Lipid‐Bound Oligosaccharides in Insects

Abstract

Membrane preparations from immature stages of the fruit fly Ceratitis capitata catalyze the transfer of mannose from GDP-['4C]mannose into lipid-linked oligosaccharides. These compounds behave as polyprenyl derivatives and their formation is stimulated by the addition of an acidic glycolipid fraction isolated from insects.The mannose-labeled oligosaccharides are attached to the poly-isoprenol by a pyrophosphoryl linkage and can be released by mild acid hydrolysis. The trisaccharide lipid has been partially characterized. The results indicate that the compound is polyprenyl-pyrophosphate-N,N'-diacetylchitobiose-mannose.Incubation of dolichyl phosphate [14C]mannose or lower 14C-labeled oligosaccharide lipids with unlabeled GDP-mannose and the insect enzyme leads to the labeling of a higher lipid-bound oligosaccharide.When UDP-N-a~etyl['~C]glucosamine was incubated with insect membranes a '4C-labeled chitobiosyl lipid was synthesized. If unlabeled GDP-mannose was also present, the I4C label appeared in the trisaccharide and higher oligosaccharide lipids.Preliminary evidence indicates that the insect polyprenyl oligosaccharides described here might participate in glycoprotein biosynthesis.The role of polyprenylphosphates as glycosyl carriers in eucaryotic tissues is now well established [l-61. Evidence is being accumulated showing that dolichyl-bound saccharides are intermediates in the glycosylation of proteins in vertebrate [7 -141 plant [15,16] and fungal [17-211 systems. First, GlcNAc from UDP-GlcNAc is transferred to dolichyl-P. Then, N-acetylglucosamine and mannose are sequentially added from their respective sugar nucleotides yielding Dol-P-P-(GlcNAc)2-Man [22] which can undergo additional mannosylations [S, 11,221. Some of these mannose residues probably came from Dol-PMan which, in turn, is formed through the reaction of dolichyl-P with GDP-Man 123 -251. The last step is the transfer of the oligosaccharide from dolichyl-P-P derivatives to the polypeptide chains. The transfer of ['4C]mannose-labeled oligosaccharides has been described in yeast [26] and in vertebrate systems [8 -l l , 27,281.The amino acid involved in the protein-saccharide linkage is presumably asparagine because the sequence N,N'-diacetylchitobiose-mannose has always been found linked to it [29]. ~_ _ _Abbreviations. GlcNAc, N-acetylglucosamine; Dol, dolichyl ; Man, mannose; dolichyl-P. dolichyl moiiophosphate; dolichyl-P-P, dolichyl diphosphate.Enzyme. Streptomyes griseus protease or streptococcal proteinase (EC 3.4.22.10).It has been recently reported that dolichol [30,31], dolichyl phosphate [32] and dolichyl derivatives [33,34] occur in pluricellular invertebrates, but little is known about the mechanisms involved in protein glycosylation in those metazoan phyla [35].We previously reported the transfer of glucose, mannose and N-acetylglucosamine from their respective nucleotides to an endogenous polyprenyl phosphate catalyzed ip2 vitro by an insect extract [33,36]. These reactions could be stimulated by the addition of insect or pig li...

Lipid profiles as indicators of functional senescence in the medfly

“…The pellet was re-extracted with chloroform: methanol (3:2), centrifuged at 7000 g for 20 min, and the supernatant was mixed with the acetone extract and dried. Total lipids were then solubilized and washed according to a modified Folch's method (Quesada-Allué and Belocopitow, 1978;Folch et al, 1957). In a preliminary assay, the lipid extracts were fractionated into the various classes of lipids by absorption chromatography in silicic acid columns according to Kates (1986).…”