Mapping the adhesive domains of the myelin Po protein.
The Po protein holds PNS myelin compact at the intraperiod line by homophilic interactions of its single immunoglobulin (Ig)-like domain. Using transfected Chinese hamster ovary (CHO) cells expressing Po we can monitor this adhesion in vitro and have shown that the cells expressing Po when incubated as a single-cell suspension form large aggregates, whereas control-transfected cells do not. To precisely map the domains of Po responsible for Po:Po-mediated membrane adhesion, the ability of a number of antibodies raised to peptides corresponding to segments of the Ig-domain of Po, and the ability of the Po-peptides themselves, to inhibit aggregation was assessed. Both antibodies to Po-peptide, SDNGT, corresponding to amino acids Po 91-95, and the peptide itself, were able to block adhesion completely. Furthermore, within this Po sequence, amino acids Asp 92 and Gly 94 are conserved in a large number of V-like Ig-domains. To determine if these two amino acids are important for Po-mediated adhesion, the nucleotides coding for Asp 92 and Gly 94 were mutated to encode glutamate and alanine, respectively. Although the mutated Po reached the surface in transfected CHO cells and was glycosylated, the cells did not aggregate. These results suggest that the sequence SDNGT in the extracellular domain of Po is important for adhesion. In addition, antibodies to a second sequence, Po 74-82, and the peptide itself, also partially inhibited Po: Po-mediated adhesion indicating that there is more than one adhesive domain on Po-protein.
Zhang
K
K
Department of Biological Sciences, Hunter College of the City University of New York, New York 10021, USA.
Merazga
Y
Y
Filbin
M T
MT
D000268:Adhesiveness; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002448:Cell Adhesion; D017124:Conserved Sequence; D006224:Cricetinae; D019084:Fluorescent Antibody Technique, Indirect; D005784:Gene Amplification; D008565:Membrane Proteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009154:Mutation; D018993:Myelin P0 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D017933:Peripheral Nervous System / Q000378:metabolism*; D014162:Transfection
D008565:Membrane Proteins; D018993:Myelin P0 Protein