Phylogenetic analyses identify 10 classes of the protein disulfide isomerase family in plants, including single-domain protein disulfide isomerase-related proteins

Protein disulfide isomerases (PDIs) are molecular chaperones that contain thioredoxin (TRX) domains and aid in the formation of proper disulfide bonds during protein folding. To identify plant PDI-like (PDIL) proteins, a genome-wide search of Arabidopsis (Arabidopsis thaliana) was carried out to produce a comprehensive list of 104 genes encoding proteins with TRX domains. Phylogenetic analysis was conducted for these sequences using Bayesian and maximum-likelihood methods. The resulting phylogenetic tree showed that evolutionary relationships of TRX domains alone were correlated with conserved enzymatic activities. From this tree, we identified a set of 22 PDIL proteins that constitute a well-supported clade containing orthologs of known PDIs. Using the Arabidopsis PDIL sequences in iterative BLAST searches of public and proprietary sequence databases, we further identified orthologous sets of 19 PDIL sequences in rice (Oryza sativa) and 22 PDIL sequences in maize (Zea mays), and resolved the PDIL phylogeny into 10 groups. Five groups (I-V) had two TRX domains and showed structural similarities to the PDIL proteins in other higher eukaryotes. The remaining five groups had a single TRX domain. Two of these (quiescin-sulfhydryl oxidase-like and adenosine 5'-phosphosulfate reductase-like) had putative nonisomerase enzymatic activities encoded by an additional domain. Two others (VI and VIII) resembled small single-domain PDIs from Giardia lamblia, a basal eukaryote, and from yeast. Mining of maize expressed sequence tag and RNA-profiling databases indicated that members of all of the single-domain PDIL groups were expressed throughout the plant. The group VI maize PDIL ZmPDIL5-1 accumulated during endoplasmic reticulum stress but was not found within the intracellular membrane fractions and may represent a new member of the molecular chaperone complement in the cell.     

Phylogenetic relationship of Bombyx mori protein disulfide isomerase

A cDNA that encodes protein disulfide isomerase was isolated from Bombyx mori (bPDI), in which an open reading frame of 494 amino acids contained two PDI-typical thioredoxin active sites of WCGHCK and an ER retention signal of the KDEL motif at its C-terminal. The bPDI protein shared less than 55% of the amino acid sequence homology with other reported PDIs. bPDI is most genetically similar to the D. melanogaster PDI. The most serious evolutional diversity was observed between the metazoa and nematoda through PDI evolutional processing. Although bPDI shows a relatively low amino acid homology with other PDIs, in which both sites of the two thioredoxin active sites and the endoplasmic reticulum (ER) retention signal are completely conserved, it was successfully recognized by anti-rat PDI antibodies. This suggests that bPDI may have the activity of a protein isomerase and a chaperone.     

Channel catfish (Ictalurus punctatus) protein disulphide isomerase, PDIA6: molecular characterization and expression regulated by bacteria and virus inoculation

Protein disulfide isomerases (PDIs) are thought to aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Currently, increasing evidence suggests PDIs play an important role in host cell invasion and they are relevant targets for the host immune response. However the roles of specific PDIs in teleosts are little known. Here, we characterized the Protein disulfide isomerase family A, member 6 (PDIA6) from channel catfish, Ictalurus punctatus (named as ccPDIA6). The catfish ccPDIA6 gene was homologous to those of other vertebrate species with 13 exons and 12 introns. The consensus full-length ccPDIA6 cDNA contained an ORF of 1320 bp encoding a putative protein of 439 amino acids. It had a 19 amino acid signal peptide and two active thioredoxin-like domains. Sequence of phylogenic analysis and multiple alignments showed that ccPDIA6 was conserved throughout vertebrate evolution. Southern blot analysis suggested the presence of one copy of the ccPDIA6 gene in the catfish genome. Tissue distribution shows that ccPDIA6 was expressed in all examined tissues at the mRNA level. When using the aquatic zoonotic pathogens such as Edwardsiella tara, Streptococcus iniae, and channel catfish reovirus （CCRV） to challenge channel catfish, ccPDIA6 expression was significant changed in immune-related tissues such as head kidney, intestine, liver and spleen. The results suggested that ccPDIA6 might play an important role in the immunity of channel catfish. This is the first report that the PDI gene may be involved in fish host defense against pathogen infection.     

The evolutionary origins of eukaryotic protein disulfide isomerase domains: new evidence from the Amitochondriate protist Giardia lamblia

A phylogenetic analysis of protein disulfide isomerase (PDI) domain evolution was performed with the inclusion of recently reported PDIs from the amitochondriate protist Giardia lamblia, yeast PDIs that contain a single thioredoxin-like domain, and PDIs from a diverse selection of protists. We additionally report and include two new giardial PDIs, each with a single thioredoxin-like domain. Inclusion of protist PDIs in our analyses revealed that the evolutionary history of the endoplasmic reticulum may not be simple. Phylogenetic analyses support common ancestry of all eukaryotic PDIs from a thioredoxin ancestor and independent duplications of thioredoxin-like domains within PDIs throughout eukaryote evolution. This was particularly evident for Acanthamoeba PDI, Dictyostelium PDI, and mammalian erp5 domains. In contrast, gene duplication, instead of domain duplication, produces PDI diversity in G. lamblia. Based on our results and the known diversity of PDIs, we present a new hypothesis that the five single-domain PDIs of G. lamblia may reflect an ancestral mechanism of protein folding in the eukaryotic endoplasmic reticulum. The PDI complement of G. lamblia and yeast suggests that a combination of PDIs may be used as a redox chain analogous to that known for bacterial Dsb proteins.     

Molecular structure of a yeast gene, PDI1, encoding protein disulfide isomerase that is essential for cell growth.

A genomic DNA clone for protein disulfide isomerase (PDI) of Saccharomyces cerevisiae was isolated by hybridization with synthesized oligonucleotide probes based on a partial amino acid sequence of yeast PDI. The introduction of a multiple copy plasmid carrying this fragment into yeast caused a tenfold increase in PDI specific activity and in the amount of PDI antigen in the extract. The gene on this fragment was named PDI1. The nucleotide sequence of the gene predicts a polypeptide of 522 amino acids with about 30% identity to mammalian PDIs. The predicted amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum retention signal of yeast (HDEL), and two putative active site sequences of PDI (WCGHCK). The predicted polypeptide is acidic and contains five putative glycosylation sites, consistent with the molecular properties of the purified yeast PDI [T. Mizunaga et al. (1990) J. Biochem. 108, 846-851]. The PDI1 gene was mapped on chromosome III. A gene disruption experiment revealed that the PDI1 gene is essential for cell growth.     

Distinct roles of protein disulfide isomerase and P5 sulfhydryl oxidoreductases in multiple pathways for oxidation of structurally diverse storage proteins in rice

In the rice (Oryza sativa) endosperm, storage proteins are synthesized on the rough endoplasmic reticulum (ER), in which prolamins are sorted to protein bodies (PBs) called type-I PB (PB-I). Protein disulfide isomerase (PDI) family oxidoreductase PDIL2;3, an ortholog of human P5, contains a conserved structural disulfide in the redox-inactive thioredoxin-like (TRX) domain and was efficiently targeted to the surface of PB-I in a redox active site-dependent manner, whereas PDIL1;1, an ortholog of human PDI, was localized in the ER lumen. Complementation analyses using PDIL1;1 knockout esp2 mutant indicated that the a and a' TRX domains of PDIL1;1 exhibited similar redox activities and that PDIL2;3 was unable to perform the PDIL1;1 functions. PDIL2;3 knockdown inhibited the accumulation of Cys-rich 10-kD prolamin (crP10) in the core of PB-I. Conversely, crP10 knockdown dispersed PDIL2;3 into the ER lumen. Glutathione S-transferase-PDIL2;3 formed a stable tetramer when it was expressed in Escherichia coli, and the recombinant PDIL2;3 tetramer facilitated α-globulin(C79F) mutant protein to form nonnative intermolecular disulfide bonds in vitro. These results indicate that PDIL2;3 and PDIL1;1 are not functionally redundant in sulfhydryl oxidations of structurally diverse storage proteins and play distinct roles in PB development. We discuss PDIL2;3-dependent and PDIL2;3-independent oxidation pathways that sustain disulfide bonds of crP10 in PB-I.     

Truncation of a Protein Disulfide Isomerase,PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance in Arabidopsis thaliana

Pollen tubes must navigate through different female tissues to deliver sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here, we show that certain T-DNA insertions in Arabidopsis thaliana PDIL2-1, a protein disulfide isomerase (PDI), have reduced seed set, due to delays in embryo sac maturation. Reciprocal crosses indicate that these mutations acted sporophytically, and aniline blue staining and scanning electron microscopy showed that funicular and micropylar pollen tube guidance were disrupted. A PDIL2-1-yellow fluorescent protein fusion was mainly localized in the endoplasmic reticulum and was expressed in all tissues examined. In ovules, expression in integument tissues was much higher in the micropylar region in later developmental stages, but there was no expression in embryo sacs. We show that reduced seed set occurred when another copy of full-length PDIL2-1 or when enzymatically active truncated versions were expressed, but not when an enzymatically inactive version was expressed, indicating that these T-DNA insertion lines are gain-of-function mutants. Our results suggest that these truncated versions of PDIL2-1 function in sporophytic tissues to affect ovule structure and impede embryo sac development, thereby disrupting pollen tube guidance.     

玉米蛋白质二硫键异构酶（PDI）基因的特征和表达

蛋白质二硫键异构酶（PDI）对蛋白的折叠和二硫键的形成起重要的作用.此外,PDI还执行许多其他的生物功能,是1个多功能酶. 本文通过研究玉米中1个PDI基因的特征和表达,探讨它的功能作用. 玉米中的PDI基因编码513个氨基酸.同源分析表明,该基因和水稻、小麦的PDI基因聚为一类,有很高的蛋白相似性.蛋白结构分析表明,该基因具有明显的PDI基因的结构特点,包括硫氧还蛋白活性位点(CGHC)以及内质网定位信号(KDEL).Northern杂交分析显示,该基因在发育种子的表达量高,同时受干旱、冷、ABA和盐等逆境胁迫诱导表达.PDI与GFP融合表达研究基因的亚细胞定位,表明该基因定位在除细胞膜外的细胞质和细胞器上.     

A novel eukaryotic selenoprotein in the haptophyte alga Emiliania huxleyi

The diversity of selenoproteins raises the question of why many life forms require selenium. Especially in photosynthetic organisms, the biochemical basis for the requirement for selenium is unclear because there is little information on selenoproteins. We found six selenium-containing proteins in a haptophyte alga, Emiliania huxleyi, which requires selenium for growth. The 27-kDa protein EhSEP2 was isolated, and its cDNA was cloned. The deduced amino acid sequence revealed that EhSEP2 is homologous to protein disulfide isomerase (PDI) and contains a highly conserved thioredoxin domain. The nucleotide sequence contains an in-frame TGA codon encoding selenocysteine at the position corresponding to the cysteine residue in the reaction center of known PDIs. However, no typical selenocysteine insertion sequence was found in the EhSEP2 cDNA. The EhSEP2 mRNA level was related to the abundance of selenium. E. huxleyi possesses a novel PDI-like selenoprotein and may have a novel type of selenocysteine insertion machinery.     

Characterization of the full-length sequences of phospholipase A2 induced during flower development

The suppression subtractive hybridization (SSH) method was used to isolate developmentally regulated genes during carnation flower maturation. Carnation flower maturation-related clones obtained by the SSH were serially assigned as CFMI (carnation flower maturation-induced) clones. Northern blot analysis showed that several CFMI clones were differentially expressed during flower development. One of the clones, CFMI-3, showed similarity to various animal secretory phospholipases A2 (PLA2). Since little is known about PLA2 gene sequence in plant species, the CFMI-3 clone was selected for further characterization by sequence analysis. Full sequence analysis reveals that the CFMI-3 contains a Ca2+ binding domain, a PLA2 active site, and 12 conserved Cys residues, which is a distinct characteristic of PLA2. Amino acid sequence alignment of CFMI-3 to various putative plant PLA2 confirmed that the CFMI-3 cDNA is the full-length putative PLA2 cDNA identified in plant species.     

Characterization of two protein disulfide isomerases from the endocytic pathway of bloodstream forms of Trypanosoma brucei

Proteins from the endocytic pathway in bloodstream forms of Trypanosome brucei are modified by the addition of linear poly-N-acetyllactosamine side chains, which permits their isolation by tomato lectin affinity chromatography. Antibodies against this tomato lectin binding fraction were employed to screen a cDNA expression library from bloodstream forms of T. brucei. Two cDNAs were prominent among those selected. These cDNAs coded for two putative protein disulfide isomerases (PDIs) that respectively contained one and two double-cysteine redox-active sites and corresponded to a single domain PDI and a class 1 PDI. Assays of the purified recombinant proteins demonstrated that both proteins possess isomerase activity, but only the single domain PDI had a reducing activity. These PDIs possess a number of unusual features that distinguish them from previously characterized PDIs. The expression of both is developmentally regulated, they both co-localize with markers of the endocytic pathway, and both are modified by N-glycosylation. The larger PDI possesses N-glycans containing poly-N-acetyllactosamine, a modification that is indicative of processing in the Golgi and suggests the presence of a novel trafficking pathway for PDIs in trypanosomes. Although generally PDIs are considered essential, neither activity appeared to be essential for the growth of trypanosomes, at least in vitro.     

用质粒pUC18在大肠杆菌中表达人蛋白质二硫键异构酶

 用质粒ｐＵＣ１８在大肠杆菌中表达人蛋白质二硫键异构酶高音,王志珍（中国科学院生物物理研究所,生物大分子国家重点实验室,北京１００１０１）蛋白质二硫键异构酶（ｐｒｏｔｅｉｎｄｉｓｕｌｆｉｄｅｉｓｏｍｅｒａｓｅ,ＰＤＩ）催化蛋白质分子内天然二硫键的形成,...     

A leucine-rich repeat protein of carrot that exhibits antifreeze activity

A gene encoding an antifreeze protein (AFP) was isolated from carrot (Daucus carota) using sequence information derived from the purified protein. The carrot AFP is highly similar to the polygalacturonase inhibitor protein (PGIP) family of apoplastic plant leucine-rich repeat (LRR) proteins. Expression of the AFP gene is rapidly induced by low temperatures. Furthermore, expression of the AFP gene in transgenic Arabidopsis thaliana plants leads to an accumulation of antifreeze activity. Our findings suggest that a new type of plant antifreeze protein has recently evolved from PGIPs.     

Calcium-independent phospholipase A(2): structure and function

The classical Ca(2+)-independent phospholipase A(2) enzyme, now known as Group VIA PLA(2), was initially purified and characterized from the P388D(1) macrophage-like cell line. The corresponding cDNA was subsequently cloned from a variety of sources, and it is now known that multiple splice variants of the enzyme are expressed, some of which may act as negative regulators of the active enzyme. Group VIA PLA(2) has a consensus lipase motif (GTSTG) containing the catalytic serine, is 85-88 kDa, and exists in an aggregated form. The enzyme contains multiple ankyrin repeats, which may play a role in oligomerization. The Group VIA enzyme exhibits lysophospholipase activity as well as phospholipase A(2) activity, and it is capable of hydrolyzing a wide variety of phospholipid substrates. A major function of Group VIA PLA(2) is to mediate phospholipid remodeling, but the enzyme may play other roles as well. Other Ca(2+)-independent PLA(2) enzymes have more recently been identified, and it may be possible to discriminate between the various Ca(2+)-independent PLA(2) enzymes based on sequence or inhibitor-sensitivity. However, the physiological functions of the newly identified enzymes have yet to be elucidated.