Abstract
The laminin alpha1 chain is a subunit of laminin-1, a heterotrimeric basement membrane protein. The LG4-5 module at the C terminus of laminin alpha1 contains major binding sites for heparin, sulfatide, and alpha-dystroglycan and plays a critical role in early embryonic development. We previously identified active synthetic peptides AG73 and EF-1 from the sequence of laminin alpha1 LG4 for binding to syndecan and integrin alpha2beta1, respectively. However, their activity and functional relationship within the laminin-1 and LG4 as well as the functional relation between these sites and alpha-dystroglycan binding sites in LG4 are not clear. To address these questions, we created mutant recombinant LG4 proteins containing alanine substitutions within the AG73 (M1), EF-1 (M2, M3), and alpha-dystroglycan binding sites (M4, M5) and analyzed their activities. We found that recombinant proteins rec-M1 and rec-M5, containing mutations within M1 and M5, respectively, did not bind heparin or lymphoid cell lines expressing syndecans. These results suggest that LG4 binds to heparin and syndecans through M1 and M5. Rec-M1 and rec-M5 reduced fibroblast attachment, whereas mutant rec-M2 and rec-M3 retained cell attachment activity but did not promote cell spreading. Fibroblast attachment to rec-LG4 was inhibited by heparin but not by integrin antibodies. Spreading of fibroblasts on rec-LG4 was inhibited by anti-integrin alpha2 and beta1 but not by anti-integrin alpha1 and alpha6. These results suggest that the M1 and M5 sites are necessary for cell attachment on LG4 through syndecans and that the EF-1 site is for cell spreading activity through integrin alpha2beta1. In contrast, laminin-1-mediated fibroblast attachment and spreading were not inhibited by heparin or anti-integrin alpha2. Our findings indicate that LG4 has a unique function distinct from laminin-1 and suggest that laminin alpha1 LG4-5 may also be produced by a proteolytic cleavage in certain tissues where it exerts its activity.
Highlights
Laminins are heterotrimeric basement membrane proteins that exert multiple biological functions through interactions with extracellular matrix molecules and with cell surface receptors
Spreading of fibroblasts on recombinant wild-type LG4 protein (rec-LG4) was inhibited by anti-integrin ␣2 and 1 but not by anti-integrin ␣1 and ␣6. These results suggest that the M1 and M5 sites are necessary for cell attachment on LG4 through syndecans and that the EF-1 site is for cell spreading activity through integrin ␣21
We found that peptide AG73 (RKRLQVQLSIRT, residues 2719 –2730) from LG4 is active for cell attachment, promotes neurite outgrowth, and binds to syndecans, a membrane-associated heparan sulfate proteoglycan (12–16), and peptide EF-1 (DYATLQLQEGRLHFMFDLG, residues 2747–2765), which consists of -sheet strands with their connecting loop region of LG4, binds to integrin ␣21 (17)
Summary
Cells and Culture—293 EBNA cells (Invitrogen) and human foreskin fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen). The expression vector for the LG4 module, pCEP4-MulPURD, is a modified vector of pCEP4-WT-His (33), a derivative of pCEP4 (Invitrogen), and contains a BM-40 signal peptide, a hexahistidine tag, and a multicloning site along with the cytomegalovirus promoter and enhancer In this vector the original hygromycin resistance gene was replaced by the puromycin resistance sequence from the pPUR vector (BD Bioscience). Cell Attachment Assay—96-Well round-bottomed microtiter plates (Immulon-2HB) were coated with recombinant proteins or laminin-1 from Engelbreth-Holm-Swarm (EHS) tumor (Sigma) in 50 l of D-PBS overnight at 4 °C and blocked for 60 min at RT with 200 l of heat-denatured (30 min at 70 °C) Dulbecco’s modified Eagle’s medium containing 1% bovine serum albumin (Sigma, 1% blocking solution) and washed twice with 0.1% blocking solution. Cells were incubated with anti-vinculin or integrin antibody in D-PBS, 0.05% Tween 20, and 7% MOM protein concentrate (Vector Laboratories, Inc.) to 10 g/ml for 60 min at RT or 4 °C overnight. Cells were mounted with VECTASHIELD (Vector Laboratories) and examined under a LSM510 fluorescent confocal microscope (Carl Zeiss)
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