Normal Human Keratinocytes Bind to the α3LG4/5 Domain of Unprocessed Laminin-5 through the Receptor Syndecan-1

Abstract

Basal keratinocytes of the epidermis adhere to their underlying basement membrane through a specific interaction with laminin-5, which is composed by the association of ␣3, ␤3, and ␥2 chains. Laminin-5 has the ability to induce either stable cell adhesion or migration depending on specific processing of different parts of the molecule. One event results in the cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) of the ␣3 chain. In this study, we recombinantly expressed the human ␣3LG4/5 fragment in mammalian cells, and we show that this fragment induces adhesion of normal human keratinocytes and fibrosarcoma-derived HT1080 cells in a heparan-and chondroitin sulfate-dependent manner. Immunoprecipitation experiments with Na 2 35 SO 4 -labeled keratinocyte and HT1080 cell lysates as well as immunoblotting experiments revealed that the major proteoglycan receptor for the ␣3LG4/5 fragment is syndecan-1. Syndecan-4 from keratinocytes also bound to ␣3LG4/5. Furthermore we could show for the first time that unprocessed laminin-5 specifically binds syndecan-1, while processed laminin-5 does not. These results demonstrate that the LG4/5 modules within unprocessed laminin-5 permit its cell binding activity through heparan and chondroitin sulfate chains of syndecan-1 and reinforce previous data suggesting specific properties for the precursor molecule.Laminins (LNs) 1 are extracellular matrix glycoproteins composed of ␣, ␤, and ␥ chains assembled into a cross-shaped heterotrimer (␣␤␥) by forming a triple-stranded coiled-coil structure through their ␣-helical domains. At present, 15 heterotrimers termed LN-1 to LN-15 have been described with different subunit composition selected from five individual ␣ chains (␣1-␣5), three ␤ chains (␤1-␤3), and three ␥ chains (␥1-␥3) (1, 2). All LN ␣ chains comprise a large globular domain in their carboxylterminal region (G domain), which consists of five homologous globular subdomains of about 200 amino acids each (LG1-LG5). LN-5, with chain composition ␣3␤3␥2, is a component of basement membranes underlying specialized epithelia with secretory or protective function (3). In skin, LN-5 is synthesized by keratinocytes initially as a high molecular mass precursor protein of 460 kDa of which the ␣3 and ␥2 chains undergo specific processing to smaller forms after being secreted and deposited into the extracellular matrix (ECM) (4, 5). Processing of the ␣3 chain consists of cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) (6, 7). An additional cleavage within the aminoterminal domain IIIa of the ␣3 chain occurs subsequently and may also be important for LN-5 function (8, 9). Processing of the ␥2 chain occurs at the amino terminus with loss of a 50-kDa domain (9 -11). Processed LN-5 is the major component of anchoring filaments in skin (12) where it mediates cell adhesion via interaction of the ␣3 carboxyl-terminal LG1-3 triplet domain with both ␣ 3 ␤ 1 and ␣ 6 ␤ 4 integrins (13-16). Several studies have suggested that processed LN-5 functions both in the nuclea...

Laminin-332-Integrin Interaction: A Target For Cancer Therapy?

“…For example, the globular (G) domain at the carboxy-terminal of the laminin α chain interacts with cell-surface receptors such as integrin, syndecan, and α-dystroglycan [13,[22][23][24]. In regard to the α3 laminin subunit, its G domain interacts with two integrins (α3β1 and α6 4 integrin) and syndecan [25,26].…”

“…The processing site is a spacer region between G3 and G4 at the amino acids Gln(1337)-Asp(1338) [77]. The G4/G5 fragment that is released is believed to stimulate cell migration [25].…”

Integrin Regulation of Epidermal Functions in Wounds

“…60 Integrin a 6 b 4 is a major transmembrane component of hemidesmosomes, which are the intermediate filamentassociated structures on the basal surfaces of keratinocytes that anchor the epidermis to the dermis. 10,61 In this context, a 6 b 4 is thought to play primarily a structural role with little or no signal transduction function.…”

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

“…Binding of laminin 1 (α1β1γ1) onto the surface of a 'competent' cell is dependent on the G4/5 domain of the laminin α1 chain (Li et al, 2003;Li et al, 2002;Smirnov et al, 2002). G4/5 in precursor α3 200 and soluble G4/5 bind heparin (Nguyen et al, 2000a) and as a result candidate receptors for cell surface binding of the precursor α3 200 chain of lam5 include sulfatecontaining syndecans (Okamoto et al, 2003;Utani et al, 2001), dystroglycans (Hohenester et al, 1999;Winder, 2001) and glycolipids (Hall et al, 1997;Taraboletti et al, 1990). Integrin α3β1 was suggested to incorporate lam5 into its In epidermal wounds, precursor laminin 5 (α3β3γ2) is deposited in the provisional basement membrane (PBM) before other BM components.…”