Abstract
Two lectin-resistant mutants derived from Madin Darby canine kidney cells, with constitutive alterations in the asparagine-linked carbohydrate moieties, retained the characteristic structural and functional epithelial polarity of the parental cells. A ricin-resistant cell line was unable to incorporate galactose-sialic acid into glycoproteins and, from the pattern of cross-resistance to other lectins, appears to be different from previously described lines resistant to this lectin: the mutation in a concanavalin A-resistant line results, probably, in the production of defective carbohydrate cores of glycoproteins. In spite of glycosylation defects which result in an increased electrophoretic mobility of many cellular glycoproteins, both mutants retained the typical asymmetric structure of the plasma membrane (microvilli on the apical surface, junctional elements on the basolateral surface), functional tight junctions, and unidirectional active transport of electrolytes and water. These results suggest that glycoproteins with terminal galactose-sialic acid moieties are not critically involved in the development and maintenance of polarity in epithelial cells. The mutant cells, particularly the ricin-resistant line, exhibited, however, morphological and electrophysiological changes which suggest a quantitative effect of the mutations on intracellular traffic of membranes and tight junction formation. The cell lines described in this paper, the first lectin-resistant mutants of epithelial lineage, should prove useful tools for studying the peculiarities of glycosylating pathways in polarized cells.
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Selected References
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- Cereijido M., Ehrenfeld J., Meza I., Martínez-Palomo A. Structural and functional membrane polarity in cultured monolayers of MDCK cells. J Membr Biol. 1980;52(2):147–159. doi: 10.1007/BF01869120. [DOI] [PubMed] [Google Scholar]
- Cereijido M., Robbins E. S., Dolan W. J., Rotunno C. A., Sabatini D. D. Polarized monolayers formed by epithelial cells on a permeable and translucent support. J Cell Biol. 1978 Jun;77(3):853–880. doi: 10.1083/jcb.77.3.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chapman A., Fujimoto K., Kornfeld S. The primary glycosylation defect in class E Thy-1-negative mutant mouse lymphoma cells is an inability to synthesize dolichol-P-mannose. J Biol Chem. 1980 May 25;255(10):4441–4446. [PubMed] [Google Scholar]
- Chapman A., Trowbridge I. S., Hyman R., Kornfeld S. Structure of the lipid-linked oligosaccharides that accumulate in class E Thy-1-negative mutant lymphomas. Cell. 1979 Jul;17(3):509–515. doi: 10.1016/0092-8674(79)90259-9. [DOI] [PubMed] [Google Scholar]
- Edelson P. J., Cohn Z. A. Effects of concanavalin A on mouse peritoneal macrophages. I. Stimulation of endocytic activity and inhibition of phago-lysosome formation. J Exp Med. 1974 Nov 1;140(5):1364–1386. doi: 10.1084/jem.140.5.1364. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Green R. F., Meiss H. K., Rodriguez-Boulan E. Glycosylation does not determine segregation of viral envelope proteins in the plasma membrane of epithelial cells. J Cell Biol. 1981 May;89(2):230–239. doi: 10.1083/jcb.89.2.230. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hyman R. Studies on surface antigen variants. Isolation of two complementary variants for Thy 1.2. J Natl Cancer Inst. 1973 Feb;50(2):415–422. doi: 10.1093/jnci/50.2.415. [DOI] [PubMed] [Google Scholar]
- Leighton J., Brada Z., Estes L. W., Justh G. Secretory activity and oncogenicity of a cell line (MDCK) derived from canine kidney. Science. 1969 Jan 31;163(3866):472–473. doi: 10.1126/science.163.3866.472. [DOI] [PubMed] [Google Scholar]
- Lenard J., Compans R. W. The membrane structure of lipid-containing viruses. Biochim Biophys Acta. 1974 Apr 8;344(1):51–94. doi: 10.1016/0304-4157(74)90008-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Louvard D. Apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4132–4136. doi: 10.1073/pnas.77.7.4132. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meiss H. K., Basilico C. Temperature sensitive mutants of BHK 21 cells. Nat New Biol. 1972 Sep 20;239(90):66–68. doi: 10.1038/newbio239066a0. [DOI] [PubMed] [Google Scholar]
- Misfeldt D. S., Hamamoto S. T., Pitelka D. R. Transepithelial transport in cell culture. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1212–1216. doi: 10.1073/pnas.73.4.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parodi A. J., Leloir L. F. The role of lipid intermediates in the glycosylation of proteins in the eucaryotic cell. Biochim Biophys Acta. 1979 Apr 23;559(1):1–37. doi: 10.1016/0304-4157(79)90006-6. [DOI] [PubMed] [Google Scholar]
- Revel J. P., Wolken K. Electronmicroscope investigations of the underside of cells in culture. Exp Cell Res. 1973 Mar 30;78(1):1–14. doi: 10.1016/0014-4827(73)90031-1. [DOI] [PubMed] [Google Scholar]
- Richardson J. C., Simmons N. L. Demonstration of protein asymmetries in the plasma membrane of cultured renal (MDCK) epithelial cells by lactoperoxidase-mediated iodination. FEBS Lett. 1979 Sep 15;105(2):201–204. doi: 10.1016/0014-5793(79)80611-0. [DOI] [PubMed] [Google Scholar]
- Robbins P. W., Hubbard S. C., Turco S. J., Wirth D. F. Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins. Cell. 1977 Dec;12(4):893–900. doi: 10.1016/0092-8674(77)90153-2. [DOI] [PubMed] [Google Scholar]
- Robertson M. A., Etchison J. R., Robertson J. S., Summers D. F., Stanley P. Specific changes in the oligosaccharide moieties of VSV grown in different lectin-resistnat CHO cells. Cell. 1978 Mar;13(3):515–526. doi: 10.1016/0092-8674(78)90325-2. [DOI] [PubMed] [Google Scholar]
- Rodriguez Boulan E., Pendergast M. Polarized distribution of viral envelope proteins in the plasma membrane of infected epithelial cells. Cell. 1980 May;20(1):45–54. doi: 10.1016/0092-8674(80)90233-0. [DOI] [PubMed] [Google Scholar]
- Rodriguez Boulan E., Sabatini D. D. Asymmetric budding of viruses in epithelial monlayers: a model system for study of epithelial polarity. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5071–5075. doi: 10.1073/pnas.75.10.5071. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roseman S. The synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion. Chem Phys Lipids. 1970 Oct;5(1):270–297. doi: 10.1016/0009-3084(70)90024-1. [DOI] [PubMed] [Google Scholar]
- Schlesinger S., Gottlieb C., Feil P., Gelb N., Kornfeld S. Growth of enveloped RNA viruses in a line of chinese hamster ovary cells with deficient N-acetylglucosaminyltransferase activity. J Virol. 1975 Jan;17(1):239–246. doi: 10.1128/jvi.17.1.239-246.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stanley P., Caillibot V., Siminovitch L. Selection and characterization of eight phenotypically distinct lines of lectin-resistant Chinese hamster ovary cell. Cell. 1975 Oct;6(2):121–128. doi: 10.1016/0092-8674(75)90002-1. [DOI] [PubMed] [Google Scholar]
- Tabas I., Schlesinger S., Kornfeld S. Processing of high mannose oligosaccharides to form complex type oligosaccharides on the newly synthesized polypeptides of the vesicular stomatitis virus G protein and the IgG heavy chain. J Biol Chem. 1978 Feb 10;253(3):716–722. [PubMed] [Google Scholar]
- Wright J. A. Evidence for pleiotropic changes in lines of Chinese hamster ovary cells resistant to concanavalin A and phytohemagglutinin-P. J Cell Biol. 1973 Mar;56(3):666–675. doi: 10.1083/jcb.56.3.666. [DOI] [PMC free article] [PubMed] [Google Scholar]