Isolation and properties of a soluble sialidase from the culture fluid of Chinese hamster ovary cells

Glycobiology. 1993 Oct;3(5):455-63. doi: 10.1093/glycob/3.5.455.

Abstract

A soluble sialidase that can degrade recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells has been isolated and purified to near homogeneity from the cell culture fluid of this host. Purification of approximately 34,000-fold was carried out using conventional purification techniques including sequential DEAE-Sepharose and S-Sepharose ion-exchange chromatography, followed by hydrophobic interaction chromatography with Phenyl-Toyopearl. Final purification was achieved by heparin-agarose and chromatofocusing chromatography. The minimum molecular weight of the sialidase on SDS-PAGE was approximately 43,000 Da. When the final preparation was examined under non-denaturing conditions, 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 carbohydrate diversity since no carbohydrates were detected on the purified protein. The enzyme degraded a variety of sialyl-conjugate substrates, at a pH optimum of 5.9, including intact glycoproteins, oligosaccharides and gangliosides with a 4-fold preference for 2,3- versus 2,6-linked sialic acid residues. With ganglioside substrates, internally linked sialic acid residues were not cleaved by the enzyme. Delineation of this enzyme from the lysosomal and plasma membrane sialidases was made using inhibition studies with C-9 substituted 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2- enonic acid derivatives. The enzyme was identified in several CHO cell lines by immunoblotting using antiserum raised against a synthetic peptide based on amino acid sequence of a fragment derived by trypsin digestion of the purified sialidase.(ABSTRACT TRUNCATED AT 250 WORDS)

MeSH terms

  • Amino Acid Sequence
  • Animals
  • CHO Cells / enzymology*
  • Carbohydrate Sequence
  • Cricetinae
  • Enzyme Stability
  • Glycoconjugates / metabolism
  • Hydrogen-Ion Concentration
  • Immunoblotting
  • Isoelectric Point
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • N-Acetylneuraminic Acid* / analogs & derivatives*
  • Neuraminidase / antagonists & inhibitors
  • Neuraminidase / isolation & purification*
  • Neuraminidase / metabolism
  • Peptide Fragments / genetics
  • Peptide Fragments / immunology
  • Peptide Fragments / isolation & purification
  • Sialic Acids / chemistry
  • Sialic Acids / pharmacology
  • Substrate Specificity

Substances

  • Glycoconjugates
  • Peptide Fragments
  • Sialic Acids
  • 2-deoxy-2,3-dehydro-N-acetylneuraminic acid
  • Neuraminidase
  • N-Acetylneuraminic Acid