Kinetic analysis of L1 homophilic interaction: role of the first four immunoglobulin domains and implications on binding mechanism.
L1 is a cell adhesion molecule of the immunoglobulin (Ig) superfamily, critical for central nervous system development, and involved in several neuronal biological events. It is a type I membrane glycoprotein. The L1 ectodomain, composed of six Ig-like and five fibronectin (Fn) type-III domains, is involved in homophilic binding. Here, co-immunoprecipitation studies between recombinant truncated forms of human L1 expressed and purified from insect Spodoptera frugiperda Sf9 cells, and endogenous full-length L1 from human NT2N neurons, showed that the L1 ectodomain (L1/ECD) and L1/Ig1-4 interacted homophilically in trans, contrary to mutants L1/Ig1-3 and L1/Ig2-Fn5. All mutants were correctly folded as evaluated by combination of far-UV CD and fluorescence spectroscopy. Surface plasmon resonance analysis showed comparable dissociation constants of 116 +/- 2 and 130 +/- 6 nm for L1/ECD-L1/ECD and L1/ECD-L1/Ig1-4, respectively, whereas deletion mutants for Ig1 or Ig4 did not interact. Accordingly, in vivo, Sf9 cells stably expressing L1 were found to adhere only to L1/ECD- and L1/Ig1-4-coated surfaces. Furthermore, only these mutants bound to HEK293 cells overexpressing L1 at the cell surface. Enhancement of neurite outgrowth, which is the consequence of signaling events caused by L1 homophilic binding, was comparable between L1/ECD and L1/Ig1-4. Altogether, these results showed that domains Ig1 to Ig4 are necessary and sufficient for L1 homophilic binding in trans, and that the rest of the molecule does not contribute to the affinity under the conditions of the current study. Furthermore, they are compatible with a cooperative interaction between modules Ig1-Ig4 in a horseshoe conformation.
Gouveia
Ricardo M
RM
Instituto de Tecnologia Química e Biológica, Avenida da República, Apartado 127, 2780-157 Oeiras, Portugal.
Gomes
Cláudio M
CM
Sousa
Marcos
M
Alves
Paula M
PM
Costa
Júlia
J
D000818:Animals; D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry*; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D039842:Neural Cell Adhesion Molecule L1 / Q000737:chemistry*; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence / Q000379:methods; D018411:Spodoptera / Q000378:metabolism; D020349:Surface Plasmon Resonance
D007136:Immunoglobulins; D039842:Neural Cell Adhesion Molecule L1