a1,6-Fucosyltransferase (Fut8), an enzyme that catalyzes the introduction of α1,6 core fucose to the innermost N-acetylglucosamine residue of the N-glycan, has been implicated in the development, immune system, and tumorigenesis. We found that α1,6-fucosyltransferase and E-cadherin expression levels are significantly elevated in primary colorectal cancer samples. Interestingly, low molecular weight population of E-cadherin appeared as well as normal sized E-cadherin in cancer samples. To investigate the correlation between α1,6-fucosyltransferase and E-cadherin expression, we introduced α1,6-fucosyltransferase in WiDr human colon carcinoma cells. It was revealed that the low molecular weight population of E-cadherin was significantly increased in α1,6-fucosyltransferase-transfected WiDr cells in dense culture, which resulted in an enhancement in cell-cell adhesion. The transfection of mutated a1,6-fucosyltransferase with no enzymatic activity had no effect on E-cadherin expression, indicating that core fucosylation is involved in the phenomena. In α1,6-fucosyltransferase knock down mouse pancreatic acinar cell carcinoma TGP49 cells, the expression of E-cadherin and E-cadherin dependent cellcell adhesion was decreased. The introduction of α1,6-fucosyltransferase into kidney epithelial cells from α1,6-fucosyltransferase -/-mice restored the expression of E-cadherin and E-cadherin-dependent cell-cell adhesion. Based on the results of lectin blotting, peptide Nglycosidase F treatment, and pulse-chase studies, it was demonstrated that the low molecular weight population of E-cadherin contains peptide N-glycosidase F insensitive sugar chains, and the turnover rate of E-cadherin was reduced in α1,6-Fucosyltransferase transfectants. Thus, it was suggested that core fucosylation regulates the processing of oligosaccharides and turnover of E-cadherin. These results suggest a possible role of core fucosylation in the regulation of cell-cell adhesion in cancer. (Cancer Sci 2009; 100: 888-895) I t is generally accepted that glycosylation affects many properties of glycoproteins, including their conformation, flexibility, and hydrophilicity. As a result, it regulates protein sorting, stability, and protein-protein interactions.(1-5) N-Glycans have a common core structure, and their branching patterns are determined by glycosyltransferases.(6,7) Fut8 is an enzyme that catalyzes the introduction of α1,6 core fucose on the asparagine-branched N-acetylglucosamine residue of the chitobiose unit of complextype N-glycans. (8,9) Fut8 has been investigated especially in terms of oncogenesis, since the α1,6-fucosylation of α-fetoprotein is a well-known marker of hepatocellular carcinoma. (10) In previous studies, our group reported that Fut8 expression is markedly enhanced in several types of cancer cell lines (11) rat hepatoma tissues (12) and in ovarian serous adenocarcinoma cells.E-cadherin is a 120 kDa type I membrane protein, which belongs to the class of calcium-dependent cell adhesion molecules. (14) It mediates cell-cell adhesi...

Cardiac-type sarco(endo)plasmic reticulum Ca(2)-ATPase (SERCA2a) plays a major role in cardiac muscle contractility. Phospholamban (PLN) regulates the function of SERCA2a via its Ser(16)-phosphorylation. Since it has been proposed that the Ser/Thr residues on cytoplasmic and nuclear proteins are modified by O-linked N-acetylglucosamine (O-GlcNAc), we examined the effect of O-GlcNAcylation on PLN function in rat adult cardiomyocytes. Studies using enzymatic labeling and co-immunoprecipitation of wild type and a series of mutants of PLN showed that PLN was O-GlcNAcylated and Ser(16) of PLN might be the site for O-GlcNAcylation. In cardiomyocytes treated with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), the O-GlcNAcylation was significantly increased compared to non-treated cells. Simultaneously, Ser(16)-phosphorylation of PLN was reduced. In Chinese hamster ovary cells where PLN cDNA and O-GlcNAc transferase siRNA were co-transfected, the Ser(16)-phosphorylation of PLN was significantly increased compared to controls. The same results were observed in heart homogenates from diabetic rats. In a co-immunoprecipitation of PLN with SERCA2a, the physical interaction between the two proteins was increased in PUGNAc-treated cardiomyocytes. Unlike non-treated cells, the activity of SERCA2a and the profiles of calcium transients in PUGNAc-treated cardiomyocytes were not significantly changed even after treatment with catecholamine. These data suggest that PLN is O-GlcNAcylated to induce the inhibition of its phosphorylation, which correlates to the deterioration of cardiac function. This might define a novel mechanism by which PLN regulation of SERCA2a is altered under conditions where O-GlcNAcylation is increased, such as those occurring in diabetes.

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Phospholamban (PLN) is an effective inhibitor of the sarco(endo) plasmic reticulum Ca 2+ ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [ 35 S]methionine pulse-chase experiments, together with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. This reduction in PLN levels was functionally correlated with an increased rate of SERCA2a activity, accounting for an inotropic effect of metformin. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3, became increasingly colocalized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at the K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1-null mouse hearts was associated with increased PLN levels determined by immunoblots and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.selective autophagy | ubiquitinylation | protein degradation P hospholamban (PLN) is a 52-amino acid peptide located in the sarcoplasmic reticulum (SR) membrane in cardiac, slowtwitch skeletal, and smooth muscle, where it exists as a monomer or pentamer. Whereas monomeric PLN physically interacts with sarco(endo)plasmic reticulum Ca 2+ ATPase type 2a (SERCA2a) to antagonize its function, pentameric PLN complexes are thought to be a reservoir of inactive PLN (1-3). The physical interaction between SERCA2a and PLN reduces the apparent affinity of SERCA2a for Ca 2+ , thereby making SERCA2a less active in transporting Ca 2+ from the cytoplasm to the lumen of the SR at the same concentration of cytoplasmic Ca 2+. The physical interaction between the two proteins is regulated by phosphorylation of PLN at Ser16 by protein kinase A or at Thr17 by Ca 2+ /calmodulin-dependent protein kinase II (2). Phosphorylation of PLN reduces its affinity for SERCA2a, thereby increasing SERCA2a activity (2). Evidence from transgenic mice also supports the inhibitory function of PLN. Although targeted PLN deletion enhances baseline cardiac performance, cardiac-specific overexpression of superinhibitory forms of PLN leads to decreases in the affinity of SERCA2a for Ca 2+ (2). These observations underscore the primary role of PLN as a regulator of SERCA2a activity and, therefore, as a crucial regulator of cardiac contractility. PLN inhibition of SERCA2a can be reversed by either external (i.e., activation of β-adrenergic receptors) or internal (i.e., increased...