Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of a family 101 glycoside hydrolase from Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is a serious human pathogen that is responsible for a wide range of diseases including pneumonia, meningitis, septicemia, and otitis media. Full virulence of this bacterium is reliant on carbohydrate processing and metabolism as revealed by biochemical and genetic studies. One carbohydrate processing enzyme is a family 101 glycoside hydrolase (SpGH101) responsible for catalyzing the liberation of galactosyl β1,3-N-acetyl-D-galactosamine (Galβ1,3GalNAc) α-linked to serine or threonine residues from mucin-type glycoproteins. The 124 kDa catalytic module of this enzyme (SpGH101CM) was cloned and overproduced in Escherichia coli and purified. Crystals were obtained in spacegroup P2₁ and diffracted to a resolution of 2.0Å with unit cell dimensions of a = 81.86 b = 88.91 c = 88.77′, and β = 112.46°. The SpGH101CM also qualitatively displayed good activity on the synthetic substrate p-nitrophenyl-2-acetamido-2-deoxy-3-O-(β-D-galactopyranosyl)-α-D-galactopyranoside which is consistent with the classification of this enzyme as an endo-α-N-acetylgalactosaminidase.

1. Introduction

Pneumonia is an acute inflammatory illness of the lungs that is caused by a variety of bacteria and viruses as well as certain fungi and protozoans. Streptococcus pneumoniae is a Gram-positive encapsulated diplococcus that is a major causative agent of pneumonia. There are over 90 serotypes of this bacterium which are defined based on the variable composition of S. pneumoniae’s polysaccharide capsule. S. pneumoniae is a human commensal that colonizes the nasopharynx of approximately ~40% of individuals asymptomatically and has no environmental niche ( ▸). The innate and adaptive immune system typically prevents colonization from becoming disease but host-pathogen homeostasis can be altered leading to not only pneumonia but also meningitis, septicaemia, and otitis media ( ▸). Invasive S. pneumoniae infections cause more deaths than any other bacteria and are the fifth leading cause of death worldwide ( ▸). The importance of S. pneumoniae as a pathogen is driving studies of its virulence factors and other aspects of the host-pathogen interaction in the hopes that this will ultimately aid in developing new strategies to deal with infections caused by this bacterium.

Genome sequencing, signature-tagged mutagenesis, and other biochemical and genetic studies have revealed S. pneumoniae’s reliance on carbohydrate processing and metabolism for full virulence of the bacterium ( ▸; ▸; ▸; ▸). One component of S. pneumoniae’s extracellular, cell-wall attached armory of enzymes is an endo-α-N-acetylgalactosaminidase, which catalyzes the liberation of galactosyl β1,3-N-acetyl-D-galactosamine (Galβ1,3GalNAc) α-linked to serine or threonine residues from mucin-type glycoproteins. Though the identity of the gene encoding this protein has remained unreported in the literature, recent identification and characterization of proteins with very similar activity have suggested that the activity of S. pneumoniae is attributable to the hypothetical protein SP_0368 from S. pneumoniae TIGR4 ( ▸; ▸). This hypothetical protein is classified as a family 101 glycoside hydrolase along with other known endo-α-N-acetylgalactosaminidases (http://www.cazy.org/) ( ▸). Currently, no three-dimensional structure has been determined for a family 101 glycoside hydrolase. In this communication, we report the cloning, recombinant production, crystallization and preliminary X-ray diffraction data of a 124 kDa fragment of the S. pneumoniae hypothetical protein SP_0368 (here the catalytic module fragment is called SpGH101CM), which harbours endo-α-N-acetylgalactosaminidase activity.

2. Materials and methods

2.1. Cloning, production and purification of SpGH101

The gene fragment encoding the GH101 catalytic module was PCR amplified from Streptococcus pneumoniae TIGR4 genomic DNA (ATCC BAA-334D) using the following oligonucleotide primers 5′-GGC AGC CAT ATG GAA AAA GAA ACA GGT CCT G -3′ and 5′-GGA TCC CTC GAG TTA CAA CAT CTT ACC TG-3′. The PCR amplified gene fragment was obtained using standard PCR methods using Phusion High-Fidelity DNA Polymerase (New England Biolabs). The product was digested with NdeI and XhoI restriction endonucleases and ligated to like digested pET-28a (+) (Novagen) using standard cloning procedures. The resultant plasmid encodes the polypeptide, residues 317-1425 of the unprocessed sequence, preceded by MGSSHHHHHHSSGLVPRGSH, an N-terminal six-histidine tag followed by a thrombin protease cleavage site

The SpGH101 catalytic module was produced in four litre cultures of Escherichia coli BL21 Star (DE3) (Invitrogen) in Luria-Bertani media containing 50 μg ml⁻¹ kanamycin (Sigma). Cells were harvested using centrifugation and were resuspended in 30 mL 25% sucrose in 20 mM Tris HCl, pH 8.0. 10 mg of lysozyme was added to the resuspended cells and stirred for 10 min. 60 mL of 1% deoxycholate, 1% Triton X-100, 20mM Tris HCl, pH 7.5, 100 mM NaCl, was then added to the cells and stirred for an additional 10 min. Finally, 0.5 mg of DNase (Sigma) and 5 mM MgCl₂ was added to the lysed cells and allowed to spin for another 10 min. Cell debris was pelleted using centrifugation at 27 000 × g for 45 minutes. The polypeptide was purified from cell-free extract using immobilized metal affinity chromatography (IMAC). The supernatant was loaded onto a nickel resin (Sigma His-Select) and protein elution began with a step-wise gradient of imidazole. The purity of fractions was assessed using SDS-PAGE and those deemed to be greater than 95% pure were pooled The pooled polypeptides were concentrated and exchanged into 20 mM Tris HCl, pH 8.0, in a stirred ultra-filtration unit (Amicon) using a 10K molecular weight cut-off (MWCO) membrane (Filtron). Protein was further purified by size exclusion chromatography using Sephacryl S-200 (GE biosciences) in 20 mM Tris HCl, pH 8.0. The concentration of purified protein was determined by UV absorbance at 280 nm using a calculated molar extinction coefficient of 240 420 M⁻¹ cm⁻¹.

2.2. Crystallization and X-ray data collection

Prior to crystallization, SpGH101 catalytic module was concentrated to 15 mg ml⁻¹ in 20 mM Tris HCl, pH 8.0. Crystals were grown within one week by adding 1μL of 25% polyethylene glycol (PEG) 1500 (Hampton Research) to 1μL of protein using the hanging-drop vapour diffusion method at 19°C. Removal of the six-histidine tag was unnecessary for crystallization. Crystals were cryoprotected in 1μL of 33% PEG 1500 supplemented with 6% MPD (Hampton Research) and flash cooled directly in a nitrogen gas stream at 113 K. Diffraction experiments were performed on a ‘home-beam’ Micromax 002 X-ray source, Osmic Blue Optics, Oxford Cryo 700 System, and R-Axis IV++ area detector. 410 images were collected every 0.5° with an exposure time of 2 min and d*TREK was used for data processing ( ▸).

3. Results and discussion

SpGH101 is a large and multi-modular protein, as is common for glycoside hydrolases, comprising 1767 amino acids, three definable domains or modules sandwiched by an N-terminal secretion signal peptide and a C-terminal LPXTG cell-wall attachment motif. The first module following the signal peptide comprises 278 amino acids and is of unknown identity. The following 317-1425 amino acids comprise the catalytic domain of SpGH101, here called SpGH101CM, and neighboring the catalytic module is a carbohydrate-binding module. In an effort to characterize the structure of this S. pneumoniae protein we cloned the gene fragment that we predicted to contain the catalytic module (SpGH101CM), recombinantly produced the 1109 amino acid 124 kDa polypeptide in E. coli, and purified it in high yields near 30 mg/L of culture. The resulting polypeptide qualitatively displayed good activity on the synthetic substrate p-nitrophenyl-2-acetamido-2-deoxy-3-O-(β-D-galactopyranosyl)-α-D-galactopyranoside (Toronto Research Chemical Inc.). This was consistent with SpGH101’s classification as an endo-α-N-acetylgalactosaminidase and strongly suggests that this hypothetical protein is indeed the same previously characterized endo-α-N-acetylgalactosaminidase from S. pneumoniae that is available in commercial preparations and has been referred to as EngSP ( ▸; ▸).

Diffraction quality crystals of SpGH101CM were grown within 1–2 weeks of setting up the crystallization experiment ( ▸). The crystals diffracted to a resolution of 2.0 Å and were in the spacegroup P2₁ with unit cell dimensions of a = 81.86 b = 88.91 c = 88.77′, and β = 112.46° ( ▸). Analysis of the asymmetric unit contents indicated that it contains only one 124 kDa SpGH101CM molecule with a predicted solvent content of ~49%. Native Patterson and self-rotation function analyses did not reveal any peaks above background, consistent with their being a single molecule of SpGH101CM in the asymmetric unit.

Figure 1.

Crystals of GH101 catalytic module from S. pneumoniae TIGR4 grown in 25% polyethylene glycol 1500.

Table 1.

Data-collection statistics

Values in parentheses are for the highest resolution bin
Wavelength ( )	1.5418
Spacegroup	P21
Unit cell dimensions (′,°)	a = 81.86 b = 88.91 c = 88.77, β = 112.46
Resolution range (′)	19.98 – 2.00 (2.07 – 2.00)
Total number of reflections	300953
Number of unique reflections	76040
Average redundancy	3.96 (3.88)
% completeness	95.8 (93.1)
Rmerge†	0.113 (0.398)
Reduced chisquared	0.98 (1.08)
(I/sigI)	7.9 (3.1)

^†R_merge = Σ|I_obs − I_ave|/ΣI_ave.

Structural studies of family 101 glycoside hydrolases have been lacking, likely due to their generally very large size, which makes them recalcitrant to crystallization. We have dissected SpGH101 into a smaller fragment that retains catalytic activity and readily crystallizes. These crystals are of sufficient quality to enable the determination of a high resolution crystal structure of this protein. Determining the 3-dimensional structure of this enzyme will not only help determine the structure and catalytic mechanism of this particular enzyme but will also give considerable insight into this uncharacterized family of glycoside hydrolases.

Note added in revision: during the review of this manuscript ▸ published the 2.9 Å resolution crystal structure of a protein that comprised residues 4 – 1567 of SpGH101 from S. pneumoniae R6. Our crystals are of a different form, higher quality, and diffract to substantially higher resolution, and will thus be of utility in studying the structural basis of substrate and inhibitor recognition by this protein. Structure solution by molecular replacement is ongoing.