Phosphorylation of arylsulphatase A occurs through multiple interactions with the UDP-N-acetylglucosamine-1-phosphotransferase proximal and distal to its retrieval site by the KDEL receptor.
Phosphorylation of oligosaccharides of the lysosomal enzyme arylsulphatase A (ASA), which accumulate in the secretions of cells that mis-sort most of the newly synthesized lysosomal enzymes due to a deficiency of mannose 6-phosphate receptors, was found to be site specific. ASA residing within the secretory route of these cells contains about one third of the incorporated [2-3H]mannose in phosphorylated oligosaccharides. Oligosaccharides carrying two phosphate groups are almost 2-fold less frequent than those with one phosphate group and only a few of the phosphate groups are uncovered. Addition of a KDEL (Lys-Asp-Glu-Leu) retention signal prolongs the residence time of ASA within the secretory route 6-fold, but does not result in more efficient phosphorylation. In contrast, more than 90% of the [2-3H]mannose incorporated into secreted ASA (with or without a KDEL retention signal) is present in phosphorylated oligosaccharides. Those with two phosphate groups are almost twice as frequent as those with one phosphate group and most of the phosphate groups are uncovered. Thus, ASA receives N-acetylglucosamine 1-phosphate groups in a sequential manner at two or more sites located within the secretory route proximal and distal to the site where ASA is retrieved by the KDEL receptor, i.e. proximal to the trans-Golgi. At each of these sites up to two N-acetylglucosamine 1-phosphate groups can be added to a single oligosaccharide. Of several drugs known to inhibit transit of ASA through the secretory route only the ionophore monensin had a major inhibitory effect on phosphorylation, uncovering and sialylation. (+info)
Subcellular localization of mannose 6-phosphate glycoproteins in rat brain.
The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification of N-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties. (+info)
Disruption of ligand binding to the insulin-like growth factor II/mannose 6-phosphate receptor by cancer-associated missense mutations.
The insulin-like growth factor II/mannose 6-phosphate receptor (IGF2R) carries out multiple regulatory and transport functions, and disruption of IGF2R function has been implicated as a mechanism to increase cell proliferation. Several missense IGF2R mutations have been identified in human cancers, including the following amino acid substitutions occurring in the extracytoplasmic domain of the receptor: Cys-1262 --> Ser, Gln-1445 --> His, Gly-1449 --> Val, Gly-1464 --> Glu, and Ile-1572 --> Thr. To determine what effects these mutations have on IGF2R function, mutant and wild-type FLAG epitope-tagged IGF2R constructs lacking the transmembrane and cytoplasmic domains were characterized for binding of insulin-like growth factor (IGF)-II and a mannose 6-phosphate-bearing pseudoglycoprotein termed PMP-BSA (where PMP is pentamannose phosphate and BSA is bovine serum albumin). The Ile-1572 --> Thr mutation eliminated IGF-II binding while not affecting PMP-BSA binding. Gly-1449 --> Val and Cys-1262 --> Ser each showed 30-60% decreases in the number of sites available to bind both (125)I-IGF-II and (125)I-PMP-BSA. In addition, the Gln-1445 --> His mutant underwent a time-dependent loss of IGF-II binding, but not PMP-BSA binding, that was not observed for wild type. In all, four of the five cancer-associated mutants analyzed demonstrated altered ligand binding, providing further evidence that loss of IGF2R function is characteristic of certain cancers. (+info)
Mannose 6-Phosphate/Insulin-like growth factor II receptor mediates internalization and degradation of leukemia inhibitory factor but not signal transduction.
Leukemia inhibitory factor (LIF) is a multifunctional cytokine belonging to the interleukin-6 subfamily of helical cytokines, all of which use the glycoprotein (gp) 130 subunit for signal transduction. The specific receptor for LIF, gp190, binds this cytokine with low affinity and is also required for signal transduction. We have recently reported that glycosylated LIF produced by transfected Chinese hamster ovary cells also binds to a lectin-like receptor, mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGFII-R) (Blanchard, F., Raher, S., Duplomb, L., Vusio, P., Pitard, V., Taupin, J. L., Moreau, J. F., Hoflack, B., Minvielle, S., Jacques, Y., and Godard, A. (1998) J. Biol. Chem. 273, 20886-20893). The present study shows that (i) mannose 6-phosphate-containing LIF is naturally produced by a number of normal and tumor cell lines; (ii) other cytokines in the interleukin-6 family do not bind to Man-6-P/IGFII-R; and (iii) another unrelated cytokine, macrophage-colony-stimulating factor, is also able to bind to Man-6-P/IGFII-R in a mannose 6-phosphate-sensitive manner. No functional effects or signal transductions mediated by this lectin-like receptor were observed in various biological assays after LIF binding, and mannose 6-phosphate-containing LIF was as active as non-glycosylated LIF. However, mannose 6-phosphate-sensitive LIF binding resulted in rapid internalization and degradation of the cytokine on numerous cell lines, which suggests that Man-6-P/IGFII-R plays an important role in regulating the amounts of LIF available in vivo. (+info)
Altered ligand binding by insulin-like growth factor II/mannose 6-phosphate receptors bearing missense mutations in human cancers.
The M6P/IGF2R gene, encoding the insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptor (IGF2R), is frequently inactivated during carcinogenesis. M6P/IGF2R is postulated to be a tumor suppressor gene due to its ability to bind and degrade the mitogen IGF-II, promote activation of the growth inhibitor transforming growth factor beta, and regulate the targeting of lysosomal enzymes. In this study, we determined the effects of four M6P/IGF2R missense mutations associated with loss of heterozygosity in hepatocellular and breast cancers on the ligand binding properties of full-length membrane-bound receptors. Site-directed mutagenesis was used to prepare COOH-terminal, c-myc epitope-tagged human IGF2R cDNA expression constructs bearing point mutations that lead to the substitutions I1572T, G1464E, G1449V, and Q1445H, all of which are located in the receptor's extracytoplasmic domain. Ligand binding was measured in plasma membranes from 293T cells expressing full-length receptors. No binding of 125I-IGF-II to I1572T mutant receptors was observed. Binding to G1449V mutant receptors was decreased by 50% relative to wild-type (WT). However, IGF-II binding to the G1464E and Q1445H mutant receptors was equivalent to WT when plasma membranes were assayed immediately after preparation. The phosphomannosylated pseudoglycoprotein pentamannose 6-phosphate-BSA (PMP-BSA) was synthesized as a ligand for the M6P binding site. Binding of 125I-PMP-BSA was equivalent to WT for the I1572T, G1464E, and Q1445H mutations, but there was a 60% reduction in PMP-BSA binding to the G1449V mutant receptor. Thus, several missense mutations in M6P/IGF2R disrupt the ligand binding functions of the intact IGF2R, lending further support to the hypothesis that the M6P/IGF2R is a tumor suppressor gene. (+info)
Identification of cation-independent mannose 6-phosphate receptor/insulin-like growth factor type-2 receptor as a novel target of autoantibodies.
Two human monoclonal autoantibodies, B-33 and B-24, were generated from the B cells of a patient with scleroderma. Both monoclonal antibodies (mAbs) were composed of mu and lambda chains, and recognized cytoplasmic vesicular structures by indirect immunofluorescence on Hep-2 cell line slides, although mAb B-24 showed an additional diffuse cytoplasmic staining pattern. By Western blot, mAb B-24 exhibited a polyreactive-like binding pattern, whereas mAb B-33 failed to recognize any electroblotted Hep-2 antigen. The polyreactive versus monospecific behaviour of mAbs B-24 and B-33 was further confirmed by enzyme-linked immunosorbent assay (ELISA) with a variety of foreign and autoantigens. The N-terminal sequence of a protein band isolated by affinity chromatography with mAb B-33 was identical to that of cation-independent mannose 6-phosphate receptor (CI-MPR), also known as the insulin-like growth factor type-2 receptor (IGF-2R). Immunofluorescence experiments on Hep-2 cell line slides demonstrated a striking co-localization between the staining pattern exhibited by these mAbs and the pattern obtained using a goat anti-CI-MPR serum, indicating the recognition by B-24 and B-33 of a structure located predominantly in late endosomes. Sequence analysis of the V-region gene segments of B-33 and B-24 showed both to be identical, except for the existence of a point mutation in B-33 located in the H-complementarity-determining region 3 (H-CDR3) (position 100D), which produces a non-conservative replacement of Gly by Ser. This single replacement appears to be responsible for the dramatic change in reactivity of human mAb B-33. The data shown here provide new evidence of the critical role played by the H-CDR3 region in distinguishing a polyspecific from a monospecific antibody. A population study demonstrated the existence of immunoglobulin G (IgG) reactivity against CI-MPR/IGF-2R in serum specimens from five individuals with different pathological conditions, thus indicating that this molecule is a potential target for the human autoimmune response. (+info)
Carbohydrate-deficient glycoprotein syndrome type 1A: expression and characterisation of wild type and mutant PMM2 in E. coli.
We have identified the PMM2 genotypes of 22 unrelated Danish patients with carbohydrate-deficient glycoprotein syndrome type 1A: R141H/F119L (18), R141H/C192G (1), F119L/F119L (1), F119L/G117R (1) and D223E/T237R (1). The lack of patients homozygous for R141H is statistically highly significant, but unexplained. In order to investigate the effect of PMM2 mutations on phosphomannomutase (PMM2) activity, PMM2-cDNA was cloned into a pET3a vector. Following introduction of mutations into PMM2-cDNA by site-specific mutagenesis, wild type and mutant PMM2-cDNA were expressed in E. coli Bl21(DE3) cells, and the activity of PMM2 was determined by an enzymatic assay using mannose 1-phosphate as substrate. Recombinant R141H, G117R, and T237R PMM2 had no detectable catalytic activity, and the F119L PMM2 had 25% of the activity of the wild type. The activity of the C192G and D223E PMM2 was in the normal range, but the affinity for their substrate was lower, and the proteins were more sensitive to increased temperatures. Each patient has at least one mutation which retains residual PMM2 activity. Our results support the hypotheses that a genotype conveying residual PMM2 catalytic activity is required for survival, and that homozygosity for R141H impairs PMM2 to a degree incompatible with life. (+info)
Enzymatic reduction of disulfide bonds in lysosomes: characterization of a gamma-interferon-inducible lysosomal thiol reductase (GILT).
Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter- and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin, but the mechanism of lysosomal reduction is unknown. We describe here a lysosomal thiol reductase optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro. The active site, determined by mutagenesis, consists of a pair of cysteine residues separated by two amino acids, similar to other enzymes of the thioredoxin family. The enzyme is a soluble glycoprotein that is synthesized as a precursor. After delivery into the endosomal/lysosomal system by the mannose 6-phosphate receptor, N- and C-terminal prosequences are removed. The enzyme is expressed constitutively in antigen-presenting cells and induced by IFN-gamma in other cell types, suggesting a potentially important role in antigen processing. (+info)