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Background Alzheimer's disease (AD) is a major form of dementia. Many evidence-based clinical trials have been performed, but no effective treatment has yet been developed. This suggests that our understanding of AD patho-mechanisms is still insufficient. In particular, the pathological roles of posttranslational modifications including glycosylation have remained poorly understood, but recent advances in glycobiology technology have gradually revealed that sugar modifications of AD-related molecules are profoundly involved in the onset and progression of this disease. Scope of review We summarize the roles of N -glycans in AD pathogenesis and progression, particularly focusing on key AD-related molecules, including amyloid precursor protein (APP), α-, β-, and γ-secretases, and tau. Major conclusions Biochemical, genetic and pharmacological studies have gradually revealed how N -glycans regulate AD development and progression through functional modulation of the key glycoproteins. These findings suggest that further glycobiology approaches in AD research will reveal novel glycan-based drug targets and early biomarkers of AD. However, N -glycan structures of these molecules in physiological and disease conditions and their precise functions are still largely unclear. Deeper glycobiology studies will be needed to reveal how AD pathology is regulated by glycosylation. General significance It is now known that N -glycans play significant roles in AD development. However, specific pathological functions of particular glycan epitopes on each AD-related glycoprotein are still poorly understood. Future glycobiology studies with more sensitive glycoproteomic techniques and a wider variety of chemical glycosylation inhibitors could contribute to the development of novel glycan-based AD therapeutics. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa. [ABSTRACT FROM AUTHOR]

It is well known that biosynthesis of glycans takes place in organ- and tissue-specific manners and glycan expression is controlled by various factors including glycosyltransferases. The expression mechanism of glycosyltransferases, however, is poorly understood. Here we investigated the expression mechanism of a brain-specific glycosyltransferase, GnT-IX (N-acetylglucosaminyltransferase IX, also designated as GnT-Vb), which synthesizes branched O-mannose glycan. Using an epigenetic approach, we revealed that the genomic region around the transcriptional start site of the GnT-IX gene was highly associated with active chromatin histone marks in a neural cell-specific manner, indicating that brain-specific GnT-IX expression is under control of an epigenetic "histone code." By EMSA and ChIP analyses we identified two regulatory proteins, NeuroD1 and CTCF that bind to and activate the GnT-IX promoter. We also revealed that GnT-IX expression was suppressed in CTCF- and NeuroD1-depleted cells, indicating that a NeuroD1- and CTCF-dependent epigenetic mechanism governs brain-specific GnT-IX expression. Several other neural glycosyltransferase genes are also found to be regulated by epigenetic histone modifications. This is the first report demonstrating a molecular mechanism at the chromatin level underlying tissue-specific glycan expression. [ABSTRACT FROM AUTHOR]

Members of the P4 family of P-type ATPases (P4-ATPases) are believed to function as phospholipid flippases in complex with CDC50 proteins. Mutations in the human class 1 P4-ATPase gene ATP8B1 cause a severe syndrome characterized by impaired bile flow (intrahepatic cholestasis), often leading to end-stage liver failure in childhood. In this study, we determined the specificity of human class 1 P4-ATPase interactions with CDC50 proteins and the functional consequences of these interactions on protein abundance and localization of both protein classes. ATP8B1 and ATP8B2 co-immunoprecipitated with CDC50A and CDC50B, whereas ATP8B4, ATP8A1, and ATP8A2 associated only with CDC50A. ATP8B1 shifted from the endoplasmic reticulum (ER) to the plasma membrane upon coexpression of CDC50A or CDC50B. ATP8A1 and ATP8A2 translocated from the ER to the Golgi complex and plasma membrane upon coexpression of CDC50A, but not CDC50B. ATP8B2 and ATP8B4 already displayed partial plasma membrane localization in the absence of CDC50 coexpression but displayed a large increase in plasma membrane abundance upon coexpression of CDC50A. ATP8B3 did not bind CDC50A and CDC50B and was invariably present in the ER. Our data show that interactions between CDC50 proteins and class 1 P4-ATPases are essential for ER exit and stability of both subunits. Furthermore, the subcellular localization of the complex is determined by the P4-ATPase, not the CDC50 protein. The interactions of CDC50A and CDC50B with multiple members of the human P4-ATPase family suggest that these proteins perform broader functions in human physiology than thus far assumed. [ABSTRACT FROM AUTHOR]

Antiangiogenesis therapies are now part of the standard repertoire of cancer therapies, but the mechanisms for the proliferation and survival of endothelial cells are not fully understood. Although endothelial cells are covered with a glycocalyx, little is known about how endothelial glycosylation regulates endothehal functions. Here, we show that α2,6-sialic acid is necessary for the cell-surface residency of platelet endothehial cell adhe- sion molecule (PECAM), a member of the immunoglobulin superfamily that plays multiple roles in cell adhesion, mechani- cal stress sensing, antiapoptosis, and angiogenesis. As a possible underlying mechanism, we found that the homophilic interactions of PECAM in endothehial cells were dependent on α2,6-sialic acid. We also found that the absence of α2,6-sialic acid down regulated the tyrosine phosphoryIation of PECAM and recruitment of Src homology 2 domain-containing protein-tyrosine phosphatase 2 and rendered the cells more prone to mitochondrion-dependent apoptosis, as evaluated using PECAM-deficient endothehial cells. The present findings open up a new possibility that modulation of glycosylation could be one of the promising strategies for regulating angiogenesis. [ABSTRACT FROM AUTHOR]

β-Site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, Aβ, and is implicated in triggering the pathogenesis of Alzheimer disease. We previously re- ported that BACE1 cleaved rat β-galactoside α2,6-sialyltransferase (ST6Gal I) that was overexpressed in COS cells and that the NH2 terminus of ST6Gal I secreted from the cells (E41 form) was Glu41. Here we report that BACE1 gene knock-out mice have one third as much plasma ST6Gal I as control mice, indicating that BACE1 is a major protease which is responsible for cleaving ST6Gal I in vivo. We also found that BACEl-transgenic mice have increased level of ST6Gal I in plasma. Secretion of ST6Gal I from the liver into the plasma is known to be up-regulated during the acute-phase response. To investigate the role of BACE1 in ST6Gal I secretion in vivo, we analyzed the levels of BACE1 mRNA in the liver, as well as the plasma levels of ST6Gal I, in a hepatopathological model, i.e. Long-Evans Cinnamon (LEC) rats. This rat is a mutant that spontaneously accumulates copper in the liver and incurs hepatic damage. LEC rats exhibited simultaneous increases in BACE1 mRNA in the liver and in the E41 form of the ST6Gal I protein, the BACE1 product, in plasma as early as 6 weeks of age, again suggesting that BACE1 cleaves ST6Gal I in vivo and controls the secretion of the E41 form. [ABSTRACT FROM AUTHOR]

Reports on the cleavage of the sialyltransferase (A-ST6Gal I) fusion protein by the protease BACE1-Fc chimera protein. Suggestion of an initial cleavage product of three amino acids by BACE1; Revelation of the truncated nature of the amino acids by exopeptidase activity; Likelihood of an initial cleave of ST6Gal I between leu[sup37] and Gln[sup38] by BACE1; Removal of the three amino acid sequence by exopeptidase.

The enzymes involved in glycosaminoglycan chain biosynthesis are mostly Golgi resident proteins, but some are secreted extracellularly. For example, the activities of heparan sulfate 6-O-sulfotransferase (HS6ST) and heparan sulfate 3-O-sulfotransferase are detected in the serum as well in the medium of cell lines. However, the biological significance of this is largely unknown. Here we have investigated by means of monitoring green fluorescent protein (GFP) fluorescence how C-terminally GFP-tagged HS6STs that are stably expressed in CHO-K1 cell lines are secreted/shed. Brefeldin A and monensin treatments revealed that the N-terminal hydrophobic domain of HS6ST3 is processed in the endoplasmic reticulum or cis/medial Golgi. Treatment of HS6ST3-GFP-expressing cells with various protease inhibitors revealed that the cell-permeable β-secretase inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal (Z-VLL-CHO) specifically inhibits HS6ST secretion, although this effect was specific for HS6ST3 but not for HS6ST1 and HS6ST2. However, Z-VLL-CHO treatment did not increase the molecular size of the HS6ST3-GFP that accumulated in the cell. Z-VLL-CHO treatment also induced the intracellular accumulation of SP-HS6ST3(-TMD)-GFP, a modified secretory form of HS6ST3 that has the preprotrypsin leader sequence as its N-terminal hydrophobic domain. Diminishment of β-secretase activity by coexpressing the amyloid precursor protein of a Swedish mutant, a potent β-secretase substrate, also induced intracellular HS6ST3-GFP accumulation. Moreover, Z-VLL-CHO treatment increased the 6-O-sulfate (6S) levels of HS, especially in the disaccharide unit of hexuronic acid-GIcNS(6S). Thus, the HS6ST3 enzyme in the Golgi apparatus and therefore the 6-O sulfation of heparan sulfates in the cell are at least partly regulated by β-secretase via an indirect mechanism. [ABSTRACT FROM AUTHOR]

Background Langerin, a C-type lectin receptor (CLR) expressed in a subset of dendritic cells (DCs), binds to glycan ligands for pathogen capture and clearance. Previous studies revealed that langerin has an unusual binding affinity toward 6-sulfated galactose (Gal), a structure primarily found in keratan sulfate (KS). However, details and biological outcomes of this interaction have not been characterized. Based on a recent discovery that the disaccharide L4, a KS component that contains 6-sulfo-Gal, exhibits anti-inflammatory activity in mouse lung, we hypothesized that L4-related compounds are useful tools for characterizing the langerin-ligand interactions and their therapeutic application. Methods We performed binding analysis between purified long and short forms of langerin and a series of KS disaccharide components. We also chemically synthesized oligomeric derivatives of L4 to develop a new high-affinity ligand of langerin. Results We show that the binding critically requires the 6-sulfation of Gal and that the long form of langerin displays higher affinity than the short form. The synthesized trimeric (also designated as triangle or Tri) and polymeric (pendant) L4 derivatives displayed over 1000-fold higher affinity toward langerin than monomeric L4. The pendant L4, but not the L4 monomer, was found to effectively transduce langerin signaling in a model cell system. Conclusions L4 is a specific ligand for langerin. Oligomerization of L4 unit increased the affinity toward langerin. General significance These results suggest that oligomeric L4 derivatives will be useful for clarifying the langerin functions and for the development of new glycan-based anti-inflammatory drugs. [ABSTRACT FROM AUTHOR]

Abstract: Idiopathic normal pressure hydrocephalus (iNPH) is an elderly dementia caused by abnormal metabolism in the cerebrospinal fluid (CSF). The tap test is the current basis for confirming iNPH, but it shows very low sensitivity, indicating that many patients who might be cured by a shunt operation will be missed. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we found two transferrin isoforms: one had a unique N-glycan (Tf-1) whereas the other had N-glycan similar to that of serum transferrin (Tf-2). Glycan analyses revealed that Tf-1 had branching (biantennary) asialo- and agalacto-complex type N-glycans (N-acetylglucosamine [GlcNAc]-terminated glycans), which carried bisecting β1,4-N-acetylglucosamine and core α1,6-fucose. To examine glycoform whether changes in iNPH, we introduced the Tf-2/Tf-1 ratio as a diagnostic index, which minimized blot-to-blot variations in measurement. The Tf-2/Tf-1 ratios of iNPH patients are significantly higher than those of controls (p = 0.0019) and Alzheimer''s patients (p = 0.0010). This suggests that the Tf-2/Tf-1 ratio could distinguish iNPH from Alzheimer''s disease, and possibly other dementias. In conclusion, glycoform analysis has diagnostic potential in neurological diseases. [Copyright &y& Elsevier]

We previously demonstrated that a deficiency in core fucosylation caused by the genetic disruption of α1,6-fucosyltransferase (Fut8) leads to lethal abnormalities and the development of emphysematous lesions in the lung by attenuation of TGF-β1 receptor signaling. Herein, we investigated the physiological relevance of core fucosylation in the pathogenesis of emphysema using viable heterozygous knock-out mice (Fut8+/-) that were exposed to cigarette smoke (CS). The Fut8+/- mice exhibited a marked decrease in FUT8 activity, and matrix metalloproteinase (MMP)-9 activities were elevated in the lung at an early stage of exposure. Emphysema developed after a 3-month CS exposure, accompanied by the recruitment of large numbers of macrophages to the lung. CS exposure substantially and persistently elevated the expression level of Smad7, resulting in a significant reduction of Smad2 phosphorylation (which controls MMP-9 expression) in Fut8+/- mice and Fut8-deficient embryonic fibroblast cells. These in vivo and in vitro studies show that impaired core fucosylation enhances the susceptibility to CS and constitutes at least part of the disease process of emphysema, in which TGF-β-Smad signaling is impaired and the MMP-mediated destruction of lung parenchyma is up-regulated. [ABSTRACT FROM AUTHOR]

BACE1 (β-site amyloid precursor protein-cleaving enzyme-1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, amyloid β-peptide, and has been implicated in triggering the pathogenesis of Alzheimer disease. We showed previously that BACE1 cleaves β-galactoside α2,6-sialyltransferase I (ST6Gal I) to initiate its secretion, but it remained unclear how BACE1 affects the cellular level of α2,6-sialylation. Here, we found that BACE1 overexpression in Hep3B cells increased the sialylation of soluble secreted glycoproteins, but did not affect cell-surface sialylation. The sialylation of soluble glycoproteins was not increased by ST6Gal I overexpression alone, but was increased by co-overexpression of ST6Gal I and BACE1 or by expression of the soluble form of ST6Gal I, suggesting that soluble ST6Gal I produced by BACE1 plays, at least in part, a role in the sialylation of soluble glycoproteins. We also found that plasma glycoproteins from BACE1-deficient mice exhibited reduced levels of α2,6-sialylation compared with those from wild-type mice. We propose a novel regulatory mechanism in which cleavage and secretion of ST6Gal I enhance the sialylation of soluble glycoprotein substrates. [ABSTRACT FROM AUTHOR]

Recently, we reported a novel substrate-based octapeptide BACE1 inhibitor KMI-008 containing hydroxymethylcarbonyl (HMC) isostere as a transition-state mimic. Using KMI-008 as a lead compound, a small-sized and highly potent BACE1 inhibitor KMI-370 (IC50=3.4 nM) was designed and synthesized. [Copyright &y& Elsevier]