Identification of Biologically Active Sequences in the Laminin α4 Chain G Domain

Abstract

Laminins are a family of trimeric extracellular matrix proteins consisting of ␣, ␤, and ␥ chains. So far five different laminin ␣ chains have been identified. The laminin ␣4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin ␣4 chain. Here we have focused on the biological functions of the laminin ␣4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-␣4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-␣4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-␣4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVIS-LYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-␣4G. These results suggest that the four active sites are involved in the biological functions of the laminin ␣4 chain G domain. Furthermore, rec-␣4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways.Laminins, a family of extracellular matrix proteins, consist of three different subunits, ␣, ␤, and ␥ chains. So far, five ␣, three ␤, and three ␥ chains have been identified, and at least 15 isoforms (laminin 1-15) are formed by various combinations of each subunit (1-4). Laminins have diverse biological activities including promotion of cell adhesion, migration, neurite outgrowth, angiogenesis, and tumor metastasis (5). More than 20 receptors have been reported for these laminin molecules (6). Several active sites on laminin-1 have been identified using proteolytic fragments, recombinant proteins, and synthetic peptides (7,8). Previously, we screened for cell adhesive sequences on laminin-1 using 673 overlapping synthetic peptides covering the entire protein (9 -12). Most of the active peptides were localized in the globular domains and found to play a critical role in binding to cell surface receptors in a peptide-and cell type-specific manner (13,14). Several peptides were found to interact with integrins and syndecans (15-18). Some of the peptides promoted neurite outgrowth, angiogenesis, and tumor metastasis (19 -22).The laminin ␣ chains are generally large (M r ϭ 400,000) and contain a C-terminal globular domain consisting of five globular modules LG1-LG5. The laminin ␣4 chain lacks the N-terminal short arm and is expressed in cells of mesenchymal origin, such as...

Extracellular Matrix Organization, Structure, and Function

“…However, this glycoprotein may influence valve cell types differently. In vivo and in vitro studies have shown that laminin interacts with endothelial and epithelial cells, and can help maintain physiological functionality of the cells [97,100,101]. However, VICs cultured on laminin have been found to support high quantities of calcific nodule formation in the presence of TGF-β, when compared to subendothelial ECM components collagen type I and fibronectin [19,102].…”

“…In addition to their structural contributions to the basement membrane, laminins are essential for proper biological activity. These glycoproteins have been shown to promote cell adhesion, migration, differentiation, and maintenance of cellular phenotype [94,97,98]. Dysfunction in laminin expression has been linked to diseases with improper tissue formation such as muscular dystrophy, epidermolysis bullosa, and various nephritic syndromes [94,99].…”

Biological Activities of Homologous Loop Regions in the Laminin α Chain G Domains

“…The A3G75 site (KNSFMALYLSKG, human laminin ␣ 3 chain 1411-1422) promoted syndecan-2-and syndecan-4-mediated cell attachment and neurite outgrowth (29,30). The A4G82 (TLFLAH-GRLVFM, mouse laminin ␣ 4 chain 1514 -1525) sequence showed heparin binding and cell attachment activity (31,32). These active sequences are in the homologous region of the LG4 modules of the ␣ 3 and ␣ 4 chains.…”

“…The crystal structure of the laminin ␣ 2 LG5 module has been prepared and found to consist of 14 ␤ strands (A-N strands) (35). The homologous regions of A3G75 and A4G82 are located on the connecting loop region between the E and F strands (14,29,30,32). Additionally, mutagenesis analysis using recombinant LG4 proteins with serine or alanine substitutions of basic residues (Lys 1421 and Arg 1423 , human laminin ␣ 3 chain; His 1519 and Arg 1521 , mouse laminin ␣ 4 chain) in the E-F loop regions suggested that the loop regions are critical for the biological activity of the recombinant proteins (29,31).…”

The Requirement of the Glutamic Acid Residue at the Third Position from the Carboxyl Termini of the Laminin γ Chains in Integrin Binding by Laminins

“…By analogy with the identification of the Arg-Gly-Asp (RGD) cell-adhesive motif in fibronectin, many attempts have been made to identify specific sequences mimicking the integrin binding activity of laminins. However, neither recombinant fragments of the G domain nor synthetic peptides modeled after the sequences in the G domain have shown any significant cell-adhesive activity comparable with that of intact laminins (15)(16)(17)(18). Previously, we found that deletion of the LG4-5 modules did not compromise the ability of laminin-511 4 (␣5␤1␥1) to bind ␣3␤1/␣6␤1 integrins, but further deletion up to the LG3 module resulted in the loss of its integrin binding ability (19).…”