Functional Characterization of Atherosclerosis-Associated Ser128Arg and Leu554Phe E-Selectin Mutations

Abstract The cellular adhesion molecule E-selectin is expressed on activated endothelial cells, and is involved in the process of adherence of blood cells to vessel endothelium in inflammatory events such as atherosclerosis. In a recent study we found a Ser128Arg mutation in the EGF domain as well as a Leu554Phe mutation in the membrane domain of E-selectin. We also established increased frequencies of both mutations among young patients with severe coronary atherosclerosis. In the present study we investigated the influence of these mutations on cell adhesion and on the release of soluble E-selectin. Mutants were created by site-directed mutagenesis and COS cells were transfected with E-selectin, either wild-type or mutant. Antibody-binding studies and cell-adhesion assays were performed on transfected COS cells and on interleukin-1 beta-stimulated HUVECs. Soluble E-selectin in supernatants of wild type and Leu554Phe mutant-transfected COS cells was measured by ELISA. We discovered significant differences in the strength of HL-60 cell adhesion for the Ser128Arg mutant: in comparison with the wild type, the strength of adhesion to the mutant was reduced on transfected COS cells (P < 0.01) as well as on stimulated HUVECs (P < 0.01). Significantly diminished release of soluble E-selectin was detected for the Leu554Phe membrane domain mutant, in comparison with the wild type. In summary, the mutations studied here influence the E-selectin function in vitro and may be considered as one of the risk factors involved in the complex pathogenesis of atherosclerosis.

[1]  D. Wagner,et al.  P-selectin and E-selectin , 2001 .

[2]  D. Wagner,et al.  Leukocyte-endothelium adhesion molecules in atherosclerosis. , 1998, The Journal of laboratory and clinical medicine.

[3]  C. Haslett,et al.  Potential pro‐inflammatory effects of soluble E‐selectin upon neutrophil function , 1998, European journal of immunology.

[4]  E. Boerwinkle,et al.  Circulating adhesion molecules VCAM-1, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases: the Atherosclerosis Risk In Communities (ARIC) study. , 1997, Circulation.

[5]  K. Miwa,et al.  Soluble E-selectin, ICAM-1 and VCAM-1 levels in systemic and coronary circulation in patients with variant angina. , 1997, Cardiovascular research.

[6]  J. W. Shaw,et al.  The white blood cell adhesion molecule E-selectin predicts restenosis in patients with intermittent claudication undergoing percutaneous transluminal angioplasty. , 1997, Circulation.

[7]  C. Bode,et al.  Circulating vascular cell adhesion molecule-1 correlates with the extent of human atherosclerosis in contrast to circulating intercellular adhesion molecule-1, E-selectin, P-selectin, and thrombomodulin. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[8]  S. Watson,et al.  L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes: interactions between the lectin, epidermal growth factor, and consensus repeat domains of the selectins determine ligand binding specificity. , 1996, Journal of immunology.

[9]  K. Ley,et al.  Molecular mechanisms of leukocyte recruitment in the inflammatory process. , 1996, Cardiovascular research.

[10]  D. Scott,et al.  Single Amino Acid Residues in the E- and P-selectin Epidermal Growth Factor Domains Can Determine Carbohydrate Binding Specificity* , 1996, The Journal of Biological Chemistry.

[11]  C. Ballantyne,et al.  Levels of Soluble Cell Adhesion Molecules in Patients With Dyslipidemia , 1996 .

[12]  A. Gotto,et al.  Levels of soluble cell adhesion molecules in patients with dyslipidemia. , 1996, Circulation.

[13]  A. Blann,et al.  von Willebrand Factor, Soluble P-Selectin, Tissue Plasminogen Activator and Plasminogen Activator Inhibitor in Atherosclerosis , 1995, Thrombosis and Haemostasis.

[14]  T. Kogan,et al.  A Single Amino Acid Residue Can Determine the Ligand Specificity of E-selectin (*) , 1995, The Journal of Biological Chemistry.

[15]  K. Kawazoe,et al.  Concentration of soluble vascular cell adhesion molecule‐1 (VCAM‐1) correlated with expression of VCAM‐1 mRNA in the human atherosclerotic aorta , 1995, Coronary artery disease.

[16]  B. Reinhold,et al.  Myeloglycan, a series of E-selectin-binding polylactosaminolipids found in normal human leukocytes and myelocytic leukemia HL60 cells. , 1995, Biochemical and biophysical research communications.

[17]  D. Schaper,et al.  Low-density lipoprotein induces vascular adhesion molecule expression on human endothelial cells. , 1995, Hypertension.

[18]  D. Parums The Distribution of Adhesion Molecules in Normal and Atherosclerotic Aarteries and Aortas , 1995 .

[19]  J. Sedlák,et al.  Sialylated Lewis(x) and Lewis(a) determinants expression on human neoplastic cell lines: immunocytometric study with the 5th workshop monoclonal antibodies. , 1995, Neoplasma (Bratislava).

[20]  K. Kopple,et al.  The soluble form of E-selectin is an asymmetric monomer. Expression, purification, and characterization of the recombinant protein. , 1994, The Journal of biological chemistry.

[21]  A. Blann,et al.  Circulating Endothelial Cell/Leukocyte Adhesion Molecules in Atherosclerosis , 1994, Thrombosis and Haemostasis.

[22]  M. Bevilacqua,et al.  Endothelial-leukocyte adhesion molecules in human disease. , 1994, Annual review of medicine.

[23]  M. Davies,et al.  The expression of the adhesion molecules ICAM‐1, VCAM‐1, PECAM, and E‐selectin in human atherosclerosis , 1993, The Journal of pathology.

[24]  A. Gearing,et al.  Circulating adhesion molecules in disease. , 1993, Immunology today.

[25]  C. Gahmberg,et al.  The vascular E-selectin binds to the leukocyte integrins CD11/CD18. , 1993, Glycobiology.

[26]  A. Becker,et al.  Adhesion molecules on the endothelium and mononuclear cells in human atherosclerotic lesions. , 1992, The American journal of pathology.

[27]  L. Lasky,et al.  Selectins: interpreters of cell-specific carbohydrate information during inflammation. , 1992, Science.

[28]  L. Presta,et al.  Identification of an E-selectin region critical for carbohydrate recognition and cell adhesion [published erratum appears in J Cell Biol 1993 Feb;120(4):1071] , 1992, The Journal of cell biology.

[29]  D. Wagner,et al.  P-selectin and E-selectin. Distinct but overlapping leukocyte ligand specificities. , 1992, The Journal of biological chemistry.

[30]  A. Gown,et al.  Various cell types in human atherosclerotic lesions express ICAM-1. Further immunocytochemical and immunochemical studies employing monoclonal antibody 10F3. , 1992, The American journal of pathology.

[31]  J. Coucher,et al.  Expression of intercellular adhesion molecule-1 in atherosclerotic plaques. , 1992, The American journal of pathology.

[32]  R. Hynes,et al.  Contact and adhesive specificities in the associations, migrations, and targeting of cells and axons , 1992, Cell.

[33]  L. Needham,et al.  Structural and functional studies of the endothelial activation antigen endothelial leucocyte adhesion molecule-1 using a panel of monoclonal antibodies. , 1991, Journal of immunology.

[34]  S. Bixler,et al.  Expression and functional characterization of a soluble form of endothelial-leukocyte adhesion molecule 1. , 1991, Journal of immunology.

[35]  S. Wright,et al.  Endothelial-leukocyte adhesion molecule 1 stimulates the adhesive activity of leukocyte integrin CR3 (CD11b/CD18, Mac-1, alpha m beta 2) on human neutrophils , 1991, The Journal of experimental medicine.

[36]  Timothy A. Springer,et al.  Adhesion receptors of the immune system , 1990, Nature.

[37]  J. Gamble,et al.  Prevention of activated neutrophil adhesion to endothelium by soluble adhesion protein GMP140. , 1990, Science.

[38]  I. Weissman,et al.  The mouse lymph node homing receptor is identical with the lymphocyte cell surface marker Ly-22: Role of the EGF domain in endothelial binding , 1990, Cell.

[39]  F. Greenwood,et al.  Preparation of Iodine-131 Labelled Human Growth Hormone of High Specific Activity , 1962, Nature.

[40]  G. Scatchard,et al.  THE ATTRACTIONS OF PROTEINS FOR SMALL MOLECULES AND IONS , 1949 .