Phosphoglucose-containing glycoproteins in intracellular trafficking and neuronal recognition.
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Animals↗
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Reciprocal modulation of binding of lysosomal enzymes and insulin-like growth factor-II (IGF-II) to the mannose 6-phosphate/IGF-II receptor.
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Animals↗
Insulin-like growth factor II: complexity of biosynthesis and receptor binding.
Insulin-like growth factor II (IGF-II) belongs to the insulin family of peptides and acts as a growth factor in many fetal tissues and tumors. The gene expression of IGF-II is initiated at three different promoters which gives rise to multiple transcripts. In a human rhabdomyosarcoma cell line IN 157 IGF-II mRNAs of 6.0-kb, 4.8-kb, and 4.2-kb are present. Fractionation of cellular extracts on sucrose gradients and Northern blot analysis showed that only the 4.8-kb mRNA was associated with polysomes, whereas the other transcripts cosedimented with monosomal particles. This suggests that only the 4.8-kb mRNA is translated to IGF-II. The cell line secretes two forms of immunoreactive and bioactive IGF-II to the medium of molecular size 10 kd and 7.5 kd which may be involved in autocrine control of cell growth. IGF-II binds to two receptors on the surface of many cell types: the IGF-I receptor and the mannose-6-phosphate (Man-6-P)/IGF-II receptor. There is consensus that the cellular effects of IGF-II are mediated by the IGF-I receptor via activation of its intrinsic tyrosine kinase. The Man-6-P/IGF-II receptor is involved in endocytosis of lysosomal enzymes and IGF-II. In selected cell types, however, Man-6-P induces cellular responses. We have studied rat brain neuronal precursor cells where Man-6-P acted as a mitogen suggesting that phosphomannosylated proteins may act as growth factors via the Man-6-P/IGF-II receptor. In conclusion, the gene expression and mechanism of action of IGF-II is very complex suggesting that its biological actions can be regulated at different levels including the transcription, translation, posttranslational processing, receptor binding and intracellular signalling.
Animals↗
Targeting of proteins to the lysosome.
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Animals↗
Cathepsin D: a protease involved in breast cancer metastasis.
Cathepsin D is an acidic lysosomal protease present in all cells. In estrogen receptor positive and negative breast cancer cell lines, the mRNA coding for pro-cathepsin D is overexpressed and sorting and maturation of the pro-enzyme are altered, via possibly saturation of the Man-6-P/IGF-II receptor, leading to accumulation of the active proteinase in large endosomes and to secretion of the precursor (52K protein). In MCF7 cells, the cathepsin D mRNA is induced directly and transcriptionally by estrogens and indirectly by growth factors. In patients, there is a significant correlation between high cathepsin D concentrations in the cytosol of primary breast cancer and development of metastasis. This marker is independent of other prognostic factors and appears to be particularly useful in axillary node-negative tumors. Transfection of a human cDNA cathepsin D expression vector under the control of SV40 promoter increases the metastatic potential of 3YA1-Ad12 rat tumorigenic cells when intravenously injected into nude mice. The mechanism of cathepsin D-induced metastasis is currently unknown. These results indicate that overexpression of cathepsin D might facilitate breast cancer metastasis, suggesting new possible therapeutic approaches.
Breast Neoplasms↗
The effects of sucrose loading on lysosomal hydrolases.
The addition of 88 mM sucrose to the culture medium of human skin fibroblasts from normal subjects caused remarkable increase in the intracellular lysosomal hydrolase activities. The mechanism of this induction by sucrose loading was carefully studied with several fibroblast strains of different inherited lysosomal storage disorders. In single lysosomal hydrolase defect such as GM1-gangliosidosis, mannosidosis and Sandhoff disease, no induction of the deficient hydrolase was found with 88 mM sucrose loading. In contrast, sucrose loading caused normalization of intracellular lysosomal hydrolase activities in I-cell disease fibroblasts and cytoplasmic inclusion materials disappeared. Subsequent investigations reveal that I-cell disease cells are classified into three subgroups by the degree of hydrolase induction by sucrose loading; a high responding, an intermediate responding and a no-response group. The heterogeneity may be based upon different induction by sucrose loading of the enzyme, probably the residual phosphotransferase which is involved in the processing steps of lysosomal enzyme molecules. With the addition of mannose-6-phosphate and 10 mM NH4Cl to cultured skin fibroblasts, it was shown that sucrose loading caused increased synthesis of lysosomal enzyme proteins. The result of the test with 2,4-dinitrophenol suggests that sucrose is indeed pinocytosed by cultured human skin fibroblasts and localized in lysosomes and that this event is the essential factor to trigger the induction of lysosomal hydrolases. Simultaneous loading of both invertase and sucrose in cultured cells caused no induction of alpha-mannosidase activity. This result indicates that invertase is also pinocytosed, reaches the lysosomes and hydrolyzes sucrose in the lysosomes. Lysosomal overloading with sucrose resulted in induction of lysosomal hydrolases and invertase blocked the induction of alpha-mannosidase activity. However, some induction still exists in beta-galactosidase and alpha-fucosidase activity. Thus it is very likely that the induction of lysosomal hydrolases demands a complicated process. In this article, we investigated the effects of sucrose on the lysosomal hydrolases in cultured human skin fibroblasts of several inherited lysosomal storage disorders and normal subjects and discuss the possible mechanism of the induction of lysosomal hydrolase activities by sucrose loading.
2,4-Dinitrophenol↗
Dual anomeric specificity of phosphomannoisomerase assessed by 2D phase sensitive 13C EXSY NMR.
The reversible conversion between D-mannose 6-phosphate and D-fructose 6-phosphate catalyzed by yeast phosphomannoisomerase was studied by phase sensitive 2D 13C-(1H) EXSY NMR spectroscopy at 100.623 MHz, using 13C enriched substrates in the C2 position of the D-hexose 6-phosphates. The unique pair of isomerization cross-peaks observed in the 2D EXSY map correlates the 13C2 resonances of the beta-anomers of both D-[2-13C]-mannose 6-phosphate and D-[213C]-fructose 6-phosphate. This indicates that phosphomannoisomerase specifically catalyzes the reversible conversion between beta-D-mannose 6-phosphate and beta-D-fructose 6-phosphate. Since phosphoglucoisomerase was recently found to catalyze specifically the interconversion of alpha-D-glucose 6-phosphate and beta-D-fructose 6-phosphate, the beta-anomer of the ketohexose ester could be directly channeled in a multi-enzyme system involving phosphoglucoisomerase, phosphomannoisomerase and phosphofructokinase.
Fructosephosphates↗
Preliminary characterization of a Chinese hamster ovary cell glycosylation mutant isolated by screening for low intracellular lysosomal enzyme activity.
A novel screening procedure was developed for isolating Chinese hamster ovary cell mutants altered in the early steps of the biosynthesis of asparagine-linked glycoproteins. This procedure identifies cells with low intracellular levels of two lysosomal hydrolases, beta-glucuronidase and alpha-iduronidase. One mutant cell line isolated in this way, CHB 11-1-3, has low intracellular levels of seven lysosomal enzymes as compared to wild-type cells. Although CHB 11-1-3 synthesizes mannosylphosphoryldolichol and [Man]5[NAcGlcNH2]2-P-P-lipid, it fails to utilize these lipid intermediates to make normal amounts of [Glc]3[Man]9[NAcGlcNH2]2-P-P-lipid. As a consequence of this glycosylation defect, this mutant transfers oligosaccharides of a different structure than wild type to the lysosomal enzyme beta-hexosaminidase. In addition, it underglycosylates its proteins.
Animals↗
Glucose phosphotransferase and intracellular trafficking.
Glycoproteins containing phosphodiester-linked glucose residues have recently been described. The synthesis of this structure occurs due to the intact transfer of alpha glucose-1-phosphate from UDP-glucose and is catalyzed by the enzyme glucose phosphotransferase (GlcPTase). The endogenous acceptors for GlcPTase have been characterized as to molecular weight following incubation of selected homogenates with (beta 32P)UDP-glucose. These glycoproteins are distinct from the lysosomal hydrolases recognized by the GlcNAc phosphotransferase. The transfer of 32P from (beta 32P)UDP-Glc can also be detected when the nucleotide sugar is microinjected into the cytoplasm of individual neurons in Aplysia. The phosphorylated acceptors in this system seem to be predominantly two glycoproteins that are subjected to rapid axoplasmic transport. The possible role of this post-translational modification in the intracellular trafficking of a subset of newly synthesized glycoproteins is discussed.
Animals↗
Metabolic actions of insulin-like growth factor II in cultured adult rat hepatocytes are not mediated through the insulin-like growth factor II receptor.
Short- and long-term regulation of hepatic carbohydrate metabolism by insulin-like growth factor II was studied in primary cultures of adult rat hepatocytes and compared to the metabolic potency of insulin. Insulin-like growth factor II stimulated glycogen synthesis from [14C]glucose, uptake of [3H]aminoisobutyric acid and [14C]lactate formation from [14C]glucose up to three-fold. Basal glycogenolysis was inhibited to about 10%, and glucagon-activated glycogenolysis was blocked completely. The enzymatic activity of glucokinase and pyruvate kinase was induced two-fold, the glucagon-dependent induction of phosphoenolpyruvate carboxykinase was antagonized. Compared to insulin, half-maximal responses required up to 50 times higher insulin-like growth factor II concentrations ranging from 10-20 nmol/l. A similar difference was observed for binding affinity of insulin-like growth factor II to the insulin receptor. The interaction with the insulin-like growth factor II/mannose 6-phosphate (IGF-II/Man-6-P) receptor was examined by studying 125I-insulin-like growth factor II binding and uptake of lysosomal enzymes. The affinity of insulin-like growth factor II to the IGF-II/Man-6-P receptor was considerably higher than for the insulin receptor. Antibodies against the IGF-II/Man-6-P receptor did not affect metabolic responses to insulin-like growth factor II, while binding to its receptor and the receptor-mediated endocytosis of arylsulphatase A were strongly inhibited. Thus, in adult rat liver insulin-like growth factor II appeared to exert metabolic actions not via interaction with its own receptor but through low affinity binding to hepatic insulin receptors.
Aminoisobutyric Acids↗
Involvement of the macrophage mannose-6-phosphate receptor in the recognition of Leishmania mexicana amazonensis.
Significant differences were found in the ability of resident mouse peritoneal macrophages to ingest amastigote and promastigote forms of Leishmania mexicana amazonensis. Differences in the association index of the parasites to the macrophages were also found between infective and non-infective promastigotes. Evidence was obtained suggesting that the macrophage receptor, which recognizes mannose-6-phosphate-containing units found in lysosomal enzymes, is involved in the association with the macrophage of promastigotes, but not of amastigotes. Addition of mannose-6-phosphate, its structural analogue fructose-1-phosphate, Hansenula holstii phosphomannan or the mannose-6-phosphate-containing lysosomal enzyme alpha-D-mannosidase to the interaction medium, markedly inhibits the association of the parasites with macrophages.
Animals↗
Diagnosis of Hunter's syndrome carriers; radioactive sulphate incorporation into fibroblasts in the presence or fructose 1-phosphate.
Mutual correction of co-cultivated fibroblasts from patients with Hunter's and Hurler's syndrome could be inhibited by either fructose 1-phosphate or mannose 6-phosphate. In the presence of fructose 1-phosphate a 50% mixture of fibroblasts from a patient with Hunter's syndrome and a normal homozygous individual showed an increased 35S-sulphate incorporation into acid mucopolysaccharides. When fibroblast cultures from one obligate and two possible carriers of Hunter's syndrome were tested for 35S-sulphate incorporation, the cultures showed either twice the normal 35S-sulphate incorporation into acid mucopolysaccharides in the presence of fructose 1-phosphate or an abnormally high incorporation in the presence as well as in the absence of the sugar phosphate.
Adult↗
Changes in the glucose-6-phosphatase complex in hepatomas.
Hepatomas tend to have a decreased glucose-6-phosphatase activity. We have observed phenotypic stability for this change in Morris hepatomas transplanted in rats. To determine if this decrease is selective for translocase functions or the hydrolase activity associated with glucose-6-phosphatase, we have compared activities in liver and hepatomas with glucose-6-phosphate or mannose-6-phosphate as substrates and with intact or histone-disrupted microsomes. In five out of seven subcutaneously transplanted rat hepatoma lines, the microsomal mannose-6-phosphatase activity was lower than in preparations from liver of normal or tumor-bearing rats. With liver microsomes and with most hepatoma microsomes, preincubation with calf thymus histones caused a greater increase in mannose-6-phosphatase than in glucose-6-phosphatase activity. In studies with liver and hepatoma microsomes there were similar increases in mannose-6-phosphatase activity with total calf thymus histones and arginine-rich histones. A smaller increase was seen with lysine-rich histones. The effect of polylysine was similar to the action of lysine-rich histones. There was only a small effect with protamine at the same concentration (1 mg/ml). Rat liver or hepatoma H1 histones gave only about half the activation seen with core nucleosomal histones. Our data suggested that microsomes of rat hepatomas tend to have decreased translocase and hydrolase functions of glucose-6-phosphatase relative to activities in untransformed liver.
Animals↗
Role of cytoskeleton and surface lectins in Giardia duodenalis attachment to Caco2 cells.
An in vitro model of Giardia duodenalis and the Caco2 cell line enable the study of parameters that could play a part in trophozoite attachment. We explored the role of membranous lectins of G. duodenalis in attachment-inhibition studies using carbohydrates in solution. Attachment rates were reduced by 14% and 23% in the presence of 100 mmol/l mannose-6-phosphate and glucose, respectively, as compared with control values. No significant modification was observed after trophozoite trypsinization at room temperature or at 37 degrees C. The inhibitory effects of colchicine (35%) and nocodazole (70%) suggest a primordial role of the cytoskeleton; microtubules appear to be the principal effectors of trophozoite fixation. Scanning electron microscopy revealed circular imprints on the Caco2 brush border after trophozoite detachment. The mechanisms of attachment of G. duodenalis to intestinal enterocyte-like cells in culture are thus essentially of the mechanical or hydrodynamic type; surface lectins would appear to intervene in the specificity for duodenal cells.
Animals↗
Studies on natural, antibody-dependent, and interleukin-2-activated killer-cell activity of a patient with mucolipidosis III as a test of the mannose-6-phosphate lytic acceptor hypothesis.
The natural (NK), antibody-dependent (K), and interleukin-2-generated (LAK) killer-cell activity of a patient with mucolipidosis III (ML III; an autosomal recessive defect in UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase) was studied to determine whether or not the defect in the phosphorylation of lysosomal enzyme mannose residues resulted in a failure of target-cell lysis, as would be predicted from recent studies showing NK inhibition by phosphorylated sugars, especially mannose-6-phosphate. The patient was studied in parallel with normal donors known to be at the high and low extremes of NK activity. The following results were obtained: NK activity was markedly elevated against K562, Molt-4, human fibroblasts, HL-60, and MeWo to levels approximately one to two times that of our previous highest donor and five times the mean of normal donors previously tested. Interleukin-2-generated killer-cell activity and antibody-dependent cell-mediated cytotoxicity against antibody-coated P815 cells were normal and increased, respectively. HNK-1-positive cells were normal in frequency (7.3 +/- 1.7%), while lytic conjugates were proportional to activity (3.9 +/- 0.6 vs 2.7 +/- 0.4% for the "high" donor), and this was attributable to an increased proportion of lytic cells. The addition of fresh serum from the ML III patient had no effect on the NK activity of normal donors and the effects of preincubation with interferon (enhancement), monensin (inhibition), fructose-6-phosphate (inhibition), and mannose-6-phosphate (inhibition) were identical to those seen using lymphocytes from normal donors. Studies on the NK activity of the parents and two normal female siblings showed that the father's NK activity was high, the mother's was low, and both siblings' was intermediate but low. The data obtained suggest that the inability of lymphocytes to phosphorylate lysosomal enzyme mannose residues had no effect on NK-, K-, or LAK-cell function and that the mechanism of target-cell lysis is independent of either a mannose-6-phosphate-bearing lytic moiety or a mannose-6-phosphate-dependent ligand mechanism.
Adult↗
[Mannose-6-phosphate receptors: their role in the transport of lysosomal proteins].
Targetting of newly synthesized lysosomal enzymes to lysosomes requires mannose 6-phosphate receptors. These receptors shuttle between the Golgi apparatus, an organelle of the secretory route, and acidic prelysosomal organelles, which are part of the endocytic route. Vectorial transport of the ligands is ensured by the pH dependence of ligand binding. Ligands are bound at near-neutral pH in the Golgi apparatus and released in the acidic prelysosomal organelles.
Animals↗
Biosynthesis of lysosomal enzymes in cells of the End3 complementation group conditionally defective in endosomal acidification.
Various aspects of lysosome biogenesis have been studied in Chinese hamster ovary (CHO) cells of the End3 complementation group (designated G.7.1 cells), which display a temperature-sensitive defect in the acidification of endosomes, but not lysosomes. In G.7.1 and normal wild-type cells grown at the permissive temperature (34 degrees C), the lysosomal enzymes alpha-glucosidase and cathepsin D were synthesized as high-molecular-weight precursors that subsequently underwent intracellular proteolytic processing to yield lower molecular weight mature forms. The mature forms of the enzymes were retained in cells, and small amounts of each precursor were secreted. However, in G.7.1 cells grown at the restrictive temperature (41 degrees C), there was a massive and inappropriate oversecretion of lysosomal enzyme precursors, which resulted in very little of the mature forms being processed and retained by the cells. This mistargeting of lysosomal enzymes was not due to an absence of phosphorylated oligosaccharides on the enzymes, nor to a defect in mannose 6-phosphate (Man6P) receptors. However, it was found that whereas G.7.1 cells had the same number of cell surface Man6P receptors at 34 degrees C and 41 degrees C, the rate of accumulation and degradation of Man6P-containing ligands was about two to three times more rapid in cells maintained at the permissive temperature. There did not appear to be any gross changes in Golgi function as the oligosaccharides of alpha-glucosidase and the Man6P receptor were processed in a similar fashion at both 34 degrees C and 41 degrees C. In addition to these studies, electron microscopic observations revealed that at 41 degrees C, G.7.1 cells accumulated inclusion-type bodies reminiscent of those found in I-cell disease fibroblasts. Thus, the biochemical and electron microscopic results on G.7.1 cells provide further evidence that acidified endosomes are important for the biogenesis of lysosomes.
Animals↗
Normal exocytosis and endocytosis of lysosomal beta-hexosaminidase in a case of alpha 1-antitrypsin deficiency.
Secretion of lysosomal beta-hexosaminidase by cultivated skin fibroblasts and receptor-mediated endocytosis of leucocyte beta-hexosaminidase from a patient by cultivated non-parenchymal rat liver cells and skin fibroblasts were similar to that of a control proband. The results suggest normal oligosaccharide side chains of high mannose type on lysosomal enzymes in alpha 1-antitrypsin (AAT) deficiency.
Animals↗