Cloning of Trypanosoma cruzi trans-Sialidase and Expression in Pichia pastoris

Trypanosoma cruzi, the agent causing Chagas' disease, expresses an enzyme that transfers sialic acids among glycoproteins and glycolipids both from the host cell surface and its own surface. This enzyme, called trans-sialidase, is different from higher eukaryotic sialyltransferases in that it does not accept cytidine 5′-monophospho-N-acetylneuraminic acid as a donor substrate. Also, the common glycosyltransferase structure is not present. To study this enzyme, an active member was cloned and expressed in higher eukaryotic cells. Expression of recombinant enzyme was achieved in the methylotrophic yeast Pichia pastoris. The N-terminal fusion of a secretion signal and the C-terminal addition of an epitope tag resulted not only in high expression levels, but also enabled easy detection and purification. Using P. pastoris, we obtained about 5 mg of enzymatically active trans-sialidase per liter of induced culture medium.     

Only some members of a gene family in Trypanosoma cruzi encode proteins that express both trans-sialidase and neuraminidase activities.

Trypomastigotes, the blood stage form of the human parasite Trypanosoma cruzi, contain an enzyme on their surface, trans-sialidase, which catalyses the transfer of sialic acid from host glycoconjugates to acceptors on its own cell surface. At least a subset of the sialic acid-bearing acceptor molecules are involved in parasite invasion of host cells, an essential step in the life cycle of the parasite. Another trypomastigote surface enzyme that affects host cell invasion is neuraminidase and recent evidence suggests that both trans-sialidase and neuraminidase activities may be expressed by the same proteins on the parasite surface. We describe here the isolation and expression of several members of a trans-sialidase--neuraminidase gene family from T.cruzi. One of the isolated genes does indeed encode a protein with both trans-sialidase and neuraminidase activities, while other members of the gene family encode closely related proteins that express neither enzymatic activity. Chimeric protein constructs combining different portions of active and inactive genes identified a region of the gene necessary for enzymatic activity. Sequence analysis of this portion of the gene revealed a limited number of amino acid differences between the predicted active and inactive gene products.     

Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-alpha-D-mannosidase for N-glycan engineering in Pichia pastoris

Therapeutic glycoprotein production in the widely used expression host Pichia pastoris is hampered by the differences in the protein-linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human-compatible N-glycans in this organism is the conversion of the yeast-type high-mannose glycans to mammalian-type high-mannose and/or complex glycans. In this perspective, we have co-expressed an endoplasmic reticulum-targeted Trichoderma reesei 1,2-alpha-D-mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans-sialidase. Analysis of the N-glycans of the two purified proteins showed a >85% decrease in the number of alpha-1,2-linked mannose residues. Moreover, the human-type high-mannose oligosaccharide Man(5)GlcNAc(2) was the major N-glycan of the glyco-engineered trans-sialidase, indicating that N-glycan engineering can be effectively accomplished in P. pastoris.     

Identification of a second catalytically active trans-sialidase in Trypanosoma brucei

The procyclic stage of Trypanosoma brucei is covered by glycosylphosphatidylinositol (GPI)-anchored surface proteins called procyclins. The procyclin GPI anchor contains a side chain of N-acetyllactosamine repeats terminated by sialic acids. Sialic acid modification is mediated by trans-sialidases expressed on the parasite’s cell surface. Previous studies suggested the presence of more than one active trans-sialidases, but only one has so far been reported. Here we cloned and examined enzyme activities of four additional trans-sialidase homologs, and show that one of them, Tb927.8.7350, encodes another active trans-sialidase, designated as TbSA C2. In an in vitro assay, TbSA C2 utilized α2-3 sialyllactose as a donor, and produced an α2-3-sialylated product, suggesting that it is an α2-3 trans-sialidase. We suggest that TbSA C2 plays a role in the sialic acid modification of the trypanosome cell surface.     

trans-sialidase of Trypanosoma cruzi: location of galactose-binding site(s).

Trypanosoma cruzi expresses a trans-sialidase on its surface, which catalyzes the transfer of sialic acid from mammalian host glycans to its own surface glycoproteins. It has been proposed that the enzyme consists of three domains prior to a long C-terminal repeating sequence that is not required for enzyme activity. The first of these domains shares significant sequence identity with bacterial sialidases which catalyse the hydrolysis of sialic acid. Here we report the sequence of the N-terminal domains of the TS19y trans-sialidase gene, which was expressed in bacteria with the same specific activity as natural enzyme of T. cruzi. Various deletion mutants of TS19y, without the C-terminal tandem repeat, have been cloned and expressed and their trans-sialidase and sialidase activities measured. These experiments show that all three N-terminal domains are required for full trans-sialidase activity, though only the first is necessary for sialidase activity. Some transferase activity is observed, however, even with the shortest construct comprising the first N-terminal domain. Deletion mutants to probe the role of the N-terminal residues of the first domain suggest that the first 33 residues are also required for trans-sialidase activity, but not for sialidase activity. Molecular modelling of the first N-terminal domain of TS19y based on our structures of bacterial sialidases and site-directed mutations suggests the location of a galactose-binding site within this domain.     

A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells.

When trypomastigotes of T. cruzi emerge from cells of the mammalian host, they contain little or no sialic acids on their surfaces. However, rapidly upon entering the circulation, they express a unique cell surface trans-sialidase activity. This enzyme specifically transfers alpha (2-3)-linked sialic acid from extrinsic host-derived macromolecules to parasite surface molecules, leading to the assembly of Ssp-3, a trypomastigote-specific epitope. The T. cruzi trans-sialidase does not utilize cytidine 5' monophospho-N-acetylneuraminic acid as a donor substrate, but readily transfers sialic acid from exogenously supplied alpha (2-3)-sialyllactose. Monoclonal antibodies that recognize sialic acid residues of Ssp-3 inhibit attachment of trypomastigotes to host cells, suggesting that the unusual trans-sialidase provides Ssp-3 with structural features required for target cell recognition.     

Procyclic Trypanosoma brucei expresses separate sialidase and trans-sialidase enzymes on its surface membrane

The procyclic stage of Trypanosoma brucei in the insect vector expresses a surface-bound trans-sialidase (TbTS) that transfers sialic acid from glycoconjugates in the environment to glycosylphosphatidylinositol-anchored proteins on its surface membrane. RNA interference against TbTS abolished trans-sialidase activity in procyclic cells but did not diminish sialidase activity, suggesting the presence of a separate sialidase enzyme for hydrolyzing sialic acid. A search of the T. brucei genome sequence revealed seven other putative genes encoding proteins with varying similarity to TbTS. RNA interference directed against one of these proteins, TbSA C, greatly decreased the sialidase activity but had no effect on trans-sialidase activity. The deduced amino acid sequence of TbSA C shares only 40% identity with TbTS but conserves most of the relevant residues required for catalysis. However, the sialidase has a tryptophan substitution for a tyrosine at position 170 that is crucial in binding the terminal galactose that accepts the transferred sialic acid. When this same tryptophan substitution in the sialidase was placed into the recombinant trans-sialidase, the mutant enzyme lost almost all of its trans-sialidase activity and increased its sialidase activity, further confirming that the gene and protein identified correspond to the parasite sialidase. Thus, in contrast to all other trypanosomes analyzed to date that express either a trans-sialidase or a sialidase but not both, T. brucei expresses these two enzymatic activities in two separate proteins. These results suggest that African trypanosomes could regulate the amount of critical sialic acid residues on their surface by modulating differential expression of each of these enzymes.     

trans-Sialidase from Trypanosoma cruzi binds host T-lymphocytes in a lectin manner

Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, expresses on its surface an uncommon membrane-bound sialidase, known as trans-sialidase. trans-Sialidase is the product of a multigene family encoding both active and inactive proteins. We report here that an inactive mutant of trans-sialidase physically interacts with CD4(+) T cells. Using a combination of flow cytometry and immunoprecipitation techniques, we identified the sialomucin CD43 as a counterreceptor for trans-sialidase on CD4(+) T cells. Using biochemical, immunological, and spectroscopic approaches, we demonstrated that the inactive trans-sialidase is a sialic acid-binding protein displaying the same specificity required by active trans-sialidase. Taken together, these results suggest that inactive members of the trans-sialidase family can physically interact with sialic acid-containing molecules on host cells and could play a role in host cell/T. cruzi interaction.     

Trypanosoma rangeli sialidase: cloning, expression and similarity to T.cruzi trans-sialidase

Sialidases are hydrolytic enzymes present from virus to highereukaryotes, catalyzing the removal of sialic acid from glycoconjugates.Some protozoa Trypanosomatidae secrete high levels of sialidaseinto the medium. We have now purified the secreted sialidasefrom Trypanosoma rangeli Its N-terminal sequence reveals 100%identity with the corresponding region of the trans-sialidasefrom T.cruzi Trans-sialidase, although homologous to viral andbacterial sialidases, displays a novel sialyltransferase activityand is involved in host cell invasion. Several homologous trans-sialidase-likegenes were cloned from genomic DNA of T.rangeli, and groupedin three subfamilies. Active siali-dase-encoding genes werefound in one of them. The re-combinant sialidase shows similarproperties to those of the native enzyme, including undetectabletrans-sialidase activity. Nevertheless, it has an overall identityof 68.9% with the catalytic domain of T.cruzi trans-sialidase,increasing to 86.7% admitting conservative substitutions. Onlythree other eukaryotic sialidases have been previously cloned,none of them showing significant homology to trans-sialidase.The isolation of a highly similar sialidase is relevant to furtheridentify the molecular determinants allowing trans-sialidaseactivity. As a first approach, chimeric constructs between sialidaseand trans-sialidase were generated, one of them rendering asialidase with three times lower K_m than the natural enzyme. eukaryotic sialidase gene family glycosidase parasite sialic acid     

Trans-sialidase-like sequences from Trypanosoma congolense conserve most of the critical active site residues found in other trans-sialidases

Trypanosoma congolense is the agent of Nagana, the trypanosomiasis in African ruminants. Trypanosomes express an enzyme called trans-sialidase, which is believed to play an important role in maintaining pathogenicity of the parasites. Thus far, only two complete trans-sialidase sequences have been characterised, one from the American trypanosome T. cruzi and one from the African trypanosome T. brucei brucei. Although the crystal structure of T. cruzi trans-sialidase has recently been published [Buschiazzo et al., Mol. Cell 10 (2002), pp. 757-768], a number of questions concerning the exact transfer mechanism remain unanswered. The availability of further trans-sialidase sequences will ensure a better understanding of how transfer activity can be achieved and will provide the opportunity to develop highly specific, structure-based trans-sialidase inhibitors. Utilising a PCR-based approach two different trans-sialidase gene copies from T. congolense were identified, which share only 50% identity with each other, but show significant similarity with known viral, bacterial and trypanosomal sialidases and trans-sialidases. In both partial sequences most of the critical active site residues common to other trypanosomal sialidases and trans-sialidases are conserved. This is further illustrated by modelling the active site of the longer of the two partial gene sequences.     

The trans-sialidase from Trypanosoma cruzi triggers apoptosis by target cell sialylation

The trans-sialidase, a modified sialidase that transfers sialyl residues among macromolecules, is a unique enzymatic activity expressed by some parasitic trypanosomes being essential for their survival in the mammalian host and/or in the insect vector. The enzyme from Trypanosoma cruzi, the agent of Chagas disease, is found in blood and able to act far from the infection site by inducing apoptosis in cells from the immune system. A central and still unsolved question is whether trans-sialidase-mediated addition or removal of sialic acid to/from host acceptor molecules is the event associated with the apoptosis induced by the enzyme. Here we show that lactitol, a competitive inhibitor that precluded the transference of the sialyl residue to endogenous acceptors but not the hydrolase activity of the enzyme, prevented ex vivo and in vivo the apoptosis caused by the trans-sialidase. By lectin histochemistry, the transference of sialyl residue to the cell surface was demonstrated in vivo and found associated with the apoptosis induction. The sialylation of the CD43 mucin, a key molecule involved in trans-sialidase-apoptotic process, was readily detected and also prevented by lactitol on thymocytes. Therefore, lesions induced by trans-sialidase on the immune system are due to the sialylation of endogenous acceptor molecules.     

Enzymatically inactive trans-sialidase from Trypanosoma cruzi binds sialyl and beta-galactopyranosyl residues in a sequential ordered mechanism

Host/parasite interaction mediated by carbohydrate/lectin recognition results in the attachment to and invasion of host cells and immunoregulation, enabling parasite replication and establishment of infection. Trypanosoma cruzi, the protozoan responsible for Chagas disease, expresses on its surface a family of enzymatically active and inactive trans-sialidases. The parasite uses the active trans-sialidase for glycoprotein sialylation in an unusual trans-glycosylation reaction. Inactive trans-sialidase is a sialic acid-binding lectin that costimulates host T cells through leucosialin (CD43) engagement. The co-mitogenic effect of trans-sialidase can be selectively abrogated by N-acetyllactosamine, suggesting the presence of an additional carbohydrate binding domain for galactosides, in addition to that for sialic acid. Here we investigated the interaction of inactive trans-sialidase in the presence of beta-galactosides. By using NMR spectroscopy, we demonstrate that inactive trans-sialidase has a beta-galactoside recognition site formed following a conformational switch induced by sialoside binding. Thus prior positioning of a sialyl residue is required for the beta-galactoside interaction. When an appropriate sialic acid-containing molecule is available, both sialoside and beta-galactoside are simultaneously accommodated in the inactive trans-sialidase binding pocket. This is the first report of a lectin recognizing two distinct ligands by a sequential ordered mechanism. This uncommon binding behavior may play an important role in several biological aspects of T. cruzi/host cell interaction and could shed more light into the catalytic mechanism of the sialic acid transfer reaction of enzymatically active trans-sialidase.     

The trans-sialidase from Trypanosoma cruzi efficiently transfers alpha-(2-->3)-linked N-glycolylneuraminic acid to terminal beta-galactosyl units

The trans-sialidase from Trypanosoma cruzi (TcTS), the agent of Chagas' disease, is a unique enzyme involved in mammalian host-cell invasion. Since T. cruzi is unable to synthesize sialic acids de novo, TcTS catalyzes the transfer of alpha-(2-->3)-sialyl residues from the glycoconjugates of the host to terminal beta-galactopyranosyl units present on the surface of the parasite. TcTS also plays a key role in the immunomodulation of the infected host. Chronic Chagas' disease patients elicit TcTS-neutralizing antibodies that are able to inhibit the enzyme. N-Glycolylneuraminic acid has been detected in T. cruzi, and the trans-sialidase was pointed out as the enzyme involved in its incorporation from host glycoconjugates. However, N-glycolylneuraminic acid alpha-(2-->3)-linked-containing oligosaccharides have not been analyzed as donors in the T. cruzi trans-sialidase reaction. In this paper we studied the ability of TcTS to transfer N-glycolylneuraminic acid from Neu5Gc(alpha2-->3)Gal(beta1-->4)GlcbetaOCH(2)CH(2)N(3) (1) and Neu5Gc(alpha2-->3)Gal(beta1-->3)GlcNAcbetaOCH(2)CH(2)N(3) (2) to lactitol, N-acetyllactosamine and lactose as acceptor substrates. Transfer from 1 was more efficient (50-65%) than from 2 (20-30%) for the three acceptors. The reactions were inhibited when the enzyme was preincubated with a neutralizing antibody. K(m) values were calculated for 1 and 2 and compared with 3'-sialyllactose using lactitol as acceptor substrate. Analysis was performed by high-performance anion-exchange (HPAEC) chromatography. A competitive transfer reaction of compound 1 in the presence of 3'-sialyllactose and N-acetyllactosamine showed a better transfer of Neu5Gc than of Neu5Ac.     

Structural basis of sialyltransferase activity in trypanosomal sialidases

The intracellular parasite Trypanosoma cruzi, the etiological agent of Chagas disease, sheds a developmentally regulated surface trans-sialidase, which is involved in key aspects of parasite-host cell interactions. Although it shares a common active site architecture with bacterial neuraminidases, the T.cruzi enzyme behaves as a highly efficient sialyltransferase. Here we report the crystal structure of the closely related Trypanosoma rangeli sialidase and its complex with inhibitor. The enzyme folds into two distinct domains: a catalytic beta-propeller fold tightly associated with a lectin-like domain. Comparison with the modeled structure of T.cruzi trans-sialidase and mutagenesis experiments allowed the identification of amino acid substitutions within the active site cleft that modulate sialyltransferase activity and suggest the presence of a distinct binding site for the acceptor carbohydrate. The structures of the Trypanosoma enzymes illustrate how a glycosidase scaffold can achieve efficient glycosyltransferase activity and provide a framework for structure-based drug design.     

NMR spectroscopic and molecular modeling investigations of the trans-sialidase from Trypanosoma cruzi

Nuclear magnetic resonance (NMR) spectroscopy was used to investigate the transfer of sialic acid from a range of sialic acid donor compounds to acceptor molecules, catalyzed by Trypanosoma cruzi trans-sialidase (TcTS). We demonstrate here that NMR spectroscopy is a powerful tool to monitor the trans-sialidase enzyme reaction for a variety of donor and acceptor molecules. The hydrolysis or transfer reactions that are catalyzed by TcTS were also investigated using a range of N-acetylneuraminosyl-based donor substrates and asialo acceptor molecules. These studies showed that the synthetic N-acetylneuraminosyl donor 4-methylumbelliferyl alpha-d-N-acetylneuraminide (MUN) is hydrolyzed by the enzyme approximately 3-5 times faster than either the disaccharide Neu5Acalpha(2,3)Galbeta1Me or the trisaccharide Neu5Acalpha(2,3)Lacbeta1Me. In the transfer reaction, we show that Neu5Acalpha(2,3)Lacbeta1Me is the most favorable substrate for TcTS and is a better substrate than the naturally-occurring N-acetylneuraminosyl donor alpha1-acid glycoprotein. In the case of MUN as the donor molecule, the transfer of Neu5Ac to different acceptors is significantly slower than when other N-acetylneuraminosyl donors are used. We hypothesize that when MUN is bound by the enzyme, the orientation and steric bulk of the umbelliferyl aglycon moiety may restrict the access for the correct positioning of an acceptor molecule. AutoDock studies support our hypothesis and show that the umbelliferyl aglycon moiety undergoes a strong pi-stacking interaction with Trp-312. The binding properties of TcTS towards acceptor (lactose) and donor substrate (Neu5Ac) molecules have also been investigated using saturation transfer difference (STD) NMR experiments. These experiments, taken together with other published data, have clearly demonstrated that lactose in the absence of other coligands does not bind to the TcTS active site or other binding domains. However, in the presence of the sialic acid donor, lactose (an asialo acceptor) was observed by NMR spectroscopy to interact with the enzyme's active site. The association of the asialo acceptor with the active site is an absolute requirement for the transfer reaction to proceed.     

Relevance of the diversity among members of the Trypanosoma cruzi trans-sialidase family analyzed with camelids single-domain antibodies

The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system.     

球孢白僵菌丝氨酸蛋白酶基因CDEP-1在毕赤酵母中的表达

我们从球孢白僵菌中克隆了丝氨酸蛋白酶Pr1类基因CDEP-1。为明确CDEP-1的功能、评价其在害虫生物防治中的潜力,需要大量制备具有生物活性的CDEP-1编码蛋白。由于大肠杆菌系统表达真核基因存在产物复性困难的问题,本文利用毕赤酵母系统来表达CDEP-1。结果表明,CDEP-1可在毕赤酵母中高效的分泌表达,而且产物活性高,甲醇诱导48h后上清液中的酶活即可达到38,266U/L。诱导表达的上清液经浓缩后进行凝胶过滤层析,得到了CDEP-1的初纯品,蛋白质含量为50mg/L。将纯化的蛋白酶CDEP-1免疫家兔,制备了CDEP-1的抗血清。Westernblotting分析表明,制备的抗血清可特异性地检测CDEP-1。     

Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum)

ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology.     

Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena

Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task. We simplified the protocol for protein expression by P. pastoris and scaled it down to 0.5-ml cultures. Optimising standard growth conditions and procedures, programmed cell death and necrosis of P. pastoris in microscale cultures were diminished. Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants. Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.     

Protein kinase A catalytic subunit interacts and phosphorylates members of trans-sialidase super-family in Trypanosoma cruzi

Protein kinase A (PKA) has been suggested as a regulator of stage differentiation in Trypanosoma cruzi. Using a yeast two-hybrid system we have begun to characterize the downstream substrates of T. cruzi PKA. We identified several members of the trans-sialidase super family by this approach. Immunoprecitation demonstrated that a TcPKAc monoclonal antibody was able to pull-down proteins recognized by trans-sialidase antibodies as well as a SA85-1.1 antibody and vice versa. An in vitro phosphorylation assay demonstrated that PKA phosphorylated the recombinant protein of an active trans-sialidase. In addition, a phospho-(Ser/Thr) PKA substrate antibody detected bands on immunoblot analysis of trans-sialidase antibody precipitated proteins from parasite lysate and the media of L₆E₉ myoblasts infected with trypomastigotes as well as from a SA85-1.1 antibody precipitated proteins from parasite lysate. Immunofluorescence analysis suggested that some TcPKAc localizes to the plasma membrane surface of trypomastigotes. The identified trans-sialidases have PKA consensus phosphorylation sites located near the endoplasmic reticulum retention motif in the N-terminal. These data support that PKA phosphorylates trans-sialidase super family members in vivo.