National Pharmaceutical Biotechnology Centre, BioResearch, Ireland1, and Department of Microbiology, Moyne Institute of Preventive Medicine2, Trinity College, Dublin 2, Ireland
Author for correspondence: Peter Owen. Tel: +353 1 6081188. Fax: +353 1 6799294. e-mail: powen{at}tcd.ie
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ABSTRACT |
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Keywords: Streptococcus equi subsp. equi, fibrinogen-binding protein, ligand-binding domain
Abbreviations: CD, circular dichroism; Fg, fibrinogen; FgBP, fibrinogen-binding protein; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight
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INTRODUCTION |
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Streptococcus equi subsp. equi, a group C streptococcus, is the causative agent of strangles, a highly contagious and debilitating upper respiratory tract disease of the family Equidae. The disease is initially characterized by nasal discharge and fever followed by swelling and abscess formation in local lymph nodes (Timoney, 1993 ). In a recent publication, we described the purification of a fibrinogen-binding protein (termed FgBP) from the cell wall of S. equi subsp. equi. FgBP was shown to have an apparent Mr 220000 when analysed by SDS-PAGE, reacted with convalescent horse serum and was protective in a small animal model against lethal S. equi challenge (Meehan et al., 1998a
). The sequence of the corresponding gene (fbp) was determined and was shown to encode a protein of 534 amino acids (Mr 58344) which possessed some structural and sequence similarities to other streptococcal cell wall proteins. Of note were characteristic signal sequence and cell wall/membrane-spanning domains, two blocks (A and B) of degenerate repeated sequences and a high probability of
-helical coiled-coil structure over about 60% of the molecule. The sequence was identical, with the exception of six amino acids, to the M-like protein (SeM) described by Timoney et al. (1997)
for a different isolate of S. equi subsp. equi. There is evidence to suggest that, like streptococcal M proteins, FgBP (SeM) is antiphagocytic by limiting C3b deposition on the bacterial cell surface, that antiserum to the protein is opsonic and that Fg binding may enhance the ability of S. equi subsp. equi to resist killing by equine neutrophils (Boschwitz & Timoney, 1994a
, b
; Timoney et al., 1997
).
Previously, we described the overexpression and purification of a recombinant FgBP truncate which lacked the signal sequence and C-terminal cell wall/membrane domain and showed that this recombinant protein behaved in an analogous fashion to wild-type FgBP in terms of Fg binding, seroreactivity and protective immunogenic potential (Meehan et al., 1998a , b
). In the present communication, we describe the construction of a panel of FgBP truncates, show that the Fg-binding domain is localized in the N-terminal region of mature FgBP, and that the efficiency of Fg binding is dependent on temperature and source of Fg. We also provide evidence from MS analysis of recombinant and wild-type FgBP that the native protein may be covalently linked at its C-terminus to the streptococcal cell wall.
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METHODS |
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Recombinant DNA techniques.
Genomic DNA was isolated from S. equi TW by a modification of the method of Yu & Ferretti (1989) as described previously (Meehan et al., 1998a
). Plasmid DNA was purified from E. coli by a modified alkaline lysis method (Feliciello & Chinali, 1993
). DNA restriction digestions, ligations and transformations were carried out by standard methods (Sambrook et al., 1989
).
Biochemical procedures.
SDS-PAGE was performed using either 12·5% (w/v) or 7·5% (w/v) polyacrylamide separating gels and a 4·5% (w/v) polyacrylamide stacking gel (Laemmli, 1970 ). Samples were routinely heated for 3 min at 100 °C in Laemmli sample buffer (Laemmli, 1970
) prior to electrophoresis. Proteins were detected by staining with Coomassie brilliant blue. Mr values were determined from the relative mobilities of 15 standard marker proteins (Gibco-BRL; BenchMark protein ladder).
Protein concentration was estimated by a modification (Dulley & Grieve, 1975 ) of the Lowry method using BSA as standard. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS was performed as follows by Dr Len C. Packman, Dept Biochemistry, Cambridge, UK. FgBP samples (0·5 µl) dissolved in dilute (1050 mM) sodium phosphate buffer (pH 7·2) or 0·1% (v/v) trifluoroacetic acid/50% (v/v) acetonitrile were mixed with 0·5 µl matrix solution [sinapinic acid (10 mg ml-1) in 50% (v/v) acetonitrile containing 0·1% (v/v) trifluoroacetic acid] and dried onto sample slides at 2530 °C in a fan-ventilated drying box. Samples were washed twice with 4 µl water to remove salts, then dried, and a further 0·5 µl matrix was added. As necessary to improve the signal, samples were rewashed and further matrix solution was applied. An empirical dilution series of the protein sample (550-fold) was tested to ascertain the sample conditions necessary to give the optimal mass spectral signal. Mass analysis was carried out on a Kratos MALDI 4 time-of-flight mass spectrometer following the manufacturers recommendations. Masses were determined using rabbit aldolase (Mr 39211) and horse myoglobin (Mr 16951) as internal calibrants. Errors were minimized by averaging at least five mass determinations.
Immunological and affinity procedures.
Western immunoblotting was performed as described previously (Caffrey et al., 1988 ) using 5% (w/v) dried skimmed milk as blocking reagent and rabbit anti-FgBP (1:10000 dilution; Meehan et al., 1998a
). Affinity-purified horseradish-peroxidase-labelled goat anti-rabbit IgG H+L (ICN) was used as localizing antibody and 4-chloro-1-naphthol as developing reagent. The procedure for Fg-affinity blotting after electrotransfer from SDS-PAGE was as described by Meehan et al. (1998a)
. For dot Fg-affinity blots, solutions containing FgBP truncates were subjected to doubling dilutions and 150 µl aliquots (containing 1000·05 pmol) were transferred onto nitrocellulose using the Bio-Rad Bio-Dot apparatus. After the blots had dried, they were blocked, incubated with labelled horse Fg and developed as described for Fg-affinity electroblots (Meehan et al., 1998a
).
ELISA tests were carried out using standard procedures (Newell et al., 1988 ). For analysis of FgBP binding to Fgs from different animal species, wells of microtitre plates were coated overnight with 50 µl Fg solutions, ranging in concentration from 10 to 0·0195 µg per ml 0·1 M sodium carbonate buffer (pH 9·6). Wells were blocked with 4% (w/v) dried skimmed milk, incubated with FgBP (1·25 µg ml-1), followed by rabbit anti-FgBP antibodies (1:5000 dilution) and peroxidase-labelled anti-rabbit IgG (1:5000 dilution; ICN). For analysis of Fg binding at different temperatures and to determine the concentration of ligand resulting in 50% saturation of receptor, wells were coated with 25 ng FgBP1, blocked with 1% (w/v) BSA and probed with doubling dilutions of horseradish-peroxidase-labelled horse/human Fg as appropriate. For these experiments, blocking was performed in two stages: (1) incubation at 22 °C for 30 min followed by (2) incubation for 30 min at one of several temperatures (4, 22, 30, 37 or 45 °C). Subsequent washing and incubation steps were carried out at the chosen temperature. All ELISA tests were developed at room temperature using 3,3',5,5'-tetramethylbenzidine as substrate and the A450 was measured.
Purification of FgBP.
FgBP was purified from S. equi as described previously (Meehan et al., 1998a ). Briefly, this involved lysis of S. equi cells by passage through a French pressure cell (221 MPa) and harvesting of cell envelopes by centrifugation (45000 g, 1 h, 4 °C). Cell envelopes were then digested overnight with mutanolysin (800 U ml-1) in the presence of a cocktail of protease inhibitors. Following centrifugation (45000 g, 1 h, 4 °C), proteins, including FgBP, which were released from the cell wall were recovered in the supernatant fraction. FgBP was then purified from this preparation by horse Fg-affinity chromatography (Meehan et al., 1998a
).
Expression and purification of FgBP truncates.
Oligonucleotide primers (Genosys) complementary to selected fbp sequence were used to amplify fbp fragments. Restriction enzyme cleavage sites (BamHI or XmaI) were engineered into the 5' end of all primers. A stop codon was also engineered directly after the restriction enzyme cleavage site at the 5' end of reverse primers. One hundred microlitre PCR reactions were performed in cloned pfu polymerase buffer (Stratagene) containing 5 U cloned pfu polymerase, 250 ng forward and reverse primers, 250 µM dNTPs and 100 ng pFP21 (recombinant pQE30 which expresses truncate FgBP1) or 500 ng S. equi genomic DNA. Amplification conditions consisted of 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 2 min/1 kb of product followed by a final extension at 72 °C for 10 min. DNA was then purified using Wizard PCR preps (Promega), cleaved with appropriate restriction enzymes and cloned into pQE30. Recombinant plasmids were transformed into E. coli XL-1 Blue.
Hexahistidyl-tagged FgBP truncates were purified by metal chelate affinity chromatography using a modification of the method described by Meehan et al. (1998a) . E. coli containing the recombinant pQE30 plasmids was grown, harvested and lysed by passage through a French pressure cell (221 MPa) as previously described. The soluble cytoplasmic/periplasmic fraction was applied to a nickel-iminodiacetic acid column, which was then washed extensively with binding buffer containing 40 mM imidazole until the A280 of the effluent reached zero. Hexahistidyl-tagged FgBP truncates were recovered using an 80400 mM gradient of imidazole in binding buffer. Most of the purified proteins were then extensively dialysed against several changes of 50 mM sodium phosphate (pH 7·2) containing decreasing concentrations (0·5, 0·2 and 0·1 M) of NaCl. Some of the smaller truncates tended to precipitate at lower NaCl concentrations. Accordingly, these were ultimately dialysed against either 0·2 M NaCl (FgBP68) or 0·5 M NaCl (FgBP9). Where necessary, purified proteins were concentrated by ultrafiltration using an Amicon PM10 filter.
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RESULTS |
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Wall association of FgBP
Several lines of evidence strongly support the view that FgBP is covalently linked to peptidoglycan in situ. Firstly, it can only be released from the purified cell walls following digestion of the latter with the muralytic enzyme mutanolysin. Secondly, it possesses an LPSTG cell-wall sorting sequence (Meehan et al., 1998a ). In staphylococci, there is formal evidence that such sequences are proteolytically cleaved and that a new amide bond is formed between Thr of the sorting sequence and the uncross-linked peptide crossbridge of peptidoglycan (Navarre & Schneewind, 1999
; Ton-That et al., 1997
). Finally and importantly, MALDI-TOF MS analysis of wild-type FgBP (purified following mutanolysin digestion) supports this general scenario. Thus the Mr of FgBP1 estimated by mass spectrometry (Mr 49540±22) was in excellent agreement with that predicted from consideration of sequence (49541). However, the estimated Mr of wild-type FgBP purified following mutanolysin digestion of the cell wall was determined to be 52440±90. This is at considerable variance with that predicted for the primary translation product after N-terminal processing of the signal sequence at N37 (Mr 54597; Meehan et al., 1998a
) or after additional C-terminal processing at T503 in the cell wall sorting motif (Mr 51467). On the other hand, it is consistent with a FgBP molecule (predicted Mr 52486) which had been processed at both its N- and C-termini (i.e. N37T503) and amide-linked at T503 to the dialanyl crossbridge of the putative murein subunit AAK(A)QAMurNAcGlcNAc (Schleifer & Kandler, 1972
). However, additional experimentation is required to formally prove this point.
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DISCUSSION |
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An N-terminal localization of Fg binding for FgBP is reminiscent of the reported binding of Fg to other streptococcal proteins such as the M proteins of serotypes 1, 3 and 5, and the FAI protein. However, sequence comparison of the relevant domains of these and other similar proteins has failed to reveal any regions of significant homology or recognized motif structures. It seems that Fg is capable of binding to some of the most variable regions of these proteins (kesson et al., 1994
; Kehoe, 1994
; Reichardt et al., 1997
; Talay et al., 1996
). In these respects, the mechanism of Fg binding to S. equi FgBP is clearly distinct from that displayed by the staphylococcal Fg-binding proteins ClfA, ClfB and Efb. These latter proteins have been shown to bind preferentially to one or more of the three Fg chains in a manner which probably involves Ca2+ ions and a cation-binding EF-hand-like motif (McDevitt et al., 1997
; Meehan et al., 1998a
; Ní Eidhin et al., 1998
; OConnell et al., 1998
; Palma et al., 1998
).
Of probable relevance to the mechanism of Fg binding are computer predictions of structure. These suggest that (a) residues 3745 at the extreme N-terminus of wild-type FgBP (and FgBP19) exist in a random coil structure, whilst the balance of the molecule is largely (over 84%) -helical; and (b) 60% of the mature protein (from residues 166 to 475 of the precursor protein) possesses an extremely high probability (95100%) of
-helical coiled-coil structure, with zero to very low probability of such structures in the first 110 residues (residues 37147; Meehan et al., 1998a
). Ongoing structural studies of key FgBP truncates by circular dichroism (CD) have confirmed many of these predictions. Thus FgBP1 shows almost 100%
-helical content by CD using the CONTIN secondary structure estimation procedure (Provencher & Glockner, 1981
), and shows a molar ellipticity ([
222]/[
208·6]=1·06) similar to that observed for other coiled coils (M. Meehan and others, unpublished data). Based on these observations and the ligand-binding profiles detailed above, it is tempting to envisage critical Fg-binding domain(s) located in the less structured non-coiled-coil N-terminal aspect of the FgBP dimer, with
-helical coiled-coil sequences to the flanking (C-terminal) side providing, in part, a stabilizing structure which anchors and places constraints on the relative positioning of the non-coiled-coil single strands. In this model, excessive C-terminal truncation might serve to alter the gross structure of the FgBP molecule, either prior to or following interaction with its macromolecular ligand, leading to loss of effective interaction with Fg. Alternatively, Fg might interact with both the N-terminal and coiled-coil aspects of FgBP. Indeed, the coiled-coil region apparently necessary for full Fg binding (approx. 27 heptads, or 52 helical turns) is substantially greater than the minimum length generally considered necessary for the formation and stabilization of coiled-coil structures (three to four heptads; Su et al., 1994
; Lau et al., 1984
). Perhaps, as has been suggested for other systems, the coiled-coil structure of FgBP interacts in zipper-like fashion with the coiled-coil structure of Fg (
kesson et al., 1994
; Kehoe, 1994
; Reichardt et al., 1997
). This might serve to facilitate/stabilize interactions at the distal N-terminal site (or vice versa) and enhance binding efficiency. Both A and B repeated sequences of S. equi FgBP are present in the region of predicted coiled-coil structure (see Fig. 1
). Of these, the degenerate B repeats are unlikely to be involved in binding since they are absent in truncate FgBP3, which displays wild-type Fg-binding efficiency. In contrast, the A repeats are clearly positioned within the region of the molecule required for full Fg binding. However, the A repeats are neither essential nor sufficient for Fg binding since they are absent in FgBP5, which still retains some, albeit weak, binding activity and are present in truncates (FgBP1316) which are devoid of detectable activity. Whatever the precise mechanisms involved, it would appear that sites at the immediate N-terminus of FgBP and an extended region within the coiled-coil section of the molecule are both required for optimum Fg-binding activity. Biophysical studies should allow more critical appraisal of the role of coiled-coil structure in binding.
The observation that FgBP binds ligand more efficiently at lower temperatures has also been noted for some class C M proteins of group A streptococci. This in turn has been correlated with the stability of the -helical coiled-coil structure and a tendency for these proteins to unfold to random coil monomers at higher temperatures (
kerström et al., 1992
; Nilson et al., 1995
; Cedervall et al., 1997
). A similar explanation seems likely for FgBP since ongoing CD analysis has shown FgBP1 to become progressively more unfolded in response to increased temperature over the range 880 °C and to be about 0, 16 and 30% unfolded at 8, 37 and 45 °C, respectively (M. Meehan and others, unpublished data).
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ACKNOWLEDGEMENTS |
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Received 18 November 1999;
revised 31 January 2000;
accepted 8 February 2000.