The 19 kDa Protein from Mycobacterium avium subspecies paratuberculosis Is a Glycolipoprotein

This study characterizes the 19 kDa protein expressed by Mycobacterium avium subspecies paratuberculosis (MAP) as a glycolipoprotein, providing the foundation for future experiments regarding its antigenicity and role in disease pathogenicity. We have previously shown that a 4.8 kb insert from MAP will produce a 16 kDa recombinant protein when expressed in Escherichia coli and 19 kDa recombinant protein when expressed in M. smegmatis (smeg19K). The difference of 3 kDa in size of these expressed proteins may be related to post translational modifications that occur in Mycobacterium species. We hypothesized that smeg19K is a glycolipoprotein since BLAST analysis revealed approximately 76% amino acid identity between the MAP 19 kDa protein and a known lipoglycoprotein, the 19 kDa protein of M. tuberculosis. This prediction was confirmed by the following positive staining of smeg19K with Sudan Black 4B, a postelectrophoresis dye used to stain for lipids. Smeg19K has also stained positively for glycosylation with the lectin concavalin A, a highly specific stain for mannose residues. As expected, treatment with tunicamycin (an antibiotic known to inhibit N-glycosylation) and treatment with deglycosylation assay (non-specific for mannose), showed no reduction in size of 19 kDa glycolipoprotein.


Introduction
The genus Mycobacterium has well recognized pathogenic species.M. tuberculosis and M. leprae are the causative agents of infectious diseases, tuberculosis and leprosy, respectively [1], then M. avium and M. intracellulare, opportunistic pathogens, are associated with infection in hosts such as patients with AIDS [2,3].The development and improvement needed for therapeutics and vaccines for these microorganisms have led to the genetic characterization of mycobacterium antigens, and to the identification of highly conserved protein families [4].Despite decades of research, not much is known about the M. avium subsp.paratuberculosis (MAP or M. paratuberculosis) proteins.However, with the genome sequence of M. paratuberculosis identified [5], molecular biology techniques can be used to study different proteins/antigens expressed by this microorganism.
Previously in our laboratory, a genomic library of M. paratuberculosis was constructed in E. coli.Colonies were screened for gene expression using immunoblot and rabbit anti-M.paratuberculosis antibody.Clones specific to M. paratuberculosis were identified when they reacted negatively with rabbit anti-M.tuberculosis, rabbit anti-M.leprae and rabbit anti-M.bovis and rabbit anti-M.avium subspecies avium antibodies.pMptb#28 recombinant clone contained the insert of 4.8 kb and encoded for a single protein of 16 kDa.Surprisingly, M. paratuberculosis lacked a 16 kDa protein band when screened with rabbit anti-M.paratuberculosis antibodies.When the 4.8 kb BamH I fragment was cloned in M. smegmatis by using a pNEZ6.3shuttle plasmid, the expressed product was estimated at 19 kDa protein (Smeg19k) similar to the size observed in extracts from M. paratuberculosis.This present study focused on the characterization of the 19 kDa protein expressed by M paratuberculosis regarding posttranslational modification.
Recent studies have shown some post-translational modification in mycobacterial antigens such as acylation and glycosylation [6][7][8], which is lacking in the majority of Gram negative bacteria such as E. coli.In acylated proteins, the lipid can attach through cysteine amino acid residues, while in glycoproteins, the carbohydrate moiety attaches via its anomeric carbon through a glycosidic link to the functional group −OH of a serine or threonine amino acid residue (referred to as O-linked), or through an N-glycosyl link to the amide nitrogen of an asparagine amino acid residue (N-linked) [9].
We propose smeg19K is a glycolipoprotein because there could be a homologue relation to the 19 kDa protein expressed by M. tuberculosis, known to be a glycolipoprotein [10].BLAST analysis has revealed approximately 76% homology in amino acid identity between M. tuberculosis and M. paratuberculosis's 19 kDa protein [10].Young and Garbe, in 1991, showed through detergent phase separation and metabolic labeling that the 19 kDa of M. tuberculosis is a lipoprotein that constitutes an important target of the innate immune response [11].Acylation is believed to occur at amino acids 19 -24 [12].Another study by Garbe et al., in 1993, published evidence for glycosylation in M. tuberculosis based on the ability of the 19 kDa lipoprotein to bind lectin concavalin A (ConA).ConA is highly specific for mannose residues linked via their anomeric carbon in α position [13].Herrmann et al., in 1996, concluded that 5 threonine residues at positions 13, 14, 15, 19 and 20, of M. tuberculosis 19 kDa played a role in lectin ConA binding [8].
It is known that covalent modification of proteins with acyl or glycosyl moieties alter immunogenicity and/or pathogenicity.Some lipoproteins have been identified as major antigens from Treponema pallidum, Haemophilus influenzae and Mycoplasma hyorhinis based on their hydrophobicity during phase partitioning in the detergent Triton-Xll4 [14,15], and by metabolic labeling with fatty acid precursors [14,16,17].In addition, glycoprotein antigens have been found in pathogenic mycobacteria [7,18,19].The ultimate goal of this study is to characterize the posttranslational modification of the 19 kDa M. paratuberculosis protein, which can play a role in its antigenicity.The discovery of effective vaccines to prevent Johne's disease requires identification of antigens and their ability to modulate the immune response causing acquired protective immunity.

Bacterial Strains and Growth Conditions
E. coli strain Top 10 purchased from Invitrogen (Invitrogen Corp., Carlsbad, CA) and its recombinants were grown in 5 mL Luria Britani (LB) broth media containing 1% w/v tryptone, 0.5% w/v yeast extract and 1% w/v NaCl.This broth was supplemented with 12.5 μl ampicillin (Sigma) at a final concentration of 50 μg/mL.These cultures were incubated at 37˚C until growth was observed.Samples were aliquoted into 1 mL tubes, centrifuged for 5 min at 7000 rpm, and cell pellet was stored at −80˚C.The E. coli clone containing the plasmid pMptb#28 and E. coli pcDNAII were obtained from our genomic library as previously described [20].M. smegmatis and M. smegmatis recombinant clone (SMEG19K) were grown in BBL MGIT Mycobacterium growth indicator tube (110 μl of fluorescent indicator and 4 mL broth: modified middlebrook 7H9 broth base, casein peptone).MGIT tube was supplemented with 5 μl kanamycin at a final concentration of 20 μg/mL.Samples were aliquoted into 1 mL tubes, centrifuged for 5 min at 7000 rpm, and cell pellet was stored at −80˚C.M. tuberculosis and M. paratuberculosis were previously grown in our lab from stock solutions and stored at −80˚C.

SDS-PAGE and Western Blot Analysis
Sonicated samples were boiled in the presence of SDS and β-mercaptoethanol.Electrophoresis was performed under reducing conditions by the method of Laemmli [21] with gels containing a 6% stack over a 12% resolving gel.Proteins were visualized by silver nitrate [22].
Proteins extracts, subjected to SDS-PAGE, were transferred to nitrocellulose membranes (Bio-Rad).For visualization of recombinant expression, blots were staining with rabbit hyperimmune anti-M.paratuberculosis serum (1:10,000) at 4˚C overnight (Sigma).Blot was then incubated with 1:10,000 diluted Goat anti-rabbit IgG-peroxidase conjugates for 2 hours at room temperature (Sigma).Color development on the membrane was visualized using opti-4CN diluent solution substrate (Bio-Rad).

Carbohydrate Analysis 1)
The procedure for staining of nitrocellulose blots with peroxidase-conjugated concavalin A (ConA) was very similar to that used for immuno-staining [7].Color development was visualized using Opti-4CN diluent solution substrate as described by the manufacturer (BioRad).
2) M. smegmatis recombinant clone (SMEG 19K) was subcultured into a BBL MGIT (Mycobacterium Growth Indicator Tube) and placed in non-motile incubator at 37˚C for 5 days or until visible growth.Tunicamycin powder (Sigma) was dissolved completely in 5 mL of DMSO to a stock concentration of 10 mg/mL, and working solution of 1 mg/mL tunicamycin was prepared.Different concentration of antibiotic and harvested SMEG-19K subclone were added to new MGIT tubes and tubes were placed in rotator incubator at 37˚C until visible growth observed.

Acylation Analysis
Following electrophoresis, gels were stained in Sudan Black 4B solution (SB) (Sigma).First, gels were fixed in 40% v/v methanol, 10% v/v acetic acid, 50% v/v Millipore water for 30 min at room temperature.Then, gels were stained overnight with a 60% solution of SB in ethanol.Gels were destained in 3 changes of 30% ethanol, 15% acetic acid, and 65% Millipore water [23].

Results
The expression of 19 kDa protein in cell extracts from M. smegmatis 19 kDa recombinant, and M. tuberculosis was confirmed by immunoblot using M. smegmatis adsorbed rabbit anti-M.paratuberculosis sera for M. tuberculosis (Figure 1, lane 3), for M. smegmatis (Figure 1, lane 5).No binding was observed against protein extracts from non-recombinant M. smegmatis (Figure 1, lane 4).Additionally, there was immunoreactivity in protein extracts from E. coli recombinant protein (Figure 1, lane 9) bound antibody at 16 kDa and not from non-recombinant E. coli (Figure 1, lane 7).

Evaluation of Smeg19K Protein for
Carbohydrate Content

By ConA Staining
ConA is a lectin that specifically binds to α-D-glucose and α-D-mannose and identifies O-glycosylation.Samples were evaluated following staining an immunoblot with conA-peroxidase conjugated.We included, as shown in Figure 2  and similar amount of protein present.M. smegmatis recombinant samples treated with tunicamycin at a concentration of 2, 6, 10 μg/mL were stained with conA-peroxidase conjugate (Figure 3, lanes 5 -7) and showed binding to conA at 19 kDa molecular weight.M. tuberculosis (Figure 3, lane 2) and M. smegmatis recombinant without tunicamycin treatment (Figure 3, lane 4) were used as positive controls to show binding at 19 kDa.Again, M. smegmatis showed no binding to conA at 19 kDa (Figure 3, lane 3).spiked with Bovine fetuin glycoprotein control and then subject to enzymatic deglycosylation.The Fetuin glycolprotein has shifted in size as expected following the treatment illustrating absence of any inhibitors.The 19 kDa protein was not affected by the deglycosylation treatment (Figure 4, lane 5).

Evaluation of Smeg19K Protein for Lipid Content
Presence of lipid was assessed by staining with Sudan Black, a lipophilic dye, post-gel electrophoresis.As shown in Figure 5  shown).M. tuberculosis 19 kDa was used as positive control for lipoprotein (Figure 5, lane 2).
In addition, M. smegmatis recombinant samples treated with tunicamycin at concentrations of 2, 4, 6, 8 μg/mL were stained with Sudan black post-electrophoresis (data not shown).These samples stained positive, indicating presence of lipid.

Discussion
Covalent modification in prokaryotic proteins is less common than in eukaryotic proteins but influences the overall structure thus affecting protein function.Numerous virulence factors of mycobacterial proteins have been found to be covalently modified [24].Understanding the structure of proteins can allow for immunological characterization, which can further lead to vaccine or drug development.
The objective of this study was to characterize the 19 kDa M. paratuberculosis protein as glycosylated, acylated, or both.We chose to express the 19 kDa protein in a rapidly growing mycobacterial homologue host, M. Smegmatis, because M. paratuberculosis is a very fastidious grower and requires specific growth conditions.
BLAST analysis of amino acid sequence revealed approximately 76% homology in amino acid identity between M. tuberculosis and M. paratuberculosis's 19 kDa protein [10].Potential sites of glycosylation in M. paratuberculosis included a set of serine residues, found in same region, where M. tuberculosis 19 kDa protein has threonine residues implicated in O-glycosylation [8].Unfortunately, there is no sequence motif indicative of possible O-glycosylation, as it has been found for N-glycosylation in eukaryotic and some prokaryotic proteins.This consensus motif includes Ser/Thr-Xaa-Asn, in which Xaa, with exception of proline, can be any amino acid and it has been found in M. tuberculosis and Clostridium thermocellum [25].In addition to threonine and serine clusters involved in carbohydrate attachment, adjacent proline and alanine residues can have local conformational effects in O-glycosylation [26][27][28][29].
The expression of the recombinant 19 kDa antigen of M. paratuberculosis in M. smegmatis, is O-glycosylated, as evidenced by ConA binding (Figure 2).M. tuberculosis known 19 kDa glycolipoprotein [11][12][13] was used as positive control.The 19 kDa recombinant protein expressed in E. coli system showed no evidence of glycosylation; similar to what was observed when the recombinant 19 kDa protein of M. tuberculosis was cloned in E. coli [7].Since lectin conA is highly specific for α-mannose residues [8], mannose could be attached to serine residues through an O-glycosidic bond; similar to mannose residues attached to threonine residues in 19 kDa of M. tuberculosis [8].To confirm the of 19 kDa M. paratuberculosis recombinant protein, samples were immunoblotted against M. smegmatis adsorbed rabbit anti-M.paratuberculosis sera.Results showed the presence of 19 kDa protein from M. tuberculosis and 19 kDa recombinant protein in M. smegmatis.No binding between antibody and M. smegmatis was observed at 19 kDa.As expected, the E. coli recombinant protein bound antibody at 16 kDa while E. coli had no binding at 16 kDa.
Treatment of M. smegmatis recombinant proteins with tunicamycin supports that 19 kDa M. paratuberculosis is O-glycosylated, as evidence by conA staining.M. smegmatis recombinant proteins showed no band shift from 19 kDa to a lower molecular weight (Figure 3) when subjected to different concentration of tunicamycin (0.25, 0.50, 1, and 2 μg/mL) [30].The experiment was repeated at higher concentrations up to 10 ug/mL to rule out the possibility the resistance to lower antibiotic concentrations.Results showed no band shift at 19 kDa for the treated M. smegmatis recombinants.Additionally, high concentrations of antibiotic affected cell metabolism, as observed with decreased thickness of bands.Silver stain of SDS-gels containing these samples, showed similar amount of protein content present for all of the treated M. smegmatis recombinants.Furthermore, M. smegmatis recombinant samples treated with tunicamycin, at a concentration of 2, 6, 10 μg/mL, were stained with conAperoxidase conjugate and showed binding to conA at 19 kDa molecular weight (Figure 3).M. tuberculosis and M. smegmatis recombinant without tunicamycin treatment were used as positive controls to show binding to conA of 19  The second objective of this study was to characterize if acylation occurred.Comparison of amino acid sequence between 19 kDa of M. paratuberculosis and M. tuberculosis, showed conservation of a cysteine residue at amino acid 22 across both species.Presence of highly conserved six-residue consensus sequences for acylation in M. tuberculosis, 19-LSGCSS-24, and in M. avium subsp.Intracellulare, 19-ISGSG-24, located adjacent to hydrophobic signal peptide, and biochemical evidence [29,31], suggest that the 19 kDa protein from M. paratuberculosis, 19-VSGCSS-24, undergoes acylation and subsequent cleavage of signal peptide prior to secretion of the mature lipoprotein across cell membrane.Confirmation that 19 kDa M. paratuberculosis is lipoprotein was defined by staining with Sudan black dye post-gel electrophoresis (Figure 5).Both, M. smegmatis and M. smegmatis recombinant, stained positive with lipophilic dye.However, level of expression of M. smegmatis recombinant is higher, as observed in a thicker band.Silver stain of samples, showed similar amount of protein present for both protein extracts.BLAST analysis comparing 19 kDa sequence of M. paratuberculosis to M. smegmatis resulted in 48% homology in amino acid identity and discovery that M. smegmatis has endogenous lipoprotein.E. coli recombinant and E. coli stained positively with Sudan black at 19 kDa weight.Immunoblot against M. smegmatis adsorbed rabbit anti-M.paratuberculosis sera confirmed this 19 kDa band belongs to an endogenous E. coli protein, and not to E. coli recombinant of 16 kDa (data not shown).Immunoblot also showed M. smegmatis recombinant protein present at 19 kDa and no 19 kDa protein was recognized with antibody for M. smegmatis.M. tuberculosis 19 kDa was used as positive control for lipoprotein.
In this study, the data illustrates that the 19 kDa M. paratuberculosis is lipoglycosylated.Confirmation that 19 kDa protein is glycosylated will require structural evidence of covalent sugar-protein linkage.Future studies on the 19 kDa protein of M. paratuberculosis should exploit the availability of rapidly growing homologue mycobacterial hosts to study biological function and immunological significance.Furthermore, to confirm if the serine residues of the amino acid sequence play a role in O-glycosylation, site-directed mutagenesis should be performed.Substitution of serine residues with nonpolar amino acid residues such as valine or alanine, which cannot support glycosylation, will show which serine residues are involved with ConA binding.Herrmann et al., in 1996, studied the 19 kDa protein expressed by M. tuberculosis.Site-directed mutagenesis substituted suspected threonine residues near N-terminus with valine residues, and showed that these amino acid residues played a role in conA binding [8].Since the recombinant 19 kDa antigen from M. tuberculosis is expressed at 16 kDa in E. coli in comparison to M. smegmatis recombinant [7]; threonine substitutions had a very slight effect on molecular size of 16 kDa in E. coli [8].Also, these recombinants showed no detectable conA binding [8].To study acylation, another homologue host without endogenous 19 kDa should be used.
The importance of this study was to characterize the possible posttranslational modification of the 19 kDa MAP protein which can play a role in its antigenicity.Studies on M. tuberculosis 19 kDa glycolipoprotein shows this antigen stimulates both a T and B cell response as well as induces a number of T H 1 cytokines.It has been shown to be immunodominant in both mice [12,32,33], and in humans [34,35].It stimulates a CD4+ T cell proliferation and the release of IL-2, IFN-Y, and IL-12 [11,36].Glycosylation modification inhibits innate immune response, such as the release of TNF-α, IL-6, and IL-10 from macrophages, while not inhibiting antibody binding [7,19,23].More importantly, experimental infection and staining of macrophages have shown that this 19 kDa protein is secreted by live M. tuberculosis residing within phagolysosomal compartment [37][38][39][40].

Conclusion
Collectively, our study clearly illustrates for the first time that M. paratuberculosis 19 kDa expressed protein is experimentally confirmed to be a lipoglycosylated protein.Furthermore, this posttranslational modification includes O-glycosylation and mannose as possible sugar subunit.The data confirm observation reported in 19 kDa from M. tuberculosis.The significance of this study may aid in investigating the pathogenicity of M. paratuberculosis infection in animals and humans.
, lane 2; protein extracts from M. tuberculosis as a positive control for glycosylation of the 19 kDa.
kDa protein.Again, M. smegmatis showed no binding to conA at 19 kDa.