Abstract
Ethanol may cause fetal alcohol spectrum disorders (FASD) in part by inhibiting cell adhesion mediated by the L1 neural cell adhesion molecule. Azialcohols photolabel Glu-33 and Tyr-418, two residues that are predicted by homology modeling to lie within 2.8 Å of each other at the interface between the Ig1 and Ig4 domains of L1 (Arevalo, E., Shanmugasundararaj, S., Wilkemeyer, M. F., Dou, X., Chen, S., Charness, M. E., and Miller, K. W. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 371-375). Using transient transfection of NIH/3T3 cells with wild type (WT-L1) and mutated L1, we found that cysteine substitution of both residues (E33C/Y418C-L1) significantly increased L1 adhesion above levels observed for WT-L1 or the single cysteine substitutions E33C-L1 or Y418C-L1. The reducing agent β-mercaptoethanol (βME) reversibly decreased the adhesion of E33C/Y418C-L1, but had no effect on WT-L1, E33C-L1, or Y418C-L1. Thus, disulfide bond formation occurs between Cys-33 and Cys-418, confirming both the close proximity of these residues and the importance of Ig1-Ig4 interactions in L1 adhesion. Maximal ethanol inhibition of cell adhesion was significantly lower in cells expressing E33C/Y418C-L1 than in those expressing WT-L1, E33C-L1, or Y418C-L1. Moreover, the effects of βME and ethanol on E33C/Y418C-L1 adhesion were non-additive. The cutoff for alcohol inhibition of WT-L1 adhesion was between 1-butanol and 1-pentanol. Increasing the size of the alcohol binding pocket by mutating Glu-33 to Ala-33, increased the alcohol cutoff from 1-butanol to 1-decanol. These findings support the hypothesis that alcohol binding within a pocket bordered by Glu-33 and Tyr-418 inhibits L1 adhesion by disrupting the Ig1-Ig4 interaction.
Highlights
Ethanol may cause fetal alcohol spectrum disorders (FASD) in part by inhibiting cell adhesion mediated by the L1 neural cell adhesion molecule
Effect of Cysteine Substitution at Glu-33 and Tyr-418 on L1 Adhesion—To determine whether Glu-33 and Tyr-418 are close enough to form a functional disulfide bond [40], we created human L1 (hL1) constructs with cysteine substitutions at Glu-33 (E33C-L1), Tyr-418 (Y418C-L1), or both sites (E33C/Y418CL1) and transiently transfected NIH/3T3 fibroblast cells with wild-type L1 (WT-L1) and mutant-L1 plasmid DNA
Three principal findings of this study support the hypothesis that alcohol inhibits L1 adhesion partly by interacting with a binding site at the domain interface between Ig1 and Ig4 of the L1-extracellular domain (ECD)
Summary
Mutagenesis—Several L1 mutants were generated from a vector containing wild-type human L1 (hL1) cDNA gene (RSLEϩ) kindly provided by Dr Patricia Maness (University of North Carolina; Chapel Hill, NC). Inhibition of L1 adhesion was calculated as [100 (1- (the ratio of L1 adhesion in the presence and absence of inhibitors))] [7, 29] These mutational analyses were performed using transfected NIH/ 3T3 cells. Cells were fixed in 4% paraformaldehyde, blocked with PBS supplemented with 5% bovine serum (PBS/BS), and stained overnight at room temperature with mouse L1 mAb 5G3 in PBS/BS, washed three times with PBS, incubated for 3 h at room temperature with goat anti-mouse antibody conjugated with Alexa Fluor 594 (Invitrogen) in PBS/BS, and washed again with PBS. Flow Cytometry—Cells cultured in T75 flasks or 6-well plates were detached in PBS supplemented with 2 mM EDTA and separated into a single-cell suspension by trituration. Nonlinear least squares analysis was done with Igor (Wave Metrics Inc)
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