Identification of Active Sequences in the L4a Domain of Laminin 伪5 Promoting Neurite Elongation

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• 作者：Fumihiko Katagiri ; Misuzu Sudo ; Takayuki Hamakubo ; Kentaro Hozumi ; Motoyoshi Nomizu ; Yamato Kikkawa
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：June 19, 2012
• 年：2012
• 卷：51
• 期：24
• 页码：4950-4958
• 全文大小：552K
• 年卷期：v.51,no.24(June 19, 2012)
• ISSN：1520-4995

文摘

Laminin 伪5 is an extracellular matrix protein containing multiple domains implicated in various biological processes, such as embryogenesis and renal function. In this study, we used recombinant proteins and synthetic peptides to identify amino acid residues within the short arm region of 伪5 that were critical for neurite outgrowth activity. The short arm of 伪5 contains three globular domains (LN, L4a, and L4b) and three rodlike elements (LEa, LEb, and LEc). Recombinant proteins comprised of the 伪5 short arm fused with a Fc tag produced in 293 cells were assayed for PC12 (pheochromocytoma) cell adhesion and neurite outgrowth activities. Although it did not have cell attachment activity, neurite outgrowth was promoted by the recombinant protein. To narrow the region involved in neurite outgrowth activity, two truncated recombinant proteins were produced in 293 cells. A recombinant protein lacking L4a and LEb lost activity. Furthermore, we synthesized 78 partially overlapping peptides representing most of the amino acid sequences of L4a and LEb. Of the peptides, A5鈥?6 [mouse laminin 伪5 928鈥?39 (TSPDLFRLVFRY) in L4a] exhibited neurite outgrowth activity. Mutagenesis studies showed that Phe⁹³³ and Arg⁹³⁴ were involved in neurite outgrowth activity. Moreover, inhibition assays using anti-integrin monoclonal antibodies showed that neurite outgrowth on the 伪5 short arm was partially mediated by integrin 伪1尾1. However, the antibodies to integrin 伪1 and 尾1 did not inhibit neurite elongation on the A5鈥?6 peptide. These results suggest that in addition to cellular interactions with the active site in the L4a domain, the binding of integrin 伪1尾1 seems to modulate neurite elongation on the short arm of 伪5.