MOLECULAR-CLONING AND EXPRESSION OF CDNA-ENCODING RAT SKELETAL-MUSCLECYTOSOLIC SIALIDASE

We have isolated a cDNA clone encoding the cytosolic sialidase of rat skeletal muscle. Degenerate oligonucleotides, based on amino acid sequ ence data for the purified enzyme, were used as primers to amplify fra gments of the gene from rat skeletal muscle cDNA by the polymerase cha in reaction. The amplified cDNA fragment was then applied as probe to screen a rat skeletal muscle cDNA library. The longest cDNA clone thus isolated was incomplete at the 5'-end, and therefore an amplified cDN A from the 5'-end portion of the gene was further generated by polymer ase chain reaction. These two cDNAs were used to construct a cDNA enco ding the entire sequence of rat sialidase. The composite sequence enco des an open reading frame of 379 amino acids that include all sequence d peptides. Although the deduced amino acid sequence is not largely si milar to those of bacterial and parasite sialidases, it contains two A sp blocks, the conserved sequence of the sialidases from these microor ganisms. When the cDNA was inserted into an expression vector followed by transformation in Escherichia coli, sialidase activity appeared in the cell extract. The sialidase could be completely immunoprecipitate d by antiserum against the cytosolic sialidase of rat skeletal muscle.