Catalytic preference of Salmonella typhimurium LT2 sialidase for N-acetylneuraminic acid residues over N-glycolylneuraminic acid residues

In a comparison of sialidase activities toward N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), we found that Salmonella typhimurium LT2 sialidase (STSA) hardly cleaved 4-methylumbelliferyl Neu5Gc (4MU-Neu5Gc). The kcat/Km value of STSA for 4MU-Neu5Gc was found to be 110 times lower than that for 4-methylumbelliferyl Neu5Ac (4MU-Neu5Ac). Additionally, STSA had remarkably weak ability to cleave α2-3-linked-Neu5Gc contained in gangliosides and equine erythrocytes. In silico analysis based on first-principle calculations with transition-state analogues suggested that the binding affinity of Neu5Gc2en is 14.3 kcal/mol more unstable than that of Neu5Ac2en. The results indicated that STSA preferentially cleaves Neu5Ac residues rather than Neu5Gc residues, which is important for anyone using this enzyme to cleave α2-3-linked sialic acids.


Introduction
Sialidases remove sialic acid from sialoglycoconjugates and are expressed in many species such as bacteria, viruses, fungi, protozoa, invertebrates and mammals [ 1 -3 ]. The Salmonella typhimurium LT2 sialidase (STSA) cleaves sialic acid residues of glycoproteins and gangliosides efficiently and has kinetic preference for sialyl α2-3 linkages over sialyl α2-6 linkages [ 4 , 5 ]. The catalytic mechanism of STSA for the high specificity toward sialyl α2-3 linkages has been estimated from high-resolution structure analysis by X-ray crystallography [ 6 ]. Due to this kinetic preference, STSA has been used for determination of sialyl α2-3 linkage [ 7 ] and for detailed investigations regarding the roles of sialyl α2-3-linked oligosaccharides [ 8 ].
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Synthesis of 4MU-Neu5Gc
The synthetic scheme is shown in Fig. 1 . For the synthesis of Nsubstituted sialoside [ 15 ], compound 2 [ 16 ] was acylated by Boc 2 O and 4-dimethylaminopyridine in THF to give N,N -Boc,Ac analogue 3 of sialic acid in 74% yield. Selective N,O -deacetylation of 3 with sodium methoxide gave N -Boc derivative 4 in 78% yield, which was deprotected by CF 3 CO 2 H and subsequently submitted to N -acylation of the resulting free amino group with acetylglycoloyl chloride and NEt 3 in MeOH to give the corresponding N -acetylglycolyl glycoside 5 in 69% yield in two steps. Finally, treatment of 5 with 0.1 M NaOH-MeOH (1:1) gave 4MU-Neu5Gc 1 in 49% yield.

Measurement of enzyme units
Enzyme units of AUSA and STSA, unless otherwise noted, were determined by incubation of these enzymes in 80 mM sodium acetate buffer (pH 6.0) containing 10 μM 4MU-Neu5Ac, 80 mM NaCl and 0.8 mg / ml BSA at 37 • C. One unit was defined as the amount of enzyme that catalyzed the release of 1 μmol of sialic acid for 1 min.

Hydrolysis of sialylglycans in erythrocytes
Erythrocytes were prepared from equine blood (Kohjin Bio, Saitama, Japan) by repeated suspension and centrifugation at 2000 rpm for 5 min in phosphate buffered saline (PBS). The erythrocytes (0.5-50%, v / v) were incubated in PBS (100 μl, pH 6.0) containing 1 mU / ml AUSA or 1 mU / ml STSA for 180 min at 37 • C. After centrifugation at 2000 rpm for 5 min at 4 • C, the supernatant containing released sialic acid was collected.

Quantitative analysis of sialic acid
Fluorometric determination of Neu5Ac and Neu5Gc was performed by high-performance liquid chromatography (HPLC) as previously described [ 18 ]. For precolumn fluorescence derivatization, 7.0 mM DMB solution containing 1.0 M β-mercaptoethanol and 18 mM sodium hydrosulfite was added to 10 μl of free sialic acidcontaining solutions, and incubated for 2.5 h at 60 • C. The mixtures of DMB-derivatized sialic acid were analyzed by HPLC [LC-2000Plus series, Jasco, Tokyo, Japan; C 18 column, Tosoh, Tokyo, Japan; mobile phase, methanol / water (25:75, v / v), flow rate of 1.2 ml / min]. Fluorescence was monitored at excitation and emission wavelengths of 373 and 448 nm, respectively.

In silico analysis
The 3D structure of STSA for all sequence alignments was generated by homology modeling as a template of the X-ray crystal structure (PDB ID: 2SIM) using the Molecular Operating Environment (MOE) program package (MOE 2011.10, Chemical Computing Group, Montreal, QC, Canada) [ 19 ]. Docking simulations of Neu5Ac2en or Neu5Gc2en to the STSA structure were carried out by the MOE-Dock method [ 20 ]. The binding affinity was evaluated using the correlated fragment molecular orbital (FMO) calculations at the RI-MP2 / cc-pVDZ level [ 21 ]. All FMO calculations were performed on 2.93GHz Nehalem 8Core 7 CPUs (56CPUs) cluster system using the Parallelized ab initio Calculation System based on FMO (PAICS) program (available from http: // www.paics.net ) [ 22 ].

Kinetic parameters of STSA
It has been reported that Neu5Gc α2-3Gal β1-4Glc-pyridylamine was cleaved with a high concentration of STSA [ 7 ]. Our preliminary data also showed that 4MU-Neu5Gc was hydrolyzed slightly with 10 mU / ml STSA. To measure the kinetic parameters of STSA and AUSA for the hydrolysis of 4MU-Neu5Gc and 4MU-Neu5Ac, both substrates were cleaved with STSA (1 −10 mU / ml) and AUSA (1 mU / ml) in a pH 6.0 buffer solution at 37 • C ( Fig. 3 ). The cleavage of 4MU-Neu5Gc with AUSA was inhibited with 300 μM DANA, indicating that 4MU-Neu5Gc measured sialidase activity specifically.
The K m (mM) and k cat / K m (M −1 s −1 ) values were calculated using the Lineweaver-Burk plot ( Table 1 ). The K m values of STSA and AUSA for 4MU-Neu5Gc were 8.4-times and 2.5-times higher, respectively, than those for 4MU-Neu5Ac. The K m value of STSA for 4MU-Neu5Ac in our measurement was 0.37 mM, which is close to the K m value (0.25 mM) measured by Hoyer et al. under similar conditions [ 4 ]. The k cat / K m value of STSA for 4MU-Neu5Gc was 110-times lower than that for 4MU-Neu5Ac. On the other hand, the k cat / K m value of AUSA for 4MU-Neu5Gc was only two-times lower than that for 4MU-Neu5Ac. These results indicated that STSA had low affinity toward and weak ability for cleaving 4MU-Neu5Gc compared with 4MU-Neu5Ac.

Enzyme activity of STSA toward Neu5Gc-containing sialylglycans in equine erythrocytes
Equine erythrocytes contain Neu5Gc in gangliosides and glycoproteins. Suzuki et al. reported that Neu5Gc was the only molecular species of sialic acid contained in equine erythrocyte membranes [ 32 ]. The ganglioside from equine erythrocytes was shown to be composed of Neu5Gc as Neu5Gc-GM 3 or 4-O -acetyl-Neu5Gc-GM 3 but with little Neu5Ac [ 33 -35 ]. Neu5Gc-containing glycoproteins were also detected in equine erythrocytes as a 68-kDa protein band by Western blotting analysis [ 36 ]. The linkage form of sialic acid in equine erythrocytes was analyzed using lectins, suggesting that equine erythrocytes contained sialic acid (Sia) α2-3Gal-linkage abundantly but little Sia α2-6Gal-linkage [ 37 ]. Thus, equine erythrocytes would contain a large amount of the Neu5Gc α2-3Gal structure.
We analyzed the hydrolytic potential of STSA toward Neu5Gccontaining sialylglycans in equine erythrocytes. Equine erythrocytes The asterisks indicate significant differences ( * P < 0.05, *** P < 0.001; t -test) from the amount of released Neu5Gc with AUSA. (0.5-50%, v / v) were treated with STSA and AUSA at 37 • C in a pH 6.0 buffer solution. The amount of Neu5Gc cleaved with STSA was remarkably small compared to the amount cleaved with AUSA, suggesting that STSA hardly hydrolyzes Neu5Gc α2-3Gal structure in equine erythrocytes ( Fig. 5 ).

In silico analysis for the catalytic mechanism of STSA
To explore the origin of substrate specificity of STSA, we performed in silico analysis for the STSA complex with Neu5Ac / Neu5Gc based on first-principles ( ab initio ) calculations using the FMO method, which can correctly evaluate interactions between a substrate and hydrophilic / hydrophobic amino acid residues in a protein. We docked transition-state analogues, Neu5Gc2en and Neu5Ac2en, to the STSA structure and evaluated the binding affinity of them to STSA. As a result, the binding affinity of Neu5Gc2en is 14.3 kcal / mol more unstable than that of Neu5Ac2en ( Table 2 ). The positions of the hydroxyl and carboxyl groups of Tyr307 and Arg309 were changed by 1.18 and 0.85 Å , respectively, due to steric hindrance of the hydroxymethyl group of Neu5Gc in the binding site ( Fig. 6 ). Tyr307 and Arg309 have been pointed out as the key residues of recognition for the difference between sialyl α2-3 and sialyl α2-6-linkeages. Neu5Ac2en can make the salt-bridge interaction with Arg309 more effectively than Neu5Gc2en.
In conclusion, in addition to its kinetic preference for sialyl α2-3 linkage over sialyl α2-6 linkage, STSA has kinetic preference for Neu5Ac residue over Neu5Gc residue. The amino acid residues that recognize sialyl α2-3 linkage in STSA also play crucial roles in selective cleavage for the molecular species of sialic acid. STSA is useful for preferentially removing α2-3-linked sialic acids. For usage of STSA, it is necessary to pay attention to the low ability to cleave Neu5Gc. Our finding indicates that STSA is also useful for biologically confirming molecular species of the sialic acid linked to galactose by the α2-3 linkage.