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J. Biol. Chem., Vol. 280, Issue 15, 14370-14377, April 15, 2005

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Regulation of Biological Activity and Matrix Assembly of Laminin-5 by COOH-terminal, LG4–5 Domain of 3 Chain^*

Yoshiaki Tsubota, Chie Yasuda, Yoshinobu Kariya, Takashi Ogawa, Tomomi Hirosaki¶, Hiroto Mizushima||, and Kaoru Miyazaki**

From the Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University and the Kihara Memorial Yokohama Foundation for the Advancement of Life Sciences, 641-12 Maioka-cho, Totsuka-ku, Yokohama 244-0813, Japan

The basement membrane protein laminin-5 (LN5; 332) undergoes specific proteolytic processing of the 190-kDa 3 chain to the 160-kDa form after the secretion, releasing its COOH-terminal, LG4–5 domain. To clarify the biological significance of this processing, we tried to express a recombinant precursor LN5 with a 190-kDa 3 chain (pre-LN5), in which the cleavage sequence Gln-Asp was changed to Ala-Ala by point mutation. When the wild-type and mutated LN5 heterotrimers were expressed in HEK293 cells, the wild-type 3 chain was completely cleaved, whereas the mutated 3 chain was partially cleaved at the same cleavage site (Ala-Ala). pre-LN5 was preferentially deposited on the extracellular matrix, but this deposition was effectively blocked by exogenous heparin. This suggests that interaction between the LG4–5 domain and heparan sulfate proteoglycans on the cell surface and/or extracellular matrix is important in the matrix assembly of LN5. Next, we purified both pre-LN5 and the mature LN5 with the processed, 160-kDa 3 chain (mat-LN5) from the conditioned medium of the HEK293 cells and compared their biological activities. mat-LN5 showed higher activities to promote cell adhesion, cell scattering, cell migration, and neurite outgrowth than pre-LN5. These results indicate that the proteolytic removal of LG4–5 from the 190-kDa 3 chain converts the precursor LN5 from a less active form to a fully active form. Furthermore, the released LG4–5 fragment stimulated the neurite outgrowth in the presence of mat-LN5, suggesting that LG4–5 synergistically enhances integrin signaling as it is released from the precursor LN5.

Received for publication, November 18, 2004 , and in revised form, January 24, 2005.

^* This work was supported in part by grants-in-aid from Yokohama City Collaboration of Regional Entities for the Advancement of Technological Excellence, Japan Science and Technology Agency, and 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.

^¶ Present address: Discovery Research Laboratories II, Minase Research Institute, Ono Pharmaceutical Co. Ltd., Osaka 618-8585, Japan.

^|| Present address: Division of Cell Biology, Research Institute for Microbial Disease, Osaka University, Osaka 565-0871, Japan.

^** To whom correspondence should be addressed. Tel.: 81-45-820-1905; Fax: 81-45-820-1901; E-mail: miyazaki{at}yokohama-cu.ac.jp.

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