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

Abstract

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 [␣(I)] 2 ␤ 2 , [␣(II)] 2 ␤ 2 , and [␣(III)] 2 ␤ 2 tetramers with identical ␤ subunits. We generated mice with targeted inactivation of the P4ha1 gene encoding the catalytic ␣ 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.

D-2-hydroxyglutarate produced by mutant IDH1 perturbs collagen maturation and basement membrane function

“…5A), implying that Hyl-mediated glycosylation was impaired. Similar phenotypes have been observed in tissues of mice deficient for P4ha1 (the a subunit of C-P4H I) or Plod3 (Rautavuoma et al 2004;Holster et al 2007). Thus, we propose that the excess D2HG present due to the activity of the mutated Idh1 enzyme impairs Hyp-and Hyl-mediated modification of collagen proteins, preventing their maturation.…”

“…When the activities of these enzymes are suppressed, newly synthesized collagens are not properly modified, leading to the accumulation of unfolded proteins in the ER (Marutani et al 2004;Rautavuoma et al 2004;Holster et al 2007). Because the presence of these abnormal proteins in bulk triggers an ER stress response that leads to apoptosis (Ron and Walter 2007), we hypothesized that such a response might be induced by the impaired collagen maturation in Nes-KI mutants.…”

4‐Chloroprolines: Synthesis, conformational analysis, and effect on the collagen triple helix

“…Both Caenorhabditis elegans and mice lacking P4H experience embryonic morbidity due to weakened collagen superstructures. 15,16 Why is proline hydroxylation essential to the formation of a stable ECM? It is known that collagens containing a high fraction of Hyp in the Yaa position are particularly stable, 17,18 but the physicochemical basis for this stability was unclear.…”

Biological role of prolyl 3-hydroxylation in type IV collagen

“…They develop at the expected Mendelian ratio up to E9.5, indicating that type IV collagen is not essential during early development but is essential for basement membrane stability (12). Even mice lacking prolyl 4-hydroxylase develop up to E9.5, indicating that collagens are generally not required for development to this stage (13).…”