Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium.
PLoS One. 2012;7(3):e34199. doi: 10.1371/journal.pone.0034199. Epub 2012 Mar 27.
Carboxypeptidase M (CPM) targets the basic amino acids arginine and lysine present at the C-terminus of peptides or proteins. CPM is thought to be involved in inflammatory processes. This is corroborated by CPM-mediated trimming and modulation of inflammatory factors, and expression of the protease in inflammatory environments. Since the function of CPM in and beyond inflammation remains mainly undefined, the identification of natural substrates can aid in discovering the (patho)physiological role of CPM. CCL1/I-309, with its three C-terminal basic amino acids, forms a potential natural substrate for CPM. CCL1 plays a role not only in inflammation but also in apoptosis, angiogenesis and tumor biology. Enzymatic processing differently impacts the biological activity of chemokines thereby contributing to the complex regulation of the chemokine system. The aim of the present study was to investigate whether (i) CCL1/I-309 is prone to trimming by CPM, and (ii) the biological activity of CCL1 is altered after C-terminal proteolytic processing. CCL1 was identified as a novel substrate for CPM in vitro using mass spectrometry. C-terminal clipping of CCL1 augmented intracellular calcium release mediated by CCR8 but reduced the binding of CCL1 to CCR8. In line with the higher intracellular calcium release, a pronounced increase of the anti-apoptotic activity of CCL1 was observed in the BW5147 cellular model. CCR8 signaling, CCR8 binding and anti-apoptotic activity were unaffected when CPM was exposed to the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid. The results of this study suggest that CPM is a likely candidate for the regulation of biological processes relying on the CCL1-CCR8 system.
羧肽酶 M（CPM）靶向存在于肽或蛋白质 C 末端的碱性氨基酸精氨酸和赖氨酸。CPM 被认为参与炎症过程。CPM 介导的炎症因子的修剪和调节，以及蛋白酶在炎症环境中的表达，证实了这一点。由于 CPM 在炎症内外的功能仍主要未定义，因此天然底物的鉴定有助于发现 CPM 的（病理）生理作用。CCL1/I-309 带有三个 C 末端碱性氨基酸，是 CPM 的潜在天然底物。CCL1 不仅在炎症中发挥作用，而且在细胞凋亡、血管生成和肿瘤生物学中发挥作用。酶切处理会对趋化因子的生物学活性产生不同的影响，从而有助于趋化因子系统的复杂调节。本研究的目的是研究（i）CCL1/I-309 是否容易被 CPM 修剪，以及（ii）CCL1 的 C 末端蛋白水解处理后其生物学活性是否改变。本研究使用质谱法在体外鉴定 CCL1 是 CPM 的一种新型底物。CCL1 的 C 末端剪接增强了 CCR8 介导的细胞内钙释放，但降低了 CCL1 与 CCR8 的结合。与细胞内钙释放增加相一致，在 BW5147 细胞模型中观察到 CCL1 的抗凋亡活性显著增加。当 CPM 暴露于羧肽酶抑制剂 DL-2-巯基甲基-3-胍基-乙基硫代丙酸时，CCR8 信号、CCR8 结合和抗凋亡活性不受影响。本研究结果表明，CPM 可能是依赖 CCL1-CCR8 系统的生物学过程的调节候选物。
