Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface

A. M. Schmidt, M. Vianna, M. Gerlach, J. Brett, J. Ryan, J. Kao, C. Esposito, H. Hegarty, W. Hurley, Matthias Clauss, F. Wang, Y. C E Pan, T. C. Tsang, D. Stern

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Abstract

Nonenzymatic glycosylation of proteins, as occurs at an accelerated rate in diabetes, can lead to the formation of advanced glycosylation end products of proteins (AGEs), which can bind to endothelial cells, thereby altering cellular function in a manner which could contribute to the pathogenesis of diabetic angiopathy. In this report, we describe the isolation of two endothelial cell surface-associated proteins which mediate, at least in part, the interaction of AGEs with endothelium. Based on pilot studies demonstrating AGE binding activity with comparable characteristics in bovine endothelial cell and lung extracts, the material from lung was sequentially subjected to chromatography on hydroxylapatite, fast protein liquid chromatography Mono S, and gel filtration. Two distinct polypeptides, ≃35 and ≃80 kDa, were purified to homogeneity, each of which bound AGEs as demonstrated by competitive binding assays using cellular binding proteins immobilized on a plastic surface. NH2-terminal sequence analysis indicated that the ≃35-kDa protein was novel, whereas the NH2-terminal sequence of the ≃80-kDa protein was identical to that of lactoferrin. Immunocytologic studies using polyclonal antibody prepared to each of the purified polypeptides demonstrated the presence of immunoreactive material on the surface of bovine endothelial cells maintained under serum-free conditions. Furthermore, immunoelectron microscopic studies with antibodies to the ≃35- and ≃80-kDa AGE-binding proteins conjugated to different size colloidal gold particles confirmed the presence of the target antigens on the cell surface and suggested that they were closely associated. IgG purified from polyclonal antisera to either the 35- or 80-kDa AGE-binding proteins blocked the binding of 125I-AGE-albumin to the cell surface. These results indicate that endothelial cells express specific cell surface molecules which mediate AGE- endothelial interaction. These polypeptides represent a novel class of cell surface acceptor molecules for glucose-modified proteins which may promote degradation and/or transcytosis of the ligand, and modulation of cellular function.

Original languageEnglish (US)
Pages (from-to)14987-14997
Number of pages11
JournalJournal of Biological Chemistry
Volume267
Issue number21
StatePublished - 1992
Externally publishedYes

Fingerprint

Advanced Glycosylation End Products
Endothelial cells
Glycosylation
Carrier Proteins
Endothelial Cells
Lung
Proteins
Peptides
Transcytosis
Diabetic Angiopathies
Gold Colloid
Lactoferrin
Competitive Binding
Antibodies
Durapatite
Surface Antigens
Cell Extracts
Protein Binding
Liquid Chromatography
Plastics

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)
  • Molecular Biology
  • Cell Biology

Cite this

Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface. / Schmidt, A. M.; Vianna, M.; Gerlach, M.; Brett, J.; Ryan, J.; Kao, J.; Esposito, C.; Hegarty, H.; Hurley, W.; Clauss, Matthias; Wang, F.; Pan, Y. C E; Tsang, T. C.; Stern, D.

In: Journal of Biological Chemistry, Vol. 267, No. 21, 1992, p. 14987-14997.

Research output: Contribution to journalArticle

Schmidt, AM, Vianna, M, Gerlach, M, Brett, J, Ryan, J, Kao, J, Esposito, C, Hegarty, H, Hurley, W, Clauss, M, Wang, F, Pan, YCE, Tsang, TC & Stern, D 1992, 'Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface', Journal of Biological Chemistry, vol. 267, no. 21, pp. 14987-14997.
Schmidt, A. M. ; Vianna, M. ; Gerlach, M. ; Brett, J. ; Ryan, J. ; Kao, J. ; Esposito, C. ; Hegarty, H. ; Hurley, W. ; Clauss, Matthias ; Wang, F. ; Pan, Y. C E ; Tsang, T. C. ; Stern, D. / Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 21. pp. 14987-14997.
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abstract = "Nonenzymatic glycosylation of proteins, as occurs at an accelerated rate in diabetes, can lead to the formation of advanced glycosylation end products of proteins (AGEs), which can bind to endothelial cells, thereby altering cellular function in a manner which could contribute to the pathogenesis of diabetic angiopathy. In this report, we describe the isolation of two endothelial cell surface-associated proteins which mediate, at least in part, the interaction of AGEs with endothelium. Based on pilot studies demonstrating AGE binding activity with comparable characteristics in bovine endothelial cell and lung extracts, the material from lung was sequentially subjected to chromatography on hydroxylapatite, fast protein liquid chromatography Mono S, and gel filtration. Two distinct polypeptides, ≃35 and ≃80 kDa, were purified to homogeneity, each of which bound AGEs as demonstrated by competitive binding assays using cellular binding proteins immobilized on a plastic surface. NH2-terminal sequence analysis indicated that the ≃35-kDa protein was novel, whereas the NH2-terminal sequence of the ≃80-kDa protein was identical to that of lactoferrin. Immunocytologic studies using polyclonal antibody prepared to each of the purified polypeptides demonstrated the presence of immunoreactive material on the surface of bovine endothelial cells maintained under serum-free conditions. Furthermore, immunoelectron microscopic studies with antibodies to the ≃35- and ≃80-kDa AGE-binding proteins conjugated to different size colloidal gold particles confirmed the presence of the target antigens on the cell surface and suggested that they were closely associated. IgG purified from polyclonal antisera to either the 35- or 80-kDa AGE-binding proteins blocked the binding of 125I-AGE-albumin to the cell surface. These results indicate that endothelial cells express specific cell surface molecules which mediate AGE- endothelial interaction. These polypeptides represent a novel class of cell surface acceptor molecules for glucose-modified proteins which may promote degradation and/or transcytosis of the ligand, and modulation of cellular function.",
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T1 - Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface

AU - Schmidt, A. M.

AU - Vianna, M.

AU - Gerlach, M.

AU - Brett, J.

AU - Ryan, J.

AU - Kao, J.

AU - Esposito, C.

AU - Hegarty, H.

AU - Hurley, W.

AU - Clauss, Matthias

AU - Wang, F.

AU - Pan, Y. C E

AU - Tsang, T. C.

AU - Stern, D.

PY - 1992

Y1 - 1992

N2 - Nonenzymatic glycosylation of proteins, as occurs at an accelerated rate in diabetes, can lead to the formation of advanced glycosylation end products of proteins (AGEs), which can bind to endothelial cells, thereby altering cellular function in a manner which could contribute to the pathogenesis of diabetic angiopathy. In this report, we describe the isolation of two endothelial cell surface-associated proteins which mediate, at least in part, the interaction of AGEs with endothelium. Based on pilot studies demonstrating AGE binding activity with comparable characteristics in bovine endothelial cell and lung extracts, the material from lung was sequentially subjected to chromatography on hydroxylapatite, fast protein liquid chromatography Mono S, and gel filtration. Two distinct polypeptides, ≃35 and ≃80 kDa, were purified to homogeneity, each of which bound AGEs as demonstrated by competitive binding assays using cellular binding proteins immobilized on a plastic surface. NH2-terminal sequence analysis indicated that the ≃35-kDa protein was novel, whereas the NH2-terminal sequence of the ≃80-kDa protein was identical to that of lactoferrin. Immunocytologic studies using polyclonal antibody prepared to each of the purified polypeptides demonstrated the presence of immunoreactive material on the surface of bovine endothelial cells maintained under serum-free conditions. Furthermore, immunoelectron microscopic studies with antibodies to the ≃35- and ≃80-kDa AGE-binding proteins conjugated to different size colloidal gold particles confirmed the presence of the target antigens on the cell surface and suggested that they were closely associated. IgG purified from polyclonal antisera to either the 35- or 80-kDa AGE-binding proteins blocked the binding of 125I-AGE-albumin to the cell surface. These results indicate that endothelial cells express specific cell surface molecules which mediate AGE- endothelial interaction. These polypeptides represent a novel class of cell surface acceptor molecules for glucose-modified proteins which may promote degradation and/or transcytosis of the ligand, and modulation of cellular function.

AB - Nonenzymatic glycosylation of proteins, as occurs at an accelerated rate in diabetes, can lead to the formation of advanced glycosylation end products of proteins (AGEs), which can bind to endothelial cells, thereby altering cellular function in a manner which could contribute to the pathogenesis of diabetic angiopathy. In this report, we describe the isolation of two endothelial cell surface-associated proteins which mediate, at least in part, the interaction of AGEs with endothelium. Based on pilot studies demonstrating AGE binding activity with comparable characteristics in bovine endothelial cell and lung extracts, the material from lung was sequentially subjected to chromatography on hydroxylapatite, fast protein liquid chromatography Mono S, and gel filtration. Two distinct polypeptides, ≃35 and ≃80 kDa, were purified to homogeneity, each of which bound AGEs as demonstrated by competitive binding assays using cellular binding proteins immobilized on a plastic surface. NH2-terminal sequence analysis indicated that the ≃35-kDa protein was novel, whereas the NH2-terminal sequence of the ≃80-kDa protein was identical to that of lactoferrin. Immunocytologic studies using polyclonal antibody prepared to each of the purified polypeptides demonstrated the presence of immunoreactive material on the surface of bovine endothelial cells maintained under serum-free conditions. Furthermore, immunoelectron microscopic studies with antibodies to the ≃35- and ≃80-kDa AGE-binding proteins conjugated to different size colloidal gold particles confirmed the presence of the target antigens on the cell surface and suggested that they were closely associated. IgG purified from polyclonal antisera to either the 35- or 80-kDa AGE-binding proteins blocked the binding of 125I-AGE-albumin to the cell surface. These results indicate that endothelial cells express specific cell surface molecules which mediate AGE- endothelial interaction. These polypeptides represent a novel class of cell surface acceptor molecules for glucose-modified proteins which may promote degradation and/or transcytosis of the ligand, and modulation of cellular function.

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