Naoko Norioka

Proteomics-based identification of α-enolase as a tumor antigen in non-small lung cancer

Autoantibodies against tumor antigens represent one type of biomarker that may be assayed in serum for detection of cancer and monitoring of disease progression. In the present study, we used a proteomics‐based approach to identify novel tumor antigens in non‐small cell lung cancer (NSCLC). By combining two‐dimensional electrophoresis, western blotting, mass spectrometry and enzyme‐linked immunosorbent assay technology, we detected autoantibodies against α‐enolase in a subset of NSCLC patients’ sera. When ‘Mean ODhealthy control sera + 3 SDhealthy control sera’ was used as the cut‐off point, the prevalence of this autoantibody was 27.7% in patients with NSCLC (26 of 94), 1.7% in healthy control subjects (1 of 60), and not detectable in sera from 15 patients with small cell lung cancer, 18 patients with gastrointestinal cancer and nine patients with Mycobacterium avium complex infection of lung. Immunohistochemical staining showed that expression of α‐enolase was increased in cancer tissues of NSCLC patients, and flow cytometric analysis confirmed the expression of α‐enolase at the surface of cancer cells. The combined detection of autoantibodies against α‐enolase, carcinoembryonic antigen and cytokeratin 19 fragment (CYFRA21‐1) enhanced sensitivity for the diagnosis of NSCLC. Therefore, autoantibodies against α‐enolase may constitute a promising biomarker for NSCLC. (Cancer Sci 2007; 98: 1234–1240)

Potential Role for Purple Acid Phosphatase in the Dephosphorylation of Wall Proteins in Tobacco Cells

It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as α-xylosidase and β-glucosidase. The dephosphorylation and phosphorylation of recombinant α-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of α-xylosidase and β-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls.

Xyloglucan for Generating Tensile Stress to Bend Tree Stem

In response to environmental variation, angiosperm trees bend their stems by forming tension wood, which consists of a cellulose-rich G (gelatinous)-layer in the walls of fiber cells and generates abnormal tensile stress in the secondary xylem. We produced transgenic poplar plants overexpressing several endoglycanases to reduce each specific polysaccharide in the cell wall, as the secondary xylem consists of primary and secondary wall layers. When placed horizontally, the basal regions of stems of transgenic poplars overexpressing xyloglucanase alone could not bend upward due to low strain in the tension side of the xylem. In the wild-type plants, xyloglucan was found in the inner surface of G-layers during multiple layering. In situ xyloglucan endotransglucosylase (XET) activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, began at the inner surface layers S1 and S2 and was retained throughout G-layer development, while the incorporation of xyloglucan heptasaccharide (XXXG) for wall loosening occurred in the primary wall of the expanding zone. We propose that the xyloglucan network is reinforced by XET to form a further connection between wall-bound and secreted xyloglucans in order to withstand the tensile stress created within the cellulose G-layer microfibrils.

Activin A Binds to Perlecan through Its Pro-region That Has Heparin/Heparan Sulfate Binding Activity

Activin A, a member of the transforming growth factor-β family, plays important roles in hormonal homeostasis and embryogenesis. In this study, we produced recombinant human activin A and examined its abilities to bind to extracellular matrix proteins. Recombinant activin A expressed in 293-F cells was purified as complexes of mature dimeric activin A with its pro-region. Among a panel of extracellular matrix proteins tested, recombinant activin A bound to perlecan and agrin, but not to laminins, nidogens, collagens I and IV, fibronectin, and nephronectin. The binding of recombinant activin A to perlecan was inhibited by heparin and high concentrations of NaCl and abolished by heparitinase treatment of perlecan, suggesting that activin A binds to the heparan sulfate chains of perlecan. In support of this possibility, recombinant activin A was capable of directly binding to heparin and heparan sulfate chains. Site-directed mutagenesis of recombinant activin A revealed that clusters of basic amino acid residues, Lys259-Lys263 and Lys270-Lys272, in the pro-region were required for binding to perlecan. Interestingly, deletion of the peptide segment Lys259-Gly277 containing both basic amino acid clusters from the pro-region did not impair the activity of activin A to stimulate Smad-dependent gene expressions, although it completely ablated the perlecan-binding activity. The binding of activin A to basement membrane heparan sulfate proteoglycans through the basic residues in the pro-region was further confirmed by in situ activin A overlay assays using frozen tissue sections. Taken together, the present results indicate that activin A binds to heparan sulfate proteoglycans through its pro-region and thereby regulates its localization within tissues.

Nephronectin binds to heparan sulfate proteoglycans via its MAM domain.

Nephronectin is a basement membrane protein comprising five N-terminal epidermal growth factor (EGF)-like repeats, a central linker segment containing an Arg-Gly-Asp (RGD) motif and a C-terminal meprin-A5 protein-receptor protein tyrosine phosphatase μ (MAM) domain. Nephronectin has been shown to interact with α8β1 integrin through the central linker segment, but its interactions with other molecules remain to be elucidated. Here, we examined the binding of nephronectin to a panel of glycosaminoglycan (GAG) chains. Nephronectin bound strongly to heparin and chondroitin sulfate (CS)-E and moderately to heparan sulfate (HS), but failed to bind to CS-A, CS-C, CS-D, dermatan sulfate and hyaluronic acid. Deletion of the MAM domain severely impaired the binding of nephronectin to heparin but not CS-E, whereas deletion of the EGF-like repeats reduced its binding to CS-E but not heparin, suggesting that nephronectin interacts with CS-E and heparin through the EGF-like repeats and MAM domain, respectively. Consistent with these results, nephronectin bound to agrin and perlecan, which are heparan sulfate proteoglycans (HSPGs) in basement membranes, in HS-dependent manners. Site-directed mutagenesis of the MAM domain revealed that multiple basic amino acid residues in the putative loop regions were involved in the binding of the MAM domain to agrin. The binding of nephronectin to basement membrane HSPGs was further confirmed by in situ nephronectin overlay assays using mouse frozen tissue sections. Taken together, these findings indicate that nephronectin is capable of binding to HSPGs in basement membranes via the MAM domain, and thereby raise the possibility that interactions with basement membrane HSPGs may be involved in the deposition of nephronectin onto basement membranes.

Sequence comparison of the 5' flanking regions of Japanese pear (Pyrus pyrifolia) S-RNases associated with gametophytic self-incompatibility

Abstract Genomic clones of 2.8 kb, 4.3 kb and 6.5 kb for the S2-, S3- and S5-RNases of Japanese pear(Pyrus pyrifolia), respectively, were isolated and sequenced. Comparison of the 5’-flanking regions of these genes with the same region of the S4-RNase gene indicated that a highly similar region of approximately 200 bp exists in the regions just upstream of the putative TATA boxes of the four Japanese pear S-RNase genes. This suggests the presence of cis-regulatory element(s) in this region.

Proteomic analysis of autoantigens associated with systemic lupus erythematosus: Anti-aldolase A antibody as a potential marker of lupus nephritis.

To screen for autoantibodies associated with systemic lupus erythematosus (SLE), we used proteomic approaches combining 2‐D PAGE and Western blot analysis, followed by protein identification by LC‐MS/MS analysis, resulting in the identification of aldolase A as a novel autoantigen in SLE. ELISA showed the prevalence of anti‐aldolase A antibodies to be 29.3% in SLE, 8.2% in rheumatoid arthritis, 18.1% in polymyositis and absent in healthy controls. Furthermore, 43.4% of SLE patients suffering from nephritis showed anti‐aldolase A autoantibodies, which was significantly higher than the prevalence for those without nephritis (11.1%). In lupus nephritis, there are few reliable diagnostic methods, other than urinalysis. Therefore, these results indicate that autoantibodies against aldolase A may serve as an alternative clinical biomarker of SLE associated with nephritis.

Cloning and sequencing of cDNA encoding ribonuclease F1 from Fusarium moniliforme.

Two cDNA clones encoding ribonuclease F1 (EC 3. 1. 27. 3) have been isolated using a probe prepared by polymerase chain reaction with primers designed on the basis of amino acid sequence of the enzyme. They derived probably from the same gene and contained 393‐base pair open reading frame encoding 131 amino acid residues (Mr 13,606) including a putative 25‐residue signal peptide. The sequences of 43‐base pair 5′‐untranslated region and 125‐base pair 3′‐untranslated region including a poly(A) tail of 25 nucleotides were also elucidated. Homology analyses showed that cDNA for ribonuclease F1 has 65% homology to that for ribonuclease T1 in the coding region. At the preprotein level, they share 53% homology.