Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells

Abstract

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide-lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…The quenching method that was modified from previous reports provides reliable results of the instantaneous steady state levels of LLO in cell lines (Jones et al, 2010) or tissues (Gao and Lehrman, 2006) It is well known that glucose starvation alters the LLO composition. In 25 mM glucose culture, LLO composition is consistent with previous studies (Jones et al, 2010;Quellhorst et al, 1999;Rearick et al, 1981), showing M9-P-P-Dol as the primary LLO found in CHO-K1 cells, along with small amounts of Glc2Man9GlcNAc2, Glc1Man9GlcNAc2, and Man9GlcNAc2 due to the possible reason that an addition of glucose residues onto the fully mannosylated LLO (Man9GlcNAc2-P-P-Dol) is the rate determined step (Jones et al, 2010). Glucose starvation can induce an accumulation of truncated LLO intermediates in the early stage of N-glycosylation, such as M5 to M2 (Rearick et al, 1981), which is consistent with LLO pools extracted from cells grown in 0-15 mM glucose.…”

“…Cells were seeded in high density in media containing glucose from 0-25 mM, and incubated for 24 h. In chapter 3 we found that cells in culture with an initial glucose concentration 15 mM had been in a glucose depleted state for nearly 4 h. Cells in lower glucose concentration (<15 mM) were No evidence has been found between extracellular galactosidase and galactose content (Gramer et al, 2011;Jones et al, 2010;Quellhorst et al, 1999;Rearick et al, 1981). The results showed a linear correlation between the time period of glucose depletion during 24 h and galactosylation and sialylation indexes, suggesting that the decrease in galactosylation and sialylation could be elicited by depletion of activated monosacharides as dolichol phosphate or nucleotide derivates, mostly glucose-derived substrates.…”

“…One other very important note is that glucose-starved cells produce truncated LLO intermediates, which are less favorable to OST compared to fully glucosylated donors (Gao et al, 2005;Kelleher et al, 2003;Turco et al, 1977). An accumulation of truncated LLO intermediates can result in the addition of premature N-glycans onto the nascent polypeptides at a low rate or leaving Asn residue completely empty (Jackson et al, 1989;Quellhorst et al, 1999;Sharma et al, 1981). Both premature N-glycans and hypoglycosylation may have an effect on the N-glycosylation profile of the final product, which will be investigated in the next experiments in this thesis.…”

“…There are several reports of non-commercialized Mabs from CHO cells with high GI values around 4.5 (Costa et al, 2013), although the intrinsic difference between cell lines remains unclear. For Mab production, no evidence showed a relationship between extracellular galactosidase and external galactose content (Gramer et al, 2011;Quellhorst et al, 1999). However, it is clear that the extent of galactosylation varies with the culture conditions of the producer cell, as well as environmental factors that may change significantly during the course of a batch culture (Gramer et al, 2011).…”

Defects in the N-Linked Oligosaccharide Biosynthetic Pathway in a Trypanosoma brucei Glycosylation Mutant

“…Instead, it comigrated with an R f similar to that of Man 9 GlcNAc 2 (Fig. 5A, lane 2), the species found in MI8-5, a CHO mutant defective in glucosylation of the N-glycan precursor (28) and with an authentic nonradioactive Man 9 GlcNAc 2 standard. The lack of OSL glucosylation is consistent with OSL structures in other trypanosomatids (24).…”