Cation-independent mannose 6-phosphate and 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments.

The distribution of the cation-independent mannose 6-phosphate and 78 kDa receptors was studied in postnuclear subcellular fractions from two rat liver cell lines. ELISA assays revealed that the mannose 6-phosphate receptor is enriched in the light buoyant Percoll fractions that contain Golgi structures and early endosomes. Most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient and smaller amounts in the endosomal fractions. The high-density compartment is denser than lysosomes, contains LAMP2 but not LIMPII or acid hydrolases, and is not disrupted with glycyl-l-phenylalanine 2-naphthylamide, a substrate for cathepsin C that selectively disrupts lysosomes. Immunofluorescence microscopy studies indicate no colocalization of the 78 kDa receptor with the mannose 6-phosphate receptor or LIMPII. Mannose 6-phosphate-independent endocytosed beta-glucuronidase was found in the lysosomal, the early and late endosomal fractions. These fractions were immunoadsorbed in columns containing antibodies against the 78 kDa receptor. Only the endocytosed beta-glucuronidase present in the early and late endosomal fractions is associated to immunoadsorbed vesicles. In these vesicles, LAMP2 was detected but no LIMPII or the mannose 6-phosphate receptor. Results obtained suggest that the 78 kDa receptor is found along the endocytic pathway, but in vesicles different from the cation-independent mannose 6-phosphate receptor.

Phosphomannosyl receptors on the surface of spermatozoa from the cauda epididymis of the rat.

This study demonstrates that beta-glucuronidase from rat preputial glands binds with high affinity to spermatozoa from the cauda epididymis. The binding was calcium-independent and was inhibited by mannose-6-phosphate, but not by other phosphorylated or non-phosphorylated sugars. Binding was also inhibited by alpha-mannosidase from Dictyostelium discoideum, an enzyme known to have mannose-6-phosphate as the ligand. From solubilized sperm membranes, a protein of > 200 kDa and one of 45 kDa, were absorbed to a column of D. discoideum enzyme and to a phosphomannan column respectively, and eluted with mannose-6-phosphate. According to histochemical observations at the light and the electron microscopic level, gold particles coated with the enzyme became bound to the external surface of the plasmalemma in the acrosomal region of caudal spermatozoa. Similar labelling was observed using gold particles coated with antibodies against the rat 300 kDa phosphomannosyl receptor. The existence of phosphomannosyl receptors on the sperm plasma membrane, and our previous demonstration of the presence of affinity sites for epididymal beta-galactosidase on these gametes which is inhibited by phosphofructosyl derivatives, suggest strongly that maturing spermatozoa could be a target for glycosidases secreted into the lumen of the cauda epididymis, which then become bound to these cells via different ligand-receptor systems.