Identification of new autoantigens for primary biliary cirrhosis using human proteome microarrays.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and is considered to be an autoimmune disease. Autoantibodies are important tools for accurate diagnosis of PBC. Here, we employed serum profiling analysis using a human proteome microarray composed of about 17,000 full-length unique proteins and identified 23 proteins that correlated with PBC. To validate these results, we fabricated a PBC-focused microarray with 21 of these newly identified candidates and nine additional known PBC antigens. By screening the PBC microarrays with additional cohorts of 191 PBC patients and 321 controls (43 autoimmune hepatitis, 55 hepatitis B virus, 31 hepatitis C virus, 48 rheumatoid arthritis, 45 systematic lupus erythematosus, 49 systemic sclerosis, and 50 healthy), six proteins were confirmed as novel PBC autoantigens with high sensitivities and specificities, including hexokinase-1 (isoforms I and II), Kelch-like protein 7, Kelch-like protein 12, zinc finger and BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1. To facilitate clinical diagnosis, we developed ELISA for Kelch-like protein 12 and zinc finger and BTB domain-containing protein 2 and tested large cohorts (297 PBC and 637 control sera) to confirm the sensitivities and specificities observed in the microarray-based assays. In conclusion, our research showed that a strategy using high content protein microarray combined with a smaller but more focused protein microarray can effectively identify and validate novel PBC-specific autoantigens and has the capacity to be translated to clinical diagnosis by means of an ELISA-based method.

Isolation, characterization and expression analysis of a leaf-specific phosphoenolpyruvate carboxylase gene in Oryza sativa.

Suppression subtractive hybridization was carried out to enrich gene fragments over-expressed in rice leaves by subtraction to rice roots, from which two identical cDNA fragments were identified to encode putative phosphoenolpyruvate carboxylase. Then the corresponding full-length cDNA (Osppc) is isolated by RT-PCR and sequenced, which indicates an open reading frame of 2895bp is contained. Its deduced protein is encoded in 10 exons and shows high similarity to many other plant PEPCs. Comparing with maize and bacterial PEPCs, it is revealed that OSPPC shares many conserved domains and active sites that responsible for the structure, activity and regulation of this enzyme. Phylogenetic analysis demonstrates that OSPPC is grouped with C3 form PEPCs of wheat, maize and sorghum, which is consistent with the classification of rice. And a putative promoter element is predicted with DOF binding box, CAAT box and TATA box in the 5'-flanking sequence of Osppc gene. Moreover, Quantitative RT-PCR analyses are performed in hybrid rice and its parents, which show that Osppc is specifically expressed in leaf including leaf vein and sheath.

Cloning, characterization and prokaryotic expression of cytosolic malate dehydrogenase from Oryza sativa.

cDNA sequences of malate dehydrogenase (MDH) were cloned from various species, and MDH was identified to play an important role in cell energy metabolism. Here, we present the isolation and characterization of its homologue (OscMDH) in Oryza sativa. Comparison of the results to the genome details indicated that OscMDH consisted of seven exons. Sequence alignment showed that the deduced amino acid sequence of OscMDH shared a significant similarity with cMDH protein in Zea mays, as well as with other cMDH gene products. The different expression patterns of OscMDH in different tissues revealed that OscMDH mRNA was highly transcribed in either young panicle or immature seed, while its abundance was much low in green leaf and root. A nearly 56-kDa fusion protein generated by adding a Trx-His-tag at the N-terminal of OscMDH was induced by IPTG in Escherichia coli BL21 and an obvious MDH activity was detected in the protein by native PAGE analysis. All these results suggest that OscMDH encodes a cytosolic MDH in rice.

cDNA cloning and expression analysis of the rice (Oryza sativa L.) RNase L inhibitor.

A subtractive cDNA library was constructed using rice (Oryza sativa L.) callus cDNA as driver and differentiating callus cDNA as tester. A novel cDNA fragment encoding RNase L inhibitor (RLI) was isolated by screening the subtractive library, which had a higher expression level in differentiating callus than in callus. The full-length cDNA of rice-RLI was obtained by the method of rapid amplification of cDNA ends, which contained a 1812-bp open reading frame encoding a 604 amino acid polypeptide. Homologous analysis showed that rice-RLI contained the conserved motifs (two repeated P-loops, two ATP-binding boxes and an iron-sulfur binding motif). The fluorescence quantitative PCR analysis showed that it was a constitutive expressed gene but up-regulated in abiotic stress (low temperature and NaCl treatment) and down-regulated under the treatments of NAA and IAA.

Cloning and prokaryotic expression of a cDNA encoding a putative mitochondrial malate dehydrogenase in Oryza sativa.

Malate dehydrogenase (MDH) has been characterized as a key player in oxaloacetate (OAA) biosynthesis mechanism in citrate acid cycle that generates reducing powers for further assimilation in the whole cell. Here we present the cloning, characterization and prokaryotic expression of a putative Mdh (OsmMDH) in Oryza sativa. Sequence alignment shows that there is a high homology between the deduced amino acid sequence of OsmMDH and MDH portein in Eucalyptus gunnii (80%), as well as between the deduced amino acid sequence of OsmMDH and other MDHs. Moreover, pI and the mitochondrial location of OsmMDH are predicted. The tissue-specific expression pattern of OsmMDH reveals that it is abundant in young panicle and immature seed, while its expression level is mush lower in leaf and root. Its expression in E. coli BL21 as a fusion gene is studied further.

Cloning and characterization of a novel Hsp100/Clp gene (osClpD) from Oryza sativa.

A novel osClpD gene, encoding a highly conservative ClpD subfamily member, was first isolated and characterized from Oryza sativa. The full-length cDNA of osClpD gene was 3140 bp and contained a 2884 bp open reading frame encoding a 938 amino acid protein. The phylogenetic tree and blast search showed that OSClpD belonged to the ClpD subfamily of the Hsp100/Clp family, and contained all protein motifs characteristic for the ClpD subfamily of Hsp100/Clp proteins. The real-time quantitative PCR analysis proved that it was inducible by water deficit and temperature stress in vegetative tissues.