过氧化物酶(PXDN)是一种分泌的血红素过氧化物酶,通过过氧化氢和溴化物的中间次溴酸(HOBr)合成,催化细胞外基质(ECM)内IV胶原的氧化交联,但最近的研究结果也表明,PXDN对ECM蛋白进行了替代修饰,包括将溴化物掺入酪氨酸残基。在这项工作中,我们试图从小鼠畸胎癌PFHR9细胞中鉴定HOBr或PXDN介导的ECM中酪氨酸溴化的主要靶蛋白。我们从PFHR9细胞中检测到61种含有溴酪氨酸(BrY)的肽,代表HOBr修饰的ECM中的23种蛋白质,其中层粘连蛋白表现出最突出的溴酪氨酸掺入。此外,我们还发现层粘连蛋白α1,层粘连蛋白β1和肾小管间质性肾炎抗原样(TINAGL1)在未经治疗的PFHR9细胞中含有BrY,这取决于PXDN。我们将这些分析扩展到健康小鼠和实验性肺纤维化小鼠的肺组织,以及从人类受试者获得的肺组织。对富含ECM的小鼠肺组织提取物的分析表明,博来霉素诱导的纤维化中,有83种ECM蛋白升高,其中包括各种胶原蛋白和层粘连蛋白,PXDN同样,纤维化小鼠肺组织中PXDN和层粘连蛋白α/β1的mRNA和蛋白表达增强,以及在用转化生长因子β1(一种促纤维化生长因子)刺激的小鼠骨髓来源的巨噬细胞或人成纤维细胞中。我们鉴定了11种含有BrY的ECM蛋白,包括胶原蛋白IVα2,胶原蛋白VIα1,TINAGL1和各种层粘连蛋白,在健康和小鼠纤维化肺组织中,尽管在纤维化期间,层粘连蛋白的酪氨酸溴化的相对程度并未显着增加。最后,我们还在人肺组织中鉴定出7种含有BrY的ECM蛋白,再次包括胶原IVα2、胶原VIα1和TINAGL1。总之,这项工作证明了几种溴酪氨酸修饰的ECM蛋白的存在,可能涉及PXDN,即使在正常的肺组织中,提示这些修饰的潜在生物学功能。
Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61
bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent
bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin β1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/β1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor β1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several
bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.