Neuraminidase 1 Research Articles

Inhibition of neuraminidase-1 sialidase activity by interfering peptides impairs insulin receptor activity in vitro and glucose homeostasis in vivo

Neuraminidases also called sialidases are glycosidases which catalyze the removal of terminal sialic acid residues from glycoproteins, glycolipids and oligosaccharides. Mammalian Neuraminidase-1 (NEU-1) participates in regulation of cell surface receptors such as insulin receptor (IR), epithelial growth factor receptor, low density lipoprotein receptor and toll like receptor 4. At the plasma membrane, NEU-1 can be associated with the elastin-binding protein and the carboxypeptidase protective protein/cathepsin A to constitute the elastin receptor complex. In this complex, NEU-1 is essential for elastogenesis, signal transduction through this receptor and for biological effects of the elastin-derived peptides on atherosclerosis, thrombosis, insulin resistance, non-alcoholic steatohepatitis and cancers. This is why research teams are developing inhibitors targeting this sialidase. Previously, we developed interfering peptides to inhibit the dimerization and the activation of NEU-1. In this study, we investigated the effects of these peptides on IR activation in vitro and in vivo. Using cellular overexpression and endogenous expression models of NEU-1 and IR (COS-7 and HepG2 cells respectively), we have shown that interfering peptides inhibit NEU-1 dimerization and sialidase activity which results in a reduction of IR phosphorylation. These results demonstrated that NEU-1 positively regulates IR phosphorylation and activation in our conditions. In vivo, biodistribution study showed that interfering peptides are well distributed in mice. Treatment of C57Bl/6 mice during eight weeks with interfering peptides induces a hyperglycemic effect in our experimental conditions. Altogether, we report here that inhibition of NEU-1 sialidase activity by interfering peptides decreases IR activity in vitro and glucose homeostasis in vivo.

Gene therapy corrects the neurological deficits of mice with sialidosis.

Patients with sialidosis (mucolipidosis type I) type I typically present with myoclonus, seizures, ataxia, cherry-red spots, and blindness because of mutations in the neuraminidase 1 (NEU1) gene. Currently, there is no treatment for sialidosis. In this study, we developed an adeno-associated virus (AAV)-mediated gene therapy for a Neu1 knockout (Neu1-/-) mouse model of sialidosis. The vector, AAV9-P3-NP, included the human NEU1 promoter, NEU1 cDNA, IRES, and CTSA cDNA. Untreated Neu1-/- mice showed astrogliosis and microglial LAMP1 accumulation in the nervous system, including brain, spinal cord, and dorsal root ganglion, together with impaired motor function. Coexpression of NEU1 and protective protein/cathepsin A (PPCA) in neonatal Neu1-/- mice by intracerebroventricular injection, and less effective by facial vein injection, decreased astrogliosis and LAMP1 accumulation in the nervous system and improved rotarod performance of the treated mice. Facial vein injection also improved the grip strength and survival of Neu1-/- mice. Therefore, cerebrospinal fluid delivery of AAV9-P3-NP, which corrects the neurological deficits of mice with sialidosis, could be a suitable treatment for patients with sialidosis type I. After intracerebroventricular or facial vein injection of AAV vectors, NEU1 and PPCA are expressed together. PPCA-protected NEU1 is then sent to lysosomes, where β-Gal binds to this complex to form a multienzyme complex in order to execute its function.

Lysosomal sialidase NEU1, its intracellular properties, deficiency, and use as a therapeutic agent.

Neuraminidase 1 (NEU1) is a lysosomal sialidase that cleaves terminal α-linked sialic acid residues from sialylglycans. NEU1 is biosynthesized in the rough endoplasmic reticulum (RER) lumen as an N-glycosylated protein to associate with its protective protein/cathepsin A (CTSA) and then form a lysosomal multienzyme complex (LMC) also containing β-galactosidase 1 (GLB1). Unlike other mammalian sialidases, including NEU2 to NEU4, NEU1 transport to lysosomes requires association of NEU1 with CTSA, binding of the CTSA carrying terminal mannose 6-phosphate (M6P)-type N-glycan with M6P receptor (M6PR), and intralysosomal NEU1 activation at acidic pH. In contrast, overexpression of the single NEU1 gene in mammalian cells causes intracellular NEU1 protein crystallization in the RER due to self-aggregation when intracellular CTSA is reduced to a relatively low level. Sialidosis (SiD) and galactosialidosis (GS) are autosomal recessive lysosomal storage diseases caused by the gene mutations of NEU1 and CTSA, respectively. These incurable diseases associate with the NEU1 deficiency, excessive accumulation of sialylglycans in neurovisceral organs, and systemic manifestations. We established a novel GS model mouse carrying homozygotic Ctsa IVS6 + 1g/a mutation causing partial exon 6 skipping with simultaneous deficiency of Ctsa and Neu1. Symptoms developed in the GS mice like those in juvenile/adult GS patients, such as myoclonic seizures, suppressed behavior, gargoyle-like face, edema, proctoptosis due to Neu1 deficiency, and sialylglycan accumulation associated with neurovisceral inflammation. We developed a modified NEU1 (modNEU1), which does not form protein crystals but is transported to lysosomes by co-expressed CTSA. In vivo gene therapy for GS and SiD utilizing a single adeno-associated virus (AAV) carrying modNEU1 and CTSA genes under dual promoter control will be created.

Impact of Age on Hemostasis of Patients with Immune Thrombocytopenia

Introduction Incidence of primary immune thrombocytopenia (ITP) has two peaks: one in children and young adult and one in the elderly. Management of older patients represents a challenge, because older age is associated with increased frailty, comorbidities, polypharmacy, and worse outcome. Moreover, we cannot rule out the effect of old age on hemostasis of individuals without ITP. Objectives We aimed to compare characteristics of hemostasis in healthy controls and in patients with ITP stratified according to their age in ≤65 years old (yo) and >65 yo, in order to determine if old age modifies hemostasis and if there is an increased risk of bleeding in older patients with ITP. Methods This is a prospective project that was approved by the Ethics Committee from Hospital Universitario La Paz. Informed consent was signed before sampling. ITP patients (n=100≤65 yo and n=68>65 yo) and healthy controls (n=100≤65 yo and n=28>65 yo) were recruited. Patients with ITP and participants with uncontrolled hypertension, artery disease, abnormal hepatic or renal function tests, a diagnosis of a bleeding disorder or thrombopathy, treatment with drugs that could affect hemostasis or a history of thrombotic episodes were excluded. We evaluated platelet activation markers (TRAP and ADP-induced activation of fibrinogen receptor determined through PAC1-binding, and TRAP-induced P-selectin exposure with anti-P-selectin antibody); active caspase-3, -7, -8 and -9; surface loss of sialic acid determined by the binding of Ricinus Communis Agglutinin I (RCA) to galactose residues; and presence of GlcNAc residues on platelet's surface determined by the binding of Wheat Germ Agglutinin (WGA). Neuraminidase 1 (NEU1) associated to membrane of quiescent platelets were tested with anti-NEU1-Alexa 546 antibody. All samples were analyzed by flow cytometry. Thrombin generation was measured in platelet-poor plasma by Calibrated automated thrombogram (CAT) and coagulation was triggered by proper recalcification and the addition (final concentrations) of 1 pmol/l of recombinant human tissue factor and 4 µmol/l of phospholipid mixture (PPP-Reagent LOW). The following parameters were measured: Lagtime (= time when 10 nmol/l thrombin is formed); peak height (PH = maximum thrombin concentration reached); and endogenous thrombin potential (ETP = area under the thrombin-concentration-vs-time curve) were calculated with the Thrombinoscope software package (Thrombinoscope BV). Statistical analyses were performed with GraphPrism 6.0. Comparisons between controls vs ITP patients with similar range of age; controls ≤65 vs >65 yo; and ITP patients ≤65 vs >65 yo were performed with 2 way ANOVA and p<0.05 were considered as significant. Results Table 1 shows results obtained. There was no difference in age between controls and ITP groups with the same age range. Platelets from patients with ITP had a low ability to be activated when compared to their corresponding control group, and impairment in activation of fibrinogen receptor was even more evident for the older group of ITP patients.This fact was not due to a reduction in the number of fibrinogen receptors. Caspases 3, 8 and 9 were higher in ITP patients than their age-matched controls. Moreover, caspase 3 was higher in older ITP than in patients ≤65 yo. Regarding analyses of glycoside residues, platelets from patients with ITP had less sialic acid and more GlcNAc residues on their surface when compared with the corresponding controls. Surprisingly, sialic content in platelets from controls > 65yo was higher than in the controls ≤65 yo, and this fact is in accordance to the lowest content of NEU1 associated to their membrane. Plasma from ITP patients had a higher capacity to generate thrombin when compared with their age-matched control group. Nevertheless, both groups (control and ITP)> 65 yo generated less thrombin than the groups ≤65 yo. No differences were observed in other CAT parameters. Conclusion Platelets from patients with ITP are less functional than those from control groups. This impairment seemed to be more pronounced in platelets from patients >65 yo, putting forward the possibility of an increased risk of bleeding in older patients with ITP. This study also demonstrates that old age might modify hemostasis even in individuals without ITP. Work supported by Instituto de Salud Carlos III (ISCIII) (PI19/00772, PI22/01489), co-funded by the European Union.

Smart Nanomedicine Targeting Endocytosis Mediated by Cancer Cell Surface Neuraminidase‐1

Human neuraminidase‐1 (NEU1) plays a much more profound function in human cancers than previously considered. It is demonstrated that cancer cell surface NEU1 is a desired gatekeeper for an innovative anticancer therapeutic nanomedicine enabling active drug‐targeting delivery by specific endocytosis into the cytoplasm. Nanosome, an antiadhesive nanoparticular shuttle, carrying multiple suicide substrates for NEU1 confers potent and universal inhibitory effects on the proliferation of human cancer cells, such as hepatocellular carcinoma (HCC) (HepG2, IC50 = 13.5 nM), lung cancer (A549, IC50 = 9.57 nM), and colon cancer (HT‐29, IC50 = 11.1 nM), in which irreversible inactivation of cell surface NEU1 is essential for the intracellular trafficking and subsequent lysosomal membrane permeabilization by nanosomal aggregation due to the formation of “sialidase corona” through irreversible inactivation of NEU1–NEU4 residing in lysosome. Nanomedicine targeting membrane‐tethered NEU1 allows efficient delivery of hydrophobic sorafenib (Nexavar), a RAF family kinase inhibitor for the treatment of advanced renal cell carcinoma and unresectable HCC at the recommended dose of 400 mg orally twice daily, into endolysosome, resulting in a potent and sustainable inhibition (IC50 = 3.1–6.2 nM at 24–96 h after coincubation) against HepG2 cell growth.

The expression of neuraminidase-1 in Ewing sarcoma and its impact on sarcoma cell proliferation and migration

Objective: To investigate the expression of neuraminidase-1 (NEU1) in Ewing sarcoma (ES) tissue and its effect on the proliferation and migration of ES cells. Methods: To obtain datasets of ES from the National Center for Biotechnology Information's High-Throughput Gene Expression Omnibus (GEO) for the analysis of NEU1 expression in ES; to acquire ES patient dataset from the International Cancer Genome Consortium (ICGC) database and apply Kaplan-Meier survival analysis to investigate the relationship between NEU1 and the prognosis of ES patients; adopting both univariate and multivariate Cox regression analysis to determine whether NEU1 is a prognostic factor for ES; adopting the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation to analyze the potential mechanism of NEU1 in regulating the malignant biological behavior of ES; adopting the real-time fluorescence quantitative polynucleotide chain reaction (RT-qPCR) to verify the expression of NEU1 in the human bone marrow mesenchymal stem cells (hBMSC) and the ES cell line RD-ES; adopting the transfection technology to knock down the expression of NEU1 in ES cell lines and divide them into two groups: shRNA-NEU1 and shRNA-NC to explore the effects of altered NEU1 expression on ES malignant behavior; adopting the cell counting kit (CCK-8) and cell clone formation experiment to detect the proliferation ability of two groups of cells; adopting the scratch healing experiment to test the cell migration ability of the two groups. Results: We retrieved and analyzed data from the GEO database, including GSE17674 (44 ES tissues and 18 normal tissues) and GSE17679 (87 ES tissues and 18 normal tissues), and found that NEU1 expression was significantly higher in ES tissues compared to normal control tissues (P<0.001). The complete gene expression and clinical information of 56 ES patients obtained from the ICGC database revealed that the ES patients with high NEU1 expression (n=28) had a significantly lower overall survival rates at different time points compared to those with low NEU1 expression (n=28) (HR=2.830, 95%CI:1.324-6.051, P=0.005). Univariate analysis indicated that NEU1 could impact ES patient prognosis (HR=1.049, 95%CI: 1.008-1.092, P=0.019), and multivariate analysis further suggested that NEU1 could serve as a risk factor for ES prognosis (HR=1.087, 95%CI: 1.028-1.148, P=0.003). KEGG results show that MAPK signaling pathway and cell adhesion molecule signaling pathway were potential mechanisms regulating the malignant process of ES. The RT-qPCR results showed that the expression level of NEU1 in the RD-ES cell line is significantly higher than that in the control cell hBMSC (2 184.23±527.32 vs 1.00±0.08, P<0.001). The CCK-8 experiment results show that the proliferation of RD-ES cells in the NEU1 knockdown group was lower than that in the control group at 24, 48, and 72 hours (0.494±0.126 vs 0.696±0.118, 0.657±0.096 vs 1.142±0.182, 1.053±0.064 vs 1.980±0.146, all P<0.001). The results of single cell clone formation experiment show that the number of colony formation in the low expression NEU1 group was significantly lower than that in the control group (184.2±123.9 vs 362.8±78.0, P=0.021). The cell scratch healing experiment finds that the average scratch distance of the NEU1 knockdown group was significantly lower than that of the control group (19.6%±5.7% vs 56.0%±7.6%, P<0.001). Conclusion: NEU1 may be a prognostic factor in ES, and its abnormal expression in ES can affect the proliferation and migration ability of the ES cells, leading to poor prognosis in ES patients.

Severe kidney dysfunction in sialidosis mice reveals an essential role for neuraminidase 1 in reabsorption.

Sialidosis is an ultra-rare multisystemic lysosomal disease caused by mutations in the neuraminidase 1 (NEU1) gene. The severe type II form of the disease manifests with a prenatal/infantile or juvenile onset, bone abnormalities, severe neuropathology, and visceromegaly. A subset of these patients present with nephrosialidosis, characterized by abrupt onset of fulminant glomerular nephropathy. We studied the pathophysiological mechanism of the disease in 2 NEU1-deficient mouse models, a constitutive Neu1-knockout, Neu1ΔEx3, and a conditional phagocyte-specific knockout, Neu1Cx3cr1ΔEx3. Mice of both strains exhibited terminal urinary retention and severe kidney damage with elevated urinary albumin levels, loss of nephrons, renal fibrosis, presence of storage vacuoles, and dysmorphic mitochondria in the intraglomerular and tubular cells. Glycoprotein sialylation in glomeruli, proximal distal tubules, and distal tubules was drastically increased, including that of an endocytic reabsorption receptor megalin. The pool of megalin bearing O-linked glycans with terminal galactose residues, essential for protein targeting and activity, was reduced to below detection levels. Megalin levels were severely reduced, and the protein was directed to lysosomes instead of the apical membrane. Together, our results demonstrated that desialylation by NEU1 plays a crucial role in processing and cellular trafficking of megalin and that NEU1 deficiency in sialidosis impairs megalin-mediated protein reabsorption.

Neuraminidase 1 promotes renal fibrosis development in male mice

The functions of the influenza virus neuraminidase has been well documented but those of the mammalian neuraminidases remain less explored. Here, we characterize the role of neuraminidase 1 (NEU1) in unilateral ureteral obstruction (UUO) and folic acid (FA)-induced renal fibrosis mouse models. We find that NEU1 is significantly upregulated in the fibrotic kidneys of patients and mice. Functionally, tubular epithelial cell-specific NEU1 knockout inhibits epithelial-to-mesenchymal transition, inflammatory cytokines production, and collagen deposition in mice. Conversely, NEU1 overexpression exacerbates progressive renal fibrosis. Mechanistically, NEU1 interacts with TGFβ type I receptor ALK5 at the 160-200aa region and stabilizes ALK5 leading to SMAD2/3 activation. Salvianolic acid B, a component of Salvia miltiorrhiza, is found to strongly bind to NEU1 and effectively protect mice from renal fibrosis in a NEU1-dependent manner. Collectively, this study characterizes a promotor role for NEU1 in renal fibrosis and suggests a potential avenue of targeting NEU1 to treat kidney diseases.

Platelet Desialylation Is a Novel Mechanism and Therapeutic Target in Daboia siamensis and Agkistrodon halys Envenomation-Induced Thrombocytopenia.

Venom-induced thrombocytopenia (VIT) is one of the most important hemotoxic effects of a snakebite, which is often associated with venom-induced consumptive coagulopathy (VICC). Refractory thrombocytopenia without significant coagulation abnormalities has also been reported after envenomation by some viperid snakes; however, the mechanisms are not well understood and therapeutic strategies are lacking. Here, we found that patients injured by Daboia siamensis or Agkistrodon halys snakes, who were resistant to standard antivenom treatment, had developed coagulopathy-independent thrombocytopenia. Venoms from these viperid snakes, rather than from the elapid snake (Bungarus multicinctus), induced platelet surface expression of neuraminidase-1 (NEU-1), and significantly increased the desialylation of the glycoproteins on human platelets. The desialylated platelets caused by viperid snake venoms were further internalized by macrophages, which resulted in reduced platelet numbers in peripheral blood. Importantly, neuraminidase inhibitor significantly decreased viper venom-induced platelet desialylation, therefore inhibiting platelet phagocytosis by macrophages, and alleviating venom-induced thrombocytopenia. Collectively, these findings support an important role for desialylated platelet clearance in the progression of viper envenomation-induced, coagulopathy-independent thrombocytopenia. Our study demonstrates that the neuraminidase inhibitor may be a potential therapy or adjuvant therapy to treat snakebite-induced thrombocytopenia.

Emerging Therapeutic Targets for Heart Failure.

Heart failure is a global epidemic that affects at least 26 million individuals globally and is becoming more prevalent. Despite advances in treatment strategies, survival and symptom management in individuals with heart failure remain exceptionally low. This review discusses emerging targets for the treatment of heart failure. Recently, a number of targets are being investigated as prospective treatment possibilities for heart failure. These include targets like Runx1 transcription factor (RUNX1), milk fact globule-EFG factor 8 (MFGE8) protein and enzymes such as neuraminidase 1 (NEU1), G protein-coupled receptor kinase 5 (GRK5), G protein-coupled oestrogen receptor 1 (GPER1), urotensin-II receptor (UTR), cluster of differentiation 47 (CD47) and relaxin receptor 1 (RXFP1). On a worldwide level, heart failure is a developing epidemic with substantial morbidity and death. The number of individuals diagnosed with chronic heart failure is rising, and it is anticipated to surge by 46% by 2030. Appropriate heart failure treatment can have the greatest influence on prolonging patients' lives in the coming year. Targets discussed in this review may provide new therapeutic approaches for the treatment of heart failure.

Sialidase NEU1 May Serve as a Potential Biomarker of Proliferation, Migration and Prognosis in Melanoma

BackgroundMelanoma is a kind of malignant tumor with high mortality originating from melanocytes. It is urgent to find new molecular biomarkers for prognosis and new treatment methods for melanoma. As an important molecule of sialidase family, neuraminidase-1 (NEU1) has been found to play an important role in regulating the occurrence and progression of tumors, but the role of NEU1 in melanoma is not sure.MethodsThe expression level of NEU1 in melanoma and normal tissues was evaluated by analyzing the expression data from ONCOMINE, UALCAN and GEPIA database. The mutation, copy number alteration and gene correlation of NEU1 in melanoma were evaluated by analyzing the melanoma data from cBioPortal database The protein expression levels of NEU1 were further validated by immunohistochemical (IHC) staining data from The Human Protein Atlas database. The melanoma data in TIMER 2.0 database were used to analyze the correlation between NEU1 expression and immune cell infiltration. The proliferative and migratory abilities of melanoma cells were examined by cell proliferation and migration assay in vitro and nude mice.ResultsWe discovered that NEU1 was highly expressed in melanoma samples compared with normal samples. The alteration frequency of NEU1 in melanoma patients reached 18%, and most of them were “mutation” type. The expression of NEU1 was positively correlated with the overall survival of patients with melanoma. The expression of NEU1 was positively correlated with the expression of proliferation marker CDK2 and epithelial-mesenchymal transition marker CD44 and negatively correlated with the expression of apoptosis marker CASP3 and CASP8. Moreover, the expression level of NEU1 was related to the infiltration of immune cells in melanoma. Knockdown of NEU1 attenuated the in vitro proliferative and migratory abilities of melanoma cells, as well as in vivo tumor progression of melanoma cells.ConclusionsThese findings suggest that NEU1 may play a key role in the development of melanoma and may be used as a prognostic target of melanoma.

NEU1-A Unique Therapeutic Target for Alzheimer's Disease.

Neuraminidase 1 (NEU1) is considered to be the most abundant and ubiquitous mammalian enzyme, with a broad tissue distribution. It plays a crucial role in a variety of cellular mechanisms. The deficiency of NEU1 has been implicated in various pathological manifestations of sialidosis and neurodegeneration. Thus, it is a novel therapeutic target for neurodegenerative changes in the Alzheimer’s brain. However, to manipulate NEU1 as a therapeutic target, it is imperative to understand that, although NEU1 is commonly known for its lysosomal catabolic function, it is also involved in other pathways. NEU1 is involved in immune response modulation, elastic fiber assembly modulation, insulin signaling, and cell proliferation. In recent years, our knowledge of NEU1 has continued to grow, yet, at the present moment, current data is still limited. In addition, the unique biochemical properties of NEU1 make it challenging to target it as an effective therapeutic option for sialidosis, which is a rare disease but has an enormous patient burden. However, the fact that NEU1 has been linked to the pathology of Alzheimer’s disease, which is rapidly growing worldwide, makes it more relevant to be studied and explored. In the present study, the authors have discussed various cellular mechanisms involving NEU1 and how they are relevant to sialidosis and Alzheimer’s disease.

Reversal of neuroinflammation in novel GS model mice by single i.c.v. administration of CHO-derived rhCTSA precursor protein

Galactosialidosis (GS) is a lysosomal cathepsin A (CTSA) deficiency. It associates with a simultaneous decrease of neuraminidase 1 (NEU1) activity and sialylglycan storage. Central nervous system (CNS) symptoms reduce the quality of life of juvenile/adult-type GS patients, but there is no effective therapy. Here, we established a novel GS model mouse carrying homozygotic Ctsa IVS6+1g→a mutation causing partial exon 6 skipping with concomitant deficiency of Ctsa/Neu1. The GS mice developed juvenile/adult GS-like symptoms, such as gargoyle-like face, edema, proctoprosia due to sialylglycan accumulation, and neurovisceral inflammation, including activated microglia/macrophage appearance and increase of inflammatory chemokines. We produced human CTSA precursor proteins (proCTSA), a homodimer carrying terminal mannose 6-phosphate (M6P)-type N-glycans. The CHO-derived proCTSA was taken up by GS patient-derived fibroblasts via M6P receptors and delivered to lysosomes. Catalytically active mature CTSA showed a shorter half-life due to intralysosomal proteolytic degradation. Following single i.c.v. administration, proCTSA was widely distributed, restored the Neu1 activity, and reduced the sialylglycans accumulated in brain regions. Moreover, proCTSA suppressed neuroinflammation associated with reduction of activated microglia/macrophage and up-regulated Mip1α. The results show therapeutic effects of intracerebrospinal enzyme replacement utilizing CHO-derived proCTSA and suggest suppression of CNS symptoms.

Antiplatelet antibody predicts platelet desialylation and apoptosis in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a bleeding disorder caused by dysregulated B- and T- cell functions, which lead to platelet destruction. A well-recognized mechanism of ITP pathogenesis involves anti-platelet and anti-megakaryocyte antibodies recognizing membrane glycoprotein (GP) complexes, mainly GPIb/IX and GPIIb/IIIa. In addition to the current view of phagocytosis of the opsonised platelets by splenic and hepatic macrophages via their Fc γ receptors, antibody-induced platelet desialylation and apoptosis have also been reported to contribute to ITP pathogenesis. Nevertheless, the relationship between the specific thrombocytopenic mechanisms and various types of anti-platelet antibodies has not been established. In order to ascertain such association, we used sera from 61 ITP patients and assessed the capacity of anti-platelet antibodies to induce neuraminidase 1 (NEU1) surface expression, RCA-1 lectin binding and loss of mitochondrial inner membrane potential on donors’ platelets. Sera from ITP patients with detectable antibodies caused significant platelet desialylation and apoptosis. Anti-GPIIb/IIIa antibodies appeared more capable of causing NEU1 surface translocation while anti-GPIb/IX complex antibodies resulted in a higher degree of platelet apoptosis. In ITP patients with anti-GPIIb/IIIa antibodies, both desialylation and apoptosis were dependent on FcγRIIa signaling rather than platelet activation. Finally, we confirmed in a murine model of ITP that destruction of human platelets induced by anti-GPIIb/IIIa antibodies can be prevented with the NEU1 inhibitor oseltamivir. A collaborative clinical trial is warranted to investigate the utility of oseltamivir in the treatment of ITP.

The Elastin Receptor Complex: An Emerging Therapeutic Target Against Age-Related Vascular Diseases.

The incidence of cardiovascular diseases is increasing worldwide with the growing aging of the population. Biological aging has major influence on the vascular tree and is associated with critical changes in the morphology and function of the arterial wall together with an extensive remodeling of the vascular extracellular matrix. Elastic fibers fragmentation and release of elastin degradation products, also known as elastin-derived peptides (EDPs), are typical hallmarks of aged conduit arteries. Along with the direct consequences of elastin fragmentation on the mechanical properties of arteries, the release of EDPs has been shown to modulate the development and/or progression of diverse vascular and metabolic diseases including atherosclerosis, thrombosis, type 2 diabetes and nonalcoholic steatohepatitis. Most of the biological effects mediated by these bioactive peptides are due to a peculiar membrane receptor called elastin receptor complex (ERC). This heterotrimeric receptor contains a peripheral protein called elastin-binding protein, the protective protein/cathepsin A, and a transmembrane sialidase, the neuraminidase-1 (NEU1). In this review, after an introductive part on the consequences of aging on the vasculature and the release of EDPs, we describe the composition of the ERC, the signaling pathways triggered by this receptor, and the current pharmacological strategies targeting ERC activation. Finally, we present and discuss new regulatory functions that have emerged over the last few years for the ERC through desialylation of membrane glycoproteins by NEU1, and its potential implication in receptor transactivation.

Neuraminidase1 Inhibitor Protects Against Doxorubicin-Induced Cardiotoxicity via Suppressing Drp1-Dependent Mitophagy

Anthracyclines, such as doxorubicin (DOX), are among the effective chemotherapeutic drugs for various malignancies. However, their clinical use is limited by irreversible cardiotoxicity. This study sought to determine the role of neuraminidase 1 (NEU1) in DOX-induced cardiomyopathy and the potential cardio-protective effects of NEU1 inhibitor oseltamivir (OSE). Male Sprague–Dawley (SD) rats were randomized into three groups: control, DOX, and DOX + OSE. NEU1 was highly expressed in DOX-treated rat heart tissues compared with the control group, which was suppressed by OSE administration. Rats in the DOX + OSE group showed preserved cardiac function and were protected from DOX-induced cardiomyopathy. The beneficial effects of OSE were associated with the suppression of dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and mitophagy. In detail, the elevated NEU1 in cardiomyocytes triggered by DOX increased the expression of Drp1, which subsequently enhanced mitochondrial fission and PINK1/Parkin pathway-mediated mitophagy, leading to a maladaptive feedback circle towards myocardial apoptosis and cell death. OSE administration selectively inhibited the increased NEU1 in myocardial cells insulted by DOX, followed by reduction of Drp1 expression, inhibition of PINK1 stabilization on mitochondria, and Parkin translocation to mitochondria, thus alleviating excessive mitochondrial fission and mitophagy, alleviating subsequent development of cellular apoptotic process. This work identified NEU1 as a crucial inducer of DOX-induced cardiomyopathy by promoting Drp1-dependent mitochondrial fission and mitophagy, and NEU1 inhibitor showed new indications of cardio-protection against DOX cardiotoxicity.

In Cellulo Crystallization of Human Neuraminidase 1 and Biological Roles of N-Glycans.

Human neuraminidase 1 (NEU1) is a lysosomal glycosidase that cleaves the terminal sialic acids of sialylglycoconjugates. NEU1 is biosynthesized in the endoplasmic reticulum (ER) lumen as an N-glycosylated protein. NEU1 also associates with cathepsin A (CTSA) in ER, migrates to lysosomes, and exerts catalytic activity. Extraordinary in cellulo crystallization of NEU1 protein in ER despite carrying three N-glycans per molecule at N186, N343, and N352, respectively, were observed when the single human NEU1 gene was overexpressed in mammalian cells. In this study, we first purified the NEU1 from the isolated crystals produced by the HEK293 NEU1-KO cell transiently overexpressing the normal NEU1 and found that the N-glycans were high-mannose or complex types carrying terminal sialic acids. The result suggests that a part of NEU1 crystals were formed or transported to the Golgi apparatus. Second, we compared the effects of single amino acid substitution at the N-sequons, including N186Q, N343Q, and N352Q, each one N-glycan reduction from one NEU1 molecule. We demonstrated that N186Q mutant protein with low enzyme activity and formed a few amounts of smaller crystals. The N343Q mutant exhibited half of the normal intracellular activity, but the numbers and sizes of crystals were almost the same as those of normal NEU1. The N352Q mutant exhibited almost the same activity as the normal enzyme. The numbers of the N352Q crystals were smaller than those of normal NEU1. According to these findings, the N186Q NEU1 protein should have lower stability in ER due to abnormal folding. The second N-glycan at the N343-sequon has little effect on self-aggregation of NEU1. The third N-glycan at the N352-sequon contributes to the self-aggregation of NEU1. We also demonstrated that the three NEU1 mutants associate with the relatively excessive CTSA and migrate to lysosomes.

Discovery of NEU1 as a candidate renal biomarker for proliferative lupus nephritis chronicity

ObjectiveProteomic approach was applied to identify candidate biomarkers of chronicity in patients with proliferative lupus nephritis (LN), and their clinicopathological significance and prognostic values were investigated.MethodsThis study recruited 10 patients...

Neuraminidase 1 Exacerbating Aortic Dissection by Governing a Pro-Inflammatory Program in Macrophages.

Inflammation plays an important role in aortic dissection (AD). Macrophages are critically involved in the inflammation after aortic injury. Neuraminidases (NEUs) are a family of enzymes that catalyze the cleavage of terminal sialic acids from glycoproteins or glycolipids, which is emerging as a regulator of macrophage-associated immune responses. However, the role of neuraminidase 1 (NEU1) in pathological vascular remodeling of AD remains largely unknown. This study sought to characterize the role and identify the potential mechanism of NEU1 in pathological aortic degeneration. After β-aminopropionitrile monofumarate (BAPN) administration, NEU1 elevated significantly in the lesion zone of the aorta. Global or macrophage-specific NEU1 knockout (NEU1 CKO) mice had no baseline aortic defects but manifested improved aorta function, and decreased mortality due to aortic rupture. Improved outcomes in NEU1 CKO mice subjected to BAPN treatment were associated with the ameliorated vascular inflammation, lowered apoptosis, decreased reactive oxygen species production, mitigated extracellular matrix degradation, and improved M2 macrophage polarization. Furthermore, macrophages sorted from the aorta of NEU1 CKO mice displayed a significant increase of M2 macrophage markers and a marked decrease of M1 macrophage markers compared with the controls. To summarize, the present study demonstrated that macrophage-derived NEU1 is critical for vascular homeostasis. NEU1 exacerbates BAPN-induced pathological vascular remodeling. NEU1 may therefore represent a potential therapeutic target for the treatment of AD.

The sialidase NEU1 directly interacts with the juxtamembranous segment of the cytoplasmic domain of mucin-1 to inhibit downstream PI3K-Akt signaling

The extracellular domain (ED) of the membrane-spanning sialoglycoprotein, mucin-1 (MUC1), is an in vivo substrate for the lysosomal sialidase, neuraminidase-1 (NEU1). Engagement of the MUC1-ED by its cognate ligand, Pseudomonas aeruginosa-expressed flagellin, increases NEU1-MUC1 association and NEU1-mediated MUC1-ED desialylation to unmask cryptic binding sites for its ligand. However, the mechanism(s) through which intracellular NEU1 might physically interact with its surface-expressed MUC1-ED substrate are unclear. Using reciprocal coimmunoprecipitation and in vitro binding assays in a human airway epithelial cell system, we show here that NEU1 associates with the MUC1-cytoplasmic domain (CD) but not with the MUC1-ED. Prior pharmacologic inhibition of the NEU1 catalytic activity using the NEU1-selective sialidase inhibitor, C9-butyl amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid, did not diminish NEU1-MUC1-CD association. In addition, glutathione-S-transferase (GST) pull-down assays using the deletion mutants of the MUC1-CD mapped the NEU1-binding site to the membrane-proximal 36 aa of the MUC1-CD. In a cell-free system, we found that the purified NEU1 interacted with the immobilized GST-MUC1-CD and the purified MUC1-CD associated with the immobilized 6XHis-NEU1, indicating that the NEU1-MUC1-CD interaction was direct and independent of its chaperone protein, protective protein/cathepsin A. However, the NEU1-MUC1-CD interaction was not required for the NEU1-mediated MUC1-ED desialylation. Finally, we demonstrated that overexpression of either WT NEU1 or a catalytically dead NEU1 G68V mutant diminished the association of the established MUC1-CD binding partner, PI3K, to MUC1-CD and reduced downstream Akt kinase phosphorylation. These results indicate that NEU1 associates with the juxtamembranous region of the MUC1-CD to inhibit PI3K-Akt signaling independent of NEU1 catalytic activity.