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Protein Sci. Sep 2005; 14(9): 2236–2245.
PMCID: PMC2253470

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

Abstract

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.

Keywords: Mycobacterium tuberculosis, high activity binding peptides, receptor–ligand interaction, Mycobacterium tuberculosis complex

Mycobacterium tuberculosis is a facultative intracellular mycobacterium phagocyted by monocytes and macrophages. This mycobacterium is able to multiply within certain phagosomes which do not fuse with lysosomes. Phagocytosis represents the critical first step in the M. tuberculosis–phagocyte interaction, involving microbial ligands and phagocyte surface receptors (Stokes and Speert 1995; Fenton and Vermeulen 1996; Frattazzi et al. 2000). M. tuberculosis bacilli are thought to enter the macrophage via specific binding to several distinct cell surface molecules; the precise route of pathogen entry is likely to determine the ultimate fate of bacilli within the macrophage (Fenton and Vermeulen 1996). Phagocytosis of two virulent strains (Erdman and H37Rv) and an attenuated H37Ra strain is mediated by complement protein C3 activation products on bacterial surface and complement receptors (Schlesinger 1993; Schlesinger et al. 1994; Fenton and Vermeulen 1996). M. tuberculosis also binds nonopsonically to the mannose receptor, presumably via surface-expressed mannosylated glyco-conjugates that may include lipoarabinomannan (Stokes and Speert 1995; Schlesinger et al. 1996; Kang and Schlesinger 1998).

M. tuberculosis binding to human alveolar macrophages is also significantly enhanced by opsonization with surface protein (McDonough and Kress 1995; Bermudez and Goodman 1996; Ferguson et al. 1999). The major membrane protein from M. tuberculosis virulent strain peripheral membrane was reported in 1992; this 19-kDa protein contains antigenic determinants recognized by sera from a majority of patients having pulmonary tuberculosis (Lee et al. 1992).

Mycobacterium habana provides resistance against M. tuberculosis infection in mice; protective antigens are distributed between both peripheral and integral membrane compartments (Chaturvedi et al. 1999).

A large number of proteins have been reported following the deciphering of the M. tuberculosis genome (Cole et al. 1998); they have been classified as being potential membrane surface proteins (http://www.sanger.ac.uk/Projects/M_tuberculosis/Gene_list/CDS/Rv2536.shtml) but whose role still remains unknown. As we thus wanted to understand the possible role of some of these proteins, we first carried out a preliminary screening by selecting just those whose encoding genes were exclusive to the M. tuberculosis complex and not present in atypical mycobacteria. After that screening, we thus checked which genes were being transcribed in M. tuberculosis complex strains, especially H37Rv and H37Ra strains. PCR has revealed Rv2536 to be a gene belonging exclusively to the tuberculosis complex; it has also been seen to be present in different isolates from tuberculosis patients but absent in atypical mycobacteria. We have also found that the gene is transcribed in some M. tuberculosis complex strains, such as M. H37Rv, M. H37Ra, M. bovis BCG, and M. africanum. Rabbits immunized with Rv2536 protein polymeric peptides have produced antibodies recognizing a 25-kDa protein present in Mycobacterium tuberculosis lysate and membrane fraction. Binding assays were also done for identifying Rv 2536 protein binding sequences regarding binding to human cell-lines susceptible to M. tuberculosis infection. Two U937 cell high activity binding peptides (U937-HABPs) were identified in the present study: peptides 11206 (141AKAPVRHHGLAAEHERAADTY160) and 11207 (161DVFSAVRADDSPTGEMQVAQY180). A549 cell high activity binding peptides (A549-HABPs) 11207 and 11208 (181PEAQTAAVATVEREAPTEVIY 200) were also identified. Our results suggested that HABP 11207 interacted with A549 and U937 cell membrane proteins.

Results

Rv2536 protein has been classified as being a potential membrane protein (http://www.sanger.ac.uk/Projects/M_tuberculosis/Gene_list/CDS/Rv2536.shtml); its sequence contains 230 amino acids and it has a molecular weight of 24,627 kDa. PCR experiments with genomic mycobacterial DNA have shown the presence of Rv2536 gene in all Mycobacterium tuberculosis complex strains (Fig. 1A [triangle]) and M. tuberculosis clinical isolates (data not shown). This gene was not found in any of the 19 atypical Mycobacterium strains tested by these techniques (Fig. 1B [triangle]). As shown by the 354-bp gene product in the RTPCR assays, Rv2536 is being transcribed in M. tuberculosis H37Rv and H37Ra, M. bovis BCG, and M. africanum, but not in M. bovis or M. microtti (Fig. 1C [triangle]). Figure 1D [triangle] shows the positive control for mRNA transcription in all these strains (the rpoB gene). Rv2536 gene polymorphism within the amplified region was analyzed in the different mycobacterial strains by automatic DNA sequencing. No variation was observed, suggesting that the encoded protein is highly conserved (data not shown).

Figure 1.
(A) PCR assays. Rv2536 354 bp PCR product is shown in 1% agarose gel and amplified over mycobacterium genomic DNA. (Lane 1) 1 Kbase Molecular Weight Markers (Gibco), (lane 2) M. tuberculosis H37Rv, (lane 3) M. tuberculosis H37Ra, (lane 4) M. bovis, (lane ...

Just-adsorbed sera from rabbits (labeled 7, 31, and 43) immunized with polymer peptide 25664 (peptide 11209 monomer) recognized inoculated peptide in ELISA assays (data not shown). Antibody obtained from second and final bleeding (P-II and P-III) presented high recognition by the inoculum. Immunoassays were thus done with serum obtained from second and final bleeding (PII and P-III), adsorbed sera (P-II and P-III) from rabbits inoculated with polymer peptide 25664 specifically recognized a 25-kDa band in M. tuberculosis sonicate by Western blot assays (Fig. 2 [triangle]). Sera from rabbits immunized with peptides 25562 and 25568 did not present recognition of the inoculated peptide in ELISA assays, nor did it present recognition of any protein from M. tuberculosis in immunoblotting assays.

Figure 2.
Rabbits were immunized with polymer peptide 25564 (peptide 11209 analog). The three adsorbed serum (P-II and P-III) from rabbits (7, 31, and 43) immunized with polymer peptide recognized 25564, a band which was very close to 25 kDa when compared to M. ...

U937 and A549 cell binding assays using nonoverlapped 20-amino-acid-long peptides were done by incubating radiolabeled peptide (0–100 nM) with U937 or A549 cells in the presence (nonspecific binding) or absence (total binding) of the same nonradiolabeled (cold) peptide (Fig. 3A,C,E [triangle]). The foregoing allowed specific binding activity to be calculated; this was defined as being total binding minus nonspecific binding (Garcia et al. 2002, 2003, 2004; Puentes et al. 2004) (Fig. 3 B,D,F [triangle]). Binding assays allowed specific U937 cell binding activity curves to be obtained for the twelve peptides from the Rv2536 protein where three different types of behavior were observed: peptides presenting high specific binding activity, those presenting nonspecific binding activity, and peptides presenting no binding activity at all. Peptides 11206 (141AKAPVRHHGLAAEHERAADTY160) and 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific U937 cell binding activity (radiolabeled peptide binding was inhibited by the same unlabeled peptide) (Fig. 3A,B [triangle]), while peptide 11208 (181PEAQTAAVATVEREAPTEVIY200) presented high nonspecific U937 cell binding activity (this bound to cells but there was noninhibition with the same nonradiolabeled peptide) (Fig. 3C,D [triangle]). Peptides 11204 (Fig. 3E,F [triangle]), 11200, 11203, 11204, 11205, 11209, and 11210 did not present U937 cell binding activity (Fig. 4 [triangle]). Peptides 11199, 11201, and 11202 presented low specific U937 cell binding activity.

Figure 3.
Binding assays. Peptide U937 cell binding assays for peptides 11206 (A), 11208 (C), and 11204 (E) are shown. Cells incubated with different 125I-labeled peptide concentrations (total binding, •). Cells incubated with 125I-labeled peptide in the ...
Figure 4.
Peptides’ specific U937 and A549 cell binding activity. The number, the amino acid sequence, and the position in Rv2536 protein of each synthesized peptide used in the binding assays can be seen. The underlined Tyr residues (Y) are those which ...

A549 cell binding assays revealed that peptides 11207 and 11208 presented high specific binging to these cells while peptides 11202, 11204, 11206, 11209, and 11210 presented low specific A549 cell binding activity. Peptides which did not present specific binding activity to these cells were: 11199, 11200, 11201, 11203, and 11205 (Fig. 4 [triangle]). No peptides were found having nonspecific high activity binding to A549 cells.

CD spectra were taken for knowing the secondary structural elements of HABPs 11206, 11207, and 11208 (5 μMin 30% TFE-water). The results obtained revealed that HABP 11206, 11207, and 11208 circular dichroism curves were different. HABP 11206 presented a θλ value close to 0 at 190 nm, while HABPs 11207 and 11208 presented approximate θλ values of ~40 and 60, respectively, at 190 nm. HABP 11206 presented minimum troughs at 202 and 224 nm and HABPs 11207 and 11208 presented minimum troughs at 205 and 223 nm.

U937 cell saturation assays were only done for U937-HABPs 11206 and 11207 and A549 cell saturation assays were only done with A549-HABP 11207 (as peptide 11208 chemical synthesis presented low yield it was not possible to carry out A549 cell saturation assays). The curves obtained in saturation assays showed that U937-HABPs 11206 and 11207 had 2300 and 1600 nM affinity constants, respectively. Binding sites per cell calculated for these U937-HABPs were 650,000 and 640,000 binding sites, respectively (Fig. 5A,B [triangle]; Table 11).). A549 cell saturation assays with A549-HABP 11208 presented a 530-nM affinity constant and 150,000 binding sites per cell (Fig. 5C [triangle]; Table 11).). Hill coefficients were >1 in all cases (Table 11).

Table 1.
Affinity constants, Hill coefficients, and number of binding sites calculated for the receptor–ligand interaction between M. tuberculosis Rv2536 protein HABPs from human cell-lines
Figure 5.
Saturation assays. The saturation curves resulted from graphing the concentration of specifically bound 125I-HABP cf free 125I-HABP. Affinity constants and maximum number of sites per cell were obtained from these curves. The Hill plot is placed inside ...

HABPs 11206 and 11207 were used in cross-linking assays using both types of cells. The results from these assays showed that this peptide specifically bound to U937 and A549 cell membrane proteins. Peptide 11207 specifically bound to membrane proteins having a weight of ~50 kDa (U937 cells) and 45 kDa (A549 cells), calculated by SDS-PAGE (Fig. 6A,B [triangle], respectively). HABP 11206 presented the same behavior in U937 cell cross-linking assays as peptide 11207 (presenting specific recognition for the 50-kDa protein), while it presented no binding to any membrane protein in A549 cell cross-linking assays.

Figure 6.
Cross-linking assays. A549 or U937 cells were incubated with 125-I11207 in the presence and the absence of the same unlabeled peptide. Membranes were obtained by lysis and run in an acrylamide gel by SDS-PAGE. The figure shows total binding (lane 1) and ...

Discussion

The complete Mycobacterium tuberculosis genome sequence was reported in 1998 (Cole et al. 1998); this work describes a great number of putative membrane proteins whose function is still unknown. One such protein is Rv 2536, which has been classified as a potential membrane protein. We thus carried out PCR assays with mycobacteria belonging to the M. tuberculosis complex and 19 atypical mycobacteria to determine this gene’s presence or absence in M. tuberculosis and other mycobacteria. The results show that the fragment gene was only present in M. tuberculosis complex mycobacteria, suggesting that the encoded protein is exclusive to this complex (see Fig. 1A,B [triangle]). RT-PCR assays showed that this gene is differentially transcribed in M. tuberculosis H37Rv, H37Ra, M. bovis BCG, and M. africanum but is not transcribed in M. bovis or M. microti (Fig. 1C [triangle]). Mycobacterium tuberculosis proteins are characterized by being highly conserved; analyzing sequences from different clinical isolates has shown that this gene does not have any change in its 354 nucleotide sequence, indicating its high conservation (data not shown). The Rv2536 protein presented 100% homology with probable conserved transmembrane protein in M.bovis AF2122/ 97 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=31619312) and 63.2% identity with putative M. leprae Q9CCS2|ML0520 membrane protein (Cole et al. 2001). When comparing amino acid sequences from the putative M. leprae Q9CCS2|ML0520 membrane protein with those from M. tuberculosis Rv2536 protein it was found that all HABPs (11206, 11207, and 11208) were localized in regions presenting variability.

Knowing that the gene was exclusively present in the M. tuberculosis complex and transcribed, New Zealand white rabbits were immunized with polymeric peptides whose sequences belonged to protein Rv2536 and their anti-peptide sera were evaluated against mycobacterial sonicate (H3Rv strain, ATCC 27294) in a blot to find out whether Rv 2536 protein was expressed in M. tuberculosis H37Rv (ATCC 27294). The results showed that the adsorbed serum (P-II and P-III) from the three rabbits immunized with polymer peptide 25664 specifically recognized a 25-kDa band. The weight of the 25-kDa protein recognized by adsorbed serum (P-II and P-III) from rabbits immunized with polymeric peptides from Rv2536 protein agreed with the 24.6-kDa molecular weight reported for the Rv2536 protein (http://www.sanger.ac.uk/Projects/M_tuberculosis/Gene_list/CDS/Rv2536.shtml) (Cole et al. 1998) (Fig. 2 [triangle]), suggesting that this protein is expressed in the virulent M. tuberculosis strain.

Based on previous results and following our methodology (used extensively in prior experiments to recognize other HABPs in other microbes), we managed to identify different types of binding sequences to two different human cell-lines which are susceptible to M. tuberculosis infection. Our results show that two U-937HABPs (11206 and 11207) and two A549-HABPs (11207 and 11208) were found (Fig. 4 [triangle]). These three consecutive peptides were located in the C-terminal region, suggesting the presence of a binding region in the C-terminal extreme lying between residues A141 and I200 (Fig. 4 [triangle]). Peptide 11206 presented high U937 cell binding activity while the same peptide presented low specific A549 cell binding activity. Peptide 11208 bound with high specific activity to A549 cells while the same peptide had high nonspecific U937 cell binding activity. The foregoing suggests that these peptides’ binding activity just depended on specific receptor molecules present in each type of cell (Fig. 4 [triangle]). Peptide 11207 bound to both types of cell with high specific binding activity, suggesting that this was possibly due to the presence of the same receptor in both classes of cell (U937 and A549 cells) or that it bound to different receptors characteristic for each cell-line.

U937 cell binding assays using Rv2536 protein peptides revealed that 25% of those peptides assayed (11199, 11201, and 11202; 3/12) had low specific binding activity, while 50% of the peptides did not bind to these cells. A549 cell binding assays revealed that 33% of the peptides presented low specific binding activity, while 40% of them did not bind to A549 cells. The foregoing indicates that only 16% of the peptides covered by the protein bound with high specific activity in both cases, suggesting that this protein’s C-terminal extreme is involved in binding to human cell-lines.

The results of the CD assays for HABPs 11206, 11207, and 11208 suggested that HABP 11206 secondary structure was formed by α-helix and random coil structural elements. HABP 11207 and 11208 CD spectra indicated that these peptides presented a greater percentage of random coil structure, even though they possessed some α-helix structural elements (Fig. 7 [triangle]). HABPs 11207 and 11208 presented notable differences in their A549 and U937 cell binding activity, suggesting that such binding activity depends on amino acid sequence. Peptides’ secondary structural elements mainly depend on each particular peptide’s amino acid sequence; such structural elements might possibly be influencing peptides’ functional properties at a given moment.

Figure 7.
Dichroism assays. This figure shows graphs for the DCs of the HABPs 11206, 11207, and 11208 (TFE-H2O 30%); all peptides presented a curve characteristic of α-helix and random coil conformation.

The curves obtained from saturation assays involving U937-HABP and A549-HABP revealed that the two peptides’ binding was saturable, independent of the cell line used (Fig. 5 [triangle]). Peptides 11206 and 11207 bound with low affinity to U-937 cells, since they presented micro-molar affinity constants. However, these peptides presented high binding capacity, peptide 11206 (640,000 binding sites cell) being the one presenting the greatest U937 cell binding capacity. Saturation assays involving peptide 11207 binding to A549 cells revealed that this peptide bound with high affinity (Kd 530 nM) to A549 cells while binding with low affinity (Kd 1600 nM) to U937 cells. Hill coefficients calculated for the interaction between peptide 11207 and the cells were 1.3 (U937) and 3.0 (A549), suggesting that a simple receptor–ligand interaction was being presented in the first case and a receptor–ligand interaction with positive cooperativity in the second one. However, in spite of differences in affinity and Hill coefficient values, peptide 11207 presented similar binding capacity to both types of cells (Table 11).

Cross-linking assays revealed that peptide 11207 bound specifically to a 50-kDa membrane protein in U937 cells and to a 45-kDa membrane protein in A549 cells. Figure 6 [triangle] shows that the binding of 125I-11207 (lane 1) to both types of cells was inhibited by the presence of nonradiolabeled peptide 11207, indicating that the interaction was specific. These results suggest that peptide 11207 specifically bound to membrane proteins from U937 and A549 cells (Fig. 6 [triangle]). The specificity is evident from the results obtained from cross-linking assays using HABP 11206 where the same behavior was seen for HABPs 11206 and 11207 in U937 cell cross-linking assays, while HABP 11206 did not bind to any A549 cell membrane protein (data not shown).

The results of saturation and cross-linking assays done with peptide 11207 suggest that peptide 11207 could have different receptor–ligand interactions with each cell line. Peptide 11207 was possibly binding to different molecules in each type of cell-line and receptor–ligand interaction affinity may have been mainly due to the receptor’s intrinsic characteristics and not to its cell concentration. It was possibly binding to a common receptor present in both types of cell but the receptor–ligand interaction was being governed by the microenvironment surrounding the receptor on the cell surface. However, more studies are needed for completely defining the receptor or receptors involved in the receptor–ligand interaction between peptide 11207 and U937 and A549 cells.

Materials and methods

Mycobacterial strains

The ATCC and Trudeau Mycobacterial Collection (TMC) were the sources of the mycobacterial strains used, whose codes are the following: M. tuberculosis H37Rv (ATCC 27294), M. tuberculosis H37Ra (ATCC 25177), M. bovis (ATCC 19210), M. bovis BCG (ATCC 27291; Pasteur sub-strain), M. africanum (ATCC 25420), M. microti (Pasteur strain) (kindly donated by Dr. F. Portaels, Institute of Tropical Medicine, Belgium), M. flavescens (ATCC 14474), M. fortuitum (ATCC 6841), M. szulgai (ATCC 65799), M. peregrinum (ATCC 14467), M. phlei (ATCC 11758), M. scrofulaceum (ATCC 19981), M. avium (ATCC 25291), M. smegmatis (ATCC 14468), M. nonchromogenicum (ATCC 19530), M. simiae (TMC 1595), M. intracellulare (ATCC 13950), M. gastri (TMC 115754), M. kansasii (ATCC 12478), M. diernhoferi (NT 2301), M. gordonae (ATCC 14470), M. marinum (ATCC927), M. terrae (ATCC15755), M. chelonae subsp. chelonae (ATCC 35752), M. chelonae subsp. abscesus (ATCC 19977), M. xenopi (ATCC 35841), M. vaccae (ATCC 15483), M. xenopi (ATCC 35841), and 10 M. tuberculosis clinical isolates obtained from different specimens from patients attending the TB Program at either the San Juan de Dios Hospital or the Santa Clara Hospital, both in Bogotá . Such patients from different areas in Colombia had been diagnosed as being Tb cases by clinical, radiological, and laboratory parameters. All mycobacterial strains were grown for 5–15 d in Middlebrook 7H9 and Middlebrook 7H10 cultures.

Chromosomal DNA extraction

Chromosomal DNA was isolated using the method previously described by Maharias et al. (1996). DNA was precipitated with 0.6-volume 2-propanol. The pellet was washed with 70% ethanol and suspended in 1× TE (Parra et al. 1991; del Portillo et al. 1991).

PCR conditions and primers

PCR amplifications were done in a thermal-cycler (Perkin Elmer Gene Amp PCR system 9600) using 100 ng genomic DNA from different mycobacterial strains with 40 μL PCR master mix (50 mMKCl, 10 mM Tris-HCl [pH8.3], 1.5mM MgCl2, 0.1 mM each deoxynucleoside triphosphate, 0.4 mM each oligonucleotide primer, and 1.5 U Taq DNA polymerase,Promega). DNA was denatured for 5 min at 94°C. Reaction mixture was brought to the annealing temperature at 58°C with sense 5′-GGGGTA CCACTGCTCCCGCCATCT-3′; and anti-sense 5′-AAAACT GCAGTTGTCAAATGAGAGCCG-3′; primers for 15 sec. An extension cycle was carried out at 72°C for 15 sec and a further denaturing cycle, at 94°C for 30 sec. The amplifications were performed for 30 cycles, with a final 5-min extension cycle at 72°C.

RNA isolation

The different mycobacteria were harvested by centrifuging at 12,000g for 15 min at 4°C. Sodium azide (10 mM) was added to the culture just before harvesting. The supernatant was decanted rapidly, and the cell pellet was suspended in 2 mL of cold lysis buffer per each 200 mg wet weight of bacteria (Katoch and Cox 1986; Alland et al. 1998) and sonicated twice for 15 min. Two volumes of Trizol (Gibco Technologies) were then added and extraction was done according to manufacturer’s instructions. The pellet was suspended in 100 μL distilled water and stored in aliquots at −80°C.

Reverse transcription-PCR (RT-PCR)

Total RNA was quantified by spectrometer (Genquant, Pharmacia), treated with RNAse-free DNAse RQ1 at 37°C for 3 h, precipitated with isopropanol, washed with 70% ethanol and suspended in distilled water. M. tuberculosis H37Rv DNA was included as DNAse Q activity control (1U/μg DNA). Target RNA was reverse-transcribed in a single tube containing distilled water and 10 μg/mL random primers (GIBCO). This mixture was incubated for 10 min at 70°C. Next, 1X RT buffer (0.14MKCl, 8 mM MgCl2, 50 mM Tris-HCl [pH 8.1]), 10 mM dithiothreitol (DTT), 0.5 mM dNTPs, 40 U Human Placenta Ribonuclease Inhibitor (PRO-MEGA) were added on ice. 200 U M-MLV reverse transcriptase (GIBCO) was then added in a 30 μL final volume. This mixture was kept at 37°C for 1 h. The enzyme was finally denatured for 5 min at 95°C. PCR was carried out as described above. The rpoB gene was used as transcription positive control. RpoB-1 (forward) (5′-TCAAGGAGAAGC GCTACGA-3′) and RpoB-2 (reverse) (5′-GGATGTTGAT CAGGGTCTGC-3′) rpoB gene (Rv0667) primers were used. This gene, encoding RNA polymerase subunit B, is present in all mycobacterial strains (Lee et al. 2000); it is also one of the genes implicated in its metabolism. DNAse-Q treated M. tuberculosis H37Rv was used as cDNA synthesis negative control. Distilled water and M. tuberculosis H37Rv DNA were used as PCR negative and positive controls, respectively.

Cell culture

U937 and A549 cells (ATCC) were kept in culture using RPMI 1640–10% FCS at 37°C and 5% CO2. The U937 cells were collected and washed with PBS. The A549 cells were treated with 0.1% PBS-EDTA, collected and washed with PBS. The cells were collected during logarithmic growth phase and counted in Neubauer chamber and the cell viability was minimal 90%.

Peptides

Twelve 20-mer-long nonoverlapped peptides, covering the complete sequence of a putative membrane protein Rv2536 (MTCY159.20c; this sequence can be found at http://www.sanger.ac.uk/Projects/M_tuberculosis/Gene_list/CDS/Rv2536.shtml) (Cole et al. 1998), were synthesized by solid-phase multiple peptide system (t-Boc strategy) (Merrifield 1963). The peptides were lyophilized and purified by RP-HPLC and then characterized by MALDI-TOF mass spectrometry. One Tyr residue was added at the C-terminal to those peptides which did not contain it in their native sequences to enable 125I-radiolabeling (Garcia et al. 2002, 2003, 2004; Puentes et al. 2004). The polymeric peptides (for rabbit immunization) were obtained by using the following methodology: Monomer peptide sequences containing glycine–cysteine residues at the carboxy and N-terminals were synthesized by solid-phase methodology (Merrifield 1963). The synthesized peptide was lyophilized and dissolved in water, then oxidized at pH 7.5 by slowly passing oxygen through it until a negative reaction was achieved with Ellman reagent. The polymeric peptides were analyzed by Size Exclusion Chromatography (SEC).

Radiolabeling

Purified peptides (2 nmol) were radiolabeled with 5 μL Na125I (100 mci/mL, ICN) and 0.3 μmol chloramine-T in a 25 μL final volume for 15 min (Garcia et al. 2002, 2003, 2004; Puentes et al. 2004). The reaction was stopped with 0.3 μmol sodium metabisulphite (Yamamura et al. 1978). The radiolabeled peptides were passed through a Sephadex G-10 column; specific activity was between 40–50 μCi/nmol.

Binding assays

A549 or U937 cells (2 × 106 cells/μL) were incubated with different radiolabeled-peptide concentrations (0–100 nM) in the presence or absence of unlabeled peptide (40 μM) for 90 min at 4°C (Garcia et al. 2002, 2003, 2004; Puentes et al. 2004). An aliquot of this reaction mixture was passed through a 60:40 dioctylftalate-dibutylftalate cushion (1.015 g/mL), spinning at 13,200 rpm for 1.5 min, and cell-associated radioactivity was quantified. The binding assays were performed in triplicate (Fig. 1 [triangle]).

Saturation assays

The saturation binding assay was adapted from previously reported assays (Garcia et al. 2002, 2003, 2004; Puentes et al. 2004). Briefly, A549 or U937 cells (2 × 106 cells/μL) were incubated (90 min, 4°C) with radiolabeled HABPs (125I-HABP) using a broad range of concentrations (0–3000 nM) in the presence or absence of unlabeled peptide (40 μM). An aliquot of supernatant was taken after a 90-min incubation for quantifying the concentration of free 125I-HABP. A fraction of reaction mixture was then taken and passed through a 60:40 dioctylftalate-dibutylftalate cushion (1.015 g/mL), spinning at 13,200 rpm for 1.5 min. Cell-associated radioactivity was quantified. The saturation assays were performed in triplicate. Affinity constant values, Hill coefficients and the number of binding sites were determined using saturation curves (Garcia et al. 2002, 2003, 2004; Puentes et al. 2004).

Cross-linking assays

U937 or A549 cell cross-linking assays were done according to Lopez et al. (2003). Briefly, U937 or A549 cell binding assays were done with as 125I-HABP 11207, in the presence and absence of nonradiolabeled peptide, as previously described for the binding assays. Following incubation, the cells were washed three times with 3 mL HBS and centrifuged at 12,000 rpm for 5 min. The pellet was then treated with 500 μL of 25 μM BS3 (PIERCE), for 20 min at 4°C. The reaction was stopped with 40 nM Tris-HCl (pH 7.4) and washed again with HBS. Then cells were then treated with lysis buffer (Triton 1%, iodoacetamide 10 mM, SDS 5%, EDTA 100 mM, PMSF 10 mM). The obtained membrane proteins were solubilized in Laemmli buffer and separated in SDS-PAGE. Those proteins cross-linked with radiolabeled peptide were exposed on BioRad Imaging Screen K (BioRad Molecular Imager FX; BioRad Quantity One Quantitation Software) for 7 d and the apparent molecular weight was determined by using 6.4–198 kDa molecular weight markers (BIO-RAD).

Bacterial culture

H37Rv Mtb strain (ATCC 27291) was cultured in Middlebrook 7H10 agar for 20 d, harvested, and suspended in Middlebrook 7H9 broth and grown for 7 d. The suspension was briefly sonicated (20W for 5 sec), vortex agitated for 1 min, and then centrifuged for 1 min at 500g. The supernatant was removed and the concentration was adjusted to 107 bacteria/ mL by using a McFarland turbimetric standard. The bacterial suspension was stained with Ziehl-Neelsen stain and observed under light microscopy.

Mycobacterial sonicate

Ten grams (wet weight) of mycobacteria were suspended in 20 mL phosphate-buffered saline (PBS 1X) containing a final concentration of 1 mM PMSF, 1 mM EDTA, and 1 μg/mL leupeptin and 1 μg/mL Pepsatin A. Sonication was performed in a Branson Sonifier twice for 15 min with amplitude set at 4 (output) and 90% duty cycle. Sonicate was centrifuged at 650g for 20 min. The supernatant was then centrifuged at 36,000g for 45 min at 4°C to obtain the total sonicate in the new supernatant. A fraction of this total sonicate was later centrifuged at 150,000g for 2 h at 4°C so as to obtain the membrane protein fraction from the pellet. The protein concentration in different fractions was determined with bicinchoninic acid assay (Pierce) and stored in aliquots at −70°C until needed.

Rabbit immunization

Groups of three New Zealand strain rabbits (previously determined to be nonreactive to M. tuberculosis sonicate by Western blot) per peptide were injected on days 0, 20, and 40 with either 500 μg 25562 (CGKASPDPDRRQDLAMTWLLAGYGC) or 25568 (CGIYTGGPINELLTTFAAFTALIGC), or 25664 (CGRTTESDTPTEVIRTDTEADQGC) polymerized peptide (corresponding to monomer 11202, 11204, and 11209 peptides’ amino acid sequences, respectively) mixed with Freund’s Incomplete Adjuvant. Bleeding was carried out on days 0 (PI), 60 (P-II), and 90 (P-III), and sera were collected. Immunizations and bleeding were performed in accordance with Colombian Ministry of Public Health handing procedures for animals. The Rv2536 protein peptide sequences chosen for immunizing rabbits were obtained by using epitope prediction software (15 mers [15 aa] for MHC type II only) downloaded from http://www.syfpeithi.de. Peptides were evaluated with different DRβ1 alleles and those were chosen as follows: presenting greater score and not having a high specific binding sequence (due to previous work done in our laboratory, suggesting that these regions are poorly immunogenic or antigenic) (Espejo et al. 2001; Purmova et al. 2002).

Rabbit sera adsorption with E. coli and M. smegmatis sonicates

M. smegmatis proteins were obtained from the 5-d Middlebrook 7H9 broth culture, washed, suspended, sonicated for 10 min (as described above) and centrifuged for 10 min at 4500g. E. coli (DH5α) strain proteins were obtained from overnight culture in Luria Bertani medium and washed, suspended, and sonicated for 2 min at 4°C and 10min at 4500g. Both pellets were suspended in 0.1 M NaHCO3 (pH 8.3) (coupling buffer). The suspended lysates were collected and used individually for coupling to CNBr-activated Sepharose 4B (Pharmacia Biotech), according to manufacturer’s recommendations. Each rabbit serum (preimmune and immune) was preadsorbed with E. coli–Sepharose and M. smegmatis–Sepharose affinity columns to eliminate crossreactivity. Briefly, 5 mL of each serum were added to 4 mL lysate-Sepharose affinity column and left in a gentle rotating/ shaking mode for 20 min at room temperature. This procedure was done twice using a new lysate-Sepharose affinity column each time (Garcia et al. 2004).

SDS-PAGE and immunoblotting

Proteins from M. tuberculosis sonicate were separated in a discontinuous SDS-PAGE system, using a 10% to 20% (w/v) acrylamide gradient. A total of 500 μg/mL sonicate was loaded per gel and then transferred to nitrocellulose membrane (Hybond 203c, Pharmacia) using the semidry blotting technique (Kyhse-Andersen 1984). Commercial molecular mass markers (NEB) were used for calibration. The filters were incubated with a 1:100 dilution of the adsorbed sera obtained from rabbits immunized with peptides 25562, 25564, and 25564 in TBST (0.02 M Tris-HCl [pH 7.5], 0.05 M NaCl, 1% Tween 20) and 5% skim milk. Incubation for 1 h with 1:3,000 alkaline phosphatase conjugated anti-rabbit IgG antibody (ICN) was carried out after five TBST washes. The reaction was developed with NBT/BCIP (KPL).

Circular dichroism assays

CD spectra for each of the HABPs were recorded at 20°C on Jasco J-810 spectro-polarimeter at wavelengths ranging from 260 to 190 nm in 1.00-cm cells. Each spectrum was obtained (0.1 mM peptides in water or in aqueous 30% TFE solutions) from averaging three scans taken at 20 nm/min scan rate with 1-nm spectral bandwidth, corrected for baseline. The results were expressed as mean residue ellipticity [Θ], the units being degrees × cm2 × dmol−1 according to the [Θ]= Θλ/(100lcn) function, where Θλ is measured ellipticity, l is optical pathlength, c is peptide concentration, and n is the number of amino acid residues contained in the sequence.

Acknowledgments

This research project was supported by the Colombian President’s Office and the Colombian Ministry of Public Health. The collaboration of our chemistry and immunology sections is greatly appreciated, as is that of the whole staff at the Institute of Immunology. We especially thank Jason Garry for translating and correcting the manuscript.

Abbreviations

  • HABP, high activity binding peptide
  • BS3, bis-sulfosuccinimidyl suberate
  • HBS, hepes buffer saline
  • SEC, size exclusion chromatography

Notes

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051526305.

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