Probing molecular function of trypanosomal sialidases: single point mutations can change substrate specificity and increase hydrolytic activity

Abstract

Sialidases are present on the surface of several trypanosomatid protozoan parasites. They are highly specific for sialic acid linked in alpha-(2,3) to a terminal beta-galactose and include the strictly hydrolytic enzymes and trans-sialidases (sialyl-transferases). Based on the structural comparison of the sialidase from Trypanosoma rangeli and the trans-sialidase from T. cruzi (the agent of Chagas' disease in humans), we have explored the role of specific amino acid residues sought to be important for substrate specificity. The substitution of a conserved tryptophanyl residue in the two enzymes, Trp312/313-Ala, changed substrate specificity, rendering the point mutants capable to hydrolyze both alpha-(2,3)- and alpha-(2,6)-linked sialoconjugates. The same mutation abolished sialyl-transferase activity, indicating that transfer (but not hydrolysis) requires a precise orientation of the bound substrate. The exchange substitution of another residue that modulates oligosaccharide binding, Gln284-Pro, was found to significantly increase the hydrolytic activity of sialidase, and residue Tyr119 was confirmed to be part of a second binding site for the acceptor substrate in trans-sialidase. Together with the structural information, these results provide a consistent framework to account for the unique enzymatic properties of trypanosome trans-sialidases.

Procyclic Trypanosoma brucei Expresses Separate Sialidase and trans-Sialidase Enzymes on Its Surface Membrane

“…Nevertheless, when we assayed the activity of TbTS recombinant protein bearing the Y191W substitution, we observed that this amino acid replacement almost completely abolished trans-sialidase activity, although slightly increasing the sialidase activity. A similar effect was observed by replacing Tyr-119 with serine in TcTS (17,18), indicating that this residue is critical for the trans-sialylation reaction in both trypanosomal trans-sialidases. Watts et al (30) postulate that the replacement of the side chain of Tyr-119 of T. cruzi trans-sialidase by Ser-120 in T. rangeli sialidase could be responsible for the difference in the positioning of the glycerol of sialic acid observed by comparing the covalent intermediates of both enzymes.…”

“…To obtain the 5Ј-UTR sequence, first-strand cDNA was prepared with the Superscript II system using an internal primer (5Ј-CACACTTAAGCATCCCCTCGT-3Ј) of TbSA C. Reverse transcription-PCR was carried out with Taq DNA polymerase (Invitrogen) and primers for the T. brucei 39-nucleotide 5Ј-spliced leader sequence as forward (5Ј-AAC-GCTATTATTAGAACAGTTTCTGTACT-3Ј) and the one used for first-strand synthesis as reverse. To obtain the 3Ј-UTR sequence, reverse transcription-PCR was performed with Superscript II (Invitrogen) using the oligonucleotide anchor-(dT) 18 (5Ј-GCGACTCCGCGGCCGCG(T) 18 -3Ј). PCR was conducted on the first-strand product using the anchor-(dT) 18 as the reverse primer.…”

“…To obtain the 3Ј-UTR sequence, reverse transcription-PCR was performed with Superscript II (Invitrogen) using the oligonucleotide anchor-(dT) 18 (5Ј-GCGACTCCGCGGCCGCG(T) 18 -3Ј). PCR was conducted on the first-strand product using the anchor-(dT) 18 as the reverse primer. The forward primer was 5Ј-TACTTTA-CCTGTTGATGGGTCT-3Ј.…”

“…However, in the absence of acceptors of sialic acid in the milieu, they are able to release free sialic acid and thus are, to a much lower extent, hydrolytic enzymes. A comparison of the crystal structures of TcTS and TrSA (17) and information derived from several mutagenesis approaches (16,18,19) show that trypanosomal sialidases and trans-sialidases share a similar active site architecture in which several amino acid residues critical for enzyme function are conserved. In the T. cruzi and T. rangeli enzymes, a conserved tryptophan residue (Trp-313) was shown to be implicated in the binding of substrate and to be necessary for the specificity of the enzyme for ␣-(2,3)-linkage sialic acid (18).…”

Trypanosoma rangeli sialidase: lack of activity in stocks from Panama and other regions

“…This becomes of greater interest in view of recent biochemical evidence (Cremona et al 1999, Paris et al 2001) which suggests that T. rangeli SA molecule, as in T. cruzi TSA, is present in active and inactive forms.…”