Globular domains 4/5 of the laminin alpha3 chain mediate deposition of precursor laminin 5.
In epidermal wounds, precursor laminin 5 (alpha3beta3gamma2) is deposited in the provisional basement membrane (PBM) before other BM components. Precursor laminin 5 contains G4/5 globular domains at the carboxyl terminus of the alpha3 chain. Here, the function of G4/5 was evaluated in deposition of laminin 5. Soluble laminin 5, secreted by keratinocytes in culture, is cleaved by an endogenous protease releasing G4/5. Thrombin, a serum protease, cleaves G4/5 indistinguishably from endogenous protease. Soluble human precursor laminin 5, but not cleaved laminin 5, was bound and deposited by mouse keratinocytes null for mouse alpha3 chain (alpha3-/- MKs). The deposition rescued adhesion and spreading and survival. In a model for PBM assembly, precursor laminin 5 was deposited along fibronectin fibrils at the junction between co-cultures of keratinocytes and fibroblasts. In both models, the deposition of precursor laminin 5 was inhibited by removal of G4/5 with thrombin. To confirm that G4/5 participates in deposition, the human LAMA3A gene was modified to produce alpha3 chains either without or with G4/5 that cannot be cleaved. Both precleaved and noncleavable alpha3 isoforms were expressed in alpha3-/- MKs, where they deposited sufficiently to rescue adhesion via integrins alpha3beta1 and alpha6beta4. Despite this similarity, noncleavable laminin 5 was at least threefold more efficiently deposited than precleaved isoform. We conclude that the G4/5 domain in the alpha3 chain facilitates deposition of precursor laminin 5 into the PBM in epidermal wounds.
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue, Seattle, WA 98109, USA.
Randy O
RO
Sigle
Susana G
SG
Gil
Mallar
M
Bhattacharya
Maureen C
MC
Ryan
Tai-Mei
TM
Yang
Tod A
TA
Brown
Ariel
A
Boutaud
Yuko
Y
Miyashita
John
J
Olerud
William G
WG
Carter
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism* / Q000473:pathology; D002448:Cell Adhesion / Q000502:physiology*; D049109:Cell Proliferation; D018920:Coculture Techniques; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D039201:Integrin alpha3beta1 / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism* / Q000473:pathology; D007797:Laminin / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D013917:Thrombin / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology
D005353:Fibronectins; D039201:Integrin alpha3beta1; D007797:Laminin; C095567:laminin alpha 3; D013917:Thrombin