Accumulation of pentamannose oligosaccharides in human mononuclear leukocytes by action of swainsonine, an inhibitor of glycoprotein processing.
Swainsonine, a known inhibitor of the alpha-mannosidase II involved in processing of asparagine-linked glycoproteins, causes accumulation of hybrid-type oligosaccharide-containing glycoproteins in mammalian cells. Swainsonine augments lymphokine-activated, killer-cell induction at suboptimal doses of interleukin-2; the amount needed to increase LAK activity is 100-1000 fold higher than required to completely inhibit mannosidase II. Human mononuclear lymphocytes, when treated with these relatively high (58 microM) concentrations of swainsonine showed a 3-4 fold increase in D-[3H]mannose incorporation into the glycan as compared to glycans of untreated cells. Analysis indicated accumulation of high-mannose type, free oligosaccharides in the soluble fractions of the cell. Chromatographic analysis of glycan obtained by D-[2-3H]mannose labeling of human mononuclear lymphocytes showed synthesis of a new oligosaccharide, at 58 microM of swainsonine, that contained 36% of the total radioactivity incorporated into the glycan (oligosaccharide pool). This oligosaccharide fraction was resistant to hydrolysis by endoglycosidase H, endoglycosidase F, O and N-glycanase, but was susceptible to cleavage by Jack bean alpha-mannosidase and was bound > 90% to concanavalin A-Sepharose. A similar chromatographic elution profile was obtained from glycans labeled with D-[2-3H]mannose from mouse B16F10 melanoma and baby hamster kidney cells subsequent to swainsonine treatment. Methylation analysis of free oligosaccharides obtained from MNL revealed the presence of a pentamannose. These results indicate the accumulation of a free high-mannose oligosaccharide rather than expected hybrid-type structure on treatment of cells with relatively high concentrations of swainsonine.
Kang
M S
MS
Marion Merrell Dow Research Institute, Cincinnati, OH 45215.
Bowlin
T L
TL
Vijay
I K
IK
Sunkara
S P
SP
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D006224:Cricetinae; D006020:Glycopeptides / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism*; D008361:Mannosidases / Q000037:antagonists & inhibitors; D008546:Melanoma, Experimental / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D017026:Swainsonine / Q000494:pharmacology*; D043323:alpha-Mannosidase
D006020:Glycopeptides; D009844:Oligosaccharides; C028569:oligomannoside; D008361:Mannosidases; D043323:alpha-Mannosidase; D008358:Mannose; D017026:Swainsonine