Validating divergent ORF annotation of the Mycobacterium leprae genome through a full translation data set and peptide identification by tandem mass spectrometry.

Mycobacterium leprae has undergone extensive degenerative evolution, with a large number of pseudogenes. It is also the organism with the greatest divergence between gene annotations from independent institutes. Therefore, M. leprae is a good model to verify the currently predicted coding sequence regions between different annotations, to identify new ones and to investigate the expression of pseudogenes. We submitted a total extract of the bacteria isolated from Armadillo to Gel-LC-MS/MS using a linear quadrupole ion trap-Orbitrap mass spectrometer. Spectra were analyzed using the Leproma (1614 genes and 1133 pseudogenes) and TIGR (5446 genes) databases and a database containing the full genome translation. We identified a total of 1046 proteins, including five proteins encoded by previously predicted pseudogenes, which upon closer inspection appeared to be proper genes. Only 11 of the additional annotations by TIGR were verified. We also identified six tryptic peptides from five proteins from regions not considered to be coding sequences, in addition to peptides from two unannotated gene candidates that overlap with other genes. Our data show that the Leproma annotation of M. leprae is quite accurate, and there were no peptide observations corresponding to true pseudogenes, except for a new gene candidate, overlapping with an essential enolase on the complementary strand.

TMP21 regulates Abeta production but does not affect caspase-3, p53, and neprilysin.

The presenilin (PS)-dependent gamma-secretase activity refers to a high molecular mass-complex including, besides PS1 or PS2, three other proteins recently identified, namely nicastrin, Aph-1, and Pen-2. This proteolytic complex has been shown to contribute to both gamma- and epsilon-cleavages of the beta-amyloid precursor protein (betaAPP), thereby generating beta-amyloid peptides (Abeta) and the APP intracellular domain (AICD), respectively. TMP21, a member of the p24 cargo protein family, was recently shown to interact with PS complexes. Interestingly, TMP21 modulates gamma-secretase-mediated Abeta production but does not regulate epsilon-secretase-derived AICD formation [F. Chen, H. Hasegawa, G. Schmitt-ulms, T. Kawarai, C. Bohm, T. Katayama, Y. Gu, N. Sanjo, M. Glista, E. Rogaeva, Y. Wakutami, R. Pardossi-Piquard, X. Ruan, A. Tandon, F. Checler, P. Marambaud, K. Hansen, D. Westaway, P. St. George-Hyslop, P. Fraser, TMP21 is a presenilin complex component that modulates gamma- but not epsilon-secretase activities, Nature 440 (2006) 1208-1212]. Here we investigate the functional incidence of the over-expression or depletion of TMP21 on both intracellular and secreted Abeta recoveries and AICD-associated phenotypes. First we confirm that TMP21 depletion yields increased levels of secreted Abeta40. However, we demonstrate that both staurosporine-stimulated caspase-3 activation, p53 and neprilysin expression and activity were not affected by TMP21 over-expression or depletion. Overall, our functional data further reinforce the view that TMP21 behaves as a regulator of gamma- but not epsilon-cleavages generated by PS-dependent gamma-secretase complex.

The Mycobacterium leprae hsp65 displays proteolytic activity. Mutagenesis studies indicate that the M. leprae hsp65 proteolytic activity is catalytically related to the HslVU protease.

The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (beta-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P(1), although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli HslVU protease suggested two putative threonine catalytic groups, one in the N-domain (T(136), K(168), and Y(264)) and the other in the C-domain (T(375), K(409), and S(502)). Mutagenesis studies showed that the replacement of K(409) by A caused a complete loss of the proteolytic activity, whereas the mutation of K(168) to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T(375), K(409), and S(502) at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.

Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae.

The cell wall of pathogenic mycobacteria is abundant with complex glycolipids whose roles in disease pathogenesis are mostly unknown. Here, we provide evidence for the involvement of the specific trisaccharide unit of the phenolic glycolipid-1 (PGL-1) of Mycobacterium leprae in determining the bacterial predilection to the peripheral nerve. PGL-1 binds specifically to the native laminin-2 in the basal lamina of Schwann cell-axon units. This binding is mediated by the alpha(2LG1, alpha2LG4, and alpha2LG5 modules present in the naturally cleaved fragments of the peripheral nerve laminin alpha2 chain, and is inhibited by the synthetic terminal trisaccharide of PGL-1. PGL-1 is involved in the M. leprae invasion of Schwann cells through the basal lamina in a laminin-2-dependent pathway. The results indicate a novel role of a bacterial glycolipid in determining the nerve predilection of a human pathogen.

Structural requirements of peptide and MHC for DR(alpha, beta 1*0401)-restricted T cell antigen recognition.

We identified functionally important regions of the DR(alpha, beta 1*0401) peptide binding site and present a model of bound peptide. DR(alpha, beta 1*0401)-restricted T cell recognition and peptide binding of Mycobacterium leprae (ML) peptide 38-50 and overlapping peptides from the 18-kDa heat-shock protein were analyzed. ML38-50 is unusual in its restricted binding pattern, binding to only one of five DR4 subtypes and no other DR molecules tested. Amino acid substitutions were introduced into ML38-50 and the DR(alpha, beta 1*0401) peptide binding site at positions likely to influence peptide-MHC or peptide- or MHC-TCR interactions. Peptide binding, T cell proliferation, and computer modeling studies suggest that residues 39F, 42E, and 44D of ML38-50 interact with pockets 1, 4, and 6, respectively, of the peptide binding site. Only DR(alpha, beta 1*0401) substitutions at residues in pockets 4 or 7 prevented binding of ML38-50, while multiple substitutions at other positions negatively affected its T cell recognition. In contrast, T cell recognition of some high affinity ML peptides that overlapped ML38-50, and contained N-terminal extensions, was only abolished with pocket 4 substitutions. An inverse correlation of peptide affinity for DR(alpha, beta 1*0401) with negative effects of MHC substitutions on T cell recognition of the overlapping ML peptides was observed. Thus, some regions, such as pocket 4, dominantly influence T cell recognition of multiple DR(alpha, beta 1*0401)-binding peptides. However, each DR(alpha, beta 1*0401)-binding peptide appears to have unique properties that determine the outcome of its MHC-peptide interactions and the relative importance of other polymorphic pockets.

Identification of an antigenic domain on Mycobacterium leprae protein antigen 85B, which is specifically recognized by antibodies from patients with leprosy.

Sixty-three overlapping 15-oligomer peptides covering the 30-kDa protein antigen 85B of Mycobacterium leprae were tested by ELISA to identify epitopes recognized by human antibodies. Serum samples from patients with lepromatous leprosy (LL) reacted mainly with peptides comprising amino acid regions (AA) 206-230, 251-280, and 291-325. Sera of patients with active tuberculosis who responded to the native 30-kDa antigen did not recognize these peptides. The antibody-binding specificity to the defined B cell regions was evaluated in a blind study with 71 serum samples from patients and household contacts living in Ethiopia where leprosy is endemic. The peptide of AA 256-280 was recognized by 88% of LL patients, 15% of patients with tuberculoid leprosy, and none of the contacts. These findings suggest that there are major linear B cell epitopes on the M. leprae 30-kDa protein that are recognized by lepromin-negative LL patients, whereas lepromin-positive patients respond preferentially to conformational epitopes.

Mechanism of interaction of the 85B secreted protein of Mycobacterium bovis with fibronectin.

The 85B protein of Mycobacterium bovis is a member of the secreted antigen 85 complex, which has been identified in a number of pathogenic mycobacteria. The 85 complex contains three components with molecular masses of 30 to 32 kDa which share the property of binding to fibronectin, a large glycoprotein present in plasma. To investigate this activity we have expressed the M. bovis 85B antigen as a recombinant protein and studied its interaction with human fibronectin. Fibronectin bound to the immobilized 85B protein in a solid-phase enzyme-linked immunosorbent assay (ELISA) and in the fluid phase in a radioimmunoassay using 125I-labelled 85B protein. In addition, fibronectin reacted with immobilized 85B in immunoblots and vice versa. Fibronectin also bound to three fragments of a cyanogen bromide digest of 85B which were subsequently identified by N-terminal sequencing. These fragments contained fibronectin-reactive peptides identified in ELISAs utilizing a set of 28 overlapping 20-mer peptides encompassing the 85B sequence. Further studies showed that the 85B protein reacted with a 32-kDa polypeptide from a limited tryptic digest of fibronectin which was identified as the collagen-binding domain. This region was confirmed as the 85B binding site by the fact that gelatin but not heparin inhibited the binding of fibronectin to 85B. These data indicate that the 85B-fibronectin interaction involves the binding of multiple regions of the 85B protein to the collagen-binding domain of fibronectin.

Immunological and functional characterization of proteins of the Mycobacterium tuberculosis antigen 85 complex using synthetic peptides.

As tuberculosis re-emerges as an important health problem worldwide, new drugs, better diagnostic tests and vaccines are being sought. In order to identify potentially useful peptides for the development of a synthetic vaccine against tuberculosis, immunological and functional studies were performed using proteins of the antigen 85 complex. Western blot (immuno-blot) analysis and a lymphoproliferation study was used to investigate the B- and T-cell immune response of tuberculosis patients, healthy household contacts and normal controls to proteins of the Mycobacterium tuberculosis antigen 85 complex. Peptides derived from the 85A amino acid sequence were synthesized and used in fibronectin-binding and in ELISA assays. A peptide with the sequence CQPACRKAGCQTYKWEC bound to radiolabelled fibronectin in a time-dependent manner and was recognized by human sera in ELISA. This peptide was identified as a potential component of a synthetic vaccine against tuberculosis.

Evolutionary conservation of major histocompatibility complex-DR/peptide/T cell interactions in primates.

Many major histocompatibility complex (MHC) polymorphisms originate from ancient structures that predate speciation. As a consequence, members of the Mhc-DRB1*03 allelic lineage are not only present in humans but in chimpanzees and rhesus macaques as well. This emphasizes that Mhc-DRB1*03 members must have been present in a common ancestor of these primate species that lived about 30 million years ago. Due to the accumulation of genetic variation, however, alleles of the Mhc-DRB1*03 lineage exhibit species-unique sequences. To investigate the biological importance of such conservation and variation, we have studied both the binding and antigen presentation capacity of various trans-species Mhc-DRB1*03 lineage members. Here we show that p3-13 of the 65-kD heat-shock protein (hsp65) of Mycobacterium leprae and M. tuberculosis binds not only to HLA-DR17(3) but also to some chimpanzee and rhesus macaque class II-positive cells. Comparison of the corresponding human, chimpanzee, and rhesus macaque Mhc-DRB1*03 lineage members revealed the presence of uniquely shared amino acid residues, at positions 9-13 and 26-31, of the antigen-binding site that are critical for p3-13 binding. In addition it is shown that several nonhuman primate antigen-presenting cells that bind p3-13 can activate HLA-DR17-restricted T cells. Certain amino acid replacements, however, in Mhc-DRB1*03 lineage members did not influence peptide binding or T cell recognition. Therefore, these studies demonstrate that some polymorphic amino acid residues (motifs) within the antigen-binding site of MHC class II molecules that are crucial for peptide binding and recognition by the T cell receptor have been conserved for over 30 million years.

Phenylalanine hydroxylase-stimulating protein/pterin-4 alpha-carbinolamine dehydratase from rat and human liver. Purification, characterization, and complete amino acid sequence.

Phenylalanine hydroxylase-stimulating protein, also known as pterin-4 alpha-carbinolamine dehydratase (PHS/PCD), was purified from rat and, for the first time, from human liver. We obtained their complete protein primary sequence using a combination of liquid secondary ionization mass spectrometry/tandem quadrupole mass spectrometry, electrospray ionization mass spectrometry, and Edman microsequence analysis. The amino acid sequences of human and rat PHS/PCD were found to be identical. Surprisingly, the primary structure of PHS/PCD is also essentially identical to a protein of the cell nucleus, named dimerization cofactor of hepatocyte nuclear factor 1 alpha, recently reported to be involved in transcription (Mendel, D. M., Khavari, P. A., Conley, P. B., Graves, M. K., Hansen, L. P., Admon, A., and Crabtree, G. R. (1991) Science 254, 1762-1767).

Nerve lesions induced by macrophage activation.

The neuropathies associated with infectious processes, including leprosy, retroviral infections and Chagas' disease, represent the largest group of neuropathies in the world. Segmental demyelination and axonal degeneration of nerve fibres are associated with inflammatory infiltrates which contain a large number of mononuclear phagocytes. In order to learn more about the role played by macrophage activation in the nerve lesions observed in inflammatory neuropathies, we have performed a morphological study of nerves injected with products of activation of macrophages including proteolytic enzymes and cytokines (tumour necrosis factor and alpha beta-interferon). We have also studied the effects on nerve fibres of macrophages activated by ingestion of proteose-peptone, a foreign protein, and in the course of a delayed-type hypersensitivity (DTH) reaction. We have found that proteases and urokinase were potent demyelinating agents and that activated macrophages were also able to induce significant demyelination of neighbouring fibres. In contrast, injection of TNF alpha induced more severe nerve lesions consisting of axonal degeneration of the majority of nerve fibres. We thus conclude that infected macrophages which penetrate the endoneurium and macrophages activated in a DTH reaction can both cause neuropathy.

Changes in nerves and neuropeptides in skin from 100 leprosy patients investigated by immunocytochemistry.

The cutaneous innervation is now known to contain neuropeptides including substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory nerves, and vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), principally in autonomic nerves. Skin biopsies from 100 leprosy patients and equivalent areas from 50 non-leprosy controls were fixed in p-benzoquinone solution for immunofluorescence staining and in Bouin's fluid for classification of leprosy type. Antisera to the neural markers, neurofilaments, and protein gene product 9.5 (PGP 9.5), and to neuropeptides were used. Cutaneous nerves and nerve endings immunoreactive for neuropeptides, neurofilaments, and PGP 9.5 were seen in all non-leprous control cases. In leprosy, PGP 9.5- and neurofilament-immunoreactive nerve fibres were seen in all 14 cases of the indeterminate (early) type and in the majority (33/43) of lepromatous cases, but in a smaller proportion (15/43) of tuberculoid cases. Neuropeptide immunoreactivity was seen in only 2/14 of the indeterminate leprosy specimens and was completely absent in other types. This early disappearance may be of diagnostic significance. Thus, cutaneous sensory and autonomic dysfunctions in leprosy are well reflected by changes in nerve fibres and neuropeptides.