Complete sequence, recombinant analysis and binding to laminins and sulphated ligands of the N-terminal domains of laminin α3B and α5 chains

JHO Garbe, W Göhring, K Mann, R Timpl… - Biochemical …, 2002 - portlandpress.com
JHO Garbe, W Göhring, K Mann, R Timpl, T Sasaki
Biochemical Journal, 2002portlandpress.com
The N-terminal sequences of mouse laminin α3B and α5 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 (45–65kDa) from this region yielding properly
folded proteins, which were secreted from HEK-293—EBNA cells. Pepsin digestion of these
fragments yielded products of 25–35kDa, which consisted only of domain V. The αVI/V …
The N-terminal sequences of mouse laminin α3B and α5 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 (45–65kDa) from this region yielding properly folded proteins, which were secreted from HEK-293—EBNA cells. Pepsin digestion of these fragments yielded products of 25–35kDa, which consisted only of domain V. The αVI/V fragments were able to inhibit self-assembly of laminin-1, with those from the α3B and α5 chains being more active than those from α1 and α2 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 (Kd = 0.02 to > 6μM) for the binding to laminin-1. This indicated that laminins containing α3B or α5 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 α1/α2, moderate for α3B, whereas no binding was observed for α5. They all bound to heparan sulphate chains of perlecan and to sulphatides, with a lower variability in binding activity. Specific antibodies were raised against α3BVI/V and α5VI/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 α3B, β3 and γ2 chains.
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