scholarly journals Lysosomal enzyme oligosaccharide phosphorylation in mouse lymphoma cells: specificity and kinetics of binding to the mannose 6-phosphate receptor in vivo

1982 ◽  
Vol 95 (2) ◽  
pp. 536-542 ◽  
Author(s):  
CA Gabel ◽  
DE Goldberg ◽  
S Kornfeld
Keyword(s):  
Lysosomal Enzymes ◽  
Half Time ◽  
Mannose Residue ◽  
Receptor Cells ◽  
Mouse Lymphoma Cells ◽  
High Mannose ◽  
Mannose 6 Phosphate ◽  
Kinetics Of ◽  
Mouse Lymphoma

Phosphomannosyl residues on lysosomal enzymes serve as an essential component of the recognition marker necessary for binding to the mannose 6-phosphate (Man 6-P) receptor and translocation to lysosomes. The high mannose-type oligosaccharide units of lysosomal enzymes are phosphorylated by the following mechanism: N-acetylglucosamine 1-phosphate is transferred to the 6 position of a mannose residue to form a phosphodiester; then N- acetylglucosamine is removed to expose a phosphomonoester. We examined the kinetics of this phosphorylation pathway in the murine lymphoma BW5147.3 cell line to determine the state of oligosaccharide phosphorylation at the time the newly synthesized lysosomal enzymes bind to the receptor. Cells were labeled with [2-(3)H]mannose for 20 min and then chased for various times up to 4 h. The binding of newly synthesized glycoproteins to the Man 6-P receptor was followed by eluting the bound ligand with Man 6-P. Receptor-bound material was first detected at 30 min of chase and reached a maximum at 60 min of chase, at which time approximately 10 percent of the total phosphorylated oligosaccharides were associated with the receptor. During longer chase times, the total quantity of cellular phosphorylated oligosaccharides decreased with a half-time of 1.4 h, suggesting that the lysosomal enzymes had reached their destination and had been dephosphorylated. The structures of the phosphorylated aligosaccharides of the eluted ligand were then determined and compared with the phosphorylated oligosaccharides of molecules which were not bond to the receptor. The major phosphorylated oligosaccharide species present in the nonreceptor-bound material contained a single phosphosphodiester at all time examined. In contrast, receptor-bound oligosaccharides were greatly enriched in species possessing one and two phosphomonoesters. These results indicate that binding of newly synthesized lysosomal enzymes to the Man 6-P receptor occurs only after removal of the covering N- acetylglucosamine residues.

scholarly journals Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum

2013 ◽  
Vol 288 (23) ◽  
pp. 16460-16475 ◽  
Author(s):  
Linda J. Olson ◽  
Ramiro Orsi ◽  
Solana G. Alculumbre ◽  
Francis C. Peterson ◽  
Ivan D. Stigliano ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Mannose Residue ◽  
Glucosidase Ii ◽  
Mannose 6 Phosphate ◽  
First Time ◽  
Conserved Residue

Here we report for the first time the three-dimensional structure of a mannose 6-phosphate receptor homology (MRH) domain present in a protein with enzymatic activity, glucosidase II (GII). GII is involved in glycoprotein folding in the endoplasmic reticulum. GII removes the two innermost glucose residues from the Glc3Man9GlcNAc2 transferred to nascent proteins and the glucose added by UDP-Glc:glycoprotein glucosyltransferase. GII is composed of a catalytic GIIα subunit and a regulatory GIIβ subunit. GIIβ participates in the endoplasmic reticulum localization of GIIα and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal MRH domain. We determined the structure of a functional GIIβ MRH domain by NMR spectroscopy. It adopts a β-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue outside the binding pocket (Trp-409) present in GIIβ but not in other MRHs that influences GII glucose trimming activity.

Mannose 6-phosphate receptor-dependent endocytosis of lysosomal enzymes is increased in sulfatide-storing kidney cells

2009 ◽  
Vol 390 (1) ◽  
Author(s):  
Diana Klein ◽  
Afshin Yaghootfam ◽  
Ullrich Matzner ◽  
Bettina Koch ◽  
Thomas Braulke ◽  
...  
Keyword(s):  
Lysosomal Enzymes ◽  
Cell Types ◽  
Recycling Rate ◽  
Kidney Cells ◽  
Lysosomal Disorder ◽  
Renal Tubular Cells ◽  
Internalization Rate ◽  
Mannose 6 Phosphate ◽  
Renal Tubular ◽  
Kinetics Of

Abstract Metachromatic leukodystrophy is a lysosomal disorder caused by the deficiency of arylsulfatase A (ASA). This leads to the storage of the sphingolipid 3-O-sulfogalactosylceramide (sulfatide) in various cell types, such as renal tubular cells. Examination of mannose 6-phosphate receptor (MPR300)-dependent endocytosis revealed that uptake of lysosomal enzymes is more than two-fold increased in sulfatide-storing kidney cells. Expression of MPR300 and its internalization rate is increased in these cells, whereas the recycling rate is decreased. Similar alterations can be found for the transferrin receptor, indicating that sulfatide storage leads to a general alteration of the endocytotic pathway. These data allow calculating that the endosomal pool from which receptors can recycle is 1.4- to 2-fold increased in lipid-storing cells. Immunocytochemistry demonstrates that the MPR300 in lipid-storing cells does not co-localize with accumulated sulfatide, suggesting that the kinetics of internalization and recycling appear to be altered indirectly.

Kinetics of endogenously labeled plasma fibronectin: incorporation into tissues

1983 ◽  
Vol 245 (4) ◽  
pp. R564-R575
Author(s):  
D. C. Deno ◽  
T. M. Saba ◽  
E. P. Lewis
Keyword(s):  
Residence Time ◽  
Mean Residence Time ◽  
Pool Size ◽  
Soluble Form ◽  
Half Time ◽  
Plasma Fibronectin ◽  
Insoluble Form ◽  
Kinetics Of ◽  
Later Phase

Fibronectin exists in a soluble form in plasma and lymph as well as in a relatively insoluble form in tissues. The disappearance of endogenously labeled fibronectin from plasma and its subsequent extravascular localization was studied over a 38-h period in normal rats (350-400 g) utilizing plasma fibronectin labeled in vivo with 75Se-selenomethionine. For comparative analysis, 125I-albumin was utilized in these dual isotopic experiments. After the simultaneous injection of 75Se plasma fibronectin (20 nCi) and 125I-albumin (2 microCi), all measured tissues demonstrated accumulation of both radiolabeled proteins in extravascular sites. Plasma fibronectin demonstrated a rather specific and high affinity for liver and spleen, which are enriched with reticuloendothelial cells. Albumin manifested the expected washout from extravascular sites, whereas fibronectin primarily displayed retention in tissues. The plasma disappearance of 75Se-labeled fibronectin was well described by two exponentials: an early phase with a half time of 0.52 h and a later phase with a half time of 21 h. To account for tissue retention of labeled fibronectin, a two-compartment kinetics model was required that included loss from the extravascular exchangeable compartment. Analysis of the disappearance kinetics and fibronectin distribution allowed estimation of the fractional turnover rate, pool size, and mean residence time. Accordingly the total rate of loss for plasma fibronectin was 0.51-0.54 mg/h. The calculated pool size of the soluble form of fibronectin was 15.5-16.3 mg. The mean residence time for exchangeable fibronectin was 29.6-29.9 h. These findings suggest that plasma fibronectin can be incorporated within the insoluble pool of fibronectin in tissues.

scholarly journals Studies of the biosynthesis of the mannose 6-phosphate receptor in receptor-positive and -deficient cell lines.

1983 ◽  
Vol 97 (6) ◽  
pp. 1700-1706 ◽  
Author(s):  
D E Goldberg ◽  
C A Gabel ◽  
S Kornfeld
Keyword(s):  
Cell Lines ◽  
Lysosomal Enzymes ◽  
Cell Types ◽  
Receptor Protein ◽  
Complex Type ◽  
Cultured Cell ◽  
Selective Targeting ◽  
High Mannose ◽  
Mannose 6 Phosphate ◽  
Intracellular Pathway

Phosphomannosyl residues present on lysosomal enzymes are specifically recognized by the mannose 6-phosphate receptor protein. This interaction results in the selective targeting of lysosomal enzymes to lysosomes. While this pathway is operative in many cell types, we have found four cultured cell lines that are deficient in the ability to bind lysosomal enzymes containing phosphomannosyl residues to their intracellular or surface membranes (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). These cells appear to segregate lysosomal enzymes by an alternate intracellular pathway. To determine the basis for the lack of mannose 6-phosphate receptor activity in these cell lines, we studied the biosynthesis of the receptor in receptor-positive (BW5147) and receptor-deficient (P388D1 and MOPC 315) cells. The cells were labeled with [2-3H]mannose or [35S]methionine and the receptor was immunoprecipitated with an antireceptor antiserum. BW5147 cells synthesize a receptor protein whose size increases after translation/glycosylation. MOPC 315 cells produce an apparently normal receptor and degrade it rapidly. P388D1 cells fail to synthesize any detectable receptor. The receptor from BW5147 and MOPC 315 cells is a glycoprotein with both high mannose and complex asparagine-linked oligosaccharides. The complex-type units become fully sialylated and remain so during long periods of chase.

scholarly journals Recognition and receptor-mediated uptake of phosphorylated high mannose-type oligosaccharides by cultured human fibroblasts.

1983 ◽  
Vol 96 (3) ◽  
pp. 915-919 ◽  
Author(s):  
M Natowicz ◽  
D W Hallett ◽  
C Frier ◽  
M Chi ◽  
P H Schlesinger ◽  
...  
Keyword(s):  
Lysosomal Enzymes ◽  
Intracellular Transport ◽  
Specific Activity ◽  
Human Fibroblasts ◽  
Lysosomal Hydrolases ◽  
Minimal Structure ◽  
Cultured Human Fibroblasts ◽  
Specific Uptake ◽  
High Mannose ◽  
Mannose 6 Phosphate

The intracellular transport of newly synthesized lysosomal hydrolases to lysosomes requires the presence of one or more phosphorylated high mannose-type oligosaccharides per enzyme. A receptor that mediates mannose-6-PO4-specific uptake of lysosomal enzymes is expressed on the surface of fibroblasts and presumably accounts for the intracellular transport of newly synthesized enzymes to the lysosome. In this study, we examined the internalization of lysosomal enzyme-derived phosphorylated oligosaccharides by cultured human fibroblasts. Oligosaccharides of known specific activity bearing a single phosphate in monoester linkage were internalized with Kuptake of 3.2 X 10(-7) M, whereas oligosaccharides bearing two phosphates in monoester linkage were internalized with a Kuptake of 3.9 X 10(-8) M. Thus, phosphorylated high mannose-type oligosaccharides appear to be the minimal structure required for recognition and uptake by the fibroblast receptor. The finding that the Kuptake for monophosphorylated oligosaccharides is 100-fold less than the reported Ki for mannose-6-phosphate indicates that the fibroblast phosphomannosyl receptor contains a binding site that recognizes features of the oligosaccharide in addition to mannose-6-phosphate.

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