N-Acetylneuraminic acid biosynthesis in rat liver and kidney

Rat liver and kidney tissue slices incubated withN-acetyl [³H]mannosamine incorporated radioactivity into free and boundN-acetylneuraminic acid and CMP-N-acetylneuraminic acid (CMP-NeuAc). Liver and kidney also incorporated radioactivity from intravenously injected [³H]ManNAc intoN-acetylneuraminic acid and CMP-NeuAc. From the decrease in the specific radioactivity of CMP-NeuAc after a single injection ofN-acetyl[³H]mannosamine the half-life of CMP-NeuAc was determined. From this half-life and the pool size of CMP-NeuAc a synthesis rate of CMP-NeuAc was calculated, being 1.2 nmol/min/g wet weight of kidney. In previous experiments a value of 1.0 nmol/min/g wet weight was determined for liver [Ferwerdaet al. (1983) Biochem J 216: 87–92]. The synthesis rate of CMP-NeuAcin vivo was in the same range as the synthesis rate calculated from the turnover of boundN-acetylneuraminic acid, which was 2.7 and 0.4 nmol/min/g wet weight for liver and kidney respectively.The assay conditions for UDP-N-acetylglucosamine 2-epimerase andN-acetylmannosamine kinase were adapted to measure low activitiesin vitro. It appeared that the kinase activity detected in kidney can synthesizeN-acetylmannosamine6-phosphate at a rate sufficient for the observed production ofN-acetylneuraminic acidin vivo. Also a low, but measurable activity of UDP-N-acetylglucosamine 2-epimerase was detected in kidneyin vitro, suggesting that the biosynthetic pathway ofN-acetylneuraminic acid in kidney is the same as in liver. The synthesis rate ofN-acetylneuraminic acid in liver determinedin vivo is approximately 12 times slower than the maximal potential rate calculated from the activities of theN-acetylneuraminic acid (precursor-) forming enzymes as detectedin vitro. This indicates that in liverin vivo the enzymes are working far below their maximal capacity.     

Cover Image,Volume 88, Issue 5

Human pathogenic and commensal bacteria have evolved the ability to scavenge host-derived sialic acids and subsequently degrade them as a source of nutrition. Expression of the Escherichia coli yjhBC operon is controlled by the repressor protein nanR, which regulates the core machinery responsible for the import and catabolic processing of sialic acid. The role of the yjhBC encoded proteins is not known—here, we demonstrate that the enzyme YjhC is an oxidoreductase/dehydrogenase involved in bacterial sialic acid degradation. First, we demonstrate in vivo using knockout experiments that YjhC is broadly involved in carbohydrate metabolism, including that of N-acetyl-d -glucosamine, N-acetyl-d -galactosamine and N-acetylneuraminic acid. Differential scanning fluorimetry demonstrates that YjhC binds N-acetylneuraminic acid and its lactone variant, along with NAD(H), which is consistent with its role as an oxidoreductase. Next, we solved the crystal structure of YjhC in complex with the NAD(H) cofactor to 1.35 Å resolution. The protein fold belongs to the Gfo/Idh/MocA protein family. The dimeric assembly observed in the crystal form is confirmed through solution studies. Ensemble refinement reveals a flexible loop region that may play a key role during catalysis, providing essential contacts to stabilize the substrate—a unique feature to YjhC among closely related structures. Guided by the structure, in silico docking experiments support the binding of sialic acid and several common derivatives in the binding pocket, which has an overall positive charge distribution. Taken together, our results verify the role of YjhC as a bona fide oxidoreductase/dehydrogenase and provide the first evidence to support its involvement in sialic acid metabolism.     

Probing the determinants of phosphorylated sugar-substrate binding for human sialic acid synthase

N-acetylneuraminic acid (NeuNAc), the most naturally abundant sialic acid, is incorporated as the terminal residue of mammalian cell surface glycoconjugates and acts as a key facilitator of cellular recognition, adhesion and signalling. Several pathogenic bacteria similarly express NeuNAc on their cell surfaces, allowing evasion of their host's immune system. Prokaryotic NeuNAc biosynthesis proceeds via condensation of phosphoenolpyruvate (PEP) with N-acetylmannosamine (ManNAc), a reaction catalysed by the domain-swapped homodimeric enzyme, N-acetylneuraminic acid synthase (NeuNAcS). Conversely, the mammalian orthologue, N-acetylneuraminic acid 9-phosphate synthase (NeuNAc 9-PS) utilises the phosphorylated substrate N-acetylmannosamine 6-phosphate (ManNAc 6-P) in catalysis. Here we report an investigation into the determinants of substrate specificity of human NeuNAc 9-PS, using model-guided mutagenesis to delineate binding interactions with ManNAc 6-P. Modelling predicts the formation of a domain-swapped homodimer as observed for bacterial variants, which was supported by experimental small angle X-ray scattering. A number of conserved residues which may play key roles in the selection of ManNAc 6-P were identified and substituted for alanine to assess their function. Lys290 and Thr80 were identified as a putative phosphate binding pair, with the cationic lysine residue extending into the active site from the adjacent chain of the dimeric enzyme. Substitution of these residues results in a significant loss of activity and reduced affinity for ManNAc 6-P. These residues, along with the electropositive β₂α₂ loop, are likely to facilitate the PEP dependent binding and stabilisation of ManNAc 6-P. By utilising a phosphorylated sugar-substrate, the mammalian enzyme gains considerable catalytic affinity advantage over its bacterial counterpart.     

Chemical Synthesis of 4-Trifluoromethylumbelliferyl-α- -N-acetylneuraminic Acid Glycoside and Its Use for the Fluorometric Detection of Poorly Expressed Natural and Recombinant Sialidases

When compared to bacterial or viral sialidases, eukaryotic sialidases are expressed at lower levels and frequently show poor specific activities. The identification and characterization of sialidases from eukaryotes have been slowed down due to the limited sensitivity of available sialidase substrates. Therefore, we chemically synthesized a fluorogenic compound, 4-trifluoromethylumbelliferyl-α- -N-acetylneuraminic acid (CF₃MU-Neu5Ac), and tested its use as a substrate for eight different sialidases, including enzymes from viral, bacterial, and eukaryotic sources. Kinetic analysis revealed CF₃MU-Neu5Ac to be a very sensitive sialidase substrate. Furthermore, this substance proves to be perfectly suitable for thein vivoexamination of sialidases and for the detection of recombinant sialidase by means of expression cloning.     

Chemical Synthesis of 4-Trifluoromethylumbelliferyl-α-d-N-acetylneuraminic Acid Glycoside and Its Use for the Fluorometric Detection of Poorly Expressed Natural and Recombinant Sialidases

When compared to bacterial or viral sialidases, eukaryotic sialidases are expressed at lower levels and frequently show poor specific activities. The identification and characterization of sialidases from eukaryotes have been slowed down due to the limited sensitivity of available sialidase substrates. Therefore, we chemically synthesized a fluorogenic compound, 4-trifluoromethylumbelliferyl-α-d-N-acetylneuraminic acid (CF₃MU-Neu5Ac), and tested its use as a substrate for eight different sialidases, including enzymes from viral, bacterial, and eukaryotic sources. Kinetic analysis revealed CF₃MU-Neu5Ac to be a very sensitive sialidase substrate. Furthermore, this substance proves to be perfectly suitable for thein vivoexamination of sialidases and for the detection of recombinant sialidase by means of expression cloning.     

Interaction ofN-acetyl-4-, 7-, 8- or 9-deoxyneuraminic acids andN-acetyl-4-, 7- or 8-mono-epi- and-7,8-di-epineuraminic acids withN-acetylneuraminate lyase

Various deoxy- and epi-derivatives ofN-acetylneuraminic acid were synthesized and tested for their substrate properties withN-acetylneuraminate lyase fromClostridium perfringens.N-Acetyl-9-deoxyneuraminic acid is a good substrate,N-acetylneuraminic acid derivatives with epimeric configuration at C-7, C-8 or both are cleaved slowly, whileN-acetyl-4-epi-,N-acetyl-4-deoxy-,N-acetyl-7-deoxy-andN-acetyl-8-deoxyneuraminic acid are resistant to enzyme action.N-Acetyl-4-deoxyneuraminic acid andN-acetyl-4-epineuraminic acid competitively inhibit the enzyme. These studies give further insight into a mechanism proposed for the reversible cleavage of sialic acids byN-acetylneuraminate lyase.     

Indications for the enzymatic synthesis of 9-O-lactoyl-N-acetylneuraminic acid in equine liver

Fractionation of horse liver homogenate by centrifugation into heavy membranes at 10 000 × g, microsomal fraction at 105 000 × g, and the supernatant revealed sialate 9-O-lactoyltransferase activity only in the latter fraction. For the enzyme assay, the various fractions were incubated with¹⁴C labelled CMP-N-acetylneuraminic acid,N-acetylneuraminic acid and glycoconjugate-boundN-acetylneuraminic acid. Lactoylation was identified in three different TLC systems after acid hydrolysis and purification of the sialic acids in the incubation mixtures. Enzyme activity was found only in the supernatant fraction. Glycoconjugate-boundN-acetylneuraminic acid was the best substrate tested, although some lactoylation was also found when using CMP-N-acetylneuraminic acid.     

Comparison of purified acid phosphatase allozymes inDrosophila virilis: Differences in carbohydrate content and composition of the allozymes

Three acid phosphatase (EC 3.1.3.2) allozymes (ACPH¹, ACPH², and ACPH⁴) ofDrosophila virilis show different activities as measured by electrophoretic techniques. Recently, it was suggested that these differences are attributable to the variable ability of the allozymes to be incorporated into lysosomes (Narise, S.,Genet. Res. Cambr., 45:143, 1985). Immunoelectrophoresis demonstrated that the activity differences between these electrophoretic variants coincided with differences in the amount of the enzyme protein in soluble fractions but not in whole cell-free extracts. These results support the idea that acid phosphatase allozymes inD. virilis are cell-localization variants. We examined the problem by structural analysis of both the protein and the carbohydrate moieties of these allozyme glycoproteins, since lysosomal enzymes are known to become localized in lysosomes through their carbohydrate moieties. The three ACPH allozymes were purified to homogeneity from their respective homozygotes and compared with respect to amino acid composition and carbohydrate content and composition. Amino acid compositions were similar, while content and compositions of neutral sugars were significantly different. The neutral sugar content of ACPH¹ was 9.2%; that of ACPH², 21.0%; and that of ACPH⁴, 7.3%. A trace of hexosamines, but noN-acetylneuraminic acid, was found in the ACPH allozymes. Isoelectric points varied corresponding to their electrophoretic mobilities, which were not changed by treatment with alkaline phosphatase and neuraminidase.     

A novel peptidoglycan D,L‐endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence

Bacteria remodel peptidoglycan structure in response to environmental changes. Many enzymes are involved in peptidoglycan metabolism; however, little is known about their responsiveness in a defined environment or the modes they assist bacteria to adapt to new niches. Here, we focused in peptidoglycan enzymes that intracellular bacterial pathogens use inside eukaryotic cells. We identified a peptidoglycan enzyme induced by Salmonella enterica serovar Typhimurium in fibroblasts and epithelial cells. This enzyme, which shows γ‐D‐glutamyl‐meso‐diaminopimelic acid D,L‐endopeptidase activity, is also produced by the pathogen in media with limited nutrients and in resting conditions. The enzyme, termed EcgA for e ndopeptidase responding to c essation of g rowth’, is encoded in a S. Typhimurium genomic island absent in Escherichia coli. EcgA production is strictly dependent on the virulence regulator PhoP in extra‐ and intracellular environments. Consistent to this regulation, a mutant lacking EcgA is attenuated in the mouse typhoid model. These findings suggest that specialised peptidoglycan enzymes, such as EcgA, might facilitate Salmonella adaptation to the intracellular lifestyle. Moreover, they indicate that readjustment of peptidoglycan metabolism inside the eukaryotic cell is essential for host colonisation.     

Isolation and properties of a sialidase fromTrypanosoma rangeli

In the culture supernatant ofTrypanosoma rangeli, strain El Salvador, a sialidase was present with an activity of 0.1 U/mg protein as determined with the 4-methylumbelliferyl glycoside of -N-acetylneuraminic acid as substrate. This enzyme was purified about 700-fold almost to homogeneity by gel chromatography on Sephadex G-100 and Blue Sepharose, and affinity chromatographies on 2-deoxy-2,3-didehydroneuraminic acid and horse submandibular gland mucin, both immobilized on Sepharose. The pH optimum is at 5.4–5.6, and the molecular weight was determined by gel chromatography, high performance liquid chromatography and sodium dodecyl sulphate gel electrophoresis to be 70 000. The substrate specificity of the enzyme is comparable to bacterial, viral and mammalian sialidases with cleavage rates for the following substrates in decreasing order: N-acetylneuraminyl-(2–3)-lactose> N-glycoloylneuraminy-(2–3)-lactose> N-acetylneuraminyl-(2–6)-lactose >sialoglycoproteins>gangliosides>9-O-acetylated sialoglycoproteins.4-O-Acetylated derivatives are resistant towards the action of this sialidase. The enzyme activity can be inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, Hg²⁺ ions, andp-nitrophenyloxamic acid; it is not dependent on the presence of Ca²⁺ Mn²⁺ or Mg²⁺ ions.Abbreviations BSA bovine serum albumin - BSM bovine submandibular gland mucin - CMP cytidine monophosphate - EDIA ethylenediaminetetraacetic acid - ESM equine submandibular gland mucin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC high performance liquid chromatography - Lac lactose - MU-Neu5Ac 4-methylumbelliferyl glycoside of -N-acetylneuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - Neu4Ac5Gc N-glycoloyl-4-O-acetylneuraminic acid - Neu2en 2-deoxy-2,3-didehydroneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - PMSF phenylmethylsulfonyl fluoride - PSM pig submandibular gland mucin - SDS sodium dodecyl sulfate - Tris tris-(hydroxymethyl)aminomethane Dedicated to Professor Dr. Heinz Mühlpfordt on the occasion of his 65th birthday.     

Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution

The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P2₁ at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.     

Growth-promoting Effects of N-Acetylneuraminic Acid-containing Substances on Bifidobacteria

The use of N-acetylneuraminic acid, sialyl-lactose, and glyco-macropeptide by bifidobacteria and lactobacilli, and their growth-promoting effects on B. longum, B. breve, B. bifidum, and B. infantis were investigated. The data presented here suggest that fortification with N-acetylneuraminic acid-containing substances of infant formula may provide formula-fed infants with a function that human milk possesses.     

SYNTHESIS OF GLYCOPROTEINS IN BRAIN: IDENTIFICATION, PURIFICATION AND PROPERTIES OF GLYCOSYL TRANSFERASES FROM PURIFIED SYNAPTOSOMES OF GUINEA PIG CEREBRAL CORTEX

Abstract— Four glycoprotein:glycosyl transferases (a fetuin:N-acetylglucosaminyl transferase; a bovine submaxillary mucin: N-acetylgalactosaminyl transferase; a collagen: glucosyl transferase and an orosomucoid: galactosyl transferase) were purified 34-, 45-, 37- and 47-fold, respectively, from synaptosomes prepared from guinea pig cerebral cortex. Purifications were achieved by centrifugation and by column chromatography on Sephadex G-100 and G-150 of 0 , 1% (w/v) Triton X-100 extractsof the purified cerebral cortical synaptosomes. The enzymes were separated from endogenous acceptors and were highly specific for specific macromolecular acceptors; small molecules were ineffective as acceptors. The fetuin: N-acetylglucosaminyl transferase functioned only with fetuin minus N-acetylneuraminic acid, galactose and N-acetylglucosamine; the bovine submaxillary mucin: N- acetylgalactosaminyl transferase with bovine submaxillary much minus N-acetylneuraminic acid and N-acetylgalactosamine; the collagen: glucosyl transferase with collagen minus glucose; and the orosomucoid: galactosyl transferase with either orosomucoid minus N-acetylneuraminic acid and galactose or fetuin minus N-acetylneuraminic acid and galactose. Each transferase required a specific (XDP)-monosaccharide for transfer. The transferases were entirely dependent on either Mn²⁺ or Mg²⁺ for activation and Fe²⁺ and Hg²⁺ inhibited each of the four enzymes. The optimum pH's for the enzymes were: for fetuin: N-acetylglucosaminyl transferase, 7 , 4–8.0; for bovine submaxillary mucin: N-acetylgalactosaminyl transferase, 7 , 7; for collagen: glucosyl transferase, 7 , 7 and for orosomucoid: galactosyl transferase, 6 , 6. The enzymes were distributed subsynaptosomally primarily in the synaptosomal plasma membrane and in the mitochondria of the synaptosome. The respective values for K_m (μM) and V_mex (pmoles/h/mg of protein) for the transferases were: fetuin: N-acetylglucosaminyl transferase, 12 and 143; for bovine submaxillary mucin: N-acetylgalactosaminyl transferase, 25 and 166; for collagen: glucosyl transferase, 4 and 10 and for orosomucoid:galactosyl transferase, 8 and 111.     

Comparative biochemistry of bacterial N-acyl- -amino acid amidohydrolase

N-acyl- -amino acid amidohydrolases can be classified into three types based on substrate specificity. -aminoacylase has been reported to occur in a very few bacteria such as Pseudomonas, Streptomyces, and Alcaligenes. N-acyl- -aspartate amidohydrolase ( -AAase) has been reported in only Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) while N-acyl- -glutamate amidohydrolase ( -AGase) has been isolated in two stains of Pseudomonas sp. 5f-1 and Alcaligenes A-6. The physiological roles of these enzymes in these microbes are not clear. They are individually characteristic in their substrate specificities, inducer profiles, inhibitors, isoelectric points, metal dependency, and some physicochemical properties. The primary structures of all the three types of N-acyl- -amino acid amidohydrolases from Alcaligenes A-6 were determined from their nucleotide sequences. Comparison of their primary structures revealed high homology (46–56%) between the different enzymes. The three enzymes showed 26–27% sequence homology with -aminoacylases from Bacillus stearothermophilus, porcine, and human. Chemical modification and site-directed mutagenesis identified the histidyl residues essential for catalysis. The Alcaligenes N-acyl- -amino acid amidohydrolases share significant sequence similarities with some members of the urease-related amidohydrolase superfamily proposed by Holm and Sander [L. Holm, C. Sander, Proteins: Structure, Function and Genetics 28 (1997) 72].     

Conservation of structure and mechanism by Trm5 enzymes

Enzymes of the Trm5 family catalyze methyl transfer from S-adenosyl methionine (AdoMet) to the N¹ of G37 to synthesize m¹G37-tRNA as a critical determinant to prevent ribosome frameshift errors. Trm5 is specific to eukaryotes and archaea, and it is unrelated in evolution from the bacterial counterpart TrmD, which is a leading anti-bacterial target. The successful targeting of TrmD requires detailed information on Trm5 to avoid cross-species inhibition. However, most information on Trm5 is derived from studies of the archaeal enzyme Methanococcus jannaschii (MjTrm5), whereas little information is available for eukaryotic enzymes. Here we use human Trm5 (Homo sapiens; HsTrm5) as an example of eukaryotic enzymes and demonstrate that it has retained key features of catalytic properties of the archaeal MjTrm5, including the involvement of a general base to mediate one proton transfer. We also address the protease sensitivity of the human enzyme upon expression in bacteria. Using the tRNA-bound crystal structure of the archaeal enzyme as a model, we have identified a single substitution in the human enzyme that improves resistance to proteolysis. These results establish conservation in both the catalytic mechanism and overall structure of Trm5 between evolutionarily distant eukaryotic and archaeal species and validate the crystal structure of the archaeal enzyme as a useful model for studies of the human enzyme.     

Determination of sialic acids in biological fluids using reversed-phase ion-pair high-performance liquid chromatography

A simple, rapid and sensitive reversed-phase ion-pair high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in biological fluids is described. Determination of N-acetylneuraminic acid released by acidic hydrolysis, in serum, urine and saliva, and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in urine, without hydrolysis, was accomplished by injecting the sample without derivatization, into the chromatograph. Measurements were carried out isocratically within 6 min using a C₁₈ column and a mobile phase of aqueous solution of triisopropanolamine, as ion-pair reagent, 60 mM, pH 3.5 at room temperature with UV absorbance detection. The present method is reported for the first time for the determination of sialic acids in biological fluids. Recoveries in serum, urine and saliva ranged from 90 to 102% and the limits of detection were 60 nM and 20 nM for the two sialic acids, respectively. The method has been applied to normal and pathological sera from patients with breast, stomach, colon, ovarian and cervix cancers, to normal urine and urine from patient with sialuria and to normal saliva.     

Cloning and characterization of a sialidase from the filamentous fungus, <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

A gene encoding a putative sialidase was identified in the genome of the opportunistic fungal pathogen, Aspergillus fumigatus. Computational analysis showed that this protein has Asp box and FRIP domains, it was predicted to have an extracellular localization, and a mass of 42 kDa, all of which are characteristics of sialidases. Structural modeling predicted a canonical 6-bladed β-propeller structure with the model’s highly conserved catalytic residues aligning well with those of an experimentally determined sialidase structure. The gene encoding the putative Af sialidase was cloned and expressed in Escherichia coli. Enzymatic characterization found that the enzyme was able to cleave the synthetic sialic acid substrate, 4-methylumbelliferyl α-D-N-acetylneuraminic acid (MUN), and had a pH optimum of 3.5. Further kinetic characterization using 4-methylumbelliferyl α-D-N-acetylneuraminylgalactopyranoside revealed that Af sialidase preferred α2-3-linked sialic acids over the α2-6 isomers. No trans-sialidase activity was detected. qPCR studies showed that exposure to MEM plus human serum induced expression. Purified Af sialidase released sialic acid from diverse substrates such as mucin, fetuin, epithelial cell glycans and colominic acid, though A. fumigatus was unable to use either sialic acid or colominic acid as a sole source of carbon. Phylogenetic analysis revealed that the fungal sialidases were more closely related to those of bacteria than to sialidases from other eukaryotes.     

Purification and characterization of a sialidase fromClostridium chauvoei NC08596

The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg^–1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn²⁺, Mg²⁺, and Ca²⁺ and bovine serum albumin have a positive effect on the sialidase activity, while Hg²⁺, Cu²⁺, and Zn²⁺, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.Abbreviations FPLC fast protein liquid chromatography - NCTC National Collection of Type Cultures - ATCC American Type Culture Collection - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - buffer A 0.02m piperazine, 0.01m CaCl₂, pH 5.5 - buffer B 0.02m piperazine, 0.01m CaCl₂, 1.0m NaCl, pH 5.5 - buffer C 0.1m sodium acetate, 0.01m CaCl₂, pH 5.5 - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - Neu5Ac N-acetylneuraminic acid - BSM bovine submandibular gland mucin - GD1a IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer - GM1 II³Neu5Ac-GgOse₄Cer - MU-Neu4,5Ac₂ 4-methylumbelliferyl--d-N-acetyl-4-O-acetylneuraminic acid - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol bis(2-aminoethyl-ethen)-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - IEF isoelectric focusing - IEP isoelectric point     

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.