Staphylokinase Requires NH2-terminal Proteolysis for Plasminogen Activation

doi:10.1074/jbc.272.9.6067

Staphylokinase Requires NH₂-terminal Proteolysis for Plasminogen Activation*

From the ^‡ Institute for Molecular Biotechnology, 07745 Jena, Germany and the
^¶ Center for Molecular and Vascular Biology, University of Leuven, B-3000 Leuven, Belgium

^§ To whom correspondence should be addressed:
Institute for Molecular Biotechnology, 07745 Jena, Germany.

Abstract

Staphylokinase (Sak), a single-chain protein comprising 136 amino acids with NH₂-terminal sequence, forms a complex with plasmin, that is endowed with plasminogen activating properties. Plasmin is presumed to process mature (high molecular weight, HMW) Sak to low molecular weight derivatives (LMW-Sak), primarily by hydrolyzing the Lys¹⁰-Lys¹¹ peptide bond, but the kinetics of plasminogen activation by HMW-Sak and LMW-Sak are very similar. Here, the requirement of NH₂-terminal proteolysis of Sak for the induction of plasminogen activating potential was studied by mutagenesis of Lys¹⁰ and Lys¹¹ in combination with NH₂-terminal microsequence analysis of equimolar mixtures of Sak and plasminogen and determination of kinetic parameters of plasminogen activation by catalytic amounts of Sak. Substitution of Lys¹⁰ with Arg did not affect processing of the Arg¹⁰-Lys¹¹ site nor plasminogen activation, whereas substitution with His resulted in cleavage of the Lys¹¹-Gly¹² peptide bond and abolished plasminogen activation. Substitution of Lys¹¹ with Arg did not affect Lys¹⁰-Arg¹¹ processing or plasminogen activation, whereas replacement with His did not prevent Lys¹⁰-His¹¹ hydrolysis but abolished plasminogen activation. Substitution of Lys¹¹ with Cys yielded an inactive processed derivative which was fully activated by aminoethylation. Deletion of the 10 NH₂-terminal amino acids did not affect plasminogen activation, but additional deletion of Lys¹¹ eliminated plasminogen activation.

Thus generation of plasminogen activator potential in Sak proceeds via plasmin-mediated removal of the 10 NH₂-terminal amino acids with exposure of Lys¹¹ as the new NH₂ terminus. This provides a structural basis for the hypothesis, derived from kinetic measurements, that plasminogen activation by Sak needs to be primed by plasmin and a mechanism for the high fibrin selectivity of Sak in a plasma milieu.

Footnotes

↵* This work was supported by Bundes Minïsterium für Biologische Forschung Grant 0311015. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Sak

staphylokinase

Sak42D

wild-type staphylokinase with Gly³⁴, Arg³⁶, and Arg⁴³

Sak42DΔN10

Sak42DΔN11, and Sak42DΔN14, Sak42D with deletion of the 10, 11, and 14 NH₂-terminal amino acids, respectively

Sak42DAnB

Sak42D with amino acid A in position n of mature staphylokinase substituted with amino acid B

HMW-Sak

high molecular weight Sak (mature Sak with 136 amino acids)

LMW-Sak

low molecular weight Sak (Sak after removal of the 10 NH₂-terminal amino acids)

Glu-Plg

native human plasminogen with NH₂-terminal Glu

rPlg(S741A)

recombinant plasminogen with the active site Ser⁷⁴¹ mutagenized to Ala

k_a

association rate constant

k_d

dissociation rate constant

PAGE

polyacrylamide gel electrophoresis

IEF

isoelectric focusing

K_a

association equilibrium constant

S-2251

D-Val-Phe-Lys-p-nitroanilide.
- Received October 2, 1996.
- Revision received December 3, 1996.