Evolution of C, D and S-Type Cystatins in Mammals: An Extensive Gene Duplication in Primates

Cystatins are a family of inhibitors of cysteine peptidases that comprises the salivary cystatins (D and S-type cystatins) and cystatin C. These cystatins are encoded by a multigene family (CST3, CST5, CST4, CST1 and CST2) organized in tandem in the human genome. Their presence and functional importance in human saliva has been reported, however the distribution of these proteins in other mammals is still unclear. Here, we performed a proteomic analysis of the saliva of several mammals and studied the evolution of this multigene family. The proteomic analysis detected S-type cystatins (S, SA, and SN) in human saliva and cystatin D in rat saliva. The evolutionary analysis showed that the cystatin C encoding gene is present in species of the most representative mammalian groups, i.e. Artiodactyla, Rodentia, Lagomorpha, Carnivora and Primates. On the other hand, D and S-type cystatins are mainly retrieved from Primates, and especially the evolution of S-type cystatins seems to be a dynamic process as seen in Pongo abelii genome where several copies of CST1-like gene (cystatin SN) were found. In Rodents, a group of cystatins previously identified as D and S has also evolved. Despite the high divergence of the amino acid sequence, their position in the phylogenetic tree and their genome organization suggests a common origin with those of the Primates. These results suggest that the D and S type cystatins have emerged before the mammalian radiation and were retained only in Primates and Rodents. Although the mechanisms driving the evolution of cystatins are unknown, it seems to be a dynamic process with several gene duplications evolving according to the birth-and-death model of evolution. The factors that led to the appearance of a group of saliva-specific cystatins in Primates and its rapid evolution remain undetermined, but may be associated with an adaptive advantage.

半胱氨酸蛋白酶抑制剂（Cystatins）是一类半胱氨酸蛋白酶抑制剂家族，涵盖唾液型半胱氨酸蛋白酶抑制剂（D型与S型半胱氨酸蛋白酶抑制剂）以及半胱氨酸蛋白酶抑制剂C。此类抑制剂由多基因家族（CST3、CST5、CST4、CST1及CST2）编码，该家族在人类基因组中呈串联排布。已有研究报道了此类蛋白在人类唾液中的存在及其功能重要性，但它们在其他哺乳动物体内的分布情况仍不明确。本研究对多种哺乳动物的唾液开展了蛋白质组学分析，并对该多基因家族的演化过程进行了研究。蛋白质组学检测发现，人类唾液中存在S型半胱氨酸蛋白酶抑制剂（S、SA及SN亚型），大鼠唾液中则检测到半胱氨酸蛋白酶抑制剂D。演化分析结果显示，半胱氨酸蛋白酶抑制剂C编码基因存在于最具代表性的多个哺乳动物类群中，即偶蹄目（Artiodactyla）、啮齿目（Rodentia）、兔形目（Lagomorpha）、食肉目（Carnivora）及灵长目（Primates）。另一方面，D型与S型半胱氨酸蛋白酶抑制剂主要发现于灵长类动物中；尤为特别的是，S型半胱氨酸蛋白酶抑制剂的演化过程似乎极具动态性，例如在婆罗洲猩猩（Pongo abelii）基因组中，便发现了多份CST1样基因（即半胱氨酸蛋白酶抑制剂SN）的拷贝。在啮齿类动物中，此前被鉴定为D型与S型的半胱氨酸蛋白酶抑制剂类群也发生了演化。尽管其氨基酸序列差异显著，但系统发育树中的位置以及基因组排布特征表明，它们与灵长类的同类具有共同起源。上述研究结果提示，D型与S型半胱氨酸蛋白酶抑制剂诞生于哺乳动物辐射演化之前，且仅在灵长类与啮齿类动物中得以保留。尽管驱动此类半胱氨酸蛋白酶抑制剂演化的分子机制尚不清楚，但该过程似乎是一个动态过程，伴随多次基因复制事件，并遵循演化的“诞生-死亡”模型。导致灵长类动物中出现唾液特异性半胱氨酸蛋白酶抑制剂类群及其快速演化的诱因仍未明确，但推测可能与适应性优势相关。