Loss of assembly of the main basement membrane collagen, type IV, but not fibril-forming collagens and embryonic death in collagen prolyl 4-hydroxylase I null mice
Loss of assembly of the main basement membrane collagen, type IV, but not fibril-forming collagens and embryonic death in collagen prolyl 4-hydroxylase I null mice
复制标题
DOI：
10.1074/jbc.m606608200
复制
发表时间：
2007-01-26
影响因子：
4.8
通讯作者：
Myllyharju, Johanna
中科院分区：
文献类型：
--
作者：
Holster, Tiina;Pakkanen, Outi;Myllyharju, Johanna
胶原蛋白脯氨基4-羟基酶(C-P4Hs)催化4-羟基脯氨酸残基的形成，这些残基是形成三螺旋胶原分子所必需的。脊椎动物C-P4Hs I、II和III是具有相同β亚基的[α(I)](2)β(2)、[α(II)](2)β(2)和[α(III)](2)β(2)四聚体。我们建立了编码C-P4H I催化α亚单位的P4ha1基因的靶向失活小鼠，以分析其特定功能。空白小鼠在E10.5后死亡，显示出总体发育延迟，细胞内质网扩张。毛细血管壁经常破裂，但毛细血管密度不变。培养的空胚胎和成纤维细胞中的C-P4H活性水平是野生型的20%，这明显是由于另外两种同工酶的作用。空胚胎的基底膜几乎没有IV型胶原免疫荧光，电子显微镜显示基底膜破坏，而免疫电子显微镜显示基底膜中缺乏IV型胶原。在空白胚胎和培养的空白成纤维细胞中，可溶性IV型胶原的含量增加，这表明IV型胶原分子缺乏组装成不溶解的结构，可能是由于它们的羟基化不足，从而导致构象异常。相比之下，缺失的胚胎有典型的横纹图案的I型和III型胶原纤维，但直径略有增加，缺失的成纤维细胞分泌形成纤维的胶原，尽管效率低于野生型细胞。因此，无效胚胎的主要死亡原因很可能是IV型胶原的异常组装。
Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of the 4-hydroxyproline residues that are essential for the generation of triple helical collagen molecules. The vertebrate C-P4Hs I, II, and III are [alpha(I)](2)beta(2), [alpha(II)](2)beta(2), and [alpha(III)](2)beta(2) tetramers with identical beta subunits. We generated mice with targeted inactivation of the P4ha1 gene encoding the catalytic alpha subunit of C-P4H I to analyze its specific functions. The null mice died after E10.5, showing an overall developmental delay and a dilated endoplasmic reticulum in their cells. The capillary walls were frequently ruptured, but the capillary density remained unchanged. The C-P4H activity level in the null embryos and fibroblasts cultured from them was 20% of that in the wild type, being evidently due to the other two isoenzymes. Collagen IV immunofluorescence was almost absent in the basement membranes of the null embryos, and electron microscopy revealed disrupted basement membranes, while immunoelectron microscopy showed a lack of collagen IV in them. The amount of soluble collagen IV was increased in the null embryos and cultured null fibroblasts, indicating a lack of assembly of collagen IV molecules into insoluble structures, probably due to their underhydroxylation and hence abnormal conformation. In contrast, the null embryos had collagen I and III fibrils with a typical cross-striation pattern but slightly increased diameters, and the null fibroblasts secreted fibril-forming collagens, although less efficiently than wild-type cells. The primary cause of death of the null embryos was thus most likely an abnormal assembly of collagen IV.