IDENTIFICATION OF AN EPITOPE OF ALPHA-ENOLASE (A CANDIDATE PLASMINOGEN RECEPTOR) BY PHAGE DISPLAY

alpha-enolase is an ubiquitous cytoplasmic glycolytic enzyme which als o exhibits cell surface mediated functions and a structural role in th e lens of some species. An alpha-enolase related molecule (alpha-ERM) is present on the surfaces of neutrophils, monocytes and monocytoid ce lls and has the capacity to specifically bind plasminogen, suggesting that alpha-ERM may function as a plasminogen receptor. We have generat ed a monoclonal antibody (mAB), 9C12, against alpha-ERM. This mAB reac ted with both alpha-ERM and purified human alpha-enolase in Western bl otting and in enzyme linked immunosorbent assays (ELISA), mAB 9C12 det ected a cell surface associated molecule on human peripheral blood neu trophils and on U937 human monocytoid cells as assessed by fluorescenc e activated cell sorting (FACS) analyses. In addition, mAB 9C12 recogn ized an intracellular pool of alpha-enolase/alpha-ERM in permeabilized U937 cells. A phage display approach was employed to identify the alp ha-enolase epitope recognized by mAB 9C12. Random fragments of 100-300 base pairs (bp), obtained from the full length human alpha-enolase cD NA, 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 seque ncing. All of the fragments which bound to mAB 9C12 encoded the common sequence DLDFKSPDDPSRYISP, spanning amino acids 257-272 of human alph a-enolase. This sequence is located within an external loop of the mol ecule. These data indicate that this sequence contains the epitope rec ognized by mAB 9C12 and is, therefore, exposed on the cell surface, fu rther suggesting that alpha-enolase and alpha-ERM share common amino a cid sequences.