Peptide Antagonists of Ethanol Inhibition of L1-Mediated Cell-Cell Adhesion

Abstract

Ethanol inhibits cell-cell adhesion mediated by the L1 cell adhesion molecule. 1-Octanol potently antagonizes this cellular action of ethanol and also prevents ethanol-induced dysmorphology and cell death in mouse whole embryo culture. NAPVSIPQ (NAP) and SALLRSIPA (SAL) are active peptide fragments of two neuroprotective proteins: activity-dependent neuroprotective protein and activity-dependent neurotrophic factor. NAP and SAL are neuroprotective at femtomolar concentrations against a variety of neurotoxins and also prevent ethanol teratogenesis in mice. To explore the cellular basis for this action, we asked whether NAP and SAL antagonize ethanol inhibition of L1 adhesion. Aggregation assays were carried out in ethanol-sensitive, human L1-transfected NIH/3T3 cells in the absence and presence of NAP and SAL. Neither NAP nor SAL altered L1 adhesion or L1 expression; however, both peptides potently and completely antagonized the inhibition of L1 adhesion by 100 mM ethanol (EC 50 : NAP, 6 ϫ 10 Ϫ14 M; SAL, 4 ϫ 10 Ϫ11 M). NAP also antagonized ethanol inhibition of cell-cell adhesion in bone morphogenetic protein-7-treated NG108-15 cells. In L1-expressing NIH/3T3 cells, SAL antagonism was reversible and could be overcome by increasing concentrations of ethanol. In contrast, NAP antagonism was irreversible and could not be overcome by increasing agonist concentration. Two scrambled NAP peptides (ASPNQPIV and PNIQVASP) were not antagonists at concentrations as high as 10 Ϫ7 M. Thus, two structurally unrelated classes of compounds, alcohols and small polypeptides, share two common actions: antagonism of ethanol inhibition of L1-mediated cell adhesion and prevention of ethanol teratogenesis. These findings support the hypothesis that ethanol inhibition of L1 adhesion contributes to ethanol teratogenesis.

Ethanol Antagonist Peptides: Structural Specificity without Stereospecificity

“…Indirect immunofluorescence with flow cytometry showed that D-NAP or L-NAP did not change the cell surface expression of L1 (data not shown). These results are consistent with previous data showing that the alcohol antagonists 1-octanol, 1-pentanol, and the peptides L-NAP and L-SAL do not modify L1 expression or adhesion (Wilkemeyer et al, 2000(Wilkemeyer et al, , 2002a.…”

“…Cell adhesion was measured using a shortterm aggregation assay (Wilkemeyer et al, 2002a). Briefly, cells were detached and dissociated to single-cell suspension with calcium-and magnesium-free phosphate-buffered saline supplemented with 2 mM EDTA and 0.1 mg/ml DNase and diluted to 330,000 cells/ml for the NIH/3T3 cells and 250,000 cells/ml for the NG108-15 cells.…”

“…NAP is a potent and structurally specific antagonist of ethanol inhibition of L1 adhesion (Wilkemeyer et al, 2002a(Wilkemeyer et al, , 2003. To determine the stereospecificity of this interaction, we studied the antagonist properties of a NAP peptide that was composed entirely of D-amino acids (D-NAP).…”

“…NAP and SAL also antagonize ethanol inhibition of L1 at femtomolar to picomolar concentrations (NAP ethanol antagonism) (Wilkemeyer et al, 2002a); however, NAP neuroprotection and NAP ethanol antagonism show different structure-activity relations (Wilkemeyer et al, 2003). NAP neuroprotection is abolished by Ala substitution of Ser-5 or Pro-7 (P7A-NAP peptide) but is much less sensitive to Ala substitution of Ile-6.…”

“…We have identified a second class of compounds that potently antagonizes ethanol inhibition of L1 adhesion: the peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL) (Wilkemeyer et al, 2002a). These peptides are active fragments of the glial-derived activity-dependent neuroprotective protein and activity-dependent neurotrophic factor, respectively (Brenneman and Gozes, 1996;Bassan et al, 1999;Brenneman et al, 2000b).…”