Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membranes that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved throughout the animal kingdom, indicating they are important, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a new CRISPR-generated catalytic null, and found that homozygotes were mostly lethal with 13 % viable escapers. Mouse mutants also show semi-lethality, with Mendelian analysis demonstrating ∼50 % lethality and ∼50 % escapers. Despite the strong mutations, the homozygous fly and mouse escapers had low but detectable levels of Col4 crosslinking, indicating the existence of inefficient alternative crosslinking mechanisms, probably responsible for the viable escapers. Fly mutant phenotypes are consistent with decreased basement membrane stiffness. Interestingly, we found that even after basement membranes are assembled and crosslinked in wild-type animals, continuing Peroxidasin activity is required in adults to maintain tissue stiffness over time. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.

基底膜是细胞外基质的薄而坚固的片状。它们为上皮细胞、肌肉、神经和血管等组织提供机械和生化支持。基底膜的机械性能部分由胶原蛋白 IV （Col4） 赋予，胶原蛋白 IV （Col4） 是一种丰富的基底膜蛋白，通过头对头和尾对尾的相互作用形成广泛的二维网络。Col4 网络组装成基底膜后，被基质驻留酶过氧化物酶交联，形成大的共价聚合物。过氧化物酶和 Col4 交联在整个动物界中高度保守，表明它们很重要，但纯合突变小鼠具有轻微的表型。为了探索过氧化物酶的作用，我们分析了果蝇中的突变体，包括一个新的 CRISPR 产生的催化无效，发现纯合子大多是致命的，有 13% 的活逃逸者。小鼠突变体也显示出半致死性，孟德尔分析显示 ∼50% 的致死率和 ∼50% 的逃逸者。尽管突变强烈，但纯合子苍蝇和小鼠逃逸者的 Col4 交联水平较低但可检测到，表明存在低效的替代交联机制，这可能是活逃逸者的原因。果蝇突变表型与基底膜刚度降低一致。有趣的是，我们发现即使在野生型动物中组装和交联基底膜后，成人也需要持续的过氧化物酶活性才能随着时间的推移保持组织刚度。这些结果表明，过氧化物酶交联可能比以前认识到的更重要。