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.

基底膜是薄而坚固的细胞外基质片。它们为上皮、肌肉、神经和血管等组织提供机械和生化支持。基底膜的机械特性部分由 IV 型胶原蛋白 (Col4) 赋予，这是一种丰富的基底膜蛋白质，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，通过基质驻留酶过氧化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温和的表型。为了探索过氧化物酶的作用，我们分析了果蝇中的突变体，包括新的 CRISPR 生成的催化无效，并发现纯合子大多是致命的，其中 13% 的存活逃逸子。小鼠突变体也表现出半致死性，孟德尔分析表明致死率约为 50%，逃逸者约为 50%。尽管存在强烈的突变，纯合的果蝇和小鼠逃逸者的 Col4 交联水平较低但可检测到，这表明存在低效的替代交联机制，这可能是逃逸者存活的原因。果蝇突变体表型与基底膜硬度降低一致。有趣的是，我们发现即使野生型动物的基底膜组装并交联后，成人也需要持续的过氧化物酶活性才能随着时间的推移维持组织硬度。这些结果表明，过氧化酶交联可能比以前认识的更重要。