Identification of an epitope of alpha-enolase (a candidate plasminogen receptor) by phage display.
Alpha-enolase is an ubiquitous cytoplasmic glycolytic enzyme which also exhibits cell surface mediated functions and a structural role in the lens of some species. An alpha-enolase related molecule (alpha-ERM) is present on the surfaces of neutrophils, monocytes and monocytoid cells and has the capacity to specifically bind plasminogen, suggesting that alpha-ERM may function as a plasminogen receptor. We have generated a monoclonal antibody (mAB), 9C12, against alpha-ERM. This mAB reacted with both alpha-ERM and purified human alpha-enolase in Western blotting and in enzyme linked immunosorbent assays (ELISA). mAB 9C12 detected a cell surface associated molecule on human peripheral blood neutrophils and on U937 human monocytoid cells as assessed by fluorescence activated cell sorting (FACS) analyses. In addition, mAB 9C12 recognized an intracellular pool of alpha-enolase/alpha-ERM in permeabilized U937 cells. A phage display approach was employed to identify the alpha-enolase epitope recognized by mAB 9C12. Random fragments of 100-300 base pairs (bp), obtained from the full length human alpha-enolase cDNA, were cloned into the filamentous phage vector pComb3B, to generate a phage-displayed peptide library. Recombinant phages binding to mAB 9C12 were selected and their DNA inserts characterized by direct sequencing. All of the fragments which bound to mAB 9C12 encoded the common sequence DLDFKSPDDPSRYISP, spanning amino acids 257-272 of human alpha-enolase. This sequence is located within an external loop of the molecule. These data indicate that this sequence contains the epitope recognized by mAB 9C12 and is, therefore, exposed on the cell surface, further suggesting that alpha-enolase and alpha-ERM share common amino acid sequences.
Arza
B
B
Institut de Recerca Oncològica, Hospital Duran i Reynals, Barcelona, Spain.
Félez
J
J
Lopez-Alemany
R
R
Miles
L A
LA
Muñoz-Cánoves
P
P
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D005434:Flow Cytometry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry; D019151:Peptide Library; D010751:Phosphopyruvate Hydratase / Q000032:analysis / Q000276:immunology*; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000032:analysis / Q000276:immunology*; D055293:Receptors, Urokinase Plasminogen Activator; D013499:Surface Properties
D000911:Antibodies, Monoclonal; D000939:Epitopes; C524898:PLAUR protein, human; D019151:Peptide Library; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D010751:Phosphopyruvate Hydratase