title: Biochemical characterization of trans-sialidase TS1 variants from Trypanosoma congolense.
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.
authors.affiliation: Centre for Biomolecular Interactions Bremen, Department of Biology and Chemistry, University of Bremen, Germany.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Koliwer-Brandl
authors.forename: Thaddeus T
authors.initials: TT
authors.lastname: Gbem
authors.forename: Mario
authors.initials: M
authors.lastname: Waespy
authors.forename: Olga
authors.initials: O
authors.lastname: Reichert
authors.forename: Philipp
authors.initials: P
authors.lastname: Mandel
authors.forename: Eric
authors.initials: E
authors.lastname: Drebitz
authors.forename: Frank
authors.initials: F
authors.lastname: Dietz
authors.forename: Sørge
authors.initials: S
authors.lastname: Kelm
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D014644:Genetic Variation; D006020:Glycopeptides / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D014348:Trypanosoma congolense / Q000201:enzymology*; D014349:Trypanosoma cruzi / Q000201:enzymology
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; C069715:trans-sialidase; D009439:Neuraminidase