A Peptide within Laminin-2 Promotes Nerve Cell's Attachment through Syndecan

Objectives: Laminin-2, a heterotrimeric (a2b1g1) glycoprotein, regulates various cellular functions, particularly critical for basement membrane assembly and peripheral myelinogenesis in mice. However, the biologically active sequence affecting nerve cell's behavior from human laminin-2 and its cellular receptor and downstream signaling pathway are largely unknown. This study was performed to identify the biologically active sequence that promotes nerve cell's attachment from human laminin-2 and to clarify its cellular receptor and downstream signaling pathway.

Methods: We expressed the five human a2 chain globular (LG) domains as monomeric, soluble fusion proteins, and examined their biological functions and receptor. We also prepared synthetic peptides within LG1 domain and investigated their biological properties.

Results: We found here that a recombinant LG1 (rLG1) domain had nerve cell's attachment and heparin binding activities. Studies with synthetic peptides delineated the Ln2-LG1-P3 (aa 2221-2232) within LG1 as a major site for both heparin and cell binding. Cell attachment to rLG1 and Ln2-LG1-P3 was inhibited by heparitinase I and chondroitinase ABC treatment. Syndecan-1 and -4 from PC12 cells can bind rLG1, but not glypican-1. Double immunostaining assays confirmed that syndecan-1 and -4 colocalized with the rLG1 and Ln2-LG1-P3. The binding of syndecan-4 with rLG1 and Ln2-LG1-P3 regulated localization and stability of protein kinase C (PKC)-a and stimulated focal adhesion kinase (FAK) phosphorylation. Furthermore, cell behavior was significantly inhibited by a specific inhibitor of PKC-a, Gö 6976.

Conclusion: Taken together, our findings indicate that the Ln2-LG1-P3 within the human laminin-2 a2 chain is a major lignad for syndecan-1 and -4, and that syndecan-1 and -4 mediate nerve cell's attachment through PKC-a/FAK signaling pathway. This work was supported by a grant from the KOSEF through the IBEC at Seoul National University (to B.-M. M.).