Michael A. Kron

Histidyl–tRNA Synthetase and Asparaginyl–tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells

Autoantibodies to histidyl–tRNA synthetase (HisRS) or to alanyl–, asparaginyl–, glycyl–, isoleucyl–, or threonyl–tRNA synthetase occur in ∼25% of patients with polymyositis or dermatomyositis. We tested the ability of several aminoacyl–tRNA synthetases to induce leukocyte migration. HisRS induced CD4+ and CD8+ lymphocytes, interleukin (IL)-2–activated monocytes, and immature dendritic cells (iDCs) to migrate, but not neutrophils, mature DCs, or unstimulated monocytes. An NH2-terminal domain, 1–48 HisRS, was chemotactic for lymphocytes and activated monocytes, whereas a deletion mutant, HisRS-M, was inactive. HisRS selectively activated CC chemokine receptor (CCR)5-transfected HEK-293 cells, inducing migration by interacting with extracellular domain three. Furthermore, monoclonal anti-CCR5 blocked HisRS-induced chemotaxis and conversely, HisRS blocked anti-CCR5 binding. Asparaginyl–tRNA synthetase induced migration of lymphocytes, activated monocytes, iDCs, and CCR3-transfected HEK-293 cells. Seryl–tRNA synthetase induced migration of CCR3-transfected cells but not iDCs. Nonautoantigenic aspartyl–tRNA and lysyl–tRNA synthetases were not chemotactic. Thus, autoantigenic aminoacyl–tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses. Therefore, the selection of a self-molecule as a target for an autoantibody response may be a consequence of the proinflammatory properties of the molecule itself.

Interconversion between two unrelated protein folds in the lymphotactin native state

Proteins often have multiple functional states, which might not always be accommodated by a single fold. Lymphotactin (Ltn) adopts two distinct structures in equilibrium, one corresponding to the canonical chemokine fold consisting of a monomeric three-stranded β-sheet and carboxyl-terminal helix. The second Ltn structure solved by NMR reveals a dimeric all-β-sheet arrangement with no similarity to other known proteins. In physiological solution conditions, both structures are significantly populated and interconvert rapidly. Interconversion replaces long-range interactions that stabilize the chemokine fold with an entirely new set of tertiary and quaternary contacts. The chemokine-like Ltn conformation is a functional XCR1 agonist, but fails to bind heparin. In contrast, the alternative structure binds glycosaminoglycans with high affinity but fails to activate XCR1. Because each structural species displays only one of the two functional properties essential for activity in vivo, the conformational equilibrium is likely to be essential for the biological activity of lymphotactin. These results demonstrate that the functional repertoire and regulation of a single naturally occurring amino acid sequence can be expanded by access to a set of highly dissimilar native-state structures.

An immunodominant antigen of Brugia malayi is an asparaginyl-tRNA synthetase

Lymphatic filariasis is caused by infection with the filarial nematodes Brugia malayi, Brugia timori, Wuchereria bancrofti and Onchocerca volvulus which collectively infect about 200 million persons throughout the world. Protein sequence homology analysis of a major nematode antigen suggested that it was a class II aminoacyl‐tRNA synthetase. The overproduction, purification and verification that the major B. malayi antigen is an asparaginyl‐tRNA synthetase is described.

Tirandamycins from Streptomyces sp. 17944 Inhibiting the Parasite Brugia malayi Asparagine tRNA Synthetase

Lymphatic filariasis is caused by the parasitic nematodes Brugia malayi and Wuchereria bancrofti, and asparaginyl-tRNA synthetase (AsnRS) is considered an excellent antifilarial target. The discovery of three new tirandamycins (TAMs), TAM E (1), F (2), and G (3), along with TAM A (4) and B (5), from Streptomyces sp. 17944 was reported. Remarkably, 5 selectively inhibits the B. malayi AsnRS and efficiently kills the adult B. malayi parasite, representing a new lead scaffold to discover and develop antifilarial drugs.

Severe onchocercal dermatitis in the Ethiopian border region of Sudan

Onchocerca volvulus infection of the residents of two villages in the Upper Atbara River region of Sudan (63.4% prevalence) was found to be medically and socially significant with a remarkable degree of pruritus and dermal pathology present. The severest skin changes, which were often confined to certain areas of the body, were most common in the teenage group (13-19 years). Quantitation of the clinical changes showed that the most severe alterations were present in patients with the lower levels of microfilariae in the skin (0.1-6 mf mg-1) rather than in those with higher levels. Nodules containing adult O. volvulus were found in many individuals, and often in clusters in older patients. Major losses of vision due to O. volvulus were not found, although microfilariae were frequently seen in the cornea and anterior chamber. The commonest corneal change was punctate keratitis, and this was most frequently seen in the teenage group. Trachomatous changes of eyelids and corneas were also seen. The prevalence values of O. volvulus infection in the two villages were significantly increased when, in addition to microfilarial presence in skin biopsies, onchocercal signs such as punctate keratitis, nodule presence and microfilariae in the anterior chamber were taken into consideration. This illustrates the need to consider factors other than skin snip parasitology in obtaining a diagnosis. This study also emphasizes the degree of severity that onchodermatitis can reach and that low levels of dermal microfilariae can be accompanied by very extensive skin changes without any loss of vision. The necessity of considering the severity of skin changes when defining tolerable levels of onchocerciasis in a community is discussed.

A Hybrid Structural Model of the Complete Brugia malayi Cytoplasmic Asparaginyl-tRNA Synthetase

Aminoacyl-tRNA synthetases are validated molecular targets for anti-infective drug discovery because of their essentiality in protein synthesis. Thanks to genome sequencing, it is now possible to systematically study aminoacyl-tRNA synthetases from human eukaryotic parasites as putative targets for novel drug discovery. As part of a program targeting class IIb asparaginyl-tRNA synthetases (AsnRS) from the parasitic nematode Brugia malayi for anti-filarial drugs, we report the complete structure of a eukaryotic AsnRS. Metazoan and fungal AsnRS differ from their bacterial homologues by the addition of a conserved N-terminal extension of about 110 residues whose structure we have determined by solution NMR for the B. malayi enzyme. In addition, we solved by X-ray crystallography a series of structures of the catalytically active N-terminally truncated enzyme (residues 112-548), allowing the structural basis for the mechanism of asparagine activation to be elucidated. The N-terminal domain contains a structured region with a novel fold featuring a lysine-rich helix that is shown by NMR to interact with tRNA. This is connected by an unstructured tether to the remainder of the enzyme, which is highly similar to the known structure of bacterial AsnRS. These data enable a model of the complete AsnRS-tRNA complex to be constructed.

Brugia malayi Asparaginyl–Transfer RNA Synthetase Induces Chemotaxis of Human Leukocytes and Activates G-Protein–Coupled Receptors CXCR1 and CXCR2

Background. Lymphatic filariasis is a chronic human parasitic disease in which the parasites repeatedly provoke acute and chronic inflammatory reactions in the host bloodstream and lymphatics. Excretory-secretory products derived from filariae are believed to play an important role in the development of associated immunologic conditions; however, the specific mechanisms involved in these changes are not well understood. Recently, human cytoplasmic aminoacyl-transfer (t) RNA synthetases, which are autoantigens in idiopathic inflammatory myopathies, were shown to activate chemokine receptors on T lymphocytes, monocytes, and immature dendritic cells by recruiting immune cells that could induce innate and adaptive immune responses. Filarial (Brugia malayi) asparaginyl-tRNA synthetase (AsnRS) is known to be an immunodominant antigen that induces strong human immunoglobulin G3 responses.Methods. Recombinant B. malayi AsnRS was used to perform cellular function assays--for example, chemotaxis and kinase activation assays.Results. Unlike human AsnRS, parasite AsnRS is chemotactic for neutrophils and eosinophils. Recombinant B. malayi AsnRS but not recombinant human AsnRS induced chemotaxis of CXCR1 and CXCR2 single-receptor-transfected HEK-293 cell lines, blocked CXCL1-induced calcium flux, and induced mitogen-activated protein kinase.Conclusions. Our findings suggest that a filarial parasite chemoattractant protein may contribute to the development of chronic inflammatory disease and that chemokine receptors may be therapeutic targets to ameliorate parasite-induced pathology.

Expression, localization and alternative function of cytoplasmic asparaginyl-tRNA synthetase in Brugia malayi

Aminoacyl-tRNA synthetases (AARS) are a family of enzymes that exhibit primary and various secondary functions in different species. In Brugia malayi, the gene for asparaginyl-tRNA synthetase (AsnRS), a class II AARS, previously has been identified as a multicopy gene encoding an immunodominant antigen in the serum of humans with lymphatic filariasis. However, the relative level of expression and alternative functions of AARS in nematode parasites is not well understood. We searched the Filarial Genome Project database to identify the number and amino acid specificity of B. malayi AARS cDNAs to gain insight into the role of different AARS in filaria. These data showed that cytoplasmic AsnRS was present in five gene clusters, and is the most frequently represented member of the aminoacyl-tRNA synthetase family in adult B. malayi. The relative level of AsnRS transcribed in adult female B. malayi was compared to the levels of a low abundance and medium abundance AARS by quantitative real-time RT-PCR. By this method, AsnRS cDNA was 11 times greater than arginyl-tRNA synthetase and methionyl-tRNA synthetase cDNA. In situ hybridization using a B. malayi AsnRS-specific oligonucleotide probe identified abundant cytoplasmic mRNA, particularly in the hypodermis of adult B. malayi. In the absence of tRNA, AsnRS synthesizes diadenosine triphosphate, a potent regulator of cell growth in other eukaryotes. These data support the hypothesis that all AARS are not equally expressed in B. malayi and that these enzymes may demonstrate important alternative functions in filaria.

Lymphatic filariasis in the Philippines.

Lymphatic filariasis caused by Wuchereria bancrofti and Brugia malayi is endemic throughout most of the southern half of the Philippine archipelago. Economic and manpower shortages prior to 1996 made it difficult to acquire new prevalence data and vector control data concurrently from all provinces. Nevertheless, analysis of cumulative prevalence data on filariasis indicates the persistence of filariasis in each of the three major island groups - Luzon, Visayas and Mindanao - including 45 out of 77 provinces. Here, Michael Kron and colleagues summarize the prevalence data, and review host, parasite and vector characteristics relevant to the design and implementation of disease control initiatives in the Philippines planned for the year 2000.

Nematode Asparaginyl-tRNA Synthetase Resolves Intestinal Inflammation in Mice with T-Cell Transfer Colitis

ABSTRACT The therapeutic effects of a controlled parasitic nematode infection on the course of inflammatory bowel disease (IBD) have been demonstrated in both animal and human models. However, the inability of individual well-characterized nematode proteins to recreate these beneficial effects has limited the application of component immunotherapy to human disease. The nematodes that cause chronic human lymphatic filariasis, Brugia malayi and Wuchereria bancrofti, are among the parasites that induce immune suppression. Filarial lymphatic pathology has been shown to involve NF-κB pathway-dependent production of vascular endothelial growth factor (VEGF), and stimulation of VEGF expression has also been reported by interleukin 8 (IL-8) via NF-κB pathways. Previously, we have shown that the filarial asparaginyl-tRNA synthetase (rBmAsnRS) interacts with IL-8 receptors using a combination of extracellular loops that differ from those bound by IL-8. To test the hypothesis that rBmAsnRS might induce an anti-inflammatory effect in vivo, we studied the effects of rBmAsnRS in an established murine colitis model using T-cell transfer mice. T-cell transfer colitis mice treated intraperitoneally with 100 μg of rBmAsnRS four times over 2 weeks showed resolution of cellular infiltration in the colonic mucosa, along with induction of a CD8+ cellular response. In addition, rBmAsnRS induced a rise in IL-10 production from CD3+ and lipopolysaccharide (LPS)- and cytosine phosphate guanosine (CPG)-stimulated splenic cells. In summary, this work demonstrates a novel anti-inflammatory nematode protein, supports the hygiene hypothesis, and supports continued refinement of alternative immunotherapies for treatment of IBD.