Purification, crystallization and preliminary crystallographic study of neuraminidase from Vibrio cholerae and Salmonella typhimurium LT2

J. Mol. Biol.

(1992)

226,

1287-1290

Purification, Crystallization and Preliminary Crystallographic Study of Neuraminidase from Vibriu cholerae and Salmonella typhimurium LT2 Garry Taylor? Department of Biochemistry, University of Bath Claverton Down, Bath BA2 7A Y, U.K.

Eric Vimr Department of Veterinary Pathobiology College of Veterinary Medicine, University of Illinois 2001 South Lincoln Avenue, Urbana, IL 61801, U.S.A.

Elspeth Garman Laboratory of Molecular Biophysics, Rex Richards Building South Parks Road, Oxford OX1 3QU, U.K.

Graeme Laver John Curtin Medical School, The Australian National University GPO Box 334, Canberra, ACT 2601, Australia (Received

6 May

1992; accepted 7 May

1992)

The nanH genes of Vibrio cholerae and Salmonella typhimurium LT2 coding neuraminidase were cloned separately in Escherichia coli, and the expression products purified. Single crystals of the V. cholerae neuraminidase were obtained using the hanging drop vapour diffusion method with polyethylene glycol as precipitant at pH 7.2. The crystals belong to the orthorhombic space group P2,2,2,, with unit cell dimensions a = 71.9 A, b = 790 8, c = 165.7 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 2.5 A. Single crystals of the S. typhimurium neuraminidase were obtained by hanging drop with potassium phosphate as precipitant at pH 7.2. The crystals also belong to the with unit cell dimensions a = 47.4 A, b = 82.8 A, orthorhombic space group P2,2,21, c = 92.4 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 1.8 A. Keywords:

neuraminidase;

sialidase; cholera;

Neuraminidase (NA$) cleaves terminal sialic acid residues from glycoproteins, glycolipids and oligosaccharides. Neuraminidase can be found as one of t Author to whom all correspondence should addressed. $ Abbreviations used: NA, neuraminidase; MUNeuNAc, 2’-(4-methylumbelliferyl)-a-n-A’-acteylneuraminic acid; PEG, polyethylene glycol. 0022%2836/92/161287-04

$0%00/O

be

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Salmonella

two glycoproteins on the surface of influenza viruses, the other being hemagglutinin. The role of neuraminidase is thought to be in releasing progeny virus particles from infected cells as well as being involved in the breakdown of the mucosal lining of the upper respiratory tract during the initial infection. The structures of several subtypes of influenza neuraminidase are known (Varghese & Colman, 1991; Tulip et al., 1991; Burmeister et aZ., 1992). 6

1992 Academic

Press

Limited

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G. Taylor

Neuraminidase is also produced by a variety of pathogenic microorganisms, such as Clostridium perfringens, Corynebacterium diptheriae, Vibrio cholerae, Bacteroidacae, Streptococci, Salmonella typhimurium and non-pathogenic bacteria of Arthrobacter species. The tertiary structures of the bacterial neuraminidases are unknown, and there is little sequence homology between the viral and bacterial enzymes. Neuraminidase is also expressed by Trypanosoma eruzi on its surface during certain developmental stages, where it can chemically modify, by desialylation, the surfaces of myocardial and vascular endothelial cells (Pereira et al., 1991). Mammalian neuraminidases have not been characterized to any great extent, their roles appear to be in the regulation of cell proliferation, clearance of plasma proteins and the catabolism of gangliosides and glycoproteins. There is some variation in the reported molecular weights of bacterial neuraminidases, from gel filtration, with values ranging from 23,000 to 125,000 Da. There is evidence for two families dependent on a requirement of a divalent metal ion for optimal activity. Those which do not require metal have molecular weight values of around 42,000 Da: C. perfingens, C. sordelli, S. typhimurium and Micromonospora viridifaciens. These represent members of the “small” bacterial NA family with around 400 amino acid residues. There is also a significant sequence homology between these enzymes and the N-terminal domain of the membrane-bound NA of T. cruzi, where a 332 amino acid residue stretch has 30% identity with C. perfringens NA (Pereira et al., 1991). It is possible that the “small” NBS share the same threedimensional structure (Hoyer et al., 1992). Of those requiring a metal ion, NA from V. cholerae is reported to have a molecular weight of 90,000 Da, and belongs to the “large” family. The sequence shows no extensive homology with viral or “small” bacterial NA sequences (Galen et al., 1992). There is evidence for a conserved sequence motif (XXSXDXGXTWXX) within the bacterial NAs, repeated four times along the sequence (Roggentin et al., 1989). This is also found in the N-terminal domain of T. cruzi (Pereira et al., 1991; Kahn et al., 1991). It has been suggested that the bacterial and viral NAs share the same fold, based on this observed consensus (but only seen in 2 influenza A virus subtype NA sequences), predicted secondary structure, substrate specificity and size (Hoyer et 1992). However, photolabelling of the al., S. typhimurium enzyme with an azido analogue of acid, revealed 2,3-dehydro-N-acetylneuraminic labelling of a C-terminal peptide with remarkable secondary structural similarity to influenza A and Sendai virus neuraminidase (Warner et al., 1992). Proof of a sialidase superfamily awaits the structure determination of bacterial enzymes from both families. As part of a comparative study of viral, bacterial and mammalian neuraminidases, we have reported a preliminary X-ray analysis of a representative

et al. “small” NA from M. viridifaciens (Taylor et al., 1992). Here we report the purification and a preliminary X-ray analysis of a “large” NA from V. cholerae and the “small” enzyme from S. typhimurium LT2. Neuraminidase from V. cholerae plays a subtle, but significant role in t’he binding and uptake of cholera toxin by susceptible cells (Galen et al., 1992), while the enzyme from S. typhimurium appears to be unrelated to pathogenicity and may play a nutritional role (Hover et al., 1992). Thus, if the bacterial neuraminidases have any role in pathogenesis, it may be as an indirect consequence of the role of these enzymes in nutrition. since many bacteria have specific permeases for sialic acid uptake (Vimr & Troy, 1985: Hoyer et al.. 1992). (a) Purification The V. cholerae NA is predicted to be synthesized as an 85.6 kDa precursor polypeptide (Galen et al.. 1992) that is secreted into the periplasm after cleavage of a 24residue leader peptide (Vimr et al., 1988). The mature enzyme has a predicted size of 83.0 kDa (Galen et al., 1992) and is rapidly excreted into the medium by V. cholerae but is retained in the periplasm when nanH is expressed in Escheriehia coli (Vimr et al., 1988). This phenotype of cloned nanH suggested a simple purification scheme, since at least 97% of the total activity was recovered in the osmotic shock fluid (E. Vimr, unpublished results). Briefly, osmotic shock fluid was prepared from E. coli transformed wit,h pCVD364, as described (Vimr et al., 1988). The shock fluid was concentrated by precipitation with 50% (w/v) ammonium sulphate. The pellet was dissolved in a few millilitres of 10 miw-Tris (pH 7.6) and dialysed exhaustively against the same buffer containing 100 mM-NaCl. Undissolved material was removed by centrifugation and the clean liquid was concentrated to 0.5 ml using a 30 kDa cut-off microtype filtration unit (Millipore). The sample was loaded onto a 1.5 cmx60 cm column of Sephacryl-300 and fractionated by gravity with a flow rate of 0.25 ml min-I. Active fractions were detected by spotting 10 ~1 dilutions in Vibrio buffer. with 1 ~1 of 4 mM-2’-(4-methylumbelliferyl)-a-n-Nacetylneuraminic acid (MUNeuNAc) and observing activity with a hand-held ultraviolet light source, as described (Vimr et aZ., 1988). Sialidase eluted as a single, symmetrical peak at V,/ V, = 648. Similar results were obtained by fast protein liquid column. chromatography on a Superose-12 Approximately 1 mg of enzyme was recovered per litre of cultured cells, with a specific activity of 23 IU mg-. ’ measured against MUNeuNAc as substrate. Although a few contaminating bands were still evident in the preparation (Fig. 1, lane B), none was greater than 5% of the neuraminidase band. S. typhimurium LT2 neuraminidase was purified from E. coli harbouring pSX62 (Hoyer et al., 1992), essentially as described (Hoyer et al., 1991) but with

Crystallization

Figure 1. Electrophoretic profiles of bacterial neuraminidases used for crystallizations. Approximately 5 pg of V. cholerae (lane B) and purified recombinant S. typhimurium LT2 (lane C) neuraminidases were fractionated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, 12.5% separating gel, and visualized by staining with Coomassie brilliant blue. Electrophoresis conditions and molecular weight markers, numbered in kDa on the left (lane A), were as described by Hoyer et al. (1991).

the following modifications. Cells were disrupted by in 10 mM-Tris (pH 7.6) containing sonication 100 mM-NaCj and then dialysed exhaustively against the same buffer, with no salt. Next, instead of batch extraction with DEAE-Sephacel (Hoyer et al., 1991), the dialysed sample was chromatographed on a 1 cmx15cm column of DEAE-Sephacel or Q-Sepharose FF, equilibrated with the same buffer. Neuraminidase eluted in the flow-through volume, which was then dialysed against 10 mM-sodium exhaustively acetate (pH 4.8). The sample was fractionated by S-Sepharose FF chromatography, as described 4 mg of neura(Hoyer et al., 1991). Approximately minidase was recovered per litre of starting cells. The enzyme purified by this modified procedure was substantially free of the previously described contaminant at 29 kDa (Fig. 1, lane C). (b) Crystallization The largest and best diffracting crystals of V. chderae neuraminidase were obtained at a protein concentration of about 15 mg ml-’ in with NaN3 as a preserva615 M-NaCl, 001 M-call, tive, and mixed with an equal volume of 10% (w/v)

Notes

1289

PEG 3350 in 904 M-Nacl. Crystals were grown in hanging drops by vapour diffusion over a reservoir of 20% PEG 3350 in @15 M-NaCl. Crystals grew as long rods up to 1.5 mm x 92 mm x 92 mm. Previous trials had shown a variation in cell dimensions, particularly the c-axis, with variation in PEG concentration. Calcium is essential for well-ordered crystals, as it is for enzyme activity, suggesting that it may play a structural role as in the viral enzyme (Burmeister et al., 1992). Diffraction data were collected on a Siemens area detector mounted on a Siemens rotating anode X-ray source operating at 40 kV and 80 mA. Frames of data were recorded while the crystal was oscillated through 625” steps. The intensities were integrated using the XDS program, which was also used for the initial derivation of the unit cell (Kabsch, 1988a,b). The crystals belong to space group P2,2,2,, as judged by systematically absent or weak (F < 2-O 0) axial reflections. The unit cell parameters are a = 71.86 A, b = 79.02 A, e = 16569 A weight of 90 kDa (1 A = 61 nm). With a molecular for the monomer, a VM value of 2.61 A3/Da is obtained, consistent with a monomer in the asymmetric unit and within the reported range for protein crystals (Matthews, 1968). S. typhimurium neuraminidase, at a concentration of about 10 to 15 mg ml-’ in 915 iv-NaCl, with NaN, as a preservative, was mixed with an equal volume potassium phosphate (1.4 M-KH,PO$3 M-K,HPO,, in the volume ratio 2 : 1) and equalibrated as hanging drops over a reservoir potassium phosphate (1.4 M-KH,PO,$ M-K,HPO,, in the volume ratio 4 : 3). Crystals grew as prisms up to 0.5 mm in each dimension. Diffraction data were collected and processed as described above. The crystals belong to space group P2,2,2,, as judged by systematically absent or weak (F < 2.0 0) axial reflections. The unit cell parameters are a = 47.42 A, b = 82.78 8, c = 92.42 A. With a molecular weight of 42 kDa for the monomer, a Vv, value of 2.16 A3/Da is obtained, consistent with a monomer in the asymmetric unit. A search for suitable heavy-atom derivatives is in progress. E.R.V. was supported by NIH grant AI23039 from the Institute of Allergy and Infectious Diseases.

References Burmeister, W. P., Ruigrok, R. W. H. t Cusack, S. (1992). The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid. EMBO J. 11, 49-56. Galen, J. E., Ketley, J. M., Fasano, A., Richardson, S. H., Wasserman, S. S. & Kaper, J. B. (1992). Role of Vibrio cholerae neuraminidase in the function of cholera toxin. Infect. Immun. 60, 406-415. Hoyer, L. L., Roggentin, P., Schauer, R. & Vimr, E. R. (1991). Purification and properties of cloned Salmonella typhimurium LT2 sialidase with virus-

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by A. Klug