Heterologous expression and functional characterization of avian mu-class glutathione S-transferases

Hepatic glutathione S-transferases (GSTs: EC2.5.1.1.8) catalyze the detoxification of reactive electrophilic compounds, many of which are toxic and carcinogenic intermediates, via conjugation with the endogenous tripeptide glutathione (GSH). Glutathione S-transferase (GST)-mediated detoxification is a critical determinant of species susceptibility to the toxic and carcinogenic mycotoxin aflatoxin B₁ (AFB₁), which in resistant animals efficiently detoxifies the toxic intermediate produced by hepatic cytochrome P450 bioactivation, the exo-AFB₁-8,9-epoxide (AFBO). Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB₁, a condition associated with a deficiency of hepatic GST-mediated detoxification of AFBO. We have recently shown that unlike their domestic counterparts, wild turkeys (Meleagris gallopavo silvestris), which are relatively resistant, express hepatic GST-mediated detoxification activity toward AFBO. Because of the importance of GSTs in species susceptibility, and to explore possible GST classes involved in AFB₁ detoxification, we amplified, cloned, expressed and functionally characterized the hepatic mu-class GSTs tGSTM3 (GenBank accession no. JF340152), tGSTM4 (JF340153) from domestic turkeys, and a GSTM4 variant (ewGSTM4, JF340154) from Eastern wild turkeys. Predicted molecular masses of tGSTM3 and two tGSTM4 variants were 25.6 and 25.8 kDa, respectively. Multiple sequence comparisons revealed four GSTM motifs and the mu-loop in both proteins. tGSTM4 has 89% amino acid sequence identity to chicken GSTM2, while tGSTM3 has 73% sequence identity to human GSTM3 (hGSTM3). Specific activities of Escherichia coli-expressed tGSTM3 toward 1-chloro-2,4-dinitrobenzene (CDNB) and peroxidase activity toward cumene hydroperoxide were five-fold greater than tGSTM4 while tGSTM4 possessed more than three-fold greater activity toward 1,2-dichloro-4-nitrobenzene (DCNB). The two enzymes displayed equal activity toward ethacrynic acid (ECA). However, none of the GSTM proteins had AFBO detoxification capability, in contrast to recombinant alpha-class GSTs shown in our recent study to possess this important activity. In total, our data indicate that although turkey hepatic GSTMs may contribute to xenobiotic detoxification, they probably play no role in detoxification of AFBO in the liver.     

Alpha-Class Glutathione S-Transferases in Wild Turkeys (Meleagris gallopavo): Characterization and Role in Resistance to the Carcinogenic Mycotoxin Aflatoxin B1

Domestic turkeys (Meleagris gallopavo) are one of the most susceptible animals known to the toxic effects of the mycotoxin aflatoxin B₁ (AFB₁), a potent human hepatocarcinogen, and universal maize contaminant. We have demonstrated that such susceptibility is associated with the inability of hepatic glutathione S-transferases (GSTs) to detoxify the reactive electrophilic metabolite exo-AFB₁-8,9-epoxide (AFBO). Unlike their domestic counterparts, wild turkeys, which are relatively AFB₁-resistant, possess hepatic GST-mediated AFBO conjugating activity_. Here, we characterized the molecular and functional properties of hepatic alpha-class GSTs (GSTAs) from wild and domestic turkeys to shed light on the differences in resistance between these closely related strains. Six alpha-class GST genes (GSTA) amplified from wild turkeys (Eastern and Rio Grande subspecies), heritage breed turkeys (Royal Palm) and modern domestic (Nicholas strain) turkeys were sequenced, and catalytic activities of heterologously-expressed recombinant enzymes determined. Alpha-class identity was affirmed by conserved GST domains and four signature motifs. All GSTAs contained single nucleotide polymorphisms (SNPs) in their coding regions: GSTA1.1 (5 SNPs), GSTA1.2 (7), GSTA1.3 (3), GSTA2 (3), GSTA3 (1) and GSTA4 (2). E. coli-expressed GSTAs possessed varying activities toward GST substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), cumene hydroperoxide (CHP). As predicted by their relative resistance, livers from domestic turkeys lacked detectable GST-mediated AFBO detoxification activity, whereas those from wild and heritage birds possessed this critical activity, suggesting that intensive breeding and selection resulted in loss of AFB₁-protective alleles during domestication. Our observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are down-regulated, silenced, or otherwise modified by one or more mechanisms. These data may inform of possible molecular mechanisms of resistance to AFB₁, and may also have the benefit of identifying genetic markers which could be used to enhance AFB₁ resistance in modern domestic strains.     

Structure of the extracellular glutathione S-transferase OvGST1 from the human pathogenic parasite Onchocerca volvulus

Onchocerciasis or river blindness, caused by the filarial worm Onchocerca volvulus, is the world’s second leading infectious cause of blindness. In order to chronically infect the host, O. volvulus has evolved molecular strategies that influence and direct immune responses away from the modes most damaging to it. The O. volvulus GST1 (OvGST1) is a unique glutathione S-transferase (GST) in that it is a glycoprotein and possesses a signal peptide that is cleaved off in the process of maturation. The mature protein starts with a 25-amino-acid extension not present in other GSTs. In all life stages of the filarial worm, it is located directly at the parasite-host interface. Here, the OvGST1 functions as a highly specific glutathione-dependent prostaglandin D synthase (PGDS). The enzyme therefore has the potential to participate in the modulation of immune responses by contributing to the production of parasite-derived prostanoids and restraining the host’s effector responses, making it a tempting target for chemotherapy and vaccine development. Here, we report the crystal structure of the OvGST1 bound to its cofactor glutathione at 2.0 Å resolution. The structure reveals an overall structural homology to the haematopoietic PGDS from vertebrates but, surprisingly, also a large conformational change in the prostaglandin binding pocket. The observed differences reveal a different vicinity of the prostaglandin H₂ binding pocket that demands another prostaglandin H₂ binding mode to that proposed for the vertebrate PGDS. Finally, a putative substrate binding mode for prostaglandin H₂ is postulated based on the observed structural insights.     

Naturally occurring chlorophyll derivatives inhibit aflatoxin B1-DNA adduct formation in hepatoma cells

The inhibitory effects of four chlorophyll derivatives (chlorophyllide [Chlide] a and b and pheophorbide [Pho] a and b) on aflatoxin B₁ (AFB₁)-DNA adduct formation, and on the modulation of hepatic glutathione S-transferase (GST) were evaluated in murine hepatoma (Hepa-1) cells. Enzyme-linked immunosorbent assay showed that pretreatment with Chlide or Pho significantly reduced the formation of AFB₁-DNA adducts, and that Pho was the most potent inhibitor. However, wash-out prior to adding AFB₁ totally eliminated inhibition by Childe and partially eliminated inhibition by Pho, indicating that the inhibitory effect of Chlide, and to some extent Pho, was mediated through direct trapping of AFB₁. Furthermore, spectrophotometric analysis showed that Pho treatment could increase GST activity in Hepa-1 cells. These observations indicate that the chlorophyll derivatives studied may attenuate AFB₁-induced DNA damage in the Hepa-1 cell by direct trapping of AFB₁. Pho provided additional protection not only by direct trapping, but also by increasing GST activity against hepatic AFB₁ metabolites.     

A Novel Quasi-Species of Glutathione Transferase with High Activity towards Naturally Occurring Isothiocyanates Evolves from Promiscuous Low-Activity Variants

Glutathione transferases (GSTs) are known as promiscuous enzymes capable of catalyzing the conjugation of glutathione with a broad range of electrophilic substrates. A previous study based on recombinant chimeras derived from human GST M1-1 and GST M2-2 demonstrated the formation of a subset of F1 generation GSTs, which had lost high activity with substrates distinguishing parental enzymes. In the present study, the members of this subset were recombined by DNA shuffling to produce an F2 generation of GSTs. Screening of 930 bacterial clones demonstrated that 83% of recombinant enzyme variants were active with at least one of three alternative substrates: phenethyl isothiocyanate (PEITC), 1-chloro-2,4-dinitrobenzene, or p-nitrophenyl acetate. The majority had similar low activity as the parental GSTs in the F1 generation. However, 17 novel enzymes displayed high activity with PEITC. Half of these enzymes were similar to GST M1-1, which also has high activity with the same substrate, and all of these GSTs featured Tyr116/Ser210 in the active site. This group of F2 variants apparently had reverted to the GST M1-1 type. A second group of F2 variants with high PEITC activity was characterized by His116 in the active site. This category represented a new variety of GSTs, which demonstrated higher selectivity for isothiocyanate substrates than the GST M1-1 type. The different groups of GSTs can be considered as distinct molecular quasi-species, each of which comprises variant amino acid sequences. The quasi-species are structurally distinguished by active-site residues that govern their substrate selectivities. Clearly, minimal alterations of the active site can generate enzymes with highly distinctive functional properties.     

Immunosuppressive effects of aflatoxin in growing rats

The immunosuppressive potential of aflatoxin B₁ (AFB₁), the carcinogenic metabolite ofAspergillus flavus, was evaluated in growing rats. The weanling rats were subchronically exposed to 60, 300 and 600 µg AFB₁/kg body weight for four weeks on alternate days by oral feeding. Various parameters of cell mediated immunity (CMI) and humoral immunity were assessed in control and treated animals. CMI was evaluated by measuring delayed type of hypersensitivity (DTH) response and humoral by plaque forming cell (PFC) assay. The lymphoproliferative response assay for T- and B-cells was also performed. It was observed that AFB₁ selectively suppressed cell mediated immunity in growing rats. AFB₁ suppressed CMI at the 300 and 600 µg dose levels only as measured by DTH response assay. It is concluded that continuous low level exposure of aflatoxin to growing host may enhance its susceptibility to infection and tumorigenesis.Abbreviations AF Aflatoxin - AFB₁ Aflatoxin B₁ - CMI Cell mediated immunity - CPM Counts per minute - DTH Delayed type of hypersensitivity - GST Glutathione-S-transferase - LPS Lipopolysaccharide - PFC Plaque forming cell - PHA Phytohemagglutinin - SRBC Sheep red blood cells     

Developmental studies on the Sigma and Delta-1 glutathione transferases of Lucilia cuprina

The glutathione transferases (GSTs) are a large group of enzymes having both detoxication roles and specialist metabolic functions. The present work represents an initial approach to identifying some of these roles by examining the variation of specific members of the family under differing conditions. The GSTs from Lucilia cuprina have been partially purified, members of two families being isolated, by the use of glutathione immobilised on epichlorhydrin-activated Sepharose 6B. The GSTs were separated by 2D SDS-PAGE and characterised by MALDI-TOF analysis of tryptic peptides. The mass fragments were then matched against the corresponding Drosophila melanogaster and Musca domestica sequences. GSTs were identified as coming from only the Sigma and Delta classes. The multiple Delta zones appear all to be derived from the Lucilia GSTD1 isoform. The distribution of these GST proteins has been studied during different developmental stages of the insect. Delta isoforms were present in all developmental stages of L. cuprina. The Sigma GST was not detectable in the egg, was just detectable in the larval and pupal stages and was the major GST isolated in the adult. Sigma and Delta isoforms were both found in all body segments of the insect. Both isoforms appear to undergo extensive post-translational modification. Activities of the two types of protein with model substrates have been determined.     

A novel glutathione S-transferase isozyme similar to GST 8-8 of rat and mGSTA4-4 (GST 5.7) of mouse is selectively expressed in human tissues

A mouse glutathione S-transferase (GST) isozyme designated as GST 5.7 or mGSTA4-4 belongs to a distinct subclass of the α-class isozymes of GST. It is characterized by kinetic properties intermediate between the α- and π-classes of GSTs. We have recently cloned and expressed this isozyme (rec-mGSTA4-4) in E. coli and have reported its complete primary sequence (Zimniak, P. et al. (1992) FEBS Lett., 313, 173–176). Using antibodies raised against the homogenous rec-mGSTA4-4 expressed in E. coli, we now demonstrate that an ortholog of this isozyme was selectively expressed in various human tissues. The human ortholog of mGST A4-4 purified from liver had a pI value of 5.8 and constituted approx. 1.7% of total GST protein of human liver. Similar to other α-class GSTs, the N-terminus of this isozyme (GST 5.8) was also blocked. CNBr digestion of the enzyme yielded two major fragments with M_r values of 12 kDa and 6 kDa. The sequences of these two fragments showed identities in 16 out of 20 residues and 17 out of 20 residues with the corresponding sequences of its mouse ortholog (mGSTA4-4), and showed significant homologies with the rat and chicken orthologs, GST 8-8 and GST CL3. Human liver GST 5.8 showed more than an order of magnitude higher activity towards t-4-hydroxy-2-nonenal as compared to 1-chloro-2,4-dinitrobenzene. This isozyme also expressed glutathione-peroxidase activity towards fatty acid, as well as phospholipid hydroperoxidase suggesting its role in protection mechanisms against the toxicants generated during lipid peroxidation. Western blot analysis of human tissues revealed that this GST isozyme was selectively expressed in human liver, pancreas, heart, brain and bladder tissues, but absent in lung, skeletal muscle, spleen and colon.     

Proteomic identification, cDNA cloning and enzymatic activity of glutathione S-transferases from the generalist marine gastropod, Cyphoma gibbosum

Glutathione S-transferases (GST) were characterized from the digestive gland of Cyphoma gibbosum (Mollusca; Gastropoda), to investigate the possible role of these detoxification enzymes in conferring resistance to allelochemicals present in its gorgonian coral diet. We identified the collection of expressed cytosolic Cyphoma GST classes using a proteomic approach involving affinity chromatography, HPLC and nano-spray liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two major GST subunits were identified as putative mu-class GSTs; while one minor GST subunit was identified as a putative theta-class GST, apparently the first theta-class GST identified from a mollusc. Two Cyphoma GST cDNAs (CgGSTM1 and CgGSTM2) were isolated by RT-PCR using primers derived from peptide sequences. Phylogenetic analyses established both cDNAs as mu-class GSTs and revealed a mollusc-specific subclass of the GST-mu clade. These results provide new insights into metazoan GST diversity and the biochemical mechanisms used by marine organisms to cope with their chemically defended prey.     

Crystal structure of α-galactosidase from Lactobacillus acidophilus NCFM: insight into tetramer formation and substrate binding

Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite − 1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)₈-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.     

Mycotoxin-Producing Ability and Chemotype Diversity of Aspergillus Section Flavi from Soils of Peanut-Growing Regions in Iran

Invasion of crops with Aspergillus flavus may result in contamination of food and feed with carcinogenic mycotoxins such as aflatoxins (AF) and cyclopiazonic acid (CPA). In the present study, distribution and toxigenicity of Aspergillus flavus and A. parasiticus in soils of five peanut fields located in Guilan province, Northern Iran was investigated. From a total of 30 soil samples, 53 strains were isolated which all of them were finally identified as A. flavus by a combination of colony morphology, microscopic criteria and mycotoxin profiles. Chromatographic analysis of fungal cultures on yeast extract sucrose broth by tip culture method showed that 45 of the 53 A. flavus isolates (84.9 %) were able to produce either CPA or AFB₁, while eight of the isolates (15.1 %) were non-toxigenic. The amounts of CPA and AFB₁ produced by the isolates were reported in the range of 18.2–403.8 μg/g and 53.3–7446.3 μg/g fungal dry weights, respectively. Chemotype classification of A. flavus isolates based on the ability for producing mycotoxins and sclerotia showed that 43.4 % were producers of CPA, AFB₁ and sclerotia (group I), 13.2 % of CPA and AFB₁ (group II), 9.4 % of AFB₁ and sclerotia (group III), 13.2 % of AFB₁ (group IV), 5.7 % of CPA and sclerotia (group V) and 15.1 % were non-toxigenic with no sclerotia (group VI). No strain was found as producer of only CPA or sclerotia. These results indicate different populations of mycotoxigenic A. flavus strains enable to produce hazardous amounts of AFB₁ and CPA are present in peanuts field soils which can be quite important regard to their potential to contaminate peanuts as a main crop consumed in human and animal nutrition.     

Two classes of glutathione S-transferase genes with different response profiles to bacterial challenge in Venerupis philippinarum

Glutathione S-transferase (GST) is major cytosolic detoxification enzymes involved in many pathological and physiological processes. In the present study, two classes of GSTs (VpGST-1 and VpGST-2) were cloned from Venerupis philippinarum haemocytes by cDNA library and RACE approaches. Sequence alignments and phylogenetic analysis together supported that they belonged to a new member of sigma and pi classes GSTs protein family, respectively. The expression profiles of these two genes under Vibrio anguillarum challenge were investigated by quantitative RT-PCR. The bacterial challenge could significantly up-regulate the mRNA expression of both VpGST-1 and VpGST-2 with larger amplitude in VpGST-2, and the feedback speed for VpGST-2 was more rapid than that of VpGST-1. The differences in the response to bacterial challenge indicated that they were functional diversity and probably played cooperative roles in mediating the Vibrio challenge in clam.     

Prevention of cardiotoxicity of aflatoxin B1 via dietary supplementation of papaya fruit extracts in rats

The aim of the current study was to evaluate the cardioprotective ability of water (WE) and ethanolic (EE) papaya fruits extracts against cardiotoxicity induced by aflatoxin B₁ (AFB₁) in rats. Forty two female Sprague–Dawley rats were divided into six treatment groups and treated orally for 2 weeks as follow: control group, the group treated with WE (250 mg/kg b.w), the group treated with EE (250 mg/kg b.w), the group treated orally with AFB₁ (17 μg/kg b.w) and the groups treated orally with AFB₁ plus WE or EE. The results indicated that treatment with AFB₁ resulted in oxidative stress in the heart manifested by the marked increase in cardiac malondialdehyde and calcium levels accompanied with a significant decrease in cardiac total antioxidant capacity. Serum nitric oxide and sodium levels, lactate dehydrogenase and creatine kinase isoenzyme activities were significantly increased, whereas, cardiac Na⁺/K⁺-ATPase activity and serum potassium were insignificantly affected. Supplementation with WE or EE effectively ameliorated most of the changes induced by AFB₁. It could be concluded that both extracts attenuated the oxidative stress induced in heart tissue by AFB₁ and WE was more pronounced due to the higher total phenolic contents than in the EE.     

Relationship between GSTM1 and GSTT1 polymorphisms and schizophrenia: A case–control study in a Tunisian population

There is substantial evidence found in the literature that supports the fact that the presence of oxidative stress may play an important role in the pathophysiology of schizophrenia. The glutathione S-transferases (GSTs) forms one of the major detoxifying groups of enzymes responsible for eliminating products of oxidative stress. Interindividual differences observed in the metabolism of xenobiotics have been attributed to the genetic polymorphism of genes coding for enzymes involved in detoxification. Thus, in this study we investigated the association of glutathione S-transferase Mu-1 (GSTM1) and glutathione S-transferase theta-1 (GSTT1) gene deletion polymorphisms and schizophrenia in a Tunisian population. A case–control study including 138 schizophrenic patients and 123 healthy controls was enrolled. The GSTM1 and GSTT1 polymorphisms were analyzed by multiplex polymerase chain reaction (PCR). No association was found between the GSTM1 genotype and schizophrenia, whereas the prevalence of the GSTT1 active genotype was significantly higher in the schizophrenic patients (57.2%) than in the controls (45.5%) with (OR = 0.6, IC 0.37–0.99, p = 0.039). Thus, we noted a significant association between schizophrenia and GSTT1 active genotype. Furthermore, the combination of the GSTM1 and GSTT1 null genotypes showed a non-significant trend to an increased risk of schizophrenia. The present finding indicated that GSTT1 seems to be a candidate gene for susceptibility to schizophrenia in at least Tunisian population.     

Panax ginseng extract modulates oxidative stress,DNA fragmentation and up-regulate gene expression in rats sub chronically treated with aflatoxin B1 and fumonisin B1

Aflatoxins and fumonisins are important food-borne mycotoxins implicated in human health and have cytotoxic effects. The aims of the current study were to evaluate the protective role of Panax ginseng extract (PGE) against the synergistic effect of subchronic administration of aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) on DNA and gene expression in rat. Female Sprague–Dawley rats were divided into eight groups (ten rats/group) and treated for 12 weeks including the control group, the group having received AFB₁ (80 µg/kg bw), the group having received FB₁ (100 µg/kg bw), the group having received AFB₁ plus FB₁ and the groups having received PGE (20 mg/kg bw) alone or with AFB₁ and/or FB₁. At the end of experiment, liver and kidney were collected for the determination of DNA fragmentation, lipid peroxidation (LP), glutathione (GSH) contents and alterations in gene expression. The results indicated that these mycotoxins increased DNA fragmentation, LP and decreased GSH content in liver and kidney and down-regulated gene expression of antioxidants enzymes. The combined treatments with AFB₁ and/or FB₁ plus PGE suppressed DNA fragmentation only in the liver, normalized LP and increased GSH in the liver and kidney as well as up-regulated the expression of GPx, SOD1 and CAT mRNA. It could be concluded that AFB₁ and FB₁ have synergistic genotoxic effects. PGE induced protective effects against their oxidative stress and genotoxicity through its antioxidant properties.     

Potential for aflatoxin B1 and B2 production by Aspergillus flavus strains isolated from rice samples

In this study, we investigated the potential for aflatoxin B₁ (AFB₁) and B₂ (AFB₂) production in rice grain by 127 strains of Aspergillus flavus isolated from rice grains collected from China. These strains were inoculated onto rice grains and incubated at 28 °C for 21 days. AFB₁ and AFB₂ were extracted and quantified by high-performance liquid chromatography coupled with fluorescence detection. Among the tested strains, 37% produced AFB₁ and AFB₂ with levels ranging from 175 to 124 101 μg kg⁻¹ for AFB₁ and from not detected to 10 329 μg kg⁻¹ for AFB₂. The mean yields of these isolates were 5884 μg kg⁻¹ for AFB₁ and 1968 μg kg⁻¹ for AFB₂. Overall, most of the aflatoxigenic strains produced higher levels of AFB₁ than AFB₂ in rice. The obtained information is useful for assessing the risk of aflatoxin contamination in rice samples.     

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two α subunits and four β subunits with the structure of a double β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (α1, α2, β1, and β2) from T. KS-1. All of them (α1-β1, α2-β1, α1-β2, and α2-β2) exist as α₂β₄ heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the β1 subunit interacted with the chaperonins more strongly than those with the β2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.