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J. Biol. Chem., Vol. 279, Issue 7, 5268-5277, February 13, 2004

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Enzymatic and Molecular Characteristics of the Efficiency and Specificity of Exfoliative Toxin Cleavage of Desmoglein 1^*

Yasushi Hanakawa, Norman M. Schechter, Chenyan Lin, Koji Nishifuji¶, Masayuki Amagai¶, and John R. Stanley||

From the Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and the ^¶Department of Dermatology, Keio University School of Medicine, Tokyo 160-8582, Japan

Exfoliative toxins (ETs) from Staphylococcus aureus blister the superficial epidermis by hydrolyzing a single peptide bond, Glu³⁸¹–Gly³⁸², located between extracellular domains 3 and 4 of desmoglein 1 (Dsg1). Enzyme activity is dependent on the calcium-stabilized structure of Dsg1. Here we further define the characteristics of this cleavage. Kinetic studies monitoring the cleavage of Dsg1 by ETA, ETB, and ETD demonstrated k_cat/K_m values of 2–6 x 10⁴ M^–1 s^–1, suggesting very efficient proteolysis. Proteolysis by ETA was not efficiently inhibited by broad spectrum serine protease inhibitors, suggesting that the enzyme cleavage site may be inactive or inaccessible before specific binding to its substrate. Using truncated mutants of human Dsg1 and chimeric molecules between human Dsg1 and either human Dsg3 or canine Dsg1, we show that for cleavage, human-specific amino acids from Dsg1 are necessary in extracellular domain 3 upstream of the scissile bond. If these residues are canine rather than human, ETA binds, but does not cleave, canine Dsg1. These data suggest that the exquisite specificity and efficiency of ETA may depend on the enzyme's binding upstream of the cleavage site with a very specific fit, like a key in a lock.

Received for publication, October 8, 2003 , and in revised form, November 7, 2003.

^* This work was supported by grants from the National Institutes of Health and a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. 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.

Recipient of the Japanese Society for Investigative Dermatology International Fellowship Shiseido Award for 2002.

^|| To whom correspondence should be addressed: Dept. of Dermatology, University of Pennsylvania, 211 CRB, 415 Curie Blvd., Philadelphia, PA 19104. Fax: 215-573-2033; E-mail: jrstan{at}mail.med.upenn.edu.

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