The identification of lysosomal ganglioside sialidase in human cells

In this report we present evidence for the existence of a lysosomal ganglioside sialidase. The sialidase activity was solubilized by sonication and stimulated by cholate. The absence of ganglioside sialidase activity in sialidosis patients indicates that lysosomal sialidase is active towards gangliosides and glycoproteins. The plasma membranes were associated with two types of ganglioside sialidase activities, one was enhanced by cholate while the other was partially inhibited by this detergent.     

Identification of lysosomal sialidase NEU1 and plasma membrane sialidase NEU3 in human erythrocytes

The sialylation level of molecules, sialoglycoproteins and gangliosides, protruding from plasma membranes regulates multiple facets of erythrocyte function, from interaction with endothelium to cell lifespan. Our results demonstrate that: (a) Both sialidases NEU1 and NEU3 are present on erythrocyte plasma membrane; (b) NEU1 is kept on the plasma membrane in absence of the protective protein/cathepsin A (PPCA); (c) NEU1 and NEU3 are retained on the plasma membrane, as peripheral proteins, associated to the external leaflet and released by alkaline treatments; (d) NEU1 and NEU3 are segregated in Triton X‐100 detergent‐resistant membrane domains (DRMs); (e) NEU3 shows activity also at neutral pH; and (f) NEU1 and NEU3 are progressively lost during erythrocyte life. Interestingly, sialidase activity released from erythrocyte membranes after an alkaline treatment preserves its functionality and recognizes sialoglycoproteins and gangliosides. On the other hand, the weak anchorage of sialidases to the plasma membrane and their loss during erythrocyte life could be a tool to preserve the cellular sialic acid content in order to avoid the early ageing of erythrocyte and processes of cell aggregation in the capillaries. J. Cell. Biochem. 114: 204–211, 2012. © 2012 Wiley Periodicals, Inc.     

Methylumbelliferyl-N-acetylneuraminic acid sialidase in human liver

Human liver sialidase was measured using methylumbelliferyl-N-acetylneuraminic acid as a substrate.The enzyme activity was linear for only 20 min and linearity was not improved by adding albumin, CaCl₂, dithiothreitol, or Ep-459.The optimal pH was 4.5 and the apparent K_m value, approximately 0.090 mm.Without substrate addition, the enzyme was unstable at temperatures between 0 and 37°C, retaining only 35 and 5% of its activity, respectively, after $\text{8}\text{1}\text{2}\text{hr}$, but was protected by albumin at 5 mg/ml.The enzyme was more ptable when either total liver or liver homogenate was kept frozen at −20°C.Liver sialidase also retained about 70% of its activity after mechanical homogenization for 5 min.Potential inhibitors, notably, p-aminooxanilic acid, fetuin III, Triton X-100, mucin, sialyllactose, colominic acid, sodium taurocholate, N-acetylneuraminic acid, and methoxyphenyl-N-acetylneuraminic acid, were tested. Sialyllactose, methoxyphenyl-N-acetylneuraminic acid, fetuin, N-acetylneuraminic acid, and colominic acid were competitive inhibitors with K_i values of 1.12, 0.37, 0.20, 0.78, and 0.22 mm, respectively.The 0.11 m solutions of NaCl, LiCl, and KCl inhibited 20–30%, and CaCl₂ about 60%, of the enzyme activity.     

Rat-liver lysosomal sialidase. Solubilization, substrate specificity and comparison with the cytosolic sialidase

Purified liver lysosomes, prepared from rats previously injected with Triton WR-1339, exhibited sialidase activity towards sialyllactose, fetuin, submaxillary mucin (bovine) and gangliosides, and could be disrupted hypotonically with little loss in these activities. After centrifugation, the activities with sialyllactose and fetuin were largely recovered in the supernatant, demonstrating that they were originally in the intralysosomal space. The activities towards submaxillary mucin and gangliosides, on the other hand, remained in the pellet. In the supernatant, activity with fetuin or orosomucoid was markedly reduced by protease inhibitors, suggesting that proteolysis of these glycoproteins may be prerequisite to sialidase activity. The intralysosomal sialidase was solubilized from the mitochondrial-lysosomal fraction of rat liver and partially purified by Sephadex G-200, or Sephadex G-200 followed by CM-cellulose. The enzyme was maximally active at pH 4.7 with sialyllactose as substrate and had a minimum relative molecular mass of 60 000 +/- 5000 by gel filtration; it hydrolyzed a variety of sialooligosaccharides , those containing (alpha 2----3)sialyl linkages being better substrates than those with (alpha 2----6)sialyl linkages. The enzyme failed to attack submaxillary mucin and gangliosides. It was also inactive towards fetuin, orosomucoid and transferrin but capable of hydrolyzing glycopeptides from pronase digest of fetuin. In contrast to the intralysosomal sialidase, the sialidase partially purified from rat liver cytosol by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose and CM-cellulose hydrolyzed fetuin and orosomucoid to the extent about half that for sialyllactose. The enzyme was maximally active at pH 5.8 and had a relative molecular mass of approximately 60 000. It also hydrolyzed gangliosides but not submaxillary mucin.     

Properties of lysosomal beta-hexosaminidase accumulated in Niemann-Pick mouse liver

Various lysosomal acid hydrolases from tissues of Niemann-Pick mice, a mutant strain of C57BL/KsJ mice (spm/spm), were examined and compared to those from control mice. Activities of beta-hexosaminidase, beta-galactosidase, acid phosphatase, and cathepsin L were elevated in the liver and spleen of the affected mice, whereas no significant changes in beta-glucosidase and acid alpha-glucosidase were observed. Alpha-Mannosidase and neutral alpha-glucosidase activities were rather decreased in the affected mouse liver. The level of beta-hexosaminidase in the Niemann-Pick mice was raised sixfold in the liver and two- to threefold in the spleen and brain, whereas its total activity was decreased in the kidney. Sixty to ninety percent of total activity of lysosomal hydrolases was solubilized with 0.1% Triton X-100 in control mice, but most of the beta-hexosaminidase activity of the Niemann-Pick mice remained associated with the membrane fraction of liver lysosomes. The beta-hexosaminidase of the Niemann-Pick mice was appreciably stable when heated at 55 degrees C, while hydrolases of the affected mice and all of the enzymes tested in control mice were heat labile. The relative content of two beta-hexosaminidase fractions separated by DEAE-cellulose column chromatography was 8% for beta-hexosaminidase I and 92% for beta-hexosaminidase II in the case of the control mouse liver. The isozyme pattern of hexosaminidases in Niemann-Pick mice was similar to that of control enzymes. However, the beta-hexosaminidase II accumulated in Niemann-Pick mouse liver was different from that of the control in optimum pH, Km values and thermostability.     

Design, synthesis, and biological evaluation of human sialidase inhibitors. Part 1: selective inhibitors of lysosomal sialidase (NEU1)

We here report the design and synthesis of selective human lysosomal sialidase (NEU1) inhibitors. A series of amide-linked C9 modified DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) analogues were synthesized and their inhibitory activities against all four human sialidases (NEU1-NEU4) were determined. Structure-based approach was used to investigate the basis of selectivity of the compounds with experimentally observed activity. Results from the present study are found to be informative in a qualitative manner for the further design of isoform selective human sialidase inhibitors for therapeutic value.     

Properties of binding sites for chloroquine in liver lysosomal membranes

Chloroquine (CQ) is an antimalarial and antirheumatic drug that accumulates in lysosomes. We purified liver lysosomal membranes of tritosomes from albino mice injected with Triton WR 1339. The membranes were used for the binding assay with CQ in 0.01 M Tris-HCl buffer (pH 7.4). This binding was saturable, with a KD value of 6.2 microM. To understand the nature of CQ affinity, the binding was done under conditions that alter membrane structure and composition. Changes in pH, high ionic strength, and bivalent cations reversibly decreased the binding, while the effect of non-ionic detergents was partially reversed. The cationic detergent Hyamine strongly decreased the binding, and its effect was trypsin and neuraminidase had no effect. The results indicate the existence of binding sites for CQ in liver lysosomal membranes, which were strongly affected by changes of charge in the molecules involved in the binding. The treatment with the enzymes suggests that loss of polar groups of phospholipids increases the affinity of CQ by exposing protein sites located deep in the membrane, or by permiting a closer interaction between the drug and membrane lipids. CQ lysosomotropism and other effects of CQ on the lysosomal apparatus studied by other authors may be due not only to its accumulation inside the acid milieu of the lysosomes, in the same manner as other weak bases, but also to the affinity of CQ for binding sites in the lysosomal membrane.     

Mutations in sialidosis impair sialidase binding to the lysosomal multienzyme complex

Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the catabolism of sialoglycoconjugates. The disease is associated with progressive impaired vision, macular cherry-red spots, and myoclonus (sialidosis type I) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation, and hepatosplenomegaly (sialidosis type II). We analyzed the effect of the missense mutations G68V, S182G, G227R, F260Y, L270F, A298V, G328S, and L363P, which are identified in the sialidosis type I and sialidosis type II patients, on the activity, stability, and intracellular distribution of sialidase. We found that three mutations, F260Y, L270F, and A298V, which are clustered in the same region on the surface of the sialidase molecule, dramatically reduced the enzyme activity and caused a rapid intralysosomal degradation of the expressed protein. We suggested that this region might be involved in sialidase binding with lysosomal cathepsin A and/or beta-galactosidase in the multienzyme lysosomal complex required for the expression of sialidase activity. Transgenic expression of mutants followed by density gradient centrifugation of cellular extracts confirmed this hypothesis and showed that sialidase deficiency in some sialidosis patients results from disruption of the lysosomal multienzyme complex.     

Degradation of gangliosides by the lysosomal sialidase requires an activator protein.

Lysosomal sialidase, which was formerly believed to degrade only water-soluble substrates but not glycolipids, cleaves ganglioside substrates II3NeuNAc-LacCer, IV3NeuNAc, II3NeuNAc-GgOse4Cer, IV3 NeuNAc, II3(NeuNAc)2-GgOse4Cer when these are dispersed either with an appropriate detergent (taurodeoxycholate) or with the sulfatide activator protein, a physiologic lipid solubilizer required for the lysosomal hydrolysis of other glycolipids by water-soluble hydrolases. In the presence of the activator protein, time and protein dependence were linear within wide limits, while the detergent rapidly inactivated the enzyme. The disialo group of the b-series gangliosides was only poorly attacked by the enzyme when the lipids were dispersed with the activator protein, whereas in the presence of the detergent, they were hydrolyzed as fast as terminal sialic acid residues. With the appropriate assay method, significant ganglioside sialidase activity could be demonstrated in the secondary lysosome fraction of normal skin fibroblasts but not of sialidosis fibroblasts. Our results support the notion that there is only one lysosomal sialidase, which degrades both the water-soluble and the membrane-bound sialyl glycoconjugates.     

Fractionation, biochemical characterization and lysosomal phospholipases of human liver

Human liver was homogenised and fractionated by differential centrifugation, and the subcellular fractions were characterised biochemically. Absolute values and distribution patterns of protein and marker enzyme activities obtained from human liver have also been compared with those from rat liver. In addition, acid phospholipase activities have been studied in human liver. On the basis of product formation from stereo-specifically radiolabeled phosphatidylethanolamine substrates, lysosomal phospholipases A1 and A2 with optimal activities at pH 4.7 have been identified in human liver. Acid phospholipase C and lysophospholipase activities, however, were not found in human liver. Cationic amphiphilic drugs inhibited the activities of the acid phospholipases A in human and rat liver lysosomes to about the same extent.     

Neu4, a novel human lysosomal lumen sialidase, confers normal phenotype to sialidosis and galactosialidosis cells

Three different mammalian sialidases have been described as follows: lysosomal (Neu1, gene NEU1), cytoplasmic (Neu2, gene NEU2), and plasma membrane (Neu3, gene NEU3). Because of mutations in the NEU1 gene, the inherited deficiency of Neu1 in humans causes the severe multisystemic neurodegenerative disorder sialidosis. Galactosialidosis, a clinically similar disorder, is caused by the secondary Neu1 deficiency because of genetic defects in cathepsin A that form a complex with Neu1 and activate it. In this study we describe a novel lysosomal lumen sialidase encoded by the NEU4 gene on human chromosome 2. We demonstrate that Neu4 is ubiquitously expressed in human tissues and has broad substrate specificity by being active against sialylated oligosaccharides, glycoproteins, and gangliosides. In contrast to Neu1, Neu4 is targeted to lysosomes by the mannose 6-phosphate receptor and does not require association with other proteins for enzymatic activity. Expression of Neu4 in the cells of sialidosis and galactosialidosis patients results in clearance of storage materials from lysosomes suggesting that Neu4 may be useful for developing new therapies for these conditions.     

Characteristics of human intestinal acid sialidase

With methylumbelliferyl-N-acetyl-neuraminic acid (MU-NANA) as substrate, acid sialidase was determined in intestinal biopsies of children. The enzyme has an acid pH optimum, a Km value of 4 mmol/l and a pronounced thermal lability which can be partially prevented by the addition of albumin. N-acetyl-neuraminic acid (NANA) and derivatives as well as other glycoprotein and oligosaccharide sialidase substrates inhibit sialidase whereas gangliosides have no effect. This could be an indication that intestinal MU-NANA sialidase is different from ganglioside sialidase as has been reported for many other tissues.     

Inhibition of mouse liver sialidase by plant flavonoids

Flavonoids (103 species) were tested for inhibitory activity against mouse liver sialidase using sodium p-nitrophenyl-N-acetyl-alpha-D-neuraminate (PNP-NeuAc) as substrate. Isoscutellarein-8-O-glucuronide from the leaf of Scutellaria baicalensis showed most potent activity (IC50, 40 microM), and this flavone appeared to be a non-competitive inhibitor of the enzyme. This flavone inhibited the lysosomal solubilized sialidase against PNP-NeuAc and sialyllactose effectively, but not microsomal enzyme against gangliosides and colominic acid, whereas, negligible or weak inhibitory activities were observed for influenza virus sialidase, beta-galactosidase, alpha-mannosidase, and alpha-glucosidase tested. These results indicate that this flavone may be useful to elucidate the function of the lysosomal solubilized sialidase.     

Purification and characterization of cytosolic sialidase from rat liver

Sialidase has been purified from rat liver cytosol 83,000-fold by sequential chromatography on DEAE-cellulose, CM-cellulose, Blue-Sepharose, Sephadex G-200, and heparin-Sepharose. When subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the purified cytosolic sialidase moved as a single protein band with Mr = 43,000, a value similar to that obtained by sucrose density gradient centrifugation. The purified enzyme was active toward all of the sialooligosaccharides, sialoglycoproteins, and gangliosides tested except for submaxillary mucins and GM1 and GM2 gangliosides. Those substrates possessing alpha 2----3 sialyl linkage were hydrolyzed much faster than those with alpha 2----6 or alpha 2----8 linkage. The optimum pH was 6.5 for sialyllactose and 6.0 for orosomucoid and mixed brain gangliosides. The activity toward sialyllactose was lost progressively with the progress of purification but restored by addition of proteins such as bovine serum albumin. In contrast, neither reduction by purification nor restoration by albumin was observed for the activity toward orosomucoid. When mixed gangliosides were the substrate, bile acids were required for activity and this requirement became almost absolute after the enzyme had been purified extensively. Intracellular distribution study showed that about 15% of the neutral sialidase activity was in the microsomes. The enzyme could be released by 0.5 M NaCl; the released enzyme was indistinguishable from the cytosolic sialidase in properties.     

Properties of sialidase isolated from Actinomyces viscosus DSM 43798

The cell-bound sialidase of Actinomyces viscosus DSM 43798 was solubilized by mechanical cell disruption and lysozyme treatment. The enzyme was enriched 30,000-fold by cation-exchange chromatography, gel-filtration, and FPLC ion-exchange chromatography, thus obtaining 10 micrograms sialidase protein from 26 g wet cells with a specific activity of 680 U/mg protein. Since sialidase activity was also found in the culture medium, this enzyme was isolated as well, requiring the additional application of FPLC gel-filtration. Both sialidase preparations were apparently homogenous on SDS-PAGE and have similar properties. The substrate specificity of the A. viscosus sialidase was tested with 16 sialoglycoconjugates: The enzyme showed a higher activity with serum glycoproteins than with gangliosides, mucins or sialyllactoses. 4-O-Acetylated N-acetylneuraminic acid was not cleaved from equine submandibular gland mucins or serum glycoproteins in contrast to N-acetyl- and N-glycoloylneuraminic acid. 9-O-Acetyl-N-acetylneuraminic acid was released from bovine submandibular gland mucin, as confirmed by TLC. The sialidase hydrolyses alpha(2----6)-linkages more rapidly than alpha(2----8)- and alpha(2----3)-bonds. Cations, except Hg2+, or chelating agents have no influence on enzyme activity. The sialidase has a relatively high molecular mass of 150 kDa, but consists of only one unit. The enzyme is labile towards freezing and thawing, but can be stored at 4 degrees C in 0.1 M acetate buffer, pH 5.     

Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail

Sialidase (neuraminidase), encoded by the neu-1 gene in the major histocompatibility complex locus catalyzes the intralysosomal degradation of sialylated glycoconjugates. Inherited deficiency of sialidase results in sialidosis or galactosialidosis, both severe metabolic disorders associated with lysosomal storage of oligosaccharides and glycopeptides. Sialidase also plays an important role in cellular signaling and is specifically required for the production of cytokine interleukin-4 by activated T lymphocytes. In these cells, neu-1-encoded sialidase activity is increased on the cell surface, suggesting that a specific mechanism regulates sorting of this enzyme to the plasma membrane. We investigated that mechanism by first showing that sialidase contains the internalization signal found in lysosomal membrane proteins targeted to endosomes via clathrin-coated pits. The signal consists of a C-terminal tetrapeptide (412)YGTL(415), with Tyr(412) and Leu(415) essential for endocytosis of the enzyme. We further demonstrated that redistribution of sialidase from lysosomes to the cell surface of activated lymphocytes is accompanied by increased reactivity of the enzyme with anti-phosphotyrosine antibodies. We speculate that phosphorylation of Tyr(412) results in inhibition of sialidase internalization in activated lymphocytes.     

The molecular heterogeneity of purified human liver lysosomal alpha-glucosidase (acid alpha-glucosidase).

An α-glucosidase active at acid pH and presumably lysosomal in origin has been purified from human liver removed at autopsy. The enzyme has both α-1,4-glucosidase and α-1,6-glucosidase activities. The K_m of maltose for the enzyme is 8.9 mm at the optimal pH of 4.0. The K_m of glycogen at the optimal pH of 4.5 is 2.5% (9.62 mm outerchain end groups). Isomaltose has a K_m of 33 mm when α-1,6-glucosidase activity is tested at pH 4.2. The enzyme exists in several active charge isomer forms which have pI values between 4.4 and 4.7. These forms do not differ in their specific activities. Electrophoresis in polyacrylamide gels under denaturing conditions indicates that the protein is composed of two subunits whose approximate molecular weights are 88,000 and 76,000. An estimated molecular weight of 110,000 was obtained by nondenaturing polyacrylamide gel electrophoresis. When the protein was chromatographed on Bio-Gel P-200 it was separated into two partially resolved active peaks which did not differ in their charge isomer constitution or in subunit molecular weights. One peak gave a strongly positive reaction for carbohydrate by the periodic acid-Schiff method and the other did not. Both had the same specific activity. The enzyme was antigenic in rabbits, and the antibodies so obtained could totally inhibit the hydrolytic action of the enzyme on glycogen but were markedly less effective in inhibiting activity toward isomaltose and especially toward maltose. Using these antibodies it was found that liver and skeletal muscle samples from patients with the “infantile” form or with the “adult” form of Type II glycogen storage disease, all of whom lack the lysosomal α-glucosidase, do not have altered, enzymatically inactive proteins which are immunologically cross-reactive with antibodies for the α-glucosidase of normal human liver.     

Cyclic changes of sialidase in human cervical mucus

• 1.1. Sialidase activity is detectable in whole cervical mucus of normal women throughout the menstrual cycle and presents cyclic variations towards endogenous and exogenous substrates.
• 2.2. The level of sialic acid bound to the mucus increases progressively till mid-cycle and declines in the post-ovulatory phase.
• 3.3. The sialidase of the mucus probably derives from different sources and its role remains speculative.

     

Purification and characterization of a membrane-bound sialidase from pig liver

A membrane-bound sialidase in pig liver microsomes was solubilized with a nonionic detergent, IGEPAL CA630, and purified to homogeneity by sequential chromatographies on SP-Toyopearl, Butyl-Toyopearl (1st), SuperQ-Toyopearl, Hydroxyapatite, Butyl-Toyopearl (2nd), GM1-Cellulofine affinity, and sialic acid-Cellulofine affinity columns. The molecular weight of the purified enzyme was estimated to be 57 kDa on SDS-PAGE. The pH optimum was 4.8 for the activity measured using 4-methylumbelliferyl-alpha-N-acetylneuraminic acid (4MU-Neu5Ac) as the substrate. The enzyme activity was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, iodoacetamide and p-chloromercuribenzoic acid. While the enzyme could effectively hydrolyze 4MU-Neu5Ac, it failed to significantly cleave a sialic acid residue(s) from sialyllactose, glycoproteins or gangliosides at pH 4.8. These results suggest that the purified enzyme is a novel sialidase with a substrate specificity distinct from those of known membrane-bound sialidases in mammalian tissues.