Transport and processing of endocytosed lysosomal alpha-glucosidase in cultured human skin fibroblasts.
In the present study we have characterised the molecular products that arise from processing of a precursor form of alpha-glucosidase isolated from urine after endocytosis at 37 degrees C by cultured human skin fibroblasts. The urinary precursor (Mr 110 000) was processed to forms with Mr of 100 000, 80 000 and 74 000. These forms were approximately 4000 Da larger than the corresponding forms of endogenously synthesized alpha-glucosidase. Digestion of the different forms of the enzyme with endoglycosidase F showed that the differences in apparent molecular mass between the exogenous and corresponding endogenous forms were due to difference in glycosylation. Intracellular transport of endocytosed alpha-glucosidase was followed by incubating fibroblast homogenates with glycyl-L-phenylalanine-beta-naphthylamide (Gly-Phe-NH-Nap), which leads to specific lysis of lysosomes. Transport to the lysosomes was a fast process: within 45 min after endocytosis more than 50% of the enzyme was present in the lysosome. The first step in the processing of endocytosed alpha-glucosidase started in a Gly-Phe-NH-Nap-insensitive (prelysosomal) compartment, but further processing of the enzyme to lower-Mr forms was coupled to transport to the lysosomes. Processing of alpha-glucosidase after uptake at 20 degrees C was also studied. At this temperature the enzyme accumulated in an organelle with a low buoyant density, presumably the endosome; this compartment appeared to be heterogeneous, ranging in density from 1.04 g/ml to 1.08 g/ml. Under these conditions only the first step in the processing of the enzyme occurred. It is concluded that endocytosed enzyme is processed more rapidly than endogenously synthesized enzyme owing to the fact that endocytosed enzyme is transported more rapidly to the lysosomes. Furthermore, processing may start in a prelysosomal organelle.
D001692:Biological Transport; D002459:Cell Fusion; D002478:Cells, Cultured; D004705:Endocytosis; D005347:Fibroblasts / Q000201:enzymology; D005959:Glucosidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008970:Molecular Weight; D012867:Skin / Q000201:enzymology; D000520:alpha-Glucosidases / Q000378:metabolism*
D005959:Glucosidases; D000520:alpha-Glucosidases