Ag85A
Description
Mechanistic Insights
Molecular dynamics simulations reveal that Ag85A/B exhibit greater helical motion in α9 and α5 compared to Ag85C, disrupting the catalytic triad . Mutating Ag85A/B residues to those in Ag85C (e.g., M159W, G160W, F232L) enhances activity by stabilizing helix α9 and triad configuration .
Role in Immune Response and Diagnostics
Monoclonal Antibodies (mAbs)
Five mAbs (1C6, 2E6, 2F2, 3B8, 3D9) specific to Ag85A were generated using recombinant GST-Ag85A as an immunogen . These tools enable:
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Subcellular localization studies of Ag85A in Mtb.
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Diagnostic applications for tuberculosis (TB), as Ag85A induces strong humoral responses in active TB patients .
| mAb | Isotype | Reactivity | Applications |
|---|---|---|---|
| 1C6 | IgG1 | Ag85A-specific | Western blot, immunolocalization |
| 3D9 | IgG1 | Ag85A-specific | Diagnostics, drug candidate testing |
Glycosylation and Immunogenicity
Plant-produced glycosylated Ag85A (G-Ag85A) exhibits enhanced immunogenicity compared to non-glycosylated (NG-Ag85A):
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IFN-γ Production: G-Ag85A induces higher IFN-γ secretion in Mtb-infected mice .
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T Cell Proliferation: G-Ag85A-stimulated dendritic cells promote greater CD4⁺/CD8⁺ T cell expansion .
4. Recombinant Production and Biochemical Properties
Ag85A is produced via baculovirus systems in Hi-5 insect cells, yielding a glycosylated 32.8 kDa protein . Key specifications include:
| Parameter | Value |
|---|---|
| Molecular Mass | 32.8 kDa (305 aa) |
| Expression System | Baculovirus in Hi-5 cells |
| Purity | >95% (SDS-PAGE) |
| Storage | -20°C (with 0.1% HSA/BSA) |
Vaccine Applications
DNA Vaccines
DNA vaccines encoding Ag85A induce robust humoral and cell-mediated immunity:
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Antibody Titers: i.m. vaccination with Ag85A DNA achieves ~75,000 ng/ml in C57BL/10 mice .
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Isotype Profile: Predominantly IgG2a and IgG2b, indicative of Th1 responses .
Subunit Vaccines
Plant-derived G-Ag85A adjuvanted with CAF01 liposomes confers superior protection against Mtb Beijing strain HN878:
Product Specs
Q&A
What experimental approaches validate Ag85A’s dual enzymatic activities (mycolyltransferase vs. diacylglycerol acyltransferase)?
Ag85A exhibits both mycolyltransferase (MyT) and diacylglycerol acyltransferase (DGAT) activities, which are critical for cell wall biosynthesis and lipid storage . To distinguish these functions:
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MyT Activity: Use trehalose 6,6′-dimycolate (TDM) or trehalose 6′-monomycolate (TMM) as substrates in radiolabeled assays with ^14^C-mycolic acids. Monitor transesterification via thin-layer chromatography (TLC) or mass spectrometry .
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DGAT Activity: Employ diacylglycerol (DAG) and palmitoleoyl-CoA in kinetic assays. Measure triacylglycerol (TAG) production using fluorometric kits or ^14^C-acyl-CoA incorporation .
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Competitive Inhibition: Co-incubate TDM and DAG substrates to assess active-site competition. Structural modeling (e.g., molecular docking) identifies overlapping binding regions, as demonstrated by Ser126Ala mutations disrupting both activities .
How do researchers standardize Ag85A detection across heterogeneous mycobacterial strains?
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Antigen Purification: Use His-tagged recombinant Ag85A expressed in E. coli or Mycobacterium smegmatis for consistent yields .
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Antibody Specificity: Validate monoclonal antibodies (mAbs) against epitopes in Ag85A’s N-terminal domain (e.g., residues 84–167), which show minimal cross-reactivity with Ag85B/C .
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Quantitative Western Blotting: Normalize signals using housekeeping proteins (e.g., GroEL) and account for strain-specific post-translational modifications (e.g., glycosylation in plant-derived Ag85A) .
What strategies resolve contradictions in Ag85A-induced immune responses across preclinical models?
Discrepancies in Th1/Th17 polarization and protection efficacy arise from antigen formulation and adjuvant selection. Key considerations:
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Epitope Mapping: Use overlapping peptides (15–20mers) spanning Ag85A’s sequence to identify strain-specific CD4+/CD8+ T-cell epitopes. BALB/c mice predominantly recognize residues 101–120, while C57BL/6 mice respond to C-terminal regions (241–280) .
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Adjuvant Synergy: Pair Ag85A with TLR4 agonists (e.g., MPL-A) to enhance IFN-γ/IL-17 balance, as seen in subunit vaccines like Ag85A-tnPstS1 .
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Table 1: Immune Response Variability in Ag85A Vaccines
How does Ag85A glycosylation impact its immunogenicity in subunit vaccines?
N-glycosylation in plant-derived Ag85A (G-Ag85A) alters antigen processing and MHC-II presentation:
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Glycoengineering: Express Ag85A in Nicotiana benthamiana with α-1,3-fucose and β-1,2-xylose modifications. Confirm glycosylation via Endo-H/PNGase F sensitivity assays .
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Immunogenicity Trade-offs: Glycosylated Ag85A enhances dendritic cell uptake but may mask critical B-cell epitopes. Balance glycan occupancy (e.g., 60–70% occupancy) using site-directed mutagenesis (e.g., Asn-X-Ser/Thr motifs) .
What methodologies optimize Ag85A’s structural stability for crystallography studies?
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Thermal Shift Assays: Screen buffers (pH 6.5–8.0) and additives (e.g., 0.1 M trehalose) to stabilize Ag85A during crystallization .
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Cryo-EM for Flexible Regions: Resolve dynamic N-terminal domains (residues 1–50) using single-particle cryo-EM at 2.8–3.2 Å resolution .
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Molecular Dynamics (MD): Simulate substrate-binding loops (residues 120–140) to identify conformational states favoring mycolic acid vs. DAG binding .
Product Science Overview
Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis (TB), a disease that remains a significant global health challenge. One of the major secretory proteins of M. tuberculosis is Antigen 85A (Ag85A), which is part of the antigen 85 complex. This complex consists of three proteins: Ag85A, Ag85B, and Ag85C, all of which play crucial roles in the pathogenesis and immune response to TB .
Ag85A is a 30-32 kDa protein that possesses enzymatic mycolyl-transferase activity. This enzyme is involved in the synthesis of the mycobacterial cell wall by catalyzing the transfer of mycolic acids to the arabinogalactan layer, which is essential for the formation of the cell wall and the biogenesis of cord factor . The antigen 85 complex proteins are also known for their ability to bind to extracellular matrix proteins such as fibronectin and elastin .
Ag85A is highly immunogenic and has been shown to induce strong T-cell responses, particularly Th1-type immune responses, which are crucial for controlling intracellular infections like TB . This makes Ag85A a promising candidate for TB vaccine development. In fact, Ag85A has been included in several experimental TB vaccines and has shown potential in enhancing the efficacy of the Bacillus Calmette-Guérin (BCG) vaccine .
Recombinant Ag85A refers to the protein produced through recombinant DNA technology, which involves inserting the gene encoding Ag85A into a suitable expression system, such as Escherichia coli or mammalian cells. This allows for the large-scale production of Ag85A for research, diagnostic, and vaccine development purposes .
- Vaccine Development: Recombinant Ag85A is being explored as a component of new TB vaccines. It has been shown to boost the immune response when used in combination with other antigens or adjuvants .
- Diagnostic Tools: Due to its immunogenicity, recombinant Ag85A is used in various diagnostic assays to detect TB infection. These assays include enzyme-linked immunosorbent assays (ELISA), lateral flow immunoassays, and immunoblots .
- Research: Recombinant Ag85A is used in research to study the immune response to TB and to develop new therapeutic strategies .
