An alcohol binding site on the neural cell adhesion molecule L1

*Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, MA 02114;
^†Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and
^‡Veterans Affairs Boston Healthcare System and Department of Neurology, Harvard Medical School, West Roxbury, MA 02132

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved November 15, 2007 (received for review August 18, 2007)

Abstract

Prenatal ethanol exposure causes fetal alcohol spectrum disorders (FASD) in part by disrupting the neural cell adhesion molecule L1. L1 gene mutations cause neuropathological abnormalities similar to those of FASD. Ethanol and 1-butanol inhibit L1-mediated cell–cell adhesion (L1 adhesion), whereas 1-octanol antagonizes this action. To test the hypothesis that there are alcohol binding sites on L1, we used 3-azibutanol and 3-azioctanol, the photoactivatable analogs of 1-butanol and 1-octanol, to photolabel the purified Ig1–4 domain of human L1 (hL1 Ig1–4). 3-Azibutanol (11 mM), like ethanol, inhibited L1 adhesion in NIH/3T3 cells stably transfected with hL1, whereas subanesthetic concentrations of 3-azioctanol (14 μM) antagonized ethanol inhibition of L1 adhesion. 3-Azibutanol (100–1,000 μM) and 3-azioctanol (10–100 μM) photoincorporated into Tyr-418 on Ig4 and into two adjacent regions in the N terminus, Glu-33 and Glu-24 to Glu-27. A homology model of hL1 Ig1–4 (residues 33–422), based on the structure of the Ig1–4 domains of axonin-1, suggests that Glu-33 and Tyr-418 hydrogen-bond at the interface of Ig1 and Ig4 to stabilize a horseshoe conformation of L1 that favors homophilic binding. Furthermore, this alcohol binding pocket lies within 7 Å of Leu-120 and Gly-121, residues in which missense mutations cause neurological disorders similar to FASD. These data suggest that ethanol or selected mutations produce neuropathological abnormalities by disrupting the domain interface between Ig1 and Ig4. Characterization of alcohol agonist and antagonist binding sites on L1 will aid in understanding the molecular basis for FASD and might accelerate the development of ethanol antagonists.

Footnotes

^§To whom correspondence should be addressed. E-mail: mcharness{at}hms.harvard.edu
Author contributions: E.A. and S.S. contributed equally to this work; E.A. and S.S. carried out the photolabeling and mass spectrometry work; S.S. produced the homology model; M.F.W. designed the research, analyzed data, and carried out the adhesion assay; S.C. produced stable transfectants of hL1 Ig1–4-TEV-Fc-expressing cells; X.D. produced and purified hL1 Ig1–4; and M.E.C. and K.W.M. designed research, analyzed data, and wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0707815105/DC1.