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      Biochemical characterization of trans-sialidase TS1 variants from Trypanosoma congolense

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          Abstract

          Background

          Animal African trypanosomiasis, sleeping sickness in humans and Nagana in cattle, is a resurgent disease in Africa caused by Trypanosoma 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 T. cruzi are well investigated, relatively little research has been done on these enzymes of T. congolense.

          Results

          Based on a partial sequence and an open reading frame in the WTSI database, DNA sequences encoding for eleven T. congolense 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 N-glycans on fetuin catalyzed by the trans-sialidases investigated.

          Conclusions

          The identification and characterization of a trans-sialidase gene family of the African parasite T. congolense 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.

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          Most cited references 36

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          A graph-theory algorithm for rapid protein side-chain prediction.

          Fast and accurate side-chain conformation prediction is important for homology modeling, ab initio protein structure prediction, and protein design applications. Many methods have been presented, although only a few computer programs are publicly available. The SCWRL program is one such method and is widely used because of its speed, accuracy, and ease of use. A new algorithm for SCWRL is presented that uses results from graph theory to solve the combinatorial problem encountered in the side-chain prediction problem. In this method, side chains are represented as vertices in an undirected graph. Any two residues that have rotamers with nonzero interaction energies are considered to have an edge in the graph. The resulting graph can be partitioned into connected subgraphs with no edges between them. These subgraphs can in turn be broken into biconnected components, which are graphs that cannot be disconnected by removal of a single vertex. The combinatorial problem is reduced to finding the minimum energy of these small biconnected components and combining the results to identify the global minimum energy conformation. This algorithm is able to complete predictions on a set of 180 proteins with 34342 side chains in <7 min of computer time. The total chi(1) and chi(1 + 2) dihedral angle accuracies are 82.6% and 73.7% using a simple energy function based on the backbone-dependent rotamer library and a linear repulsive steric energy. The new algorithm will allow for use of SCWRL in more demanding applications such as sequence design and ab initio structure prediction, as well addition of a more complex energy function and conformational flexibility, leading to increased accuracy.
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            The history of African trypanosomiasis

            The prehistory of African trypanosomiasis indicates that the disease may have been an important selective factor in the evolution of hominids. Ancient history and medieval history reveal that African trypanosomiasis affected the lives of people living in sub-Saharan African at all times. Modern history of African trypanosomiasis revolves around the identification of the causative agents and the mode of transmission of the infection, and the development of drugs for treatment and methods for control of the disease. From the recent history of sleeping sickness we can learn that the disease can be controlled but probably not be eradicated. Current history of human African trypanosomiasis has shown that the production of anti-sleeping sickness drugs is not always guaranteed, and therefore, new, better and cheaper drugs are urgently required.
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              Identification and application of the concepts important for accurate and reliable protein secondary structure prediction.

              A protein secondary structure prediction method from multiply aligned homologous sequences is presented with an overall per residue three-state accuracy of 70.1%. There are two aims: to obtain high accuracy by identification of a set of concepts important for prediction followed by use of linear statistics; and to provide insight into the folding process. The important concepts in secondary structure prediction are identified as: residue conformational propensities, sequence edge effects, moments of hydrophobicity, position of insertions and deletions in aligned homologous sequence, moments of conservation, auto-correlation, residue ratios, secondary structure feedback effects, and filtering. Explicit use of edge effects, moments of conservation, and auto-correlation are new to this paper. The relative importance of the concepts used in prediction was analyzed by stepwise addition of information and examination of weights in the discrimination function. The simple and explicit structure of the prediction allows the method to be reimplemented easily. The accuracy of a prediction is predictable a priori. This permits evaluation of the utility of the prediction: 10% of the chains predicted were identified correctly as having a mean accuracy of > 80%. Existing high-accuracy prediction methods are "black-box" predictors based on complex nonlinear statistics (e.g., neural networks in PHD: Rost & Sander, 1993a). For medium- to short-length chains (> or = 90 residues and < 170 residues), the prediction method is significantly more accurate (P < 0.01) than the PHD algorithm (probably the most commonly used algorithm). In combination with the PHD, an algorithm is formed that is significantly more accurate than either method, with an estimated overall three-state accuracy of 72.4%, the highest accuracy reported for any prediction method.
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                Author and article information

                Journal
                BMC Biochem
                BMC Biochemistry
                BioMed Central
                1471-2091
                2011
                30 July 2011
                : 12
                : 39
                Affiliations
                [1 ]Centre for Biomolecular Interactions Bremen, Department of Biology and Chemistry, University of Bremen, Leobener Str./NW2/B2235, 28359 Bremen, Germany
                Article
                1471-2091-12-39
                10.1186/1471-2091-12-39
                3173295
                21801439
                Copyright ©2011 Koliwer-Brandl et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Research Article

                Biochemistry

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