Syndecan Binding Sites in the Laminin α1 Chain G Domain

Abstract

The laminin alpha1 chain G domain has multiple biological activities. Previously, we identified cell binding sequences in the laminin alpha1 chain G domain by screening 113 synthetic peptide-polystyrene beads for cell attachment activity. Here, we have used a recombinant protein of the laminin alpha1 G domain (rec-alpha1G) and a large set of synthetic peptides to further identify and characterize heparin, cell, and syndecan-4 binding sites in the laminin alpha1 chain G domain. The rec-alpha1G protein promoted both cell attachment and heparin binding (K(D) = 19 nM). Cell attachment to the rec-alpha1G protein was inhibited 60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparin binding to the rec-alpha1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 (IC(50) = 147 microM) and AG75 (IC(50) = 206 microM), inhibited heparin binding to rec-alpha1G. When the peptides were compared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73 also inhibited fibroblast attachment to the rec-alpha1G protein, but AG75 did not. Cell attachment to the peptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promoted cell attachment in both assays, but AG75 only showed cell attachment activity in the bead assay. Additionally, AG73, but not AG75, inhibited branching morphogenesis of mouse submandibular glands in organ culture. Furthermore, the rec-alpha1G protein bound syndecan-4, and both AG73 and AG75 inhibited this binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4 binding in the laminin alpha1 chain G domain. These sites may play a critical role in the diverse biological activities involving heparin and syndecan-4 binding.

Identification of Cell Adhesive Sequences in the N-terminal Region of the Laminin α2 Chain

“…We have reported that AG10, AG32 (TWYKI-AFQRNRK, mouse laminin ␣1 chain, residues 2370 -2381) (15,39), A2G10, and MA3G27 (NAPFPKLSWTIQ, mouse laminin ␣3 chain, residues 1851-1862) (37) interact with integrin ␣6␤1, and EF-1 binds to integrin ␣2␤1 (17). Additionally, we have also identified syndecan-binding sequences on AG73 (18,21), MA3G70 (KPRLQFSLDIQT, mouse laminin ␣3 chain, residues 2243-2254) (19), A4G82 (20), and A5G81 (AGQWHRVS-VRWG, mouse laminin ␣5 chain, residues 3337-3348) (40), CD44 binding sequences on A5G27 (41), and ␣-DG binding sequences on A2G78 and A2G80 (12). On the other hand, a major functional role of the N-terminal region of the laminin ␣ chain has been thought to be laminin-laminin assembly to form a mesh-like structure (5).…”

“…Cell Attachment Assay Using Peptide-conjugated Sepharose Beads-The synthetic peptides were coupled to CNBr-activated Sepharose 4B (GE Healthcare) as described previously (21). The peptides (200 g) were incubated with the CNBractivated Sepharose (30 mg) in 1 ml of 0.1 M NaHCO 3 , containing 0.5 M NaCl (pH 8.…”

“…The coated plate method determines morphological differences and dose dependence of cell attachment, whereas the Sepharose bead conjugation method regulates the amount of peptides. AG73, which was identified as the strongest peptide for cell attachment in the mouse ␣1 chain LG4 module, and its scrambled peptide AG73T were used as positive and negative controls, respectively (21).…”

“…Many active peptides were derived from the LG modules, and some of them were suggested to play a critical role in the specific binding to cell surface receptors. AG73 (RKRLQVQLSIRT, mouse laminin ␣1 chain, residues 2719 -2730) (15,18), A3G756 (KNSFMALYLSKGRLVFALG, mouse laminin ␣3 chain, residues 1411-1429) (19), and A4G82 (TLFLAHGRLVFM, mouse laminin ␣4 chain, residues 1514 -1525) (20) bind to syndecans and promote various biological activities, including cell adhesion (20,21), migration (20,21), neurite outgrowth (22,23), branching morphogenesis (24), and tumor growth and metastasis (25,26). EF-1 (DYATLQ-LQEGRLHFMFDLG, mouse laminin ␣1 chain, residues 2747-2765) specifically interacts with integrin ␣2␤1 and induces cell spreading, focal adhesions, and production of actin stress fibers (17).…”

Preparation and Characterization of Laminin-Derived Peptide AG73-Coated Liposomes as a Selective Gene Delivery Tool

“…AG73 peptide is as a ligand for syndecans, one of the major heparin sulfatecontaining transmembrane protein. [11][12][13] It has been reported that syndecan-2 is highly expressed in various cancer cells and play a role in angiogenesis. [14][15][16][17][18] Therefore, AG73-labeled polyethyleneglycol-modified liposomes (AG73-PEG liposomes) were prepared, which were capable of encapsulating a gene condensed by poly-L-lysine.…”