Levels Of Mannose Research Articles

Mannose is an insulin-regulated metabolite reflecting whole-body insulin sensitivity in man

Mannose is a glucose-associated serum metabolite mainly released by the liver. Recent studies have shown several unexpected pleiotropic effects of mannose including increased regulatory T cells (Tregs), prevention of auto-immune disease and ability to reduce growth of human cancer cells. We have previously shown in large cohorts that elevated serum mannose levels are associated with future development of type 2 diabetes (T2D) and cardiovascular disease. However, potential direct effects of mannose on insulin sensitivity in vivo or in vitro are unknown.We here show that administration of mannose (0.1 g/kg BW twice daily) for one week in man did not elicit negative effects on meal-modified glucose tolerance, markers of inflammation or insulin levels. Tregs number and insulin signaling in human liver cells were unchanged. These data suggest that mannose is a marker, and not a mediator, of insulin resistance. To verify this, we examined serum mannose levels during long-term euglycemic hyperinsulinemic clamps in non-diabetic and T2D individuals. Mannose was reduced by insulin infusion in proportion to whole-body insulin sensitivity. Thus, mannose is a biomarker of insulin resistance which may be useful for the early identification of diabetic individuals with insulin resistance and increased risk of its complications.

Biochemical characterization and mutational analysis of silkworm Bombyx mori β-1,4-N-acetylgalactosaminyltransferase and insight into the substrate specificity of β-1,4-galactosyltransferase family enzymes

Silkworm Bombyx mori is one of the insect hosts for recombinant protein production at academic and industrial levels. B. mori and other insect cells can produce mammalian proteins with proper posttranslational modifications, such as N-glycosylation, but the structures of N-glycans in B. mori are mainly high mannose- and paucimannose-type, while mammals also produce hybrid- and complex-type glycans. Recently, complex-type N-glycans whose structures are different from mammalian ones have been identified in some insect cell N-glycomes at very low levels compared with levels of high mannose- and paucimannose-type glycans. However, their functions and the enzymes involved in the biosynthesis of insect complex-type N-glycans are not clear, and complex-type N-glycans, except for N-acetylglucosamine-terminated glycans, are still not identified in the B. mori N-glycome. Here, we focused on the β-1,4-galactosyltransferase family (also known as glycosyltransferase family 7, GT7) that contains mammalian β-1,4-galactosyltransferase and insect β-1,4-N-acetylgalactosaminyltransferase. A gene for a GT7 protein (BmGalNAcT) from B. mori was cloned, expressed in a soluble form using a silkworm expression system, and the gene product showed strict β-1,4-N-acetylgalactosaminyltransferase activity but not β-1,4-galactosyltransferase activity. A mutation in Ile298 or Ile310, which are predicted to be located in the active site, reduced its glycosyltransferase activity, suggesting that these residues and the corresponding residues are responsible for substrate specificity of GT7. These results suggested that BmGalNAcT may be involved in the complex-type N-glycans, and moreover, bioinformatics analysis revealed that B. mori might have an extra gene for a GT7 enzyme with different specificity in addition to the known insect GT7 glycosyltransferases.

Elevated Serum Mannose Levels as a Marker of Polycystic Ovary Syndrome.

Background: Recent reports have highlighted the role of monosaccharide biosynthesis in the pathogenesis of polycystic ovary syndrome (PCOS), suggesting that these processes may serve as a biomarker in PCOS. Mannose is the main monosaccharide for protein glycosylation in mammals; however, the correlation between mannose and PCOS remains largely unknown.Materials and Methods: A total of 132 Chinese Han women were recruited at Shengjing Hospital of China Medical University. Mannose levels were measured in serum samples collected from 71 patients with PCOS (29 lean, 42 obese) and 61 control subjects (28 lean, 33 obese). Receiver operating characteristics (ROC) curves were prepared to compare the diagnostic performance of mannose and hormonal parameters, individually or in combination. Multivariate logistic regression analysis was used to assess whether serum mannose levels were associated with PCOS after adjusting for other co-variables.Results: We showed that serum mannose levels were significantly increased in PCOS patients compared with control subjects regardless of obese status, and hyperandrogenic PCOS patients had higher serum mannose levels than normo-androgenic PCOS and control subjects. In addition, serum mannose levels were significantly correlated with serum androgen levels. Mannose had an area under the curve (AUC) of 73% at a cutoff value of 225.79 ng/mL with a sensitivity of 66.2% and specificity of 73.8% for predicting PCOS. There were no differences between mannose, total testosterone, free testosterone, or dehydroepiandrosterone sulfate in the reliability of predicting PCOS using the method outlined by Hanley and McNeil. Combining mannose and total testosterone resulted in a higher AUC of 83.3%, and had moderate sensitivity (78.9%) and specificity (77%) for predicting PCOS. The positive and negative predictive values were 80% and 75.8%, respectively. Multivariate logistic regression revealed that higher serum mannose levels were strongly associated with an increased risk of PCOS (P = 0.016; odds ratio, 5.623; 95% confidence interval, 1.371–23.070).Conclusion: Taken together, substantially elevated serum mannose levels are significantly associated with PCOS, highlighting the importance of further research into the role of mannose in the pathogenesis of PCOS.

649P - Diet-derived metabolites and the risk of colorectal cancer: A nested case-control study in a population-based cohort, the Singapore Chinese health study

BackgroundDiet is an important risk factor of colorectal cancer (CRC) and affects cancer risk through its effects on colonic microbial metabolism. Few population-based studies have examined the relationship between diet-derived metabolites and the risk of CRC in the context of Asian diet, microbiota composition and anatomical subsite. MethodsWe conducted a nested case-control study of 350 incident CRC (211 colon and 139 rectal) cases and 350 matched controls within the Singapore Chinese Health Study, a prospective cohort of 63,257 men and women. Liquid and gas chromatography-mass spectrometries were used to quantify 61 plasma metabolites, including amino acids, carnitine, acylcarnitines, short chain fatty acids, bile acids, monosaccharides and organic compounds. Conditional logistic regression models were used to estimate odds ratio (OR) and 95% confidence interval (CI). ResultsCompared to controls, colon cancer cases had statistically significant higher mean levels of glutamine, arginine, tauroursodeoxycholic acid (TUDCA), taurodeoxycholic acid (TDCA), glycodeoxycholic acid (GDCA), and lower mean levels of leucine and valine. Rectal cancer cases had statistically significant higher mean levels of mannose, glucose and isobutyric acid than controls. 9 metabolites [arginine, glutamine, mannose, deoxycholic acid (DCA), GDCA, taurochenodeoxycholic acid (TCDCA), TDCA, TUDCA and betaine] were associated with an increased risk of colon cancer (all p<0.05). Valine was inversely associated with colon cancer (OR3rd vs 1st tertile 0.45; 95% CI, 0.25-0.81). 7 metabolites (glucose, mannose, propionic acid, DCA, GDCA, TDCA and glycolate) were associated with an increased risk of rectal cancer (all p<0.05). Only glucose significantly improved the risk prediction of rectal cancer. No metabolites led to an improvement in the predictive model for colon cancer. ConclusionsDysregulation of secondary bile acids, monosaccharides, amino acids and short chain fatty acids may play a role in CRC development. Further studies are required to validate our findings. Legal entity responsible for the studyThe authors. FundingNational Medical Research Council. DisclosureAll authors have declared no conflicts of interest.

A Bacterial Pathogen Senses Host Mannose to Coordinate Virulence.

SummaryBacterial pathogens are thought to activate expression of virulence genes upon detection of host-associated cues, but identification of the nature of such signals has proved difficult. We generated a genome-scale defined transposon mutant library in Edwardsiella piscicida, an important fish pathogen, to quantify the fitness of insertion mutants for intracellular growth in macrophages and in turbot (Scophthalmus maximus). These screens identified EvrA, a transcription activator that induces expression of esrB, a key virulence regulator. EvrA is directly bound and activated by mannose-6-phosphate (man-6P) derived from actively imported mannose. Mutants lacking EvrA or expressing an EvrA unable to bind man-6P were similarly attenuated in turbot. Exogenously added mannose promoted E. piscicida virulence, and high levels of mannose were detected in fish tissue. Together, these observations reveal that binding of a host-derived sugar to a transcription factor can facilitate pathogen sensing of the host environment and trigger virulence programs.

Mannoproteins from Saccharomyces cerevisiae stimulate angiogenesis by promoting the akt-eNOS signaling pathway in endothelial cells

Mannoproteins (MPs) are a major component of yeast cell walls and consist of high levels of mannose in covalent complexes with proteins. MPs complexly enhance the immune system. We previously isolated a mutant yeast, K48L3, with a higher yield of MP from its cell wall than wild-type Saccharomyces cerevisiae, YPH499. We determined that K48L3 induces the release of nitric oxide in macrophage cells. The present study reports nitric-oxide-mediated angiogenesis by MP from K48L3 and the induction of the Akt/eNOS signal pathway. Western blotting and RT-PCR were used to demonstrate that MP treatment resulted in the upregulation of p-Akt, p-eNOS, and angiogenesis-mediated gene expression. Moreover, the angiogenesis activity of the MPs was demonstrated using three angiogenesis assays, namely, a cell migration assay, a tube-forming assay, and an ex vivo aorta ring assay. Thus, this study demonstrates for the first time that MPs from S. cerevisiae K48L3 induce angiogenesis in HUVECs via the Akt-eNOS-dependent signaling pathway.

Abstract 2633: Deciphering the role of protein glycosylation in oral cancer: insights into tumor biology and treatment

Abstract Head and neck cancer is a debilitating malignancy, with the majority of cases arising in the oral cavity as oral squamous cell carcinoma (OSCC). A major driver of OSCC is the epidermal growth factor receptor (EGFR), whose activity is aberrantly upregulated in &amp;gt;80% of tumors. EGFR is highly modified with N-linked glycans; fucosylation of N-glycans interferes with EGFR dimerization and activation. Thus, post-transcriptional changes may govern EGFR activity. In OSCC, EGFR signaling converges on Wnt/β-catenin activity, known to play pivotal roles in the pathobiology of this malignancy through the interaction of nuclear β-catenin with the histone acetyltransferase CREB-binding protein (CBP). We have shown that a small molecule inhibitor of β-catenin-CBP interaction, ICG-001, interferes with OSCC proliferation and aggressive features in cellular, zebrafish and murine models. Also, OSCC-cell line derived mouse tumor xenografts exhibit reduced EGFR abundance, and genomic analyses show a positive correlation between ICG-001 and EGFR inhibition. Given that modification of EGFR with N-glycans impacts its cell-surface localization and signaling, we hypothesized that ICG-001 affects EGFR N-glycosylation. To determine the effect of inhibition of β-catenin/CBP activity on cellular N-glycosylation programs, N-glycans from CAL27 and HSC3 cells treated with ICG-001 or vehicle control were released, permethylated, and analyzed via MALDI-TOF MS. Next, EGFR glycopeptides from CAL27 and HSC3 cells treated with ICG-001 or vehicle control, were analyzed with an Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer (Thermo Scientific) using EThcD. In CAL27 cells, we observed higher levels of high mannose (less processed) N-glycans and complex fucosylated N-glycans, whereas in HSC3 cells we observed complex, afucosylated N-glycans. After ICG-001 treatment, HSC3 cells displayed higher levels of fucosylated N-glycans, suggesting that ICG-001, via inhibition of β-catenin/CBP signaling, promotes a more indolent-like glycan profile. Similarly, EGFR from CAL27 cells had highly fucosylated N-glycans, while EGFR from HSC3 cells displayed N-glycans with a paucity of fucose. Treatment of HSC3 cells with ICG-001 led to higher fucosylation, potentially inhibiting EGFR signaling. Parallel analyses of gene expression signatures in response to ICG-001 treatment in HSC-3 cells showed increased transcriptional expression of fucosyltransferases, FUT2 and FUT3. Our studies suggest that the β-catenin/CBP axis promotes EGFR signaling through downregulation of FUT2 and FUT3 expression and activity. Thus, inhibition of β-catenin/CBP signaling with ICG-001 may serve as a therapeutic approach to downregulate EGFR pro-tumorigenic activity in OSCC. Supported by NIH grants P41 GM104603 (CEC), F32 CA196157 (KBC), and by the Evans Center for Interdisciplinary Biomedical Research ARC #9950000118 (MAK). Citation Format: Kevin B. Chandler, Vanessa L. Stahl, Khalid Alamoud, Bach-Cuc Nguyen, Vinay Kartha, Khikmet Sadykov, Stefano Monti, Maria A. Kukuruzinska, Catherine E. Costello. Deciphering the role of protein glycosylation in oral cancer: insights into tumor biology and treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2633.

In-hospital metabolite changes in infective endocarditis-a longitudinal 1H NMR-based study.

Treatment of infective endocarditis (IE) is a 4-6-week provided course of intravenously administered antibiotics. The aim of this study was to investigate how serum metabolites as measured by proton nuclear magnetic resonance (1H NMR) spectroscopy are changing over time during the active phase of IE, and to see whether these metabolite changes might be used to monitor recovery in these patients. Patients hospitalized with first-time IE at Herlev Hospital, Denmark, from September 2015 to June 2017 were included. Longitudinal blood sampling was performed and serum was analyzed using 1H NMR. Orthogonal projection to latent structures discriminant analysis (OPLS-DA) was used to separate sample groups and analyze differences in metabolite profiles. Thirteen patients were included in the study (77% men, median age 62years (IQR 53-77)). All patients were cured during the hospitalization without any relapse during 6months of follow-up. We analyzed 61 serum samples (median 5 samples, range 2-8 per person) drawn in the treatment period after IE diagnosis. The main changes during the in-hospital period were decreased levels of glucose, mannose, leucine, isoleucine, phenylalanine, tyrosine, and signals from polyols and N-acetylated protein. The metabolomic changes could in contrast to the routinely used parameters CRP and leucocyte levels distinguish between the early and late stages of disease treatment. We present the first longitudinal study of 1H NMR metabolomics in patients with infective endocarditis. The metabolomic changes show a promising strength compared to routinely used clinical parameters.

Physicochemical properties, antioxidant activity and immunological effects in vitro of polysaccharides from Schisandra sphenanthera and Schisandra chinensis.

Schisandra sphenanthera and Schisandra chinensis are widely consumed either as food or for medicinal purposes. Nevertheless, no detailed comparative assessments of their physicochemical properties and biological activity have been reported. In this paper, using hot-water extraction, alcohol precipitation, and deproteinization, we obtained polysaccharidic extracts from Schisandra sphenanthera and Schisandra chinensis (denoted as SSP and SCP, respectively) and investigated their antioxidant and immunological activities. The extracts were different from each other with regard to sugar, protein, and uronic acid contents. Both extracts were mainly composed of arabinose, glucose, and galactose, but their contents varied greatly; SSP had more galacturonic acid. Compared with SCP, SSP had stronger free radical scavenging ability, protective effects on biomolecules, cellular antioxidant activity, owing to its higher protein (35.35 ± 1.73%) and uronic acid (12.81 ± 1.15%) contents. With respect to cell viability, neutral red phagocytosis, NO production, and acid phosphatase activity, SCP had stronger effects than SSP; this was largely due to its high levels of mannose, galactose, arabinose, and glucose. These results provide evidence to support the use Schisandra-derived polysaccharides for several purposes, including clinical, agricultural, and industrial applications.

Quantification of d-mannose in plasma: Development and validation of a reliable and accurate HPLC-MS-MS method

The present paper describes the development and the validation process – in compliance with the EMA guidelines – of a method based on tandem mass spectrometry coupled to liquid chromatography for the accurate quantification of mannose in human plasma samples. The quick sample preparation procedure, simplified by the absence of any derivatization step, makes the assay suitable for routine use in a clinical chemistry laboratory. The method validation yielded satisfactory selectivity, with a good separation of mannose from its epimers (glucose and galactose), linearity over the whole concentration range of interest (0.31–40 μg/mL), reproducibility with RSD <10%, and accuracy in the range 96–104%. Instrumental LLOD (0.31 μg/mL) and LLOQ (1.25 μg/mL) were good enough to detect endogenous plasma mannose levels and in agreement with recent data from the literature. Sensitivity was affected by a 5-fold dilution factor, which, if necessary, can be reduced. The method robustness was proven in >600 injections, most of them being of plasma samples, used also to assess the reference ranges in healthy subjects (9.93 ± 3.37 μg/mL) and type 2 diabetic patients (23.47 ± 6.19 μg/mL).

Fasting plasma mannose levels are associated with insulin sensitivity independent of BMI in Japanese individuals with diabetes

BackgroundRecently, an integrated network analysis has revealed dysregulation in the metabolism of mannose, a glucose epimer, in severely obese individuals without diabetes. In addition, fasting plasma mannose levels (M0) are associated with insulin resistance independent of BMI. Since the association between mannose and insulin sensitivity (IS) in those with impaired glucose tolerance remains unknown, we aimed to investigate this association in individuals without severe obesity but with varying degrees of glucose tolerance.MethodsBased on 75 g OGTT data in Japanese individuals without diabetic medication, individuals were classified as having normal glucose tolerance (NGT), impaired glucose metabolism (IGM), or diabetes (DM). In each group, 25 individuals were consecutively recruited [total 75 individuals, age: 65 ± 11 (mean ± SD); BMI: 24.9 ± 3.8 kg/m2]. QUICKI and Matsuda index (MI) were calculated as IS indices. M0 was assayed using HPLC. Normally-distributed loge-transformed (ln-) values were used for MI and leptin.ResultsIn the simple regression analysis, ln-MI was negatively correlated with BMI (NGT: r = − 0.639, IGM: r = − 0.466, DM: r = − 0.613) and ln-leptin (NGT: r = − 0.480, IGT: r = − 0.447, DM: r = − 0.593) in all 3 groups. Ln-MI was not significantly correlated with M0 in NGT (r = 0.241, P = 0.245) and IGT (r = − 0.296, P = 0.152) groups, it was moderately and negatively correlated in the DM group (r = − 0.626, P < 0.001). Similar results were obtained, when QUICKI was used instead of MI as an index of IS. In multiple regression analysis in the DM group, QUICKI (Q) and ln-MI (M) were independently predicted by BMI (Q: β = − 0.413; M: β = − 0.400) and M0 (Q: β = − 0.413, M: β = − 0.426), accounting for 51.2% (P = 0.0004) and 51.2% (P = 0.0004) of the variability, respectively, which was larger than the prediction for BMI alone (Q: 38.4%, M: 37.6%).ConclusionFasting plasma mannose was associated with IS independent of BMI in Japanese individuals with DM.

Purification, structural analysis and mechanism of murine macrophage cell activation by sulfated polysaccharides from Cystoseira indica

Sulfated polysaccharides were isolated and purified from the water extract of Cystoseira indica using DEAE Sepharose Fast Flow column to evaluate their structure and macrophage stimulating capacity. Crude and fractionated polysaccharides, CIF1 and CIF2, were mostly composed of neutral sugars (73.1%–78.6%) with relatively lower amounts of acidic sugars (1.3%–9.0%) and sulfate esters (6.9%–9.7%). The polymer chains of polysaccharides were mainly built of different levels of glucose (2.1%–30.8%), fucose (17.2%–24.4%), mannose (17.8%–20.6%) and galactose (16.7%–17.3%). The weight average molecular weight (Mw) of polysaccharides varied between 573.1 × 103 g/mol to 1146.6 × 103 g/mol. The CIF2 polysaccharide, as the most immunostimulating polysaccharide, remarkably induced the release of nitric oxide and inflammatory cytokines including TNF-α, IL-1β, IL-6 and IL-10 from RAW264.7 murine macrophage cells through NF-κB and PAMKs transduction signaling pathways via cell surface TLR4. The interconnections of sugars in CIF2 polysaccharide were complex with (1→3)-fucopyranose, (1→2,3,4)-glucopyranose, (→1)-galactopyranose, (→1)-xylopyranose, (1→2)-rhamnopyranose and (1→2,3)-mannopyranose units being the most predominant residues.

Isolation and chemical characterization of a novel immunostimulating galactofucan from freshwater Azolla filiculoides

Water-soluble polysaccharides were isolated from Azolla filiculoides to determine their chemical and structural characteristics as well as anticancer and immunostimulatory activities. Crude and fractions (F1 and F2) were mainly composed of neutral sugars (70.0–80.1%), proteins (2.1–14.2%) and uronic acids (1.2–10.8%). The polysaccharides were mostly formed of different levels of fucose (23.8–61.2%), galactose (28.5–38.7%), mannose (7.5–16.7%), xylose (13.3–13.6%), glucose (12.7–13.3%), arabinose (5.5–11.6%) and rhamnose (8.0–9.5%) units. The polysaccharide molecules contained one or more sub-fractions with average molecular weight ranging from 992 to 2162 × 103 g/mol. Crude and fractionated polysaccharides induced RAW264.7 macrophages to release pro-inflammatory mediators and cytokines including nitric oxide, IL-1β, TNF-α, IL-6, IL-10 and IL-12 through NF-κB and MAPKs signaling pathways as confirmed by the presence of p-NK-κB, p-JNK, p-ERK and p-38 proteins in the cell cytoplasm. The most immunostimulating polysaccharide, F2, consisted of alternating →3)-Fuc-(1→ and →4)-Fuc-(1 → residues.

Abstract 3486: Metabolomics in estrogen receptor or triple-negative breast cancer

Abstract Breast cancer is the most frequently diagnosed cancer in women. Metabolic syndrome and obesity may contribute to triple-negative breast cancer (TNBC) and there are obesity-related alterations in metabolites. Therefore, we pilot-tested metabolomics in ER-negative or TNBC using urine samples from 60 breast cancer cases. In total, 475 metabolites were quantified in different super-pathways: 147 in amino acid, 38 in carbohydrate, 27 in cofactors and vitamins, 12 in energy, 50 in lipid, 31 in nucleotide, 17 in peptide, and 153 in xenobiotics. Our data in 13 ER- and 47 ER+ showed that urinary beta-alanine is higher (3.35+3.73 vs. 1.82+2.26) in ER- breast cancer patients. Our data also show, the first time, that the urinary beta-alanine was higher in TNBC (3.60+4.33; N=8) compared to other subtypes (1.93+2.32; N=52). Beta-alanine is a nonessential amino acid that has previously been shown to be metabolized into carnosine, which functions as an intracellular buffer. In breast cancer cells, beta-alanine reduces both tumor cell migration and proliferation without acting in a cytotoxic fashion. It also significantly increased breast cancer cell sensitivity to doxorubicin. Our data on xanthine are also consistent with the previous study on breast tumor that slightly higher level was observed in ER- patient (1.41+1.35 vs. 1.16+0.88). The level of xanthine was significantly higher in TNBC compared to other subtypes (1.67+1.69 vs. 1.14+0.84). More importantly, our pilot data also showed that urinary mannose levels were significantly higher in ER- tumor (2.21+1.90 vs. 0.90+1.21 p=0.032) and TNBC (2.19+1.77 vs. 1.03+1.38, p=0.037), which is consistent with a previous study that reported that serum mannose levels can differentiate patients with metastatic and localized breast cancer. Metabolic drugs for cancer, such as glutaminase inhibitors, are currently under development and being tested in clinical trials. Therefore, if our data are validated, metabolomics of ER- or TNBC will contribute to innovative discoveries that will impact breast cancer prevention and precision medicine targeting metabolomic pathways. Citation Format: Jennifer J. Hu, Cristiane Takita, Eunkyung Lee, Jean Wright. Metabolomics in estrogen receptor or triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3486.

Fasting Plasma Mannose Level Is Associated with Insulin Sensitivity Independent of BMI in Japanese Individuals with Diabetes

Recently, integrated network analysis has revealed dysregulation in the metabolism of mannose, an epimer of glucose, in individuals with severe obesity who do not have significant diabetes. It was also found that fasting plasma mannose levels (M0) are associated with insulin resistance independent of BMI. Since the association between mannose and insulin sensitivity (IS) in those with impaired glucose tolerance remains unknown, we aimed to investigate the association in individuals with varying degrees of glucose tolerance. Based on data of 75g OGTT in Japanese individuals without diabetic medication, individuals were classified as normal (NGT), impaired glucose tolerance (IGT), and diabetes (DM). In each group, 25 subjects were consecutively recruited [total 75 individuals, age: 65±11 (mean±SD); BMI: 24.9±3.8]. Matsuda index (MI) was calculated as an index of IS. M0 was assayed using HPLC. Normally-distributed loge-transformed (ln-) values were used for MI and leptin. Although fasting plasma glucose (G0), HbA1c, and ln-MI were significantly different among 3 groups, age, sex, BMI, and M0 were not different. In simple regression analysis, ln-MI was negatively correlated with BMI (NGT: r=-0.639, IGT: r=-0.466, DM: r=-0.613) and ln-leptin (NGT: r=-0.480, IGT: r=-0.447, DM: r=-0.593) in all 3 groups. Interestingly, although ln-MI was not significantly correlated with M0 in NGT (r=0.241, P=0.245) and IGT (r=-0.296, P=0.152), it was moderately and negatively correlated in DM (r=-0.626, P&amp;lt;0.001). In DM, ln-MI was slightly correlated with G0 (r=-0.484, P=0.014). In multiple regression analysis in DM, ln-MI was independently predicted by BMI (β=-0.400) and M0 (β=-0.426), accounting for 51.2% (P&amp;lt;0.001) of the variability, which was larger than that in the prediction by BMI alone (37.6%). In conclusion, in our study, fasting plasma mannose was associated with insulin sensitivity independent of BMI in Japanese individuals with diabetes. Disclosure E. Amano: None. S. Funakoshi: None. K. Yoshimura: None. S. Hirano: None. S. Ohmi: None. Y. Terada: None. S. Fujimoto: None.

N-glycan signatures identified in tumor interstitial fluid and serum of breast cancer patients: association with tumor biology and clinical outcome.

Particular N‐glycan structures are known to be associated with breast malignancies by coordinating various regulatory events within the tumor and corresponding microenvironment, thus implying that N‐glycan patterns may be used for cancer stratification and as predictive or prognostic biomarkers. However, the association between N‐glycans secreted by breast tumor and corresponding clinical relevance remain to be elucidated. We profiled N‐glycans by HILIC UPLC across a discovery dataset composed of tumor interstitial fluids (TIF, n = 85), paired normal interstitial fluids (NIF, n = 54) and serum samples (n = 28) followed by independent evaluation, with the ultimate goal of identifying tumor‐related N‐glycan patterns in blood of patients with breast cancer. The segregation of N‐linked oligosaccharides revealed 33 compositions, which exhibited differential abundances between TIF and NIF. TIFs were depleted of bisecting N‐glycans, which are known to play essential roles in tumor suppression. An increased level of simple high mannose N‐glycans in TIF strongly correlated with the presence of tumor infiltrating lymphocytes within tumor. At the same time, a low level of highly complex N‐glycans in TIF inversely correlated with the presence of infiltrating lymphocytes within tumor. Survival analysis showed that patients exhibiting increased TIF abundance of GP24 had better outcomes, whereas low levels of GP10, GP23, GP38, and coreF were associated with poor prognosis. Levels of GP1, GP8, GP9, GP14, GP23, GP28, GP37, GP38, and coreF were significantly correlated between TIF and paired serum samples. Cross‐validation analysis using an independent serum dataset supported the observed correlation between TIF and serum, for five of nine N‐glycan groups: GP8, GP9, GP14, GP23, and coreF. Collectively, our results imply that profiling of N‐glycans from proximal breast tumor fluids is a promising strategy for determining tumor‐derived glyco‐signature(s) in the blood. N‐glycans structures validated in our study may serve as novel biomarkers to improve the diagnostic and prognostic stratification of patients with breast cancer.

An efficient method to control high mannose and core fucose levels in glycosylated antibody production using deoxymannojirimycin

Glycosylation on the Fc region of recombinant Immunoglobulin G (IgG) therapeutic antibodies is a critical protein quality attribute which may affect the efficacy and safety of the molecule. During the development of biosimilar therapeutics, adjustment of the glycosylation profile is required in order to match the reference innovator profile. Deoxymannojirimycin (DMJ), a known inhibitor of mannosidase, was used in this study to modulate the glycosylation pattern of antibodies. The effect of DMJ, at concentrations of 5 μM - 500 μM, on non-fucosylated glycoform levels was tested in the biosynthesis processes of two different IgG1 (IgG1 #A and IgG1 #B) using two Chinese hamster ovary (CHO) cell lines (CHO-DXB-11 and CHOK1SV, respectively) in Erlenmeyer flasks and in lab scale bioreactors. DMJ affected glycan forms in a dose response manner. At the highest concentration tested, DMJ reduced N-linked complex glycoform and core fucose levels by 15 and 14 fold, respectively, and increased high mannose level by 21 fold. 10 μM DMJ decreased IgG1 #A core fucose level in CHO-DXB-11 from 92% to 73% and increased high mannose level from 4% to 22% in Erlenmeyer flasks. Furthermore, in lab scale bioreactors, 15 μM DMJ decreased IgG1 #A core fucose level from 95% to 84% and increased high mannose level from 3% to 13%. Core fucose level of IgG1 #B in CHOK1SV was decreased from 81% to 73% using 10 μM DMJ in lab scale bioreactors while high mannose was increased from 6% to 15%. While affecting core fucose and high mannose levels, DMJ decreased maximum viable cell concentration by 16% and did not significantly affect cell productivity (less than 10%). This study demonstrated that DMJ can enable the control of core fucosylated and high mannose levels of IgG1 antibodies in a defined range.

Glycan analysis of colorectal cancer samples reveals stage-dependent changes in CEA glycosylation patterns

BackgroundCarcinoembryonic antigen (CEA) is a glycoprotein associated with colorectal cancer (CRC). While the functions of its gene and protein have been fully characterized, its post-translational modifications in the context of CRC development remain undefined.MethodsTo show the correlation between the different stages of CRC development and changes in the glycosylation patterns of CEA, we analyzed CEA in tumor tissues (CEA-T) and paired tumor-adjacent normal tissues (CEA-A) from 53 colorectal cancer patients using a high-density lectin microarray containing 56 plant lectins.ResultsWe detected higher expression levels of fucose, mannose and Thomsen–Friedenreich antigen, and lower expression levels of N-acetylgalactosamine, N-acetylglucosamine, galactose, branched and bisecting N-glycans on CEA in the tumor tissues relative to the tumor-adjacent normal tissues. Furthermore, a combinatorial assessment of 9 lectins is sufficient to distinguish CRC tumor tissues from tumor-adjacent normal tissues with 83% sensitivity and ~ 90% specificity. Moreover, the levels of N-acetylgalactosamine, mannose, galactose, N-acetylglucosamine on CEA showed a downward trend after first experiencing an increase at Stage II with the stages of CRC.ConclusionsOur insights into the changing CEA glycosylation patterns and their role in the development of CRC highlight the importance of glycan variants on CEA for early clinical detection and staging of CRC.

Exteriorisation of Mannoses on Human Erythrocyte Membrane Skeleton Provides ‘Eat Me’ Signals for Oxidatively Damaged Cells to be Cleared By Macrophages: A Pathway Mediating Hemolysis in Sickle Cell Disease

The disposal of unwanted or dying cells is a key biological process driven by the display of ‘eat me’ signals that are recognised by phagocytes. Although these markers for uptake are known to include certain lipids and proteins, the role of glycans, which are abundant on cell surfaces, remains poorly characterized. Here, an unbiased glycomic survey by mass spectroscopic analyses of glycosidase (PNGase F) digested human red blood cell (RBC) membranes identified novel N-linked high mannose structures, which are sequestered inside healthy cells with spectrin, the major protein of the internal membrane skeleton, but exteriorised when cells are damaged as a dominant signal for uptake by macrophages. A panel of lectin probes was used to demonstrate that the mannose species were available as discrete patches on the surface of RBC that had been stressed by oxidation, but undetectable on unpermeabilized untreated cells. High mannoses co-localized with alpha-spectrin on lectin/Western blots and this signal was degraded by prior incubation with glycosidases. Super resolution microscopy showed co-localisation of mannose with spectrin in membrane protrusions of oxidized RBC. Co-localization of spectrin with N-linked mannose and exteriorisation on effete cells were also observed in nucleated cells. The mannose displayed on oxidized RBC represents a novel ‘eat-me’ signal for human cells, since congeners of mannose inhibited uptake by human monocyte-derived macrophages. The mannose receptor (CD206) was identified as an important receptor in this system by the use of blocking antibody and siRNA mediated knock-down.The mechanisms underlying the hemolysis characteristic of sickle cell disease (SCD) remain to be fully defined. We therefore investigated the contribution of this new pathway. RBC from patients with SCD demonstrated remarkably high levels of surface mannose, and super resolution microscopy showed the expression again to be limited to discrete patches and co-localized with spectrin in a disrupted membrane skeletal structure. The importance of mannose exposure to uptake of sickle cells by macrophages was confirmed by inhibition by congeners of mannose and blocking antibody to CD206.The prevalence of SCD is high because of selective pressure caused by the resistance of subjects with sickle cell trait to malarial parasites, which are mainly cleared by splenic macrophages. We therefore investigated whether RBC from subjects with sickle cell trait expressed surface mannose. We show that such RBC express modestly raised levels (p<.01, n=10 (Wilcoxon rank sum)). This excess surface mannose should predispose to parasitized RBC being cleared more rapidly in infected individuals and may cause the increasingly recognised clinical manifestations of sickle cell trait.We also investigated whether the degree of mannose exposure correlated with peripheral blood count parameters. The Pearson correlation coefficients of log transformed surface mannose expression with hemoglobin levels, red cell counts and reticulocyte counts were -0.83, -0.84 and 0.73 respectively (all p values <0.0001)(n=70, 30 patients with homozygous SS, 10 with AS, 30 with AA); it was noted that cells from patients taking hydroxycarbamide, who had concomitant alpha-thalassaemia or SC disease exhibited intermediate levels.In summary, our results reveal a previously undescribed cryptic mannose exteriorization pathway, which mediates disposal of oxidatively damaged cells. The pathway is constitutively activated in SCD, where it mediates haemolysis and the degree of activation correlates well with clinical phenotypes of SCD and sickle cell trait. It thus represents a new mechanism for possible future therapeutic intervention. DisclosuresVickers:University of Aberdeen: Patents & Royalties: About to apply for patent. Barker:University of Aberdeen: Employment, Patents & Royalties: About to apply for patent. Cao:University of Aberdeen: Patents & Royalties: About to apply for patent.

PERUBAHAN KADAR RAFINOSA, GLUKOSA, MANOSA, ARABINOSA DAN SUKROSA WHEAT POLLARD AKIBAT LAMA STEAM DAN PENAMBAHAN AIR YANG BERBEDA

Wheat pollard is a waste of the wheat industry and many contain non starch polisacharida (NSP) that interfere with the chicken digestive tract. NSP in wheat pollard can be removed by heating, so it can be used by microbes in the digestive tract of poultry. Utilization of NSP by microbes will result in short chain fatty acids (SCFA), namely acetic acid, propionate and butyrate which have a positive effect on the digestive tract of poultry. The purpose of this study is to examine changes in levels of rafinose, glucose, mannose, arabinose and sucrose in wheat pollard into a source of functional feed for poultry. The benefit of research is to know the technique / manufacture of functional feed material from wheat pollard. The material used in the research is wheat pollard and aquades by using autoclave tool. The design used was a complete randomized design of 2x3 factorial pattern with 3 replications. The first factor is the duration of steam 30 and 60 minutes while the second factor is the addition of water (0, 30 and 60%). The results showed that the addition of water and the duration of different steam strongly influenced (p &lt;0.001) and had interactions on rafinose, mannose, arabinose, sucrose and glucose parameters. The mean of water addition factor increased rafinose level from 1.11+0.03% to 29+0.02% while steam duration decreased raffinose content from 1.99+0.01% to 1.90+0.04%. Glucose levels increased in water addition factor and steam duration from 0.25+0.01% to 1.06+0.12% and 0.54+0.03% to 0.63+0.07%. The mean of water addition factor decreased the mannose level from 0.19+0.01% to 0.06+0.02% and the steam duration increased the manosa level from 0.09+0.01% to 0.15+0.01%. Levels of arabinose increased in water addition factor and steam duration from 0.30+0.03% to 0.98+0.01% and 0.54+0.02% to 0.70+0.04%. The average water addition decreased the sucrose content from 1.80+0.09% to 1.55+0.04% and the steam duration increased the sucrose content from 1.48+0.04% to 1.72+0.03%. The research conclusions that the steam treatment and the addition of water greatly affect the changes of rafinose, glucose, mannose, arabinose and sucrose in wheat pollard and potentially as functional feed. Recommendations that can be given by researchers is the source of feed ingredients that have high starch content should be processed before it is given to poultry so it has a functional value and improve the digestibility of the feed material.