Changes in the amount of nucleotide sugars in aged mouse tissues.

Aging affects tissue glycan profiles, which may alter cellular functions and increase the risk of age-related diseases. Glycans are biosynthesized by glycosyltransferases using the corresponding nucleotide sugar, and the availability of nucleotide sugars affects glycosylation efficiency. However, the effects of aging on nucleotide sugar profiles and contents are yet to be elucidated. Therefore, this study aimed to investigate the effects of aging on nucleotide sugars using a new LC-MS/MS method. Specifically, the new method was used to determine the nucleotide sugar contents of various tissues (brain, liver, heart, skeletal muscle, kidney, lung, and colon) of male C57BL/6NCr mice (7- or 26-month-old). Characteristic age-associated nucleotide sugar changes were observed in each tissue sample. Particularly, there was a significant decrease in UDP-glucuronic acid content in the kidney of aged mice and a decrease in the contents of several nucleotide sugars, including UDP-N-acetylgalactosamine, in the brain of aged mice. Additionally, there were variations in nucleotide sugar profiles among the tissues examined regardless of the age. The kidneys had the highest concentration of UDP-glucuronic acid among the seven tissues. In contrast, the skeletal muscle had the lowest concentration of total nucleotide sugars among the tissues; however, CMP-N-acetylneuraminic acid and CDP-ribitol were relatively enriched. Conclusively, these findings may contribute to the understanding of the roles of glycans in tissue aging.

Tocotrienols Prevent the Decline of Learning Ability in High-Fat, High-Sucrose Diet-Fed C57BL/6 Mice.

Obesity has been increasing worldwide and is well-known as a risk factor for cognitive decline. It has been reported that oxidative stress in the brain is deeply involved in cognitive dysfunction in rodent models. While there are many studies on oxidation in the liver and adipose tissue of obese mice, the relationship between obesity-induced cognitive dysfunction and brain oxidation has not been elucidated. Here, we show that obesity induced by a high-fat, high-sucrose diet (HFSD) alters cognitive function in C57BL/6 male mice, and it may involve the acceleration of brain oxidation. Tocotrienols (T3s), which are members of the vitamin E family, can prevent HFSD-induced cognitive changes. To elucidate these mechanisms, respiratory metabolism, locomotor activity, temperature around brown adipose tissue, and protein profiles in the cerebrum cortex were measured. Contrary to our expectation, respiratory metabolism was decreased, and temperature around brown adipose tissue was increased in the feeding of HFSD. The proteins that regulate redox balance did not significantly change, but 12 proteins, which were changed by HFSD feeding and not changed by T3s-treated HFSD compared to control mice, were identified. Our results indicated that HFSD-induced obesity decreases mouse learning ability and that T3s prevent its change. Additionally, feeding of HFSD significantly increased brain oxidation. However, further study is needed to elucidate the mechanisms of change in oxidative stress in the brain by obesity.

A characteristic N-glycopeptide signature associated with diabetic cognitive impairment identified in a longitudinal cohort study.

BACKGROUND: Identifying a biomarker for the decline in cognitive function in patients with diabetes is important. Therefore, we aimed to identify the N-glycopeptides on plasma proteins associated with diabetic cognitive impairment in participants in a longitudinal study using N-glycoproteomics. METHODS: We used samples from the 3-year SONIC (Septuagenarians, Octogenarians, Nonagenarians Investigation with Centenarians) longitudinal cohort study of older Japanese people in the general population. First, we placed the participants with diabetes into two groups: those that did or did not have cognitive decline over a 6-year period. Next, their plasma protein profiles were compared between baseline and the 6-year time point using two-dimensional fluorescence difference gel electrophoresis. Finally, an N-glycoproteomic study of the focused proteins was performed using an enrichment technique and liquid chromatography-tandem mass spectrometry. RESULTS: Approximately 500 N-glycopeptides, derived from 18 proteins, were identified in each sample, from among which we identified the N-glycopeptides that were associated with diabetic cognitive impairment using multivariate analysis. We found that N-glycopeptides with sialylated tri- or tetra-antennary glycans on alpha-2-macroglobulin, clusterin, serum paraoxonase/arylesterase 1, and haptoglobin were less abundant, whereas 3-sialylated tri-antennary N-glycopeptides on serotransferrin were more abundant. CONCLUSION: N-glycopeptides with sialylated multi-antennary glycans comprise a characteristic signature associated with diabetic cognitive impairment. GENERAL SIGNIFICANCE: The characterized N-glycopeptides represent potential biomarker candidates for diabetic cognitive impairment.

Proteolytic ectodomain shedding of muscle-specific tyrosine kinase in myasthenia gravis.

Autoantibodies to muscle-specific tyrosine kinase (MuSK) proteins at the neuromuscular junction (NMJ) cause refractory generalized myasthenia gravis (MG) with dyspnea more frequently than other MG subtypes. However, the mechanisms via which MuSK, a membrane protein locally expressed on the NMJ of skeletal muscle, is supplied to the immune system as an autoantigen remains unknown. Here, we identified MuSK in both mouse and human serum, with the amount of MuSK dramatically increasing in mice with motor nerve denervation and in MG model mice. Peptide analysis by liquid chromatography-tandem-mass spectrometry (LC-MS/MS) confirmed the presence of MuSK in both human and mouse serum. Furthermore, some patients with MG have significantly higher amounts of MuSK in serum than healthy controls. Our results indicated that the secretion of MuSK proteins from muscles into the bloodstream was induced by ectodomain shedding triggered by neuromuscular junction failure. The results may explain why MuSK-MG is refractory to treatments and causes rapid muscle atrophy in some patients due to the denervation associated with Ab-induced disruption of neuromuscular transmission at the NMJ. Such discoveries pave the way for new MG treatments, and MuSK may be used as a biomarker for other neuromuscular diseases in preclinical studies, clinical diagnostics, therapeutics, and drug discovery.

A chemical probe for proteomic analysis and visualization of intracellular localization of lysine-succinylated proteins.

Protein acylation is a vital post-translational modification that regulates various protein functions. In particular, protein succinylation has attracted significant attention because of its potential relationship with various biological events and diseases. In this report, we establish a new method for the comprehensive detection and analysis of potentially succinylated proteins using a chemical tagging technology. The newly synthesized alkyne-containing succinyl substrate successfully labeled lysine residues of proteins through intracellular metabolic labeling independent of other acylation pathways such as protein malonylation. Furthermore, reporter molecules such as biotin moieties and fluorescent dyes were conjugated to alkyne-tagged succinylated proteins via Click reactions, permitting enrichment for proteomic analysis and fluorescence imaging of the labeled proteins. We successfully analyzed and identified numerous potential succinylated proteins associated with various biological processes using gel electrophoresis, proteomic and bioinformatic analyses, and their visualization in cells.

Diagnostic potential of serum extracellular vesicles expressing prostate-specific membrane antigen in urologic malignancies.

We aimed to develop a sandwich ELISA to detect prostate-specific membrane antigen (PSMA) on small extracellular vesicles (EVs) using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) as a capture molecule for EVs and to evaluate its diagnostic potential in urologic malignancies. First, we optimized the conditions for sandwich ELISA measuring the PSMA level on EVs captured from serum by Tim4 and found that the use of highly-purified EVs released from Tim4 that had captured EVs in serum reduced the background. Second, we confirmed its validity by studying mouse xenograft model for prostate cancer (PC). Lastly, we measured PSMA-EVs in serum of patients with urologic malignancies. The PSMA-EV levels were significantly higher in metastatic PC and castration-resistant PC (CRPC) patients than in therapy-naïve PC patients. In renal cell carcinoma (RCC) patients, PSMA-EVs were elevated in those with metastasis compared with those without metastasis, which may reflect the development of the neovasculature positive for PSMA in tumors. In conclusion, we developed a sandwich ELISA for detection of PSMA-EVs using highly-purified EVs isolated from serum by Tim4. Our results suggest that PSMA-EVs may be useful to diagnose and monitor not only PC but also RCC and possibly other hypervascular solid tumors.

PCYT2 synthesizes CDP-glycerol in mammals and reduced PCYT2 enhances the expression of functionally glycosylated α-dystroglycan.

α-Dystroglycan (α-DG) is a highly glycosylated cell-surface protein. Defective O-mannosyl glycan on α-DG is associated with muscular dystrophies and cancer. In the biosynthetic pathway of the O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP) transfer ribitol phosphate (RboP). Previously, we reported that FKTN and FKRP can also transfer glycerol phosphate (GroP) from CDP-glycerol (CDP-Gro) and showed the inhibitory effects of CDP-Gro on functional glycan synthesis by preventing glycan elongation in vitro. However, whether mammalian cells have CDP-Gro or associated synthetic machinery has not been elucidated. Therefore, the function of CDP-Gro in mammals is largely unknown. Here, we reveal that cultured human cells and mouse tissues contain CDP-Gro using liquid chromatography tandem-mass spectrometry (LC-MS/MS). By performing the enzyme activity assay of candidate recombinant proteins, we found that ethanolamine-phosphate cytidylyltransferase (PCYT2), the key enzyme in de novo phosphatidylethanolamine biosynthesis, has CDP-Gro synthetic activity from glycerol-3-phosphate (Gro3P) and CTP. In addition, knockdown of PCYT2 dramatically reduced cellular CDP-Gro. These results indicate that PCYT2 is a CDP-Gro synthase in mammals. Furthermore, we found that the expression of functionally glycosylated α-DG is increased by reducing PCYT2 expression. Our results suggest an important role for CDP-Gro in the regulation of α-DG function in mammals.

Proteome analysis demonstrates involvement of endoplasmic reticulum stress response in human myocardium with subclinical left ventricular diastolic dysfunction.

AIM: Heart failure is increasing in Japan, in particular that with preserved ejection fraction (HFpEF) prevalent in older-aged patients. The purpose of this study was to investigate the pathophysiology during the early stage of left ventricular (LV) diastolic dysfunction by the quantitative proteome analysis of human myocardium. METHODS: Among 331 post-mortem autopsy patients, we selected 23 patients (aged 79 ± 9.6 years) with echocardiographic data and without major comorbidities, except hypertension. Cryopreserved autopsy tissue of the LV myocardium was subjected to proteome analysis. LV diastolic function was evaluated by echocardiographic data. Thirteen patients were classified into the impaired diastolic function (IDF) group, and 10 the normal cardiac function group. We performed comparative proteome analysis between the IDF and normal groups by isobaric tags for relative and absolute quantitation (iTRAQ) using nano-liquid chromatography-tandem mass spectrometry. RESULTS: The iTRAQ-based proteome analysis revealed 57 differentially expressed proteins in the IDF group. Molecular network analysis of differentially expressed proteins indicated that endoplasmic reticulum (ER) stress was a potentially important event. Furthermore, the expressions of proteins associated with the ER stress response, such as glucose-regulated protein 78 kDa, inositol-requiring kinase 1α and spliced X-box binding protein 1, were significantly decreased in the IDF group. CONCLUSIONS: This study suggested that reduced ER stress responses were involved during the early stage of LV diastolic dysfunction. Geriatr Gerontol Int ••; ••: ••-•• Geriatr Gerontol Int 2021; 21: 577-583.

DJ-1 is indispensable for the S-nitrosylation of Parkin, which maintains function of mitochondria.

The DJ-1 gene, a causative gene for familial Parkinson's disease (PD), has been reported to have various functions, including transcriptional regulation, antioxidant response, and chaperone and protease functions; however, the molecular mechanism associated with the pathogenesis of PD remains elusive. To further explore the molecular function of DJ-1 in the pathogenesis of PD, we compared protein expression profiles in brain tissues from wild-type and DJ-1-deficient mice. Two-dimensional difference gel electrophoresis analysis and subsequent analysis using data mining methods revealed alterations in the expression of molecules associated with energy production. We demonstrated that DJ-1 deletion inhibited S-nitrosylation of endogenous Parkin as well as overexpressed Parkin in neuroblastoma cells and mouse brain tissues. Thus, we used genome editing to generate neuroblastoma cells with DJ-1 deletion or S-nitrosylated cysteine mutation in Parkin and demonstrated that these cells exhibited similar phenotypes characterized by enhancement of cell death under mitochondrial depolarization and dysfunction of mitochondria. Our data indicate that DJ-1 is required for the S-nitrosylation of Parkin, which positively affects mitochondrial function, and suggest that the denitrosylation of Parkin via DJ-1 inactivation might contribute to PD pathogenesis and act as a therapeutic target.

Crystal structures of fukutin-related protein (FKRP), a ribitol-phosphate transferase related to muscular dystrophy.

α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.

Fumarate accumulation involved in renal diabetic fibrosis in Goto-Kakizaki rats.

The Goto-Kakizaki (GK) rat is a spontaneous animal model of type 2 diabetes and early stage of diabetic nephropathy. However, the pathophysiological mechanisms contributing to the progression of diabetic nephropathy in GK rats remain unclear. Kidneys from 15-week old male diabetic GK/Jcl rats and age-matched Wistar rats, which have the same genetic background as GK rats, were used. Proteomic analyses of GK and Wistar kidneys were performed using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Differentially expressed proteins in GK rats were subjected to pathway analysis, and expression levels of hypoxia inducible factor 1α (HIF-1α) and transforming growth factor-ß1 (TGF-ß1), and fumarate accumulation in GK kidneys were examined. Azan staining and immunohistochemical staining of α-smooth muscle actin were performed in relation to fibrosis in GK kidneys. Proteomic analysis using 2D-DIGE, analysis of fumarate content, and expression analysis of HIF-1α, TGF-ß1, and α-smooth muscle actin of GK rat's kidney, suggested the mechanism of fibrosis characterized as two stages in diabetic nephropathy of GK rats. Abnormalities of glucose metabolism such as elevated levels of 2-oxoglutarate dehydrogenase and reduction of fumarate hydratase caused the accumulation of fumarate followed by the upregulation of HIF-1α and TGF-ß1 leading to fibrosis in diabetic nephropathy. Alterations in proteins involved in the tricarboxylic acid cycle are associated with fibrosis through fumarate accumulation in diabetic nephropathy of GK rats.

Prevention of calpain-dependent degradation of STK38 by MEKK2-mediated phosphorylation.

Serine-threonine kinase 38 (STK38) is a member of the protein kinase A (PKA)/PKG/PKC-family implicated in the regulation of cell division and morphogenesis. However, the molecular mechanisms underlying STK38 stability remain largely unknown. Here, we show that treatment of cells with either heat or the calcium ionophore A23187 induced STK38 degradation. The calpain inhibitor calpeptin suppressed hyperthermia-induced degradation or the appearance of A23187-induced cleaved form of STK38. An in vitro cleavage assay was then used to demonstrate that calpain I directly cleaves STK38 at the proximal N-terminal region. Deletion of the N-terminal region of STK38 increased its stability against hyperthermia. We further demonstrated that the MAPKK kinase (MAP3K) MEKK2 prevented both heat- and calpain-induced cleavage of STK38. MEKK2 knockdown enhanced hyperthermia-induced degradation of STK38. We performed an in vitro MEKK2 assay and identified the key regulatory site in STK38 phosphorylated by MEKK2. Experiments with a phosphorylation-defective mutant demonstrated that phosphorylation of Ser 91 is important for STK38 stability, as the enzyme is susceptible to degradation by the calpain pathway unless this residue is phosphorylated. In summary, we demonstrated that STK38 is a calpain substrate and revealed a novel role of MEKK2 in the process of STK38 degradation by calpain.

Comparative Glycomic Analysis of Sialyl Linkage Isomers by Sialic Acid Linkage-Specific Alkylamidation in Combination with Stable Isotope Labeling of α2,3-Linked Sialic Acid Residues.

Sialic acids form the terminal sugars in glycan chains on glycoproteins via α2,3, α2,6, or α2,8 linkages, and structural isomers of sialyl linkages play various functional roles in cell recognition and other physiological processes. We recently developed a novel procedure based on sialic acid linkage-specific alkylamidation via lactone ring opening (aminolysis-SALSA). Herein, we have investigated an isotope labeling of α2,3-linked sialic acid residues (iSALSA) using amine hydrochloride salts. One limitation of SALSA using amine hydrochloride salts may be solved by adding only tert-butylamine (t-BA) as an acid scavenger, and comparative and quantitative glycomic analyses can be performed using iSALSA. We also developed quantitative glycomic analysis using dual isotope-labeled glycans by derivatizing with aminooxy-functionalized tryptophanylarginine methyl ester (aoWR) and iSALSA at the reducing and nonreducing end, respectively. Furthermore, we demonstrate that the amount of α2,3-linked sialoglycans in serum are altered during liver fibrosis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography MS (LC/MS) analyses. We revealed that the ratio of A33,6,6 to A3F3,6,6 was gradually decreased along with liver fibrosis progression. Therefore, these glycan alterations are potential diagnostic markers of nonalcoholic steatohepatitis (NASH) fibrosis progression.

O-GlcNAcylation and phosphorylation of ß-actin Ser199 in diabetic nephropathy.

The function of actin is regulated by various posttranslational modifications. We have previously shown that in the kidneys of nonobese type 2 diabetes model Goto-Kakizaki rats, increased O-GlcNAcylation of ß-actin protein is observed. It has also been reported that both O-GlcNAcylation and phosphorylation occur on Ser199 of ß-actin. However, their roles are not known. To elucidate their roles in diabetic nephropathy, we examined the rat kidney for changes in O-GlcNAcylation of Ser199 (gS199)-actin and in the phosphorylation of Ser199 (pS199)-actin. Both gS199- and pS199-actin molecules had an apparent molecular weight of 40 kDa and were localized as nonfilamentous actin in both the cytoplasm and nucleus. Compared with the normal kidney, the immunostaining intensity of gS199-actin increased in podocytes of the glomeruli and in proximal tubules of the diabetic kidney, whereas that of pS199-actin did not change in podocytes but decreased in proximal tubules. We confirmed that the same results could be observed in the glomeruli of the human diabetic kidney. In podocytes of glomeruli cultured in the presence of the O-GlcNAcase inhibitor Thiamet G, increased O-GlcNAcylation was accompanied by a concomitant decrease in the amount of filamentous actin and in morphological changes. Our present results demonstrate that dysregulation of O-GlcNAcylation and phosphorylation of Ser199 occurred in diabetes, which may contribute partially to the causes of the morphological changes in the glomeruli and tubules. gS199- and pS199-actin will thus be useful for the pathological evaluation of diabetic nephropathy.

Age-associated proteomic alterations in human aortic media.

AIM: Human vascular senescence, which mainly occurs in media, is not completely understood. Here, we used proteomic approaches to investigate age-associated changes in human aortic media with the goal of understanding the molecular mechanisms underlying vascular senescence. METHOD: Cryopreserved autopsy samples of aortic media from older-aged (aged 70-100 years, n = 25), middle-aged (aged 49-68 years, n = 24), and young (aged 21-39 years, n = 12) subjects were collected. We used two proteomic techniques, two-dimensional differential gel electrophoresis and isobaric tags for relative and absolute quantitation, and we subjected differentially-expressed proteins among age groups to immunohistochemical analyses. RESULTS: Proteomic analyses showed that the expression of lactadherin, which produces medin, was elevated in aortic media of older-aged individuals. Immunohistochemical and Congo red staining showed that lactadherin and apolipoprotein E were deposited, and that amyloidosis was enhanced in older-aged aortic media. Furthermore, the markers of oxidative damage (8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal) were significantly elevated in aortic media of middle-aged or older-aged individuals. The immunohistochemical expression of anti-oxidant proteins (thioredoxin and extracellular superoxide dismutase) was also high in middle-aged and older-aged groups. Oxidative damage might induce the disruption of smooth muscle cells, resulting in the decrement of α-actin, a highly-expressed protein in smooth muscle cells, and matrix remodeling, in which several proteins associated with extracellular matrix were altered with aging. CONCLUSIONS: Proteomic approaches showed that the elevated expression of lactadherin might contribute to amyloid deposition, enhancement of oxidative stress, induction of antioxidant proteins and matrix remodeling in older-aged aortic media. Geriatr Gerontol Int 2019; 19: 1054-1062.

Identification of Diketopiperazine-Containing 2-Anilinobenzamides as Potent Sirtuin 2 (SIRT2)-Selective Inhibitors Targeting the "Selectivity Pocket", Substrate-Binding Site, and NAD+-Binding Site.

The NAD+-dependent deacetylase SIRT2 represents an attractive target for drug development. Here, we designed and synthesized drug-like SIRT2-selective inhibitors based on an analysis of the putative binding modes of recently reported SIRT2-selective inhibitors and evaluated their SIRT2-inhibitory activity. This led us to develop a more drug-like diketopiperazine structure as a "hydrogen bond (H-bond) hunter" to target the substrate-binding site of SIRT2. Thioamide 53, a conjugate of diketopiperazine and 2-anilinobenzamide which is expected to occupy the "selectivity pocket" of SIRT2, exhibited potent SIRT2-selective inhibition. Inhibition of SIRT2 by 53 was mediated by the formation of a 53-ADP-ribose conjugate, suggesting that 53 is a mechanism-based inhibitor targeting the "selectivity pocket", substrate-binding site, and NAD+-binding site. Furthermore, 53 showed potent antiproliferative activity toward breast cancer cells and promoted neurite outgrowth of Neuro-2a cells. These findings should pave the way for the discovery of novel therapeutic agents for cancer and neurological disorders.

Proteomic study on neurite responses to oxidative stress: search for differentially expressed proteins in isolated neurites of N1E-115 cells.

Reactive oxygen species attack several living organs and induce cell death. Previously, we found axonal/dendrite degeneration before the induction of cell death in hydrogen peroxide-treated neuroblastoma: N1E-115 cells and primary neurons. This phenomenon may be connected with membrane oxidation, microtubule destabilization and disruption of intracellular calcium homeostasis. However, its detailed mechanisms are not fully understood. Here, we identified proteins after treatment with hydrogen peroxide using isolated neurites by liquid chromatography-matrix-assisted laser desorption/ionization-time of flight/time of flight analysis. Twenty-one proteins were increased after treatment with hydrogen peroxide. Specifically, 5 proteins which were secretogranin-1, heat shock protein family D member 1, Brain acid soluble protein 1, heat shock 70-kDa protein 5 and superoxide dismutase 1, were identified of all experiments and increased in isolated neurites of hydrogen peroxide-treated cells compared to the controls. Furthermore, secretogranin-1 and heat shock protein family D member 1 protein expressions were significantly increased in normal aged and Alzheimer's transgenic mice brains. These results indicate that secretogranin-1 and heat shock protein family D member 1 might contribute to reactive oxygen species-induced neurite degeneration. Both proteins have been related to neurodegenerative disorders, so their study may shed light on neurite dysfunction.

CDP-glycerol inhibits the synthesis of the functional O-mannosyl glycan of α-dystroglycan.

α-Dystroglycan (α-DG) is a highly glycosylated cell-surface laminin receptor. Defects in the O-mannosyl glycan of an α-DG with laminin-binding activity can cause α-dystroglycanopathy, a group of congenital muscular dystrophies. In the biosynthetic pathway of functional O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP), whose mutated genes underlie α-dystroglycanopathy, sequentially transfer ribitol phosphate (RboP) from CDP-Rbo to form a tandem RboP unit (RboP-RboP) required for the synthesis of the laminin-binding epitope on O-mannosyl glycan. Both RboP- and glycerol phosphate (GroP)-substituted glycoforms have recently been detected in recombinant α-DG. However, it is unclear how GroP is transferred to the O-mannosyl glycan or whether GroP substitution affects the synthesis of the O-mannosyl glycan. Here, we report that, in addition to having RboP transfer activity, FKTN and FKRP can transfer GroP to O-mannosyl glycans by using CDP-glycerol (CDP-Gro) as a donor substrate. Kinetic experiments indicated that CDP-Gro is a less efficient donor substrate for FKTN than is CDP-Rbo. We also show that the GroP-substituted glycoform synthesized by FKTN does not serve as an acceptor substrate for FKRP and that therefore further elongation of the outer glycan chain cannot occur with this glycoform. Finally, CDP-Gro inhibited the RboP transfer activities of both FKTN and FKRP. These results suggest that CDP-Gro inhibits the synthesis of the functional O-mannosyl glycan of α-DG by preventing further elongation of the glycan chain. This is the first report of GroP transferases in mammals.

Characteristic glycopeptides associated with extreme human longevity identified through plasma glycoproteomics.

BACKGROUND: Glycosylation is highly susceptible to changes of the physiological conditions, and accordingly, is a potential biomarker associated with several diseases and/or longevity. Semi-supercentenarians (SSCs; older than 105 years) are thought to be a model of human longevity. Thus, we performed glycoproteomics using plasma samples of SSCs, and identified proteins and conjugated N-glycans that are characteristic of extreme human longevity. METHODS: Plasma proteins from Japanese semi-supercentenarians (SSCs, 106-109 years), aged controls (70-88 years), and young controls (20-38 years) were analysed by using lectin microarrays and liquid chromatography/mass spectrometry (LC/MS). Peak area ratios of glycopeptides to corresponding normalising peptides were subjected to orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, plasma levels of clinical biomarkers were measured. RESULTS: We found two lectins such as Phaseolus vulgaris, and Erythrina cristagalli (ECA), of which protein binding were characteristically increased in SSCs. Peak area ratios of ECA-enriched glycopeptides were successfully discriminated between SSCs and controls using OPLS-DA, and indicated that tri-antennary and sialylated N-glycans of haptoglobin at Asn207 and Asn211 sites were characterized in SSCs. Sialylated glycans of haptoglobin are a potential biomarker of several diseases, such as hepatocellular carcinoma, liver cirrhosis, and IgA-nephritis. However, the SSCs analysed here did not suffer from these diseases. CONCLUSIONS: Tri-antennary and sialylated N-glycans on haptoglobin at the Asn207 and Asn211 sites were abundant in SSCs and characteristic of extreme human longevity. GENERAL SIGNIFICANCE: We found abundant glycans in SSCs, which may be associated with human longevity.

Contribution of the exosome-associated form of secreted endoplasmic reticulum aminopeptidase 1 to exosome-mediated macrophage activation.

Macrophages secrete endoplasmic reticulum aminopeptidase 1 (ERAP1) in response to lipopolysaccharide (LPS) and interferon (IFN)-γ to enhance their phagocytic and nitric oxide (NO) synthetic activities. In this study, we found that a subset of secreted ERAP1 bound to exosomes released from LPS/IFN-γ-treated murine RAW264.7 macrophages compared to untreated cells. ERAP1-bound exosomes enhanced phagocytic and NO synthetic activities of macrophages more efficiently than free ERAP1 and exosomes derived from untreated cells. Deletion of the exon 10 coding sequence in ERAP1 gene resulted in loss of binding to exosomes. By comparing the activities of exosomes derived from wild-type and ERAP1 gene-deficient RAW264.7 cells, we observed that ERAP1 contributed to the exosome-dependent phagocytosis and NO synthesis of the cells. Upon stimulation of RAW264.7 cells with LPS/IFN-γ, TNF-α, IFN-γ, and CCL3 were also associated with the released exosomes. Analyses of cytokine function revealed that while CCL3 in the exosomes was crucial to the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributed to the enhancement of NO synthesis. These results suggest that treatment with LPS/IFN-γ alters the physicochemical properties of exosomes released from macrophages in order to facilitate association with ERAP1 and several cytokines/chemokines. This leads to exosome-mediated enhancement of macrophage functions. It is possible that packaging effector molecules into exosomes upon inflammatory stimuli, facilitates the exertion of effective pathophysiological functions on macrophages. Our data provide the first evidence that ERAP1 associated with exosomes plays important roles in inflammatory processes via activation of macrophages.