Abstract
Skizzle (SkzL), secreted by Streptococcus agalactiae, has moderate sequence identity to streptokinase and staphylokinase, bacterial activators of human plasminogen (Pg). SkzL binds [Glu]Pg with low affinity (K(D) 3-16 mum) and [Lys]Pg and plasmin (Pm) with indistinguishable high affinity (K(D) 80 and 50 nm, respectively). Binding of SkzL to Pg and Pm is completely lysine-binding site-dependent, as shown by the effect of the lysine analog, 6-aminohexanoic acid. Deletion of the COOH-terminal SkzL Lys(415) residue reduces affinity for [Lys]Pg and active site-blocked Pm 30-fold, implicating Lys(415) in a lysine-binding site interaction with a Pg/Pm kringle. SkzL binding to active site fluorescein-labeled Pg/Pm analogs demonstrates distinct high and low affinity interactions. High affinity binding is mediated by Lys(415), whereas the source of low affinity binding is unknown. SkzL enhances the activation of [Glu]Pg by urokinase (uPA) approximately 20-fold, to a maximum rate indistinguishable from that for [Lys]Pg and [Glu]Pg activation in the presence of 6-aminohexanoic acid. SkzL binds preferentially to the partially extended beta-conformation of [Glu]Pg, which is in unfavorable equilibrium with the compact alpha-conformation, thereby converting [Glu]Pg to the fully extended gamma-conformation and accelerating the rate of its activation by uPA. SkzL enhances [Lys]Pg and [Glu]Pg activation by single-chain tissue-type Pg activator, approximately 42- and approximately 650-fold, respectively. SkzL increases the rate of plasma clot lysis by uPA and single-chain tissue-type Pg activator approximately 2-fold, confirming its cofactor activity in a physiological model system. The results suggest a role for SkzL in S. agalactiae pathogenesis through fibrinolytic enhancement.
Highlights
JULY 2, 2010 VOLUME 285 NUMBER 27 immune-compromised patients [1, 2]
Activation of the fibrinolytic system is a mechanism of bacterial pathogenesis employed by many bacteria through expression of cell-surface plasminogen (Pg)-binding proteins and the secretion of Pg activators
A recent study indicated that Pg activation on the surface of S. agalactiae occurs through the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-bound Pg complex, facilitating Group B streptococci (GBS) virulence and promoting bacterial dissemination in a murine model [10]
Summary
Protein Purification and Characterization—[Glu]Pg, [Lys]Pg, and Pm (all carbohydrate form 2) were prepared as described previously [41, 60, 61]. [5F]FFR-[Lys]Pg, [5F]FFR-[Glu]Pg, and [5F]FFR-Pm were generated by a thiol-specific reaction with 5-(iodoacetamido)fluorescein following inactivation of the SKinduced active site in [Lys]Pg and [Glu]Pg with N␣-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl essentially as described previously (39 – 41, 62). Competitive binding experiments with 100 nM [5F]-SkzL in the absence and presence of fixed concentrations of wtSkzL or SkzL⌬K415 to [Lys]Pg and FFR-Pm were performed under identical conditions In these titrations, mixtures of [5F]-SkzL and either wtSkzL or SkzL⌬K415 were preincubated for 10 min at 25 °C prior to titration with [Lys]Pg or FFR-Pm. Following correction for buffer blank and probe dilution (Յ10%), data were expressed as the fractional change in the initial fluorescence, (Fobs Ϫ Fo)/Fo ϭ ⌬F/Fo. Titrations were analyzed by nonlinear least squares fitting of the quadratic binding Equation 1,. Direct titrations of wtSkzL binding to [5F]FFR-[Lys]Pg and [5F]FFR-Pm performed without polarizers revealed an apparent anisotropy decrease, representing a second distinct binding interaction These data were analyzed by nonlinear least squares fitting of the sum of the solution to the quadratic equation (Equation 1) as ⌬F1/Fo, with n ϭ 1 for the high affinity interaction, and a hyperbola for the weak interaction with the assumption that [L]free Ϸ [L]o for this interaction shown in Equation 2,. Results are reported as time to half-clot lysis as a function of SkzL concentration and analyzed by Equation 1, where [P]o was 1.6 M, representing the dilution-corrected plasma Pg concentration of 2.0 M [67]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
