Abstract
The substrate specificity, physico-chemical, and kinetic properties of the trans-sialidase from Trypanosoma cruzi have been investigated. The enzyme demonstrates activity towards a wide range of saccharide, glycolipid, and glycoprotein acceptors which terminate with a beta-linked galactose residue, and synthesizes exclusively an alpha 2-3 sialosidic linkage. Oligosaccharides which terminate in Gal beta 1-4(Fuc alpha 1-3)GlcNAc, Gal beta 1-3(Fuc alpha 1-4)GlcNAc, or Gal alpha 1- are not acceptor-substrates. The enzyme utilizes alpha 2,3-linked sialic acid when the donor species is an oligosaccharide and can also transfer, at a low rate, sialic acid from synthetic alpha-sialosides such as p-nitrophenyl-alpha-N-acetylneuraminic acid, but NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)Glc is not a donor-substrate. The trans-sialidase has an apparent pH optimum of 7.9 and a temperature optimum of 13 degrees C. The kinetic properties of the enzyme suggest that the trans-sialylation reaction may occur via a rapid equilibrium random or steady-state ordered mechanism. A method for immobilizing the enzyme is described together with examples of its use for the synthesis of oligosaccharide and glycoprotein precursors of sialyl-Lewis and sialyl-Lewis.
Highlights
The substrate specificity, physico-chemical, and ki- and that the active site for both activities resides at the N netic properties of the trans-sialidase from Trypano- terminus of the molecule, a region which is known to have soma cruzi havebeen investigated
A method for immobilizing the enzyme is described together with examples of its use for the synthesis of oligosaccharide and glycoprotein precurcolostrum using the method described by Vehet al. [15] and converted into the sodium salt by passsage overDowex AG 5OW-X12 (Na+ form).The sialyl-Lewis’tetrasaccharide,NeuAcol2-3Gal~l-4(Fucal3)Glc,was prepared by the enzymatic fucosylation of 2,3-sialyllactose using partially purified human milk a-3,4-fucosyltransferase[16] and was isolated by chromatography on DowexAG1-X2 [15] using an eluant of40mM pyridinium acetate, pH 5.0
Trypomastigotes of Trypanosoma cruzi express a develop- water (3:2:1, v/v) following treatment of sialylparagloboside with mentally regulated trans-sialidase [1,2,3] that is anchored to Clostridium perfringens neuraminidase. 2-O-(p-Nitrophenyl)-N-acethe plasma membrane of the parasite via a glycosylphospha- tyl-a-neuraminic acid was from Seikagaku Kogyo
Summary
Purification and Properties of trans-Sialidase trans-Sialidase was purified 1000-fold to apparent homogeneity in a single immunoaffinity chromatography step using an immobilized monoclonal antibody, TCN-2, whichrecognizes a 12-amino acid tandem repeat (D-S-S-A-H-G-T-P-ST-P-A,peptide TR) that is present at the C terminus of the enzyme [18].A synthetic peptide having this sequence was used to elute the trans-sialidase. The specificityof this peptide was demonstrated by the failure of a similar peptide, which lacked threonine at position 7 (peptide TR-t7), to displace trans-sialidase from the affinity column (see Fig. 1).After fast protein liquid chromatography (to remove the peptide ligand), the purified trans-sialidase had a specific activity of 100units/
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