Conservation of the extended substrate specificity profiles among homologous granzymes across species

Granzymes are a family of structurally related serine proteases involved in immunity. To date four out of five human granzymes have been assigned orthologues in mice; however for granzyme H, no clear murine orthologue has been suggested and its role in cytotoxicity remains controversial. In this study, we demonstrate that granzyme H is an inefficient cytotoxin. Besides analyzing the substrate specificity profile of granzyme H by means of substrate phage display, we assessed human granzyme H and mouse granzyme C cleavage susceptibility on a proteome-wide level. The extended specificity profiles of granzyme C and granzyme H (i.e. beyond P4-P4') match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes since granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can be conferred simply by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto generally underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility. Bio-IT: The spectra were searched with Mascot Daemon 2.2. The following search parameters were used. Peptide mass tolerance was set at 0.2 Da and peptide fragment mass tolerance at 0.1 Da; with the ESI-QUAD-TOF as selected instrument for peptide fragmentation rules for the Q-TOF Premier data. Endoproteinase semiArg-C/P (i.e., no restriction towards arginine-proline cleavage) was set as enzyme allowing one missed cleavage. Variable modifications were pyroglutamate formation of N-terminal glutamine, pyrocarbamidomethyl formation of N-terminal alkylated cysteine, deamidation of asparagine, acetylation or tri-deuteroacetylation of the alpha-N-terminus. Fixed modifications were methionine oxidation (sulfoxide), carbamidomethyl for cysteine, tri-deuteroacetylation of lysine and for identifying heavy labelled peptides, [13C6] or [13C615N4]-arginine were additionally set as fixed modifications. The spectra and identifications were stored in ms_lims

颗粒酶（Granzymes）是一类结构同源的丝氨酸蛋白酶家族，参与机体免疫应答。截至目前，5种人类颗粒酶中有4种已在小鼠中鉴定出同源基因；然而针对颗粒酶H（granzyme H），目前尚未发现明确的小鼠同源基因，其在细胞毒性中的作用也存在争议。本研究证实，颗粒酶H是一种低效的细胞毒素。除通过底物噬菌体展示技术分析颗粒酶H的底物特异性谱外，我们还在蛋白质组层面评估了人类颗粒酶H与小鼠颗粒酶C的切割敏感性。颗粒酶C与颗粒酶H的扩展特异性谱（即超出P4-P4'位点的范围）与此前报道的颗粒酶B（granzyme B）一致。我们进一步证明，这类扩展特异性谱在多种颗粒酶中具有保守性：仅需将颗粒酶H/C特异性切割位点的P1位残基突变为颗粒酶B偏好的天冬氨酸，即可赋予该位点被颗粒酶B切割的能力。综上，本研究结果表明，蛋白酶-底物的扩展接触界面在调控切割敏感性中具有保守但此前普遍未被重视的特异性决定作用。

生物信息学分析：使用Mascot Daemon 2.2对质谱谱图进行检索，采用以下搜索参数：肽质量容忍度设为0.2道尔顿，肽段碎片质量容忍度设为0.1道尔顿；针对Q-TOF Premier质谱数据，选择电喷雾电离四极杆飞行时间质谱仪（ESI-QUAD-TOF）作为肽段碎裂规则的适配仪器。选用半精氨酸-C/P内切蛋白酶（Endoproteinase semiArg-C/P，即对精氨酸-脯氨酸切割无限制）作为酶解酶，允许1个漏切位点。可变修饰包括：N端谷氨酰胺的焦谷氨酸形成、N端烷基化半胱氨酸的焦氨基甲酰化修饰、天冬酰胺的脱酰胺修饰、α-N端的乙酰化或三氘代乙酰化修饰。固定修饰包括：甲硫氨酸氧化（亚砜）、半胱氨酸的氨基甲酰甲基化、赖氨酸的三氘代乙酰化；为鉴定重标肽段，额外将[13C6]或[13C615N4]-精氨酸设为固定修饰。质谱谱图与肽段鉴定结果存储于ms_lims中。