MYELIN PO PROTEIN MUTATED AT CYS21 HAS A DOMINANT-NEGATIVE EFFECT ON ADHESION OF WILD-TYPE PO

The homophilic adhesion of the peripheral nervous system myelin protei n, Po, holds myelin compact at the extracellular leaflets. Po carries a single immunoglobulin (Ig)-like domain that is stabilized by a disul fide bond between Cys21 and Cys98, We showed previously that Po mutate d at Cys21 to Ala (C21A Po), unlike wild type Po, when it was expresse d in Chinese hamster ovary (CHO) cells after transfection, was not adh esive. To determine whether C21A Po could influence the adhesion of wi ld type Po, cells that already expressed wild type Po and that were sh own to be adhesive were retransfected with plasmids containing C21A Po , After selection in hygromycin, clones that coexpressed wild type Po and Cys21-mutated Po were identified by polyacrylamide gel electrophor esis in the presence and absence of beta-mercaptoethanol, because only in coexpressors will there be two forms of Po of different apparent m olecular weights under nonreducing conditions. Two clones that coexpre ssed wild type Po and C21A Po and a third clone, which, although it wa s resistant to hygromycin, expressed only wild type Po, were chosen fo r further study. An enzyme-linked immunosorbent assay of fixed, unperm eabilized cells showed that all of these clones expressed approximatel y equivalent amounts of Po at the cell surface. However, in an aggrega tion adhesion assay, only the cells that expressed wild type Po alone formed large aggregates and dropped in total particle number with time . The cells that coexpressed wild type and C21A Po did not form aggreg ates, and their adhesive properties were indistinguishable from the co ntrol transfected cells, which did not express Po. These results sugge st that C21A Po has a dominant-negative effect on adhesion of wild typ e Po. J, Neurosci. Res.53:1-6, 1998, (C) 1998 Wiley-Liss, Inc.