外的控制水平。总之,这项研究为需要多种 C-P4H 同工酶生成能够组装成完整细胞外基质结构的胶原蛋白分子提供了分子水平的解释。

更新日期:2023-12-09
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