Impact of a human CMP-sialic acid transporter on recombinant glycoprotein sialylation in glycoengineered insect cells.
Insect cells are widely used for recombinant glycoprotein production, but they cannot provide the glycosylation patterns required for some biotechnological applications. This problem has been addressed by genetically engineering insect cells to express mammalian genes encoding various glycoprotein glycan processing functions. However, for various reasons, the impact of a mammalian cytosine-5'-monophospho (CMP)-sialic acid transporter has not yet been examined. Thus, we transformed Spodoptera frugiperda (Sf9) cells with six mammalian genes to generate a new cell line, SfSWT-4, that can produce sialylated glycoproteins when cultured with the sialic acid precursor, N-acetylmannosamine. We then super-transformed SfSWT-4 with a human CMP-sialic acid transporter (hCSAT) gene to isolate a daughter cell line, SfSWT-6, which expressed the hCSAT gene in addition to the other mammalian glycogenes. SfSWT-6 cells had higher levels of cell surface sialylation and also supported higher levels of recombinant glycoprotein sialylation, particularly when cultured with low concentrations of N-acetylmannosamine. Thus, hCSAT expression has an impact on glycoprotein sialylation, can reduce the cost of recombinant glycoprotein production and therefore should be included in ongoing efforts to glycoengineer the baculovirus-insect cell system. The results of this study also contributed new insights into the endogenous mechanism and potential mechanisms of CMP-sialic acid accumulation in the Golgi apparatus of lepidopteran insect cells.
Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA.
Hideaki
H
Mabashi-Asazuma
Xianzong
X
Shi
Christoph
C
Geisler
Chu-Wei
CW
Kuo
Kay-Hooi
KH
Khoo
Donald L
DL
Jarvis
D000818:Animals; D002460:Cell Line; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006595:Hexosamines / Q000378:metabolism; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000235:genetics / Q000378:metabolism; D033704:Nucleotide Transport Proteins / Q000378:metabolism; D027361:Organic Anion Transporters / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000378:metabolism
D006023:Glycoproteins; D006595:Hexosamines; D033704:Nucleotide Transport Proteins; D027361:Organic Anion Transporters; D011994:Recombinant Proteins; C491081:SLC35A1 protein, human; D027981:Symporters; C421224:sialic acid transport proteins; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D019158:N-Acetylneuraminic Acid; C002022:N-acetylmannosamine