Increased LAMP-2 polylactosamine glycosylation is associated with its slower Golgi transit during establishment of a polarized MDCK epithelial monolayer.

Nabi, Ivan R.; Rodríguez-Boulan, Enrique

doi:10.1091/mbc.4.6.627

Cited by 26 publications

(17 citation statements)

References 59 publications

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“…All cell culture reagents were obtained from GIBCO͞Life Technologies (Grand Island, NY) unless otherwise indicated. RPE-J cells were maintained in DMEM supplemented with 4% CELLECT Gold FBS (ICN), nonessential amino acids, glutamine, and penicillin͞ streptomycin at 32°C in a 95% air͞5% CO 2 environment as described (21,22). All assays were carried out on cells seeded at a density of 3.5 ϫ 10 5 cells͞cm 2 on Matrigel-coated 1.2-cmdiameter Transwell filters.…”

Section: Methodsmentioning

confidence: 99%

Saturation of, and competition for entry into, the apical secretory pathway

Marmorstein¹

2000

Proceedings of the National Academy of Sciences

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To investigate mechanisms of apical sorting in the secretory pathway of epithelial cells, we expressed varying amounts of the 165 amino acid isoform of vascular endothelial growth factor (VEGF 165) and transforming growth factor ␤1 (TGF-␤1) via replication defective adenoviruses. Apical sorting of both proteins was efficient at low expression levels but saturated or was reversed at high expression levels. High expression levels of TGF-␤1 were effective at competing VEGF 165 out of the apical pathway; however, VEGF165 did not compete out TGF-␤1. Tunicamycin inhibition experiments showed that the apical polarity of VEGF165 was independent of N-glycosylation. We conclude that the apical sorting of these two molecules is a saturable, signal-mediated process, involving competition for apical sorting receptors. The sorting of the two proteins does not appear to involve N-glycans as sorting signals, or lectin sorters. The observations are particularly relevant to gene therapy because they demonstrate that overexpression of a transgene can result in undesirable missorting of the encoded protein.

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Section: Methodsmentioning

confidence: 99%

Saturation of, and competition for entry into, the apical secretory pathway

Marmorstein¹

2000

Proceedings of the National Academy of Sciences

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“…Cells were fixed with 3% paraformaldehyde for 15 minutes, washed with PBS supplemented with 0.1 mM Ca 2+ and 1 mM Mg 2+ (PBS-CM) and then incubated for 15 minutes with PBS-CM supplemented with 0.2% BSA to reduce non-specific binding and 0.07% saponin to permeabilize cellular membranes. AC17 anti-LAMP-2 antibody (Nabi et al, 1991;Nabi and Rodriguez-Boulan, 1993) was used to determine the cellular distribution of LAMP-2 using Alexa 488 goat anti-mouse IgG as secondary antibody. Intracellular cholesterol was visualized by filipin labelling (1:25 of a stock solution of 10 mg/ml in DMSO).…”

Section: Immunofluorescencementioning

confidence: 99%

The lipid composition of autophagic vacuoles regulates expression of multilamellar bodies

Lajoie

Guay

Dennis

et al. 2005

Journal of Cell Science

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Multilamellar bodies (MLBs) are responsible for surfactant secretion in type II alveolar cells but also accumulate in other cell types under pathological conditions, including cancer and lysosomal storage diseases such as Niemann-Pick C (NPC), a congenital disease where defective cholesterol transport leads to its accumulation in lysosomes. Mv1Lu type II alveolar cells transfected with Golgi β1,6 N-acetylglucosaminyltransferase V (Mgat5), enhancing the polylactosamine content of complex-type N-glycans, exhibit stable expression of MLBs whose formation requires lysosomal proteolysis within dense autophagic vacuoles. MLBs of Mgat5-transfected Mv1Lu cells are rich in phospholipids and have low levels of cholesterol. In Mv1Lu cells treated with the NPC-mimicking drug U18666A, cholesterol-rich MLBs accumulate independently of both Mgat5 expression and lysosomal proteolysis. Inhibition of autophagy by blocking the PI 3-kinase pathway with 3-methyladenine prevents MLB formation and results in the accumulation of non-lamellar, acidic lysosomal vacuoles. Treatment with 3-methyladenine inhibited the accumulation of monodansylcadaverine, a phospholipid-specific marker for autophagic vacuoles, but did not block endocytic access to the lysosomal vacuoles. Induction of autophagy via serum starvation resulted in an increased size of cholesterol-rich MLBs. Although expression of MLBs in the Mv1Lu cell line can be induced by modulating lysosomal cholesterol or protein glycosylation, an autophagic contribution of phospholipids is critical for the formation of concentric membrane lamellae within late lysosomal organelles.

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“…After fixation, the cells were rinsed extensively with PBS, pH 7.4, supplemented with 0.1 mM Ca 2ϩ and 1 mM Mg 2ϩ (PBS/CM), and then incubated for 15 min with PBS/CM containing 0.5% BSA at room temperature to reduce nonspecific binding. LAMP-2 distribution was determined using the AC17 anti-LAMP-2 antibody (Nabi et al, 1991;Nabi and Rodriguez-Boulan, 1993) followed by FITC-or Texas Red-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA). Detection of the distribution of L-PHA reactivity was performed using rhodamine-conjugated L-PHA (E-Y Laboratories, San Mateo, CA).…”

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confidence: 99%

“…Increased expression of polylactosamine and the associated Lewis and blood group antigens are carcinoma markers (Fukuda, 1985;Hakamori, 1989). Modified expression of polylactosamine is also associated with cellular differentiation of various cell types (Spillmann and Finne, 1987;Youakim et al, 1989;Amos and Lotan, 1990;Lee et al, 1990;Tuo et al, 1992;Nabi and Rodriguez-Boulan, 1993). The decreasing polylactosamine glycosylation of the lysosomal LAMP glycoproteins in cultured epithelial cells with time in culture is modulated independently of glycosyltransferase activities (Brockhausen et al, 1991;Nabi and Dennis, 1998), and polylactosamine glycosylation has been shown to be regulated by the Golgi residence time of the protein (Wang et al, 1991;Nabi and Rodriguez-Boulan, 1993;Nabi and Dennis, 1998).…”

mentioning

confidence: 99%

“…Modified expression of polylactosamine is also associated with cellular differentiation of various cell types (Spillmann and Finne, 1987;Youakim et al, 1989;Amos and Lotan, 1990;Lee et al, 1990;Tuo et al, 1992;Nabi and Rodriguez-Boulan, 1993). The decreasing polylactosamine glycosylation of the lysosomal LAMP glycoproteins in cultured epithelial cells with time in culture is modulated independently of glycosyltransferase activities (Brockhausen et al, 1991;Nabi and Dennis, 1998), and polylactosamine glycosylation has been shown to be regulated by the Golgi residence time of the protein (Wang et al, 1991;Nabi and Rodriguez-Boulan, 1993;Nabi and Dennis, 1998). Increased GlcNAc-TV expression is associated with increased polylactosamine glycosylation in oncogenically transformed and undifferentiated cell lines (Yamashita et al, 1985;Heffernan et al, 1989;Yousefi et al, 1991), indicating that GlcNAc-TV expression levels can regulate the expression of polylactosamine oligosaccharides.…”

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confidence: 99%

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Biogenesis of Multilamellar Bodies via Autophagy

Hariri

Millane

Guimond

et al. 2000

MBoC

164

142

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Transfection of Mv1Lu mink lung type II alveolar cells with ␤1-6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the ␤1-6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella. INTRODUCTIONMultilamellar bodies (MLBs) are membrane-bound cellular organelles, which vary in size from 100-2400 nm, are composed of concentric membrane layers, and frequently exhibit an electron-dense core. MLBs are found in numerous cell types where they function in lipid storage and secretion (Schmitz and Mü ller, 1991). In lung type II alveolar cells, MLBs function as secretory granules whose exocytosis results in the deposition of the tubular myelin forms of surfactant on the surface of the alveolae (Hatasa and Nakamura, 1965;Ryan et al., 1975;Williams, 1977). The surfactant film over the alveolar epithelium regulates the surface tension at the air-cell interface and protects the alveola from collapse during respiration (Haagman and van Golde, 1991).Although the secretory function of MLBs in type II alveolar cells is well established, the precise mechanism of MLB biogenesis remains unclear. Autoradiographic studies of murine type II alveolar cells of mouse lungs showed that although phospholipids labeled with [ 3 H]choline are delivered directly from the Golgi to the MLB, proteins metabolically labeled with [ 3 H]leucine are visualized within multivesicular bodies before delivery to MLBs (Chevalier and Collet, 1972). Surfactant proteins A, B, and C are delivered via multivesicular bodies to MLBs, and multivesicular bodies are proposed as th...

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Increased LAMP-2 polylactosamine glycosylation is associated with its slower Golgi transit during establishment of a polarized MDCK epithelial monolayer.

Cited by 26 publications

References 59 publications

Saturation of, and competition for entry into, the apical secretory pathway

Saturation of, and competition for entry into, the apical secretory pathway

The lipid composition of autophagic vacuoles regulates expression of multilamellar bodies

Biogenesis of Multilamellar Bodies via Autophagy

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