Dataset Information

Synthesis of a Glycosylphosphatidylinositol Anchor Derived from Leishmania donovani That Can Be Functionalized by Cu-Catalyzed Azide-Alkyne Cycloadditions.

ABSTRACT: A flexible assembly strategy has been developed for the synthesis of Leishmania donovani GPI anchors that bear a clickable alkyne tag. This strategy is based on the use of the 2-naphthylmethyl (Nap) ethers and levulinoyl (Lev) ester for permanent protection of hydroxyls. Removal of seven Nap ethers by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone made it possible to prepare GPIs having an alkyne functionality that could be modified by Cu(I)-catalyzed [3 + 2] cycloadditions to install tags for imaging studies.

SUBMITTER: Ding N

PROVIDER: S-EPMC5588658 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Synthesis of a Glycosylphosphatidylinositol Anchor Derived from Leishmania donovani That Can Be Functionalized by Cu-Catalyzed Azide-Alkyne Cycloadditions.

Ding Ning N Li Xiuru X Chinoy Zoeisha S ZS Boons Geert-Jan GJ

Organic letters 20170711 14

A flexible assembly strategy has been developed for the synthesis of Leishmania donovani GPI anchors that bear a clickable alkyne tag. This strategy is based on the use of the 2-naphthylmethyl (Nap) ethers and levulinoyl (Lev) ester for permanent protection of hydroxyls. Removal of seven Nap ethers by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone made it possible to prepare GPIs having an alkyne functionality that could be modified by Cu(I)-catalyzed [3 + 2] cycloadditions to install tags for imagin ...[more]

PMID: 28696125

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Project description:O-linked β-N-acetylglucosamine (O-GlcNAc) is emerging as an essential protein post-translational modification in a range of organisms. It is involved in various cellular processes such as nutrient sensing, protein degradation, gene expression, and is associated with many human diseases. Despite its importance, identifying O-GlcNAcylated proteins is a major challenge in proteomics. Here, using peracetylated N-azidoacetylglucosamine (Ac4 GlcNAz) as a bioorthogonal chemical handle, we described a gel-based mass spectrometry method for the identification of proteins with O-GlcNAc modification in A549 cells. In addition, we made a labeling efficiency comparison between two modes of azide-alkyne bioorthogonal reactions in click chemistry: copper-catalyzed azide-alkyne cycloaddition (CuAAC) with Biotin-Diazo-Alkyne and stain-promoted azide-alkyne cycloaddition (SPAAC) with Biotin-DIBO-Alkyne. After conjugation with click chemistry in vitro and enrichment via streptavidin resin, proteins with O-GlcNAc modification were separated by SDS-PAGE and identified with mass spectrometry. Proteomics data analysis revealed that 229 putative O-GlcNAc modified proteins were identified with Biotin-Diazo-Alkyne conjugated sample and 188 proteins with Biotin-DIBO-Alkyne conjugated sample, among which 114 proteins were overlapping. Interestingly, 74 proteins identified from Biotin-Diazo-Alkyne conjugates and 46 verified proteins from Biotin-DIBO-Alkyne conjugates could be found in the O-GlcNAc modified proteins database dbOGAP (http://cbsb.lombardi.georgetown.edu/hulab/OGAP.html). These results suggested that CuAAC with Biotin-Diazo-Alkyne represented a more powerful method in proteomics with higher protein identification and better accuracy compared to SPAAC. The proteomics credibility was also confirmed by the molecular function and cell component gene ontology (GO). Together, the method we reported here combining metabolic labeling, click chemistry, affinity-based enrichment, SDS-PAGE separation, and mass spectrometry, would be adaptable for other post-translationally modified proteins in proteomics.

Project description:Visceral leishmaniasis is a deadly infectious disease caused by Leishmania donovani, a kinetoplastid parasite for which no licensed vaccine is available. To identify potential vaccine candidates, we systematically identified genes encoding putative cell surface and secreted proteins essential for parasite viability and host infection. We identified a protein encoded by LdBPK_061160 which, when ablated, resulted in a remarkable increase in parasite adhesion to tissue culture flasks. Here, we show that this phenotype is caused by the loss of glycosylphosphatidylinositol (GPI)-anchored surface molecules and that LdBPK_061160 encodes a noncatalytic component of the L. donovani GPI-mannosyltransferase I (GPI-MT I) complex. GPI-anchored surface molecules were rescued in the LdBPK_061160 mutant by the ectopic expression of both human genes PIG-X and PIG-M, but neither gene could complement the phenotype alone. From further sequence comparisons, we conclude that LdBPK_061160 is the functional orthologue of yeast PBN1 and mammalian PIG-X, which encode the noncatalytic subunits of their respective GPI-MT I complexes, and we assign LdBPK_061160 as LdPBN1. The LdPBN1 mutants could not establish a visceral infection in mice, a phenotype that was rescued by constitutive expression of LdPBN1. Although mice infected with the null mutant did not develop an infection, exposure to these parasites provided significant protection against subsequent infection with a virulent strain. In summary, we have identified the orthologue of the PBN1/PIG-X noncatalytic subunit of GPI-MT I in trypanosomatids, shown that it is essential for infection in a murine model of visceral leishmaniasis, and demonstrated that the LdPBN1 mutant shows promise for the development of an attenuated live vaccine. IMPORTANCE Visceral leishmaniasis is a deadly infectious disease caused by the parasites Leishmania donovani and Leishmania infantum. It remains a major global health problem, and there is no licensed highly effective vaccine. Molecules that are displayed on the surface of parasites are involved in host-parasite interactions and have important roles in immune evasion, making vaccine development difficult. One major way in which parasite surface molecules are tethered to the surface is via glycophosphatidylinositol (GPI) anchors; however, the enzymes required for all the biosynthetic steps in these parasites are not known. Here, we identified the enzyme required for an essential step in the GPI anchor-biosynthetic pathway in L. donovani, and we show that while parasites lacking this gene are viable in vitro, they are unable to establish infections in mice, a property we show can be exploited to develop a live genetically attenuated parasite vaccine.

Dataset Information

Synthesis of a Glycosylphosphatidylinositol Anchor Derived from Leishmania donovani That Can Be Functionalized by Cu-Catalyzed Azide-Alkyne Cycloadditions.

Publications

Synthesis of a Glycosylphosphatidylinositol Anchor Derived from Leishmania donovani That Can Be Functionalized by Cu-Catalyzed Azide-Alkyne Cycloadditions.

Similar Datasets

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