Showing papers by "Yoshiki Yamaguchi published in 2011"

Modulation of E-cadherin function and dysfunction by N-glycosylation.

Abstract: Several mechanisms have been proposed to explain the E-cadherin dysfunction in cancer, including genetic and epigenetic alterations Nevertheless, a significant number of human carcinomas have been seen that show E-cadherin dysfunction that cannot be explained at the genetic/epigenetic level A substantial body of evidence has appeared recently that supports the view that other mechanisms operating at the post-translational level may also affect E-cadherin function The present review addresses molecular aspects related to E-cadherin N-glycosylation and evidence is presented showing that the modification of N-linked glycans on E-cadherin can affect the adhesive function of this adhesion molecule The role of glycosyltransferases involved in the remodeling of N-glycans on E-cadherin, including N-acetylglucosaminyltransferase III (GnT-III), N-acetylglucosaminyltransferase V (GnT-V), and the α1,6 fucosyltransferase (FUT8) enzyme, is also discussed Finally, this review discusses an alternative functional regulatory mechanism for E-cadherin operating at the post-translational level, N-glycosylation, that may underlie the E-cadherin dysfunction in some carcinomas

Sorting of GPI-anchored proteins into ER exit sites by p24 proteins is dependent on remodeled GPI

Abstract: Glycosylphosphatidylinositol (GPI) anchoring of proteins is a posttranslational modification occurring in the endoplasmic reticulum (ER). After GPI attachment, proteins are transported by coat protein complex II (COPII)-coated vesicles from the ER. Because GPI-anchored proteins (GPI-APs) are localized in the lumen, they cannot interact with cytosolic COPII components directly. Receptors that link GPI-APs to COPII are thought to be involved in efficient packaging of GPI-APs into vesicles; however, mechanisms of GPI-AP sorting are not well understood. Here we describe two remodeling reactions for GPI anchors, mediated by PGAP1 and PGAP5, which were required for sorting of GPI-APs to ER exit sites. The p24 family of proteins recognized the remodeled GPI-APs and sorted them into COPII vesicles. Association of p24 proteins with GPI-APs was pH dependent, which suggests that they bind in the ER and dissociate in post-ER acidic compartments. Our results indicate that p24 complexes act as cargo receptors for correctly remodeled GPI-APs to be sorted into COPII vesicles.

Brain endothelial cells produce amyloid-β from amyloid precursor protein 770 and preferentially secrete the O-glycosylated form

Abstract: early events in Alzheimer disease. Specifically, it has been showed that Aß oligomers induce a decrease in dendritic spines, synaptic loss and plasticity alterations that underlie to the cognitive damage. However, the molecular mechanisms activated by Aß oligomers leading to synaptic dysfunction and loss, have not been completely explained. In our laboratory we described that Aß fibers induce c-Abl activation and regulates the neuronal death and cytoskeleton pathology. The c-Abl kinase is present in both pre and post synaptic structures and regulates the PSD-95 clustering. Besides has been described that the tyrosine kinase receptor ephrine A4 (EphA4) interacts with c-Abl and this interaction allows the reciprocal tyrosine phosphorylation. Methods: We treated wild type and EphA4-/hippocampal neurons (15 DIV) and synaptoneurosomes preparations with synthetic Aß42 oligomers and evaluated c-Abl and EphA4 signaling. Results: Here we show that c-Abl is involved in the synaptotoxicity induced by Aß oligomers. The Aß42 oligomers induce the c-Abl activation in dendritic spines of hippocampal neurons and in rat brain isolated synaptoneurosomes. Besides we observed that Aß42 oligomers increase the phospho tyrosine levels in EphA4 and the interaction between c-Abl and EphA4. Conclusions: Our data suggest Aß oligomers induce the c-Abl kinase activation through the EphA4 and this could be connected with synaptic demise.

N-Glycosylation profiling of recombinant mouse extracellular superoxide dismutase produced in Chinese hamster ovary cells

Abstract: Extracellular superoxide dismutase (EC-SOD), the major SOD isoenzyme in biological fluids, is known to be N-glycosylated and heterogeneous as was detected in most glycoproteins However, only one N-glycan structure has been reported in recombinant human EC-SOD produced in Chinese hamster ovary (CHO) cells Thus, a precise N-glycan profile of the recombinant EC-SOD is not available In this study, we report profiling of the N-glycan in the recombinant mouse EC-SOD produced in CHO cells using high-resolution techniques, including the liberation of N-glycans by treatment with PNGase F, fluorescence labeling by pyridylamination, characterization by anion-exchange, normal and reversed phase-HPLC separation, and mass spectrometry We succeeded in identifying 26 different types of N-glycans in the recombinant enzyme The EC-SOD N-glycans were basically core-fucosylated (983% of the total N-glycan content), and were high mannose sugar chain, and mono-, bi-, tri-, and tetra-antennary complex sugar chains exhibiting varying degrees of sialylation Four of the identified N-glycans were uniquely modified with a sulfate group, a Lewis(x) structure, or an α-Gal epitope The findings will shed new light on the structure-function relationships of EC-SOD N-glycans

Glycoproteins and Antibodies: Solution NMR Studies

Abstract: Carbohydrate chains covalently attached to proteins influence their physical and biological properties and mediate molecular recognition processes involved in a variety of biological events. Therefore, protein glycosylation has become a crucial factor in the design and development of biopharmaceuticals including antibody drugs. NMR spectroscopy offers valuable tools for studying conformations, dynamics, and interactions of the carbohydrate moieties of glycoproteins in solution at atomic resolution. Stable-isotope labeling of glycoproteins including their glycans is essential to reap the full benefit of NMR approaches. General strategies of stable-isotope labeling of glycoproteins have been developed using the Fc portion of immunoglobulin G (IgG) as a model system. The glycoprotein glycan can be metabolically isotope-labeled by employing mammalian or other eukaryotic cells as glycoprotein production vehicles, whereas in vitro enzymatic remodeling of the glycans enables selective isotope labeling at the nonreducing terminal sugar residues. These methods have provided useful spectroscopic probes for NMR characterization of the conformational dynamics of the sugar and polypeptide chains of IgG-Fc glycoproteins in pathological and immunological contexts. Stable-isotope-assisted NMR structural glycobiology will evolve further in conjunction with more sophisticated chemoenzymatic and molecular biology techniques along with theoretical approaches. Keywords: glycoprotein; antibody; oligosaccharide; NMR; stable-isotope labeling