Purification, characterization, and cDNA cloning of a novel acidic endoglycoceramidase from the jellyfish, Cyanea nozakii

Endoglycoceramidase (EC 3.2.1.123) is an enzyme capable of cleaving the gly cosidic linkage between oligosaccharides and ceramides in various glycosphi ngolipids, We report here the purification, characterization, and cDNA clon ing of a novel endoglycoceramidase from the jellyfish, Cyanea nozakii. The purified enzyme showed a single protein band estimated to be 51 kDa on SDS- polyacrylamide gel electrophoresis. The enzyme showed a pH optimum of 3.0 a nd was activated by Triton X-100 and Lubrol PX but not by sodium taurodeoxy cholate. This enzyme preferentially hydrolyzed gangliosides, especially GT1 b and GQ1b, whereas neutral glycosphingolipids were somewhat resistant to h ydrolysis by the enzyme. A full-length cDNA encoding the enzyme was cloned by 5'- and 3'-rapid amplification of cDNA ends using a partial amino acid s equence of the purified enzyme. The open reading frame of 1509 nucleotides encoded a polypeptide of 503 amino acids including a signal sequence of 25 residues and six potential N-glycosylation sites. Interestingly, the Asn-Gl u-Pro sequence, which is the putative active site of Rhodococcus endoglycoc eramidase, was conserved in the deduced amino acid sequences. This is the f irst report of the cloning of an endoglycoceramidase from a eukaryote.