[Show abstract][Hide abstract] ABSTRACT: The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.
Full-text · Article · Oct 2015 · Glycoconjugate Journal
[Show abstract][Hide abstract] ABSTRACT: Recently, we identified a novel liver fibrosis glycobiomarker, Wisteria floribunda agglutinin (WFA)-reactive colony stimulating factor 1 receptor (WFA(+) -CSF1R), using a glycoproteomics-based strategy. The aim of this study was to assess the value of measuring WFA(+) -CSF1R levels for the prognosis of carcinogenesis and outcome in liver cirrhosis (LC) patients with hepatitis C virus (HCV). WFA(+) -CSF1R and Total-CSF1R levels were measured in serum samples from 214 consecutive HCV-infected patients to evaluate their impact on carcinogenesis and the survival of LC patients. Serum WFA(+) -CSF1R levels were significantly higher in LC patients than chronic hepatitis (CH) patients (p < 0.001). The AUC of WFA(+) -CSF1R for predicting overall survival, calculated by time-dependent ROC analysis, was 0.691 and the HR (per 1-SD increase) was 1.80 (95% CI, 1.23-2.62, p < 0.001). Furthermore, the survival rate of LC patients with high WFA(+) -CSF1R levels (≥ 310 ng/ml) was significantly worse than those with lower levels (p < 0.01). The AUC of WFA(+) /total-CSF1R percentage (WFA(+) -CSF1R%) for predicting the cumulative carcinogenesis rate was 0.760, with an HR of 1.66 (95% CI 1.26-2.20, p < 0.001). In fact, the carcinogenesis rate was significantly higher in LC patients with a high WFA(+) -CSF1R% (≥ 35%, p = 0.006). Assessing serum levels of WFA(+) -CSF1R has diagnostic value for predicting carcinogenesis and the survival of LC patients. This article is protected by copyright. All rights reserved.
No preview · Article · Oct 2015 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: 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 synthesized by α1,3-fucosyltransferase 9 (Fut9). Fut9is 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)eavored 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.
Preview · Article · Aug 2015 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: Measurement of Wisteria floribunda agglutinin-positive human Mac-2 binding protein (WFA+-M2BP) in serum was recently shown to be a noninvasive method to assess liver fibrosis. The aim of this study was to evaluate the utility of serum WFA+-M2BP values to predict the development of hepatocellular carcinoma (HCC) in patients who achieved a sustained virological response (SVR) by interferon treatment. For this purpose, we retrospectively analyzed 238 patients with SVR who were treated with interferon in our department. Serum WFA+-M2BP values were measured at pre-treatment (pre-Tx), post-treatment (24 weeks after completion of interferon; post-Tx), the time of HCC diagnosis, and the last clinical visit. Of 238 patients with SVR, HCC developed in 16 (6.8%) patients. The average follow-up period was 9.1 years. The cumulative incidence of HCC was 3.4% at 5 years and 7.5% at 10 years. The median pre-Tx and post-Tx WFA+-M2BP values were 1.69 (range: 0.28 to 12.04 cutoff index (COI)) and 0.80 (range: 0.17 to 5.29 COI), respectively. The WFA+-M2BP values decreased significantly after SVR (P < 0.001). The median post-Tx WFA+-M2BP value in patients who developed HCC was significantly higher than that in patients who did not (P < 0.01). Multivariate analysis disclosed that age (> 60 years), sex (male), pre-Tx platelet count (< 15.0×103/μL), and post-Tx WFA+-M2BP (> 2.0 COI) were associated with the development of HCC after SVR.
Post-Tx WFA+-M2BP (> 2.0 COI) is associated with the risk for development of HCC among patients with SVR. The WFA+-M2BP values could be a new predictor for HCC after SVR.
[Show abstract][Hide abstract] ABSTRACT: AimsWisteria floribunda agglutinin (WFA)-positive human Mac-2-binding protein (WFA+-M2BP) is a new glycol marker related to liver fibrosis. The aim of the present study was to evaluate WFA+-M2BP as a predictor of hepatocellular carcinoma (HCC) development in patients with chronic hepatitis C.Methods
This case–control study included 14 patients with chronic hepatitis C who developed HCC and 52controls, matched for age, gender, and fibrosis stage. WFA+-M2BP was measured at biopsy and follow-up. Time zero was set at the date of liver biopsy.ResultsWFA+-M2BP increased stepwise with progression of liver fibrosis (p < 0.001). Cumulative incidence of HCC development was significantly higher in patients with WFA+-M2BP ≥4.2 (p < 0.001) or in those with time-course changes in WFA+-M2BP (ΔWFA+-M2BP/year) ≥0.3 (p = 0.03). Multivariate analyses demonstrated that WFA+-M2BP ≥4.2 [hazard ratio (HR): 4.1, 95% confidence interval (CI): 1.1–15, p = 0.04], ΔWFA+-M2BP/year ≥0.3 (HR: 5.5, 95% CI: 1.5–19, p = 0.008), and AFP ≥10 ng/ml (HR: 4.7, 95% CI: 1.1–19, p = 0.03) were independent predictive factors of HCC development. Based on these data, we developed a simple scoring system to predict HCC development using these three factors. Using these scores, patients were classified into four groups; cumulative incidence of HCC development significantly increased with increasing scores (p < 0.001).ConclusionsWFA+-M2BP measurements and time-course changes in WFA+-M2BP can be used to identify patients at high risk of HCC development. Real-time monitoring of WFA+-M2BP can be a novel predictor of HCC development. This article is protected by copyright. All rights reserved.
No preview · Article · Jan 2015 · Hepatology Research
[Show abstract][Hide abstract] ABSTRACT: Polylactosamine (poly-N-acetyllactosamine) is a fundamental structure of glycans present on glycoproteins and glycolipids and plays important roles in many biological processes, both directly and indirectly. Nonetheless, the detailed functions of polylactosamine are still not fully understood. Recently, knockout mouse strains lacking genes related to the biosynthesis of polylactosamine have been generated, and analysis of the resulting phenotypes is ongoing. This section introduces some of the polylactosamine functions in the immune system.
[Show abstract][Hide abstract] ABSTRACT: Histopathological classification of lung cancer has important implications in the application of clinical practice guidelines and the prediction of patient prognosis. Thus, we focused on discovering glycobiomarker candidates to classify the types of lung cancer tissue. First, we performed lectin microarray analysis of lung cancer tissue specimens and cell lines and identified Aleuria aurantia lectin (AAL), Hippeastrum hybrid lectin (HHL), and Concanavalia ensiformis agglutinin (ConA) as lectin probes specific to non-small cell lung carcinoma (NSCLC). LC-MS-based analysis was performed for the comprehensive identification of glycoproteins and N-linked glycosylation sites using lectin affinity capture of NSCLC-specific glycoforms of glycoproteins. This analysis identified 1092 AAL-bound glycoproteins (316 gene symbols) and 948 HHL/ConA-bound glycoproteins (279 gene symbols). The lectin microarray-assisted verification using 15 lung cancer cell lines revealed the NSCLC-specific expression of fibronectin. The glycosylation profiling of fibronectin indicated that the peanut agglutinin (PNA) signal appeared to differentiate two NSCLC types, adenocarcinoma and large cell carcinoma, whereas the protein expression level was similar between these types. Our glycoproteomics approach together with the concurrent use of an antibody and lectin is applicable to the quantitative and qualitative monitoring of variations in glycosylation of fibronectin specific to certain types of lung cancer tissue.
No preview · Article · Sep 2014 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: In this study, we selected 181 nematode glycogenes that are orthologous to human glycogenes and examined their RNAi phenotypes.
The results are deposited in the Caenorhabditis elegans Glycogene Database (CGGDB) at AIST, Tsukuba, Japan. The most prominent RNAi phenotypes observed are disruptions of cell cycle
progression in germline mitosis/meiosis and in early embryonic cell mitosis. Along with the previously reported roles of chondroitin
proteoglycans, glycosphingolipids and GPI-anchored proteins in cell cycle progression, we show for the first time that the
inhibition of the functions of N-glycan synthesis genes (cytoplasmic alg genes) resulted in abnormal germline formation, ER stress and small body size phenotypes. The results provide additional
information on the roles of glycoconjugates in the cell cycle progression mechanisms of germline and embryonic cells.
[Show abstract][Hide abstract] ABSTRACT: The Wisteria floribunda agglutinin-positive human Mac-2-binding protein (WFA+-M2BP) was recently shown to be a liver fibrosis glycobiomarker with a unique fibrosis-related glyco-alteration. We evaluated the ability of WFA+-M2BP to predict the development of hepatocellular carcinoma (HCC) in patients who were infected with the hepatitis C virus (HCV). A total of 707 patients who had been admitted to our hospital with chronic HCV infection without other potential risk factors were evaluated to determine the ability of WFA+-M2BP to predict the development of HCC; factors evaluated included age, sex, viral load, genotypes, fibrosis stage, aspartate and alanine aminotransferase levels, bilirubin, albumin, platelet count, alpha-fetoprotein (AFP), WFA+-M2BP and the response to interferon therapy. Serum WFA+-M2BP levels were significantly increased according to the progression of liver fibrosis stage (p < 0.001). In each distinctive stage of fibrosis (F0/1, F2, F3, and F4), the risk of development of HCC was increased according to the elevation of WFA+-M2BP. Multivariate analysis identified age ≥ 57 years, F4, AFP ≥ 20 ng/mL, WFA+-M2BP ≥ 4, and WFA+-M2BP 1 - 4 as well as the response to interferon (no therapy vs. SVR) as independent risk factors for the development of HCC. The time-dependent areas under the receiver operating characteristic curve demonstrated that the WFA+-M2BP assay predicted the development of HCC with higher diagnostic accuracy than AFP. Conclusion: WFA+-M2BP can be applied as a useful surrogate marker for the risk of HCC development, in addition to liver biopsy. (Hepatology 2014;)
[Show abstract][Hide abstract] ABSTRACT: Carbohydrate structures, including Lewis X (Lex), which is not synthesized in mutant mice that lack α1,3-fucosyltransferase 9 (Fut9−/−), are involved in cell–cell recognition and inflammation. However, immunological alteration in Fut9−/− mice has not been studied. Thus, the inflammatory response of Fut9−/− mice was examined using the highly neurovirulent mouse hepatitis virus (MHV) JHMV srr7 strain. Pathological study revealed that inflammation induced in the brains of Fut9−/− mice after infection was more extensive compared with that of wild-type mice, although viral titers obtained from the brains of mutant mice were lower than those of wild-type mice. Furthermore, the reduction in cell numbers in the spleens of wild-type mice after infection was not observed in the infected Fut9−/− mice. Although there were no clear differences in the levels of cytokines examined in the brains between Fut9−/− and wild-type mice except for interferon-β (IFN-β) expression, some of those in the spleens, including interferon-γ (IFN-γ), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), showed higher levels in Fut9−/− than in wild-type mice. Furthermore, Fut9−/− mice were refractory to the in vivo inoculation of endotoxin (LPS) compared with wild-type mice. These results indicate that Lex structures are involved in host responses against viral or bacterial challenges.
No preview · Article · May 2014 · Pathology International
[Show abstract][Hide abstract] ABSTRACT: Recently, a novel marker, hyperglycosylated Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA(+)-M2BP), was developed for liver fibrosis using the glycan "sugar chain"-based immunoassay; however, the feasibility of WFA(+)-M2BP for assessing liver fibrosis has not been proven with clinical samples of hepatitis.
Serum WFA(+)-M2BP values were evaluated in 200 patients with chronic liver disease who underwent histological examination of liver fibrosis. The diagnostic accuracy of WFA(+)-M2BP values was compared with various fibrosis markers, such as ultrasound based-virtual touch tissue quantification (VTTQ), magnetic resonance imaging based-liver-to-major psoas muscle intensity ratio (LMR), and serum markers, including hyaluronic acid, type 4 collagen, and aspartate transaminase to platelet ratio index (APRI).
Serum WFA(+)-M2BP levels in patients with fibrosis grades F0, F1, F2, F3, and F4 had cutoff indices 1.62, 1.82, 3.02, 3.32, and 3.67, respectively, and there were significant differences between fibrosis stages F1 and F2, and between F2 and F3 (P < 0.01). The area under the receiver operating characteristic curves for the diagnosis of fibrosis (F ≥ 3) using serum WFA(+)-M2BP values (0.812) was almost comparable to that using VTTQ examination (0.814), but was superior to the other surrogate markers, including LMR index (0.766), APRI (0.694), hyaluronic acid (0.683), and type 4 collagen (0.625) (P < 0.01 each).
Serum WFA(+)-M2BP values based on a glycan-based immunoassay is an accurate, reliable, and reproducible method for the assessment of liver fibrosis. This approach could be clinically feasible for evaluation of beneficial therapy through the quantification of liver fibrosis in hepatitis patients if this measurement application is commercially realized.
Preview · Article · Mar 2014 · Journal of Gastroenterology
[Show abstract][Hide abstract] ABSTRACT: Epithelial ovarian cancer (EOC) is often asymptomatic and thus diagnosed at advanced stages with a poor prognosis. False-negative results for the conventional marker CA125 frequently occur in cases of clear cell carcinoma (CCC), a type of EOC; therefore, it is necessary to develop biomarkers with greater sensitivity. We previously reported a strategy to discover glyco-biomarker candidates by combined lectin microarray and IGOT-LC/MS analysis. We have now optimized this strategy for discovering EOC biomarkers. Glycopeptides possessing cancerous glycans were enriched from the ascites fluids and culture supernatants of cancer cell lines with a fucose-binding lectin, AAL. IGOT-LC/MS analysis of CCC samples yielded 144 candidate glycoproteins. We selected WFA by lectin microarray as the optimal lectin to distinguish EOC from gastric and colon cancer. The candidates were narrowed by western analysis of the WFA-bound fraction of ascites fluids. One of the final candidates, WFA-reactive ceruloplasmin, produced higher signals in the ascites fluids of EOC patients, including CCC, in comparison to the benign samples, while CA125 levels were comparable in the sandwich ELISA. Thus, our glycoproteomic strategy featuring efficient enrichment of glycans with disease-related alterations is applicable to various diseases.
No preview · Article · Feb 2014 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: The importance of diagnosis and therapies for liver cirrhosis (LC) is indisputable. Thus, a reliable method for monitoring the progression of liver fibrosis and resultant LC is urgently needed. Previously, using a lectin-assisted glycoproteomic method, we identified 26 serum glycoproteins as promising glycobiomarker candidates for monitoring the progression of liver diseases [Kaji et al. J. Proteome Res. (2013)]. In this study, we identified colony stimulating factor 1 receptor (CSF1R) as a promising LC marker candidate, and then established Wisteria floribunda agglutinin (WFA)-reactive CSF1R (WFA(+)-CSF1R) as a novel possible glycobiomarker candidate by utilizing a glycoproteomics-based strategy [Narimatsu et al. FEBS J (2010)]. The serum level of WFA(+)-CSF1R in patients with hepatitis C virus (HCV)-infected liver disease was measured by an antibody-lectin sandwich ELISA. In a proof-of-concept experiment of the strategy preceding to future clinical studies, LC patients showed a high serum WFA(+)-CSF1R level in selected samples (P = 1.3E-17). This result suggests WFA(+)-CSF1R is a possible biomarker candidate for evaluation of LC. Our results verified feasibility of this strategy for glycobiomarker development.
No preview · Article · Jan 2014 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: Lewis X (LeX, Galβ1–4(Fucα1–3)GlcNAc) is a carbohydrate epitope that is present at the nonreducing terminus of sugar chains of glycoproteins
and glycolipids, and is abundantly expressed in several stem cell populations. LeX antigen can be used in conjunction with fluorescence-activated cell sorting to isolate neurosphere-forming neural stem cells
(NSCs) from embryonic mouse brains. However, its function in the maintenance and differentiation of stem cells remains largely
unknown. In this study, we examined mice deficient for fucosyltransferase 9 (Fut9), which is thought to synthesize most, if not all, of the LeX moieties in the brain. We found that the number of NSCs was increased in the brain of Fut9−/− embryos, suggesting that Fut9-synthesized LeX is dispensable for the maintenance of NSCs. Another α1,3-fucosyltransferase gene, fucosyltransferase 10 (Fut10), is expressed in the ventricular zone of the embryonic brain. Overexpression of Fut10 enhanced the self-renewal of NSCs. Conversely, suppression of Fut10 expression induced the differentiation of NSCs and embryonic stem cells. In addition, knockdown of Fut10 expression in the cortical ventricular zone of the embryonic brain by in utero electroporation of Fut10-miRNAs impaired the radial migration of neural precursor cells. Our data suggest that Fut10 is involved in a unique α1,3-fucosyltransferase
activity with stringent substrate specificity, and that this activity is required to maintain stem cells in an undifferentiated
Preview · Article · Aug 2013 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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)]. 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). Based on 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 a digest 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) 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.
No preview · Article · Apr 2013 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: We have cloned many genes of β1,3-glycosyltransferases, which transfer sugars via a β1,3-linkage, and have characterized their biological functions. Among β1,3- glycosyltransferases, β1,3-N-acetylglucosaminyltransferases (β3GnTs) synthesize a unique carbohydrate structure known as "polylactosamine (poly-N-acetyllactosamine)". Polylactosamine is carried on N- and O-glycans, and on glycolipids. Polylactosamine structures are considered to be integral components serving as a fundamental structure and backbone for carbohydrate structures. However, most of their biological functions are still unknown. To investigate the in vivo function of polylactosamine on glycoconjugates, we generated and analyzed two mouse lines of β1,3-Nacetylglucosaminyltransferase (B3gnt)-deficient (B3gnt2-/- or B3gnt5-/-) mice lacking the polylactosamine structure. First, to investigate the in vivo function of polylactosamine on glycoproteins, we analyzed gene knockout mice lacking B3gnt2, which synthesizes polylactosamine on glycoproteins. In B3gnt2-/- mice, glycan analysis demonstrated that the amount of long polylactosamine chains on N-glycan was greatly reduced in the tissues of B3gnt2-/- mice. We also examined immunological responses in B3gnt2-/- mice. B3gnt2-/- lymphocytes showed hyperactivation via TCR/CD28 or BCR stimulation. Next, to investigate the in vivo function of polylactosamine on glycosphingolipids (glycolipid), we analyzed B3gnt5-/- mice lacking lacto/neolacto-series glycolipids. B3gnt5-/- B cells showed an abnormality of glycolipid-enriched microdomains (GEMs; also known as glycolipid rafts) and showed hyperactivation via BCR-related molecules in GEMs, as compared with wild-type (WT) B cells. Polylactosamine deficiency seems to be involved in the immunological disorders observed in these mice. Taken together, these studies suggest that the polylactosamine chain is a putative immune regulatory factor that presumably suppresses excessive responses during immune reactions and has an important biological role in the immune system.
No preview · Article · May 2012 · Trends in Glycoscience and Glycotechnology
[Show abstract][Hide abstract] ABSTRACT: Fucose (Fuc)-containing glycoconjugates play important roles in numerous physiological and pathological processes. Given the biological importance of post-translational glycosylation, a specific and robust strategy for the identification of fucosylated glycoproteins is highly desirable. In this study, we demonstrate an alternative way of labeling of fucosylated structures by metabolic engineering, using a chemoenzymatic approach. In this approach, the activities of Bacteroides fragilis 9343 L-fucokinase/guanosine-5'-diphosphate-Fuc pyrophosphorylase and human α1,3-fucosyltransferase 9 are combined in a Namalwa cellular model. Interestingly, this system could be applied to labeling of alkyne-modified fucosylated glycoproteins. N-Glycan site mapping and identification were done using an in vitro selective chemical ligation reaction and isotope-coded glycosylation site-specific tagging, subsequent to liquid chromatography-tandem mass spectrometry analysis. This work illustrates the use of a click chemistry-based strategy combined with a glycoproteomic technique to get further insight into the pattern of Fuc-mediated biological processes and functions.
[Show abstract][Hide abstract] ABSTRACT: It is known that mutant mice of the beta-1,3-N-acetylglucosaminyltransferase gene (beta3Gn-T5) respond well to T-cell dependent and independent antigens. Here, we examined the effectiveness of anti-ganglioside antibody generation by immunization of beta3Gn-T5 mutant mice with liposome-embedded glycosphingolipids such as GD1a and GT1b. Consequently, the mutant mice showed a more efficient generation of anti-GD1a or anti-GT1b antibodies than wild-type mice in an enzyme-linked immunosorbent assay using sera during immunization. Thus, the beta3Gn-T5 deficient mutant mice proved more responsive than wild-type mice to not only protein antigens, but also to carbohydrates in glycolipids. Furthermore, about 50% of monoclonal antibodies generated using splenocytes of the immunized mutant mice were of the IgG class. Besides general high responsiveness to proteins and glycolipids, it could be expected that the mutant mice of beta3Gn-T5 would be useful in the generation of monoclonal antibodies towards lacto-/neolacto-series glycolipids, since these mutants lack lacto-/neolacto-series glycolipids. In fact, they showed a good serum response in immuno-fluorescence assay with cultured living cells when immunized by glycolipids extracted from ovarian cancer cell lines. These results suggested that beta3Gn-T5 mutant mice are useful for the generation of anti-glycolipid antigens with lacto-/neolacto-core structures expressed in cancer cells.
No preview · Article · Aug 2011 · Nagoya journal of medical science
[Show abstract][Hide abstract] ABSTRACT: Chondroitin sulfate (CS) is a glycosaminoglycan, consisting of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues, and plays important roles in development and homeostasis of organs and tissues. Here, we generated and analyzed mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT-1). Csgalnact1(-/-) mice were viable and fertile but exhibited slight dwarfism. Biochemically, the level of CS in Csgalnact1(-/-) cartilage was reduced to ∼50% that of wild-type cartilage, whereas its chain length was similar to wild-type mice, indicating that CSGalNAcT-1 participates in the CS chain initiation as suggested in the previous study (Sakai, K., Kimata, K., Sato, T., Gotoh, M., Narimatsu, H., Shinomiya, K., and Watanabe, H. (2007) J. Biol. Chem. 282, 4152-4161). Histologically, the growth plate of Csgalnact1(-/-) mice contained shorter and slightly disorganized chondrocyte columns with a reduced volume of the extracellular matrix principally in the proliferative layer. Immunohistochemical analysis revealed that the level of both aggrecan and link protein 1 were decreased in Csgalnact1(-/-) cartilage. Western blot analysis demonstrated an increase in processed forms of aggrecan core protein. These results suggest that CSGalNAcT-1 is required for normal levels of CS biosynthesis in cartilage. Our observations suggest that CSGalNAcT-1 is necessary for normal levels of endochondral ossification, and the decrease in CS amount in the growth plate by its absence causes a rapid catabolism of aggrecan.
No preview · Article · Feb 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Chondroitin sulfate (CS) is a glycosaminoglycan, consisting of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues, and plays important roles in development and homeostasis of organs and
tissues. Here, we generated and analyzed mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT-1). Csgalnact1−/− mice were viable and fertile but exhibited slight dwarfism. Biochemically, the level of CS in Csgalnact1−/− cartilage was reduced to ∼50% that of wild-type cartilage, whereas its chain length was similar to wild-type mice, indicating
that CSGalNAcT-1 participates in the CS chain initiation as suggested in the previous study (Sakai, K., Kimata, K., Sato,
T., Gotoh, M., Narimatsu, H., Shinomiya, K., and Watanabe, H. (2007) J. Biol. Chem. 282, 4152–4161). Histologically, the growth plate of Csgalnact1−/− mice contained shorter and slightly disorganized chondrocyte columns with a reduced volume of the extracellular matrix principally
in the proliferative layer. Immunohistochemical analysis revealed that the level of both aggrecan and link protein 1 were
decreased in Csgalnact1−/− cartilage. Western blot analysis demonstrated an increase in processed forms of aggrecan core protein. These results suggest
that CSGalNAcT-1 is required for normal levels of CS biosynthesis in cartilage. Our observations suggest that CSGalNAcT-1
is necessary for normal levels of endochondral ossification, and the decrease in CS amount in the growth plate by its absence
causes a rapid catabolism of aggrecan.
No preview · Article · Feb 2011 · Journal of Biological Chemistry