乳清酸性蛋白(WAP)四硫化物核心蛋白(WFDC)在先天免疫的调节中具有重要作用,抗微生物功能,和抑制粘膜表面的炎性蛋白酶。最近证明了WFDC蛋白,前列腺基质20(ps20),由WFDC1基因编码,是一种有效的生长抑制因子,并与其他WFDC蛋白共享调节伤口愈合过程和对病毒感染的免疫反应的能力。然而,ps20在蛋白质水平上仍然相对未表征。使用一组ps20抗体进行蛋白质印迹(WB),ELISA和免疫亲和纯化,我们与世隔绝,生化表征和测试ps20制剂的三种生物学特性:(i)与糖胺聚糖(GAG)的相互作用(ii)抑制细胞增殖,和(iii)转谷氨酰胺酶2(TG2)介导的PS20与纤连蛋白的交联,与伤口愈合有关的过程。我们在此表明,ps20制剂包含多种分子形式,包括全长ps20(解析为约27kDa),缺乏aa113-140的外显子3截短形式(约22kDa),以及可变数量的推定裂解的较低MW(约15-17kDa)物种。含有这些形式的混合物的未标记的纯化ps20制剂在显著抑制前列腺细胞增殖方面具有生物活性。我们表明,较低LMW形式的ps20产生的一种机制是通过组织蛋白酶L(CL)裂解,并确认CL在C端切割ps20,但这并不抑制其生长抑制功能。然而,CL裂解消除了ps20和固相纤连蛋白之间的相互作用。因此,我们首次证明,缺乏C端免疫原性表位的LMW形式的ps20可以通过CL裂解产生,并且这种裂解会损害多聚化和潜在的与ECM交联的能力,但不是ps20抑制细胞增殖的能力。我们认为ps20像其他WFDC蛋白一样可以与GAG和ECM相关。此外,我们建议PS20的翻译后加工和裂解是产生功能性蛋白质种类所必需的,和TG2介导的交联和CL裂解形成ps20调节装置的组分。
Whey-acidic-protein (WAP) four-disulphide core (WFDC) proteins have important roles in the regulation of innate immunity, anti-microbial function, and the inhibition of inflammatory proteases at mucosal surfaces. It was recently demonstrated that the WFDC protein, prostate stromal 20 (
ps20), encoded by the WFDC1 gene, is a potent growth inhibitory factor, and shares with other WFDC proteins the ability to modulate wound healing processes and immune responses to viral infections. However,
ps20 remains relatively uncharacterised at the protein level. Using a panel of ps20 antibodies for western-blotting (WB), ELISA and immunoaffinity purification, we isolated, biochemically characterised and tested ps20 preparations for three biological properties: (i) interactions with glycosaminoglycans (GAG) (ii) inhibition of cell proliferation, and (iii) transglutaminase2 (TG2) mediated crosslinking of ps20 to fibronectin, a process implicated in wound healing. We show herein that
ps20 preparations contain multiple molecular forms including full-length
ps20 (resolving at ≈27 kDa), an exon 3 truncated form (≈22 kDa) that lacks aa113-140, and variable amounts of a putatively cleaved lower MW (≈15-17 kDa) species. Untagged purified ps20 preparations containing a mixture of these forms are biologically active in significantly suppressing prostate cell proliferation. We show that one mechanism by which lower LMW forms of ps20 arise is through cathepsin L (CL) cleavage, and confirm that CL cleaves ps20 at the C-terminus, but this does not inhibit its growth inhibitory function. However, CL cleavage abrogated the interaction between
ps20 and solid-phase fibronectin. Therefore, we demonstrate for the first time that LMW forms of ps20 that lack a C-terminal immunogenic epitope can arise through CL cleavage and this cleavage impairs multimerisation and potential capacity to cross-link to ECM, but not the capacity of ps20 to inhibit cell proliferation. We propose that
ps20 like other WFDC proteins can become associated with GAGs and the ECM. Furthermore, we suggest post-translational processing and cleavage of ps20 is required to generate functional protein species, and TG2 mediated crosslinking and CL cleavage form components of a ps20 regulatory apparatus.