牛肾中豆类蛋白的纯化、分子克隆、免疫组化定位及膜联蛋白II和维生素d结合蛋白1的降解

Takuya Yamane , Keisuke Takeuchi , Yoshio Yamamoto , Yao-Hua Li , Manabu Fujiwara , Katuji Nishi , Sho Takahashi , Iwao Ohkubo
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引用次数: 46

摘要

从牛肾中纯化出豆科蛋白(天冬酰胺内肽酶)。在β-巯基乙醇存在下,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳计算得到纯化酶的分子量为34000。该酶能快速水解底物Z-Ala-Ala-Asn-MCA,并受到n-乙基马来酰亚胺、对氯苯磺酸、Hg2+和Cu2+的强烈抑制。该酶前26个残基的氨基酸序列为Gly-Gly-Lys-His-Trp-Val-Val-Ile-Val-Ala-Gly-Ser-Asn-Gly-Gln-Tyr-Asn-Tyr-Arg-His-Gln-Ala-Phe-Ala-Asp-His-。该序列与猪肾豆科蛋白n端序列高度同源。利用源自大鼠豆科蛋白的DNA探针筛选牛肾皮质cDNA文库,确定了牛肾皮质cDNA的结构和氨基酸序列。该cDNA全长1934 bp,编码区有433个氨基酸。在免疫组织化学研究中,该酶在大鼠肾近端小管中被强烈染色。已知维生素d结合蛋白是存在于近端小管中的巨噬蛋白的配体,被牛肾豆科蛋白以有限的蛋白水解方式切割。这些结果表明,豆蔻素有助于近端小管细胞吸收大分子的加工。该酶还在Asn31和Phe32之间的位置切割了牛膜联蛋白II的n端合成肽(gly24 - ser - val - lys - ala - tyr - thr30 - asn - ph - asp - ala - glu35 - arg - asp37)。膜联蛋白II的氨基末端结构域具有p11亚基结合位点和pp60src和蛋白激酶c的磷酸化位点,这表明豆类在膜联蛋白II的失活和降解中起重要作用,而膜联蛋白II在核内体/溶酶体的受体循环室中含量丰富。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Legumain from bovine kidney: its purification, molecular cloning, immunohistochemical localization and degradation of annexin II and vitamin D-binding protein1

Legumain (asparaginyl endopeptidase) was purified to homogeneity from bovine kidneys. The molecular mass of the purified enzyme was calculated to be 34 000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in the presence of β-mercaptoethanol. The enzyme rapidly hydrolyzed the substrate Z-Ala-Ala-Asn-MCA and was strongly inhibited by N-ethylmaleimide, p-chloromercuribenzene-sulfonic acid, Hg2+ and Cu2+. The amino acid sequence of the first 26 residues of the enzyme was Gly-Gly-Lys-His-Trp-Val-Val-Ile-Val-Ala-Gly-Ser-Asn-Gly-Gln-Tyr-Asn-Tyr-Arg-His-Gln-Ala-Phe-Ala-Asp-His-. This sequence is highly homologous to the sequences in the N-terminal of pig kidney legumain. We screened a bovine kidney cortex cDNA library using a DNA probe that originated from rat legumain, and we determined the bovine kidney cDNA structure and deduced the amino acid sequence. The cDNA is composed 1934 bp and encodes 433 amino acids in the coding region. The enzyme was strongly stained in the proximal tubules of the rat kidney in an immunohistochemical study. Vitamin D-binding protein which is known to be a ligand to megalin existing in the proximal tubules, was cleaved in a limited proteolytic manner by bovine kidney legumain. These results suggested that legumain contributes to the processing of macromolecules absorbed by proximal tubule cells. The enzyme also cleaved an N-terminal synthetic peptide of bovine annexin II (Gly24-Ser-Val-Lys-Ala-Tyr-Thr30-Asn-Phe-Asp-Ala-Glu35-Arg-Asp37) at a position between Asn31 and Phe32. The amino-terminal domain of annexin II has p11 subunit binding sites and phosphorylation sites for both pp60src and protein kinase C. This suggests that legumain plays an important role in inactivation and degradation of annexin II, which is abundant in the receptor-recycling compartments of endosomes/lysosomes.

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