PROTEOLYTICALLY ACTIVE STREPTOCOCCAL PYROGENIC EXOTOXIN-B CLEAVES MONOCYTIC CELL UROKINASE RECEPTOR AND RELEASES AN ACTIVE FRAGMENT OF THE RECEPTOR FROM THE CELL-SURFACE

Urokinase plasminogen activator (u-PA) receptor (u-PAR) is a glycosyl- phosphatidylinositol anchored membrane protein that promotes pericellu lar proteolysis and cellular migration. This investigation demonstrate s that u-PAR is a substrate for the proteolytically active form of str eptococcal pyrogenic exotoxin B (SPE B), a potent virulence factor sec reted by Streptococcus pyogenes. Treatment of U937 monocyte like cells with SPE B decreased specific I-125-labeled single-chain u-PA binding by up to 85%. Cysteine proteinase inhibitors neutralized SPE B withou t affecting the activity of phosphatidylinositol-specific phospholipas e C. Due to decreased u-PA binding, SPE B-treated U937 cells expressed decreased activity against a u-PA-specific fluorogenic substrate and plasminogen. SPE B released single-chain u-PA that was noncovalently b ound to U937 cells or cross-linked to cellular receptors with bis(sulf osuccinimidyl) suberate. The mass of the released u-PA-receptor comple x was 100 kDa, Western blot analysis confirmed that the u-PA receptor that was cleaved by SPE B is u-PAR. After deglycosylation, the mass of SPE B-released u-PAR was 35 kDa, slightly smaller than the phosphatid ylinositol-specific phospholipase C-derived form of this receptor. SPE B-released u-PAR retained the ability to bind u-PA, as determined by u-PA affinity chromatography. We conclude that SPE B may inhibit u-PA binding to monocytic cells by at least two mechanisms: (i) by decreasi ng the level of functional cell surface u-PAR and (ii) by releasing a soluble form of u-PAR that competes with the cellular receptor for lig and.