Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor
Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor
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DOI：
10.1110/ps.051551105
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发表时间：
2005-09
期刊：
影响因子：
8
通讯作者：
Ying-Xia Zhang;R. Lai;Wen-Hui Lee;Yun Zhang
中科院分区：
文献类型：
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作者：
Ying-Xia Zhang;R. Lai;Wen-Hui Lee;Yun Zhang
从大蹼铃蟾（Bombina maxima）皮肤中分离纯化了一种新的胰蛋白酶抑制剂。从皮肤构建的cDNA文库中获得编码蛋白质的全长cDNA。序列分析表明，该蛋白实际上包含三个保守的白蛋白结构域。随后纯化B.maximaserum白蛋白，并通过基于PCR的克隆从青蛙肝脏进一步获得其编码cDNA。在来自皮肤和血清的白蛋白序列中仅发现两个氨基酸变异。然而，皮肤蛋白与血清蛋白的区别在于血红素B（0.95 mol/mol蛋白）的结合。与牛血清白蛋白不同，B。极大白蛋白有效地抑制胰蛋白酶。它与胰蛋白酶以1：1的摩尔比紧密结合。皮肤和血清蛋白的平衡解离常数（KD）分别为1.92 × 10−9 M和1.55 × 10−9 M。B。maxima白蛋白通过由二硫键（Cys 53-Cys 62）形成的暴露环与胰蛋白酶形成非共价复合物，所述二硫键包括易断裂键Arg 58（P1）-His 59（P1′）。在测定条件下，未观察到对凝血酶、糜蛋白酶、弹性蛋白酶和枯草杆菌蛋白酶的抑制作用。免疫组化显示B.最大白蛋白广泛分布在皮肤的上皮层细胞膜周围和真皮的海绵层内，这表明它在皮肤生理功能中起重要作用，例如水经济、代谢物交换和新陈代谢调节。
A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full‐length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumindomains. B.maximaserum albumin was subsequently purified, and its coding cDNA was further obtained by PCR‐based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1:1 molar ratio. The equilibrium dissociation constants (KD) obtained for the skin and the serum proteins were 1.92 × 10−9 M and 1.55 × 10−9 M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys53–Cys62), which comprises the scissile bond Arg58(P1)–His59(P1′). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.