An alternative hypothesis of cellular transport of lysosomal enzymes in fibroblasts. Effect of inhibitors of lysosomal enzyme endocytosis on intra- and extra-cellular lysosomal enzyme activities

Abstract

Recapture of lysosomal enzymes secreted by fibroblasts was inhibited by growing the cells in the presence of either free or immobilized antibodies against lysosomal enzymes or in the presence of phosphorylated carbohydrates known to interact with the cell-surface receptors for lysosomal enzymes. The following results were obtained. 1. Conditions that prevent recapture of released lysosomal enzymes increase the rate of extracellular accumulation of these enzymes up to twice that of controls. 2. Growing cells for 12 days in the presence of 0.5 mM-mannose 6-phosphate, which decreases ,B-N-acetylglucosaminidase endocytosis to less than 10% of that of controls, has no effect on the intracellular activity of this and four other lysosomal enzymes. 3. Growing cells for 4 days in the presence of 50mM-mannose 6-phosphate, which is a 1000-fold higher concentration than that required for 50% inhibition of lysosomal enzyme endocytosis, leads to a 4-fold increase in extracellular fl-N-acetylglucosaminidase accumulation and a decrease in intracellular enzyme. These results give evidence that, in fibroblasts, transfer of lysosomal enzymes into lysosomes does not require secretion before a receptor-mediated recapture [Hickman & Neufeld (1972) Biochem. Biophys. Res. Commun. 49,[992][993][994][995][996][997][998][999]. We propose that (a) lysosomal enzymes are present in a receptor-bound form in those vesicles that fuse with the cell membrane, (b) the major part of the lysosomal enzyme cycles via the cell surface in a receptor-bound form and (c) only a minor part of the lysosomal enzyme is released into the extracellular space during its life cycle.

Characterization of a membrane-associated receptor from bovine liver that binds phosphomannosyl residues of bovine testicular beta-galactosidase.

“…Phosphomannosyl receptors of the same molecular weight and binding specificity as the purified bovine liver receptor were demonstrated in human skin fibroblasts, Chinese hamster ovary cells, and rat hepatocytes. We conclude from these results that the binding protein isolated from bovine liver membranes is the phosphomannosyl receptor thought to be involved in the absorptive endocytosis of extracellular lysosomal enzymes (2-5, 11-15) and in the delivery of newly synthesized lysosomal enzymes to lysosomes (6)(7)(8).…”

“…This process is thought to be mediated by a specific cell surface receptor that recognizes phosphomannosyl residues on oligosaccharide chains of the enzymes (2)(3)(4)(5). Recognition of lysosomal enzymes by a phosphomannosyl receptor has been proposed as an essential step for the delivery of newly synthesized lysosomal enzymes to lysosomes (6)(7)(8). Direct evidence for the existence of phosphomannosyl receptors has been obtained by demonstration of the reversible binding of a-L-iduronidase to the cell surface ofhuman skin fibroblasts (9) and by the binding of P3-glucuronidase to fibroblast cell membranes (10).…”

Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts.

“…Fig. 1 24 mM sodium acetate/0.012% sodium azide, pH 5.5, and 0.02% bovine serum albumin for 24 hr at 37'C. No 3-glucuronidase catalytic activity was lost during this incubation.…”

The Phosphomannosyl Recognition System for Intracellular and Intercellular Transport of Lysosomal Enzymes

“…As discussed earlier, the suggestion that free, secreted enzyme is a transport intermediate [22] seems to hold true for only a small fraction of the total enzyme pool [93]. This is demonstrated by 1) the lack of a precursor-product relationship between extracellular and intracellular enzyme in pulse chase experiments [89, 931, and 2) failure to deplete significantly intralysosomal enzyme levels by agents known to interfere with uptake, including Man-6-P [8, 53, 891 or immobilized antibodies [94]. It thus appears more likely that most newly synthesized acid hydrolases bind to phosphomannosyl enzyme receptors on endomembranes and are transported to lysosomes by vesicles which bud off from the endoplasmic reticulum or GERL [44, 53-55, 59, 601.…”