Department of Cell and Molecular Biology, Uppsala University, Uppsala, The Biomedical Center, Box 596, SE-751 24, Uppsala, Sweden.
Department of Medical Biochemistry and Microbiology, BMC, Box 589, SE-751 23, Uppsala, Sweden.
Dev Comp Immunol. 2022 Mar;128:104324. doi: 10.1016/j.dci.2021.104324. Epub 2021 Nov 23.
Granzyme B (GzmB) is primarily expressed by mammalian cytotoxic T cells and serves as one of the key components in the defense against viral infection by the induction of apoptosis in virus infected cells. By direct cell to cell contact and delivery into target cells by perforin, cytotoxic T cells activate apoptosis through the action of GzmB by both caspase-dependent and -independent pathways. In search for early ancestors of GzmB we have in the current study identified and characterized a GzmB homologue from a reptile, the Chinese alligator. This enzyme is encoded from the same locus as the mammalian counterparts, the chymase locus. Phage display analysis of the cleavage specificity of the recombinant alligator enzyme (named MCP1A-like) shows that it is a relatively strict Glu-ase, with strong preference for glutamic acid in the P1 position of a substrate. The majority of mammalian GzmB:s are, in marked contrast to the alligator enzyme, relatively strict Asp-ases. The alligator enzyme also showed strong preference for Ala, Pro and Gly in the P2 position and Val in the P3 position indicating that it has a narrow specificity, similar to the mammalian counterparts. Analysis of the three amino acids forming the substrate binding pocket (S1 pocket) in three amphibian homologues to MCP1A-like, from the frogs Xenopus laevis and Xenopus tropicalis, shows that these amphibian enzymes have similar substrate binding pocket as their mammalian counterparts. This finding, together with the apparent lack of GzmB homologs in fish, indicates that the ancestor of GzmB did appear with the amphibians at the base of tetrapod evolution. This study is a first step in a larger effort to understand the evolutionary processes involved in shaping anti-viral immunity in non-mammalian vertebrates.
颗粒酶 B（GzmB）主要由哺乳动物细胞毒性 T 细胞表达，是诱导病毒感染细胞凋亡以抵抗病毒感染的关键成分之一。通过直接的细胞间接触和穿孔素将其递送至靶细胞，细胞毒性 T 细胞通过依赖和不依赖半胱天冬酶的途径激活 GzmB 诱导细胞凋亡。为了寻找 GzmB 的早期祖先，我们在本研究中从爬行动物——扬子鳄中鉴定和表征了一种 GzmB 同源物。这种酶与哺乳动物的同源物糜酶基因座编码在同一基因座上。重组扬子鳄酶（命名为 MCP1A-like）的酶切特异性噬菌体展示分析表明，它是一种相对严格的 Glu-ase，在底物的 P1 位置对谷氨酸具有强烈的偏好。与扬子鳄酶相比，大多数哺乳动物 GzmB:s 是相对严格的 Asp-ase。扬子鳄酶在 P2 位置也强烈偏好 Ala、Pro 和 Gly，在 P3 位置偏好 Val，表明其具有狭窄的特异性，类似于哺乳动物的同源物。对三种两栖动物同源物（来自青蛙非洲爪蟾和 Xenopus tropicalis）中 MCP1A-like 的三个氨基酸形成的底物结合口袋（S1 口袋）进行分析表明，这些两栖动物酶具有与其哺乳动物同源物相似的底物结合口袋。这一发现，再加上鱼类中似乎缺乏 GzmB 同源物，表明 GzmB 的祖先确实在四足动物进化的基础上与两栖动物一起出现。本研究是了解非哺乳动物脊椎动物抗病毒免疫形成中涉及的进化过程的更大努力的第一步。
