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J. Biol. Chem., Vol. 283, Issue 3, 1580-1587, January 18, 2008

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The Lack of Binding of VEK-30, an Internal Peptide from the Group A Streptococcal M-like Protein, PAM, to Murine Plasminogen Is due to Two Amino Acid Replacements in the Plasminogen Kringle-2 Domain^*

Qihua Fu, Mariana Figuera-Losada, Victoria A. Ploplis, Sara Cnudde, James H. Geiger, Mary Prorok, and Francis J. Castellino¹

From the W. M. Keck Center for Transgene Research and the Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 and the Department of Chemistry, Michigan State University, East Lansing, Michigan 48824

VEK-30, a 30-amino acid internal peptide present within a streptococcal M-like plasminogen (Pg)-binding protein (PAM) from Gram-positive group-A streptococci (GAS), represents an epitope within PAM that shows high affinity for the lysine binding site (LBS) of the kringle-2 (K2) domain of human (h)Pg. VEK-30 does not interact with this same region of mouse (m)Pg, despite the high conservation of the mK2- and hK2-LBS. To identify the molecular basis for the species specificity of this interaction, hPg and mPg variants were generated, including an hPg chimera with the mK2 sequence and an mPg chimera containing the hK2 sequence. The binding of synthetic VEK-30 to these variants was studied by surface plasmon resonance. The data revealed that, in otherwise intact Pg, the species specificity of VEK-30 binding in these two cases is entirely dictated by two K2 residues that are different between hPg and mPg, namely, Arg-220 of hPg, which is a Gly in mPg, and Leu-222 of hPg, which is a Pro in mPg, neither of which are members of the canonical K2-LBS. Neither the activation of hPg, nor the enzymatic activity of its activated product, plasmin (hPm), are compromised by replacing these two amino acids by their murine counterparts. It is also demonstrated that hPg is more susceptible to activation to hPm after complexation with VEK-30 and that this property is greatly reduced as a result of the R220G and L222P replacements in hPg. These mechanisms for accumulation of protease activity on GAS likely contribute to the virulence of PAM⁺-GAS strains and identify targets for new therapeutic interventions.

Received for publication, June 20, 2007 , and in revised form, November 7, 2007.

^* This work was supported by Grants HL013423 (to F. J. C.), GM0638947 (to J. H. G.), and HL073750 (to V. A. P.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Table S1.

¹ To whom correspondence should be addressed: W. M. Keck Center for Transgene Research, 230 Raclin-Carmichael Hall, University of Notre Dame, Notre Dame, IN 46556. Tel.: 574-631-9152; Fax: 574-631-8017; E-mail: fcastell{at}nd.edu.

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