Human Sialidase Research Articles

Initial characterization of human thymocyte sialidase activity: Evidence that this enzymatic system is not altered during the course of T-cell maturation

1. 1. The sialidase activity of human thymocyte was examined by a fluorogenic assay. 2. 2. These studies revealed that human thymocyte sialidase activity is essentially acid-active and membrane-bound since 59.6% and 33% of the total activity was recovered in the lysosome-enriched and microsomal fractions, respectively. 3. 3. A weak activity was also detected in the cytosolic fraction. 4. 4. However, the acidic optimum pH of this soluble sialidase was at variance with the general concept of mammalian soluble sialidases which are known to be optimally active at more neutral pH. 5. 5. This acidic soluble sialidase seems to be a general characteristic of the human T-cell lineage since examination of mature circulating T-cells revealed that they contain a soluble sialidase activity similar to that observed in thymocytes. 6. 6. Analysis of mature and immature thymocyte subpopulation obtained by differential PNA agglutination indicated that this enzymatic system was not altered during the course of thymic maturation. 7. 7. These results suggest that unlike in T-cell activation where changes in the level of sialidase activity were shown to influence the extent of cell surface sialylation and thereby the cell physiology, this enzymatic system seems not to be involved in the fluctuation of cell surface sialic acid content observed during thymic maturation.

Further Evidence that Human Lysosomal Sialidase is not Derived from Prosaposin. Prosaposin Biosynthesis and Ganglioside Sialidase Studies in Prosaposin- and Sialidase-Deficient Fibroblast Lines

Human lysosomal sialidase has been considered by Potier et al. (Potier M., Lamontagne S., Michaud L., Tranchemontagne J. (1990) Biochem Biophys Res Commun 173, 449-456) to be a processing product of prosaposin, the common precursor of the saposin proteins A, B, C, and D that function as activators in the lysosomal degradation of sphingolipids. We tested this hypothesis on cultured fibroblasts of patients with prosaposin deficiency, a neurolipidosis caused by a complete lack of synthesis of the prosaposin protein, by determining their lysosomal and, for comparison, their plasma membrane sialidase activities. Using both the natural substrate ganglioside GM3 and the synthetic compound 4-methylumbelliferyl neuraminate, we found the lysosomal sialidase activity in the prosaposin-deficient cells to be in the normal control range; normal values were also found for the plasma membrane sialidase. In fibroblasts from patients with a genetic deficiency of the lysosomal sialidase (sialidosis), on the other hand, biosynthesis and processing of prosaposin were unimpaired. Our findings therefore show no precursor/product relationship between prosaposin and the lysosomal or the plasma membrane sialidase.

Human erythrocyte sialidase is linked to the plasma membrane by a glycosylphosphatidylinositol anchor and partly located on the outer surface

Treatment of human erythrocyte ghosts with phosphatidylinositol-phospholipase C (PIPLC) from Bacillus cereus liberated the ghost-linked sialidase. Maximal release of sialidase (about 70% of total) was achieved by incubating ghosts at 37 degrees C for 60 min, at pH 6.0, with PIPLC (PIPLC total units/ghost protein ratio, 4.5 each time) added at the beginning of incubation and every 15 min (four subsequent additions). Liberated sialidase was fully resistant to at least four cycles of rapid freezing and thawing and to storage at 4 degrees C for at least 48 h. The liberated enzyme had an optimal activity at pH 4.2, degraded ganglioside GD1a better than methylumbelliferyl N-acetylneuraminic acid (about fourfold), and gave a Km value of 2.56 x 10(-4) M and an apparent Vmax of 2.22 mU per mg protein on GD1a. Treatment of intact erythrocytes with PIPLC (PIPLC total units/erythrocyte protein ratio, 8), under conditions where haemolysis was practically negligible, caused liberation of 10-12% of membrane linked sialidase, indicating that the enzyme is, at least in part, located on the outer surface of the erythrocyte membrane. It is concluded that the erythrocyte membrane sialidase is anchored by a glycosylphosphatidylinositol structure sensitive to PIPLC action, and is partly located on the outer surface.

Human placental sialidase complex: Characterization of the 60 kDa protein that cross-reacts with anti-saposin antibodies

Sialidase isolated from human placenta is associated with several proteins including acid β-galactosidase, carboxypeptidase, N-acetyl-α-galactosaminidase, and others. These proteins are thought to form an aggregated complex during isolation of sialidase. One of the proteins of 60 kDa was recently identified by Potier et al. (Biochem. Biophys. Res. Comm. 173, 449–456, 1990) as a sialidase protein; this protein also cross-reacted with anti-prosaposin antibodies. We have isolated this protein and from the following evidence identified it as a heavy chain component of immunoglobulin G and not sialidase or a derivative of prosaposin. On gel filtration HPLC, sialidase activity and the 60 kDa protein were clearly separated from one another. The 60 kDa protein cross-reacted not only with antibodies raised against human saposins A, C, and D, but also with second antibody (goat anti-rabbit immunoglobulin G antibody) alone. This 60 kDa protein strongly cross-reacted with anti-human immunoglobulin G antibodies. The sequence of the initial 15 amino acids from the N-terminus of the 60 kDa protein was identical to the sequence of an immunoglobulin G heavy chain protein Tie (γ1).

Molecular cloning of a full-length cDNA for human α-N-acetylgalactosaminidase (α-galactosidase B)

In the process of molecular cloning of cDNA for proteins associated with a purified human placental sialidase fraction, we discovered one of the proteins with apparent molecular weight of 46 kDa is in reality α-N-acetylgalactosaminidase. The full lengh cDNA, pcD-HS1204, codes for 358 amino acids with the first 17 residues representing a putative signal peptide. The predicted amino acid sequence shows striking homology with human α-galactosidase A and yeast α-galactosidase. The substrate specificities as well as the behavior of the 46 kDa protein on hydroxylapatite chromatography confirmed that the 46 kDa protein is in reality α-N-acetylgalactosaminidase.

Human Placental Sialidase: Further Purification and Characterization

An acid sialidase [EC 3.2.1.18] has been purified from human placenta by means of successive procedures including extraction, Con A-Sepharose adsorption, ammonium sulfate precipitation, activation, p-aminophenyl thio-beta-D-galactoside-CH-Sepharose (PATG-Sepharose) affinity chromatography and high-performance liquid chromatography on a Shim pack Diol 300 column. The purified enzyme liberated sialic acid residues from sialooligosaccharides, sialoglycoproteins, and gangliosides. In particular, gangliosides GM3, GD1a, and GD1b were hydrolyzed much faster than alpha (2-3) and alpha (2-6)sialyllactoses, and sialoglycoproteins by the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme gave five protein bands with molecular weight of 78,000 (78K), 64,000 (64K), 46,000 (46K), 30,000 (30K), and 20,000 (20K). Rabbit antisera were raised against 78K and 46K proteins, and the two antibodies were specifically reactive with the respective component on immunoblot analysis. Both anti-78K protein and anti-46K protein antisera could precipitate sialidase activity. It is likely that the 78K protein and 46K protein are sub-components which are essential for sialidase activity.

Human placental sialidase: partial purification and characterization.

A sialidase [EC 3.2.1.18] has been partially purified from human placenta by means of procedures comprising Con A-Sepharose adsorption, ammonium sulfate precipitation, sucrose density gradient centrifugation, and high-pressure liquid chromatography on a Shim pack Diol 300 column. On high-pressure liquid chromatography, most of the beta-galactosidase that comigrated with the sialidase on sucrose density gradient centrifugation was removed. The sialidase was purified 3,600-fold from the preparation obtained by Con A-Sepharose adsorption. The enzyme liberated the sialic acid residues from (alpha 2-3) and (alpha 2-6) sialyllactose, colomic acid, fetuin, and transferrin, but not from bovine submaxillary mucin. The enzyme also hydrolyzed gangliosides GM3, GD1a, and GD1b in the presence of sodium cholate as a detergent, but GM1 and GM2 were less susceptible to the enzyme. The optimum pHs for 4-methylumbelliferyl-N-acetylneuraminate, sialyllactose, fetuin, and GM3 lay between 4.0 and 5.0.

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, CaCl 2, dithiothreitol, or Ep-459. The optimal pH was 4.5 and the apparent K m value, approximately 0.090 m m. Without substrate addition, the enzyme was unstable at temperatures between 0 and 37°C, retaining only 35 and 5% of its activity, respectively, after 8 1 2 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 m m, respectively. The 0.11 m solutions of NaCl, LiCl, and KCl inhibited 20–30%, and CaCl 2 about 60%, of the enzyme activity.