First Author | Garbe JH | Year | 2002 |
Journal | Biochem J | Volume | 362 |
Issue | Pt 1 | Pages | 213-21 |
PubMed ID | 11829758 | Mgi Jnum | J:75014 |
Mgi Id | MGI:2159549 | Doi | 10.1042/0264-6021:3620213 |
Citation | Garbe JH, et al. (2002) Complete sequence, recombinant analysis and binding to laminins and sulphated ligands of the N-terminal domains of laminin alpha3B and alpha5 chains. Biochem J 362(Pt 1):213-21 |
abstractText | The N-terminal sequences of mouse laminin alpha3B and alpha5 chains have been completed and demonstrate the presence of a signal peptide followed by a complete laminin N-terminal (LN) module (domain VI). These signal peptides were released after recombinant production of larger fragments comprising domains VI/V (45-65 kDa) from this region yielding properly folded proteins, which were secreted from HEK-293-EBNA cells. Pepsin digestion of these fragments yielded products of 25-35 kDa, which consisted only of domain V. The alphaVI/V fragments were able to inhibit self-assembly of laminin-1, with those from the alpha3B and alpha5 chains being more active than those from alpha1 and alpha2 chains. Domain V fragments, however, showed a reduced activity, indicating the major contribution of the LN module in inhibition. These interactions were confirmed by surface-plasmon-resonance assays demonstrating moderate affinities (K(d)=0.02 to >6 microM) for the binding to laminin-1. This indicated that laminins containing alpha3B or alpha5 chains should also be able to form non-covalent networks by polymerization. The LN modules also showed heparin binding in affinity chromatography, which was strongest for alpha1/alpha2, moderate for alpha3B, whereas no binding was observed for alpha5. They all bound to heparan sulphate chains of perlecan and to sulphatides, with a lower variability in binding activity. Specific antibodies were raised against alpha3BVI/V and alpha5VI/V and were shown to stain basement membrane zones in various mouse tissues. These antibodies also allowed the identification of a new laminin assembly form 5B consisting of alpha3B, beta3 and gamma2 chains. |