Globular domains 4/5 of the laminin α3 chain mediate deposition of precursor laminin 5

Abstract

In epidermal wounds, precursor laminin 5 (α3β3γ2) 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 α3 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 α3 chain (α3–/– 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 α3 chains either without or with G4/5 that cannot be cleaved. Both precleaved and noncleavable α3 isoforms were expressed in α3–/– MKs, where they deposited sufficiently to rescue adhesion via integrins α3β1 and α6β4. 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 α3 chain facilitates deposition of precursor laminin 5 into the PBM in epidermal wounds.

Fluorescently tagged laminin subunits facilitate analyses of the properties, assembly and processing of laminins in live and fixed lung epithelial cells and keratinocytes

“…3). Moreover, this localization is identical to that observed when skin cells are processed for indirect immunofluorescence using laminin-332 antibodies, as shown by a number of different workers (see, for example, Sehgal et al, 2006;Sigle et al, 2004)). …”

“…First, it has been suggested that the LG4-5 domains of the α3 laminin subunit mediate deposition of laminin-332 into matrix (Sigle et al, 2004). This is not the case in HEKs and BEP2D cells since α3ΔLG4-5 is incorporated into their extracellular matrix.…”

Topical application of laminin-332 to diabetic mouse wounds

“…Additionally, the LM-332 contains a mix of both the precursor LM-332 as well as the cleaved LM-332. We have previously reported that Western blots indicated that the protein was primarily the cleaved LM-332 with only small amounts of precursor LM-332 (32). In normal human acute wounds, both precursor LM-332 and its mRNA have been detected in migrating KCs for up to 7 days.…”

“…Unfortunately, soluble purified precursor LM-332 is not currently available and when present even in small quantity, may be quickly cleaved (32). Sigle et al 2004 have engineered and expressed a non-cleavable LM-332 molecule by eliminating the cleavage site between the G4/ G5 segment and the remainder of the α chain (32). This approach bypasses the issue of rapid cleavage of LM-332, but this genetically engineered molecule is almost completely deposited by the KC's, precluding its release to culture medium and collection in sufficient quantities to test on experimental wounds.…”

Interaction of Syndecan and α6β4 Integrin Cytoplasmic Domains

“…The LG4,5 domain is typically cleaved and degraded immediately after LN332 secretion. However, its expression persists in keratinocytes migrating at the edge of epidermal wounds (61) and in a high percentage of squamous cell carcinomas (62), suggesting a possible connection between its expression and cell invasion, potentially by the Sdc1-dependent activation of ␣6␤4 integrin described here. Indeed, squamous carcinoma cells engineered to express the LG4,5 domain showed increased tumorigenesis in vivo (62).…”