Structure and Function Relationship of Trans-Sialidases from Trypanosoma congolense

The study presented here addresses structural and functional relations of trans-sialidases (TS) from the African parasite Trypanosoma congolense and their biochemical characterisation. TS are unusual enzymes found in the flagellate protozoan parasite Trypanosoma and catalyse the stereo and region specific transfer of terminal sialic acid from donor sialo-glycoconjugates to terminal galactose residues of suitable acceptor substrates, resulting in alpha-2,3-sialylated glycoconjugates. Major research on trypanosomal TS has been done on Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. However, only little has been known about TS from the African Trypanosoma congolense TS, the prevalent causative agent of African animal Trypanosomiasis (AAT) in livestock and domestic animals also termed nagana

An application of linear programming to minimum fuel optimal control

Application of linear programming to minimum fuel optimal contro

Biochemical characterization of trans-sialidase TS1 variants from Trypanosoma congolense

<p>Abstract</p> <p>Background</p> <p>Animal African trypanosomiasis, sleeping sickness in humans and Nagana in cattle, is a resurgent disease in Africa caused by <it>Trypanosoma </it>parasites. Trans-sialidases expressed by trypanosomes play an important role in the infection cycle of insects and mammals. Whereas trans-sialidases of other trypanosomes like the American <it>T. cruzi </it>are well investigated, relatively little research has been done on these enzymes of <it>T. congolense</it>.</p> <p>Results</p> <p>Based on a partial sequence and an open reading frame in the WTSI database, DNA sequences encoding for eleven <it>T. congolense </it>trans-sialidase 1 variants with 96.3% overall amino acid identity were amplified. Trans-sialidase 1 variants were expressed as recombinant proteins, isolated and assayed for trans-sialylation activity. The purified proteins produced α2,3-sialyllactose from lactose by desialylating fetuin, clearly demonstrating their trans-sialidase activity. Using an HPLC-based assay, substrate specificities and kinetic parameters of two variants were characterized in detail indicating differences in substrate specificities for lactose, fetuin and synthetic substrates. Both enzymes were able to sialylate asialofetuin to an extent, which was sufficient to reconstitute binding sites for Siglec-4. A mass spectrometric analysis of the sialylation pattern of glycopeptides from fetuin revealed clear but generally similar changes in the sialylation pattern of the <it>N</it>-glycans on fetuin catalyzed by the trans-sialidases investigated.</p> <p>Conclusions</p> <p>The identification and characterization of a trans-sialidase gene family of the African parasite <it>T. congolense </it>has opened new perspectives for investigating the biological role of these enzymes in Nagana and sleeping sickness. Based on this study it will be interesting to address the expression pattern of these genes and their activities in the different stages of the parasite in its infection cycle. Furthermore, these trans-sialidases have the biotechnological potential to be used for enzymatic modification of sialylated glycoconjugates.</p

Cooperativity of catalytic and lectin-like domain of Trypanosoma congolense trans-sialidase modulates its catalytic activity

Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids (Sia) from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Enzymes comprise a N-terminal catalytic domain (CD) followed by a lectin-like domain (LD). Most work on trypanosomal TS has been done on enzymatic activities focusing on the CD of TS from Trypanosoma cruzi (causing Chagas disease in Latin America), subspecies of Trypanosoma brucei, (causing human sleeping sickness in Africa) and Trypanosoma congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS)-LD binds to several carbohydrates, such as 1,4-β-mannotriose. In this study we investigated the influence of TconTS3-LD on Sia transfer efficiency of TconTS1a-CD by swapping domains. in silico analysis on structure models of TconTS enzymes revealed the potential of domain swaps between TconTS1a and TconTS3 without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear Sia transfer activity, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolytic release of free Neu5Ac as a side product was suppressed resulting in increased transfer efficiency. Presence of 1,4-β-mannotriose during TS reactions modulates enzyme activities enhancing transfer efficiency possibly due to occupation of the binding site in TconTS1a-LD. Interestingly this effect was in the same range as that observed when swapping TconTS1a-CD and TconTS3-LD. In summary, this study demonstrate the proof-of-principle for swapping CDs and LDs of TconTS and that TconTS3-LD influences enzymatic activity of TconTS1a-CD providing evidence that LDs play pivotal roles in modulating activities and biological functions of TconTS and possibly other TS

Struktur- und Funktionsbeziehung der Trans-Sialidasen aus Trypanosoma congolense

The study presented here addresses structural and functional relations of trans-sialidases (TS) from the African parasite Trypanosoma congolense and their biochemical characterisation. TS are unusual enzymes found in the flagellate protozoan parasite Trypanosoma and catalyse the stereo and region specific transfer of terminal sialic acid from donor sialo-glycoconjugates to terminal galactose residues of suitable acceptor substrates, resulting in alpha-2,3-sialylated glycoconjugates. Major research on trypanosomal TS has been done on Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. However, only little has been known about TS from the African Trypanosoma congolense TS, the prevalent causative agent of African animal Trypanosomiasis (AAT) in livestock and domestic animals also termed nagana

Biophysical characterization and structural determination of the potent cytotoxic Psathyrella asperospora

International audienceA lectin with strong cytotoxic effect on human colon cancer HT29 and monkey kidney VERO cells was recently identified from the Australian indigenous mushroom Psathyrella asperospora and named PAL. We herein present its biochemical and structural analysis using a multidisciplinary approach. Glycan arrays revealed binding preference towards N-acetylglucosamine (GlcNAc) and, to a lesser extent, towards sialic acid (Neu5Ac). Submicromolar and millimolar affinity was measured by surface plasmon resonance for GlcNAc and NeuAc, respectively. The structure of PAL was resolved by X-ray crystallography, elucidating both the protein's amino acid sequence as well as the molecular basis rationalizing its binding specificity

Investigation of Group A Streptococcal Interactions with Host Glycan Structures Using High-Throughput Techniques: Glycan Microarray Analysis Using Recombinant Protein and Whole Cells

Glycans, also known as carbohydrates, are abundant upon cell surfaces, where they often mediate host-pathogen interactions. The specific recognition of host glycans by pathogenic lectins is an important process that allows the adherence of bacteria to the host epithelial surface in many species, including Group A Streptococcus (GAS). Glycan microarrays present a sensitive, high-throughput approach for identifying novel lectin-glycan interactions and can be applied in the context of whole bacteria or purified bacterial proteins

Investigation of Group A Streptococcal interactions with host glycan structures using high-throughput techniques: glycan microarray analysis using recombinant protein and whole cells

Glycans, also known as carbohydrates, are abundant upon cell surfaces, where they often mediate host-pathogen interactions. The specific recognition of host glycans by pathogenic lectins is an important process that allows the adherence of bacteria to the host epithelial surface in many species, including Group A Streptococcus (GAS). Glycan microarrays present a sensitive, high-throughput approach for identifying novel lectin-glycan interactions and can be applied in the context of whole bacteria or purified bacterial proteins