Homology modeling of human sialidase enzymes NEU1, NEU3 and NEU4 based on the crystal structure of NEU2: Hints for the design of selective NEU3 inhibitors
Introduction
Sialidases (E.C.3.2.1.18) also known as neuraminidases belong to a group of glycohydrolytic enzymes, which remove sialic acid residues from a variety of sialoglycoconjugates. They are widely distributed among the different classes of organisms such as viruses, bacteria, protozoa and vertebrates [1]. Sialidases are thought to be involved in various biological processes like infection, proliferation, differentiation, catabolism, signal transduction, antigenic properties and inter–intra cell interactions [2].
Several mammalian sialidases have been cloned and characterized at the molecular level. In humans, four types of sialidases are known and have been classified based on their subcellular localization as intra lysosomal (NEU1) [3], [4], cytosolic (NEU2) [5], [6], plasma membrane (NEU3) [7], [8] and lysosomal or mitochondrial membrane (NEU4) [9]. A comparison of human sialidases with respect to their location, substrate specificity, function and the changes they undergo in cancer cells has been described elsewhere in literature [10]. In addition to subcellular localization, these sialidases also differ in substrate preferences, pH required for optimum activity and also in immunological properties. Despite their different locations and substrate specificities, all human sialidases have highly conserved active site residues, the F/YRIV/P motif in the N-terminal part and the Asp boxes (consensus S/TXD(X)GXTW/F present as three to five repeat in the protein) similar to their viral and bacterial counter parts [11]. These results indicate a monophyletic origin of the sialidases and thus giving the basis for molecular modeling studies to elucidate the structure–function relationships between the family members. The comparative biochemistry and molecular biology of human sialidases has been reviewed excellently elsewhere [2].
Lysosomal sialidase (NEU1) possesses narrow substrate specificity for oligosaccharides, glycopeptides and a synthetic substrate 4MU-Neu5Ac (4-methylumbelliferyl-N-acetylneuraminic acid) [12] and is involved in lysosomal catabolism of sialoglycoconjugates by collaborating with lysosomal proteases or endoglycosidases [13]. Lysosomal storage diseases like sialidosis and galactosialidosis caused by NEU1 deficiency, interferes with the pathways for degradation of sialylated glycoconjugates [3], [14]. In addition to this NEU1 has been proposed to be involved in cellular signaling during immune responses as well as monocytes differentiation [15], [16]. Cytosolic sialidase (NEU2) is active against oligosaccharides, glycopeptides and gangliosides [17]. In mammals, it has been implicated in myotube formation [18]. The exact mechanism of myoblast differentiation through its natural substrate remains obscure but it is claimed to decrease the GM3 ganglioside content, associated with the cytoskeleton [19]. Plasma membrane sialidase (NEU3) hydroyzes gangliosides specifically (except GM1 and GM2) in presence of non-ionic surfactant (Triton X-100) [8], [20], [21]. Since gangliosides are abundant on the plasma membrane and are known modulators of several surface events like cell differentiation, cell proliferation and signal transduction, NEU3 is expected to be involved in cell surface functions by modulating gangliosides [22]. NEU3 involvement in neural differentiation has also been described in literature [23]. Lysosomal or mitochondrial membrane sialidase (NEU4) has broad specificity, active against all major sialoglycoconjugates and has been implicated in the catabolism of glycolipids [11], [24]. A recent report reveals the possibility of NEU4 being involved in apoptosis pathway at the mitochondrial level by regulating ganglioside GD3 [24]. Further functions of NEU4 are yet to be explored.
Further, there is also some correlation between sialidase expression and the alteration in sialyation levels during malignant transformation of cells [10], [22]. In murine, overexpression of lysosomal sialidase in cancer cells showed suppression of metastasis and tumor progression as well as increased sensitivity to apoptosis. Cytosolic sialidase overexpression in melanoma and colonadenocarcinoma cells of mouse has been inversely correlated with their invasiveness and metastatic potential, which is associated with a decrease in sialyl Lewis X and GM3 as well. Although it has been reported that NEU3 is not associated with metastatic potential and invasiveness, certain human cancer cells showed increased expression of NEU3 [25]. This increased expression resulted in inhibition of apoptosis accompanied by increased expression of Bcl2, followed by decreased expression of caspase. Inhibitory role of NEU3 in apoptosis is proposed to be mediated by accumulation of a possible sialidase product lactosyl ceramide (Lac-Cer), which induces Bcl2 expression or by rapid degradation of GD3 [25]. In addition, NEU3 mediated gangliosides depletion results in the activation of integrin-induced kinase/Akt, followed by deactivation of caspase-9 in SCCl2 cells [26]. A recent review discusses distinct aspects of NEU3 inhibition and its relevance in the cure of cancer [22]. NEU3 is also found to be involved in insulin signaling in two ways [27]. First is the negative regulation of insulin signaling by associating with the Grb2 protein and second is the suppression of insulin receptor (IR) phosphorylation through the modulation of gangliosides. Taken together, these anomalous functions of NEU3 are different from the possible regulatory functions of other human sialidases. So selective NEU3 inhibition may be a useful approach in cancer and diabetes therapy or in any case would be a valuable tool for exploring differential functions of human sialidases. The sialic acid transition-state analogue, DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) is available as a weak inhibitor of sialidase enzymes, but it is not selective for NEU3. Structure-based approach in this regard may provide hints on how to exploit the non-selective inhibitor substituents that extend out of the active site pocket or ‘de novo design’ for isoform-selective inhibitor design. Structure homology and sequence alignment methods have been very useful in structure-based design approaches and can tackle the challenge of selectivity. Moreover, the success story of the structure-based drug design of viral sialidase inhibitors for flu fever through computational analysis giving the impetus for our efforts in this subject.
In the present study, we report homology models of NEU1, NEU3 and NEU4 using the crystal structure of NEU2. Using homology models, we have studied active site and its vicinity of human sialidases, which can be exploited for the design of selective NEU3 inhibitors.
Section snippets
Methods
All computations and simulations were carried out on an Intel P4 based Microsoft windows 2000 workstation using Discovery Studio Modeling 1.1 Package (Accelrys) [28].
Homology models of NEU1, NEU3 and NEU4
There are many examples where homology modeling techniques have supported the drug discovery process especially in the target identification and/or validation, lead identification as well as lead optimization with respect to potency and selectivity [41]. The extent of information derived from the homology model depends on the quality of the model. Since the accuracy of the homology model is related to the degree of sequence identity and similarity between the template and target, template
Conclusion
Aberrant expression of sialidases and its possible significance in cancer has been studied. Recent progress in cloning and characterization of sialidases at molecular level has revealed the molecular mechanisms of alterations during carcinogenesis. In particular, increased expression of plasma membrane associated sialidase (NEU3) in cancer cells leads to protection against apoptosis, probably via modulation of gangliosides. NEU3 over expression is also implicated in the development of insulin
Acknowledgements
The first author (SM) is grateful to Ministry of Education, Culture, Sports, Science and Technology, Government of Japan (MEXT) for the financial support, No. 042271. This work was supported in part by Grants-in-Aid (No. 17101007) for Scientific Research from MEXT and by CREST of JST (Japan Science and Technology Corporation) to MK. The authors are thankful to Ms. Subashree and Mrs. Sativa for many valuable discussions during the preparation of manuscript.
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2019, Matrix BiologyCitation Excerpt :Although significant advances have been achieved in the search for Neu-1 inhibitors by using analogs of the broad spectrum sialidase inhibitor 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) [59], identification of more selective inhibitors of membrane Neu-1 sialidase activity is hampered by our poor knowledge on Neu-1 structure and membrane topology. In the absence of crystallographic data, homology models of the mammalian sialidase family have been developed based on the crystal structure of Neu-2 [60], with Neu-1 being predicted to have the typical structure of sialidases: a β-propeller composed of four anti-parallel β-sheets organized in six blades. However, recent studies on Neu-1 [61] and Neu-3 [62], the second plasma membrane sialidase, rather suggest that these sialidases have the characteristics of an integral membrane protein.
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2018, Bioorganic and Medicinal ChemistryCitation Excerpt :Additionally, the model for NEU3 was modified to eliminate unreasonable conformations using Modeller in Chimera. We built a homology model for NEU1 also using Modeller in Chimera and the alignment reported by Magesh, et al.29,42 The alignments for the four isoenzymes are shown in Fig. 2. Residues 1–53 in NEU1 and 290–367 in NEU4 have no homology to NEU2 and were removed before running MD simulations.
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2015, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Because the human NEU2 protein is the only mammalian sialidase of which structure has been solved to date, we selected its crystal structure as a template [13]. Although NEU2 shares lower sequence homology to NEU1 than other vertebrate sialidases, human NEU1 and NEU2 still are significantly similar (28% identity), and catalytically critical residues and motifs are conserved in both [14]. Homology modeling predicted that NEU1 forms a canonical sialidase architecture that is composed of many β-strands and has the catalytic site at the central cavity [15].