Crystal Structure of the Human Cytosolic Sialidase Neu2

Gangliosides play key roles in cell differentiation, cell-cell interactions, and transmembrane signaling. Sialidases hydrolyze sialic acids to produce asialo compounds, which is the first step of degradation processes of glycoproteins and gangliosides. Sialidase involvement has been implicated in some lysosomal storage disorders such as sialidosis and galactosialidosis. Neu2 is a recently identified human cytosolic sialidase. Here we report the first high resolution x-ray structures of mammalian sialidase, human Neu2, in its apo form and in complex with an inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). The structure shows the canonical six-blade β-propeller observed in viral and bacterial sialidases with its active site in a shallow crevice. In the complex structure, the inhibitor lies in the catalytic crevice surrounded by ten amino acids. In particular, the arginine triad, conserved among sialidases, aids in the proper positioning of the carboxylate group of DANA within the active site region. The tyrosine residue, Tyr334, conserved among mammalian and bacterial sialidases as well as in viral neuraminidases, facilitates the enzymatic reaction by stabilizing a putative carbonium ion in the transition state. The loops containing Glu111 and the catalytic aspartate Asp46 are disordered in the apo form but upon binding of DANA become ordered to adopt two short α-helices to cover the inhibitor, illustrating the dynamic nature of substrate recognition. The N-acetyl and glycerol moieties of DANA are recognized by Neu2 residues not shared by bacterial sialidases and viral neuraminidases, which can be regarded as a key structural difference for potential drug design against bacteria, influenza, and other viruses. Gangliosides play key roles in cell differentiation, cell-cell interactions, and transmembrane signaling. Sialidases hydrolyze sialic acids to produce asialo compounds, which is the first step of degradation processes of glycoproteins and gangliosides. Sialidase involvement has been implicated in some lysosomal storage disorders such as sialidosis and galactosialidosis. Neu2 is a recently identified human cytosolic sialidase. Here we report the first high resolution x-ray structures of mammalian sialidase, human Neu2, in its apo form and in complex with an inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). The structure shows the canonical six-blade β-propeller observed in viral and bacterial sialidases with its active site in a shallow crevice. In the complex structure, the inhibitor lies in the catalytic crevice surrounded by ten amino acids. In particular, the arginine triad, conserved among sialidases, aids in the proper positioning of the carboxylate group of DANA within the active site region. The tyrosine residue, Tyr334, conserved among mammalian and bacterial sialidases as well as in viral neuraminidases, facilitates the enzymatic reaction by stabilizing a putative carbonium ion in the transition state. The loops containing Glu111 and the catalytic aspartate Asp46 are disordered in the apo form but upon binding of DANA become ordered to adopt two short α-helices to cover the inhibitor, illustrating the dynamic nature of substrate recognition. The N-acetyl and glycerol moieties of DANA are recognized by Neu2 residues not shared by bacterial sialidases and viral neuraminidases, which can be regarded as a key structural difference for potential drug design against bacteria, influenza, and other viruses. Sialidases, or neuraminidases (EC 3.2.1.18), are glycohydrolytic enzymes widely distributed among species (1Saito M. Yu R.K. Rosenberg A. Biology of the Sialic Acids. Plenum Press, New York1995: 261-313Crossref Google Scholar). The removal of sialic acids on the non-reducing termini of complex carbohydrates by sialidases is the first step of degradation of glycolipids or glycoproteins. Furthermore, sialidases are considered to be involved in various biological events such as infection processes, antigenic expression, differentiation, signal transduction, or intercellular interactions (2Schauer R. Kelm S. Reuter G. Roggentin P. Shaw L. Rosenberg A. Biology of the Sialic Acids. Plenum Press, New York1995: 7-67Crossref Google Scholar). Mammalian sialidases are classified into three categories based on their cellular localizations: cytosolic, lysosomal, and plasma membrane-bound (3Monti E. Bassi M.T. Bresciani R. Civini S. Croci G.L. Papini N. Riboni M. Zanchetti G. Ballabio A. Preti A. Tettamanti G. Venerando B. Borsani G. Genomics. 2004; 83: 445-453Crossref PubMed Scopus (94) Google Scholar), with exceptions in the nucleus (4Akita H. Miyagi T. Hata K. Kagayama M. Histochem. Cell Biol. 1997; 107: 495-503Crossref PubMed Scopus (21) Google Scholar) and in the extracellular medium (5Sweeley C.C. Adv. Lipid Res. 1993; 26: 235-252PubMed Google Scholar, 6Warner T.G. Chang J. Ferrari J. Harris R. McNerney T. Bennett G. Burnier J. Sliwkowski M.B. Glycobiology. 1993; 3: 455-463Crossref PubMed Scopus (56) Google Scholar, 7Gramer M.J. Goochee C.F. Chock V.Y. Brousseau D.T. Sliwkowski M.B. Biotechnology. 1995; 13: 692-698Crossref PubMed Scopus (87) Google Scholar). Recently, the human cytosolic sialidase Neu2 has been cloned (8Monti E. Preti A. Rossi E. Ballabio A. Borsani G. Genomics. 1999; 57: 137-143Crossref PubMed Scopus (87) Google Scholar). The protein is expressed at a low level in skeletal muscle, liver, and brain cells. Rodent Neu2 enzymes are involved in the differentiation from myoblasts to myotubes of rat L6 (9Sato K. Miyagi T. Biochem. Biophys. Res. Commun. 1996; 221: 826-830Crossref PubMed Scopus (65) Google Scholar) and mouse C2C12 (10Fanzani A. Giuliani R. Colombo F. Zizioli D. Presta M. Preti A. Marchesini S. FEBS Lett. 2003; 547: 183-188Crossref PubMed Scopus (50) Google Scholar) cells. Unfortunately, the precise nature of their natural substrate(s) in the process of myotube formation and maintenance remains unclear. Transfection of NEU2 cDNA to highly invasive and metastatic murine B16 melanoma cells led to suppression of pulmonary metastasis (11Tokuyama S. Moriya S. Taniguchi S. Yasui A. Miyasaki J. Orikasa S. Miyagi T. Int. J. Cancer. 1997; 73: 410-415Crossref PubMed Scopus (59) Google Scholar). A major change detectable in Neu2 transfected cells was a decrease in ganglioside GM3, 1The abbreviations used are: GM3, N-acetylneuraminylgalactosylceramide; DANA, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid; r.m.s.d., root mean square deviation; NANA, N-acetylneuraminic acid; MES, 4-morpholineethanesulfonic acid. 1The abbreviations used are: GM3, N-acetylneuraminylgalactosylceramide; DANA, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid; r.m.s.d., root mean square deviation; NANA, N-acetylneuraminic acid; MES, 4-morpholineethanesulfonic acid. concordant with an increase in lactosylceramide content. Several lines of evidence have indicated the association of glycosphingolipids with the cytoskeleton (11Tokuyama S. Moriya S. Taniguchi S. Yasui A. Miyasaki J. Orikasa S. Miyagi T. Int. J. Cancer. 1997; 73: 410-415Crossref PubMed Scopus (59) Google Scholar, 12Chan K.F. Liu Y. Glycobiology. 1991; 1: 193-203Crossref PubMed Scopus (28) Google Scholar). In particular, the GM3 ganglioside has been suggested to interact with microtubules and intermediate filaments (13Gillard B.K. Thurmon L.T. Marcus D.M. Glycobiology. 1993; 3: 57-67Crossref PubMed Scopus (69) Google Scholar). Thus, Neu2 might act on GM3 leading to the alteration of the cytoskeleton functions, consistent with the decreased invasiveness of transfected melanoma cells (11Tokuyama S. Moriya S. Taniguchi S. Yasui A. Miyasaki J. Orikasa S. Miyagi T. Int. J. Cancer. 1997; 73: 410-415Crossref PubMed Scopus (59) Google Scholar). Similar results were obtained using highly metastatic sublines of mouse colon adenocarcinoma 26 in which endogenous Neu2 sialidase levels are inversely correlated with the metastatic capacities (14Sawada M. Moriya S. Saito S. Shineha R. Satomi S. Yamori T. Tsuruo T. Kannagi R. Miyagi T. Int. J. Cancer. 2002; 97: 180-185Crossref PubMed Scopus (75) Google Scholar). Neu2 overexpression led to a marked inhibition of lung metastasis, invasion, and cell motility, as well as a decrease of sialyl Lewis X and, again, of GM3 cellular contents. Thus, desialylation of these target molecules including GM3 by cytosolic sialidase is somehow related to suppression of metastasis. The atomic structure of the human cytoplasmic sialidase may not only give further insights into the molecular mechanisms of its substrate recognition but also will provide important clues to understanding the catalytic mechanism of mammalian sialidases. Here we report the first crystal structures of a mammalian sialidase, human Neu2, in its free form and in complex with an inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA, Fig. 1c). The core of Neu2 folds as a six-bladed β-propeller, typical of viral and bacterial sialidases. Interaction between Neu2 and DANA shows similarities with bacterial and viral counterparts but also exhibits some differences in the active site arrangement and dynamic nature of the loops containing residues responsible for catalysis and substrate recognition.Fig. 1Sialic acid and inhibitors. Schematic structure of a sialylgalactose (a), NANA (b), and DANA (c). In the case of the sialylgalactose, the dotted line indicates the bond hydrolyzed by the sialidase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Expression and Purification—The coding region of Neu2 was subcloned into pGEX-2T vector (Amersham Biosciences) and the recombinant protein produced by Escherichia coli cells was purified in three steps, affinity and anion and cation exchange chromatography as described previously (15Tringali C. Papini N. Fusi P. Croci G. Borsani G. Preti A. Tortora P. Tettamanti G. Venerando B. Monti E. J. Biol. Chem. 2004; 279: 3169-3179Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Approximately 2.4 mg of Neu2 at a concentration of 1.8 mg·ml-1 was obtained from 1 liter of culture. Crystallization and Soaking of the Crystals—The purified protein was concentrated to 10 mg·ml-1 in 400 mm sodium chloride and 50 mm MES buffer, pH 6.7. Crystals were grown in hanging drops at 289 K. For the apo form Neu2, the reservoir solution contained 100 mm sodium/potassium phosphate buffer, pH 6.2, 2.5 m NaCl, and the hanging drops contained 1:1 mixtures of the protein solution and the precipitants. Crystals grew in 1-2 days to a size of ∼500 × 200 × 50 μm3. The crystals were mounted in nylon loops (Hampton research) and flash-frozen at 100 K in a cryoprotectant solution containing 20% glycerol in addition to the precipitant solution. Crystals belong to the space group R3, with unit cell dimensions a = b = 145.6 Å, c = 64.6 Å, α = β = 90°, γ = 120°, and contain one protein per asymmetric unit. “Sugar-induced form” crystals were prepared by soaking apo form crystals in the reservoir solution additionally containing 50 μm of one of the following, monosaccharides, galactose, glucose, or maltose, for 4 h. These crystals were handled in the same way as in the apo form crystals. Cell dimensions of the crystals soaked with sugar are equivalent to those of the apo form crystals. Soaking apo form crystals in solutions containing inhibitors, N-acetylneuraminic acid (NANA, Fig. 1b) or DANA, either dissolved the crystals after a few days or did not give clear electron density of the bound ligands. Co-crystallization trials of Neu2 with either of the inhibitors using the same crystallization condition as the apo form gave rise to cracked crystals and the resulting crystal structure did not show any density of the inhibitors. Further screening for other crystallization conditions proved to be successful with DANA; crystals of the Neu2-DANA complex were obtained with a reservoir solution containing 100 mm HEPES buffer, pH 7.0, 65% 2-methyl-2,4-pentanediol, and 100 mm guanidine hydrochloride. Hanging drops were prepared at 289 K as a 1:1 mixture of the protein solution and the precipitant. The molar ratio of Neu2 to DANA in the protein solution was 1:50. The crystals grew in 20 days to an approximate size of 100 × 50 × 50 μm3. They were frozen in liquid nitrogen without additional cryoprotectant. The crystals of Neu2-DANA contain two complex molecules per asymmetric unit and belong to the space group C2 with the unit cell dimensions of a = 157.6 Å, b = 74.0 Å, c = 77.2 Å, α = β = 90°, γ = 90.6°. Data Collection and Structure Determination—X-ray diffraction data were collected at the resolutions of 1.75, 1.49, and 2.85 Å at the Photon Factory AR-NW12 and BL6A beam lines for the apo, maltose-induced (referred as sugar-induced hereafter), and the DANA complex, respectively. The data were reduced and scaled using program HKL2000 (16Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38231) Google Scholar) and CCP4 programs (17Potterton E. Briggs P. Turkenburg M. Dodson E. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1131-1137Crossref PubMed Scopus (1039) Google Scholar). The apo form structure of Neu2 was solved by the molecular replacement method using CNS (18Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16909) Google Scholar) and CCP4 programs, with the atomic coordinates of a bacterial sialidase of Micromonospora viridifaciens as a search model (Protein Data Bank code 1EUS), despite the low sequence identity between them, 27%. Structures of the sugar-induced form and Neu2-DANA complex were solved by the molecular replacement method using the apo form structure as a search model. Initial model building of the Neu2 apo form was carried out by automated model building with the program ARP/wARP (19Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2559) Google Scholar). Further model building and refinement of the apo form Neu2 and that of the complex molecules were carried out manually with programs O (20Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12997) Google Scholar) and Turbo FRODO (21Roussel A. Cambillau C. Silicon Graphics Geometry Silicon Scholar) and refinement with the CNS programs are in the model the protein structure was with those of other sialidases using programs of the CCP4 were by a of the programs J. Crystallogr. 1991; Google Scholar), A. K. B. Struct. 1991; PubMed Scopus Google Scholar), W.L. DeLano Scholar), and 1995; PubMed Scopus Google Scholar). coordinates and structure have been in the Data Bank and and refinement apo sugar-induced in are for the high resolution in are for the high resolution is the of and is the mean in are for the high resolution in are for the high resolution dimensions = b = c = = b = c = = b = = β = and was using of data from of protein of of two molecules of DANA and molecules of not in are for in are for for and residues from region region in are for the high resolution is the of and is the mean and was using of data from two molecules of DANA and molecules of not in are for in are for for and residues in a Structure and structures residues and for the apo form Neu2, residues and for the sugar-induced and residues and for the Neu2-DANA complex respectively. for the Neu2 apo the sugar-induced and the Neu2-DANA complex are and with residues in the Neu2 Neu2 apo form a typical six-bladed β-propeller with 26 and α-helices The structure of the β-propeller is to those of bacterial sialidases and viral neuraminidases G. Struct. Biol. 1996; 6: PubMed Scopus Google Scholar) despite low sequence between of the is of with the exceptions of and which are into two The and termini have residues of the β-propeller and are to α-helices are in the loops between two and or at the and In three typical of sialidases P. B. J. L. R. J. 6: PubMed Scopus Google Scholar) are in the and of the and crevice at the of the β-propeller an core for the enzymatic catalysis and the residues at the of the crevice B. C. S. R.W. Structure 1993; 1: Full Text PDF PubMed Scopus Google Scholar) the of the β-propeller, an to the of the catalytic site is also in sialidases from other such as A. S. G. J. PubMed Scopus Google Scholar), (Protein Data Bank code (Protein Data Bank code and M. viridifaciens (Protein Data Bank and and might act as an additional mechanism by binding such as and which are to Neu2 (15Tringali C. Papini N. Fusi P. Croci G. Borsani G. Preti A. Tortora P. Tettamanti G. Venerando B. Monti E. J. Biol. Chem. 2004; 279: 3169-3179Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, T. Hata K. A. T. J. 1993; PubMed Scopus Google Scholar). Neu2 loops are disordered on the of the apo form one Glu111 important for the substrate binding and the other Asp46 for the Soaking the apo form crystals with such as galactose, glucose, or the into Unfortunately, is electron density for the despite various of and For soaking in and of dissolved the crystals after Neu2-DANA two in the asymmetric unit of Neu2-DANA crystals show only structural Thus, only one of them, A and in the Data Bank will be in structure is to that of the apo form Neu2 of Å at the resolution of 2.85 Å, the electron density of DANA was clear in the active site DANA lies in a with 10 amino acids of the active site and The binding is by a of and interactions with additional interactions binding of DANA, the two which were disordered in the apo become ordered to form two and and cover the inhibitor in the active site In particular, Asp46 of and Glu111 of the to the inhibitor mammalian sialidases have been including from human (3Monti E. Bassi M.T. Bresciani R. Civini S. Croci G.L. Papini N. Riboni M. Zanchetti G. Ballabio A. Preti A. Tettamanti G. Venerando B. Borsani G. Genomics. 2004; 83: 445-453Crossref PubMed Scopus (94) Google Scholar). In we have that Neu2 also folds as a six-bladed β-propeller and the same active site a In a sequence of Neu2 with the other human sialidases that also as with the conserved residues the active M. G. C. P. B. G. R. E. and S. the other have been to target sialidases, in the neuraminidases, based on the x-ray structures B. M. L. Chem. Int. 2003; PubMed Scopus Google Scholar). a structural of Neu2 might on the catalytic mechanism of the human sialidases and give additional clues for a of inhibitors based on the difference between human sialidases and bacterial and viral in and and and of an important in the crystal and the active site In the apo and sugar-induced group into the active site of the and inhibitors from the of soaking of the apo form crystals in inhibitor containing the other in the Neu2-DANA complex structure group the in the crystal by with the same of the In the is to the active site of the the inhibitor and the loops and involved in the inhibitor the active upon between the complex and sugar-induced structures shows a of by Å, Glu111 to the active illustrating the of the residues of the catalytic in the same as in the apo with an of Å for the and in the active on viral neuraminidases a catalytic mechanism the sialic acid is first recognized by the arginine and hydrolyzed by an B. C. S. R.W. Structure 1993; 1: Full Text PDF PubMed Scopus Google Scholar). these we a binding a substrate the catalytic out of the 10 residues Asp46 and the sialic acid by and interactions the catalytic site an Asp46 and Glu111 from their the two loops containing Asp46 and Glu111 in with the sialic acid and cover the catalytic site Asp46 is at an from the C2 of the sialic acid for the reaction to Glu111 three with the of the glycerol of and Tyr334, conserved in sequence as well as in space among sialidases, the of DANA by The of the three is as the arginine triad, which the carboxylate its group in to the C2 a target of at a of with active site residues of the bacterial the arginine and show high structural similarities The that Neu2, the first human sialidase structure has been also the same active site the of a of sialidases despite the low sequence to the bacterial and viral the between Neu2, bacterial sialidases, and viral neuraminidases the of DANA, three residues and positioning of the inhibitor by with the and on the structure of potential inhibitors of the have been and B. M. L. Chem. Int. 2003; PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar). The residues described are not shared by bacterial sialidases and viral neuraminidases and can be as a key difference for drug design against bacteria, influenza, and other viruses. some sialidases have a residue, as to at the to Glu111 to DANA by interactions, which to the substrate For a cytosolic sialidase from cells sequence identity with has acid and residues at the with and is to hydrolyze N-acetylneuraminic acid as well as acid J. M. Res. PubMed Scopus Google Scholar). the acid of Neu2 is at the of which ordered upon inhibitor binding and is by and these results a of the protein for of by acid and residues and the as the glycerol of the inhibitor are for its recognition. Further structural and are way to and to the difference in of the active site for inhibitors against viral and bacterial counterparts with against human sialidases. are a structure, to the of and are in such as and sialidases P. B. J. L. R. J. 6: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). In the Neu2 structure, two and are identified residues important for A square between the two in an of only Å A which we previously as a conserved of E. Preti A. C. Ballabio A. Borsani G. Glycobiology. 1999; PubMed Scopus Google Scholar), was and with an of Å with the two other A the structure at the of three conserved and in of the three of Neu2 apo In the structure of Neu2-DANA complex, the is not to the low resolution of the data the of the remains the same as that of the apo In Neu2, the are on the of the catalytic crevice In or is between the The residues of the show a high sequence with of conserved at The two and of Neu2, by amino the same sequence of containing the previously sequence was PubMed Scopus Google Scholar) but with clear of biological roles of the in The sequence of the in Neu2, the sequence of as for A screening may give further insights into the of these structural for on the as well as and for their and on the structure