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J. Biol. Chem., Vol. 282, Issue 19, 14645-14654, May 11, 2007

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The Sulfate Groups of Chondroitin Sulfate- and Heparan Sulfate-containing Proteoglycans in Neutrophil Plasma Membranes Are Novel Binding Sites for Human Leukocyte Elastase and Cathepsin G^*

Edward J. Campbell and Caroline A. Owen¹

From the Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115 and Department of Internal Medicine, University of Utah Health Sciences Center and Intermountain Health Care, Salt Lake City, Utah 84132

Human leukocyte elastase (HLE) and cathepsin G (CG) are expressed at high levels on the surface of activated human neutrophils (PMN) in catalytically active but inhibitor-resistant forms having the potential to contribute to tissue injury. Herein we have investigated the mechanisms by which HLE and CG bind to PMN plasma membranes. ¹²⁵I-Labeled HLE and CG bind to PMN at 0 °C in a saturable and reversible manner (K_D = 5.38 and 4.36 x 10^-7 M and 11.5 and 8.1 x 10⁶ binding sites/cell, respectively). Incubation of PMN with radiolabeled HLE and CG in the presence of a 200-fold molar excess of unlabeled HLE, CG, myeloperoxidase, lactoferrin, proteinase 3, phenylmethylsulfonyl fluoride (PMSF)-inactivated HLE, or PMSF-inactivated CG inhibited binding of radiolabeled ligands. This indicates that these PMN granule proteins share binding sites on PMN and that functional active sites of HLE and CG are not required for their binding to PMN. The sulfate groups of heparan sulfate- and chondroitin sulfate-containing proteoglycans are the PMN binding sites for HLE and CG since binding of HLE and CG to PMN was inhibited by incubating PMN with 1) trypsin, chondroitinase ABC, and heparitinases, but not other glycanases, and 2) purified chondroitin sulfates, heparan sulfate, and other sulfated molecules, but not with non-sulfated glycans. Thus, heparan sulfate- and chondroitin sulfate-containing proteoglycans are low affinity, high volume PMN surface binding sites for HLE and CG, which are well suited to bind high concentrations of active serine proteinases released from degranulating PMN.

Received for publication, August 31, 2006 , and in revised form, March 20, 2007.

^* This work was supported by United States Public Health Service Grants HL63137 and HL086814 (to C. A. O.), a Career Investigator Award from the Massachusetts Thoracic Society (to C. A. O.), an American Thoracic Society/Pulmonary Fibrosis Foundation Research Grant (to C. A. O.), an American Heart Association North East Affiliate Research Award (to C. A. O.), and a Clinical Innovator Award from the Flight Attendants Medical Research Institute (to C. A. O.). 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 Figs. 1 and 2.

¹ To whom correspondence should be addressed: Division of Pulmonary and Critical Care Medicine, 905 Thorn Bldg., Brigham and Women's Hospital, 75 Francis St., Boston, MA, 02115. Tel.: 617-525-6665; Fax: 617-232-4623; E-mail: cowen{at}rics.bwh.harvard.edu.

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