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 pericellular proteolysis and cellular migration. This investigation demonstrates that u-PAR is a substrate for the proteolytically active form of streptococcal pyrogenic exotoxin B (SPE B), a potent virulence factor secreted by Streptococcus pyogenes. Treatment of U937 monocyte-like cells with SPE B decreased specific 125I-labeled single-chain u-PA binding by up to 85%. Cysteine proteinase inhibitors neutralized SPE B without affecting the activity of phosphatidylinositol-specific phospholipase 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 bound to U937 cells or cross-linked to cellular receptors with bis(sulfosuccinimidyl) suberate. The mass of the released u-PA-receptor complex 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 phosphatidylinositol-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 decreasing 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 ligand.
Wolf
B B
BB
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
Gibson
C A
CA
Kapur
V
V
Hussaini
I M
IM
Musser
J M
JM
Gonias
S L
SL
D001426:Bacterial Proteins; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005098:Exotoxins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D013297:Streptococcus pyogenes; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D001426:Bacterial Proteins; D005098:Exotoxins; D008565:Membrane Proteins; C524898:PLAUR protein, human; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; C117980:SpeA protein, Streptococcus pyogenes; C027951:erythrogenic toxin; D010738:Type C Phospholipases; D010450:Endopeptidases; D014568:Urokinase-Type Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate