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Regulation of Mature ADAM17 by Redox Agents for L-Selectin Shedding¹

Yue Wang^*, Amy H. Herrera^*, Ying Li^2,*, Kiran K. Belani and Bruce Walcheck^3,*

^* Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, MN 55108; and Children’s Hospital of Minnesota, Minneapolis, MN 55404

L-selectin is constitutively expressed by neutrophils and plays a key role in directing these cells to sites of inflammation. Upon neutrophil activation, L-selectin is rapidly and efficiently down-regulated from the cell surface by ectodomain shedding. We have directly shown that A disintegrin and metalloprotease 17 (ADAM17) is a primary and nonredundant sheddase of L-selection by activated neutrophils in vivo. Following cell activation, intracellular signals lead to the induction of ADAM17’s enzymatic activity; however, the target of this inducer mechanism remains unclear. Our study provides evidence of an activation mechanism that involves the extracellular region of the mature form of cell surface ADAM17 and not its intracellular region. We demonstrate that the catalytic activity of purified ADAM17 lacking a prodomain and its intracellular region is diminished under mild reducing conditions by DTT and enhanced by H₂O₂ oxidation. Moreover, H₂O₂ reversed ADAM17 inhibition by DTT. The treatment of neutrophils with H₂O₂ also induced L-selectin shedding in an ADAM17-dependent manner. These findings suggest that thiol-disulfide conversion occurring in the extracellular region of ADAM17 may be involved in its activation. An analysis of ADAM17 revealed that within its disintegrin/cysteine-rich region are two highly conserved, vicinal cysteine sulfhydryl motifs (cysteine-X-X-cysteine), which are well-characterized targets for thiol-disulfide exchange in various other proteins. Using a cell-based ADAM17 reconstitution assay, we demonstrate that the cysteine-X-X-cysteine motifs are critical for L-selectin cleavage. Taken together, our findings suggest that reduction-oxidation modifications of cysteinyl sulfhydryl groups in mature ADAM17 may serve as a mechanism for regulating the shedding of L-selectin following neutrophil stimulation.

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 This work was supported by Grant HL61613 from the National Institutes of Health.

² Current address: Schering-Plough Research Institute, Kenilworth, NJ 07033.

³ Address correspondence and reprint requests to Dr. Bruce Walcheck, University of Minnesota, 295j AS/VM Building, 1988 Fitch Avenue, St. Paul, MN 55108. E-mail address: walch003{at}umn.edu

⁴ Abbreviations used in this paper: ADAM, A disintegrin and metalloprotease; CGD, chronic granulomatous disease; DHR123, dihydrorhodamine123; DPI, diphenyleneiodonium sulfate; PDI, protein disulfide isomerase; redox, reduction-oxidation; ROS, reactive oxygen species; SOD, superoxide dismutase; TIMP3, tissue inhibitor of metalloproteinase 3.

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