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 protein, Po, holds myelin compact at the extracellular leaflets. Po carries a single immunoglobulin (Ig)-like domain that is stabilized by a disulfide bond between Cys21 and Cys98. We showed previously that Po mutated at Cys21 to Ala (C21A Po), unlike wild type Po, when it was expressed in Chinese hamster ovary (CHO) cells after transfection, was not adhesive. To determine whether C21A Po could influence the adhesion of wild type Po, cells that already expressed wild type Po and that were shown 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 electrophoresis in the presence and absence of beta-mercaptoethanol, because only in coexpressors will there be two forms of Po of different apparent molecular weights under nonreducing conditions. Two clones that coexpressed wild type Po and C21A Po and a third clone, which, although it was resistant to hygromycin, expressed only wild type Po, were chosen for further study. An enzyme-linked immunosorbent assay of fixed, unpermeabilized cells showed that all of these clones expressed approximately equivalent amounts of Po at the cell surface. However, in an aggregation 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 aggregates, and their adhesive properties were indistinguishable from the control transfected cells, which did not express Po. These results suggest that C21A Po has a dominant-negative effect on adhesion of wild type Po.
Zhang
K
K
Department of Biological Sciences, Hunter College of the City University of New York, New York 10021, USA.
Filbin
M T
MT
D000818:Animals; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology*; D003001:Cloning, Molecular; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics; D003711:Demyelinating Diseases / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005110:Extracellular Space / Q000378:metabolism; D005799:Genes, Dominant / Q000235:genetics; D008970:Molecular Weight; D009154:Mutation; D018993:Myelin P0 Protein / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology
D015815:Cell Adhesion Molecules; D018993:Myelin P0 Protein; D003545:Cysteine