Sugar‐specific endocytosis of glycoproteins by Lewis lung carcinoma cells

Roche, Annie‐Claude; Barzilay, M.; Midoux, Patrick; Junqua, Simone; Sharon, Nathan; Monsigny, Michel

doi:10.1002/jcb.240220302

Cited by 117 publications

(45 citation statements)

References 20 publications

(7 reference statements)

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“…Neoglycoproteins were prepared by coupling glycosylphenyl-isothiocyanate to BSA; fluoresceinylated neoglycoproteins were obtained by using fluorescein isothiocyanate as described (Roche et al, 1983;Monsigny et al, 1984). The fluoresceinylated glycoproteins were purified by gel filtration on Trisacryl GF05 using n-butanol/ water (5:95) and freeze dried.…”

Section: Immunofluorescence Microscopymentioning

confidence: 99%

ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins.

Itin

Roche

Monsigny

et al. 1996

MBoC

166

148

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Based on sequence homologies with leguminous lectins, the intermediate compartment marker proposed to be a member of a putative new class of animal lectins associated with the secretory pathway. Independently, a promyelocytic protein, MR60, was purified by mannose-column chromatography, and a cDNA was isolated that matched MR60 peptide sequences. This cDNA was identical to that of and homologies with the animal lectin family of the galectins were noticed. Not all peptide sequences of MR60, however, were found in ERGIC-53, raising the possibility that another protein associated with ERGIC-53 may possess the lectin activity. Here, we provide the first direct evidence for a lectin function of ERGIC-53. Overexpressed ERGIC-53 binds to a mannose column in a calcium-dependent manner and also co-stains with mannosylated neoglycoprotein in a morphological binding assay. By using a sequential elution protocol we show that ERGIC-53 has selectivity for mannose and low affinity for glucose and GlcNAc, but no affinity for galactose. To experimentally address the putative homology of ERGIC-53 to leguminous lectins, a highly conserved protein family with an invariant asparagine essential for carbohydrate binding, we substituted the corresponding asparagine in ERGIC-53. This mutation, as well as a mutation affecting a second site in the putative carbohydrate recognition domain, abolished mannosecolumn binding and co-staining with mannosylated neoglycoprotein. These findings establish ERGIC-53 as a lectin and provide functional evidence for its relationship to leguminous lectins. Based on its monosaccharide specificity, domain organization, and recycling properties, we propose ERGIC-53 to function as a sorting receptor for glycoproteins in the early secretory pathway.

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Section: Immunofluorescence Microscopymentioning

confidence: 99%

ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins.

Itin

Roche

Monsigny

et al. 1996

MBoC

166

148

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“…On this basis, mannosylated glycoproteins could potentially be used to target and enhance ON uptake by the AMs. Targeted delivery of several cytotoxic drugs and antiviral agents by glycoproteins specific to macrophages has been demonstrated previously (14,15). Moreover, 6-phosphomannosylated glycoproteins have also been used to target antisense ONs to peritoneal macrophages (16).…”

mentioning

confidence: 99%

Antisense Inhibition of Silica-induced Tumor Necrosis Factor in Alveolar Macrophages

Rojanasakul¹,

Weissman

Shi

et al. 1997

Journal of Biological Chemistry

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Tumor necrosis factor-␣ (TNF␣) has been shown to play an important role in the pathogenesis of silicotic fibrosis. In this study, antisense oligonucleotides targeted to TNF␣ mRNA were used to inhibit silica-induced TNF␣ gene expression in alveolar macrophages. To achieve macrophage-specific oligonucleotide delivery, a molecular conjugate consisting of mannosylated polylysine that exploits endocytosis via the macrophage mannose receptor was used. Complexes were formed between the mannosylated polylysine and oligonucleotides and added to the cells in the presence of silica. Enzyme-linked immunoadsorbent assay showed that the complex consisting of the conjugate and antisense oligomer effectively inhibited TNF␣ production, whereas the oligomer alone had much less effect. Reverse transcriptase-polymerase chain reaction analysis revealed that the reduction in TNF␣ secretion was associated with specific ablation of targeted TNF␣ mRNA. The conjugate alone or conjugate complexed with inverted or sense sequence oligonucleotide had no effect. The promoting effect of the conjugate on antisense activity was shown to be due to enhanced cellular uptake of the oligomer via mannose receptor-mediated endocytosis. Cells lacking mannose receptors showed no susceptibility to the conjugate treatment. These results indicate that effective and selective inhibition of macrophage TNF␣ expression can be achieved using the antisense mannosylated polylysine system.

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“…Radiolabeled ligands allowed a sensitive determination of the number of receptors, but the study of the uptake and of the fate of internalized ligands involves subcellular fractionation, which is a time-consuming experiment. Fluorescence methods, such as quantitative spectrofluorimetry involving the lysis of the cells (38,441 and a method involving two excitation wavelengths (20,34,48), have been developed to study the endocytosis process; the latter method also allows the measure of the pH of the endocytic vesicles. The fluorescein fluorescence is very dependent on the pH (3,4,26), so the cell fluorescence upon internalization of fluorescein-labeled ligand has been demonstrated to decrease upon entry in acidic compartments (31,33).…”

mentioning

confidence: 99%

“…They measured the kinetics of ligand internalization into acidic endosomes and lysosomes by determining the cellassociated fluorescence, either in the presence or in the absence of a pH equilibrating agent (e.g., chloroquine). The present method was set up by studying the endocytosis of fluorescein-labeled a-glucosylated bovine serum albumin (BSA) previously proved to bind a sugar-binding receptor present on Lewis lung carcinoma (3LL) cells (38). The method takes into account the fluorescence properties of fluorescein-labeled ligands and various properties of cell compartments: the luminal part of endosomes and lysosomes is acidic, the fluorescence of fluorescein bound to a protein is lower than that of free fluorescein, the fluorescence of the ligand depends upon the number of fluorophore molecules bound to a protein and upon its pH environment.…”

mentioning

confidence: 99%

Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow cytometry

1987

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The fluorescence properties of the fluorescein residues bound to a protein are used to analyze by flow cytometry the neoglycoproteins' endocytosis mediated by membrane lectins of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein is dependent on the number of fluorophore molecules bound to a protein molecule and the pH of the environmental medium. The mean fluorescence intensity of a fluorescein molecule bound to a protein decreases when the number of fluorescein residues per protein molecule increases. However, after proteolytic digestion, the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The binding of fluorescein-labeled a-glucosylated serum albumin to 3LL cells at 4'C can easily be determined by flow cytometry because under these conditions the environmental pH is neutral, and the neoglycoprotein is not degraded. When the cells are incubated at 37OC in the presence of a fluorescein-labeled neoglycoprotein, the fluorescence intensity of a cell is low because of the low pH of endosomes and lysosomes but is increased upon a postincubation at 4'C in the presence of monensin, a protonlsodium ionophore. The extent of the proteolytic digestion of an endocytosed neoglycoprotein can be assessed by comparing, upon a monensin postincubation at 4OC, the high cell-associated fluorescence of cells incubated in the absence of leupeptin (an inhibitor of lysosomal proteases) and the relatively low fluorescence intensity of cells incubated in the presence of leupeptin.Key terms: Monensin, leupeptin, tumor cells, Lewis lung carcinoma, endogenous lectins A variety of molecules (proteins, glycoproteins, hormones, toxins, and viruses) are recognized by specific receptors on the surface of cells and are brought into cells by receptor-mediated endocytosis (35,36,42,50). During this process, ligands are internalized into endocytic vesicles derived from the plasma membrane. Although the exact pathway followed by the ligand is not known, the key step is a rapid acidification of endocytic vesicles (52), allowing the dissociation of ligands from their receptors (51). The fate of internalized material markedly differs from one to another ligand, but when internalized ligand-receptor complexes are dissociated, ligands are usually delivered to lysosomes where proteolysis occurs. During the past few years, the endocytosis and the catabolism of ligands such as asialoglycoproteins (1,6,(17)(18)(19)39), mannose terminated glycoproteins (41,49), alpha-2-macroglobulin (231, low density lipoprotein (7), transferrin (10,47), immune complexes (14,25), antibodies (28), and epidermal growth factor (2,s) have been studied by biochemical and morphological methods using radiolabeled ligands. Radiolabeled ligands allowed a sensitive determination of the number of receptors, but the study of the uptake and of the fate of internalized ligands involves subcellular fractionation, which is a time-consuming experiment. Fluorescence methods, such as quantitative ...

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Sugar‐specific endocytosis of glycoproteins by Lewis lung carcinoma cells

Cited by 117 publications

References 20 publications

ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins.

ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins.

Antisense Inhibition of Silica-induced Tumor Necrosis Factor in Alveolar Macrophages

Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow cytometry

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