Dataset Information

Human Sialidase Neu3 is S-Acylated and Behaves Like an Integral Membrane Protein.

ABSTRACT: Membrane-bound sialidase Neu3 is involved in the catabolism of glycoconjugates, and plays crucial roles in numerous biological processes. Since the mechanism of its association with membranes is still not completely understood, the aim of this work was to provide further information regarding this aspect. Human Neu3 was found to be associated with the plasma membrane and endomembranes, and it was not released from the lipid bilayer under conditions that typically release peripheral membrane proteins. By different experimental approaches, we demonstrated that its C-terminus is exposed to the cytosol while another portion of the protein is exposed to the extracellular space, suggesting that Neu3 possesses the features of a transmembrane protein. However, in silico analysis and homology modeling predicted that the sialidase does not contain any ?-helical transmembrane segment and shares the same ?-propeller fold typical of viral and bacterial sialidases. Additionally, we found that Neu3 is S-acylated. Since this post-translational modification is restricted to the cytosolic side of membranes, this finding strongly supports the idea that Neu3 may contain a cytosolic-exposed domain. Although it remains to be determined exactly how this sialidase crosses the lipid bilayer, this study provides new insights about membrane association and topology of Neu3.

SUBMITTER: Rodriguez-Walker M

PROVIDER: S-EPMC5482835 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Human Sialidase Neu3 is S-Acylated and Behaves Like an Integral Membrane Protein.

Rodriguez-Walker Macarena M Daniotti Jose L JL

Scientific reports 20170623 1

Membrane-bound sialidase Neu3 is involved in the catabolism of glycoconjugates, and plays crucial roles in numerous biological processes. Since the mechanism of its association with membranes is still not completely understood, the aim of this work was to provide further information regarding this aspect. Human Neu3 was found to be associated with the plasma membrane and endomembranes, and it was not released from the lipid bilayer under conditions that typically release peripheral membrane prot ...[more]

PMID: 28646141

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Project description:Neuraminidases (NAs) are critical virulence factors for several microbial pathogens. With a highly conserved catalytic domain, a microbial NA "superfamily" has been proposed. We previously reported that murine polymorphonuclear leukocyte (PMN) sialidase activity was important in leukocyte trafficking to inflamed sites and that antibodies to Clostridium perfringens NA recognized a cell surface molecule(s), presumed to be a sialidase of eukaryotic origin on interleukin-8-stimulated human and murine PMNs. These antibodies also inhibited cell sialidase activity both in vitro and, in the latter instance, in vivo We therefore hypothesized that mammalian sialidases share structural homology and epitopes with microbial NAs. We now report that antibodies to one of the isoforms of C. perfringens NA, as well as anti-influenza virus NA serum, recognize human NEU3 but not NEU1 and that antibodies to C. perfringens NA inhibit NEU3 enzymatic activity. We conclude that the previously described microbial NA superfamily extends to human sialidases. Strategies designed to therapeutically inhibit microbial NA may need to consider potential compromising effects on human sialidases, particularly those expressed in cells of the immune system.IMPORTANCE We previously reported that sialidase activity of human neutrophils plays a critical role in the host inflammatory response. Since the catalytic domains of microbial neuraminidases are highly conserved, we hypothesized that antibodies against Clostridium perfringens neuraminidase might inhibit mammalian sialidase activity. Before the recognition of four mammalian sialidase (Neu) isoforms, we demonstrated that anti-C. perfringens neuraminidase antibodies inhibited human and murine sialidase activity in vivo and in vitro We now show that the antibodies to microbial neuraminidase (C. perfringens and influenza virus) recognize human NEU3, which is important for neural development and cell signaling. Since many microbes that infect mucosal surfaces express neuraminidase, it is possible that the use of sialidase inhibitors (e.g., zanamivir), might also compromise human sialidase activity critical to the human immune response. Alternatively, sialidase inhibitors may prove useful in the treatment of hyperinflammatory conditions.

Project description:BACKGROUND: Large surface loops contained within compact protein structures and not involved in catalytic process have been proposed as preferred regions for protein family evolution. These loops are subjected to lower sequence constraints and can evolve rapidly in novel structural variants. A good model to study this hypothesis is represented by sialidase enzymes. Indeed, the structure of sialidases is a ?-propeller composed by anti-parallel ?-sheets connected by loops that suit well with the rapid evolving loop hypothesis. These features prompted us to extend our studies on this protein family in birds, to get insights on the evolution of this class of glycohydrolases. RESULTS: Gallus gallus (Gg) genome contains one NEU3 gene encoding a protein with a unique 188 amino acid sequence mainly constituted by a peptide motif repeated six times in tandem with no homology with any other known protein sequence. The repeat region is located at the same position as the roughly 80 amino acid loop characteristic of mammalian NEU4. Based on molecular modeling, all these sequences represent a connecting loop between the first two highly conserved ?-strands of the fifth blade of the sialidase ?-propeller. Moreover this loop is highly variable in sequence and size in NEU3 sialidases from other vertebrates. Finally, we found that the general enzymatic properties and subcellular localization of Gg NEU3 are not influenced by the deletion of the repeat sequence. CONCLUSION: In this study we demonstrated that sialidase protein structure contains a surface loop, highly variable both in sequence and size, connecting two conserved ?-sheets and emerging on the opposite site of the catalytic crevice. These data confirm that sialidase family can serve as suitable model for the study of the evolutionary process based on rapid evolving loops, which may had occurred in sialidases. Giving the peculiar organization of the loop region identified in Gg NEU3, this protein can be considered of particular interest in such evolutionary studies and to get deeper insights in sialidase evolution.

Dataset Information

Human Sialidase Neu3 is S-Acylated and Behaves Like an Integral Membrane Protein.

Publications

Human Sialidase Neu3 is S-Acylated and Behaves Like an Integral Membrane Protein.

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