Beta-glucuronidase binding to human fibroblast membrane receptors.

Abstract. Endocytosis of acid hydrolases via the cell surface mannose 6-phosphate (Man 6-P) receptor results in the delivery of the enzymes to lysosomes. To examine the fate of the ligand-associated phosphorylated high mannose oligosaccharides, we have analyzed the asparagine-linked oligosaccharides attached to I~-glucuronidase after uptake and processing by Man 6-P receptor-positive mouse L cells. ~Gluc-uronidase, double-labeled with [2-3H]mannose and [35S]methionine, was isolated from the growth medium of mouse P388D1 cells. 80% of the [3H]mannose associated with the secreted enzyme was recovered as high mannose-type oligosaccharides, and 24-37 % of these units were phosphorylated. Three species of phosphorylated oligosaccharides were identified; high mannose-type units containing either one or two phosphomonoesters, and hybrid-type units containing one phosphomonoester and one sialic acid residue. After endocytosis by the L cells, the 13-glucuronidase molecules migrated faster on an SDS gel, suggesting that the enzymes had been processed within lysosomes. Examination of the cell-associated I~-glucuronidase molecules indicated that: (a) the percentage of phosphorylated oligosaccharides remained comparable to the input form of the enzyme, even after a 24-h chase period, (b) the presence of a single species of phosphorylated oligosaccharide that contained one phosphomonoester, and (c) the positioning of the phosphate within the intracellular monophosphorylated species was comparable to the positioning of the phosphate within the two phosphomonoester species originally secreted by the P388D~ cells. Therefore, the internalized 13-glucuronidase molecules undergo a limited dephosphorylation; oligosaccharides containing two phosphomonoesters are converted to monophosphorylated species, but the one phosphomonoester forms are conserved. A comparison of the phosphorylated oligosaccharides recovered from ligands internalized by the L cells at 37 ° and 20°C indicated that: (a) molecules internalized at 20°C retain a higher percentage of phosphorylated structures; and (b) at both temperatures the predominant phosphorylated oligosaccharide contains a single phosphomonoester group. The results indicate that the Man 6-P recognition marker persists after endocytosis and delivery to lysosomes and support the possibility that the limited dephosphorylation of the oligosaccharides may occur en route to these organelles.T HE mannose 6-phosphate (Man 6-P) ~ receptor is an important component in the delivery of newly synthesized acid hydrolases to lysosomes (32, 37). Lysosomal enzyme biogenesis begins in the rough endoplasmic reticulum, where the enzymes attain asparagine-linked oligosaccharides during their translation (5,27,34). Upon transfer to the Golgi apparatus, the enzymes are phosphorylated by UDP-N-acetylglucosamine/lysosomal enzyme N-acetylglucosaminylphosphotransferase to generate a phosphodiester linkage between the C-1 position of N-acetylglucosamine and the C-6 position of mannose residues 1. Abbreviations used in this ...

Section: Generality Of the Processingmentioning

confidence: 99%

Mannose 6-phosphate receptor-mediated endocytosis of acid hydrolases: internalization of beta-glucuronidase is accompanied by a limited dephosphorylation.

Gabel¹,

Foster²

1986

“…Binding to membranes was also measured by displacement. Membranes were prepared as previously described (29) with the single exception that membranes were collected by centrifugation at 100,000 g for 60 rain. Membranes were treated on ice for 30 rain in buffer (25 mM Tris, 0.25 M sucrose, 5 mM EDTA) pH 7.0, with 0.5% saponin and 5 mM mamaose 6-phosphate added (29), then pelleted, resuspended and washed by centrifugation at 100,000 g for 60 rain; 1 nag total cell protein yielded -200/~g washed membranes.…”

Section: Isolation Of Mutantsmentioning

confidence: 99%

“…Membranes were prepared as previously described (29) with the single exception that membranes were collected by centrifugation at 100,000 g for 60 rain. Membranes were treated on ice for 30 rain in buffer (25 mM Tris, 0.25 M sucrose, 5 mM EDTA) pH 7.0, with 0.5% saponin and 5 mM mamaose 6-phosphate added (29), then pelleted, resuspended and washed by centrifugation at 100,000 g for 60 rain; 1 nag total cell protein yielded -200/~g washed membranes. Samples containing 30 ~tg of membrane protein were incubated with 2.0 × 105 clam of ammonia-induced secretions in the above buffer in a final volume of 80/al for 60 rain on ice; 75/d of the assay mixture was Layered onto buffer containing 100 mg/ml bovine serum albumin (BSA), then centrifuged at 100,000 g for 20 rain at 4°C in a Beckman Airfuge (Beckman Instruments, Inc., Spinco Div., Palo Alto, CA) to separate bound from free ligand (30).…”

Section: Isolation Of Mutantsmentioning

confidence: 99%

Mutant Chinese hamster ovary cells pleiotropically defective in receptor-mediated endocytosis.

Robbins¹,

Peng²,

Marshall³

1983

112

Populations of Chinese hamster ovary cells selected for resistance to diphtheria toxin were found to be highly enriched for mutants deficient in the uptake of lysosomal hydrolases via the mannose 6-phosphate receptor. One doubly defective mutant, DTF 1-5-1, exhibited increased resistance to Sindbis virus, although it was able to bind and internalize virus normally. Normal production of virus was obtained when, subsequent to virus binding, the mutant was exposed for 2 rain to acidic pH. Similarly, a shift to acidic pH increased the sensitivity of DTF 1-5-1 to diphtheria toxin 12-fold. Decreased uptake of lysosomal hydrolases by the mutant correlated with decreased mannose 6-phosphate receptor activity at the cell surface; results of lactoperoxidase-catalyzed iodination indicated that the surface-associated receptor was present but inactive on DTF 1-5-1. Total mannose 6-phosphate receptor activity was also decreased in the mutant and this decrease was reflected by increased secretion of lysosomal hydrolases. The phenotype of DTF 1-5-1 resembles in many ways that of cells treated with ammonia. We suggest that the defect in DTF 1-5-1 stems from an inability to deliver virus, diphtheria toxin, and lysosomal hydrolases to an acidic compartment. Other ligands may be endocytosed through a different pathway since the defect of DTF 1-5-1 did not decrease the endocytosis of ricin, modeccin, or Pseudomonas toxin and had minimal effects on uptake and degradation of low density lipoprotein.

“…However, some uncleaved precursor molecules often accumulate extracellularly (24). Many cell types contain specific membrane receptors that recognize mannose-6-phosphate residues on the N-linked oligosaccharide side chains of lysosomal enzymes (12,14,27,37). Several lines of evidence have pointed to the involvement of these receptors in the capture of newly synthesized enzymes and subsequent targeting to their intraceUular destination as mature forms in lysosomes (13,35,42).…”

mentioning

confidence: 99%

Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium discoideum.

Mierendorf¹,

Cardelli²,

Dimond³

1985

In Dictyostelium discoideum, the lysosomal enzyme a-mannosidase is first synthesized as an N-glycosylated precursor of Mr 140,000. After a 20-30-min lag period, up to 30% of the precursor molecules are rapidly secreted, whereas the rest remain cellular and are proteolytically processed (tt/2 = 8 min) to mature subunits of Mr 58,000 and 60,000. The secreted precursor is modified more extensively than the cellular form, as is revealed by differences in size, charge, and sensitivity to endoglycosidase H. Subcellular fractionation has shown that, following synthesis in the rough endoplasmic reticulum, the precursor is transported to a low density membrane fraction that contains Golgi membranes. Proteolytic processing takes place in these vesicles, since newly cleaved mature enzyme, but no precursor, co-fractionates with Iysosomes. Under conditions that disrupt vesicular membranes, the precursor remains associated with the membrane fraction, whereas the newly processed mature enzyme is soluble. Proteolytic cleavage of the precursor thus coincides with the release of the mature enzyme into the lumen of a lysosomal compartment. These findings suggest a possible mechanism for lysosomal targeting that involves the specific association of enzyme precursors with Golgi membranes.