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Complete sequence, recombinant analysis and binding to laminins and sulphated ligands of the N-terminal domains of laminin alpha 3B and alpha 5 chains

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons77989

Garbe,  J. H. O.
Former Research Groups, Max Planck Institute of Biochemistry, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons78355

Mann,  K.
Mann, Matthias / Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Max Planck Society;
Huber, Robert / Structure Research, Max Planck Institute of Biochemistry, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons78797

Timpl,  R.
Former Research Groups, Max Planck Institute of Biochemistry, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons78606

Sasaki,  T.
Former Research Groups, Max Planck Institute of Biochemistry, Max Planck Society;

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Zitation

Garbe, J. H. O., Göhring, W., Mann, K., Timpl, R., & Sasaki, T. (2002). Complete sequence, recombinant analysis and binding to laminins and sulphated ligands of the N-terminal domains of laminin alpha 3B and alpha 5 chains. Biochemical Journal, 362, 213-221.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0010-6FB8-C
Zusammenfassung
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 property 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 muM) 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.