Activation of Human Lysosomal Sialidase1

Hiraiwa, Masao; Yamauchi, Toyoaki; Tsuji, Shoji; Nishizawa, Masatoyo; Miyatake, Tadashi; Oyanagi, Kiyomitsu; Ikuta, Fusahiro; Uda, Yutaka

doi:10.1093/oxfordjournals.jbchem.a124274

Cited by 15 publications

(9 citation statements)

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“…In contrast other researchers hypothesized that activation of sialidase in itro involves its proteolytic processing [39,40]. To understand the molecular mechanism associated with the activation process we performed the Western blotting of sialidase in the samples obtained in the process of the 120 min incubation of the concentrated glycoprotein fraction at 37 mC (Figure 2c), which increased the specific activity of sialidase from 0n94 mU\mg to 3n5 mU\mg (Figure 2d).…”

Section: Figure 4 Electron Micrograph Of Normal Fibroblasts Transfectmentioning

confidence: 90%

Molecular mechanism of lysosomal sialidase deficiency in galactosialidosis involves its rapid degradation

Vinogradova

Michaud

Mezentsev

et al. 1998

Biochemical Journal

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Galactosialidosis is an inherited lysosomal storage disease caused by the combined deficiency of lysosomal sialidase and β-galactosidase secondary to the deficiency of cathepsin A/protective protein, which is associated with sialidase and β-galactosidase in a high-molecular weight (1.27 MDa) complex. Clinical phenotypes of patients as well as the composition of compounds which are stored in patient's tissues implicate sialidase deficiency as the underlying pathogenic defect. The recent cloning and sequencing of lysosomal sialidase [Pshezhetsky, Richard, Michaud, Igdoura, Wang, Elsliger, Qu, Leclerc, Gravel, Dallaire and Potier (1997), Nature Genet. 15, 316-320] allowed us to study the molecular mechanism of sialidase deficiency in galactosialidosis. By Western blotting, using antibodies against the recombinant human enzyme, and by NH2-terminal sequencing, we showed that sialidase is synthesized as a 45.5 kDa precursor and after the cleavage of the 47-amino acid signal peptide and glycosylation becomes a 48.3 kDa mature active enzyme present in the 1.27 kDa complex. Transgenic expression of sialidase in cultured skin fibroblasts from normal controls and from galactosialidosis patients, followed by immunofluorescent and immunoelectron microscopy showed that in both normal and affected cells the expressed sialidase was localized on lysosomal and plasma membranes, but the amount of sialidase found in galactosialidosis cells was ~ 5-fold reduced. Metabolic labelling studies demonstrated that the 48.3 kDa mature active form of sialidase was stable in normal fibroblasts (half-life ~ 2.7 h), whereas in galactosialidosis fibroblasts the enzyme was rapidly converted (half-life ~ 30 min) into 38.7 and 24 kDa catalytically inactive forms. Altogether our data provide evidence that the molecular mechanism of sialidase deficiency in galactosialidosis is associated with abnormal proteolytic cleavage and fast degradation.

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Section: Figure 4 Electron Micrograph Of Normal Fibroblasts Transfectmentioning

confidence: 90%

Molecular mechanism of lysosomal sialidase deficiency in galactosialidosis involves its rapid degradation

Vinogradova

Michaud

Mezentsev

et al. 1998

Biochemical Journal

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“…The activation occurs in a time-and pH-dependent fashion, suggesting involvement of certain enzymatic modification(s) in the activation process. Activation of neuraminidase did not occur in a liver sample of a patient with galactosialidosis [96], raising a possibility that the deficiency of the cathepsin A activity might have influenced the activation process. A recent study demonstrated that neuraminidase bound to cathepsin A precursor, shortly after its synthesis, was transported to lysosomes.…”

Section: Galactosialidosis and Activation Of Human Lysosomal Neuraminmentioning

confidence: 93%

“…Human lysosomal neuraminidase in a partially purified preparation is activated by brief incubation under acidic pH condition [84,[93][94][95][96]. The activation occurs in a time-and pH-dependent fashion, suggesting involvement of certain enzymatic modification(s) in the activation process.…”

Section: Galactosialidosis and Activation Of Human Lysosomal Neuraminmentioning

confidence: 99%

Cathepsin A/protective protein: an unusual lysosomal multifunctional protein

Hiraiwa¹

1999

Cellular and Molecular Life Sciences (CMLS)

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Cathepsin A/protective protein [3.4.16.5], carboxypeptidase A, is a lysosomal serine protease with structural homology to yeast (Saccharomyces cerevisiae) carboxypeptidase Y. Cathepsin A is a member of the alpha/beta hydrolase fold family and has been suggested to share a common ancestral relationship with other alpha/beta hydrolase fold enzymes, such as cholinesterases. Several lines of evidence indicate that cathepsin A is a multicatalytic enzyme with deamidase and esterase in addition to carboxypeptidase activities. Cathepsin A was recently identified in human platelets as deamidase. In vitro, it hydrolyzes a variety of bioactive peptide hormones including tachykinins, suggesting that extralysosomal cathepsin A plays a role in regulation of bioactive peptide functions. Recent reports emphasize the lysosomal protective function of cathepsin A rather than its protease function. The protective function of cathepsin A is distinct from its catalytic function. Human lysosomal beta-galactosidase and neuraminidase exist as a high molecular weight enzyme complex, in which there is a 54-kDa glycoprotein termed 'lysosomal protective protein'. Based on cell culture studies, protective protein was found to protect both beta-galactosidase and neuraminidase from intralysosomal proteolysis by forming a multienzyme complex and was shown to be deficient in patients with galactosialidosis, a combined deficiency of beta-galactosidase and neuraminidase. Molecular cloning and gene expression studies have disclosed that protective protein is cathepsin A. The cathepsin A precursor has the potential to restore both beta-galactosidase and neuraminidase activities in fibroblasts from patients with galactosialidosis. Cathepsin A knockout mice showed a phenotype similar to human galactosialidosis and the deficient phenotype found in the mutant mice was corrected by transplanting erythroid precursor cells overexpressing cathepsin A. Collectively, these findings demonstrate the significance of cathepsin A as a key molecule in the onset of galactosialidosis and also highlight the therapeutic potential of the cathepsin A precursor for patients with galactosialidosis.

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“…5B), we do not know whether these trace level contaminants contribute to the distinctive pattern of endothelial barrier disruption. We did not use protease inhibitors in our preparations because of the concern that protease inhibitors can affect neuraminidase activity, as was previously described by Hiraiwa and colleagues (9). However, to rule out influence of other factors in this preparation, we heat-inactivated the enzyme as described above for the neuraminidase from Clostridium perfringens.…”

Section: Sialic Acids Contribute To the Glycocalyx Of Paecs Andmentioning

confidence: 99%

Terminal sialic acids are an important determinant of pulmonary endothelial barrier integrity

Cioffi

Pandey²,

Alvarez

et al. 2012

American Journal of Physiology-Lung Cellular and Molecular Phys

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The surface of vascular endothelium bears a glycocalyx comprised, in part, of a complex mixture of oligosaccharide chains attached to cell-surface proteins and membrane lipids. Importantly, understanding of the structure and function of the endothelial glycocalyx is poorly understood. Preliminary studies have demonstrated structural differences in the glycocalyx of pulmonary artery endothelial cells compared with pulmonary microvascular endothelial cells. Herein we begin to probe in more detail structural and functional attributes of endothelial cell-surface carbohydrates. In this study we focus on the expression and function of sialic acids in pulmonary endothelium. We observed that, although pulmonary microvascular endothelial cells express similar amounts of total sialic acids as pulmonary artery endothelial cells, the nature of the sialic acid linkages differs between the two cell types such that pulmonary artery endothelial cells express both α( 2 , 3 )- and α( 2 , 6 )-linked sialic acids on the surface (i.e., surficially), whereas microvascular endothelial cells principally express α( 2 , 3 )-linked sialic acids. To determine whether sialic acids play a role in endothelial barrier function, cells were treated with neuraminidases to hydrolyze sialic acid moieties. Disruption of cell-cell and cell-matrix adhesions was observed following neuraminidase treatment, suggesting that terminal sialic acids promote endothelial barrier integrity. When we measured transendothelial resistance, differential responses of pulmonary artery and microvascular endothelial cells to neuraminidase from Clostridium perfringens suggest that the molecular architecture of the sialic acid glycomes differs between these two cell types. Collectively our observations reveal critical structural and functional differences of terminally linked sialic acids on the pulmonary endothelium.

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Activation of Human Lysosomal Sialidase1

Cited by 15 publications

References 0 publications

Molecular mechanism of lysosomal sialidase deficiency in galactosialidosis involves its rapid degradation

Molecular mechanism of lysosomal sialidase deficiency in galactosialidosis involves its rapid degradation

Cathepsin A/protective protein: an unusual lysosomal multifunctional protein

Terminal sialic acids are an important determinant of pulmonary endothelial barrier integrity

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