Biologically active sequences in the mouse laminin alpha3 chain G domain.
The laminin alpha3 chain is mainly expressed at the skin, and its C-terminal G domain has a critical role in multiple biological functions. We screened for biologically active sites on the mouse laminin alpha3 chain G domain using 107 synthetic peptides on coated plates and conjugated to Sepharose beads with HT1080 human fibrosarcoma cells, HaCaT human skin keratinocyte cells, and human dermal fibroblasts (HDFs). Eleven peptides exhibited cell attachment activity with respect to the peptide-coated plates and/or peptide-Sepharose beads. MA3G28 (WTIQTTVDRGLL) strongly binds to HaCaT cells. Four peptides promoted PC12 cell neurite outgrowth. Heparin inhibited attachment of HDFs to eight peptides on the coated plates. In contrast, EDTA significantly inhibited attachment of HDFs to MA3G27 (NAPFPKLSWTIQ) and MA3G28 but had no effect on the attachment of the other peptides. HDF cells formed well-organized actin stress fibers and focal contacts with vinculin accumulation on MA3G27. Additionally, attachment of HDFs to MA3G27 was inhibited by anti-alpha6 and anti-beta1 integrin antibodies, suggesting that MA3G27 promotes alpha6beta1 integrin-mediated cell adhesion. MA3G57 (NQRLASFSNAQQS) exhibited cell attachment activity only in the peptide bead assay. MA3G57 conjugated to a chitosan membrane promoted HDF attachment and spreading with well-organized actin stress fibers. The anti-beta1 integrin antibody partially inhibited attachment of HDFs to the MA3G57-chitosan membrane, suggesting that the MA3G57 site is involved in beta1 integrin-mediated cell attachment. These active sites are likely important in the biological activities of the laminin alpha3 chain G domain and would be useful for the study of molecular mechanisms of laminin-receptor interactions.
Urushibata
Shunsuke
S
Laboratory of Clinical Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan.
Katagiri
Fumihiko
F
Takaki
Shu
S
Yamada
Yuji
Y
Fujimori
Chikara
C
Hozumi
Kentaro
K
Kikkawa
Yamato
Y
Kadoya
Yuichi
Y
Nomizu
Motoyoshi
M
D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007150:Immunohistochemistry; D007797:Laminin / Q000737:chemistry* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D016716:PC12 Cells; D051381:Rats
D007797:Laminin; C095567:laminin alpha 3