Mammalian homologue of the calcium-sensitive phosphoglycoprotein, parafusin

Three specific antipeptide antibodies and oligonucleotide probes synthesized to internal sequences of parafusin have been used to search for mammalian counterpart(s) of this protein. Parafusin is an exocytic-sensitive phosphoglycoprotein from a unicellular eukaryote Paramecium that was recently cloned and sequenced. Western and Southern blot analyses, polymerase chain reaction (PCR) and reverse transcriptase coupled PCR (RT-PCR) techniques have been used to examine rat liver and pancreas, human pancreas and a murine pancreatic β-cell line (βTC3) arising in transgenic mice. The parafusin-specific antibodies showed cross-reaction with a protein at ~ 63 kDa in 4 tissues, whereas a phosphoglucomutase-specific antibody also detected a second band of similar molecular weight in the βTC3 cells. The presence of two bands shows that parafusin homologue(s) and phosphoglucomutase are separate entities. βTC3 cells were shown to incorporate [β³⁵]UDPGlc into the parafusin homologue in a Ca⁺⁺ sensitive manner characterisitic of parafusin. Southern blot analysis revealed that the parafusin-specific probe hybridized with restriction enzyme digests of rat DNA in distinct patterns different from those observed with a phosphoglucomutase-specific probe. Rat genomic DNA and mRNA from the βTC3 cells were used as the templates for PCR and RT-PCR using internal parafusin primers. In both cases similarly sized products were obtained which hybridized in Southern analysis with a specific parafusin probe located within the amplified region. These results indicate that a parafusin homologue exists in mammalian cells.