Abstract. African trypanosomes contain a membranebound enzyme capable of removing dimyristylglycerol from the membrane-attached form of the variable surface glycoprotein (mfVSG; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968). Although mfVSG phospholipase-C has been implicated in the removal of the VSG from the trypanosome surface (Cardoso de Almeida, M. L., and M. J. Turner, 1983, Nature (Lond.)., 302:349-352; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968), its precise function and subcellular location have not been determined. We have developed a procedure for the separation of the cell fractions and organelles of Trypanosoma brucei brucei (and other trypanosome species) by differential sucrose and isopycnic Percoll R centrifugation. These fractions were tested for mfVSG phospholipase activity using Trypanosoma brucei mfVSG labeled with 3H-myristic acid as substrate.The highest enzyme-specific activity was associated with the flagella and evidence is presented to suggest that it is localized in the flagellar pocket. Some activity was also associated with the Golgi complex. These results suggest that the mfVSG phospholipase is localized primarily in the membrane of the flagella pocket and possibly other membrane organelles derived from and associated with this structure, and may be part of the VSG-membrane recycling system in African trypanosomes.The activity of mfVSG phospholipase amongst various trypanosome species was determined. We show that, in contrast to the bloodstream forms of 1. Abbreviations used in this paper: CRD, cross-reacting determinant; GPDH, glycerol-3-phosphate dehydrogenase; LG fraction, large granule fraction; mfVSG, membrane-attached form of the variable surface glycoprotein; SG fraction, small granule fraction; sVSG, soluble form of the variable surface glycoprotein; VSG, variable surface glycoprotein. tegrity of the surface coat is known to be essential for parasite survival in the host, the purpose of this study was to investigate VSG synthesis, processing, and replacement on the cell surface. A clearer understanding of the cell biology of the VSG may suggest alternative approaches for the possible control of trypanosomiasis.The VSGs from T. brucei are proteins that contain complex carbohydrate moeities of mannose, galactose, glucosamine, and myristilated phosphotidylinositol (17,18,39) conjugated to the protein through the alpha-carboxyl group of the carboxy-terminal amino acid via an ethanolamine residue (13,28,39). The biological importance to VSG of this carbohydrate side chain is evident since it is present on all variable antigens of T. brucei and T. congolense organisms so far studied and is probably responsible for cross-reactivity observed between soluble forms of VSGs (also known as the cross-reacting determinant [CRD]) (14). The carbohydrate side chain may be involved in anchoring the VSG to the plasma membrane (15,17,18,39), and maintaining the integrity of VSG-VSG interactions within the coat ...
Protein folding in the cell is regulated by several quality-control mechanisms. Correct folding of glycoproteins in the endoplasmic reticulum (ER) is tightly monitored by the recognition of glycan signals by lectins in the ER-associated degradation (ERAD) pathway. In mammals, mannose trimming from -glycans is crucial for disposal of misfolded glycoproteins. The mannosidases responsible for this process are ER mannosidase I and ER degradation-enhancing α-mannosidase-like proteins (EDEMs). However, the molecular mechanism of mannose removal by EDEMs remains unclear, partly owing to the difficulty of reconstituting mannosidase activity Here, our analysis of EDEM3-mediated mannose-trimming activity on a misfolded glycoprotein revealed that ERp46, an ER-resident oxidoreductase, associates stably with EDEM3. This interaction, which depended on the redox activity of ERp46, involved formation of a disulfide bond between the cysteine residues of the ERp46 redox-active sites and the EDEM3 α-mannosidase domain. In a defined system consisting of recombinant proteins purified from HEK293 cells, the mannose-trimming activity of EDEM3 toward the model misfolded substrate, the glycoprotein T-cell receptor α locus (TCRα), was reconstituted only when ERp46 had established a covalent interaction with EDEM3. On the basis of these findings, we propose that disposal of misfolded glycoproteins through mannose trimming is tightly connected to redox-mediated regulation in the ER.
Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity . Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied . The Golgi complex fraction contained most of the gaIactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions . The dolichol-dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex . Although the dolicholindependent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction . Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A, suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed .
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