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J. Biol. Chem., Vol. 281, Issue 44, 33258-33267, November 3, 2006

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N-Glycosylation of the -Propeller Domain of the Integrin 5 Subunit Is Essential for 51 Heterodimerization, Expression on the Cell Surface, and Its Biological Function^*

Tomoya Isaji, Yuya Sato, Yanyang Zhao, Eiji Miyoshi, Yoshinao Wada^¶, Naoyuki Taniguchi^||, and Jianguo Gu¹

From the Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, the Department of Biochemistry, Osaka University Graduate School of Medicine, B1, 2-2 Yamadaoka, Suita, Osaka 565-0871, the ^¶Department of Molecular Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo, Izumi, Osaka 594-1101, and the ^||Department of Disease Glycomics, Research Institute for Microbial Diseases, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

The N-glycosylation of integrin 51 is thought to play crucial roles in cell spreading, cell migration, ligand binding, and dimer formation, but the underlying mechanism remains unclear. To investigate the importance of the N-glycans of this integrin in detail, sequential site-directed mutagenesis was carried out to remove single or combined putative N-glycosylation sites on the5 integrin. Removal of the putative N-glycosylation sites on the -propeller, Thigh, Calf-1, or Calf-2 domains of the 5 subunit resulted in a decrease in molecular weight compared with the wild type, suggesting that all of these domains contain attached N-glycans. Importantly, the absence of N-glycosylation sites (sites 1–5) on the -propeller resulted in the persistent association of integrin subunit with calnexin in the endoplasmic reticulum, which subsequently blocked heterodimerization and its expression on the cell surface. Interestingly, the activities for cell spreading and migration for the 5 subunit carrying only three potential N-glycosylation sites (3–5 sites) on the-propeller were comparable with those of the wild type. In contrast, mutation of these three sites resulted in a significant decrease in cell spreading as well as functional expression, although the total expression level of the 3–5 mutant on the cell surface was comparable with that of wild type. Furthermore, we found that site 5 is a most important site for its expression on the cell surface, whereas the S5 mutant did not show any biological functions. Taken together, this study reveals for the first time that the N-glycosylation on the -propeller domain of the 5 subunit is essential for heterodimerization and biological functions of 51 integrin and might also be useful for studies of the molecular structure.

Received for publication, August 14, 2006 , and in revised form, September 7, 2006.

^* This work was supported in part by Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), and the 21st Century COE program from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 81-22-727-0078; Fax: 81-22-727-0216; E-mail: jgu{at}tohoku.pharm.ac.jp.

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