Expression and characterization of rat UDP-N-acetylglucosamine: alpha-3- D-mannoside beta-1,2-N-acetylglucosaminyltransferase I in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful host for the production ofheterologous proteins through the secretory pathway. However, because ofthe potential antigenicity of mannan-type sugar chains in humans, yeastcannot be used as a host for the production of glycoprotein therapeutics.To overcome this problem, we are trying to breed a yeast which can producehybrid- or complex-type carbohydrates. UDP- N-acetylglucosamine:alpha-3-d-mannoside beta-1, 2- N-acetylglucosaminyltransferase I (GnT-I) is essential for the conversion ofhigh mannose-type N- glycans to hybrid- and complex-type ones. As yeastlacks this enzyme, we have introduced the rat GnT-I cDNA into yeast cells.The transformed yeast cells expressed GnT-I activity in vitro. Theexpressed GnT-I was localized in all organella, including the endoplasmicreticulum (ER), Golgi apparatus, and vacuole, suggesting that the mammalianGolgi retention signal of GnT-I did not function in yeast cells. Analysisof the GnT-I gene product with a c- Myc epitope tag at the C-terminuselucidates that the N - terminal region of GnT-I, including the mammalianGolgi retention signal, should be removed in the yeast ER.