ABSTRACT
In silico analysis was used to predict MHC class I and class II promiscuous epitopes and potential antigens, from 24 novel T cell antigens of Mycobacterium tuberculosis. Majority of the antigens (16/24) had high affinity peptides to both MHC class I and class II alleles and higher population coverage compared to well-proven T cell antigens ESAT-6, CFP-10 and Ag85B. Among these, highest population coverage were calculated for three novel T cell antigens Rv0733 (97.24%), Rv0462 (96.9%) and Rv2251 (96.3%). The prediction results were experimentally tested by in vitro stimulation of these novel T cell antigens with blood drawn from QuantiFERON-TB Gold In-Tube (QFT-IT) positive healthy household contacts of tuberculosis patients and pulmonary TB patients. Significantly higher level interferon-γ (IFN-γ) was observed, with these novel T cell antigens, in healthy household contacts compared to pulmonary TB subjects (p = 0.0001). In silico analysis also resulted in prediction of 36 promiscuous epitopes from the novel 24 T cell antigens. Population coverage for 4 out of the 36 promiscuous epitopes was >90% [67 VVLLWSPRS (Rv1324), 42 VVGVTTNPS (Rv1448c), 178 MRFLLSAKS (Rv0242c) and 842 IRLMALVEY (Rv3800c)]. Our results shows that these novel antigens and promiscuous epitopes identified from our analysis can further be investigated for their usefulness for subunit vaccine development.
Subject(s)
Epitope Mapping/methods , Mycobacterium tuberculosis/immunology , T-Lymphocytes/immunology , Tuberculosis Vaccines , Tuberculosis, Pulmonary/immunology , Acyltransferases/immunology , Adult , Antigens, Bacterial/immunology , Antigens, Bacterial/isolation & purification , Bacterial Proteins/immunology , Cells, Cultured , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/isolation & purification , Female , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class II/metabolism , Humans , Interferon-gamma/metabolism , Lymphocyte Activation , Male , Middle Aged , Peptide Fragments/immunology , Peptide Fragments/isolation & purification , Protein Binding , Tuberculosis, Pulmonary/prevention & controlABSTRACT
In addition to its well known sedative and teratogenic effects, thalidomide also possesses potent immunomodulatory and antiinflammatory activities, being most effective against leprosy and chronic graft-versus-host disease. The immunomodulatory activity of thalidomide has been ascribed to the selective inhibition of tumor necrosis factor alpha from monocytes. The molecular mechanism for the immunomodulatory effect of thalidomide remains unknown. To elucidate this mechanism, we synthesized an active photoaffinity label of thalidomide as a probe to identify the molecular target of the drug. Using the probe, we specifically labeled a pair of proteins of 43-45 kDa with high acidity from bovine thymus extract. Purification of these proteins and partial peptide sequence determination revealed them to be alpha1-acid glycoprotein (AGP). We show that the binding of thalidomide photoaffinity label to authentic human AGP is competed with both thalidomide and the nonradioactive photoaffinity label at concentrations comparable to those required for inhibition of production of tumor necrosis factor alpha from human monocytes, suggesting that AGP may be involved in the immunomodulatory activity of thalidomide.
Subject(s)
Orosomucoid/metabolism , Thalidomide/metabolism , Thalidomide/pharmacology , Thymus Gland/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , Affinity Labels , Alprenolol/pharmacology , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Azides/chemical synthesis , Azides/metabolism , Binding, Competitive , Cattle , Humans , Iodine Radioisotopes , Mice , Molecular Sequence Data , Monocytes/drug effects , Monocytes/physiology , Orosomucoid/chemistry , Orosomucoid/isolation & purification , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Propranolol/pharmacology , Protein Binding , Sequence Homology, Amino Acid , Thalidomide/analogs & derivatives , Thalidomide/chemical synthesis , Thymus Gland/drug effects , Tissue ExtractsABSTRACT
The gene for Aspergillus niger glucose oxidase (EC 1.1.3.4) has been cloned from both cDNA and genomic libraries using oligonucleotide probes derived from the amino acid sequences of peptide fragments of the enzyme. The mature enzyme consists of 583 amino acids and is preceded by a 22-amino acid presequence. No intervening sequences are found within the coding region. The enzyme contains 3 cysteine residues and 8 potential sites for N-linked glycosylation. The protein shows 26% identity with alcohol oxidase of Hansenuela polymorpha, and the N terminus has a sequence homologous with the AMP-binding region of other flavoenzymes such as p-hydroxybenzoate hydroxylase and glutathione reductase. Recombinant yeast expression plasmids have been constructed containing a hybrid yeast alcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogenase promoter, either the yeast alpha-factor pheromone leader or the glucose oxidase presequence, and the mature glucose oxidase coding sequence. When transformed into yeast, these plasmids direct the synthesis and secretion of between 75 and 400 micrograms/ml of active glucose oxidase. Analysis of the yeast-derived enzymes shows that they are of comparable specific activity and have more extensive N-linked glycosylation than the A. niger protein.