Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.

Abstract

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had the same structure as the lipid-linked compound, whereas in the second oligosaccharide some mannose residues had been replaced by galactose units, but both compounds migrated as did a Man7GlcNAc standard.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...

Endogenous and surface labeling of glycoconjugates from the three differentiation stages of Trypanosoma cruzi

“…The inhibition of radioactivity observed in the lower phase of the chloroform/methanol/water (3:2:1) extract in the tunicamycin experiments suggests the existence of N-linked oligosaccharide intermediates with shorter chains than those found in other glycoproteins [ 17]. Oligosaccharide transferred to proteins in C. fasciculata contained 2 N-acetylglucosamine and only 7 mannose residues [18].…”

Deletion of the Glucosidase II Gene in Trypanosoma brucei Reveals Novel N-Glycosylation Mechanisms in the Biosynthesis of Variant Surface Glycoprotein

“…The mature Dol-PP-oligosaccharide species used for transfer to protein vary according to trypanosomatid species. For example, Trypanosoma conhorini, Trypanosoma dionisii, Leptomonas samueli, Herpetomonas samuelpessoai, and Herpetomonas muscarum utilize triantennary Man 9 GlcNAc 2 -PP-Dol; Crithidia fasciculata, Crithidia harmosa, and Leishmania enriettii utilize biantennary Man 7 GlcNAc 2 -PP-Dol; Leishmania mexicana, Leishmania adleri, and Blastocrithidia culicus utilize biantennary Man 6 GlcNAc 2 -PP-Dol (15)(16)(17)(18). Trypanosoma cruzi, the causative agent of Chagas disease in the Americas, utilizes Man 9 GlcNAc 2 -PP-Dol during most of its life cycle but uses both Man 9 GlcNAc 2 -PP-Dol and Man 7 GlcNAc 2 -PP-Dol in its bloodstream trypomastigote stage (19).…”

“…14) has shown that protein N-glycosylation in several trypanosomatid parasites is aberrant (15)(16)(17)(18)(19). None of these organisms can make Dol-P-Glc and so fail to make glucosylated Dol-PP-oligosaccharide precursors.…”

Trypanosoma brucei UDP-Glucose:Glycoprotein Glucosyltransferase Has Unusual Substrate Specificity and Protects the Parasite from Stress

“…The mature dolichol-phosphate-oligosaccharide species used for transfer to protein vary according to trypanosomatid species (17,51,52,56). Therefore, in these organisms, monoglucosylated glycans are exclusively formed through UGGT-dependent glucosylation (12).…”