Thioredoxin from Brugia malayi: defining a 16-kilodalton class of thioredoxins from nematodes

Abstract

Thioredoxins are a family of small redox proteins that undergo NADPH-dependent reduction by thioredoxin reductase. This results in a supply of reducing equivalents that cells use in a wide variety of biological reactions, which include maintaining reduced forms of the enzymes important for protection against damage from high-energy oxygen radicals, the regulation of transcription factor activity, and the inhibition of apoptosis. Here we report on a new member of the thioredoxin family of proteins from the filarial nematode Brugia malayi, Bm-TRX-1, which defines a new subclass of 16-kDa thioredoxins that occur widely in nematodes, including Caenorhabditis elegans. In addition to being larger than the thioredoxins found in mammalian and bacterial species, the putative active site sequence of Bm-TRX-1, WCPPC, does not conform to the highly conserved WCGPC reported for thioredoxins from mammals to bacteria. Interestingly, an allelic form of Bm-TRX-1 was identified with an active site sequence WCPQC, which appears to be unique to the thioredoxins from filarial species. Bm-TRX-1 was between 98% and 35% identical to thioredoxins from other nematodes and approximately 20% identical to the thioredoxins from mammals and Escherichia coli. Bm-TRX-1 was constitutively transcribed throughout the B. malayi life cycle, and Bm-TRX protein was detectable in somatic extracts and excretory-secretory products from adults and microfilariae. Recombinant Bm-TRX-1 had thiodisulfide reductase activity, as measured by the reduction of insulin, and protected DNA from the nicking activity of oxygen radicals. Overexpression of Bm-TRX-1 in a human monocyte cell line negatively regulated tumor necrosis factor alpha-induced p38 mitogen-activated protein kinase activity, suggesting a possible role of the 16-kDa Bm-TRX-1 in immunomodulation.

FIG. 1.

Alignment of region surrounding the active site of the 16-kDa thioredoxin from B. malayi with comparable regions from 12-kDa class thioredoxins. The thioredoxins tested were Bm-TRX-1, B. malayi, GenBank accession AAM51563; Hs-TRX, Homo sapiens, accession XM_091674 PP27; Tb-TRX-2, Trypanosoma brucei, accession CAA07003; Hs-TRX-1, Homo sapiens, accession AAF86466; Mm-TRX-1, Mus musculus, accession NP_035790; Rn-TRX, Rattus norvegicus, accession NP_446252; Ec-TRX, Equus caballus, accession BAA37154; Ss-TRX, Sus scrofa, accession AAK60272; Sc-TRX, Saccharomyces cerevisiae, accession TXBY1; Oa-TRX, Ovis aries, accession CAA81083; Gg-TRX, Gallus gallus, accession AAA49092; Oh-TRX, Ophiophagus hannah (king cobra), accession AAK09384; Eg-TRX, Echinococcus granulosus, accession AAC14584; Pf-TRX, Plasmodium falciparum, accession CAB90828; Lm-TRX, Leishmania major, accession NP_047052; Tb-TRX-1, Trypanosoma brucei, accession CAB81781; Ecoli-TRX, Escherichia coli, accession P00274. The numbering begins with the initiating methionine for each sequence. Residues highlighted in black are an exact match to the Bm-TRX-1 sequence. Residues highlighted in red are invariant for all of the sequences tested.

FIG. 2.

Alignment of protein sequence of B. malayi thioredoxin with other 16-kDa thioredoxins derived from nematodes. Bm-TRX-1, B. malayi, GenBank accession AY117545; Wb-TRX-1, Wuchereria bancrofti, accession AY142707; Ov-TRX-1, Onchocerca volvulus, accession AY142708; Ov-TRX-2, O. volvulus, accession AY142706; Oc-TRX, O. cervacalis, accession AI363557; Ls-TRX-5, Litomosoides carinii, accession AW152732; As-TRX-1, Ascaris suum, accession BM516048; Ac-TRX, Ancylostoma canimun, accession BM077365; Ts-TRX-1, Trichinella spiralis, accession BG322259; Ce-TRX-5, Caenorhabditis elegans, accession NP_495275; Ce-TRX-6, C. elegans, accession NP_503440; Ce-TRX-8, C. elegans, accession NP_500478; Ss-TRX-1, Strongyloides stercoralis, accession BG226973; Gr-TRX-I, Globodera rostochiensis, accession BM354327; Hg-TRX-1, Heterodera glycines, accession BF013813; Mi-TRX-1, Meloidogyne incognita, accession AW783656; Pp-TRX-1, Pristionchus pacificus, accession AW097135. Residues highlighted in black are an exact match to the Bm-TRX-1 sequence. Residues highlighted in red are invariant for all of the sequences tested. The arrows indicate the positions of the intron-exon junctions found in the Bm-trx-1 genomic sequence (accession AY161166).

FIG. 3.

Bm-TRX-1 is a secreted as a 16-kDa protein by adult and larval stages. First-stage larvae (microfilariae, M) and adult (A) parasites were placed in culture for 18 h. The culture medium was concentrated, and the secreted proteins were separated on a 12% polyacrylamide gel under reducing conditions. Following Western blotting, the nitrocellulose membrane was immunostained with anti-Bm-TRX-1 antibodies. A/ES and M/ES designate lanes containing the excretory-secretory products from adult parasites and microfilariae, respectively. The mass of the recombinant Bm-TRX-1 (lane r) was 17 kDa due to the addition of a 1-kDa T7 tag peptide derived from the expression vector.

FIG. 4.

Two allelic forms of B. malayi thioredoxin catalyze reduction of insulin. The increase in turbidity measured at 650 nm was plotted against reaction time. The conditions under which the catalytic activity of the WCPPCR- and WCPQCR-encoding alleles of Bm-trx-1 were measured in the insulin reduction assay are outlined in the text. Commercially available thioredoxin from Spirulina (Spi-TRX) was used as a positive control. B. malayi-derived SXP (Bm-SXP), bovine serum albumin (BSA), and the proteins extracted from induced, pET11b-containing E. coli (pET11b) were used as negative controls. In addition, the WCPPCR form of Bm-TRX-1 was incubated with anti-Bm-TRX-1 antibodies for 1 h before being added to the reaction mixture (Bm-TRX-1 plus Ab).

FIG. 5.

(A) Bm-TRX-1 functions as an antioxidant to protect DNA from nicking. Plasmid DNA was used as a substrate for detecting DNA damage mediated by the mixed-function oxidase (MFO) system in the presence of 0, 12, 25, 50, or 100 μg of the WCPPCR form of Bm-TRX-1 (lanes 1 through 5, respectively). The extent of MFO-mediated nicking was evaluated on ethidium bromide-stained agarose gels. Recombinant B. malayi SXP (lane 6) and Spirulina thioredoxin (lane 8) served as negative and positive controls, respectively. Anti-Bm-TRX-1 was used to block the antioxidant activity of Bm-TRX-1 (lane 7). NF, nicked form; SF, supercoiled form. (B) Bm-TRX alters clotting time. Thromboplastin was suspended in distilled water and combined with rBm-TRX-1 alone (at 50, 100, or 200 μg/ml), rBm-TRX-1 (100 μg/ml) plus anti-rBm-TRX-1, or Bm-SXP (200 μg/ml) at 37°C. After 30 s, oxalated plasma was added from a pipette, and the tube was gently inverted at 37°C. The time required to form a gel was monitored and taken as the endpoint of the assay. The data are representative of triplicate experiments.

FIG. 6.

Bm-TRX-1 inhibits activation of p38 MAP kinase. The WCPPCR and WCPQCR alleles of Bm-trx-1 were cloned separately into a eukaryotic expression plasmid vector, and the plasmids were introduced into TPA-differentiated ML-1 cells. The transfected ML-1 cells were stimulated with TNF-α (100 IU/well) for 5 min, and the phosphorylation status of p38 MAP kinase was analyzed on immunostained Western blots of cell extracts. Controls included cells maintained in medium (medium control), cells exposed to the transfection regent (transfection control), and cells transfected with a plasmid containing no insert (plasmid control).