Identification and expression of NEU3, a novel human sialidase associated to the plasma membrane

Abstract

Several mammalian sialidases have been described so far, suggesting the existence of numerous polypeptides with different tissue distributions, subcellular localizations and substrate specificities. Among these enzymes, plasma-membrane-associated sialidase(s) have a pivotal role in modulating the ganglioside content of the lipid bilayer, suggesting their involvement in the complex mechanisms governing cell-surface biological functions. Here we describe the identification and expression of a human plasma-membrane-associated sialidase, NEU3, isolated starting from an expressed sequence tag (EST) clone. The cDNA for this sialidase encodes a 428-residue protein containing a putative transmembrane helix, a YRIP (single-letter amino acid codes) motif and three Asp boxes characteristic of sialidases. The polypeptide shows high sequence identity (78%) with the membrane-associated sialidase recently purified and cloned from Bos taurus. Northern blot analysis showed a wide pattern of expression of the gene, in both adult and fetal human tissues. Transient expression in COS7 cells permitted the detection of a sialidase activity with high activity towards ganglioside substrates at a pH optimum of 3.8. Immunofluorescence staining of the transfected COS7 cells demonstrated the protein's localization in the plasma membrane.

Modification of sialidase levels and sialoglycoconjugate pattern during erythroid and erytroleukemic cell differentiation

“…The increase of GD1a could be easily associated to NEU3 down-regulation, because of its high specificity toward ganglioside substrates. [19,25,57] In addition, the glycoproteins content was greatly increased and modified, giving rise to some sialylated proteins typical of the erythroid precursors. According to this data, the total sialic acid content showed a 2.5-fold increase during differentiation (Table 1).…”

“…Lysosomal sialidase (Neu1) has a catabolic role in desialylating glycoproteins and glycolipids in the lysosome [18]. Plasma membraneassociated sialidase (Neu3) is localized mainly on the cell surface [19], and its association with membrane microdomains has been demonstrated [20,21]. Neu3 acts preferentially on ganglioside substrates, and therefore modulates the sialic acid content on the cell surface [22].…”

Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR Gαi proteins and matrix metalloproteinase-9

“…Their data were based on COS-7 cells transfected with MmNeu3-HA and the cell-surface proteins were selected using biotinylated and isolated by avidin-biotin-affinity chromatography, solubilizing Neu3 from lipid bilayer by carbonate extraction or by Triton X-114 phase separation. Immunofluorescence localization experiments have been reported to show that Neu3 is localized at the plasma membrane and intracellularly in fixed, permeabilized COS-7 transfected with MmNeu3-HA [36,48] or with COS-7 cells transfected pCDNAI-NEU3 [49]. Using Western blot analyses of cell lysates from THP-1 monocytes during differentiation to macrophages, Liang et al have shown that up-regulation of Neu1 and down-regulation of Neu3, whereas Neu3 mRNA decreased by 2-3-fold as revealed by RT-PCR data [22].…”

Where catabolism meets signalling: neuraminidase 1 as a modulator of cell receptors

“…To date 4 members of the neuraminidase family (neuraminidase-1 (NEU1) [19][20][21]; neuraminidase-2 (NEU2) [22,23]; neuraminidase-3 (NEU3, also known as ganglioside sialidase) [24][25][26], and neuraminidase-4 (NEU4) [27,28]) with different, although partially overlapping tissue expression, intracellular localization and substrate specificity, have been identified in mammals. NEU1 was initially localized to the lysosomes [19,21], NEU2 to the cytosol [23,30,31] and NEU3 to caveolae microdomains of plasma membranes [32] as well as the endosomal and lysosomal membranes [33].…”