Hiroyuki Kaji

Glycoproteomic discovery of serological biomarker candidates for HCV/HBV infection-associated liver fibrosis and hepatocellular carcinoma.

We previously proposed a high-throughput strategy to discover serological biomarker candidates of cancer. This strategy focuses on a series of candidate glycoproteins that are specifically expressed in the original tissues (cells) of the target cancer and that carry glycan structures associated with carcinogenesis [Narimatsu, H., et al. FEBS J.2010, 277(1), 95-105]. Here, we examined the effectiveness of our strategy in identifying biomarkers to assess progression of liver fibrosis and for the early detection of hepatocellular carcinoma (HCC). On the basis of the results of lectin array analyses in culture media of hepatoma cell lines, we captured glycopeptides carrying AAL-ligands (fucosylated glycans) or DSA-ligands (branched glycans) from digests of culture media proteins and sera from HCC patients with a background of liver cirrhosis (LC). Glycoproteins were identified by the IGOT-LC-MS method. In all, 21 candidates were selected from 744 AAL-bound glycoproteins for further verification according to (i) their abundance in serum, (ii) their specific expression in liver, and (iii) the availability of antibodies to the glycoproteins. All selected candidates showed enhancement of AAL-reactivity in sera of HCC patients compared with that of healthy volunteers (HV). These results indicate that our glycoproteomic strategy is effective for identifying multiple glyco-biomarker candidates in a high-throughput manner.

Processing of Amyloid β-Peptides by Neutral Cysteine Protease Bleomycin Hydrolase

We studied the processing of amyloid beta-peptides (Abetas) including Abeta(1-40), Abeta(1-42) and pAbeta(3-42) by rat neutral cysteine protease bleomycin hydrolase (BH) according to the methods of SDS-PAGE, HPLC and matrix-assisted laser desorption/inonization time-of-flight mass spectrometry (MALDI-TOF MS). BH significantly processed them by novel features of its diverse activities. It initially cleaved at two sites, His(14)-Gln(15) and Phe(19)-Phe(20) degraded to short intermediates then to amino acids by aminopeptidase and/or carboxypeptidase activities. Also, full-length Abetas were clipped at the carboxyl(C)-terminal region. On the other hand, BH cleaved at only the His(14)-Gln(15) bond in pbetaA(3-42) within a short period of the reaction by endopeptidase activity, and processed the intermediates in order by carboxypeptidase activity. On processing by BH, it found that both fibrillar Abeta(1-40) and Abeta(1-42) were more resistant than non-fibrillar peptides. These results indicate that the processing specificity of BH depends upon the structure and sequence of Abetas.

Large-Scale Identification of N-Glycan Glycoproteins Carrying Lewis x and Site-Specific N-Glycan Alterations in Fut9 Knockout Mice.

The Lewis x (Le(x)) structure (Galβ1-4(Fucα1-3)GlcNAc-R) is a carbohydrate epitope comprising the stage-specific embryonic antigen-1 (SSEA-1) and CD15, and it is synthesized by α1,3-fucosyltransferase 9 (Fut9). Fut9 is expressed specifically in the stomach, kidney, brain, and in leukocytes, suggesting a specific function in these tissues. In this study, the N-linked glycan mass spectrometry profile of wild-type mouse kidney glycoproteins revealed the presence of abundant terminal fucoses, which were lost following knockout of the Fut9 gene; the terminal fucose was therefore concluded to be Le(x). These results suggested that Le(x) presence is widespread rather than being limited to specific proteins. We endeavored to comprehensively identify the Le(x) carriers in the mouse kidney. Glycopeptides carrying fucosylated glycans were collected by Aleuria aurantia lectin (AAL) affinity chromatography from kidney homogenates of wild-type and Fut9 knockout mice. The site-specific N-glycomes on the glycopeptides were subsequently analyzed by adopting a new glycoproteomic technology composed of dissociation-independent assignment of glycopeptide signals and accurate mass-based prediction of the N-glycome on the glycopeptides. Our analyses demonstrated that 24/32 glycoproteins contained the Le(x) N-glycan structure in wild-type kidney; of these, Le(x) was lost from 21 in the knockout mice. This is the first report of large-scale identification of Le(x)-carrying glycoproteins from a native sample based on the site-specific glycome analysis.

Conformation of Bilirubin Oxidase in Native and Denatured States

The conformation of bilirubin oxidase (EC 1.3.3.5) fromMyrothecium verrucaria was studied by circular dichroism (CD). The far-UV CD spectrum showed a single minimum at 215 nm and a maximum near 198 nm, suggesting the dominance ofβ-sheets. There was another negative band at 187 nm that is absent from the spectra of modelα-helix orβ-sheet. CD analysis by the method of Changet al. agreed well with the estimates based on the Chou and Fasman sequence-predictive method, but the Provencher-Glöckner method of CD analysis agreed well with the sequence-predictive method of Garnieret al. AtpH 12 the 215- and 187-nm bands completely disappeared and the protein was denatured. This denaturation was accompanied by the appearance of a large positive band at 250 nm, probably due to ionization of tyrosine residues. In 20 mM sodium dodecyl sulfate the magnitude of the 215-nm band increased, but the spectrum transformed to that of partial helices after heating at 100°C. In 6 M guanidine hydrochloride the far-UV CD spectrum was monotonic and became more negative at the lower wavelength limit (near 212 nm), suggesting that the secondary structure of the protein was disrupted. However, the near-UV CD spectrum retained residual aromatic bands even after heating at 100°C. Thus, our denaturation studies suggest that bilirubin oxidase has a rigid tertiary structure.

Engineering of recombinant Wisteria floribunda agglutinin specifically binding to GalNAcβ1,4GlcNAc (LacdiNAc)

Wisteria floribunda agglutinin (WFA) is a useful probe for distinguishing glycan structural alterations in diseases such as intrahepatic bile duct carcinoma and hepatic fibrosis; however, the gene encoding WFA has not been identified. Here, we identified the gene encoding WFA, and recombinant WFA was expressed in Escherichia coli and purified. The natural cDNA sequence obtained from wisteria seeds contained an open reading frame of 861 nucleotides encoding a WFA precursor, which included a hydrophobic signal peptide at the N-terminus, a propeptide at the C-terminus and a single cysteine (Cys) residue for dimer formation. We characterized the natural and recombinant WFA by the glycoconjugate microarray and frontal affinity chromatography. Recombinant WFA exhibited glycan binding specificity similar to that of natural WFA: both bound to Gal- and GalNAc-terminated glycans. Moreover, the engineered WFA with an amino acid substitution in Cys-272 yielded a recombinant monomeric lectin with limited binding specificity but wild-type affinity for GalNAc-terminated glycans, specifically GalNAcβ1,4GlcNAc. Thus, this engineered lectin may be applied to highly sensitive biomarker detection.

Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1

The anti-mesothelioma mAb SKM9-2 recognizes the sialylated protein HEG homolog 1 (HEG1). HEG1 is a 400 kDa mucin-like membrane protein found on mesothelioma. SKM9-2 can detect mesothelioma more specifically and sensitively than other antibodies against current mesothelioma markers; therefore, SKM9-2 would be likely useful for the precise detection and diagnosis of malignant mesothelioma. In the present study, we investigated the epitope of SKM9-2. We analyzed the binding of SKM9-2 to truncated HEG1 and candidate epitope-fused glycosylphosphatidylinositol-anchor proteins. The epitope of SKM9-2 was identified as an O-glycosylated region, 893-SKSPSLVSLPT-903, in HEG1. An alanine scanning assay of the epitope showed that SKM9-2 bound to a simple epitope in HEG1, and the SKxPSxVS sequence within the epitope was essential for SKM9-2 recognition. Mass spectrometry analysis and lectin binding analysis of soluble epitope peptides indicated that the SKM9-2 epitope, in which Ser897 was not glycosylated, contained two disialylated core 1 O-linked glycan-modified serine residues, Ser893 and Ser900. Neuraminidase treatment analysis also confirmed that the epitope in mesothelioma cells contained a similar glycan modification. The specific detection of mesothelioma with SKM9-2 can thus be performed by the recognition of sialylated glycan modification in the specific region of HEG1.

Reaction of 2-Deoxy-2-C-(3-bromoacetoxypropyl)-α-D-arabinofuranosides with Oligonucleotide1

Abstract Reaction of methyl 2-deoxy-2-C-(3-bromoacetoxypropyl)-α-D-arabinofuranosides, prepared from methyl 2,3-anhydro-α-D-ribofuranoside, with oligodeoxyribonucleotide (21mer) in acetonitrile-H2O (pH 7) and subsequent treatment with piperidine resulted in the cleavage of the nucleotide chain at the position G, A, and C.