Structural and Cell Adhesion Properties of Zebrafish Syndecan-4 Are Shared with Higher Vertebrates

Abstract

The syndecan proteoglycans are an ancient class of receptor, bearing heparan sulfate chains that interact with numerous potential ligands including growth factors, morphogens, and extracellular matrix molecules. The single syndecan of invertebrates appears not to have cell adhesion roles, but these have been described for mammalian paralogues, especially syndecan-4. This member is best understood in terms of interactions, signaling, and structure of its cytoplasmic domain. The zebrafish homologue of syndecan-4 has been genetically linked to cell adhesion and migration in zebrafish embryos, but no molecular and cellular studies have been reported. Here it is demonstrated that key functional attributes of syndecan-4 are common to both zebrafish and mammalian homologues. These include glycosaminoglycan substitution, a NXIP motif in the extracellular domain that promotes integrin-mediated cell adhesion, and a transmembrane GXXXG motif that promotes dimer formation. In addition, despite some amino acid substitutions in the cytoplasmic domain, its ability to form twisted clamp dimers is preserved, as revealed by nuclear magnetic resonance spectroscopy. This technique also showed that phosphatidylinositol 4,5-bisphosphate can interact with the zebrafish syndecan-4 cytoplasmic domain, and that the molecule in its entirety supports focal adhesion formation, and complements the murine null cells to restore a normal actin cytoskeleton identically to the rat homologue. Therefore, the cell adhesion properties of syndecan-4 are consistent across the vertebrate spectrum and reflect an early acquisition of specialization after syndecan gene duplication events at the invertebrate/early chordate boundary.Syndecans are heparan sulfate bearing type-1 transmembrane proteins intimately associated with cell adhesion and linkage to the cytoskeleton. In mammals there are four syndecan family members, syndecan-1, -2, -3, and -4. These are characterized by a short highly conserved cytoplasmic domain, a transmembrane domain, and a less conserved ectodomain that is substituted with heparan sulfate chains (for reviews, see Refs. 1-3). Syndecan-4 is expressed in nearly all cell types and tissues and is a focal adhesion component (4 -5). In fibroblasts seeded on individual fibronectin domains there is a requirement for the engagement of syndecan-4 through its heparan sulfate chains for focal adhesion formation. Cells seeded on the RGD containing integrin-binding domain of fibronectin only form focal adhesions after the addition of the Hep II fibronectin heparin-binding domain (6, 7). In the syndecan-4 knock-out mouse vascular, wound healing, and migration defects have been reported and the ability of syndecan-4 null fibroblasts to form focal adhesions in response to fibronectin fragments is compromised (8).The syndecan-4 cytoplasmic domain shares two highly conserved regions, the C1 and C2, with the other syndecan family members and they flank a variable region (V region) unique to syndecan-4. The membrane distal C2 region interacts with PDZ p...

Transmembrane Signaling Proteoglycans

“…Its V region is highly cationic (Figure 3) and provides the structural basis for the twisted clamp motif found via NMR spectroscopy (Shin et al 2001). This structure undergoes a conformational change in the presence of PI4,5P 2 that interacts primarily with some of the eight lysine residues present in each V region dimer (Lee et al 1998, Whiteford et al 2008). This combination is proposed to provide a platform for PI4,5P 2 and syndecan-4 to interact with protein kinase Cα (PKCα) in a ternary complex.…”

“…Between these regions is a single V (variable) region that is unique to each syndecan but broadly conserved across species. Those of invertebrates are longest; the shortest is that of syndecan-4, but it is conserved from zebrafish to human (Whiteford et al 2008). With respect to interactions, there is a dearth of information from invertebrates that extends even to the C2 region, which is known in vertebrate syndecans to bind several PDZ domain proteins.…”

“…The activation of β 1 integrins by syndecan-2 and -4 ectodomains is, in contrast, indirect. An NPXY motif in a small but highly conserved region of syndecan-4 is essential (Whiteford & Couchman 2006) and functional in zebrafish (Whiteford et al 2008). One or more intermediate proteins on the cell surface presumably bridge to the integrin.…”

Zebrafish: Modeling for Herpes Simplex Virus Infections

“…15 The diverse expression of the 3-OST family members in the zebrafish embryo model system (Table 1) was the main rationale to test the role of zebrafish encoded 3-OST isoforms in mediating HSV-1 infection. Using Chinese hamster ovary (CHO-K1) cells transiently expressing zebrafish encoded 3-OST-2 and 3-OST-3 isoforms resulted in HSV-1 infection.…”

High-yield Expression and Characterization of Syndecan-4 Extracellular, Transmembrane and Cytoplasmic Domains

“…23,24 This property was concerned to researchers, thus it had been well-known that structure of cytodomain of syndecan-4 was formed to multimer and this structural property dominated these functions. [25][26][27] However, this structural information was obtained only with cytoplasmic domain of syndecan-4 without other regions like transmembrane domain and ecto domain. Thus, we tried to study the more realistic structural studies of syndecan-4 that contain transmembrane, cytoplasmic, and ectoplasmic domain.…”