Chaperone function of calnexin for the folding intermediate of gp80, the major secretory protein in MDCK cells. Regulation by redox state and ATP.

To assess the participation of the 150-kDa oxygen-regulated protein (ORP150) in protein transport, its function in Madin-Darby canine kidney (MDCK) cells was studied. Exposure of MDCK cells to hypoxia resulted in an increase of ORP150 antigen and increased binding of ORP150 to GP80/clusterin (80-kDa glycoprotein), a natural secretory protein in this cell line. In ORP150 antisense transformant MDCK cells, GP80 was retained within the endoplasmic reticulum after exposure to hypoxia. Metabolic labeling showed the delay of GP80 maturation in antisense transformants in hypoxia, whereas its matured form was detected in wild-type cells, indicating a role of ORP150 in protein transport, especially in hypoxia. The affinity chromatographic analysis of ORP150 suggested its ability to bind to ATP-agarose. Furthermore, the ATP hydrolysis analysis showed that ORP150 can release GP80 at a lower ATP concentration. These data indicate that ORP150 may function as a unique molecular chaperone in renal epithelial cells by facilitating protein transport/maturation in an environment where less ATP is accessible.

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The Environmental Pollutant Cadmium Promotes Influenza Virus Replication in MDCK Cells by Altering Their Redox State

International Journal of Molecular Sciences ◽

10.3390/ijms14024148 ◽

2013 ◽

Vol 14 (2) ◽

pp. 4148-4162 ◽

Cited By ~ 20

Author(s):

Paola Checconi ◽

Rossella Sgarbanti ◽

Ignacio Celestino ◽

Dolores Limongi ◽

Donatella Amatore ◽

...

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Redox State ◽

Mdck Cells ◽

Environmental Pollutant ◽

Influenza Virus Replication

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A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells.

The Journal of Cell Biology ◽

10.1083/jcb.109.5.2145 ◽

1989 ◽

Vol 109 (5) ◽

pp. 2145-2156 ◽

Cited By ~ 311

Author(s):

M P Lisanti ◽

I W Caras ◽

M A Davitz ◽

E Rodriguez-Boulan

Keyword(s):

Recombinant Dna ◽

Mdck Cells ◽

Apical Cell ◽

Human Growth ◽

Secretory Protein ◽

Protein Domain ◽

Intestinal Cell ◽

Apical Surface ◽

Glycosyl Phosphatidylinositol ◽

Membrane Anchoring

Glycosyl-phosphatidylinositol- (GPI) anchored proteins contain a large extracellular protein domain that is linked to the membrane via a glycosylated form of phosphatidylinositol. We recently reported the polarized apical distribution of all endogenous GPI-anchored proteins in the MDCK cell line (Lisanti, M. P., M. Sargiacomo, L. Graeve, A. R. Saltiel, and E. Rodriguez-Boulan. 1988. Proc. Natl. Acad. Sci. USA. 85:9557-9561). To study the role of this mechanism of membrane anchoring in targeting to the apical cell surface, we use here decay-accelerating factor (DAF) as a model GPI-anchored protein. Endogenous DAF was localized on the apical surface of two human intestinal cell lines (Caco-2 and SK-CO15). Recombinant DAF, expressed in MDCK cells, also assumed a polarized apical distribution. Transfer of the 37-amino acid DAF signal for GPI attachment to the ectodomain of herpes simplex glycoprotein D (a basolateral antigen) and to human growth hormone (a regulated secretory protein) by recombinant DNA methods resulted in delivery of the fusion proteins to the apical surface of transfected MDCK cells. These results are consistent with the notion that the GPI anchoring mechanism may convey apical targeting information.

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Polarized GP2 secretion in MDCK cells via GPI targeting and apical membrane-restricted proteolysis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1996.270.1.g176 ◽

1996 ◽

Vol 270 (1) ◽

pp. G176-G183 ◽

Cited By ~ 1

Author(s):

B. A. Fritz ◽

A. W. Lowe

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Apical Membrane ◽

Mdck Cells ◽

Apical Cell ◽

Secretory Protein ◽

Apical Plasma Membrane ◽

Zymogen Granule ◽

Gpi Anchor ◽

Pancreatic Acini

The major zymogen granule membrane protein in the exocrine pancreas is glycoprotein 2 (GP2), a glycosyl phosphatidylinositol (GPI)-linked membrane protein. Despite its GPI anchor, GP2 is secreted into the pancreatic duct. We examined the mechanism underlying the secretion of GP2 in isolated pancreatic acini and transfected Madin-Darby canine kidney (MDCK) cells (MDCK-GP2). MDCK-GP2 cells release GP2 almost exclusively (> 95%) from the apical membrane. Using GP2 as a model, we defined a novel mechanism of polarized protein secretion in which a secretory protein is targeted via a GPI anchor to the apical plasma membrane, whereupon the mature form is released by proteolysis. Furthermore, we described two features of MDCK cells that enhance the polarized release of GP2: an apical plasma membrane-restricted distribution of the protease responsible for GP2 membrane cleavage, and a transcytotic pathway to reroute basolateral plasma membrane GP2 to the apical cell surface.

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An anchor-minus form of the polymeric immunoglobulin receptor is secreted predominantly apically in Madin-Darby canine kidney cells.

The Journal of Cell Biology ◽

10.1083/jcb.105.5.2031 ◽

1987 ◽

Vol 105 (5) ◽

pp. 2031-2036 ◽

Cited By ~ 44

Author(s):

K E Mostov ◽

P Breitfeld ◽

J M Harris

Keyword(s):

Mdck Cells ◽

Secretory Protein ◽

Site Directed Mutagenesis ◽

Polymeric Immunoglobulin Receptor ◽

Apical Surface ◽

Basal Secretion ◽

Madin Darby Canine Kidney ◽

Immunoglobulin Receptor ◽

Darby Canine Kidney ◽

Canine Kidney

The polymeric immunoglobulin receptor is expressed in a variety of polarized epithelial cells. Newly made receptor travels first to the basolateral surface. The receptor is then endocytosed, transported across the cell in vesicles, and exocytosed at the apical surface. We have now deleted the membrane spanning and cytoplasmic portions of the receptor by site-directed mutagenesis, thus converting the receptor to a secretory protein. When expressed in polarized Madin-Darby canine kidney (MDCK) cells the truncated protein is secreted at both surfaces, with a ratio of apical-to-basal secretion of 3.4. In contrast, when the exogenous secretory protein chicken lysozyme is expressed in these cells, it is released at both sides with a ratio of apical-to-basal secretion of 0.43. (Koder-Koch, C., R. Bravo, S. Fuller, D. Cutler, and H. Garoff, 1985, J. Cell Biol., 43:297-306). Lysozyme is thought to lack a signal that targets it to one surface or the other, and so its secretion may represent a default, bulk flow pathway to both surfaces. When compared with lysozyme, the truncated polymeric immunoglobulin receptor is preferentially secreted apically by a factor of 3.4:0.43 or 7.8. We suggest that the lumenal portion of the polymeric immunoglobulin receptor contains a signal that targets it to the apical surface.

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Polarized targeting of epithelial cell proteins in thyrocytes and MDCK cells

Journal of Cell Science ◽

10.1242/jcs.112.8.1247 ◽

1999 ◽

Vol 112 (8) ◽

pp. 1247-1256 ◽

Cited By ~ 1

Author(s):

D. Prabakaran ◽

R.S. Ahima ◽

J.W. Harney ◽

M.J. Berry ◽

P.R. Larsen ◽

...

Keyword(s):

Growth Hormone ◽

Mdck Cells ◽

Basolateral Membrane ◽

Human Growth ◽

Cell Types ◽

Secretory Protein ◽

Secretory Proteins ◽

Cellular Mechanisms ◽

Polarized Trafficking ◽

Zinc Addition

Polarized trafficking signals may be interpreted differently in different cell types. In this study, we have compared the polarized trafficking of different proteins expressed endogenously in primary porcine thyroid epithelial cells to similar proteins expressed in MDCK cells. As in MDCK cells, NH4Cl treatment of filter-grown thyrocytes caused mis-sorted soluble proteins to exhibit enhanced secretion to the apical medium. In independent studies, thrombospondin 1 (a thyroid basolaterally secreted protein) was secreted basolaterally from MDCK cells. Likewise, the 5′-deiodinase (a thyroid basolateral membrane protein) encoded by the DIO1 gene was also distributed basolaterally in transfected MDCK cells. Consistent with previous reports, when the secretion of human growth hormone (an unglycosylated regulated secretory protein) was examined from transfected MDCK cells, the release was nonpolarized. However, transfected thyrocytes secreted growth hormone apically in a manner dependent upon zinc addition. Moreover, two additional regulated secretory proteins expressed in thyrocytes, thyroglobulin (the major endogenous glycoprotein) and parathyroid hormone (an unglycosylated protein expressed transiently), were secreted apically even in the absence of zinc. We hypothesize that while cellular mechanisms for interpreting polarity signals are generally similar between thyrocytes and MDCK cells, thyrocytes allow for specialized packaging of regulated secretory proteins for apical delivery, which does not require glycosylation but may involve availability of certain ions as well as appropriate intracellular compartmentation.

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Sorting of a secretory protein (gp80) to the apical surface of Caco-2 cells

Journal of Cell Science ◽

10.1242/jcs.107.2.553 ◽

1994 ◽

Vol 107 (2) ◽

pp. 553-559

Author(s):

D. Appel ◽

C. Koch-Brandt

Keyword(s):

Mdck Cells ◽

Secretory Protein ◽

Secretory Proteins ◽

Apical Surface ◽

Glycoprotein Complex ◽

Polarized Secretion ◽

Gene Coding ◽

Bona Fide ◽

Exocytic Pathway ◽

Darby Canine Kidney

We have investigated the synthesis and polarized secretion of the exogenous gp80 glycoprotein complex in the human epithelial adenocarcinoma cell line, Caco-2. gp80 is secreted at the apical surface of Madin-Darby canine kidney (MDCK) cells and should, therefore, display the signal(s) required for sorting into the apical exocytic pathway. In Caco-2 cells, no bona fide secretory protein released preferentially at the apical surface has been described so far. To address the question of whether Caco-2 cells possess a machinery capable of delivery of secretory proteins at the apical surface, we stably transfected the cells with a recombinant gene coding for the gp80 glycoprotein complex. Pulse-chase analysis showed that stably transfected Caco-2 cells secrete gp80 quantitatively into the medium. In polarized layers of filter-grown Caco-2 cells, the protein was secreted predominantly at the apical surface, demonstrating the ability of the cells to efficiently sort secretory proteins directly into the apical exocytic pathway. Our results further demonstrate that the apical targeting information of gp80 recognized by MDCK cells is also recognized by Caco-2 cells.

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