Human placental neuraminidase

Abstract

Supernatant of homogenized human placenta hardly contains lysosomal neuraminidase activity. It is, however, possible to generate remarkably high activity by concentration of a partially purified glycoprotein fraction. This activity is labile to dilution, but can be stabilized by incubation at 37°C and acid pH. Using β‐galactosidase specific affinity chromatography and immunotitration, we show that the activated and stabilized human lysosomal neuraminidase exists in a complex with β‐galactosidase. Sucrose density gradient centrifugation experiments demonstrate that the neuraminidase activity is exclusively present in a high density multimeric form of β‐galactosidase. The formation of multimeric forms of β‐galactosidase is known to require a 32000‐Mr‘protective’ protein. Monospecific antibodies against this ‘protective’ protein were purified from a conventional antiserum containing a mixture of antibodies against the 64000‐Mrβ‐galactosidase protein and against the 32000‐Mr‘protective’ protein, using a nitrocellulose blot immunoaffinity purification procedure. Immunotitration experiments with these antibodies show that the 32000‐Mr‘protective’ protein is present both in association with the β‐galactosidase multimer and with the high‐density multimeric form together with neuraminidase. Our data further suggest that association of the 32000‐Mr‘protective’ protein and another yet unidentified subunit is essential for the catalytic activity of lysosomal neuraminidase. These results explain the absence of neuraminidase activity in the autosomal recessive human lysosomal storage disorder galactosialidosis, where the 32000‐Mr‘protective’ protein is known to be absent.

Molecular and structural studies of Japanese patients with sialidosis type 1

“…Few reports on human lysosomal neuraminidase have been published, because the enzyme has low stability during purification and requires PPCA for the expression of its activity (Verheijen et al 1982(Verheijen et al , 1985(Verheijen et al , 1987Hiraiwa et al 1988;van der Horst et al 1989). Recent cDNA cloning for human lysosomal neuraminidase has revealed that it encodes a 45-kDa protein with three potential N-linked glycosylation sites (Bonten et al 1996;Pshezhetsky et al 1997;Milner et al 1997).…”

“…1. 18) is an acid glycosidase that catalyzes the removal of the terminal sialic acid residues of glycoconjugates (Verheijen et al 1982(Verheijen et al , 1985(Verheijen et al , 1987Hiraiwa et al 1988;van der Horst et al 1989). Mammalian lysosomal neuraminidase is associated with protective protein/cathepsin A (PPCA; EC 3.…”

Sialidosis Presenting as Severe Nonimmune Fetal Hydrops is Associated with Two Novel Mutations in Lysosomal α-Neuraminidase

“…8,9 Sialidosis results from a defect in a-neuraminidase, which is part of a multienzyme complex within lysosomes that also includes protective protein/cathepsin A (PPCA), and the glycosidases b-galactosidase and N-acetylgalactosamine-6-sulfate sulfatase. 10,11 Association of a-neuraminidase with PPCA, and formation of the multienzyme complex assures the efficient intracellular transport and lysosomal activation of a-neuraminidase. 12,13 Sialidosis is typically classified according to clinical presentation, although this classification scheme does not correlate with specific mutations or the degree of enzyme activity.…”

Novel missense mutations in the human lysosomal sialidase gene in sialidosis patients and prediction of structural alterations of mutant enzymes

“…In mammals, the enzyme associates with acid -galactosidase (EC 3.2.1.23) and protective protein/cathepsin A (PPCA, EC 3.4.16.1) to form a multienzyme complex in lysosomes (d'Azzo et al 1982;Verheijen et al 1982Verheijen et al , 1985Hiraiwa et al 1988;van der Horst et al 1989). In paticular, association with PPCA is essential for expression of the sialidase activity (d'Azzo et al 1982;van der Spoel et al 1998).…”