Sialidases in Vertebrates: A Family Of Enzymes Tailored For Several Cell Functions*

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Abstract

This review summarizes the recent research development on vertebrate sialidase biology. Sialic acid-containing compounds play important roles in many physiological processes, including cell proliferation, apoptosis and differentiation, control of cell adhesion, immune surveillance, and clearance of plasma proteins. In this context, sialidases, the glycohydrolases that remove the terminal sialic acid at the non-reducing end of various glycoconjugates, perform an equally pivotal function. Sialidases in higher organisms are differentially expressed in cells and tissues/organs, with particular subcellular distribution and substrate specificity: they are the lysosomal (NEU1), the cytosolic (NEU2), and plasma membrane- and intracellular-associated sialidases (NEU3 and NEU4). The molecular cloning of several mammalian sialidases since 1993 has boosted research in this field. Here we summarize the results obtained since 2002, when the last general review on the molecular biology of mammalian sialidases was written. In those few years many original papers dealing with different aspects of sialidase biology have been published, highlighting the increasing relevance of these enzymes in glycobiology. Attention has also been paid to the trans-sialidases, which transfer sialic acid residues from a donor sialoconjugate to an acceptor asialo substrate. These enzymes are abundantly distributed in trypanosomes and employed to express pathogenicity, also in humans. There are structural similarities and strategic differences at the level of the active site between the mammalian sialidases and trans-sialidases. A proper knowledge of these properties may permit the design of proper anti-pathogen drugs.

Introduction

Sialidases or neuraminidases (EC 3.2.1.18; N-acylneuraminyl glycohydrolases) are a family of exo-glycosidases that catalyze the hydrolytic cleavage of non-reducing sialic acid residues1 ketosidically linked to the saccharide chains of glycoproteins and glycolipids (gangliosides) as well as to oligo- and poly-saccharides. They are widely distributed in nature, from viruses and microorganisms (such as bacteria, protozoa, and fungi) to vertebrates, but are absent in plants, insects, and yeast.2 The molecular cloning of several mammalian sialidases since 1993 and the great development that followed early afterward has been summarized in two reviews.3, 4

In vertebrates, mammalian sialidases and their target substrates have been implicated in crucial biological processes, including the regulation of cell proliferation/-differentiation, clearance of plasma proteins, control of cell adhesion, metabolism of gangliosides and glycoproteins, immunocyte function, modification of receptors, and the developmental modeling of myelin. The pivotal and diverse functions of these enzymes, many of which have not yet been discovered, probably account for the existence of four mammalian sialidases, encoded by different genes and defined as lysosomal (NEU1), cytosolic (NEU2), plasma-membrane (NEU3), and mitochondrial/-lysosomal/-intracellular membranes (NEU4). These enzymes differ in their subcellular localizations, pH optima, kinetic properties, responses to ions and detergents, and substrate specificities. There appears to be little overlap in function of the individual sialidases, even though they share a common mechanism of action.

Here we survey the data published since 2002 on vertebrate (mainly mammalian) sialidase biology. The subject is organized as follows: (i) a description of the four different sialidase forms and their functional implication; (ii) an introductory section on some in silico analysis of the sialidase gene family; (iii) a description of recent findings in lower vertebrates (teleosts); and (iv) a comparison between vertebrate sialidases and trans-sialidases, a peculiar group of sialidases that are able to catalyze the transfer of sialic acid from a donor to an acceptor substrate. These enzymes are present in specific microorganisms, and have been implicated in some mammalian infectious diseases. Finally, based on recent discoveries, we attempt to provide a comprehensive picture of the increasing complexity of the biological roles performed by this enzyme family.

Section snippets

Background

Lysosomal sialidase (NEU1) initiates the hydrolysis of sialyl-glycoconjugates by removing their terminal sialic acid residues. The enzyme is present in almost all vertebrate tissues and cell types, and functions in a multienzyme complex containing at least two other hydrolases: the glycosidase β-galactosidase (β-GAL) and the serine carboxypeptidase protective protein/cathepsin A (PPCA). Both the human and murine neuraminidase genes (NEU1 and Neu1) map within the major histocompatibility locus.

General Properties of NEU2

NEU2 was the first vertebrate sialidase characterized using the recombinant DNA techniques. The enzyme was first cloned56 from rat skeletal muscle in 1993, and subsequently a highly similar protein was cloned from CHO cells.57 Overexpression of the human cytosolic sialidase NEU2 homologue in E. coli58 and the purification of the enzyme to homogeneity permitted its detailed kinetic characterization.59 The pH optimum was 5.6, V/E and V/t had the expected features, and the V/[S] relationships

General Properties of NEU3

The membrane-associated sialidase NEU3 was first cloned from bovine brain in 1999, as a plasma membrane-associated sialidase specific for gangliosides.49 NEU3 homologues subsequently characterized at the molecular level from various animal species were found to be ubiquitously expressed and were confirmed, at least by in vitro enzyme assays, to be highly specific for gangliosides.4, 91, 92 The enzyme shows a high level of sequence identity with NEU2 and, together with NEU4 and NEU1, shares the

General Properties of NEU4

The fourth member of the mammalian sialidase family, NEU4, has been identified by searching sequence databases for entries showing homologies to human cytosolic sialidase NEU2.4, 130, 131 One peculiar feature of this enzyme is the presence of a long stretch of about 80 amino acids (aa 294–373) that appears unique among mammalian sialidases,131 and teleosts.132 Noteworthy is the fact that human NEU4 is present in two isoforms, differing in the presence or absence of a 12 amino acid stretch at

Sialidases and Cancer

As discussed in previous sections of this survey, during the past few years many of the scientific papers published on mammalian sialidases pointed out the potential involvement of sialidases in the occurrence of various kinds of tumors. A comprehensive picture of the results obtained thus far has already been presented in a series of reviews.46, 141, 142, 143, 144 Therefore, here we give only an overview of the most recent contributions. Briefly, human NEU3 is up-regulated in human colon cancer

Sialidases and Immunity

There is evidence for an involvement of sialidase(s) in the complex cellular network of immunity in higher organisms. It has been shown that, during bone marrow differentiation, myeloid precursors highly express sialoglycoconjugates. The latter are remodeled during maturation of the precursors with the concurrent release of sialic acid and gain in plasticity and mobility required to reach the peripheral circulation. Once in the peripheral circulation, the activation of neutrophils is associated

Further Evidence for Possible Functional Implications of Sialidases

Several biological processes are known to involve sialidase activity, but attribution of this activity to any of the four mammalian enzymes is still lacking. A direct link between activation of the TrkA tyrosine protein kinase receptor for nerve growth factor (NGF) and sialidase(s) has been described in PC12 rat pheochromocytoma cell-lines and primary cortical neurons.164 Upon NGF stimulation, a sialidase activity is induced that specifically removes sialic acid residues that are α–(2→3) linked

In silico Analysis of Sialidase Gene Expression Patterns

A few studies have been performed on sialidases that describe the expression of these genes at the transcriptional level. Reports presenting the identification of NEU genes in man or in the mouse mostly included RNA expression data based either on Northern Blot or RT-PCR analyses (as reviewed in Refs. 3 and 4, as well as in the original papers cited in the different sections of this article). More recent papers provide detailed expression studies of murine sialidases carried out by quantitative

Amino Acid Sequence Variants in Human Sialidases

Almost all (99.9%) nucleotide bases are identical in all humans, with the remaining 0.1% accounting for about 3 millions single-base DNA differences/polymorphisms (SNPs). While the vast majority of SNPs fall within DNA stretches between genes or in non-coding regions of genes, a more limited number of SNPs are found within coding sequences. These SNPs may fall into two categories: (i) synonymous SNPs (sSNPs) that, due to degeneracy of the genetic code, do not result in an amino acid change in

Sialidases in Teleosts

Until recently, little was known about sialidases in vertebrates other than mammals. A 2007 study describes the identification and characterization of the sialidase gene family in the zebrafish Danio rerio.132 While in mammals four genes are known to encode sialidases, in zebrafish the picture is more complex, with seven genes homologous with human sialidases (Fig. 9). In the fish a single orthologue exists for the mammalian NEU1 gene, which was named neu1. This finding was supported by the

Trans-sialidases: What Distinguishes Them from Sialidases?

A remarkable group of sialidases are the trans-sialidases (TS), found mainly in microorganisms. The groups best investigated are those from such pathogenic trypanosomes as the African Trypanosoma brucei,190T. congolense,191 and the American T. cruzi.192 The TS are glycosylphosphatidylinositol (GPI)-anchored in the cell surface of the parasites. In contrast to the “classical” sialidases, trypanosomal TS (EC 3.2.1.18) catalyze the transfer of preferably α-(2→3)-linked sialic acids from donor

Final Remarks

The cells and tissues of vertebrates, particularly mammals, contain a variety of sialic acid-carrying compounds (sialoglycoproteins, sialoglycolipids, sialo-oligosaccharides) that are mainly associated with the plasma and intracellular membranes but are also present in the cytosol and body fluids. The removal of sialic residues from sialoglycoconjugates is catalyzed by sialidases. Four different sialidases occur in vertebrates (NEU1, NEU2, NEU3, and NEU4), and each sialidase is encoded by a

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