Immunological Discrimination of Intralysosomal, Cytosolic, and Two Membrane Sialidases Present in Rat Tissues1

Abstract

Cytosolic sialidase was purified from rat skeletal muscle, and the purified enzyme migrated as a single band of Mr 43,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal antibody raised against the enzyme inhibited and immunoprecipitated rat liver cytosolic sialidase as well as the muscle enzyme but failed to cross-react with the intralysosomal sialidase of rat liver and membrane sialidases I (synaptosomal) and II (lysosomal) of rat brain. The antibody against brain membrane sialidase I (anti-I) and that against sialidase II (anti-II), which could be useful to discriminate the two enzymes, did not cross-react with the intralysosomal and cytosolic sialidases of liver. Although more than 90% of liver plasma membrane sialidase was immunoprecipitated with anti-I, only 60% of liver lysosomal membrane sialidase was immunoprecipitated with anti-II, the remainder being immunoprecipitated with anti-I. In confirmation of these data, liver lysosomal membrane exhibited two peaks of ganglioside sialidase corresponding to the membrane sialidases I and II on Aminohexyl-Sepharose chromatography while only one peak of ganglioside sialidase corresponding to sialidase I was observed for liver plasma membrane. These results indicate that the four types of rat sialidase are proteins distinct from one another and that the three kinds of antisera described above are useful for discriminating these sialidases qualitatively and probably quantitatively.

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

“…The concentration of released sialic acid was measured by phenobarbituric method [25]. The immunotitration results (Table 1) showed that antibodies raised against recombinant lysosomal sialidase precipitated all activity of lysosomal sialidase in human liver extract, but not of cytosolic or plasma membrane sialidases, thus confirming that the antibodies are specific against the lysosomal enzyme and that the lysosomal, plasma membrane and cytosolic sialidases do not have common antigenic determinants, as has been suggested [26]. The anti-sialidase antibodies did not precipitate purified GAL (not shown) but precipitated up to 8 % of GAL activity in liver extract, which probably represents the fraction of GAL associated with the lysosomal sialidase in the 1n27 MDa complex.…”

Occurrence of sialidase activity in two distinct and highly homogeneous populations of lysosomes prepared from the brain of developing mouse

“…These developmental differences, together with property differences (for example the optimal pH) may be in favour of the concept [22,23] that the various…”

Molecular pathology of NEU1 gene in sialidosis

“…Sialidase is targeted to the endosomal-lysosomal compartment as an integral membrane protein by vesicular transport, which involves association of the adapter proteins with a tyrosine-containing internalization signal at the C-terminus of the enzyme [Lukong et al, 2001]. It is likely that the transmembrane domain in the lysosome is cleaved similarly to that of acid phosphatase, resulting in the appearance in the cell of two pools of lysosomal sialidase, soluble and membraneassociated, which are both absent in cultured cells of sialidosis patients [Verheijen et al, 1983;Miyagi et al, 1990Miyagi et al, , 1992Miyagi et al, , 1993. Immunoelectron microscopy demonstrated that, in addition to the lysosomal membrane and lysosomal lumen, NEU1 sialidase is present on the plasma membrane and in intracellular (possibly endocytic) vesicles [Vinogradova et al, 1998].…”