MUC1 O-glycosylation contributes to anoikis resistance in epithelial cancer cells

Anoikis is a fundamental cellular process for maintaining tissue homeostasis. Resistance to anoikis is a hallmark of oncogenic epithelial–mesenchymal transition and is a pre-requisite for metastasis. Previous studies have revealed that the heavily glycosylated mucin protein MUC1, which is overexpressed in all types of epithelial cancer cells, prevents anoikis initiation in response to loss of adhesion. This effect of MUC1 is largely attributed to its extracellular domain that provides cell surface anoikis-initiating molecules with a ‘homing’ microenvironment. The present study investigated the influence of O-glycosylation on MUC1 extracellular domain on MUC1-mediated cell resistance to anoikis. It shows that stable suppression of the Core 1Gal-transferase (C1GT) by shRNA substantially reduces O-glycosylation in MUC1-positively transfected human colon cancer HCT116 cells and in high MUC1-expressing SW620 cells. Suppression of C1GT significantly increased anoikis of the MUC1-positive, but not MUC1-negative, cells in response to suspended culture. This effect was shown to be associated with increased ligand accessibility to cell surface anoikis-initiating molecules such as E-cadherin, integrinβ1 and Fas. These results indicate that the extensive O-glycosylation on MUC1 extracellular domain contributes to MUC1-mediated cell resistance to anoikis by facilitating MUC1-mediated prohibition of activation of the cell surface anoikis-initiating molecules in response to loss of cell adhesion. This provides insight into the molecular mechanism of anoikis regulation and highlights the importance of cellular glycosylation in cancer progression and metastasis

Discussion: behaviour of jacked and driven piles in sandy soil

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Behaviour of jacked and driven piles in sandy soil

As an alternative to conventional dynamic pile installation methods, pile jacking is an environmentally friendly technique that could become more widely accepted. Great concern has arisen over the performance of jacked piles as compared with that of driven piles. This paper describes a comprehensive field study that was aimed at investigating the differences and similarities between the behaviour of jacked H-piles and that of driven H-piles. The instrumented piles, varying in length from 32 to 55 m and having a design capacity of up to 3540 kN, were installed in residual soils whose properties are close to silty sands. The load test results indicate that the shaft resistance of jacked piles is generally suffer and stronger than that of driven piles, but the base resistance of jacked piles is weaker than that of driven piles. At a load level of twice the design capacity, the percentage of pile head load carried by base varies from 2% to 10% for jacked piles, with a mean value of 6%; for driven piles the percentage varies from 6% to 61% with a mean value of 38%. The back-calculated values of the shaft friction coefficient, β, were found to be in a range of 0.25-0.6 for both jacked and driven piles. A correlation was also observed between the ultimate shaft friction and the mean standard penetration test N value (N̄), which suggests that the shaft friction can be taken as 1.5N̄ to 2N̄ (kPa) for both jacked and driven H-piles.published_or_final_versio

Discussion: behaviour of jacked and driven piles in sandy soil

published_or_final_versio

Galectin-3 interacts with the cell surface glycoprotein CD146 (MCAM, MUC18) and induces secretion of metastasis-promoting cytokines from vascular endothelial cells

The galactoside-binding protein galectin-3 is increasingly recognized as an important player in cancer development, progression, and metastasis via its interactions with various galactoside-terminated glycans. We have shown previously that circulating galectin-3, which is increased up to 30-fold in cancer patients, promotes blood-borne metastasis in an animal cancer model. This effect is partly attributable to the interaction of galectin-3 with unknown receptor(s) on vascular endothelial cells and causes endothelial secretion of several metastasis-promoting cytokines. Here we sought to identify the galectin-3-binding molecule(s) on the endothelial cell surface responsible for the galectin-3-mediated cytokine secretion. Using two different galectin-3 affinity purification processes, we extracted four cell membrane glycoproteins, CD146/melanoma cell adhesion molecule (MCAM)/MUC18, CD31/platelet endothelial cell adhesion molecule-1 (PECAM-1), CD144/VE-cadherin, and CD106/Endoglin, from vascular endothelial cells. CD146 was the major galectin-3-binding ligand and strongly co-localized with galectin-3 on endothelial cell surfaces treated with exogenous galectin-3. Moreover, galectin-3 bound to N-linked glycans on CD146 and induced CD146 dimerization and subsequent activation of AKT signaling. siRNA-mediated suppression of CD146 expression completely abolished the galectin-3-induced secretion of IL-6 and G-CSF cytokines from the endothelial cells. Thus, CD146/MCAM is the functional galectin-3-binding ligand on endothelial cell surfaces responsible for galectin-3-induced secretion of metastasis-promoting cytokines. We conclude that CD146/MCAM interactions with circulating galectin-3 may have an important influence on cancer progression and metastasis

The intracellular domain of homomeric glycine receptors modulates agonist efficacy

Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyREM, the agonists glycine, β-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyREM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyREM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyREM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for ICD in pentameric ligand-gated channels

Antitumor lectin Sclerotium rolfsii (SRL) induces apoptosis in human colon cancer cells by activation of multiple signaling pathways; A microarray analysis

Background: TF antigen specific Sclerotium rolfsii lectin (SRL) inhibits human colon epithelial cancer HT29 cell growth by induction of apoptosis through cell surface binding and has tumor suppressing effect in vivo as reported earlier. Here we report the purification, identification and characterization of SRL binding membrane proteins from HT29 cells. Methods and Findings: Membrane proteins from HT29 cells were isolated by phase separation and purified by affinity chromatography using SRL-Sepharose4B matrix. Affinity purified proteins were subjected to in-gel and in-solution trypsin digestion, analysed by ESI-Q-TOF LC-MS and spectrum mill software. Considering the specificity of SRL towards O-glycans, the presence of O-GalNAc sites in SRL interacting proteins were tested using NetOGlyc software. Western blotting was performed to validate the MS identified proteins. A major protein band around 25kDa following in-gel trypsin digestion was identified as Keratin 1 by MS. In-solution trypsin digestion followed by MS identified 25 SRL interacting proteins namely, keratins, heat shock proteins, tubulins, pyruvate kinase M1/M2, peroxiredoxin-1, ATP synthase subunit alpha, mitochondrial, retinal dehydrogenase 1, actin, annexin-A2, prohibitin, ADP/ATP translocase-2 and alpha enolase. NetOGlyc software analysis revealed 21 proteins positive for O-glycosylation sites including keratins alone containing 27 to 50 O-GalNAc sites. Keratin 1 identified and validated by western blotting as major SRL interacting protein showed 49 O-GalNAc sites. Conclusion: SRL binding membrane proteins from human colon epithelial cancer HT29 cells have been identified and characterized. Identified proteins contain O-GalNAc sites and are known to be involved in cell survival, apoptosis and tumorigenesis. The present study provides insights in studying the mechanism of SRL induced apoptosis and to explore lectin for its clinical implications. Key words: Sclerotium rolfsii lectin; HT29 cell membrane proteins; NetOGlyc version 4.0; Q-TOF-LC/MS; Spectrum Mill. Abbreviations: SRL: Sclerotium rolfsii lectin; LC/MS: Liquid chromatography/Mass spectrometry; ESI: Electro Spray Ionization; Q-TOF: Quadrupole- Time of Flight; PTM: Post Translational Modification; ACN: Acetonitrile; CBB: Coomassie Brilliant Blue; BSA: Bovine Serum Albumin