The number of people with dementia is increasing rapidly due to the increase in the aging population. Alzheimer's disease (AD) is a type of neurodegenerative dementia caused by the accumulation of ...abnormal proteins. Genetic mutations, smoking, and several other factors have been reported as causes of AD, but alterations in glycans have recently been demonstrated to play a role in AD. Amyloid-β (Aβ), a cleaved fragment of APP, is the source of senile plaque, a pathological feature of AD. APP has been reported to undergo
- and
-glycosylation, and several Polypeptide
-acetylgalactosaminyltransferases (ppGalNAc-Ts) have been shown to have catalytic activity for the transfer of GalNAc to APP. Since
-glycosylation in the proximity of a cleavage site in many proteins has been reported to be involved in protein processing,
-glycans may affect the cleavage of APP during the Aβ production process. In this report, we describe new findings on the
-glycosylation of APP and Aβ production.
Alterations of the structure and/or amount of glycans present on proteins are associated with many diseases. We previously demonstrated that changes in N-glycans alter Aβ production. In the present ...study, we focused on the relationship between Alzheimer's disease (AD) and O-glycan, another type of glycan. The UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) family functions in the first step of mucin-type O-glycan synthesis. Analysis of the expression of GalNAc-Ts in the human brain using real-time PCR revealed that the expression of several GalNAc-Ts was altered with sporadic AD progression. Three of these GalNAc-Ts (GalNAc-T1, GalNAc-T4 and GalNAc-T6) were transfected into HEK293T cells to examine their impact on Aβ production. Transfection of GalNAc-T6 significantly reduced both Aβ1-40 and Aβ1-42 generation, but GalNAc-T1 and GalNAc-T4 only reduced Aβ1-40 generation. Although these three GalNAc-Ts exhibited enzymatic activities on soluble amyloid precursor protein (APP), the GalNAc transferase activity of GalNAc-T6 to APP was most prominent. The expression of α-secretase and β-secretase was slightly altered in the transfected cells, but the activities of α-secretase and β-secretase were not significantly altered. These data suggest that excess O-glycosylation on APP by GalNAc-T6 inhibits Aβ production.
Abstract
The deficiency of α-Klotho in mice causes phenotypes resembling human age-associated disorders at 3–4 weeks after birth and shows short lifespans of ∼2 months. One of the crucial symptoms is ...pulmonary emphysema, although α-Klotho is not expressed in the lungs. α-Klotho secreted from the kidneys is probably involved in the pathology of emphysema because kidney-specific knockout mice exhibit emphysematous structural changes. We examined whether any glycan changes in α-Klotho mouse lungs were observed, because α-Klotho is reported to have glycosidase activity. Here, we found the accumulation of heparan sulphate in the microsomal fraction of α-Klotho mouse lungs. Meanwhile, a disintegrin and metalloproteinase 17 (ADAM17) expression was decreased in α-Klotho mice. From these results, it is thought that the increase in heparan sulphate is due to insufficient cleavage of the core protein by ADAM17. Additionally, a reduction in α-Klotho and a decline of ADAM17 were also observed both in normal aged mice and in senescence marker protein-30 (SMP30) knockout mice, a mouse model of premature ageing. Thus, the decrease in ADAM17 is caused by the reduction in α-Klotho. These may be involved in the deterioration of lung function during ageing and may be associated with the pathology of pulmonary emphysema.
Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. α-dystroglycan (α-DG) is a receptor for matrix and synaptic proteins that causes ...muscular dystrophy and lissencephaly upon its abnormal glycosylation (α-dystroglycanopathies). Here we identify the glycan unit ribitol 5-phosphate (Rbo5P), a phosphoric ester of pentose alcohol, in α-DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand-binding moiety. We show that enzyme activities of three major α-dystroglycanopathy-causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain-containing (ISPD) is cytidine diphosphate ribitol (CDP-Rbo) synthase. Fukutin and fukutin-related protein are sequentially acting Rbo5P transferases that use CDP-Rbo. Consequently, Rbo5P glycosylation is defective in α-dystroglycanopathy models. Supplementation of CDP-Rbo to ISPD-deficient cells restored α-DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α-DG-associated diseases.
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•Ribitol 5-phosphate is a functional glycan unit in mammals•α-dystroglycan function requires tandem ribitol 5-phosphate structure•Muscular dystrophy proteins are involved in ribitol 5-phosphate glycosylation•Supplementation with ribitol 5-phosphate metabolites may be a therapeutic strategy
Kanagawa et al. show that ribitol 5-phophate is a functional glycan unit in mammals and that defects in its post-translational modification pathway are associated with muscular dystrophy.
Glycans are involved in many fundamental cellular processes such as growth, differentiation, and morphogenesis. However, their broad structural diversity makes analysis difficult. Glycomics via mass ...spectrometry has focused on the composition of glycans, but informatics analysis has not kept pace with the development of instrumentation and measurement techniques. We developed Toolbox Accelerating Glycomics (TAG), in which glycans can be added manually to the glycan list that can be freely designed with labels and sialic acid modifications, and fast processing is possible. In the present work, we improved TAG for large-scale analysis such as cohort analysis of serum samples. The sialic acid linkage-specific alkylamidation (SALSA) method converts differences in linkages such as α2,3- and α2,6-linkages of sialic acids into differences in mass. Glycans modified by SALSA and several structures discovered in recent years were added to the glycan list. A routine to generate calibration curves has been implemented to explore quantitation. These improvements are based on redefinitions of residues and glycans in the TAG List to incorporate information on glycans that could not be attributed because it was not assumed in the previous version of TAG. These functions were verified through analysis of purchased sera and 74 spectra with linearity at the level of R2 > 0.8 with 81 estimated glycan structures obtained including some candidate of rare glycans such as those with the N,N’-diacetyllactosediamine structure, suggesting they can be applied to large-scale analyses.
Klotho protein deficiency and aging Manya, Hiroshi; Akasaka-Manya, Keiko; Endo, Tamao
Geriatrics & gerontology international,
July 2010, Volume:
10, Issue:
s1
Journal Article
Peer reviewed
Open access
Aging is inevitable; however, the molecular mechanism of aging has not been fully elucidated. Investigations into aging are facing difficulties because aging is influenced by complex factors such as ...circumstances, living habits and genetic background. Recently, a variety of animals, such as Caenorhabditis elegans, Drosophila and mice, that have aberrations in their lifespan, have been investigated and a large number of genes related to aging have been found, one of which is α‐klotho. The α‐Klotho mouse (α‐kl−/− mouse), which has a defect of the α‐klotho gene expression, was established a decade ago. It is of great interest because the α‐kl−/− mouse shows various phenotypes resembling human aging. The relationship between aging and α‐klotho protein function is gradually becoming clear. This review covers the recent advance in α‐klotho protein research. Geriatr Gerontol Int 2010; 10 (Suppl. 1): S80–S87.
Protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is a Golgi glycosyltransferase that catalyzes the formation of the N-acetylglucosamine (GlcNAc) β1→2Man linkage of O-mannosyl ...glycan. POMGNT1 is not modified by N-glycans because there are no potential N-glycosylation sites; however, it is not clear whether POMGNT1 is modified by O-glycans. To determine whether POMGNT1 is O-glycosylated, we prepared recombinant human POMGNT1 from HEK293T cells. The recombinant POMGNT1 was recognized by Sambucus sieboldiana lectin (SSA), and sialidase digestion of POMGNT1 decreased SSA reactivity and enhanced the reactivity of Arachis hypogaea lectin (PNA). These results suggest that POMGNT1 is modified by a sialylated core-1 O-glycan. Next, we analyzed the structures of the O-glycans on POMGNT1 by β-elimination and pyrazolone-labeling methods in combination with mass spectrometry. We identified several mucin-type O-glycans containing (NeuAc)1(Hex)1(HexNAc)1, (NeuAc)2(Hex)1(HexNAc)1, and (NeuAc)2(Hex)2(HexNAc)2. To examine whether the O-glycans affect the functions and properties of POMGNT1, we compared glycosylated and non-glycosylated forms of recombinant sPOMGNT1 for their activity and surface hydrophobicity using the hydrophobic probe 1-anilino-8-naphthalene sulfonate (ANS). POMGNT1 activity and surface hydrophobicity were not affected by the presence or absence of O-glycans.
The α-Klotho mouse is an animal model that prematurely exhibits phenotypes resembling human aging owing to mutation of the α-Klotho gene. Although α-Klotho mice appear normal at birth, they begin ...showing multiple age-associated disorders after 3-4 weeks. Meanwhile, overexpression of α-Klotho extends lifespan. Therefore, α-Klotho may be involved in the aging process. The α-Klotho protein has homology to β-glucosidase and is proposed to have glycosidase activity. However, it is unclear whether glycan alterations are present in α-Klotho mice. Here we found increased levels of the non-sulfated HNK-1 glyco-epitope in the kidneys of α-Klotho mice. This phenomenon was also observed in normal aged mice. Immunohistochemical analysis demonstrated that increased non-sulfated HNK-1 glyco-epitope appeared predominantly in the outer half of the renal cortex, where α-Klotho protein is highly expressed. To clarify the cause, the expression of glucuronyltransferase S (GlcAT-S) and the activity of β-glucuronidase were also examined. The expressions of GlcAT-S were comparable in α-Klotho mice and wild-type mice, but β-glucuronidase activity was lower in α-Klotho mice than in wild-type. These results suggest that increased non-sulfated HNK-1 epitope levels in α-Klotho mice may be due to decreased β-glucuronidase activity. Taken together, α-Klotho expression was associated with expression of the non-sulfated HNK-1 epitope.
The complex of protein O-mannosyltransferase 1 (POMT1) and POMT2 catalyzes the initial step of O-mannosyl glycan biosynthesis. The mutations in either POMT1 or POMT2 can lead to Walker-Warburg ...syndrome, a congenital muscular dystrophy with abnormal neuronal migration. Here, we used three algorithms for predicting transmembrane helices to construct the secondary structural models of human POMT1 and POMT2. In these models, POMT1 and POMT2 have seven- and nine-transmembrane helices and contain four and five potential N-glycosylation sites, respectively. To determine whether these sites are actually glycosylated, we prepared mutant proteins that were defective in each site by site-directed mutagenesis. Three of the POMT1 sites and all of the POMT2 sites were found to be N-glycosylated, suggesting that these sites face the luminal side of the endoplasmic reticulum. Mutation of any single site did not significantly affect POMT activity, but mutations of all N-glycosylation sites of either POMT1 or POMT2 caused a loss of POMT activity. The loss of activity appeared to be due to the decreased hydrophilicity. These results suggest that the N-glycosylation of POMT1 and POMT2 is required for maintaining the conformation as well as the activity of the POMT1-POMT2 complex.
A defect of protein O-mannosylation causes congenital muscular dystrophy with brain malformation and structural eye abnormalities, so-called Walker-Warburg syndrome. Protein O-mannosylation is ...catalyzed by protein O-mannosyltransferase 1 (POMT1) and its homologue, POMT2. Coexpression of POMT1 and POMT2 is required to show O-mannosylation activity. Here we have shown that POMT1 forms a complex with POMT2 and the complex possesses protein O-mannosyltransferase activity. Results indicate that POMT1 and POMT2 associate physically and functionally in vivo. Recently, three mutations were reported in the POMT1 gene of patients who showed milder phenotypes than typical Walker-Warburg syndrome. We coexpressed these mutant POMT1s with POMT2 and found that none of them had any activity. However, all POMT1 mutants, including previously identified POMT1 mutants, coprecipitated with POMT2. These results indicate that the mutant POMT1s could form heterocomplexes with POMT2 but that such complexes are insufficient for enzymatic activity.
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