Recognition of Highly Branched N-Glycans of the Porcine Whipworm by the Immune System.

Glycans are key to host-pathogen interactions, whereby recognition by the host and immunomodulation by the pathogen can be mediated by carbohydrate binding proteins, such as lectins of the innate immune system, and their glycoconjugate ligands. Previous studies have shown that excretory-secretory products of the porcine nematode parasite Trichuris suis exert immunomodulatory effects in a glycan-dependent manner. To better understand the mechanisms of these interactions, we prepared N-glycans from T. suis and both analyzed their structures and used them to generate a natural glycan microarray. With this array, we explored the interactions of glycans with C-type lectins, C-reactive protein, and sera from T. suis-infected pigs. Glycans containing LacdiNAc and phosphorylcholine-modified glycans were associated with the highest binding by most of these proteins. In-depth analysis revealed not only fucosylated LacdiNAc motifs with and without phosphorylcholine moieties but phosphorylcholine-modified mannose and N-acetylhexosamine-substituted fucose residues, in the context of maximally tetraantennary N-glycan scaffolds. Furthermore, O-glycans also contained fucosylated motifs. In summary, the glycans of T. suis are recognized by both the innate and adaptive immune systems and also exhibit species-specific features distinguishing its glycome from those of other nematodes.

N-glycan antennal modifications are altered in Caenorhabditis elegans lacking the HEX-4 N-acetylgalactosamine-specific hexosaminidase.

Simple organisms are often considered to have simple glycomes, but plentiful paucimannosidic and oligomannosidic glycans overshadow the less abundant N-glycans with highly variable core and antennal modifications; Caenorhabditis elegans is no exception. By use of optimized fractionation and assessing wildtype in comparison to mutant strains lacking either the HEX-4 or HEX-5 ß-N-acetylgalactosaminidases, we conclude that the model nematode has a total N-glycomic potential of 300 verified isomers. Three pools of glycans were analyzed for each strain: either PNGase F released and eluted from a reversed-phase C18 resin with either water or 15% methanol or PNGase Ar released. While the water-eluted fractions were dominated by typical paucimannosidic and oligomannosidic glycans and the PNGase Ar-released pools by glycans with various core modifications, the methanol-eluted fractions contained a huge range of phosphorylcholine-modified structures with up to three antennae, sometimes with four N-acetylhexosamine residues in series. There were no major differences between the C. elegans wildtype and hex-5 mutant strains, but the hex-4 mutant strains displayed altered sets of methanol-eluted and PNGase Ar-released pools. In keeping with the specificity of HEX-4, there were more glycans capped with N-acetylgalactosamine in the hex-4 mutants, as compared with isomeric chito-oligomer motifs in the wildtype. Considering that fluorescence microscopy showed that a HEX-4::enhanced GFP fusion protein colocalizes with a Golgi tracker, we conclude that HEX-4 plays a significant role in late-stage Golgi processing of N-glycans in C. elegans. Furthermore, finding more "parasite-like" structures in the model worm may facilitate discovery of glycan-processing enzymes occurring in other nematodes.

Revisiting the isolation and characterisation of Entamoeba histolytica lipopeptidophosphoglycan.

The parasite Entamoeba histolytica is the cause of amoebic dysentery and liver abscess in humans. On the protozoan cell surface, a variety of glycosylated molecules are involved in the interaction with the environment, such as attachment to the colonic mucus. One of these molecules is the lipopeptidophosphoglycan (LPPG), a complex surface component with antigenic properties. Its structure is only partly known, it is a glycosylphosphatidylinositol (GPI)-linked glycoprotein with a large amount of O-glycosylation. To date, the sequence of a core protein has not been identified. In this study, we further investigated this complex surface molecule aided by the availability of the monoclonal antibody EH5, which had been raised in our laboratory. We studied the extraction of LPPG in various solvent mixtures and discovered that 2-butanol saturated water was simple and superior to other solvents used in the past. The isolated LPPG was subjected to treatment with several proteases and the Ser/Thr specific cleavage agent scandium (III) trifluoromethanesulfonate (scandium triflate). The products were probed with antibody EH5 and the blots showed that the LPPG preparation was largely resistant to standard proteases, but could be cleaved by the scandium compound. These observations could point to the existence of a Ser- or Thr-rich core protein structure.

Sulfated and sialylated N-glycans in the echinoderm Holothuria atra reflect its marine habitat and phylogeny.

Among the earliest deuterostomes, the echinoderms are an evolutionary important group of ancient marine animals. Within this phylum, the holothuroids (sea cucumbers) are known to produce a wide range of glycoconjugate biopolymers with apparent benefits to health; therefore, they are of economic and culinary interest throughout the world. Other than their highly modified glycosaminoglycans (e.g. fucosylated chondroitin sulfate and fucoidan), nothing is known about their protein-linked glycosylation. Here we used multistep N-glycan fractionation to efficiently separate anionic and neutral N-glycans before analyzing the N-glycans of the black sea cucumber (Holothuria atra) by MS in combination with enzymatic and chemical treatments. These analyses showed the presence of various fucosylated, phosphorylated, sialylated, and multiply sulfated moieties as modifications of oligomannosidic, hybrid, and complex-type N-glycans. The high degree of sulfation and fucosylation parallels the modifications observed previously on holothuroid glycosaminoglycans. Compatible with its phylogenetic position, H. atra not only expresses vertebrate motifs such as sulfo- and sialyl-Lewis A epitopes but displays a high degree of anionic substitution of its glycans, as observed in other marine invertebrates. Thus, as for other echinoderms, the phylum- and order-specific aspects of this species' N-glycosylation reveal both invertebrate- and vertebrate-like features.

Isomeric Separation and Recognition of Anionic and Zwitterionic N-glycans from Royal Jelly Glycoproteins.

Royal jelly has received attention because of its necessity for the development of queen honeybees as well as claims of benefits on human health; this product of the hypopharyngeal glands of worker bees contains a large number of proteins, some of which have been claimed to have various biological effects only in their glycosylated state. However, although there have been glycomic and glycoproteomic analyses in the past, none of the glycan structures previously defined would appear to have potential to trigger specific biological functions. In the current study, whole royal jelly as well as single protein bands were subject to off-line LC-MALDI-TOF MS glycomic analyses, complemented by permethylation, Western blotting and arraying data. Similarly to recent in-depth studies on other insect species, previously overlooked glucuronic acid termini, sulfation of mannose residues and core ß-mannosylation of the N-glycans were found; additionally, a relatively rare zwitterionic modification with phosphoethanolamine is present, in contrast to the phosphorylcholine occurring in lepidopteran species. Indicative of tissue-specific remodelling of glycans in the Golgi apparatus of hypopharyngeal gland cells, only a low amount of fucosylated or paucimannosidic glycans were detected as compared with other insect samples or even bee venom. The unusual modifications of hybrid and multiantennary structures defined here may not only have a physiological role in honeybee development, but represent epitopes recognized by pentraxins with roles in animal innate immunity.

The parasitic nematode Oesophagostomum dentatum synthesizes unusual glycosaminoglycan-like O-glycans.

O-glycosylation is probably one of the most varied sets of post-translational modifications across all organisms, but amongst the most refractory to analyze. In animals, O-xylosylation of serine residues represents the first stage in the synthesis of glycosaminoglycans, whose repeat regions are generally analyzed as fragments resulting from enzymatic or chemical degradation, whereas their core regions can be isolated by ß-elimination or endo-ß-xylosidase digestion. In the present study, we show that hydrazinolysis can be employed for release of glycosaminoglycan-type oligosaccharides from nematodes prior to fluorescent labeling with 2-aminopyridine. While various [HexNAcHexA]nGal2Xyl oligosaccharides were isolated from the model organism Caenorhabditis elegans, more unusual glycosaminoglycan-type glycans were found to be present in the porcine parasite Oesophagostomum dentatum. In this case, as judged by MS/MS before and after hydrofluoric acid or ß-galactosidase digestion, core sequences with extra galactose and phosphorylcholine residues were detected as [(±PC)HexNAcHexA]n(±PC)Galß3-(±Galß4)Galß4Xyl. Thus, hydrazinolysis and fluorescent labeling can be combined to analyze unique forms of O-xylosylation, including new examples of zwitterionic glycan modifications.

Core Richness of N-Glycans of Caenorhabditis elegans: A Case Study on Chemical and Enzymatic Release.

Despite years of research, the glycome of the model nematode Caenorhabditis elegans is still not fully understood. Certainly, data over the years have indicated that this organism synthesizes unusual N-glycans with a range of galactose and fucose modifications on the Man2-3GlcNAc2 core region. Previously, up to four fucose residues were detected on its N-glycans, despite these lacking the fucosylated antennae typical of many other eukaryotes; some of these fucose residues are capped with hexose residues as shown by the studies of us and others. There have, though, been contrasting reports regarding the maximal number of fucose substitutions in C. elegans, which in part may be due to different methodological approaches, including use of either peptide:N-glycosidases F and A (PNGase F and A) or anhydrous hydrazine to cleave the N-glycans from glycopeptides. Here we compare the use of hydrazine with that of a new enzyme (rice PNGase Ar) and show that both enable release of glycans with more sugar residues on the proximal GlcNAc than previously resolved. By use of exoglycosidase sequencing, in conjunction with high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), we now reveal that actually up to five fucose residues modify the core region of C. elegans N-glycans and that the α1,3-fucose on the reducing terminus can be substituted by an α-linked galactose. Thus, traditional PNGase F and A release may be insufficient for release of the more highly core-modified N-glycans, especially those occurring in C. elegans, but novel enzymes can compete against chemical methods in terms of safety, ease of cleanup, and quality of resulting glycomic data.

More Than Just Oligomannose: An N-glycomic Comparison of Penicillium Species.

N-glycosylation is an essential set of post-translational modifications of proteins; in the case of filamentous fungi, N-glycans are present on a range of secreted and cell wall proteins. In this study, we have compared the glycans released by peptide/N-glycosidase F from proteolysed cell pellets of three Penicillium species (P. dierckxii, P. nordicum and P. verrucosum that all belong to the Eurotiomycetes). Although the major structures are all within the range Hex(5-11)HexNAc(2) as shown by mass spectrometry, variations in reversed-phase chromatograms and MS/MS fragmentation patterns are indicative of differences in the actual structure. Hydrofluoric acid and mannosidase treatments revealed that the oligomannosidic glycans were not only in part modified with phosphoethanolamine residues and outer chain och1-dependent mannosylation, but that bisecting galactofuranose was present in a species-dependent manner. These data are the first to specifically show the modification of N-glycans in fungi with zwitterionic moieties. Furthermore, our results indicate that mere mass spectrometric screening is insufficient to reveal the subtly complex nature of N-glycosylation even within a single fungal genus.

Gender and developmental specific N-glycomes of the porcine parasite Oesophagostomum dentatum.

BACKGROUND: The porcine nodule worm Oesophagostomum dentatum is a strongylid class V nematode rather closely related to the model organism Caenorhabditis elegans. However, in contrast to the non-parasitic C. elegans, the parasitic O. dentatum is an obligate sexual organism, which makes both a gender and developmental glycomic comparison possible. METHODS: Different enzymatic and chemical methods were used to release N-glycans from male and female O. dentatum as well as from L3 and L4 larvae. Glycans were analysed by MALDI-TOF MS after either 2D-HPLC (normal then reversed phase) or fused core RP-HPLC. RESULTS: Whereas the L3 N-glycome was simpler and more dominated by phosphorylcholine-modified structures, the male and female worms express a wide range of core fucosylated N-glycans with up to three fucose residues. Seemingly, simple methylated paucimannosidic structures can be considered 'male', while methylation of fucosylated glycans was more pronounced in females. On the other hand, while many of the fucosylated paucimannosidic glycans are identical with examples from other nematode species, but simpler than the tetrafucosylated glycans of C. elegans, there is a wide range of phosphorylcholine-modified glycans with extended HexNAc2-4PC2-4 motifs not observed in our previous studies on other nematodes. CONCLUSION: The interspecies tendency of class V nematodes to share most, but not all, N-glycans applies also to O. dentatum; furthermore, we establish, for the first time in a parasitic nematode, that glycomes vary upon development and sexual differentiation. GENERAL SIGNIFICANCE: Unusual methylated, core fucosylated and phosphorylcholine-containing N-glycans vary between stages and genders in a parasitic nematode.

Accumulation of GD1α Ganglioside in MDA-MB-231 Breast Cancer Cells Expressing ST6GalNAc V.

α-Series gangliosides define a particular sub-class of glycosphingolipids containing sialic acid α2,6-linked to GalNAc residue that was isolated as a minor compound from the brain. The sialyltransferase ST6GalNAc V was cloned from mouse brain and showed α2,6-sialyltransferase activity almost exclusively for GM1b, to form GD1α and is considered as the main enzyme involved in the biosynthesis of α-series gangliosides. Recently, ST6GALNAC5 was identified as one of the genes over-expressed in breast cancer cell populations selected for their ability to produce brain metastasis. However, the capacity of human breast cancer cells to produce α-series gangliosides has never been clearly demonstrated. Here, we show by stable transfection and MS-MS analysis of total glycosphingolipids that ST6GALNAC5 expressing MDA-MB-231 breast cancer cells accumulate GD1α ganglioside (IV3Neu5Ac1, III6Neu5Ac1Gg4-Cer).

Analysis of Caenorhabditis Protein Glycosylation.

Glycoproteins result from post-translational modification of proteins by glycans attached to certain side chains, with possible heterogeneity due to different structures being possible at the same glycosylation site.In contrast to the mammalian systems, analysis of invertebrate glycans presents a challenge in analysis as there exist unfamiliar epitopes and a high degree of structural and isomeric variation between different species-Caenorhabditis elegans is no exception. Simple screening using lectins and antibodies can yield hints regarding which glycan epitopes are present in wild-type and mutant strains, but detailed analysis is necessary for determining more exact glycomic information. Here, our analytical approach is to analyze N- and O-glycans involving "off-line" RP-HPLC MALDI-TOF MS/MS. Enrichment and labeling steps facilitate the analysis of single structures and provide isomeric separation. Thereby, the "simple" worm expresses over 200 N-glycan structures varying depending on culture conditions or the genetic background.

Molecular characterisation of Entamoeba histolytica UDP-glucose 4-epimerase, an enzyme able to provide building blocks for cyst wall formation.

In the human host, the protozoan parasite Entamoeba histolytica is adapted to a non-invasive lifestyle in the colon as well as to an invasive lifestyle in the mesenterial blood vessels and the liver. This means to cope with bacteria and human cells as well as various metabolic challenges. Galactose and N-acetylgalactosamine (GalNAc) are sugars of great importance for the amoebae, they attach to the host mucus and enterocytes via their well-studied Gal/GalNAc specific lectin, they carry galactose residues in their surface glycans, and they cleave GalNAc from host mucins. The enzyme UDP-glucose 4-epimerase (GalE) works as a bridge between the galactose and glucose worlds, it can help to generate glucose for glycolysis from phagocytosis products containing galactose as well as providing UDP-galactose necessary for the biosynthesis of galactose-containing surface components. E. histolytica contains a single galE gene. We recombinantly expressed the enzyme in Escherichia coli and used a spectrophotometric assay to determine its temperature and pH dependency (37°C, pH 8.5), its kinetics for UDP-glucose (Km = 31.82 µM, Vmax = 4.31 U/mg) and substrate spectrum. As observed via RP-HPLC, the enzyme acts on UDP-Glc/Gal as well as UDP-GlcNAc/GalNAc. Previously, Trypanosoma brucei GalE and the bloodstream form of the parasite were shown to be susceptible to the three compounds ebselen, a selenoorganic drug with antioxidant properties, diethylstilbestrol, a mimic of oestrogen with anti-inflammatory properties, and ethacrynic acid, a loop diuretic used to treat oedema. In this study, the three compounds had cytotoxic activity against E. histolytica, but only ebselen inhibited the recombinant GalE with an IC50 of 1.79 µM (UDP-Gal) and 1.2 µM (UDP-GalNAc), suggesting that the two other compounds are active against other targets in the parasite. The importance of the ability of GalE to interconvert UDP-GalNAc and UDP-GlcNAc may be that the trophozoites can generate precursors for their own cyst wall from the sugar subunits cleaved from host mucins. This finding advances our understanding of the biochemical interactions of E. histolytica in its colonic environment.

Recognition of highly branched N-glycans of the porcine whipworm by the immune system.

Glycans are key to host-pathogen interactions, whereby recognition by the host and immunomodulation by the pathogen can be mediated by carbohydrate binding proteins, such as lectins of the innate immune system, and their glycoconjugate ligands. Previous studies have shown that excretory-secretory products of the porcine nematode parasite Trichuris suis exert immunomodulatory effects in a glycan-dependent manner. To better understand the mechanisms of these interactions, we prepared N-glycans from T. suis and both analyzed their structures and used them to generate a natural glycan microarray. With this array we explored the interactions of glycans with C-type lectins, C-reactive protein and sera from T. suis infected pigs. Glycans containing LacdiNAc and phosphorylcholine-modified glycans were associated with the highest binding by most of these proteins. In-depth analysis revealed not only fucosylated LacdiNAc motifs with and without phosphorylcholine moieties, but phosphorylcholine-modified mannose and N-acetylhexosamine-substituted fucose residues, in the context of maximally tetraantennary N-glycan scaffolds. Furthermore, O-glycans also contained fucosylated motifs. In summary, the glycans of T. suis are recognized by both the innate and adaptive immune systems, and also exhibit species-specific features distinguishing its glycome from those of other nematodes.

Mapping the expressed glycome and glycosyltransferases of zebrafish liver cells as a relevant model system for glycosylation studies.

The emergence of zebrafish as a model organism for human diseases was accompanied by the development of cellular model systems that extended the possibilities for in vitro manipulation and in vivo studies after cell implantation. The exploitation of zebrafish cell systems is, however, still hampered by the lack of genomic and biochemical data. Here, we lay a path toward the efficient use of ZFL, a zebrafish liver-derived cell system, as a platform for studying glycosylation. To achieve this, we established the glycomic profile of ZFL by a combination of mass spectrometry and NMR. We demonstrated that glycoproteins were substituted by highly sialylated multiantennary N-glycans, some of them comprising the unusual zebrafish epitope Galß1-4[Neu5Ac(α2,3)]Galß1-4[Fuc(α1,3)]GlcNAc, and core 1 multisialylated O-glycans. Similarly, these analyses established that glycolipids were dominated by sialylated gangliosides. In parallel, analyzing the expression patterns of all putative sialyl- and fucosyltransferases, we directly correlated the identified structures to the set of enzymes involved in ZFL glycome. Finally, we demonstrated that this cell system was amenable to metabolic labeling using functionalized monosaccharides that permit in vivo imaging of glycosylation processes. Altogether, glycomics, genomics, and functional studies established ZFL as a relevant cellular model for the study of glycosylation.

Accumulation of unusual gangliosides G(Q3) and G(P3) in breast cancer cells expressing the G(D3) synthase.

Glycosphingolipids from the ganglio-series are usually classified in four series according to the presence of 0 to 3 sialic acid residues linked to lactosylceramide. The transfer of sialic acid is catalyzed in the Golgi apparatus by specific sialyltransferases that show high specificity toward glycolipid substrates. ST8Sia I (EC 2.4.99.8, SAT-II, SIAT 8a) is the key enzyme controlling the biosynthesis of b- and c-series gangliosides. ST8Sia I is expressed at early developmental stages whereas in adult human tissues, ST8Sia I transcripts are essentially detected in brain. ST8Sia I together with b- and c-series gangliosides are also over-expressed in neuroectoderm-derived malignant tumors such as melanoma, glioblastoma, neuroblastoma and in estrogen receptor (ER) negative breast cancer, where they play a role in cell proliferation, migration, adhesion and angiogenesis. We have stably expressed ST8Sia I in MCF-7 breast cancer cells and analyzed the glycosphingolipid composition of wild type (WT) and GD3S+ clones. As shown by mass spectrometry, MCF-7 expressed a complex pattern of neutral and sialylated glycosphingolipids from globo- and ganglio-series. WT MCF-7 cells exhibited classical monosialylated gangliosides including G(M3), G(M2), and G(M1a). In parallel, the expression of ST8Sia I in MCF-7 GD3S+ clones resulted in a dramatic change in ganglioside composition, with the expression of b- and c-series gangliosides as well as unusual tetra- and pentasialylated lactosylceramide derivatives G(Q3) (II(3)Neu5Ac(4)-Gg(2)Cer) and G(P3) (II(3)Neu5Ac(5)-Gg(2)Cer). This indicates that ST8Sia I is able to act as an oligosialyltransferase in a cellular context.

Systems glycomics of adult zebrafish identifies organ-specific sialylation and glycosylation patterns.

The emergence of zebrafish Danio rerio as a versatile model organism provides the unique opportunity to monitor the functions of glycosylation throughout vertebrate embryogenesis, providing insights into human diseases caused by glycosylation defects. Using a combination of chemical modifications, enzymatic digestion and mass spectrometry analyses, we establish here the precise glycomic profiles of eight individual zebrafish organs and demonstrate that the protein glycosylation and glycosphingolipid expression patterns exhibits exquisite specificity. Concomitant expression screening of a wide array of enzymes involved in the synthesis and transfer of sialic acids shows that the presence of organ-specific sialylation motifs correlates with the localized activity of the corresponding glycan biosynthesis pathways. These findings provide a basis for the rational design of zebrafish lines expressing desired glycosylation profiles.

Alkynyl monosaccharide analogues as a tool for evaluating Golgi glycosylation efficiency: application to Congenital Disorders of Glycosylation (CDG).

The visualization of Golgi glycosylation defects in patients' cells with Congenital Disorders of Glycosylation (CDG) is challenging and necessitates the use of cumbersome glycan analysis methods that are barely adapted to clinical research. We show here that metabolic labelling of patient cells with alkyne-tagged sialic-acid (SiaNAl) enables an easy and reliable readout assay for the detection of CDG occurrence. It also provides valuable clues regarding the pathological processes by assessing the distribution of sialic acid analogues within the cells.

Assessing ER and Golgi N-glycosylation process using metabolic labeling in mammalian cultured cells.

Modifications of N-glycosylation in disease states are common and illustrate the crucial requirement of glycosylation in human biology. Mainly based on glycan permethylation and the use of mass spectrometry analysis, we can easily understand that many different methods to analyze the N-glycome have seen the day. While extremely powerful, these methods are mainly used to analyze qualitative variations of N-glycosylation of human serum proteins and do not necessarily reflect the glycosylation status of derived mammalian cultured cells. This chapter summarizes two methods that we are routinely using in our laboratory to assess the ER and Golgi N-glycosylation process. The proposed methodology allows pinpointing ER as well as Golgi glycosylation deficiencies in mammalian cultured cells. The first approach is based on direct metabolic labeling of cultured mammalian cells with [2-(3)H] mannose followed by sequential extraction and HPLC analysis of the purified oligosaccharides. The second one is based on the copper-catalyzed azide alkyne cycloaddition (CuAAC) strategy. We propose the use of alkyne-tagged sialic acid (SialNAl) to visualize the Golgi glycosylation efficiency. Their metabolic incorporation into newly synthesized glycoproteins can then be chemoselectively coupled to complementary azide-functionalized fluorophores, and visualized by using confocal laser scanning microscopy. To summarize, we present here a detailed description of our know-how in the field of ER and Golgi N-glycosylation.

Glycoinositolphospholipids from Trypanosomatids subvert nitric oxide production in Rhodnius prolixus salivary glands.

BACKGROUND: Rhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands. METHODOLOGY/PRINCIPAL FINDINGS: Here we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities. CONCLUSIONS/SIGNIFICANCE: Taken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.