ISOLATION AND PROPERTIES OF A SOLUBLE SIALIDASE FROM THE CULTURE FLUID OF CHINESE-HAMSTER OVARY CELLS

A soluble sialidase that can degrade recombinant glycoproteins express ed in Chinese hamster ovary (CHO) cells has been isolated and purified to near homogeneity from the cell culture fluid of this host. Purific ation of approximately 34 000-fold was carried out using conventional purification techniques including sequential DEAE-Sepharose and S-Seph arose ionexchange chromatography, followed by hydrophobic interaction chromatography with Phenyl-Toyopearl. Final purification was achieved by heparin-agarose and chromatofocusing chromatography. The minimum mo lecular weight of the sialidase on SDS-PAGE was approximately 43 000 D a. When the final preparation was examined under nondenaturing conditi ons, two major (pI = 6.8 and 7.0) and five minor electrophoretic forms with different isoelectric points were identified. The basis for the electrophoretic heterogeneity is not known, but it was not due to carb ohydrate diversity since no carbohydrates were detected on the purifie d protein. The enzyme degraded a variety of sialyl-conjugate substrate s, at a pH optimum of 5.9, including intact glycoproteins, oligosaccha rides and gangliosides with a 4-fold preference for 2,3- versus 2,6-li nked sialic acid residues. With ganglioside substrates, internally lin ked sialic acid residues were not cleaved by the enzyme. Delineation o f this enzyme from the lysosomal and plasma membrane sialidases was ma de using inhibition studies with C-9 substituted hydro-3,5-dideoxy-D-g lycero-D-galacto-non-2-enonic 2-enonic acid derivatives. The enzyme wa s identified in several CHO cell lines by immunoblotting using antiser um raised against a synthetic peptide based on amino acid sequence of a fragment derived by trypsin digestion of the purified sialidase. The amino acid sequence from additional peptides representing approximate ly 50% of the total protein sequence was apparently unique, giving no significant sequence similarity to other microbial sialidases or any m ammalian protein.