Disease-associated single amino acid mutation in the calf-1 domain of integrin 伪3 leads to defects in its processing and cell surface expression

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• 作者：Masashi Yamada ; Kiyotoshi Sekiguchi
• 关键词：DMEM ; Dulbecco&rsquo ; s modified Eagle&rsquo ; s medium ; ER ; endoplasmic reticulum ; mAb ; monoclonal antibody ; pAb ; polyclonal antibody ; PBS ; phosphate-buffered saline
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：29 November, 2013
• 年：2013
• 卷：441
• 期：4
• 页码：988-993
• 全文大小：1167 K

文摘

Integrin 伪3尾1, a receptor for laminins, is involved in the structural and functional organization of epithelial organs, including the lung, kidney, and skin. Recently, a missense mutation that causes substitution of Arg628 with Pro (R628P) in the calf-1 domain of human 伪3 was shown to be associated with disorders of the lung, kidney, and skin. Here, we found that the R628P mutation leads to aberrations in the posttranslational processing of 伪3. Specifically, 伪3 with the R628P mutation showed hardly any cleavage at the calf-2 domain, which usually occurs in the Golgi apparatus during the delivery of de novo-synthesized 伪3. The mutant 伪3 retained the ability to associate with integrin 尾1, but not with the tetraspanin CD151, and the bound 尾1 was a partially glycosylated immature form, the maturation of which also takes place in the Golgi apparatus. Furthermore, the cell surface expression of the mutant protein was markedly reduced. These results suggest that the R628P mutation leads to a deficit in the transport of 伪3尾1 from the ER to the Golgi apparatus. When Arg628 was mutated to Gln or Glu, instead of Pro, the resulting mutants did not display aberrations in processing or CD151 binding, indicating that the presence of Pro, rather than the absence of Arg, at amino acid residue 628 of 伪3 is important for the abnormalities in the R628P mutant. In support of this notion, a homology modeling analysis of the calf-1 domain of 伪3 showed that replacement with Pro, but not with Gln or Glu, caused partial disruption of the 尾-sheet structures. Furthermore, the ER-associated degradation of the R628P mutant was not enhanced compared with that of the wild-type protein, suggesting that the deficits in the posttranslational processing and cell surface expression of the R628P mutant are independent of the ER-associated degradation, but arise from the defect in its export from the ER. We conclude that the calf-1 domain is required for the transport of 伪3 from the ER to the Golgi apparatus to maintain the integrity of epithelial tissues, and hence the impairment of the calf-1 domain by the R628P mutation leads to severe diseases of the kidneys, lungs, and skin.