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2-Macroglobulin Capture Allows Detection of Mast Cell Chymase in Serum and Creates a Reservoir of Angiotensin II-Generating Activity1
,,¶
* Cardiovascular Research Institute and
Department of Medicine, University of California at San Francisco, San Francisco, CA 94143;
Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305; the
Veterans Health Research Institute and the
¶ Veterans Affairs Medical Center, San Francisco, CA 94121; and
|| The Laboratory of Allergic Diseases of the National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892
Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by mast cells. When secreted from degranulating cells, it can interact with a variety of circulating antipeptidases, but is mostly captured by 
2-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that 2-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used 2-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of 
1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. 2-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and it appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin-converting enzyme. These findings suggest that chymase bound to 2-macroglobulin is active, that the complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that 2-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases.
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 in part by National Institutes of Health Grant HL024136.
2 W.W.R. and S.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. George H. Caughey, Veterans Affairs Medical Center 111-D, 4150 Clement Street, San Francisco, CA 94121. E-mail address: george.caughey{at}ucsf.edu
4 Abbreviations used in this paper: ACE, angiotensin converting enzyme; 1ACT, 1-antichymotrypsin; 2M, 2-macroglobulin; AAPF-4NA, succinyl-L-Ala-Ala-Pro-Phe-4-nitroanilide; AEPF-4NA, succinyl-L-Ala-Glu-Pro-Phe-4-nitroanilide; VPF-4NA, succinyl-L-Val-Pro-Phe-4-nitroanilide; RETF-4NA, acetyl-L-Arg-Glu-Thr-Phe-4-nitroanilide; kcat, turnover number; Km, Michaelis constant; kcat/Km, specificity constant.
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