Abstract
Lec23 Chinese hamster ovary (CHO) cells have been shown to possess a unique lectin resistance phenotype and genotype compared with previously isolated CHO glycosylation mutants (Stanley, P., Sallustio, S., Krag, S. S., and Dunn, B. (1990) Somatic Cell Mol. Genet. 16, 211-223). In this paper, a biochemical basis for the lec23 mutation is identified. The carbohydrates associated with the G glycoprotein of vesicular stomatitis virus (VSV) grown in Lec23 cells (Lec23/VSV) were found to possess predominantly oligomannosyl carbohydrates that bound strongly to concanavalin A-Sepharose, eluted 3 sugar eq beyond a Man9GlcNAc marker oligosaccharide on ion suppression high pressure liquid chromatography, and were susceptible to digestion with jack bean alpha-mannosidase. Monosaccharide analyses revealed that the oligomannosyl carbohydrates contained glucose, indicating a defect in alpha-glucosidase activity. This was confirmed by further structural characterization of the Lec23/VSV oligomannosyl carbohydrates using purified rat mammary gland alpha-glucosidase I, jack bean alpha-mannosidase, and 1H NMR spectroscopy at 500 MHz. [3H]Glucose-labeled Glc3Man9GlcNAc was prepared from CHO/VSV labeled with [3H]galactose in the presence of the processing inhibitors castanospermine and deoxymannojirimycin. Subsequently, [3H]Glc2Man9GlcNAc was prepared by purified alpha-glucosidase I digestion of [3H]Glc3Man9GlcNAc. When these oligosaccharides were used as alpha-glucosidase substrates it was revealed that Lec23 cells are specifically defective in alpha-glucosidase I, a deficiency not previously identified among mammalian cell glycosylation mutants.
Highlights
Lec23 Chinesehamster ovary(CHO) cells have been sylation-defective phenotypes
A similar ratio was observed with galactose-labeledCHO/VSVglycopeptides(Fig. 1)
A qualitativelysimilar profile was obtained for Lec231VSV glycopeptideslabeled witheither ["Hlmannose or [3H]galacto~(eFig. l),consistentwiththe possibility that themajor species eluting with 10mM methyla-D-mannoside ("(10)) was of the hybrid type [14]
Summary
Materiak-~-[6-~H]Galactose(31.5 Ci/mmol), ~ - [ 2 - ~ H ] mannose by purifiae-dglucosidase. After incubation at 37"C for 18 h, the Sepharose column (0.6 X 22 cm) in ConA buffer Bound glycopeptides were eluted Jack bean a-mannosidase digestions were performed in 100 pl of with at least 4 column volumes of 10 mM MG, 10 mM MM, and 200 sodium citrate buffer (pH 4.5) at 37 "C under toluene for 96 h with a mM MM added sequentially in ConA buffer. All the fractions beyond the Vo and in the included volume of sidase 11, was prepared from [3H]Glc3MangGlcNAbcy digestion with the G-25 columns werepooled, passed through ConA to remove a-glucosidase I and purification on HPLC (see Fig. 6). Most importantfor comparisons were the structures assigned by Tsai et al [28] for carbohydrates synthesized by the yeast a-glucosidase I mutant glsl [31]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
