Structure and Function Relationship of Trans-Sialidases from Trypanosoma congolense

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. Transsialidase 1 variants were expressed as recombinant proteins, isolated and assayed for trans-sialylation activity. The purified proteins produced a2,3-sialyllactose from lactose by desialylating fetuin, clearly demonstrating their transsialidase 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. Background Animal African trypanosomiasis, called sleeping sickness in humans and Nagana in cattle, is a resurgent disease in Africa. Nagana is caused by Trypanosoma congolense (T. congolense), Trypanosoma vivax (T. vivax) and Trypanosoma brucei (T. brucei) subspecies. Most research on African trypanosomes has focused on T. brucei, whereas only few studies have been done with other African trypanosomes including T. congolense. In wild animals, these parasites cause relatively mild infections while in domestic animals they cause a severe, often fatal disease. Because of Nagana, stock farming is very difficult within the tsetse belt of Africa [1]. Although of crucial importance for their survival, cyclical transmission and hence pathogenicity of trypanosomes, trypanosomes lack the biochemical metabolic machinery synthesizing sialic acids (Sia), but use a unique enzyme, trans-sialidase (TS) to transfer Sia onto the parasites surface molecules from the environment. Structurally TS belong to the family of sialidases (SA). In contrast to the usual sialyltransferases, TS does not utilize CMP-activated Sia as monosaccharide donors, but catalyzes the transfer of carbohydrate-linked Sia to another glycan forming a new a2,3-glycosidic linkage to galactose or N-acetylgalactosamine. Whereas more detailed studies exist on the role of TS in the pathogenicity of T. cruzi, the etiologic agent of * Correspondence: skelm@uni-bremen.de Centre for Biomolecular Interactions Bremen, Department of Biology and Chemistry, University of Bremen, Leobener Str. /NW2/B2235, 28359 Bremen, Germany Koliwer-Brandl et al. BMC Biochemistry 2011, 12:39 http://www.biomedcentral. com/1471-2091/12/39 © 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.