HOMOPHILIC ADHESION OF THE MYELIN PO PROTEIN REQUIRES GLYCOSYLATION OF BOTH MOLECULES IN THE HOMOPHILIC PAIR

The myelin Po protein is glycosylated at a single site, asparagine 93, within its only immunoglobulin (Ig)-like domain. We have previously shown that Po behaves like a homophilic adhesion molecule (Filbin, M. T., F. S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. Nature (Lond.). 344:871-872). To determine if the sugar residues of this molecule contribute to its adhesiveness, the glycosylation site was eliminated by replacing asparagine 93 with an alanine, through site-directed mutagenesis of the Po cDNA. The mutated Po cDNA was transfected into CHO cells and surface expression of the mutated Po was assessed by immunofluorescence, limited trypsinization and an ELISA. A cell line was chosen which expressed approximately equivalent amounts of the unglycosylated Po (UNGPo) at the cell surface as did a cell line expressing the fully glycosylated Po (GPo); the adhesive properties of these two cell lines were compared. It was found that when a single cell suspension of the UNGPo cells were incubated, by 60 min, unlike the GPo cells, they had not formed large aggregates; they were indistinguishable from the control transfected cells. This suggests that the UNGPo protein does not behave like an adhesion molecule. To establish if only one molecule in the Po:Po homophilic pair must be glycosylated for adhesion to occur, the ability of UNGPo cells to adhere to GPo cells was assessed both qualitatively and quantitatively. The results of both types of assay imply that, indeed, both Po molecules in the homophilic pair must be glycosylated for adhesion to take place.