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.

Multilectin assay for detecting fibrosis-specific glyco-alteration by means of lectin microarray.

BACKGROUND: Despite the progress made in understanding glyco-alterations of specific glycoproteins such as α1-acid glycoprotein (AGP) associated with liver fibrosis, there has been no useful diagnostic assay with a lectin recognizing the fibrosis-specific alteration and an antibody against the core protein. We therefore developed a compatible multiple lectin-antibody sandwich immunoassay on the basis of the results obtained by the lectin microarray analysis for monitoring fibrosis. METHODS: AGP-enriched fractions derived from 0.5-µL sera of 125 patients with staging-determined fibrosis (26.4% F0-F1, 25.6% F2, 24% F3, and 23.2% F4) were subjected to systematic analysis by antibody-overlay lectin microarray. Data were analyzed to statistically relate to the degree of fibrosis progression. Additionally, we applied an optimal lectin signal set on the microarray to distinguish 45 patients with cirrhosis from 43 patients with chronic hepatitis. RESULTS: Signal patterns of the 12 selected lectins reflected fibrosis-associated glyco-alteration of AGP. Among the 12 lectins, we found a specific lectin at each stage of fibrosis (i.e., significant fibrosis, severe fibrosis, and cirrhosis) (P < 0.0001). The test for the detection of cirrhosis showed that combinational use of 3 lectins (AOL, MAL, and DSA) on the array enhanced the diagnostic value for liver cirrhosis to 95% diagnostic sensitivity and 91% diagnostic specificity. CONCLUSIONS: The multiple lectin-antibody sandwich immunoassay targeting AGP enables monitoring of disease progression in chronic hepatitis patients at risk of developing hepatocellular carcinoma.

Enhancement of metastatic ability by ectopic expression of ST6GalNAcI on a gastric cancer cell line in a mouse model.

ST6GalNAcI is a sialyltransferase responsible for the synthesis of sialyl Tn (sTn) antigen which is expressed in a variety of adenocarcinomas including gastric cancer especially in advanced cases, but the roles of ST6GalNAcI and sTn in cancer progression are largely unknown. We generated sTn-expressing human gastric cancer cells by ectopic expression of ST6GalNAcI to evaluate metastatic ability of these cells and prognostic effect of ST6GalNAcI and sTn in a mouse model, and identified sTn carrier proteins to gain insight into the function of ST6GalNAcI and sTn in gastric cancer progression. A green fluorescent protein-tagged human gastric cancer cell line was transfected with ST6GalNAcI to produce sTn-expressing cells, which were transplanted into nude mice. STn-positive gastric cancer cells showed higher intraperitoneal metastatic ability in comparison with sTn-negative control, resulting in shortened survival time of the mice, which was mitigated by anti-sTn antibody administration. Then, sTn-carrying proteins were immunoprecipitated from culture supernatants and lysates of these cells, and identified MUC1 and CD44 as major sTn carriers. It was confirmed that MUC1 carries sTn also in human advanced gastric cancer tissues. Identification of sTn carrier proteins will help understand mechanisms of metastatic phenotype acquisition of gastric cancer cells by ST6GalNAcI and sTn.

Strategy for glycoproteomics: identification of glyco-alteration using multiple glycan profiling tools.

Glycan alterations of proteins, a common feature of cancer cells, are associated with carcinogenesis, invasion and metastasis. Glycomics, the study of glycans and glycan-binding proteins in various biological systems, is an emerging field in the postgenome and postproteomics era. However, systematic and robust strategies for glycomics are still not fully established because the structural analysis of glycans, which comprise different patterns of branching, various possible linkage positions as well as monomer anomericity, is technically difficult. Here, we introduce a new strategy for glyco-alteration analysis of glycoproteins by using multiple glycan profiling tools. To understand glycan alterations of proteins by correlating the glycosyltransferase expression profile with the actual glycan structure, we systematically used three glycan profiling tools: (1) multiplex quantitative PCR (qPCR) array format for profiling the expression pattern of glycogenes, (2) lectin microarray as a multiplex glycan-lectin interaction analysis system for profiling either a pool of cell glycoproteins or a target glycoprotein, and (3) tandem mass spectrometry for identifying the glycan structure connected to a target glycoprotein. Using our system, we successfully identified glycan alterations on alpha-fetoprotein (AFP), including a novel LacdiNAc structure in addition to previously reported alterations such as alpha1,6 fucosylation.

CD43, but not P-selectin glycoprotein ligand-1, functions as an E-selectin counter-receptor in human pre-B-cell leukemia NALL-1.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL/B-precursor ALL) is characterized by a high rate of tissue infiltration. The mechanism of BCP-ALL cell extravasation is not fully understood. In the present study, we have investigated the major carrier of carbohydrate selectin ligands in the BCP-ALL cell line NALL-1 and its possible role in the extravascular infiltration of the leukemic cells. B-precursor ALL cell lines and clinical samples from patients with BCP-ALL essentially exhibited positive flow cytometric reactivity with E-selectin, and the reactivity was significantly diminished by O-sialoglycoprotein endopeptidase treatment in NALL-1 cells. B-precursor ALL cell lines adhered well to E-selectin but only very weakly to P-selectin with low-shear-force cell adhesion assay. Although BCP-ALL cell lines did not express the well-known core protein P-selectin glycoprotein ligand-1 (PSGL-1), a major proportion of the carbohydrate selectin ligand was carried by a sialomucin, CD43, in NALL-1 cells. Most clinical samples from patients with BCP-ALL exhibited a PSGL-1(neg/low)/CD43(high) phenotype. NALL-1 cells rolled well on E-selectin, but knockdown of CD43 on NALL-1 cells resulted in reduced rolling activity on E-selectin. In addition, the CD43 knockdown NALL-1 cells showed decreased tissue engraftment compared with the control cells when introduced into gamma-irradiated immunodeficient mice. These results strongly suggest that CD43 but not PSGL-1 plays an important role in the extravascular infiltration of NALL-1 cells and that the degree of tissue engraftment of B-precursor ALL cells may be controlled by manipulating CD43 expression.

Impaired interactions between mouse Eyal harboring mutations found in patients with branchio-oto-renal syndrome and Six, Dach, and G proteins.

Mutations in the EYA1 gene are responsible for branchio-oto-renal (BOR) syndrome as well as for other ocular defects. Most of the mutations are located within or in the vicinity of the EYA domain, which is highly conserved in the EYA protein family. The EYA domain is required for protein-protein interactions, which are important to the biological function of EYA proteins. To determine how EYA1 mutations cause BOR syndrome and/or ocular defects, we tested the effects of Eya1 mutations on interactions with Six. Dach, and G proteins by mammalian two-hybrid and GST-pulldown assays. Defective interactions were noted between BOR-type mutations S486P and L504R of Eya1 and Dach1, G proteins, and some Six proteins. These mutations impaired the activation of transcription from a Six-responsive gene, myogenin, with Six5. S486P and L504R showed an altered digestion pattern with trypsin, and L504R also decreased the sensitivity to V8 protease digestion and produced a peptide fragment with a different M(r). Our results suggest that defective protein-protein interactions of the mutations in the EYA domain underlie BOR syndrome and that SIX, DACH, and/or G proteins are possibly involved in the pathogenic processes.

Identification of transcriptional targets for Six5: implication for the pathogenesis of myotonic dystrophy type 1.

Myotonic dystrophy 1 (DM1) is the most common inherited neuromuscular disease in adults. The disorder, characterized by myotonia, muscle wasting and weakness, cataract, insulin resistance, and mental impairment, is caused by the expansion of an unstable CTG repeat located in the 3' untranslated region of DMPK. The repeat expansion suppresses the expression of the homeobox gene SIX5. We describe here an experimental system to identify downstream transcriptional targets of mouse Six5 in order to elucidate the role of SIX5 in the pathogenesis of DM1 and development. By overexpressing a constitutively active Six5 (VP16-Six5wt) using adenovirus-mediated gene transfer in P19 cells and subsequent expression profiling using cDNA arrays, 21 genes, whose expression level increased by the treatment, were identified as potential target genes. Genes expressed in the somites, skeletal muscles, brain and meninges comprised the majority, suggesting the role of Six5 in the development and function of mesodermal tissues and brain. We provide evidence that Igfbp5 encoding a component of IGF signaling is a direct Six5-target. Moreover, the overall expression level of Igfbp5 was decreased in Six5-deficient mouse fibroblasts, and the response of human IGFBP5 to MyoD-induced muscle conversion was altered in cells of DM1 patients. Our results not only identify Six5 as an activator that directs Igfbp5 expression but also suggest that reduced SIX5 expression in DM1 might contribute to specific aspects of the DM1 phenotype.

Six and Eya expression during human somitogenesis and MyoD gene family activation.

This report describes the characterisation of the expression profile of several myogenic determination genes during human embryogenesis. The data were obtained from axial structures and limb buds of human embryos aged between 3 and 8 weeks of development. Using in situ hybridisation to detect Pax3 and MyoD gene family mRNAs, and immunochemistry to follow Six and Eya protein accumulation, we have been able to establish the chronology of accumulation of these gene products. As in mouse, the first transcripts detected in myotomes of 3 week-old embryos are Pax3 and Myf5, followed by the expression of myogenin. MyoD appears to be activated well after Myf5, myogenin and MRF4 in the early myotome, whereas, in limb bud muscles, the presence of all four of these mRNAs is concomitant from 6 weeks. Six1, Six4 and Six5 homeoproteins are detected later than Myf5 activation. These Six homeoproteins are first observed in the cytoplasm of myogenin expressing cells. At later stages of development, Six1 and Six5, but not Six4, are translocated into the nuclei of myogenic cells, concomitantly with MyHCemb expression. Eya1 and Eya2 proteins, potential Six cofactors, were also detected in myogenin positive cells, but their accumulation was delayed and was mainly cytoplasmic. These results preclude that early activation of Myf5, myogenin and MRF4 is under the control of Six and Eya proteins, while Six and Eya proteins would be involved in later steps of myogenic differentiation.

Six1 controls patterning of the mouse otic vesicle.

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.