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J. Biol. Chem., Vol. 280, Issue 48, 39897-39906, December 2, 2005

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Effects of Spontaneous Deamidation on the Cytotoxic Activity of the Bacillus anthracis Protective Antigen^*

Gil Zomber1, Shaul Reuveny, Nissim Garti, Avigdor Shafferman¶2, and Eytan Elhanany¶

From the Departments of Biotechnology and ^¶Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel and the Casali Institute of Applied Chemistry, The Hebrew University, Jerusalem 91905, Israel

Protective antigen (PA) is a central virulence factor of Bacillus anthracis and a key component in anthrax vaccines. PA binds to target cell receptors, is cleaved by the furin protease, self-aggregates to heptamers, and finally internalizes as a complex with either lethal or edema factors. Under mild room temperature storage conditions, PA cytotoxicity decreased (t 7 days) concomitant with the generation of new acidic isoforms, probably through deamidation of Asn residues. Ranking all 68 Asn residues in PA based on their predicted deamidation rates revealed five residues with half-lives of <60 days, and these residues were further analyzed: Asn¹⁰ in the 20-kDa region, Asn¹⁶² at P₆ vicinal to the furin cleavage site, Asn³⁰⁶ in the pro-pore translocation loop, and both Asn⁷¹³ and Asn⁷¹⁹ in the receptor-binding domain. We found that PA underwent spontaneous deamidation at Asn¹⁶² upon storage concomitant with decreased susceptibility to furin. A panel of model synthetic furin substrates was used to demonstrate that Asn¹⁶² deamidation led to a 20-fold decrease in the bimolecular rate constant (k_cat/K_m) of proteolysis due to the new negatively charged residue at P₆ in the furin recognition sequence. Furthermore, reduced PA cytotoxicity correlated with a decrease in PA cell binding and also with deamidation of Asn⁷¹³ and Asn⁷¹⁹. On the other hand, neither deamidation of Asn¹⁰ or Asn³⁰⁶ nor impairment of heptamerization could be observed upon prolonged PA storage. We suggest that PA inactivation during storage is associated with susceptible deamidation sites, which are intimately involved in both mechanisms of PA cleavage by furin and PA-receptor binding.

Received for publication, August 4, 2005 , and in revised form, September 6, 2005.

^* 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.

¹ Performed this work as part of a Ph.D. thesis submitted to the Casali Institute of Applied Chemistry, The Hebrew University, Jerusalem, Israel.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, Israel Inst. for Biological Research, P. O. Box 19, Ness-Ziona 74100, Israel. Tel.: 972-8-938-1595; Fax: 972-8-940-1404; E-mail: avigdor{at}iibr.gov.il.

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