Genetic engineering of CHO cells producing human interferon-gamma by transfection of sialyltransferases

Glycoconj J. 2000 Dec;17(12):895-904. doi: 10.1023/a:1010977431061.

Abstract

Natural human interferon-gamma (hIFN-gamma) contains mainly biantennary complex-type sugar chains. We previously remodeled the branch structures of N-glycans on hIFN-gamma in Chinese hamster ovary (CHO) cells by overexpressing UDP-N-acetylglucosamine: alpha1,6-D-mannoside beta1,6-N-acetylglucosaminyltransferase (GnT-V). Normal CHO cells primarily produced hIFN-gamma having biantennary sugar chains, whereas a CHO clone, designated IM4/Vh, transfected with GnT-V, primarily produced hIFN-gamma having GlcNAcbeta1-6 branched triantennary sugar chains when sialylation was incomplete and an increase in poly-N-acetyllactosamine (Galbeta1-4GlcNAcbeta1-3)n was observed. In the present study, we introduced mouse Galbeta1-3/4GlcNAc-R alpha2,3-sialyltransferase (ST3Gal IV) and/or rat Galbeta1-4GlcNAc-R alpha2,6-sialyltransferase (ST6Gal I) cDNAs into the IM4/Vh cells to increase the extent of sialylation and to examine the effect of sialyltransferase (ST) type on the linkage of sialic acid. Furthermore, we speculated that sialylation extent might affect the level of poly-N-acetyllactosamine. We isolated four clones expressing different levels of alpha2,3-ST and/or alpha2,6-ST. The extent of sialylation of hIFN-gamma from the IM4/Vh clone was 61.2%, which increased to about 80% in every ST transfectant. The increase occurred regardless of the type of overexpressed ST, and the proportion of alpha2,3- and alpha2,6-sialic acid corresponded to the activity ratio of alpha2,3-ST to alpha2,6-ST. Furthermore, the proportion of N-glycans containing poly-N-acetyllactosamine was significantly reduced (less than 10%) in the ST transfectants compared with the parental IM4/Vh clone (22.9%). These results indicated that genetic engineering of STs is highly effective for regulating the terminal structures of sugar chains on recombinant proteins in CHO cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Carbohydrate Conformation
  • Carbohydrate Sequence
  • Chromatography, High Pressure Liquid
  • Clone Cells / enzymology
  • Clone Cells / metabolism
  • Cloning, Molecular
  • Cricetinae
  • Genetic Engineering / methods*
  • Humans
  • Interferon-gamma / biosynthesis*
  • Interferon-gamma / chemistry
  • Mice
  • Molecular Sequence Data
  • N-Acetylneuraminic Acid / metabolism
  • Polymerase Chain Reaction
  • Rats
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Sialyltransferases / genetics*
  • Sialyltransferases / metabolism*
  • Transfection

Substances

  • Recombinant Proteins
  • Interferon-gamma
  • Sialyltransferases
  • beta-D-galactoside alpha 2-6-sialyltransferase
  • beta-galactoside alpha-2,3-sialyltransferase
  • N-Acetylneuraminic Acid