Molecular cloning, expression and characterization of photoreceptor cell peripherin : the defective protein responsible for the retinal degeneration slow (rds) defect

Gregory James Connell
Peripherin, a membrane protein with an apparent molecular weight of 34 kDa, has been previously localized to the rim region of the vertebrate rod photoreceptor disk membrane using monoclonal antibodies and immunocytochemical labelling techniques. As an initial step in determining the structure and function of this protein, cDNA containing its coding sequence has been cloned and sequenced. A bovine retinal ʎgtll expression library was screened with antiperipherin monoclonal antibodies, and a 583
more » ... base pair clone was initially isolated. The remaining part of the coding sequence was obtained from subsequent rescreenings of the same library and an independent ʎgt10 library. A C-terminal CNBr fragment of peripherin was purified by immunoaffinity chromatography and reverse phase high-performance liquid chromatography. The amino acid sequence of the isolated C-terminal peptide and the N-terminal sequence analysis of immunoaffinity purified peripherin are in agreement with the cDNA sequence. At the amino acid level, the sequence of peripherin has 92.5% sequence identity to the gene proposed to be responsible for the retinal degeneration slow defect in mice (Travis et al.(1989) Nature 338, 70-73) The differences between the two sequences can be attributed to species differences. The identity of the retinal degeneration slow gene product and its intracellular localization were previously unknown. The cDNA sequence of peripherin predicts that there are possibly four transmembrane domains. On the basis of immunocytochemical studies and sequence analysis, the hydrophilic C-terminal segment containing the antigenic sites for the antiperipherin monoclonal antibodies has been localized on the cytoplasmic side of the disk membrane. There are three consensus sequences for asparagine linked glycosylation. Deglycosylation studies have indicated that at least one of these sites is utilized. The complete coding sequence of peripherin was expressed in COS-1 cells. Western blot analysis of the expressed peripherin suggest that it exis [...]
doi:10.14288/1.0100298 fatcat:2ra3t3xag5hxzj5gdlonrxp7ey