Identification of cell adhesive sequences in the N-terminal region of the laminin α2 chain.
The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.
Department of Clinical Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Kentaro
K
Hozumi
Masaya
M
Ishikawa
Takemitsu
T
Hayashi
Yuji
Y
Yamada
Fumihiko
F
Katagiri
Yamato
Y
Kikkawa
Motoyoshi
M
Nomizu
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D002448:Cell Adhesion / Q000502:physiology; D006801:Humans; D038982:Integrin alpha2beta1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007797:Laminin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016716:PC12 Cells; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D051381:Rats
D000906:Antibodies; D038982:Integrin alpha2beta1; D007797:Laminin; D010455:Peptides; C102645:laminin alpha 2