Isolation, purification, and biochemical characterization of two forms of lysosomal ß-N-acetylhexosaminidase from the invertebrate Unio.

Lysosomal hexosaminidases are glycosyl hydrolases that remove the terminal hexosamine residues of glycoconjugates. Though mammalian hexosaminidases are well characterized, the biochemical nature of these enzymes among invertebrates remains elusive. In this study, we purified two thermostable N-acetyl ß-D-hexosaminidases (hex A and B) to homogeneity from soluble extracts of whole Unio animal tissue by a combination of chromatographic procedures. Purified hex A and hex B migrated as a single protein species on native PAGE and exhibited enzyme activity. However on SDS-PAGE, hex A dissociated into two subunits of molecular masses about 75 kDa and 30 kDa respectively, while hex B showed a molecular mass of 40 kDa. Hex A and B were recognized by the affinity purified mannose 6-phosphate receptor 46 on ligand blot analysis. This specific interaction was similar to what is known for the vertebrate receptors and lysosomal enzymes. The enzymes showed different K(M) values with respect to the substrates p-nitrophenyl N-acetyl-ß-D-glucosaminide and p-nitrophenyl N-acetyl-ß-D-galactosaminide. The enzymes were thermally stable up to 80 °C and showed pH optima between 5.0 and 6.0. This is the first report on the purification of two forms of hexosaminidases from Unio.

Identification and characterization of mature beta-hexosaminidases associated with human placenta lysosomal membrane.

Hex (beta-hexosaminidase) is a soluble glycohydrolase involved in glycoconjugate degradation in lysosomes, however its localization has also been described in the cytosol and PM (plasma membrane). We previously demonstrated that Hex associated with human fibroblast PM as the mature form, which is functionally active towards G(M2) ganglioside. In the present study, Hex was analysed in a lysosomal membrane-enriched fraction obtained by purification from highly purified human placenta lysosomes. These results demonstrate the presence of mature Hex associated with the lysosomal membrane and displaying, as observed for the PM-associated form, an acidic optimum pH. When subjected to sodium carbonate extraction, the enzyme behaved as a peripheral membrane protein, whereas Triton X-114 phase separation confirmed its partially hydrophilic nature, characteristics which are shared with the PM-associated form of Hex. Moreover, two-dimensional electrophoresis indicated a slight difference in the pI of beta-subunits in the membrane and the soluble forms of the lysosomal Hex. These results reveal a new aspect of Hex biology and suggest that a fully processed membrane-associated form of Hex is translocated from the lysosomal membrane to the PM by an as yet unknown mechanism. We present a testable hypothesis that, at the cell surface, Hex changes the composition of glycoconjugates that are known to be involved in intercellular communication and signalling.