Identification of a conformational epitope induced in the kringle 5‐latent protease domain of Glu‐plasminogen Induced by its interaction with cells (833.2)

Activation of Glu‐plasminogen is markedly enhanced on cell surfaces, arming cells with the broad spectrum proteolytic activity of plasmin that promotes cell migration. This may be due to conformational changes in Glu‐plasminogen when bound to cells. To explore these conformational changes we have developed anti‐plasminogen mAbs that preferentially recognize receptor‐induced binding sites in Glu‐plasminogen. We used specific proteolysis and immunoaffinity chromatography, followed by LC‐MS‐MS to define the epitope(s) recognized by two mAbs. Plasminogen was digested with trypsin and applied to a normal mouse IgG affinity column and the flow‐through applied to either a mAb 109 or mAb 20 immunoaffinity column. The eluate from the mAb 109 column contained two peaks with masses of 1298 AMU and 1030 AMU in LC‐MS‐MS corresponding to a nonlinear epitope contained within H 494 ‐R 504 within the kringle 5 domain and L 652 ‐K 661 within the latent protease domain, respectively. For mAb 20, MALDI TOF results were consistent with an epitope contained within L 650 ‐I 663 , overlapping the mAb 109 epitope. Furthermore, mAb 109 and mAb 20 cross‐reacted in pairwise epitope mapping using surface plasmon resonance. The conformational changes detected by mAbs 109 and 20 are likely to reflect changes in Glu‐plasminogen conformation that modulate its ability to be activated on the cell surface. Grant Funding Source : Supported by HL080146 and CA166473