A Trypanosoma brucei β3 glycosyltransferase superfamily gene encodes a β1-6 GlcNAc-transferase mediating N-glycan and GPI anchor modification

The parasite Trypanosoma brucei exists in both a bloodstream form (BSF) and a procyclic form (PCF), which exhibit large carbohydrate extensions on the N-linked glycans and glycosylphosphatidylinositol (GPI) anchors, respectively. The parasite's glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are currently uncharacterized. Here, we probe the function(s) of the uncharacterized GT67 glycosyltransferase family and a β3 glycosyltransferase (β3GT) superfamily gene, TbGT10. A BSF-null mutant, created by applying the diCre/loxP method in T. brucei for the first time, showed a fitness cost but was viable in vitro and in vivo and could differentiate into the PCF, demonstrating nonessentiality of TbGT10. The absence of TbGT10 impaired the elaboration of N-glycans and GPI anchor side chains in BSF and PCF parasites, respectively. Glycosylation defects included reduced BSF glycoprotein binding to the lectin ricin and monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope present on lysosomal glycoprotein p67 that we show here consists of (-6Galβ1-4GlcNAcβ1-)≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase-digested glycopeptides isolated from BSF wild-type and TbGT10 null parasites showed a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. These data define TbGT10 as a UDP-GlcNAc:βGal β1-6 GlcNAc-transferase. The dual role of TbGT10 in BSF N-glycan and PCF GPI-glycan elaboration is notable, and the β1-6 specificity of a β3GT superfamily gene product is unprecedented. The similar activities of trypanosome TbGT10 and higher-eukaryote I-branching enzyme (EC 2.4.1.150), which belong to glycosyltransferase families GT67 and GT14, respectively, in elaborating N-linked glycans, are a novel example of convergent evolution

Neopetroside-B alleviates doxorubicin-induced cardiotoxicity via mitochondrial protection

Doxorubicin, a member of the anthracycline family, is a widely prescribed anticancer chemotherapy drug. Unfortunately, cumulative doses of doxorubicin can cause mitochondrial dysfunction, leading to acute or chronic cardiotoxicity. This study demonstrated that Neopetroside-B (NPS-B) protects cardiomyocytes in the presence of doxorubicin. NPS-B improved mitochondrial function in cardiomyocytes by increasing ATP production and oxygen consumption rates. On the other hand, NPS-B negatively influenced cancer cell lines by increasing reactive oxygen species. We analyzed NPS-B-influenced metabolites (VIP > 1.0; AUC>0.7; p  2.0) and constructed metabolite-protein enrichment, which showed that NPS-B affected uracil metabolism and NAD-binding proteins (e.g., aldehyde dehydrogenase and glutathione reductase) in cardiomyocytes. However, for the cancer cells, NPS-B decreased the NAD+/NADH balance, impairing cell viability. In a xenograft mouse model treated with doxorubicin, NPS-B reduced cardiac fibrosis and improved cardiac function. NPS-B may be a beneficial intervention to reducing doxorubicin-induced cardiotoxicity with anticancer effects

Probing Enzymes Late in the Trypanosomal Glycosylphosphatidylinositol Biosynthetic Pathway with Synthetic Glycosylphosphatidylinositol Analogues

Glycosylphosphatidylinositol (GPI)-anchored proteins are abundant in the protozoan parasite <i>Trypanosoma brucei</i>, the causative agent of African sleeping sickness in humans and the related disease Nagana in cattle, and disruption of GPI biosynthesis is genetically and chemically validated as a drug target. Here, we examine the ability of enzymes of the trypanosomal GPI biosynthetic pathway to recognize and process a series of synthetic dimannosyl-glucosaminylphosphatidylinositol analogues containing systematic modifications on the mannose residues. The data reveal which portions of the natural substrate are important for recognition, explain why mannosylation occurs prior to inositol acylation in the trypanosomal pathway, and identify the first inhibitor of the third α-mannosyltransferase of the GPI biosynthetic pathway