Removal of sialic acid from a glycoprotein in CHO cell culture supernatant by action of an extracellular CHO cell sialidase.
We have directly tested the hypothesis that Chinese hamster ovary (CHO) cell-produced glycoproteins are subject to extracellular degradation by a sialidase endogenous to the CHO cell line. Factors important to understanding the potential for extracellular degradation are addressed including the glycoprotein specificity, subcellular source, mechanism of release, and stability of the sialidase activity. The extracellular CHO cell sialidase apparently originates from the cytosol of the cells, and is released to the cell culture supernatant as a result of damage to the cellular membrane. The extracellular sialidase is active toward a variety of CHO cell-produced glycoproteins, and can hydrolyze sialic acid from the recombinant glycoprotein gp120 in the culture supernatant. While measuring the actual degradation of a glycoprotein by extracellular CHO cell sialidase can be difficult, data presented here suggest that the level of degradation can be estimated indirectly by using a more convenient fluorescent substrate, 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid, to quantify sialidase activity. Degradation by sialidase is minimized through addition of the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid to the culture supernatant. The results in this study suggest additional potential approaches for minimizing degradation by sialidase, including isolation of a sialidase-deficient CHO cell line.
Gramer
M J
MJ
Department of Chemical Engineering, Stanford University, CA 94305-5025, USA.
Goochee
C F
CF
Chock
V Y
VY
Brousseau
D T
DT
Sliwkowski
M B
MB
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003470:Culture Media; D003600:Cytosol / Q000201:enzymology; D004795:Enzyme Stability; D006023:Glycoproteins / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D019158:N-Acetylneuraminic Acid / Q000302:isolation & purification*; D009439:Neuraminidase / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D003470:Culture Media; D006023:Glycoproteins; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid