HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pethe, K. Articles by Menozzi, F. D. Search for Related Content PubMed PubMed Citation Articles by Pethe, K. Articles by Menozzi, F. D. Social Bookmarking                      What's this?

J Biol Chem, Vol. 275, Issue 19, 14273-14280, May 12, 2000

Characterization of the Heparin-binding Site of the Mycobacterial Heparin-binding Hemagglutinin Adhesin^*

Kevin Pethe^§, Marc Aumercier^¶, Emmanuelle Fort, Christophe Gatot, Camille Locht^**, and Franco D. Menozzi

From the  INSERM U447, Mécanismes Moléculaires de la Pathogénie Microbienne, Institut Pasteur de Lille, Institut de Biologie de Lille, 1 rue A. Calmette, 59019 Lille Cedex, France, ^¶ CNRS, Mécanismes du Développement et de la Cancérisation, UMR 8526, Institut de Biologie de Lille, 59019 Lille Cedex, France, and the  Computer Cell Culture Center s.a., 14 rue de la Marlette, 7140 Seneffe, Belgium

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains several lysine-rich repeats at its carboxyl-terminal end. Using truncated recombinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these repeats are responsible for heparin binding. Immunofluorescence microscopy studies revealed that their deletion abrogates binding of HBHA to human pneumocytes. Conversely, when fused to MBP, the HBHA repeats confer pneumocyte adherence properties to the hybrid protein. Treatment of pneumocytes with glycosaminoglycan-degrading enzymes showed that HBHA binding depends on the presence of heparan sulfate chains on the cell surface. The epitope of a monoclonal antibody that inhibits mycobacterial adherence to epithelial cells was mapped within the lysine-rich repeats, confirming their involvement in mycobacterial adherence to epithelial cells. Surface plasmon resonance analyses showed that recombinant HBHA binds to immobilized heparin with fast association kinetics (k_a = 5.62 (± 0.10) × 10⁵ M¹ s¹), whereas the dissociation kinetics were slower (k_d = 0.015 (± 0.002) s¹), yielding a K_D value of 26 nM. Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain the entire heparin-binding site of HBHA. The molecular characterization of the interactions of HBHA with epithelial glycosaminoglycans should help to better understand mycobacterial adherence within the lungs and may ultimately lead to new approaches for therapy or immunoprophylaxis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mycobacteria are among the most successful pathogenic microorganisms for humans. Mycobacterium tuberculosis infects over one-third of the population in the world, causing annually 10 million new cases of active tuberculosis and 3 million deaths (1, 2). Leprosy, caused by Mycobacterium leprae, remains a major health problem in developing countries (3), whereas members of the Mycobacterium avium-intracellulare complex are among the most frequent opportunistic pathogens infecting patients suffering from acquired immunodeficiency syndrome (4).

Despite the importance of mycobacterial diseases, their molecular mechanisms are still poorly understood. One of the initial and crucial events in any infectious process is the adherence of the microorganism to its target tissues (5). Mycobacteria exhibit a tropism for the lungs, and interactions of the tubercle bacillus with alveolar macrophages have been extensively documented (6-8). However, mycobacterial adhesins interacting with respiratory epithelial cells or extracellular matrix may also play a role in the infection, because these are the first host tissues encountered by mycobacteria when they are transmitted by aerosol. As described for other pathogenic bacteria (9, 10), viruses (11, 12), and parasites (13, 14), M. tuberculosis produces on its surface a heparin-binding protein involved in adherence to epithelial cells (15). Because of its capability to agglutinate rabbit erythrocytes, this adhesin was called heparin-binding hemagglutinin (HBHA).¹ HBHA is a 199-residue glycoprotein that also induces bacterial autoaggregation (15-17). Recombinant HBHA (rHBHA) produced by Escherichia coli is not glycosylated and, in contrast to the native protein, is highly sensitive to proteolytic degradation affecting essentially its carboxyl-terminal end. This part of the protein contains two different lysine-rich repeated motifs (16), named R1 and R2. R1 (KKAAPA) is directly repeated thrice between residues 160 and 177, whereas R2 (KKAAAKK) is repeated twice between amino acids 178 and 194. In this report, we show that these repeats constitute the high affinity heparin-binding site of HBHA and that they are responsible for the binding of HBHA to heparan sulfate glycosaminoglycans (GAGs) present on the surface of human pneumocytes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains, Growth Conditions, and DNA Manipulations-- Growth conditions for Mycobacterium bovis BCG (strain 1173P2; World Health Organization, Stockholm, Sweden) have been previously described (15). E. coli XL1-Blue (New England Biolabs, Beverly, MA), E. coli BL21(DE3) (Novagen, Madison, WI), and E. coli B834 (18) were grown in LB medium (19) supplemented with 100 µg/ml ampicillin or 30 µg/ml kanamycin when appropriate. Restriction enzymes, T4 DNA ligase, and other molecular biology reagents were purchased from New England Biolabs, Roche Molecular Biochemicals, or Promega (Madison, WI). Polymerase chain reactions (PCRs) were performed using a Perkin-Elmer thermal cycler model 480 (Perkin-Elmer) with 100 ng of purified plasmid DNA and 1 µg of each primer (Table I). All the PCR fragments were sequenced using an ABI PRISM Dye Terminator Cycle Sequencing kit and an ABI PRISM 377 sequencer (Perkin-Elmer). Plasmids were purified on Nucleobond AX 100 cartridges (Macherey-Nagel, Düren, Germany) according to the instructions of the manufacturer.

Plasmid Construction-- To produce rHBHA under the control of the T7 promoter, the 643-base pair NcoI-HindIII fragment of pKK-HBHA (16) was introduced into NcoI/HindIII-restricted pET-24d(+) (Novagen), and the resulting plasmid, pET-HBHA, was used to transform E. coli BL21(DE3). The gene coding for a truncated HBHA (rHBHAC) lacking amino acids 161-199 was generated by PCR using pKK-HBHA as template and the oligonucleotides HBHA (5') and HBHAC as primers (Table I). The 492-base pair PCR fragment was digested with NcoI and SacI and ligated into NcoI/SacI-restricted pKK388-1 (CLONTECH, Palo Alto, CA) and pET24d(+) to generate pKK-HBHAC and pET-HBHAC, respectively. pKK-HBHAC was used to transform E. coli XL1-Blue, and pET-HBHAC was introduced into E. coli BL21(DE3).

Genes coding for HBHA with one (rHBHAC+1R1), two (rHBHAC+2R1), or three (rHBHAC+3R1) R1 motifs were generated by PCR using pKK-HBHA as template and oligonucleotides HBHA (5') with HBHAC+1R1 or HBHAC+2/3R1 as primers. After purification and restriction by NcoI and SacI, the PCR fragments were inserted into NcoI/SacI-restricted pET-24d(+), generating pET-HBHAC+1R1, pET-HBHAC+2R1, and pET-HBHAC+3R1, respectively. These plasmids were used to transform E. coli BL21(DE3). Genes encoding maltose-binding protein (MBP) containing various combinations of HBHA repeats (MBP+3R1+2R2 and MBP+1R1+2R2) were generated as follows. First, the 3'-region of the HBHA gene coding for the repeats was amplified by PCR using pKK-HBHA and oligonucleotides E4 (5') and E4 (3'). Oligonucleotide E4 (5') has three hybridization sites on the template DNA. However, the PCR generated only two products of 222 base pairs and 186 base pairs that correspond to amino acids 161-199 (3R1+2R2) and amino acids 173-199 (1R1+2R2), respectively. After digestion with EcoRI and PstI, the PCR fragments were ligated into EcoRI/PstI-digested pMAL-c (New England Biolabs) to generate pMAL+3R1+2R2 and pMAL+1R1+2R2. These plasmids were then used to transform E. coli B834 and E. coli XL1-Blue.

Production of Recombinant Proteins in E. coli-- Recombinant HBHA with or without the carboxyl-terminal truncations, as well as MBP, MBP+3R1+2R2, and MBP+1R1+2R2 (Table II) were produced in E. coli. After transformation with the appropriate recombinant plasmid, E. coli cells were grown at 37 °C in 250 ml of LB broth supplemented with ampicillin or kanamycin. At an A₆₀₀ of 0.5, isopropylthiogalactoside was added to a final concentration of 1 mM, and incubations were continued for 4 h. The cultures were then centrifuged for 15 min at 4 °C and 7000 × g. The supernatants were discarded, and the cells were resuspended into 20 ml of phosphate-buffered saline (PBS; 140 mM NaCl, 5 mM KCl, 20 mM Na₂HPO₄, 3.5 mM KH₂PO₄). Cell suspensions were stored at 20 °C until further use.

Protein Purification-- To purify the rHBHA, frozen recombinant E. coli cell suspensions were thawed and sonicated twice for 5 min at 4 °C using a Branson Sonifier at an output of 5 delivered to a microtip. The bacterial lysates were then centrifuged at 10,000 × g for 15 min at 4 °C. The supernatants were diluted to 100 ml with PBS and then applied onto a heparin-Sepharose CL-6B (Amersham Pharmacia Biotech) column (1 × 4 cm) previously equilibrated with 100 ml of PBS. The bound material was eluted with a 0-500 mM NaCl linear gradient prepared in 80 ml of PBS. The column was regenerated by washing the gel with 30 ml of PBS containing 3 M NaCl. All chromatographic steps were carried out at room temperature using a flow rate of 1.5 ml/min. Native M. bovis BCG HBHA was purified by heparin-Sepharose chromatography as described previously (15).

To purify rHBHAC, cells from a 250-ml culture of E. coli BL21(DE3)(pET-HBHAC) were resuspended into 50 ml of 50 mM Tris-HCl (pH 8.0) and sonicated twice for 5 min at 4 °C. The soluble fraction obtained after centrifugation (7000 × g for 15 min at 4 °C) was loaded at a flow rate of 0.5 ml/min onto a DEAE-cellulose (DE 52, Whatman, Maidstone, United Kingdom) column (1 × 8 cm) equilibrated with 150 ml of 50 mM Tris-HCl (pH 8.0). rHBHAC was eluted using a linear 0-1 M NaCl gradient in 100 ml of 50 mM Tris-HCl (pH 8.0). The material eluted between 150 and 200 mM NaCl was collected, diluted twice with 50 mM Tris-HCl (pH 8.0), 4 M NaCl, and finally loaded at a flow rate of 0.5 ml/min onto a Phenyl Sepharose CL-4B (Amersham Pharmacia Biotech) column (1 × 5 cm) equilibrated with 100 ml of 50 mM Tris-HCl (pH 8.0), 2 M NaCl. rHBHAC was eluted with a negative linear 2-0 M NaCl gradient in 100 ml of 50 mM Tris-HCl (pH 8.0). The fractions eluted between 0.8 and 0.6 M NaCl contained purified rHBHAC, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) performed according to Laemmli (20) by using a 4% stacking and 12 or 15% separating gels. After electrophoresis, gels were stained with Coomassie Brilliant Blue R-250 (Merck). Protein concentrations were determined according to the method of Bradford (21), using bovine serum albumin (BSA) as a standard.

Surface Plasmon Resonance (SPR) Analysis-- The BIAcore 2000 apparatus (BIAcore AB, Uppsala, Sweden) was used for real time analysis of molecular interactions between rHBHA, rHBHAC, MBP, or MBP+3R1+2R2 and heparin. Biotinylated heparin with an average molecular mass of 5 kDa (Biotinylated-Ardeparin, Celsus Laboratories, Cincinnati, OH) was immobilized onto a streptavidin-coated sensor chip (CM5 Sensor Chip, BIAcore AB). Briefly, the streptavidin was covalently bound to the sensor chip after activation of the chip with a mixture of N-hydroxysuccinimide and N-ethyl-N'-dimethylaminopropyl carbodiimide according to the manufacturer's instructions. 10 µl of biotinylated heparin solution at 5 µg/ml in PBS were injected onto the streptavidin-coated chip, generating an immobilization signal of 250 resonance units. The protein solutions in PBS were then injected at a flow rate of 10 µl/min for 3 min at 25 °C, and binding of soluble proteins to immobilized heparin was measured in resonance units. Concentrations of purified proteins used in the SPR analyses were determined after acid hydrolysis (24 h at 110 °C in 6 N HCl) by total amino acid quantitation performed with a Beckman 6300 amino acid analyzer coupled to a high pressure liquid chromatography detection system (Beckman Gold System). The association (k_a) and the dissociation rate constants (k_d) were calculated according to the BIAevaluation software 3.0. The affinity constants were calculated from the equation K_D = k_d/k_a. rHBHA, rHBHAC, MBP, or MBP+3R1+2R2 did not bind directly to streptavidin bound to the sensor chip.

Protein Binding to Pneumocytes-- The human lung pneumocyte A549 cell line (CCL185) was obtained from the American Type Culture Collection and cultured in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% decomplemented fetal bovine serum (Life Technologies, Inc.). 18 h before use, the cells were seeded on glass coverslips at ~10⁵ cells/12-mm diameter coverslip. After two washes with 1 ml of PBS, the coverslips were incubated with 400 µl of 1 µM rHBHA, rHBHAC, MBP, or MBP+3R1+2R2 solution in PBS. After 1 h at 37 °C, the cells were washed three times with 1 ml of PBS containing 0.01% Tween 80, fixed for 10 min with 400 µl of 2% paraformaldehyde, and washed again three times with PBS containing 0.01% Tween 80. The cells were then incubated for 30 min with 400 µl of rabbit anti-rHBHA serum (diluted 500-fold) or rabbit anti-MBP serum (diluted 10,000-fold). After three washes with PBS containing 0.01% Tween 80, 400 µl of 1000-fold-diluted rhodamine-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) were added for 30 min. The cells were washed again three times with PBS containing 0.01% Tween 80 and analyzed using a Zeiss Axioplan 2 fluorescence microscope. Cells incubated with rhodamine-conjugated secondary antibodies alone served as a negative control.

HBHA Binding to Pneumocytes Treated with GAG-degrading Enzymes-- A549 pneumocytes were propagated in suspension using 293 SFM growth medium (Life Technologies, Inc.) supplemented with 4 mM L-glutamine. After 48 h of culture, 5 × 10⁵ cells were collected by low speed centrifugation, washed once with PBS, and resuspended in 1 ml of PBS supplemented with 0.1% BSA (PBS/BSA). Following the addition of 1 unit of chondroitinase ABC (Sigma) or heparinase III (Sigma), the cells were incubated for 2 h at 37 °C, washed twice with PBS/BSA, and resuspended in 1 ml of PBS containing 1 µM rHBHA. After 30 min of incubation at 4 °C followed by 3 washes with PBS/BSA, the cells were incubated for 1 h at 4 °C with 1 ml of rabbit anti-rHBHA serum diluted 500-fold. The cells were then washed thrice with PBS/BSA and incubated for 20 min at room temperature in 1 ml of fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 500-fold. After 3 final washes with PBS/BSA, the cells were fixed with 500 µl of 4% paraformaldehyde, and cell-associated fluorescence (percentage of fluorescein isothiocyanate-labeled pneumocytes) was quantified by flow cytometry using an EPICS Elite cytometer (Coulter, Hialeah, FL). Fluorescence associated with pneumocytes that were not incubated with rHBHA was used to determine nonspecific signals.

Immunoblot Analyses-- After SDS-PAGE, proteins were transferred onto nitrocellulose membranes (BA 85; Schleicher & Schuell) as described by Towbin et al. (22). Immobilized native HBHA, rHBHA, and truncated versions of rHBHA, MBP, or MBP-HBHA hybrid proteins were then probed with purified anti-HBHA monoclonal antibody (mAb) 4057D2 or 3921E4 (23), 500-fold diluted anti-HBHA rat serum (15), or 10,000-fold diluted rabbit anti-MBP serum (New England Biolabs). mAb 4057D2 was purified by cryoprecipitation (24), and mAb 3921E4 as purified by protein A-Sepharose Fast Flow (Amersham Pharmacia Biotech) chromatography. Immune complexes were developed with alkaline phosphatase-linked goat anti-mouse, anti-rat, or anti-rabbit IgG (ProtoBlot System; Promega, Madison, WI). To investigate the influence of dextran (500 kDa, Sigma), dextran sulfate (500 kDa, Sigma), and heparin (3 kDa, Sigma) on the immunoreactivities, nitrocellulose membranes bearing immobilized antigens were incubated at room temperature for 1 h in PBS containing 500 µg/ml of the polysaccharide to be tested. After two washes with PBS, the membranes were probed with mAb 3921E4 or anti-MBP serum and developed as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Dextran Sulfate Protects rHBHA from Proteolytic Degradation-- Unglycosylated rHBHA produced in E. coli undergoes rapid proteolytic degradation affecting its carboxyl-terminal lysine-rich repeats (16). This degradation correlates with a reduction of affinity for heparin, raising the possibility that these repeats are involved in heparin binding by HBHA. To test this hypothesis, dextran sulfate (500 kDa) or dextran (500 kDa) was added at 500 µg/ml to an E. coli XL1-Blue(pKK-HBHA). As shown in Fig. 1, dextran sulfate but not dextran partially protected rHBHA from proteolysis during incubation at 37 °C. A decrease in dextran sulfate concentration correlated with a reduction of the protective effect (data not shown). These observations suggest that the sulfated polysaccharide masked proteolytic cleavage sites through its binding to the carboxyl terminus of rHBHA. Low molecular mass heparin (3 kDa) did not protect rHBHA, even at concentrations up to 2 mg/ml, indicating that the length of the sulfated polysaccharide may be important to protect rHBHA from proteolytic degradation.

View larger version (88K): [in this window] [in a new window]   Fig. 1.   Inhibition by dextran sulfate of proteolysis of rHBHA produced in E. coli XL1-Blue. Cell extracts obtained by sonication of a 250-ml culture of E. coli XL1-Blue(pKK-HBHA) were incubated at 37 °C with dextran (500 kDa, left) or dextran sulfate (500 kDa, right) at a final concentration of 500 µg/ml. After 1 min (lanes A), 15 min (lanes B), 30 min (lanes C), 1 h (lanes D), and 3 h of incubation (lanes E), the proteins were analyzed by SDS-PAGE and Coomassie Blue staining. The position of rHBHA is shown by a star, and molecular mass markers expressed in kDa are indicated in the left margin.

High Level Production of rHBHA by a Protease-deficient E. coli Strain-- To circumvent the degradation of rHBHA, the protein was produced under the control of the T7 promoter in E. coli BL21(DE3), a protease-deficient strain. No significant rHBHA degradation was observed during the first 4 h in an E. coli BL21(DE3)(pET-HBHA) lysate incubated at 37 °C, whereas under the same conditions all rHBHA expressed in E. coli XL1-Blue was cleaved into a 19-kDa polypeptide (16). After 24 h of incubation, less than 50% of the rHBHA was cleaved into a 25-kDa polypeptide in the E. coli BL21(DE3)(pET-HBHA) lysate (data not shown). These results show that a protease-deficient E. coli strain allows for stable production of rHBHA. Moreover, the use of the T7 promoter instead of the trc promoter permitted a ~10-fold increase in rHBHA production, as determined by densitometric analysis of the Coomassie-stained electrophoresis gels.

rHBHAC Does Not Bind to Heparin and Is Not Recognized by mAb 3921E4-- The production of rHBHAC in E. coli XL1-Blue(pKK-HBHAC) and in E. coli BL21(DE3)(pET-HBHAC) was analyzed by SDS-PAGE and immunoblotting. Similar to full-length rHBHA, the production of rHBHAC was strongly enhanced when the recombinant gene was under the control of the T7 promoter (Fig. 2, compare lanes E and G). In both expression systems, the apparent molecular mass of rHBHAC was ~25.4 kDa, whereas its calculated molecular mass is 17.4 kDa. This difference is similar to that observed for full-length rHBHA (16), indicating that the deletion of the repeated motifs does not abolish the aberrant electrophoretic migration. Incubation of E. coli XL1-Blue(pKK-HBHAC) lysates for 1 day at 37 °C revealed that rHBHAC does not undergo degradation (data not shown), consistent with the notion that the proteolytic susceptibility of full-length rHBHA concerns the lysine-rich repeats. mAb 3921E4, which blocks the heparin-inhibitable binding of M. tuberculosis to epithelial cells (15, 16), failed to recognize rHBHAC (Fig. 2, lanes E), indicating that at least part of its epitope is located within the HBHA repeats. To determine whether these repeats are involved in the interaction of HBHA with heparin, an E. coli BL21(DE3)(pET-HBHAC) lysate was applied onto a heparin-Sepharose column. All the rHBHAC was recovered in the flow-through fraction, whereas under the same conditions, full-length rHBHA produced by E. coli BL21(DE3)(pET-HBHA) remained bound to the column. These observations indicate that the lysine-rich motifs of HBHA are involved in the interaction with heparin.

View larger version (55K): [in this window] [in a new window]   Fig. 2.   SDS-PAGE and immunoblot analyses of rHBHA and rHBHAC produced in E. coli XL1-Blue and E. coli BL21(DE3). E. coli XL1-Blue(pKK-HBHA) (lanes B and C), E. coli XL1-Blue(pKK-HBHAC) (lanes D and E), and E. coli BL21(DE3)(pET-HBHAC) (lanes F and G) lysates were analyzed before (lanes B, D, and F) and after induction by isopropylthiogalactoside (lanes C, E, and G). Lanes A contain 200 ng of purified HBHA from M. bovis BCG. After electrophoresis, the proteins were stained with Coomassie Blue R-250 (left and right panels) or transferred onto nitrocellulose membranes and probed with mAb 3921E4 (middle panel). The arrowheads indicate the positions of rHBHA (lane C) and rHBHAC (lane E). Molecular mass markers expressed in kDa are given in the left margin.

Deletions within the rHBHA Repeats Weaken the Interaction with Heparin-- To assess the contribution of the R1 repeats to the HBHA-heparin-binding activity and to further map the epitope of mAb 3921E4, rHBHAC+1R1, rHBHAC+2R1, and rHBHAC+3R1 were produced in E. coli BL21(DE3). Recombinant protein production was analyzed by SDS-PAGE and immunoblotting using mAb 3921E4 (Fig. 3). This antibody failed to recognize rHBHAC+1R1 and reacted only faintly with rHBHAC+2R1, whereas its reactivity with rHBHAC+3R1 was comparable to that observed with rHBHA, suggesting that the mAb 3921E4 epitope overlaps two successive R1 motifs. Interestingly, compared with rHBHA, the rHBHAC with 1-3 R1 motifs was less stable in E. coli BL21(DE3), suggesting that the missing carboxyl-terminal amino acids, perhaps the terminal VTQK sequence, may shield from proteolysis (Fig. 3, lanes C-E).

View larger version (62K): [in this window] [in a new window]   Fig. 3.   SDS-PAGE and immunoblot analyses of rHBHA containing truncations within the carboxyl-terminal repeats. E. coli BL21(DE3)(pET24d(+)) (lanes A), E. coli BL21(DE3)(pET-HBHA) (lanes B), E. coli BL21(DE3)(pET-HBHAC+3R1) (lanes C), E. coli BL21(DE3)(pET-HBHAC+2R1) (lanes D), E. coli BL21(DE3)(pET-HBHAC+1R1) (lanes E), and E. coli BL21(DE3)(pET-HBHAC) (lanes F) lysates were analyzed by SDS-PAGE and Coomassie Blue staining (left panel) and immunoblotting using mAb 3921E4 (right panel). Molecular mass markers expressed in kDa are shown in the left margin.

When tested for their ability to interact with heparin, rHBHAC+1R1 was found unable to bind to heparin-Sepharose, and rHBHAC+2R1 bound weakly because it could be eluted with 20 mM NaCl. The presence of the third R1 motif strengthened the interaction with heparin because elution of rHBHAC+3R1 required 100 mM NaCl. These results indicate that the R1 motifs are involved in heparin binding, but they are not sufficient for the high affinity binding observed for full-length rHBHA, suggesting that the missing 21 residues, perhaps the R2 motifs, also play a role in the interaction with heparin.

Grafting of the HBHA Repeats onto MBP Confers Heparin Binding and mAb 3921E4 Recognition to the Hybrid Protein-- To investigate whether the carboxyl-terminal repeats are sufficient for heparin binding, amino acids 161-199 (3R1+2R2) or 173-199 (1R1+2R2) of HBHA were fused to the carboxyl end of MBP. Production of the hybrid proteins MBP+3R1+2R2 and MBP+1R1+2R2 was analyzed by SDS-PAGE and immunoblotting. Both proteins were equally well recognized by mAb 3921E4, confirming that its epitope is located within the repeats. When produced in E. coli XL1-Blue, MBP+3R1+2R2 and MBP+1R1+2R2 were rapidly degraded into polypeptides of 41.5 and 40 kDa, respectively, which were resistant to further degradation and no longer recognized by mAb 3921E4 (data not shown).

The capacity of the MBP hybrid proteins to bind to sulfated polysaccharides was investigated by immunoblotting and heparin-Sepharose chromatography. As shown in Fig. 4, pretreatment of nitrocellulose membranes bearing MBP+3R1+2R2 with dextran sulfate but not with unsulfated dextran impeded the subsequent recognition by mAb 3921E4 but not that of anti-MBP antibodies, suggesting that the sulfated polysaccharide bound to the hybrid protein and masked the 3921E4 epitope. The size of the sulfated polysaccharide appeared to be important as low molecular weight heparin did not inhibit 3921E4 reactivity (data not shown). To determine whether the fusion of the HBHA repeats to MBP induces its binding to heparin, E. coli B834(pMAL-c), E. coli B834(pMAL+3R1+2R2), and E. coli B834(pMAL+1R1+2R2) lysates were subjected to heparin-Sepharose chromatography. Whereas MBP was unable to bind to heparin, MBP+3R1+2R2 bound to the column and was eluted with 300 mM NaCl (Fig. 5), a condition similar to that required for the elution of rHBHA. MBP+1R1+2R2 also bound to heparin-Sepharose but less strongly because it was eluted with 100 mM NaCl. These results indicate that the molecular determinants required for the heparin-binding activity of HBHA are all located within the carboxyl-terminal repeats and that the heparin-binding activity of HBHA can be transferred to an heterologous protein by tagging with the HBHA repeats.

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Influence of dextran and dextran sulfate on reactivity of MBP and MBP+3R1+2R2 with anti-MBP antiserum or mAb 3921E4. E. coli B834(pMAL-c) (lanes A) and E. coli B834(pMAL+3R1+2R2) (lanes B) lysates were submitted to SDS-PAGE and Coomassie Blue staining (left panel) and analyzed by immunoblotting using a rabbit anti-MBP antiserum (-MBP) or mAb 3921E4 after incubation of the membranes with dextran or dextran sulfate at 1 mg/ml in PBS. Molecular mass markers expressed in kDa are shown in the left margin.

View larger version (85K): [in this window] [in a new window]   Fig. 5.   Heparin-Sepharose chromatography of an E. coli B834(pMAL+3R1+2R2) lysate. Bacterial cells of a 250-ml culture of E. coli B834(pMAL+3R1+2R2) were sonicated in PBS. The soluble material was subjected to heparin-Sepharose chromatography and eluted using a 0-500 mM NaCl gradient. 50 µg of starting material (lane A), flow-through material (lane B), eluting material (lanes C), and material released during the regeneration step of the column using PBS containing 3 M NaCl (lanes D and E) were analyzed by SDS-PAGE and Coomassie Blue staining. Molecular mass markers expressed in kDa are shown in the left margin.

SPR Analysis of the HBHA-Heparin Interactions-- SPR was used to more precisely analyze the interaction of rHBHA and MBP+3R1+2R2 with heparin. As shown in Fig. 6, rHBHAC and MBP were unable to interact with the heparin sensor chip, whereas rHBHA and MBP+3R1+2R2 displayed comparable interaction profiles. The affinity constants calculated using protein concentrations of 200 nM were 26 (±2) and 23 (± 1) nM for rHBHA and MBP+3R1+2R2, respectively, confirming that the high affinity binding of HBHA to heparin is entirely mediated by its lysine-rich repeats. For both proteins, the interaction with heparin was characterized by fast association kinetics. However, the association rate constant for the interaction of MBP+3R1+2R2 with heparin (k_a = 6.65 (± 0.15) × 10⁵ M¹ s¹) was slightly higher than that calculated for the rHBHA-heparin interaction (k_a = 5.62 (± 0.10) × 10⁵ M¹ s¹), suggesting perhaps a slightly faster binding for the hybrid protein. No difference was observed for the dissociation rate constants between the two proteins.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   SPR analysis of the interactions of rHBHA and MPB+3R1+2R2 with immobilized heparin. Biotinylated heparin was immobilized onto the surface of sensor chips, and 30 µl of rHBHA, rHBHAC, MBP, or MBP+3R1+ 2R2 at a concentration of 200 nM was used to monitor the protein interactions with immobilized heparin in real time (A). No interaction was observed with rHBHAC and MBP. Data are expressed as relative responses in resonance units (RU) after subtraction of the background signal recorded on streptavidin-coated sensor chips. The kinetic parameters of the interactions observed with rHBHA and MBP+3R1+2R2 are summarized in B. Standard deviations were calculated from three independent experiments.

Binding of HBHA to Human Pneumocytes Is Mediated by the Carboxyl-terminal Repeats-- To test whether the HBHA repeats are involved in HBHA adherence to human pneumocytes, purified rHBHA and rHBHAC were incubated with A549 cells, and protein binding was monitored by immunofluorescence microscopy. As shown in Fig. 7, rHBHA bound to the cell surface, whereas rHBHAC did not, indicating that the repeats are required for interaction of HBHA with human pneumocytes. In this binding assay, MBP did not bind to A549 cells, whereas MBP+3R1+2R2 bound to the pneumocytes (Fig. 7), indicating that the repeats are sufficient for binding of HBHA to respiratory epithelial cells.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Immunofluorescence microscopy analysis of rHBHA and MBP+3R1+2R2 binding to human pneumocytes. A549 cells seeded onto glass coverslips were incubated with 400 µl of 1 mM rHBHA (A), rHBHAC (B), MBP+3R1+2R2 (C), or MBP (D). After washing and fixing with paraformaldehyde, the bound proteins were detected using a rabbit anti-rHBHA antiserum (A and B) or a rabbit anti-MBP antiserum (C and D) followed by incubation with rhodamine-conjugated goat anti-rabbit IgG.

Pneumocyte Heparan Sulfate Chains Act as Receptors for HBHA-- To investigate the involvement of GAGs in HBHA binding to human pneumocytes, A549 cells were treated with chondroitinase ABC or heparinase III, which cleaves chondroitin sulfate A, B, and C or heparan sulfate, respectively. Untreated cells as well as lyase-treated cells were then incubated with rHBHA, and the adhesin binding was monitored by flow cytometry. Whereas the chondroitinase ABC treatment did not significantly reduce the interaction of rHBHA with pneumocytes, the heparinase III treatment led to a reduction of 68% of rHBHA binding (Fig. 8), indicating that proteoglycans containing heparan sulfate chains serve as receptors for HBHA on the surface of human pneumocytes.

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Flow cytometry analysis of rHBHA binding to human pneumocytes treated with GAG-degrading enzymes. Untreated A549 cells and A549 cells treated with chondroitinase ABC or heparinase III were incubated with rHBHA, and protein binding was assessed by flow cytometry using a rabbit anti-rHBHA antiserum. Nonspecific antibody binding corresponding to fluorescein isothiocyanate-labeled cells observed in absence of rHBHA was 10.4% (± 0.3). Standard deviations were calculated from three independent experiments. FITC, fluorescein isothiocyanate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To mediate adherence during the early steps of infection, pathogenic microorganisms commonly interact with cell surface receptors that are normally recognized by eukaryotic ligands to mediate signal transduction, as well as cell-cell and cell-extracellular matrix interactions (25). These molecules are generally glycoproteins, and an increasing number of bacterial adhesins are now being shown to express specific lectin activity for the carbohydrate moieties of these receptors (26). Among those carbohydrates, heparan sulfate and related sulfated polysaccharides such as chondroitin sulfate are of special interest, because they are present on the surface of virtually all animal cells in the form of sulfated proteoglycans (27). Indeed, the GAG chains of proteoglycans constitute a family of receptors for various bacterial adhesins (28), including HBHA, a surface-exposed glycosylated heparin-binding adhesin produced by M. tuberculosis (15, 16).

For most adhesins, the molecular details of their interaction with GAG chains are not known. Here, we show that the carboxyl terminus of HBHA, which comprises five lysine-rich repeats (3 R1 and 2 R2), is responsible for heparin interaction. Consistent with the notion that basic amino acids are most often involved in sulfated polysaccharide binding via electrostatic interactions (29), a recombinant HBHA variant from which all the repeats were deleted was unable to bind to heparin. Progressive truncations within the HBHA repeats correlated with a reduction of affinity for heparin, and both the R1 and the R2 repeats were found to contribute to binding to the sulfated polysaccharide. When the repeats were grafted onto MBP, which by itself does not bind to heparin (30), the hybrid protein displayed the same affinity for heparin as full-length HBHA, indicating that all the molecular determinants responsible for heparin binding are located within the repeats. Because the hybrid protein bound to heparin-Sepharose, the HBHA repeats may constitute a new type of molecular tag for the production of recombinant proteins easy to be purified by heparin-Sepharose chromatography. This application may have an advantage over other tags, such as histidine or protein A tags, in that the hybrid proteins can be easily eluted under mild, nondenaturing conditions, such as increased NaCl concentrations, whereas the elution of recombinant proteins tagged by other means usually requires urea or low pH (31, 32). Furthermore, the lysine-rich repeats are highly susceptible to proteolytic degradation (16), which should facilitate the removal of the heparin-binding tag after purification of the recombinant hybrid protein. However, the presence of other heparin-binding proteins in the recombinant cell extracts may require additional steps to purify the tagged protein.

SPR analysis of rHBHA-heparin interactions revealed high affinity binding, with affinity constants in the range of values that have been reported for the GAG-binding activities of eukaryotic proteins such as fibronectin (33), antithrombin III (34), or HARP, a heparin-binding growth factor (35). The affinity of HBHA for sulfated polysaccharides is thus sufficient to mediate adherence to the surface of physiologically relevant tissues, such as the pulmonary alveolus. By comparing different heparin-binding domains of various eukaryotic proteins, Cardin and Weintraub (36) proposed two heparin-binding motifs, XBBXBX and XBBBXXBX, where B represent basic amino acids and X any other amino acids. The heparin-binding site of HBHA does not totally fit the consensus sequence of Cardin and Weintraub (36). However, the high density of lysine residues within these repeats is consistent with their role as basic amino acids interacting with the negatively charged sulfate groups on the carbohydrate receptors (29). It is striking to notice that the HBHA heparin-binding domain is composed of only three amino acids (alanine, lysine, and proline) and consists on seven di-lysine motifs separated by the tetrapetide AAPA or the tripeptides AAA or APA, which probably serve as spacers of the positive charges borne by the lysines.

The presence of proline-rich regions and repeats is known to induce aberrant electrophoretic mobility during SDS-PAGE. We have previously noticed that rHBHA displays such aberrant electrophoretic mobility (16). However, rHBHAC still migrates slower than expected, indicating that the HBHA repeats are not responsible for its aberrant electrophoretic mobility.

The epitope recognized by mAb 3921E4 mapped within the lysine-rich repeats. This mAb has been reported to inhibit adherence of BCG to Chinese hamster ovary cells (15). We show here that this epitope comprises the sequence AAPAKKAA. Deletion of this octapeptide from the HBHA protein abolished mAb 3921E4 recognition, and grafting of a peptide containing this octapeptide onto MBP resulted in the recognition of the recombinant hybrid protein by the mAb. Antibody-mediated blocking of mycobacterial binding to target cells may perhaps constitute the basis of a novel vaccine design against tuberculosis, although antibody-mediated immune responses have not been considered protective yet (37). Therefore, repeated motifs of HBHA may represent an entirely new target for vaccine development. In this respect it is important to realize that the HBHA repeats are involved in binding to human A549 pneumocytes, a more relevant target for infections by M. tuberculosis than Chinese hamster ovary cells.

In addition to mAb 3921E4, a second mAb has also been shown to inhibit BCG adherence to Chinese hamster ovary cells. This mAb, named 4057D2, only recognizes glycosylated HBHA (16). Here, we show that the glycosylation of HBHA is not important for heparin binding. Previous studies have indicated that the glycosylation protects HBHA from proteolytic degradation at the carboxyl-terminal end (16) and thus protects the heparin-binding domain from proteolytic removal. Although the glycosylation site of HBHA has not been identified yet, it is tempting to hypothesize that it is located in the vicinity of the lysine-rich repeats, and that mAb 4057D2 binding causes a steric hindrance that impedes recognition of GAGs. However, the possibility that such a steric hindrance may be caused by binding of the mAb to distal sites cannot be ruled out and certainly warrants further investigation, because the identification of the glycosylation site of HBHA will increase the molecular understanding of the structure-function relationship of this mycobacterial adhesin.

Experiments using various GAG lyases indicated that heparan sulfate chains, but not chondroitin sulfate chains, play a crucial role in HBHA binding to A549 pneumocytes. Specific recognition of GAGs by bacterial adhesins has already been described for Chlamydia trachomatis (38) and Neisseria gonorrhoeae (39). Similar to this study, N. gonorrhoeae has been shown to adhere to heparan sulfate chains on epithelial cells, whereas chondroitin sulfate chains did not appear to be involved in gonococcal adherence (39). The purification and characterization of the HBHA receptor on the surface of pneumocytes will generate new valuable information on the mycobacteria-host interactions. Given the high yield of rHBHA and the possibility to graft the heparin-binding site onto MBP, as reported in this study, such investigations are now feasible.

	ACKNOWLEDGEMENTS

We are grateful to Eve Willery for excellent assistance in DNA sequencing, Alex Veithen for the immunofluorescence microscopy, Nathalie Spruyt for help in SPR analyses, Hervé Drobecq for total amino acid analyses, Gwenola Kervoaze for the suspension cultures of A549 cells, Brigitte Quatannens for the flow cytometry analyses, Elizabeth Pradel for critically reading the manuscript, and Dominique Stéhelin for the free access to the BIAcore apparatus.

	FOOTNOTES

^* This work was supported by the Institut Pasteur de Lille, the Région Nord-Pas de Calais, and INSERM.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Holds a fellowship from the Ministère de la Recherche et de la Technologie.

^** To whom correspondence should be addressed. Tel.: 33-3-20871151; Fax: 33-3-20871158; E-mail: camille.locht@pasteur-lille.fr.

	ABBREVIATIONS

The abbreviations used are: HBHA, heparin-binding hemagglutinin; r, recombinant; GAG, glycosaminoglycan; PCR, polymerase chain reaction; MBP, maltose-binding protein; PBS, phosphate-buffered saline; BCG, Bacille de Calmette et Guérin; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; SPR, surface plasmon resonance; mAb, monoclonal antibody.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				C. Verbelen, V. Dupres, D. Raze, C. Bompard, C. Locht, and Y. F. Dufrene Interaction of the Mycobacterial Heparin-Binding Hemagglutinin with Actin, as Evidenced by Single-Molecule Force Spectroscopy J. Bacteriol., December 1, 2008; 190(23): 7614 - 7620. [Abstract] [Full Text] [PDF]

				C. Esposito, M. V. Pethoukov, D. I. Svergun, A. Ruggiero, C. Pedone, E. Pedone, and R. Berisio Evidence for an Elongated Dimeric Structure of Heparin-Binding Hemagglutinin from Mycobacterium tuberculosis J. Bacteriol., July 1, 2008; 190(13): 4749 - 4753. [Abstract] [Full Text] [PDF]

				C. Verbelen, D. Raze, F. Dewitte, C. Locht, and Y. F. Dufrene Single-Molecule Force Spectroscopy of Mycobacterial Adhesin-Adhesin Interactions J. Bacteriol., December 15, 2007; 189(24): 8801 - 8806. [Abstract] [Full Text] [PDF]

				C. Jenkins, S. J. Geary, M. Gladd, and S. P. Djordjevic The Mycoplasma gallisepticum OsmC-like protein MG1142 resides on the cell surface and binds heparin Microbiology, May 1, 2007; 153(5): 1455 - 1463. [Abstract] [Full Text] [PDF]

				A-R. Shin, K.-S. Lee, J.-S. Lee, S.-Y. Kim, C.-H. Song, S.-B. Jung, C.-S. Yang, E.-K. Jo, J.-K. Park, T.-H. Paik, et al. Mycobacterium tuberculosis HBHA Protein Reacts Strongly with the Serum Immunoglobulin M of Tuberculosis Patients. Clin. Vaccine Immunol., August 1, 2006; 13(8): 869 - 875. [Abstract] [Full Text] [PDF]

				C. Jenkins, J. L. Wilton, F. C. Minion, L. Falconer, M. J. Walker, and S. P. Djordjevic Two Domains within the Mycoplasma hyopneumoniae Cilium Adhesin Bind Heparin Infect. Immun., January 1, 2006; 74(1): 481 - 487. [Abstract] [Full Text] [PDF]

				J. R. Hayman, T. R. Southern, and T. E. Nash Role of Sulfated Glycans in Adherence of the Microsporidian Encephalitozoon intestinalis to Host Cells In Vitro Infect. Immun., February 1, 2005; 73(2): 841 - 848. [Abstract] [Full Text] [PDF]

				I. R. Henderson, F. Navarro-Garcia, M. Desvaux, R. C. Fernandez, and D. Ala'Aldeen Type V Protein Secretion Pathway: the Autotransporter Story Microbiol. Mol. Biol. Rev., December 1, 2004; 68(4): 692 - 744. [Abstract] [Full Text] [PDF]

				K. Aoki, S. Matsumoto, Y. Hirayama, T. Wada, Y. Ozeki, M. Niki, P. Domenech, K. Umemori, S. Yamamoto, A. Mineda, et al. Extracellular Mycobacterial DNA-binding Protein 1 Participates in Mycobacterium-Lung Epithelial Cell Interaction through Hyaluronic Acid J. Biol. Chem., September 17, 2004; 279(38): 39798 - 39806. [Abstract] [Full Text] [PDF]

				J. P. Bannantine, J. F. J. Huntley, E. Miltner, J. R. Stabel, and L. E. Bermudez The Mycobacterium avium subsp. paratuberculosis 35 kDa protein plays a role in invasion of bovine epithelial cells Microbiology, August 1, 2003; 149(8): 2061 - 2069. [Abstract] [Full Text] [PDF]

				S. L. Mueller-Ortiz, E. Sepulveda, M. R. Olsen, C. Jagannath, A. R. Wanger, and S. J. Norris Decreased Infectivity despite Unaltered C3 Binding by a {Delta}hbhA Mutant of Mycobacterium tuberculosis Infect. Immun., December 1, 2002; 70(12): 6751 - 6760. [Abstract] [Full Text] [PDF]

				K. Pethe, P. Bifani, H. Drobecq, C. Sergheraert, A.-S. Debrie, C. Locht, and F. D. Menozzi Mycobacterial heparin-binding hemagglutinin and laminin-binding protein share antigenic methyllysines that confer resistance to proteolysis PNAS, August 6, 2002; 99(16): 10759 - 10764. [Abstract] [Full Text] [PDF]

				S. L. Mueller-Ortiz, A. R. Wanger, and S. J. Norris Mycobacterial Protein HbhA Binds Human Complement Component C3 Infect. Immun., December 1, 2001; 69(12): 7501 - 7511. [Abstract] [Full Text] [PDF]

				K. K. Singh, X. Zhang, A. S. Patibandla, P. Chien Jr., and S. Laal Antigens of Mycobacterium tuberculosis Expressed during Preclinical Tuberculosis: Serological Immunodominance of Proteins with Repetitive Amino Acid Sequences Infect. Immun., June 1, 2001; 69(6): 4185 - 4191. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pethe, K. Articles by Menozzi, F. D. Search for Related Content PubMed PubMed Citation Articles by Pethe, K. Articles by Menozzi, F. D. Social Bookmarking                      What's this?