Complete sequence, recombinant analysis and binding to laminins and sulphated ligands of the N-terminal domains of laminin alpha3B and alpha5 chains.
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.
Garbe
Jörg H O
JH
Max-Planck-Institut für Biochemie, Klopferspitz 18A, D-82152 Munich, Germany.
Göhring
Walter
W
Mann
Karlheinz
K
Timpl
Rupert
R
Sasaki
Takako
T
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007797:Laminin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013464:Sulfuric Acids / Q000737:chemistry; D020349:Surface Plasmon Resonance
D007797:Laminin; D008024:Ligands; D011994:Recombinant Proteins; D013464:Sulfuric Acids; C097043:laminin alpha5; C095567:laminin alpha 3