SCAN: The homophilic interaction of the L1 cell adhesion molecule: kinetic analysis and the functional role of the Ig1-Ig4 domains.

SCAN

Title: The homophilic interaction of the L1 cell adhesion molecule: kinetic analysis and the functional role of the Ig1-Ig4 domains

Speaker: Ricardo Gouveia, Glycobiology Laboratory

Abstract:

L1 is a cell adhesion glycoprotein of the immunoglobulin (Ig) superfamily, critical for central nervous system development. L1 is involved in several cellular functions, such as cell adhesion, neurite elongation, axon pathfinding and synaptic plasticity. This large protein is composed by an ectodomain region with six Ig-like and five fibronectin (Fn) type-III domains with 21 potential N-glycosylation sites, a single transmembrane region and a short cytoplasmic domain. L1-mediated functions depend on its interactions with various proteins of the cell membrane and extracellular matrix, including other L1 molecules (homophilic interaction).
In this work, we sought to characterize the kinetics for L1 homophilic interaction. For this, several truncated forms of human L1 were overexpressed in insect Spodoptera frugiperda Sf9 cells using the stable expression system for a high-yield production, in the mg/L range. Purified L1 mutants were correctly folded -proteins, as evaluated by a combination of far-UV CD and fluorescence spectroscopies. Furthermore, they were found to be N-glycosylated and the N-glycans consisted mainly of paucimannosidic-type structures. Co-immunoprecipitation between L1 mutants and endogenous full-length L1 from human NT2N neurons, showed that the L1 ectodomain (L1/ECD) and L1/Ig1-4 interacted homophilicaly in trans, contrary to mutants where Ig1 or Ig4 was deleted. Surface Plasmon Resonance analysis showed comparable KD 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 expressing L1 were found to adhere only to L1/ECD and L1/Ig1-4 coated surfaces. Also, only these mutants bound to HEK293 cells overexpressing L1 at the cell surface. Neurite outgrowth enhancement, which is the consequence of signalling events caused by L1 homophilic binding, was comparable between L1/ECD and L1/Ig1-4.
Altogether, these results showed that Ig1-Ig4 domains are necessary and sufficient for L1 homophilic binding in trans. Furthermore, the data obtained is compatible with a cooperative interaction between modules Ig1-Ig4 in a horseshoe conformation.

Short CV:
1999-2004: Graduation in Biology by the Faculdade de Ciências, Universidade de Lisboa.
2003-2004: Undergraduate training at the Plant Genetic Engineering Lab, ITQB, under the supervision of Dr. Ana Sanchez and Prof. Margarida Oliveira.
2004-2005: Investigation fellowship integrated in the European Network project CellPROM, at the Glycobiology lab, ITQB, under the supervision of Dr. Júlia Costa.
Since January 2006: PhD fellowship entitled “Study of the effect of the cell adhesion glycoprotein L1 on neuronal differentiation”, in the context of the CellPROM project and financed by FCT, performed at the Glycobiology lab, ITQB, under the supervision of Dr. Júlia Costa.