We describe a simple method for efficiently labeling cell surface glycans on virtually any living animal cell. The method employs mild Periodate oxidation to generate an aldehyde on sialic acids, followed by Aniline-catalyzed oxime Ligation with a suitable tag (PAL). Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell surface glycoproteins while maintaining high cell viability. Keywordsglycoprotein; sialic acid; oxime ligation; aniline; periodate oxidation; metabolic labeling; live cell labelingThe expanding interest in glycoproteomics and the biological roles of glycoconjugates has increased efforts to develop efficient tools to label cell surface glycoproteins. Several elegant approaches have exploited metabolic labeling of cells, and even whole model organisms, using analogs of glycan precursors that carry bio-orthogonal groups (e.g. azide, alkyne, ketone or aldehyde), allowing the chemical ligation of reporter groups onto cell surface glycoconjugates 1, 2.The chemistries used for conjugation with these functional groups each have both advantages and disadvantages for use with living cells. The conjugation of azides with substituted triphenylphosphines using the Staudinger-Bertozzi ligation can be performed on living cells, but suffers from slow reaction kinetics 3 . Conjugation of azides with substituted-alkynes (or vice versa) with the Huisgen cycloaddition, or 'click chemistry', has rapid reaction kinetics, but requires a copper catalyst that is toxic to living cells4 , 5. The newly described ligation of azides with ring-strained alkynes is compatible with living cells and has rapid reaction kinetics, but requires reagents that are not currently commercially available 2 . Finally, imine (oxime or A limitation of all these methods is the need for culturing cells with a glycan precursor containing a bio-orthogonal group prior to labeling. As an alternative, aldehydes can be readily introduced into cell surface glycans by mild periodate oxidation, known for nearly 40 years to selectively oxidize the polyhydroxy side chain of sialic acids 7,8 . The recent demonstration that oxime ligation on complex biomolecules is dramatically accelerated using aniline as a nucleophilic catalyst 9-12 inspired us to explore the efficiency of this reaction with aldehydes on living cells introduced by metabolic labeling or periodate oxidation. While aniline can be efficiently used as a nucleophilic catalyst for labeling biomolecules in solution by oxime and hydrazone ligations, we chose to employ the oxime ligation, which gives a more stable product than the hydrazone ligation 13 . NIH Public AccessThe two approaches used for the introduction of aldehydes onto cell surface sialic acids for subsequent ligation with aminooxy-biotin are illustrated in Fig. 1a. Cells were subjected to mild periodate oxidation (1 mM NaIO 4 at 4 °C for 30 min; Supplementary Fig. 1) to selectively introduce an aldehyde at C-7 of sialic acid...

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The emergence of drug-resistant forms of Plasmodium falciparum emphasizes the need to develop new antimalarials. In this context, the fatty acid biosynthesis (FAS) pathway of the malarial parasite has recently received a lot of attention. Due to differences in the fatty acid biosynthesis systems of Plasmodium and man, this pathway is a good target for the development of new and selective therapeutic drugs directed against malaria. In continuation of these efforts we report cloning and overexpression of P. falciparum ␤-hydroxyacyl-acyl carrier protein (ACP) dehydratase (PffabZ) gene that codes for a 17-kDa protein. The enzyme catalyzes the dehydration of ␤-hydroxyacyl-ACP to trans-2-acyl-ACP, the third step in the elongation phase of the FAS cycle. It has a K m of 199 M and k cat /K m of 80.4 M ؊1 s ؊1 for the substrate analog ␤-hydroxybutyryl-CoA but utilizes crotonoyl-CoA, the product of the reaction, more efficiently (K m ‫؍‬ 86 M, k cat / K m ‫؍‬ 220 M ؊1 s ؊1 ). More importantly, we also identify inhibitors (NAS-91 and NAS-21) for the enzyme. Both the inhibitors prevented the binding of crotonoyl-CoA to PfFabZ in a competitive fashion. Indeed these inhibitors compromised the growth of P. falciparum in cultures and inhibited the parasite fatty acid synthesis pathway both in cell-free extracts as well as in situ. We modeled the structure of PfFabZ using Escherichia coli ␤-hydroxydecanoyl thioester dehydratase (EcFabA) as a template. We also modeled the inhibitor complexes of PfFabZ to elucidate the mode of binding of these compounds to FabZ. The discovery of the inhibitors of FabZ, reported for the first time against any member of this family of enzymes, essential to the type II FAS pathway opens up new avenues for treating a number of infectious diseases including malaria.Malaria continues to exact the highest mortality and morbidity rate next only to tuberculosis. "The scourge of the tropics," malaria is endemic to around 100 countries in the world.Approximately 500 million cases of malaria are reported every year, and around 3000 children die of malaria every day (1). Emerging resistance to chloroquine and other currently prescribed drugs limits treatment of malaria today, in particular cerebral malaria, caused by Plasmodium falciparum (2, 3). The situation definitely warrants express remedial actions: extensive research on P. falciparum to identify drug targets and, ultimately, the development of a new armamentarium of antimalarials.Our recent demonstration of the occurrence of the type II fatty acid synthesis (FAS) 1 pathway in the malaria parasite and its inhibition by triclosan, an inhibitor of the rate-limiting enzyme of type II FAS, enoyl-acyl carrier protein (ACP) reductase, proved the pivotal role played by this pathway in the survival of the malarial parasite (4, 5). The essential role of fatty acids and lipids in cell growth and differentiation and the different type (type I) of fatty acid biosynthetic pathway occurring in the human host, which is distinct from type II FAS of the malaria parasit...

Targeting of apicoplast replication and protein synthesis in the apicomplexan Toxoplasma gondii has conventionally been associated with the typical "delayed death" phenotype, characterized by the death of parasites only in the generation following drug intervention. We demonstrate that antibiotics like clindamycin, chloramphenicol, and tetracycline, inhibitors of prokaryotic protein synthesis, invoke the delayed death phenotype in Plasmodium falciparum, too, as evident from a specific reduction of apicoplast genome copy number. Interestingly, however, molecules like triclosan, cerulenin, fops, and NAS-91, inhibitors of the recently discovered fatty acid synthesis pathway, and succinyl acetone, an inhibitor of heme biosynthesis that operates in the apicoplast of the parasite, display rapid and striking parasiticidal effects. Our results draw a clear distinction between apicoplast functions per se and the apicoplast as the site of metabolic pathways, which are required for parasite survival, and thus subserve the development of novel antimalarial therapy.

In this paper, we present two complementary strategies for enrichment of glycoproteins on living cells that combine the desirable attributes of "robust enrichment" afforded by covalent-labeling techniques and "specificity for glycoproteins" typically provided by lectin or antibody affinity reagents. Our strategy involves the selective introduction of aldehydes either into sialic acids by periodate oxidation (periodate oxidation and aniline-catalyzed oxime ligation (PAL)) or into terminal galactose and N-acetylgalactosamine residues by galactose oxidase (galactose oxidase and aniline-catalyzed oxime ligation (GAL)), followed by aniline-catalyzed oxime ligation with aminooxy-biotin to biotinylate the glycans of glycoprotein subpopulations with high efficiency and cell viability. As expected, the two methods exhibit reciprocal tagging efficiencies when applied to fully sialylated cells compared with sialic acid-deficient cells. To assess the utility of these labeling methods for glycoproteomics, we enriched the PAL- and GAL-labeled (biotinylated) glycoproteome by adsorption onto immobilized streptavidin. Glycoprotein identities (IDs) and N-glycosylation site information were then obtained by liquid chromatography-tandem mass spectrometry on total tryptic peptides and on peptides subsequently released from N-glycans still bound to the beads using peptide N-glycosidase F. A total of 175 unique N-glycosylation sites were identified, belonging to 108 nonredundant glycoproteins. Of the 108 glycoproteins, 48 were identified by both methods of labeling and the remainder was identified using PAL on sialylated cells (40) or GAL on sialic acid-deficient cells (20). Our results demonstrate that PAL and GAL can be employed as complementary methods of chemical tagging for targeted proteomics of glycoprotein subpopulations and identification of glycosylation sites of proteins on cells with an altered sialylation status.