Development of a fluorescence and quencher-based FRET assay for detection of endogenous peptide:N-glycanase/NGLY1 activity.

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals) catalyzes deglycosylation of N-glycans on glycoproteins. A genetic disorder caused by mutations in the NGLY1 gene leads to NGLY1 deficiency with symptoms including motor deficits and neurological problems. Effective therapies have not been established, though, a recent study used the administration of an adeno-associated viral vector expressing human NGLY1 to dramatically rescue motor functions in young Ngly1-/- rats. Thus, early therapeutic intervention may improve symptoms arising from central nervous system dysfunction, and assay methods for measuring NGLY1 activity in biological samples are critical for early diagnostics. In this study, we established an assay system for plate-based detection of endogenous NGLY1 activity using a FRET-based probe. Using this method, we revealed significant changes in NGLY1 activity in rat brains during aging. This novel assay offers reliable disease diagnostics and provides valuable insights into the regulation of PNGase/NGLY1 activity in diverse organisms under different stress conditions.

Amino acid editing of NFE2L1 by PNGase causes abnormal mobility on SDS-PAGE.

NFE2L1 (also known as NRF1) is a member of the nuclear erythroid 2-like family of transcription factors and is critical for counteracting various types of cellular stress such as oxidative, proteotoxic or metabolic stress. This unique transcription factor is also known to undergo changes, including post-translational modifications, limited proteolysis or translocation into the nucleus, before it exerts full transcriptional activity. As a result, there are various molecular forms with distinct sizes for this protein, while the precise nature of each form remains elusive. In this study, the N-glycosylated status of NFE2L1 in cells was examined. The findings revealed that when NFE2L1 was deglycosylated by PNGase F, the size-shift on SDS-PAGE was minimal. This was in contrast to deglycosylation by Endo H, which resulted in a clear size-shift, even though N-linked GlcNAc residues remained on the protein. It was found that this unusual behavior of PNGase-deglycosylated NFE2L1 was dependent on the conversion of the glycosylated-Asn to Asp, resulting in the introduction of more negative charges into the core peptide of NFE2L1. We also demonstrate that NGLY1-mediated deglycosylation and DDI2-mediated proteolytic processing of NFE2L1 are not strictly ordered reactions. Our study will allow us to better understand the precise structures as well as biochemical properties of the various forms of NFE2L1.

O-GalNAc glycosylation determines intracellular trafficking of APP and Aß production.

A primary pathology of Alzheimer's disease (AD) is amyloid ß (Aß) deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aß precursor protein (APP). Although vascular amyloid deposits' origins remain unclear, endothelial APP expression in APP knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP's importance. Furthermore, two types of endothelial APP-highly O-glycosylated APP and hypo-O-glycosylated APP-have been biochemically identified, but only the former is cleaved for Aß production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that hypo-O-glycosylated APP is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires additional O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced Aß production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.

Endothelial expression of human amyloid precursor protein leads to amyloid ß in the blood and induces cerebral amyloid angiopathy in knock-in mice.

The deposition of amyloid ß (Aß) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer's disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770+ mice exhibited increased levels of serum Aß and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aß levels. Even though aged EC-APP770+ mice did not exhibit Aß deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (AppNL-F/NL-F) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aß deposition. We propose that these EC-APP770+:AppNL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood-brain barrier upon administration of anti-Aß antibodies.

A method for assaying peptide: N-glycanase/N-glycanase 1 activities in crude extracts using an N-glycosylated cyclopeptide.

Cytosolic peptide: N-glycanase (PNGase; NGLY1), an enzyme responsible for de-glycosylation of N-glycans on glycoproteins, is known to play pivotal roles in a variety of biological processes. In 2012, NGLY1 deficiency, a rare genetic disorder, was reported and since then, more than 100 patients have now been identified worldwide. Patients with this disease exhibit several common symptoms that are caused by the dysfunction of NGLY1. However, correlation between the severity of patient symptoms and the extent of the reduction in NGLY1 activity in these patients remains to be clarified, mainly due to the absence of a facile quantitative assay system for this enzyme, especially in a crude extract as an enzyme source. In this study, a quantitative, non-radioisotope (RI)-based assay method for measuring recombinant NGLY1 activity was established using a BODIPY-labeled asialoglycopeptide (BODIPY-ASGP) derived from hen eggs. With this assay, the activities of 27 recombinant NGLY1 mutants that are associated with the deficiency were examined. It was found that the activities of three (R469X, R458fs and H494fs) out of the 27 recombinant mutant proteins were 30-70% of the activities of wild-type NGLY1. We further developed a method for measuring endogenous NGLY1 activity in crude extracts derived from cultured cells, patients' fibroblasts, iPS cells or peripheral blood mononuclear cells (PBMCs), using a glycosylated cyclopeptide (GCP) that exhibited resistance to the endogenous proteases in the extract. Our methods will not only provide new insights into the molecular mechanism responsible for this disease but also promises to be applicable for its diagnosis.

Regulation of BMP4/Dpp retrotranslocation and signaling by deglycosylation.

During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.

Chlamydia trachomatis-infected human cells convert ceramide to sphingomyelin without sphingomyelin synthases 1 and 2.

The obligate intracellular bacterium Chlamydia trachomatis proliferates in the membranous compartment inclusion formed in host cells. The host ceramide transport protein CERT delivers ceramide from the endoplasmic reticulum to the Golgi complex for the synthesis of sphingomyelin (SM). Chlamydia trachomatis has been suggested to employ CERT to produce SM in the inclusion by host SM synthases (SMSs). Here, we found that C. trachomatis proliferates and produces infective progeny even in SMS1 and SMS2 double-knockout HeLa cells, but not in the SMS1/SMS2/CERT triple-knockout cells. Interestingly, infected cells convert ceramide to SM without host SMSs. These results suggest that C. trachomatis-infected cells can convert ceramide to SM without host SMSs after CERT-mediated transfer of ceramide to the inclusions.

A CRISPR Screen Identifies LAPTM4A and TM9SF Proteins as Glycolipid-Regulating Factors.

Glycosphingolipids (GSLs) are produced by various GSL-synthesizing enzymes, but post-translational regulation of these enzymes is incompletely understood. To address this knowledge disparity, we focused on biosynthesis of globotriaosylceramide (Gb3), the Shiga toxin (STx) receptor, and performed a genome-wide CRISPR/CAS9 knockout screen in HeLa cells using STx1-mediated cytotoxicity. We identified various genes including sphingolipid-related genes and membrane-trafficking genes. In addition, we found two proteins, LAPTM4A and TM9SF2, for which physiological roles remain elusive. Disruption of either LAPTM4A or TM9SF2 genes reduced Gb3 biosynthesis, resulting in accumulation of its precursor, lactosylceramide. Loss of LAPTM4A decreased endogenous Gb3 synthase activity in a post-transcriptional mechanism, whereas loss of TM9SF2 did not affect Gb3 synthase activity but instead disrupted localization of Gb3 synthase. Furthermore, the Gb3-regulating activity of TM9SF2 was conserved in the TM9SF family. These results provide mechanistic insight into the post-translational regulation of the activity and localization of Gb3 synthase.

Excess APP O-glycosylation by GalNAc-T6 decreases Aß 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 Aß 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 Aß production. Transfection of GalNAc-T6 significantly reduced both Aß1-40 and Aß1-42 generation, but GalNAc-T1 and GalNAc-T4 only reduced Aß1-40 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 ß-secretase was slightly altered in the transfected cells, but the activities of α-secretase and ß-secretase were not significantly altered. These data suggest that excess O-glycosylation on APP by GalNAc-T6 inhibits Aß production.

Role of Intracellular Lipid Logistics in the Preferential Usage of Very Long Chain-Ceramides in Glucosylceramide.

Ceramide is a common precursor of sphingomyelin (SM) and glycosphingolipids (GSLs) in mammalian cells. Ceramide synthase 2 (CERS2), one of the six ceramide synthase isoforms, is responsible for the synthesis of very long chain fatty acid (C20-26 fatty acids) (VLC)-containing ceramides (VLC-Cer). It is known that the proportion of VLC species in GSLs is higher than that in SM. To address the mechanism of the VLC-preference of GSLs, we used genome editing to establish three HeLa cell mutants that expressed different amounts of CERS2 and compared the acyl chain lengths of SM and GSLs by metabolic labeling experiments. VLC-sphingolipid expression was increased along with that of CERS2, and the proportion of VLC species in glucosylceramide (GlcCer) was higher than that in SM for all expression levels of CERS2. This higher proportion was still maintained even when the proportion of C16-Cer to the total ceramides was increased by disrupting the ceramide transport protein (CERT)-dependent C16-Cer delivery pathway for SM synthesis. On the other hand, merging the Golgi apparatus and the endoplasmic reticulum (ER) by Brefeldin A decreased the proportion of VLC species in GlcCer probably due to higher accessibility of UDP-glucose ceramide glucosyltransferase (UGCG) to C16-rich ceramides. These results suggest the existence of a yet-to-be-identified mechanism rendering VLC-Cer more accessible than C16-Cer to UGCG, which is independent of CERT.

Soluble amyloid precursor protein 770 is a novel biomarker candidate for acute coronary syndrome.

Most Alzheimer disease patients show deposition of amyloid ß (Aß) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid ß precursor protein (APP) 770, a different APP isoform from neuronal APP695, and that they produce amyloid ß peptide. We analyzed the glycosylation of APP770 and found that O-glycosylated sAPP770 is preferentially processed by proteases for Aß production. Because the soluble APP cleavage product sAPP is considered to be a possible marker for Alzheimer disease diagnosis, sAPP, consisting of a mixture of these variants, has been widely measured. We hypothesized that measurement of the endothelial APP770 cleavage product in patients separately from that of neuronal APP695 would enable us to discriminate between endothelial and neurological dysfunctions. Our recent findings, showing that the level of plasma sAPP770 is significantly higher in patients with acute coronary syndrome, raise the possibility that sAPP770 could be an indicator of endothelial dysfunction. In this review, we first describe the expression, glycosylation, and processing of APP770, and then discuss sAPP770 as a novel biomarker candidate of acute coronary syndrome.

Soluble amyloid precursor protein 770 is released from inflamed endothelial cells and activated platelets: a novel biomarker for acute coronary syndrome.

BACKGROUND: Separate monitoring of the cleavage products of different amyloid ß precursor protein (APP) variants may provide useful information. RESULTS: We found that soluble APP770 (sAPP770) is released from inflamed endothelial cells and activated platelets as judged by ELISA. CONCLUSION: sAPP770 is an indicator for endothelial and platelet dysfunctions. SIGNIFICANCE: How sAPP770 is released in vivo has been shown. Most Alzheimer disease (AD) patients show deposition of amyloid ß (Aß) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid ß precursor protein (APP) 770, a different APP isoform from neuronal APP695, and produce Aß. Since the soluble APP cleavage product, sAPP, is considered to be a possible marker for AD diagnosis, sAPP has been widely measured as a mixture of these variants. We hypothesized that measurement of the endothelial APP770 cleavage product in patients separately from that of neuronal APP695 would enable discrimination between endothelial and neurological dysfunctions. Using our newly developed ELISA system for sAPP770, we observed that inflammatory cytokines significantly enhanced sAPP770 secretion by endothelial cells. Furthermore, we unexpectedly found that sAPP770 was rapidly released from activated platelets. We also found that cerebrospinal fluid mainly contained sAPP695, while serum mostly contained sAPP770. Finally, to test our hypothesis that sAPP770 could be an indicator for endothelial dysfunction, we applied our APP770 ELISA to patients with acute coronary syndrome (ACS), in which endothelial injury and platelet activation lead to fibrous plaque disruption and thrombus formation. Development of a biomarker is essential to facilitate ACS diagnosis in clinical practice. The results revealed that ACS patients had significantly higher plasma sAPP770 levels. Furthermore, in myocardial infarction model rats, an increase in plasma sAPP preceded the release of cardiac enzymes, currently used markers for acute myocardial infarction. These findings raise the possibility that sAPP770 can be a useful biomarker for ACS.

Brain endothelial cells produce amyloid {beta} from amyloid precursor protein 770 and preferentially secrete the O-glycosylated form.

Deposition of amyloid ß (Aß) in the brain is closely associated with Alzheimer disease (AD). Aß is generated from amyloid precursor protein (APP) by the actions of ß- and γ-secretases. In addition to Aß deposition in the brain parenchyma, deposition of Aß in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how Aß accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aß40 and Aß42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, Ο-glycosylated APP770 was preferentially processed by both α- and ß-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770ß in human cerebrospinal fluid, this unique soluble APP770ß has the potential to serve as a marker for cortical dementias such as AD and vascular dementia.

Interleukin-1 beta up-regulates TACE to enhance alpha-cleavage of APP in neurons: resulting decrease in Abeta production.

The proinflammatory cytokine interleukin (IL)-1beta is up-regulated in microglial cells surrounding amyloid plaques, leading to the hypothesis that IL-1beta is a risk factor for Alzheimer's disease. However, we unexpectedly found that IL-1beta significantly enhanced alpha-cleavage, indicated by increases in sAPPalpha and C83, but reduced beta-cleavage, indicated by decreases in sAPPbeta and Abeta40/42, in human neuroblastoma SK-N-SH cells. IL-1beta did not significantly alter the mRNA levels of BACE1, ADAM-9, and ADAM-10, but up-regulated that of TACE by threefold. The proform and mature form of TACE protein were also significantly up-regulated. A TACE inhibitor (TAPI-2) concomitantly reversed the IL-1beta-dependent increase in sAPPalpha and decrease in sAPPbeta, suggesting that APP consumption in the alpha-cleavage pathway reduced its consumption in the beta-cleavage pathway. IL-1Ra, a physiological antagonist for the IL-1 receptor, reversed the effects of IL-1beta, suggesting that the IL-1beta-dependent up-regulation of alpha-cleavage is mediated by the IL-1 receptor. IL-1beta also induced this concomitant increase in alpha-cleavage and decrease in beta-cleavage in mouse primary cultured neurons. Taken together we conclude that IL-1beta is an anti-amyloidogenic factor, and that enhancement of its signaling or inhibition of IL-1Ra activity could represent potential therapeutic strategies against Alzheimer's disease.

Screening a series of sialyltransferases for possible BACE1 substrates.

Deposition of amyloid beta-peptide (Abeta) and neurofibrillary tangles in the brain are hallmarks of Alzheimer's disease (AD) pathogenesis. BACE1, a membrane-bound aspartic protease that cleaves amyloid precursor protein (APP) to produce Abeta, has been implicated in triggering the pathogenesis of the disease. We previously reported that BACE1 also cleaved alpha2,6-sialyltransferase (ST6Gal I) in the Golgi apparatus and induced its secretion from the cell. Since most glycosyltransferases show Golgi localization and many of these are cleaved and secreted from the cell, we hypothesized that other glycosyltransferases may also be BACE1 substrates. Here, we focused on a series of sialyltransferases as candidates for BACE1 substrates. We found that BACE1 cleaved polysialyltransferase ST8Sia IV (PST) in vitro. We further found that BACE1 overexpression in COS cells enhanced the secretion of ST3Gal I, II, III and IV, although these sialyltransferases were not cleaved by BACE1 in vitro. These results suggest that BACE1 expression affects glycosylation not only by directly cleaving glycosyltransferases but also by modifying the secretion of glycosyltransferases via some other mechanisms.

In vivo cleavage of alpha2,6-sialyltransferase by Alzheimer beta-secretase.

beta-Site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, Abeta, and is implicated in triggering the pathogenesis of Alzheimer disease. We previously reported that BACE1 cleaved rat beta-galactoside alpha2,6-sialyltransferase (ST6Gal I) that was overexpressed in COS cells and that the NH(2) terminus of ST6Gal I secreted from the cells (E41 form) was Glu(41). 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 BACE1-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.

Characterization of alpha 2,6-sialyltransferase cleavage by Alzheimer's beta -secretase (BACE1).

BACE1 is a membrane-bound aspartic protease that cleaves the amyloid precursor protein (APP) at the beta-secretase site, a critical step in the Alzheimer disease pathogenesis. We previously found that BACE1 also cleaved a membrane-bound sialyltransferase, ST6Gal I. By BACE1 overexpression in COS cells, the secretion of ST6Gal I markedly increased, and the amino terminus of the secreted ST6Gal I started at Glu(41). Here we report that BACE1-Fc chimera protein cleaved the A-ST6Gal I fusion protein, or ST6Gal I-derived peptide, between Leu(37) and Gln(38), suggesting that an initial cleavage product by BACE1 was three amino acids longer than the secreted ST6Gal I. The three amino acids, Gln(38)-Ala(39)-Lys(40), were found to be truncated by exopeptidase activity, which was detected in detergent extracts of Golgi-derived membrane fraction. These results suggest that ST6Gal I is cleaved initially between Leu(37) and Gln(38) by BACE1, and then the three-amino acid sequence at the NH(2) terminus is removed by exopeptidase(s) before secretion from the cells.