Abstract
The trypanosomatids are generally aberrant in their protein N-glycosylation pathways. However, protein N-glycosylation in the African trypanosome Trypanosoma brucei, etiological agent of human African sleeping sickness, is not well understood. Here, we describe the creation of a bloodstream-form T. brucei mutant that is deficient in the endoplasmic reticulum enzyme glucosidase II. Characterization of the variant surface glycoprotein, the main glycoprotein synthesized by the parasite with two N-glycosylation sites, revealed unexpected changes in the N-glycosylation of this molecule. Structural characterization by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical and enzymatic treatments revealed that one of the two glycosylation sites was occupied by conventional oligomannose structures, whereas the other accumulated unusual structures in the form of Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, and Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc. The possibility that these structures might arise from Glc1Man9GlcNAc2 by unusually rapid alpha-mannosidase processing was ruled out using a mixture of alpha-mannosidase inhibitors. The results suggest that bloodstream-form T. brucei can transfer both Man9GlcNAc2 and Man5GlcNAc2 to the variant surface glycoprotein in a site-specific manner and that, unlike organisms that transfer exclusively Glc3Man9GlcNAc2, the T. brucei UDP-Glc: glycoprotein glucosyltransferase and glucosidase II enzymes can use Man5GlcNAc2 and Glc1Man5GlcNAc2, respectively, as their substrates. The ability to transfer Man5GlcNAc2 structures to N-glycosylation sites destined to become Man(4-3)GlcNAc2 or complex structures may have evolved as a mechanism to conserve dolichol-phosphate-mannose donors for glycosylphosphatidylinositol anchor biosynthesis and points to fundamental differences in the specificities of host and parasite glycosyltransferases that initiate the synthesis of complex N-glycans.
Highlights
Protein N-glycosylation serves a wide variety of functions including signaling through interaction with lectins, protein stabilization, protease resistance, endocytic sorting functions, and protein folding [5,6,7]
Endo H-resistant N-Glycans Are Added to One Glycosylation Site of VSG221—Variant 221 trypanosomes were pulse-labeled in culture with [35S]methionine for 3 min, and the cell lysates were analyzed by SDSPAGE and fluorography before and after PNGase F and Endo H treatment
The results show that two forms of variant surface glycoprotein (VSG) are apparent after 3 min of pulse-labeling (Fig. 1, lanes 1 and 3); they are a weakly labeled 53-kDa unglycosylated band and a strongly labeled upper 57-kDa band that co-migrates with mature VSG221 containing 2 occupied N-glycosylation sites at Asn-263 and Asn-428 [4]
Summary
Cultivation of Trypanosomes—Bloodstream-form T. brucei genetically modified to express T7 polymerase and the tetracycline repressor protein were cultivated in HMI-9 medium containing 2.5 g/ml G418 at 37 °C in a 5% CO2 incubator as described in Wirtz et al [25]. Transformation of Bloodstream-form T. brucei—Constructs for gene replacement and ectopic expression were purified using the Qiagen Maxiprep kit, digested with NotI to linearize, precipitated, washed twice with 70% ethanol, and redissolved in sterile water. The linearized DNA was electroporated into T. brucei bloodstream cells (strain 427, variant 221) that were stably transformed to express T7 RNA polymerase and the tetracycline repressor protein under G418 selection [25]. Southern Blotting—Aliquots of genomic DNA isolated from 100 ml of bloodstream-form T. brucei cultures (ϳ2 ϫ 108 cells) were digested with various restriction enzymes. Aliquots of 8 l were used for each Southern blot experiment
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