Human NEU2 Research Articles

A Lysosome-Targeting hNEU1 Inhibitor Treats Myocardial Infarction: A Potential Therapeutic Breakthrough.

The overexpression of NEU1 has recently been certified as being associated with myocardial infarction. However, the pursuit of an efficacious human NEU1 (hNEU1) inhibitor remains challenging, and viral NEU1 (viNEU1) inhibitor drugs are significantly weaker in terms of hNEU1 inhibition. Recognizing that hNEU1 is located within the lysosome, we designed a series of lysosome-targeting compounds, derived from oseltamivir, aimed at hNEU1 inhibition. Among these compounds, OsMo exhibits the most potent activity. Our findings reveal that OsMo accumulates within lysosomes and releases its pharmacophore via enzymatic catalysis. OsMo enhances hNEU1 inhibition by accumulating pharmacophores at the target site. OsMo exhibits improved regulation of abnormal autophagy during myocardial injury, demonstrating superior efficacy in treating myocardial infarction in vivo. Furthermore, OsMo exhibits acceptable pharmacokinetic parameters. Importantly, the development of molecules with lysosome-targeting abilities represents a promising avenue for addressing myocardial injuries linked to hNEU1 overexpression.

AAV-mediated gene therapy for sialidosis

Sialidosis (Mucolipidosis I) is a glycoprotein storage disease, clinically characterized by a spectrum of systemic and neurological phenotypes. The primary cause of the disease is deficiency of the lysosomal sialidase NEU1, resulting in accumulation of sialylated glycoproteins/ oligosaccharides in tissues and body fluids. Neu1–/– mice recapitulate the severe, early-onset forms of the disease, affecting visceral organs, muscles, and the nervous system, with wide-spread lysosomal vacuolization evident in most cell types. Sialidosis is considered an orphan disorder with no therapy currently available. Here, we assessed the therapeutic potential of AAV-mediated gene therapy for the treatment of sialidosis. Neu1–/– mice were co-injected with two scAAV2/8 vectors, expressing human NEU1 and its chaperone PPCA. Treated mice were phenotypically indistinguishable from their WT controls. NEU1 activity was restored to different extent in most tissues, including the brain, heart and muscle, and visceral organs. This resulted in diminished/absent lysosomal vacuolization in multiple cell types and reversal of sialyl-oligosacchariduria. Lastly, normalization of lysosomal exocytosis in the CSF and serum of treated mice, coupled to diminished neuroinflammation, were measures of therapeutic efficacy. These findings point to AAV-mediated gene therapy as a suitable treatment for sialidosis and possibly other diseases, associated with low NEU1 expression.

Characterization of three sialidases from Danio rerio

Zebrafish encodes several sialidases belonging to the NEU3 group, the plasma membrane-associated member of the family with high specificity toward ganglioside substrates. Neu3.1, Neu3.2 and Neu 3.3 have been expressed in E. coli and purified using the pGEX-2T expression system. Although all the enzymes are expressed by bacterial cells, Neu3.1 formed insoluble aggregates that hampered its purification. Neu3.2 and Neu3.3 formed oligomers as demonstrated by gel filtration chromatography experiments. Actually, the first formed a trimer whereas the second a pentamer. Intriguingly, despite relevant degree of sequence identity and similarity, the two enzymes showed peculiar substrate specificities toward gangliosides other than GM3, two glycoproteins and two forms of sialyllactose. Using molecular modelling and the crystal structure of the human cytosolic sialidase NEU2 as a template, the 3D models of the sialidases from zebrafish have been generated. As expected, the 3D models showed the typical six blade beta-propeller typical of sialidases, with an overall highly conserved active site architecture. The differences among the three zebrafish enzymes and human NEU2 are mainly located in the loops connecting the antiparallel beta strands of the propeller core. These portions of the proteins are probably responsible for the differences observed in substrate specificities, as well as in the different subcellular localization and aggregation features observed in solution. Finally, the in silico analysis of RNA-Seq data evidenced a peculiar expression profile of the three genes during embryogenesis, suggesting different roles of these sialidases during development.

Neuraminidase 1 and its Inhibitors from Chinese Herbal Medicines: An Emerging Role for Cardiovascular Diseases.

Neuraminidase, also known as sialidase, is ubiquitous in animals and microorganisms. It is predominantly distributed in the cell membrane, cytoplasmic vesicles, and lysosomes. Neuraminidase generally recognizes the sialic acid glycosidic bonds at the ends of glycoproteins or glycolipids and enzymatically removes sialic acid. There are four types of neuraminidases, named as Neu1, Neu2, Neu3, and Neu4. Among them, Neu1 is the most abundant in mammals. Recent studies have revealed the involvement of Neu1 in several diseases, including cardiovascular diseases, diabetes, cancers, and neurological disorders. In this review, we center the attention to the role of Neu1 in cardiovascular diseases, including atherosclerosis, ischemic myocardial injury, cerebrovascular disease, congenital heart disease, and pulmonary embolism. We also summarize inhibitors from Chinese herbal medicines (CHMs) in inhibiting virus neuraminidase or human Neu1. Many Chinese herbs and Chinese herb preparations, such as Lonicerae Japonicae Flos, Scutellariae Radix, Yupingfeng San, and Huanglian Jiedu Decoction, have neuraminidase inhibitory activity. We hope to highlight the emerging role of Neu1 in humans and potentially titillate interest for further studies in this area.

Transmembrane Peptides as a New Strategy to Inhibit Neuraminidase-1 Activation.

Sialidases, or neuraminidases, are involved in several human disorders such as neurodegenerative, infectious and cardiovascular diseases, and cancers. Accumulative data have shown that inhibition of neuraminidases, such as NEU1 sialidase, may be a promising pharmacological target, and selective inhibitors of NEU1 are therefore needed to better understand the biological functions of this sialidase. In the present study, we designed interfering peptides (IntPep) that target a transmembrane dimerization interface previously identified in human NEU1 that controls its membrane dimerization and sialidase activity. Two complementary strategies were used to deliver the IntPep into cells, either flanked to a TAT sequence or non-tagged for solubilization in detergent micelles. Combined with molecular dynamics simulations and heteronuclear nuclear magnetic resonance (NMR) studies in membrane-mimicking environments, our results show that these IntPep are able to interact with the dimerization interface of human NEU1, to disrupt membrane NEU1 dimerization and to strongly decrease its sialidase activity at the plasma membrane. In conclusion, we report here new selective inhibitors of human NEU1 of strong interest to elucidate the biological functions of this sialidase.

Suppression of Neu1 sialidase delays the absorption of yolk sac in medaka (Oryzias latipes) accompanied with the accumulation of α2-3 sialo-glycoproteins

Sialidase catalyzes the removal of sialic acids from glycoconjugates. Recently, medaka sialidase Neu1 has been cloned and its enzymatic properties were investigated. Although enzymatic properties of this sialidase, such as optimal pH and substrate specificity, exhibits high similarity with human NEU1, Neu1 physiological functions in fish are still unclear. Here, to understand Neu1 significance in medaka embryogenesis, sialidase translation knockdown was carried out with one-cell stage fertilized egg using morpholino oligo injection.Neu1 exhibited desialylation of α2-3 sialic acid linkage in vitro and lysosomal localization in medaka caudal fin primary cells. Chloroquine treatment, inhibitor of lysosomal enzymes, caused an accumulation of α2-3 sialo-glycoproteins in the primary cells. During the embryogenesis neu1 mRNA level was elevated until 3.5 day post fertilization (dpf) while an initial decrease of α2-3 sialo-glycoprotein was observed around the same developmental stage. Neu1 knockdown by morpholino oligo induced some abnormal phenotypes such as delay of yolk sac absorption and small embryos. Sialidase-knockdown embryos also showed increase of heart rate in 5.5 and 6.5 dpf. Furthermore, about 37% decrease of hatching rate was observed in Neu1-MO treated embryos compared with control MO. Embryos showing severe phenotypes stopped embryogenesis at the late stage of development. Alteration of embryonic sialo-glycoproteins induced by morpholino injection was examined by lectin blotting to clarify the mechanism of abnormal development. As a result, degradation of several α2-3 sialo-glycoproteins was suppressed in Neu1-MO embryo, possibly induced by the interruption of lysosomal desialylation toward yolk glycoprotein. Our results suggest that medaka Neu1 could be crucial for embryonic development through the degradation of yolk sac nutrition.

New Insights into Molecular Organization of Human Neuraminidase-1: Transmembrane Topology and Dimerization Ability.

Neuraminidase 1 (NEU1) is a lysosomal sialidase catalyzing the removal of terminal sialic acids from sialyloconjugates. A plasma membrane-bound NEU1 modulating a plethora of receptors by desialylation, has been consistently documented from the last ten years. Despite a growing interest of the scientific community to NEU1, its membrane organization is not understood and current structural and biochemical data cannot account for such membrane localization. By combining molecular biology and biochemical analyses with structural biophysics and computational approaches, we identified here two regions in human NEU1 - segments 139–159 (TM1) and 316–333 (TM2) - as potential transmembrane (TM) domains. In membrane mimicking environments, the corresponding peptides form stable α-helices and TM2 is suited for self-association. This was confirmed with full-size NEU1 by co-immunoprecipitations from membrane preparations and split-ubiquitin yeast two hybrids. The TM2 region was shown to be critical for dimerization since introduction of point mutations within TM2 leads to disruption of NEU1 dimerization and decrease of sialidase activity in membrane. In conclusion, these results bring new insights in the molecular organization of membrane-bound NEU1 and demonstrate, for the first time, the presence of two potential TM domains that may anchor NEU1 in the membrane, control its dimerization and sialidase activity.

Lysosomal localization of Japanese medaka (Oryzias latipes) Neu1 sialidase and its highly conserved enzymatic profiles with human

Desialylation in the lysosome is a crucial step for glycoprotein degradation. The abnormality of lysosomal desialylation by NEU1 sialidase is involved in diseases of mammals such as sialidosis and galactosialidosis. Mammalian Neu1 sialidase is also localized at plasma membrane where it regulates several signaling pathways through glycoprotein desialylation. In fish, on the other hand, the mechanism of desialylation in the lysosome and functions of Neu1 sialidase are still unclear. Here, to understand the significance of fish Neu1 sialidase, neu1 gene was cloned from medaka brain and the profiles of its polypeptides were analyzed. Open reading frame of medaka neu1 consisted 1,182bp and the similarity of its deduced amino acids with human NEU1 was 57%. As this recombinant polypeptide did not show significant sialidase activity, medaka cathepsin A, known in mammals as protective protein activating Neu1, was cloned and then co-expressed with medaka Neu1 to examine whether medaka cathepsin A activates Neu1 activity. As a result, Neu1/cathepsin A showed a drastic increase of sialidase activity toward MU-NANA. Major substrate of medaka Neu1 was 3-sialyllactose and its optimal pH was 4.0. With immunofluorescence analysis, signal of overexpressed medaka Neu1 was found to coincide with Lysotracker signals (organelle marker of lysosome) and co-localized with medaka cathepsin A in fish hepatic Hepa-T1 cells. Furthermore, part of medaka Neu1 was also detected at plasma membrane. Medaka Neu1 possessed signal peptide sequence at N-terminal and incomplete lysosomal targeting sequence at C-terminus. Medaka neu1 gene was ubiquitously expressed in various medaka tissues, and its expression level was significantly higher than other sialidase genes such as neu3a, neu3b and neu4. The present study revealed the profiles of fish Neu1 sialidase and indicated its high conservation with human NEU1 for the first time, suggesting the presence of similar desialylation system in the medaka lysosome to human. Moreover, the present study showed the possibility of medaka as a model animal of human NEU1 sialidase.

Novel pH-dependent regulation of human cytosolic sialidase 2 (NEU2) activities by siastatin B and structural prediction of NEU2/siastatin B complex

Human cytosolic sialidase (Neuraminidase 2, NEU2) catalyzes the removal of terminal sialic acid residues from glycoconjugates. The effect of siastatin B, known as a sialidase inhibitor, has not been evaluated toward human NEU2 yet. We studied the regulation of NEU2 activity by siastatin B in vitro and predicted the interaction in silico. Inhibitory and stabilizing effects of siastatin B were analyzed in comparison with DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) toward 4-umbelliferyl N-acetylneuraminic acid (4-MU-NANA)- and α2,3-sialyllactose-degrading activities of recombinant NEU2 produced by E. coli GST-fusion gene expression. Siastatin B exhibited to have higher competitive inhibitory activity toward NEU2 than DANA at pH 4.0. We also revealed the stabilizing effect of siastatin B toward NEU2 activity at acidic pH. Docking model was constructed on the basis of the crystal structure of NEU2/DANA complex (PDB code: 1VCU). Molecular docking predicted that electrostatic neutralization of E111 and E218 residues of the active pocket should not prevent siastatin B from binding at pH 4.0. The imino group (1NH) of siastatin B can also interact with D46, neutralized at pH 4.0. Siastatin B was suggested to have higher affinity to the active pocket of NEU2 than DANA, although it has no C7–9 fragment corresponding to that of DANA. We demonstrated here the pH-dependent affinity of siastatin B toward NEU2 to exhibit potent inhibitory and stabilizing activities. Molecular interaction between siastatin B and NEU2 will be utilized to develop specific inhibitors and stabilizers (chemical chaperones) not only for NEU2 but also the other human sialidases, including NEU1, NEU3 and NEU4, based on homology modeling.

Nile Tilapia Neu3 sialidases: Molecular cloning, functional characterization and expression in Oreochromis niloticus

Mammalian Neu3 is a ganglioside specific sialidase. Gangliosides are involved in various physiological events such as cell growth, differentiation and diseases. Significance of Neu3 and gangliosides is still unclear in aquaculture fish species. To gain more insights of fish Neu3 sialidases, molecular cloning and characterization were carried out in tilapia (Oreochromis niloticus). A tilapia genome-wide search for orthologues of human NEU1, NEU2, NEU3 and NEU4 yielded eight putative tilapia sialidases, five of which were neu3-like and designated as neu3a, neu3b, neu3c, neu3d and neu3e. Among five neu3 genes, neu3a, neu3d and neu3e were amplified by PCR from adult fish brain cDNA with consensus sequences of 1227bp, 1194bp and 1155bp, respectively. Multiple alignments showed conserved three Asp-boxes (SXDXGXTW), YRIP and VGPG motifs. The molecular weights for Neu3a, Neu3d and Neu3e were confirmed using immunoblotting analysis as 45.9kDa, 44.4kDa and 43.6kDa, respectively. Lysate from neu3 genes transfected HEK293 cells showed sialidase activity in Neu3a towards ganglioside mix optimally at pH4.6. Using pure gangliosides as substrates, highest sialidase activity for Neu3a was observed towards GD3 followed by GD1a and GM3, but not GM1. On the other hand, sialidase activities were not observed in Neu3d and Neu3e towards various sialoglycoconjugates. Indirect immunofluorescence showed that tilapia Neu3a and Neu3d are localized at the plasma membrane, while most Neu3e showed a cytosolic localization. RT-PCR analyses for neu3a showed significant expression in the brain, liver, and spleen tissues, while neu3d and neu3e showed different expression patterns. Based on these results, tilapia Neu3 exploration is an important step towards full understanding of a more comprehensive picture of Neu3 sub-family of proteins in fish.

Chemoenzymatic synthesis of sialosides containing C7-modified sialic acids and their application in sialidase substrate specificity studies

Modifications at the glycerol side chain of sialic acid in sialosides modulate their recognition by sialic acid-binding proteins and sialidases. However, limited work has been focused on the synthesis and functional studies of sialosides with C7-modified sialic acids. Here we report chemical synthesis of C4-modified ManNAc and mannose and their application as sialic acid precursors in a highly efficient one-pot three-enzyme system for chemoenzymatic synthesis of α2–3- and α2–6-linked sialyl para-nitrophenyl galactosides in which the C7-hydroxyl group in sialic acid (N-acetylneuraminic acid, Neu5Ac, or 2-keto-3-deoxynonulosonic acid, Kdn) was systematically substituted by –F, –OMe, –H, and –N3 groups. Substrate specificity study of bacterial and human sialidases using the obtained sialoside library containing C7-modified sialic acids showed that sialosides containing C7-deoxy Neu5Ac were selective substrates for all bacterial sialidases tested but not for human NEU2. The information obtained from sialidase substrate specificity can be used to guide the design of new inhibitors that are selective against bacterial sialidases.

Tuning Mechanism-Based Inactivators of Neuraminidases: Mechanistic and Structural Insights

3-Fluorosialosyl fluorides are inhibitors of sialidases that function by the formation of a long-lived covalent active-site adduct and have potential as therapeutics if made specific for the pathogen sialidase. Surprisingly, human Neu2 and the Trypanosoma cruzi trans-sialidase are inactivated more rapidly by the reagent with an equatorial fluorine at C3 than by its axial epimer, with reactivation following the same pattern. To explore a possible stereoelectronic basis for this, rate constants for spontaneous hydrolysis of the full series of four 3-fluorosialosyl fluorides were measured, and ground-state energies for each computed. The alpha (equatorial) anomeric fluorides hydrolyze more rapidly than their beta anomers, consistent with their higher ground-state energies. However ground-state energies do not explain the relative spontaneous reactivities of the 3-fluoro-epimers. The three-dimensional structures of the two 3-fluoro-sialosyl enzyme intermediates of human Neu2 were solved, revealing key stabilizing interactions between Arg21 and the equatorial, but not the axial, fluorine. Because of changes in geometry these interactions will increase at the transition state, likely explaining the difference in reaction rates.

Tuning Mechanism‐Based Inactivators of Neuraminidases: Mechanistic and Structural Insights

3-Fluorosialosyl fluorides are inhibitors of sialidases that function by the formation of a long-lived covalent active-site adduct and have potential as therapeutics if made specific for the pathogen sialidase. Surprisingly, human Neu2 and the Trypanosoma cruzi trans-sialidase are inactivated more rapidly by the reagent with an equatorial fluorine at C3 than by its axial epimer, with reactivation following the same pattern. To explore a possible stereoelectronic basis for this, rate constants for spontaneous hydrolysis of the full series of four 3-fluorosialosyl fluorides were measured, and ground-state energies for each computed. The alpha (equatorial) anomeric fluorides hydrolyze more rapidly than their beta anomers, consistent with their higher ground-state energies. However ground-state energies do not explain the relative spontaneous reactivities of the 3-fluoro-epimers. The three-dimensional structures of the two 3-fluoro-sialosyl enzyme intermediates of human Neu2 were solved, revealing key stabilizing interactions between Arg21 and the equatorial, but not the axial, fluorine. Because of changes in geometry these interactions will increase at the transition state, likely explaining the difference in reaction rates.

A proline-rich loop mediates specific functions of human sialidase NEU4 in SK-N-BE neuronal differentiation

Human sialidase NEU4 long (N4L) is a membrane-associated enzyme that has been shown to be localized in the outer mitochondrial membrane. A role in different cellular processes has been suggested for this enzyme, such as apoptosis, neuronal differentiation and tumorigenesis. However, the molecular bases for these roles, not found in any of the other highly similar human sialidases, are not understood. We have found that a proline-rich sequence of 81 amino acids, unique to NEU4 sequence, contains potential Akt and Erk1 kinase motifs. Molecular modeling, based on the experimentally determined three-dimensional structure of cytosolic human NEU2, showed that the proline-rich sequence is accommodated in a loop, thus preserving the typical beta-barrel structure of sialidases. In order to investigate the role of this loop in neuronal differentiation, we obtained SK-N-BE neuroblastoma cells stably overexpressing either human wild-type N4L or a deletion mutant lacking the proline-rich loop. Our results demonstrate that the proline-rich region can also enhance cell proliferation and retinoic acid (RA)-induced neuronal differentiation and it is also involved in NEU4 interaction with Akt, as well as in substrate recognition, modifying directly or through the interaction with other protein(s) the enzyme specificity toward sialylated glycoprotein(s). On the whole, our results suggest that N4L could be a downstream component of the PI3K/Akt signaling pathway required for RA-induced differentiation of neuroblastoma SK-N-BE cells.

Inhibitory effects and specificity of synthetic sialyldendrimers toward recombinant human cytosolic sialidase 2 (NEU2)

Human sialidase 2 (NEU2) is a cytoplasmic sialidase that degrades sialylglycoconjugates, including glycoproteins and gangliosides, via hydrolysis of terminal sialic acids to produce asialo-type molecules. Here, we first report the inhibitory effects of a series of synthetic sialyldendrimers comprising three types [Dumbbell(1)6-S-Neu5Ac(6), Fan(0)3-S-Neu5Ac(3) and Ball(0)4-S-NeuAc(4)] toward recombinant human NEU2 in vitro. Among them, Dumbbell(1)6-S-Neu5Ac(6) exhibited the most potent inhibitory activity (concentration causing 50% inhibition (IC(50)), 0.4 ∼ 0.5 mM). In addition, NeuSLac and NeuSCel carrying thiosialyltrisaccharide moieties exhibited more potent inhibitory effects than NeuSGal and NeuSGlc carrying thiosialyldisaccharides. Docking models composed of NEU2 and the thiosialyloligosaccharide suggested that the active pocket of NEU2 prefers the second galactose-β (Galβ) to the glucose-β (Glcβ) residue in the trisaccharide structure, there being a hydrogen bond between the 4-hydroxy group of the second Galβ and the side chain of the D46 residue of NEU2. The third Glcβ residues of NeuSLac and NeuSCel were also predicted to be stabilized by hydrogen bonds with the side chains of the R21, R304, D358 and Y359 residues of NEU2. NEU2 mutants (D358A and Y359A) exhibited reduced affinity for NeuSLac carrying thiosialyltrisaccharide moieties, suggesting the significant roles of D358 and Y359 residues in recognition of thiosialyltrisaccharide moieties of NeuSLac bound in the active pocket of NEU2. Thus, the present sialyldendrimers could be utilized not only as a new class of NEU2 inhibitors but also as molecular probes for evaluating the biological functions of NEU2, including the catalytic activity and mechanism as to natural substrates carrying sialyloligosaccharides.

Sialyl Residues Modulate LPS-Mediated Signaling through the Toll-Like Receptor 4 Complex

We previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFκB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated. HEK293T cells transfected with TLR4/MD2/CD14 responded robustly to the addition of LPS; however, omission of the MD2 plasmid abrogated this response. Addition of culture supernatants from MD2 (sMD2)-transfected HEK293T cells, but not recombinant, non-glycosylated MD2 reconstituted this response. NA treatment of sMD2 enhanced the LPS response as did NA treatment of the TLR4/CD14-transfected cell supplemented with untreated sMD2, but optimal LPS-initiated responses were observed with NA-treated TLR4/CD14-transfected cells supplemented with NA-treated sMD2. We hypothesized that removal of negatively charged sialyl residues from glycans on the TLR4 complex would hasten the dimerization of TLR4 monomers required for signaling. Co-transfection of HEK293T cells with separate plasmids encoding either YFP- or FLAG-tagged TLR4, followed by treatment with NA and stimulation with LPS, led to an earlier and more robust time-dependent dimerization of TLR4 monomers on co-immunoprecipitation, compared to untreated cells. These findings were confirmed by fluorescence resonance energy transfer (FRET) analysis. Overexpression of human Neu1 increased LPS-initiated TLR4-mediated NFκB activation and a NA inhibitor suppressed its activation. We conclude that (1) sialyl residues on TLR4 modulate LPS responsiveness, perhaps by facilitating clustering of the homodimers, and that (2) sialic acid, and perhaps other glycosyl species, regulate MD2 activity required for LPS-mediated signaling. We speculate that endogenous sialidase activity mobilized during cell activation may play a role in this regulation.

Synthesis of selective inhibitors against V. cholerae sialidase and human cytosolic sialidase NEU2.

Sialidases or neuraminidases catalyze the hydrolysis of terminal sialic acid residues from sialyl oligosaccharides and glycoconjugates. Despite successes in developing potent inhibitors specifically against influenza virus neuraminidases, the progress in designing and synthesizing selective inhibitors against bacterial and human sialidases has been slow. Guided by sialidase substrate specificity studies and sialidase crystal structural analysis, a number of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA or Neu5Ac2en) analogues with modifications at C9 or at both C5 and C9 were synthesized. Inhibition studies of various bacterial sialidases and human cytosolic sialidase NEU2 revealed that Neu5Gc9N(3)2en and Neu5AcN(3)9N(3)2en are selective inhibitors against V. cholerae sialidase and human NEU2, respectively.

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies

Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in the sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. A low cellular expression level of the human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of the human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty p-nitrophenol (pNP)-tagged α2-3- or α2-6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.

Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid

In addition to sialic acid, bacteria produce several other nonulosonic acids, including legionaminic acid (Leg). This has exactly the same stereochemistry as sialic acid, with the added features of 9-deoxy and 7-amino groups. In order to explore the biological effects of replacing sialic acid residues (Neu5Ac) in glycoconjugates with Leg in its diacetylated form, diacetyllegionaminic acid (Leg5Ac7Ac), we tested CMP-Leg5Ac7Ac as a donor substrate with a selection of bacterial and mammalian sialyltransferases. The CMP-Leg5Ac7Ac was synthesized in vitro by means of cloned enzymes from the bacillosamine portion of the Campylobacter jejuni N-glycan pathway and from the Leg pathway of Legionella pneumophila. Using fluorescent derivatives of lactose, Galβ1,4GlcNAcβ and T-antigen (Galβ1,3GalNAcα) as acceptors, we tested eight different sialyltransferases and found that the Pasteurella multocida PM0188h and porcine ST3Gal1 sialyltransferases were significantly active with CMP-Leg5Ac7Ac, showing ∼60% activity when compared with CMP-Neu5Ac. The Photobacterium α2,6 sialyltransferase was weakly active, with ∼6% relative activity. The Leg5Ac7Ac-α-2,3-lactose product was then tested as a substrate with six sialidases of viral, bacterial and mammalian origin. All showed much lower activities than with the corresponding sialic acid substrate, with the influenza virus N1 being the most active and human NEU2 being the least active. These results show the feasibility of producing glycoconjugates with Leg5Ac7Ac residues as the terminal sugars, which should display novel biological properties.

Expression of Sialidase Neu2 in Leukemic K562 Cells Induces Apoptosis by Impairing Bcr-Abl/Src Kinases Signaling

Chronic myeloid leukemia is a hematopoietic stem cell cancer, originated by the perpetually "switched on" activity of the tyrosine kinase Bcr-Abl, leading to uncontrolled proliferation and insensitivity to apoptotic stimuli. The genetic phenotype of myeloid leukemic K562 cells includes the suppression of cytosolic sialidase Neu2. Neu2 transfection in K562 cells induced a marked decrease (-30% and -80%) of the mRNA of the anti-apoptotic factors Bcl-XL and Bcl-2, respectively, and an almost total disappearance of Bcl-2 protein. In addition, gene expression and activity of Bcr-Abl underwent a 35% diminution, together with a marked decrease of Bcr-Abl-dependent Src and Lyn kinase activity. Thus, the antiapoptotic axis Bcr-Abl, Src, and Lyn, which stimulates the formation of Bcl-XL and Bcl-2, was remarkably weakened. The ultimate consequences of these modifications were an increased susceptibility to apoptosis of K562 cells and a marked reduction of their proliferation rate. The molecular link between Neu2 activity and Bcr-Abl signaling pathway may rely on the desialylation of some cytosolic glycoproteins. In fact, three cytosolic glycoproteins, in the range 45-66 kDa, showed a 50-70% decrease of their sialic acid content upon Neu2 expression, supporting their possible role as modulators of the Bcr-Abl complex.