Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells

Erythropoietin (Epo), a glycoprotein that regulates the formation of erythrocytes in mammals, was produced in cultured tobacco BY2 cells (Nicotiana tabacum L. cv. Bright Yellow 2) by introducing human Epo cDNA via Agrobacterium tumefaciens-mediated gene transfer. Epo was correctly processed and subsequently penetrated the plasma membrane of tobacco cells. However, it remained attached to the cell wall and was not released into the culture medium. Although Epo produced by tobacco cells was glycosylated with N-linked oligosaccharides, these carbohydrates were smaller than those of the recombinant Epo produced in mammalian cells. Epo produced in tobacco exhibited in vitro biological activities by inducing the differentiation and proliferation of erythroid cells. However, it had no in vivo biological activities. A lectin-binding assay indicated the lack of sialic acid residues in the N-linked oligosaccharides of Epo, suggesting that Epo was removed from the circulation before it reached erythropoietic tissues.

[1]  T. Boone,et al.  Role of glycosylation on the secretion and biological activity of erythropoietin. , 1992, Biochemistry.

[2]  E. Goldwasser,et al.  Purification of human erythropoietin. , 1977, The Journal of biological chemistry.

[3]  C. H. Lin,et al.  Cloning and expression of the human erythropoietin gene. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[4]  R. Sasaki,et al.  Isolation of human erythropoietin with monoclonal antibodies. , 1987, Methods in enzymology.

[5]  G. Kahl,et al.  Foreign genes in plants: transfer, structure, expression, and applications. , 1988, Annual review of genetics.

[6]  F. Sieber,et al.  Erythroid colony formation in cultures of mouse and human bone marrow: Analysis of the requirement for erythropoietin by gel filtration and affinity chromatography on agarose‐concanavalin A , 1974, Journal of cellular physiology.

[7]  A. Cattaneo,et al.  ‘Phytoantibodies’: a general vector for the expression of immunoglobulin domains in transgenic plants , 1991, Plant Molecular Biology.

[8]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[9]  K. Yamamoto,et al.  Structural requirements for the binding of oligosaccharides and glycopeptides to immobilized wheat germ agglutinin. , 1981, Biochemistry.

[10]  Kokichi Hinata,et al.  Transgenic Rice Plants After Direct Gene Transfer into Protoplasts , 1988, Bio/Technology.

[11]  E. Fritsch,et al.  Isolation and characterization of genomic and cDNA clones of human erythropoietin , 1985, Nature.

[12]  M. Fukuda,et al.  Survival of recombinant erythropoietin in the circulation: the role of carbohydrates. , 1989 .

[13]  S. Masuda,et al.  Effects of site-directed removal of N-glycosylation sites in human erythropoietin on its production and biological properties. , 1991, The Journal of biological chemistry.

[14]  K. Janda,et al.  Evaluation of Immunoglobulins from Plant Cells , 1991, Biotechnology progress.

[15]  I. Goldstein,et al.  An examination of the topography of the saccharide binding sites of concanavalin A and of the forces involved in complexation. , 1970, Biochemistry.

[16]  P. Elzen,et al.  Production of Correctly Processed Human Serum Albumin in Transgenic Plants , 1990, Bio/Technology.

[17]  M. Bevan,et al.  Binary Agrobacterium vectors for plant transformation. , 1984, Nucleic acids research.

[18]  D. Delmer,et al.  Determination of the Pore Size of Cell Walls of Living Plant Cells , 1979, Science.

[19]  R. Sasaki,et al.  Expression of human erythropoietin in cultured tobacco cells. , 1993, Bioscience, biotechnology, and biochemistry.

[20]  J. Spivak,et al.  The in vivo metabolism of recombinant human erythropoietin in the rat. , 1989, Blood.

[21]  J. Ma,et al.  Monoclonal antibody engineering in plants , 1992, FEBS letters.

[22]  G. An,et al.  High efficiency transformation of cultured tobacco cells. , 1985, Plant physiology.

[23]  K. Drickamer.  Clearing up glycoprotein hormones , 1991, Cell.

[24]  F. Skoog,et al.  Organic Growth Factor Requirements of Tobacco Tissue Cultures , 1965 .

[25]  E. Goldwasser,et al.  The role of carbohydrate in erythropoietin action. , 1985, Endocrinology.

[26]  D. M. Lam,et al.  Expression of hepatitis B surface antigen in transgenic plants. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[27]  V. Bhavanandan,et al.  The interaction of wheat germ agglutinin with sialoglycoproteins. The role of sialic acid. , 1979, The Journal of biological chemistry.

[28]  L. Staehelin,et al.  The plant Golgi apparatus: a factory for complex polysaccharides and glycoproteins. , 1993, Trends in biochemical sciences.

[29]  A. Hiatt,et al.  Production of antibodies in transgenic plants , 1989, Nature.

[30]  M. Higuchi,et al.  Role of sugar chains in the expression of the biological activity of human erythropoietin. , 1992, The Journal of biological chemistry.

[31]  J. Schell,et al.  Transfer and function of T-DNA genes from Agrobacterium Ti and Ri plasmids in plants , 1989, Cell.

[32]  R. Sasaki,et al.  Characterization and use of monoclonal antibodies directed against human erythropoietin that recognize different antigenic determinants. , 1989, Blood.

[33]  R. Sasaki,et al.  Comparative structural study of N-linked oligosaccharides of urinary and recombinant erythropoietins. , 1988, Biochemistry.

[34]  C. Gasser,et al.  Genetically Engineering Plants for Crop Improvement , 1989, Science.

[35]  Jeff Schell,et al.  Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum , 1990, Plant Molecular Biology.

[36]  F. Maley,et al.  Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. , 1989, Analytical biochemistry.

[37]  M. Montagu,et al.  Enkephalins Prouduced in Transgenic Plants Using Modified 2S Seed Storage Proteins , 1989, Bio/Technology.

[38]  T. Nagata,et al.  Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (Liposomes) , 1981, Molecular and General Genetics MGG.

[39]  A. Dell,et al.  Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. , 1987, The Journal of biological chemistry.

[40]  M. Gross,et al.  [7] Erythropoietin: Assay and study of its mode of action , 1975 .

[41]  H. Bohnert,et al.  The use of nuclear‐encoded sequences to direct the light‐regulated synthesis and transport of a foreign protein into plant chloroplasts , 1985, The EMBO journal.

[42]  美昭 森山,et al.  Erythropoietin , 1972, Reactions Weekly.

[43]  Masaaki Goto,et al.  Production of Recombinant Human Erythropoietin in Mammalian Cells: Host–Cell Dependency of the Biological Activity of the Cloned Glycoprotein , 1988, Bio/Technology.

[44]  S. Masuda,et al.  The role of carbohydrate in recombinant human erythropoietin. , 1990, European journal of biochemistry.