Cells of the insect parasite Crithidia fasciculata incubated with ['4C]glucose were found to possess only one lipidbound oligosaccharide with solubility in chloroform/methanol/ water mixtures and net charge similar to the charges of dolichol pyrophosphate derivatives. The saccharide moiety could be released from lipid by mild acid hydrolysis. Several enzymatic and chemical treatments of the oligosaccharide indicated that the latter had the structure Mana-+Mana---Mana--[Mana-+ Mana-+Man(al--6)]Man--GlcNAc(l1--4)GlcNAc. Two labeled oligosaccharides were liberated from proteins by a sequential treatment with a protease and endo-13-N-acetylglucosaminidase H. One ofthe protein-bound oligosaccharides had the same structure as the lipid-linked compound, whereas in the second oligosaccharide some mannose residues had been replaced by galactose units, but both compounds migrated as did a Man7GlcNAc standard. These were the largest oligosaccharides obtained even after short labeling periods. It is suggested that glycosylation ofproteins in the protozoan Crithidiafasciculata does not involve glucosylated lipid-bound oligosaccharides as intermediates. It has become evident in recent years that glycosylation of asparagine residues in eukaryotic cell proteins involves dolichol pyrophosphate (DolPP)-bound oligosaccharides as intermediates (1). It has been reported that in animal tissues (2, 3), yeasts (4-6), and probably also in insects (7) and plants (8), the oligosaccharide transferred from the lipid derivative to proteins is composed of two N-acetylglucosamine, nine mannose, and three glucose residues. A report by Lehle suggests that in yeast the oligosaccharides containing two or three glucose residues are equally transferred to protein (9). However, no evidence was presented indicating that the oligosaccharides used in the assay had the same specific activity.The protein-bound oligosaccharides are then processed by loss of some of their monosaccharide constituents and addition ofother residues directly from the respective sugar nucleotides. We here report evidence suggesting that in the protozoan Crithidta fasciculata, an insect obligate parasite, the oligosaccharide transferred to protein contains two N-acetylglucosamine and seven mannose residues and that the oligosaccharide may be processed once bound to protein.MATERIALS AND METHODS Materials.[14C]Glucose (284 Ci/mol; 1 Ci = 3.7 X 1010 becquerels) was from New England Nuclear. Jack bean a-mannosidase type III and Streptomyces griseus protease type VI were purchased from Sigma and endo-,B3N-acetylglucosaminidase H (endo H), from Miles. Isolation of Lipid-Bound Oligosaccharides. C. fasciculata cells (Anopheles isolate ATCC 11745) were grown in the medium described by Bacchi et aL (10) without agar at 28°C. Six hundred milliliters of culture containing about 2 g of cells (logarithmic phase) were cooled on ice and centrifuged at 2500 X g for 10 min at 4°C. The cell pellet was resuspended in 30 ml of ice-cold minimal Eagle's medium without glucose but containing 5...

Gross morphological changes in Ceratitis capitata (Wied.) during metamorphosis within the puparium are described and temporally correlated with the appearance of main differentiation markers. A table that allows determination of physiological age of pupal and pharate adult stages is presented.

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The microsomal fraction of insects was found to contain an enzyme which transfers mannose from guanosine diphosphate mannose to an endogenous or exogenous insect lipid and to other acceptors such as dolichol monophosphate or ficaprenol monophosphate. This activity depended on the presence of Triton X-100 and magnesium ions, the optimal concentration of the latter being 10mM. The optimal temperature of the reaction was 25 degrees C and the maximal activity was obtained at pH 7.9. The mannolipid formed behaved as a monophosphodiester when chromatographed on DEAE-cellulose. Weak acid treatment of the product liberated mannose. Its behaviour both on thin layer and Sephadex G-150 chromatography would indicate the presence of a number of isoprenyl units similar to the dolichol and different from the ficaprenol derivative. Stability to phenol treatment indicated that the lipid fraction of the mannolipid is an alpha-saturated polyprenol phosphate similar to dolichol monophosphate.

Microsome enriched Ceratitis capitata extracts synthesized a glucosylated lipid linked oligosaccharide. Its properties were closely related to those of the previously described insect mannosylated dolichyl diphosphate oligosaccharides and almost the same as those of the rat liver dolichyl-diphosphate-(GlcNAc)2-(Man)9-(Glc)1-3. The saccharide moiety of the latter was transferred to an unknown endogenous protein-like acceptor by the fly extracts. These represent the first evidence of a protein glycosylation in a pluricellular invertebrate through dolichyl derivatives.