α2-Macroglobulin Capture Allows Detection of Chymase in Serum and Creates a Reservoir of Angiotensin II-Generating Activity This information is current as of September 25, 2021. Wilfred W. Raymond, Sharon Su, Anastasia Makarova, Todd M. Wilson, Melody C. Carter, Dean D. Metcalfe and George H. Caughey J Immunol 2009; 182:5770-5777; ; doi: 10.4049/jimmunol.0900127 Downloaded from http://www.jimmunol.org/content/182/9/5770

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

␣ 2-Macroglobulin Capture Allows Detection of Mast Cell Chymase in Serum and Creates a Reservoir of Angiotensin II-Generating Activity1

Wilfred W. Raymond,2* Sharon Su,2‡ Anastasia Makarova,§ Todd M. Wilson,ʈ Melody C. Carter,ʈ Dean D. Metcalfe,ʈ and George H. Caughey3*†§¶

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 . ␣ The present work shows that 2-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I,

␣ Downloaded from with activity in serum that is stable with prolonged incubation. We used 2-macroglobulin capture to develop a sensitive, mi- crotiter 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 G and . The assay detects activity in chymase-spiked serum with a threshold ϳ ␣ 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 . These findings suggest that chymase http://www.jimmunol.org/ ␣ bound to 2-macroglobulin is active, that the complex is an angiotensin-converting -resistant reservoir of an- ␣ giotensin II-generating activity, and that 2-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases. The Journal of Immunology, 2009, 182: 5770–5777.

uman mast cell chymase cleaves angiotensin I selec- vators, and it is expressed mainly on the lumenal surface of vas- tively at Phe8 to generate bioactive angiotensin II (1–4). cular endothelium. On the other hand, chymase is a chymotryptic Indeed, chymase appears to be more efficient in this re- serine that is stored in mast cell secretory gran- H 4 gard than angiotensin-converting enzyme (ACE), based on kinet- ules and is potentially inactivated shortly after exocytosis by by guest on September 25, 2021 ics of hydrolysis by purified (1). Chymase is not inacti- any of several inhibitors present in vast molar excess in extra- vated by pharmaceutical inhibitors of ACE, so it is potentially cellular fluids (6, 7). Because chymase is sequestered inside responsible for angiotensin II generated in humans treated with cells and soon encounters inhibitors when released outside, a ACE inhibitors for hypertension. Generation of angiotensin II by major role in generating bioactive angiotensin may be consid- chymase may explain the greater antihypertensive effect of ACE ered unlikely. Nonetheless, pharmacological and genetic evi- inhibitors combined with angiotensin II receptor blockers com- dence in animal models suggest that generation of angiotensin pared with ACE inhibitors alone (5). However, chymase and ACE II by non-ACE, chymase-like pathways are important in vaso- belong to different enzyme classes and are made by different cells. motor dysfunction (8), vascular proliferation and stenosis (9, ACE is an ecto-metallo-dipeptidase with few if any native inacti- 10), angiogenesis (11, 12), ventricular remodeling and infarc- tion (13, 14), aneurysm formation (15), and regulation of blood *Cardiovascular Research Institute and †Department of Medicine, University of Cal- pressure (16). Pharmaceutical efforts are under way to develop ‡ ifornia at San Francisco, San Francisco, CA 94143; Department of Pediatrics, Stan- therapeutic inhibitors of the chymase pathway for generating ford University School of Medicine, Stanford, CA 94305; the §Veterans Health Re- search Institute and the ¶Veterans Affairs Medical Center, San Francisco, CA 94121; angiotensin II and to inhibit effects of chymase that may relate and ʈThe Laboratory of Allergic Diseases of the National Institute of Allergy and to targets other than angiotensin I. The present studies were Infectious Diseases, Bethesda, MD 20892 undertaken to explore the potential for chymase to generate Received for publication January 14, 2009. Accepted for publication February 26, 2009. angiotensin II following release from mast cells into biological fluids containing chymase inhibitors. 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 Initially chymase was proposed to be inactivated mainly by ser- with 18 U.S.C. Section 1734 solely to indicate this fact. pin-class inhibitors (6). However, subsequent work revealed that 1 This work was supported in part by National Institutes of Health Grant HL024136. ␣ ␣ serpins, including 1-antichymotrypsin ( 1ACT), are better sub- 2 W.W.R. and S.S. contributed equally to this work. strates for human chymase than they are inactivators, and that 3 ␣ Address correspondence and reprint requests to Dr. George H. Caughey, Veterans much or most of chymase added to serum is inactivated by 2- Affairs Medical Center 111-D, 4150 Clement Street, San Francisco, CA 94121. E- macroglobulin (␣ M), with which the association rate constants mail address: [email protected] 2 are highly favorable (7). This contrasts with the fate of certain 4 ␣ Abbreviations used in this paper: ACE, angiotensin converting enzyme; 1ACT, ␣ ␣ ␣ other mast and leukocyte serine peptidases, including , 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- ␣ which is too large to enter the 2M cage, and and 4NA, succinyl-L-Val-Pro-Phe-4-nitroanilide; RETF-4NA, acetyl-L-Arg-Glu-Thr-Phe- 4-nitroanilide; k , turnover number; K , Michaelis constant; k /K , specificity neutrophil , which are more rapidly inactivated by plasma cat m cat m ␣ ␣ constant. serpins than by 2M (17). 2M is a major blood protein and differs www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900127 The Journal of Immunology 5771

from other circulating antipeptidases in key ways. It is nonspecific oration. Initial rates of nitroaniline release were measured spectrophoto- with regard to peptidase class (serine, aspartyl, thiol, metallo) and metrically at 410 nm and 25°C using a kinetic microplate reader (Synergy 2; BioTek). Turnover number (k ) and Michaelis constant (K ) were de- attracts peptidases with a broad range of peptide target preferences cat m termined from observed initial rates of hydrolysis over a range of substrate (18). Although it attaches covalently to peptidases via a thiol ester concentrations by nonlinear regression as implemented by GraphPad Prism ␣ that becomes reactive after cleavage of a target region in 2M, this 4 software (GraphPad Software). Active enzyme concentrations used in connection is made with a lysine on the surface of the peptidase calculating kcat from predicted maximum rates of hydrolysis were deter- and does not involve the canonical antipeptidase mechanism of mined in separate assays under standard assay conditions for which specific ␣ activity values were available, as specified in the preceding paragraph. occupying the substrate (19). Instead, 2M traps the peptidase in a cage-like structure, which is inaccessible to protein Measurement of chymase activity in chymase-spiked serum targets of the peptidase but may allow access by small substrates to the trapped peptidase. The present findings suggest that human Pilot studies compared performance of substrates in serum that had been chymase circulates bound to ␣ M, where it is active and can be spiked with human chymase, cathepsin G, or chymotrypsin. Further pilot 2 studies examined influence of assay duration and temperature of incuba- assayed in serum using a selective, newly developed substrate. The tion, salt concentration, inclusion of DMSO and detergent, substrate selec- ␣ findings further reveal that chymase captured by 2M generates tion (e.g., AEPF-4NA vs RETF-4NA), and mode of measurement (1-ml angiotensin II. This suggests that chymase, after secretion by mast cuvette vs microtiter plate). Conditions were optimized for assay of native cells, remains active longer than once thought and may circulate chymase activity in serum using the sealed, 96-well microtiter plate format ␮ bound to ␣ M, in which form it can generate angiotensin II. described in the preceding paragraph. Briefly, 20 l of serum was diluted 2 10-fold in 20 mM Tris-HCl (pH 7.9) containing 2 M NaCl, 0.05% DMSO,

0.01% Triton X-100, and 1 mM RETF-4NA. Change in A410 nm was mea- Materials and Methods sured serially in duplicate at 37°C for up to 3 h. In additional experiments, Downloaded from Materials activities of peptidases spiked into human serum (Sigma-Aldrich) at a final concentration of 10 pM were compared using similar assay conditions, Recombinant human prochymase was expressed in Trichoplusia ni cells except that change in A410 nm was measured in 1-ml cuvettes in a Genesys and purified as described (20). Mature human chymase was activated from 5 spectrophotometer (Thermo Fisher Scientific). recombinant prochymase and repurified as described (20, 21). Human ca- thepsin G and bovine ␣-chymotrypsin were purchased from MP Biomedi- cals and Sigma-Aldrich, respectively. Peptidase substrate succinyl-L-Ala- Stability of chymase and cathepsin G in serum http://www.jimmunol.org/ Ala-Pro-Phe-4-nitroanilide (AAPF-4NA) was from Sigma-Aldrich and Activity was compared in PBS (containing 0.05% DMSO and 0.01% Tri- succinyl-L-Ala-Glu-Pro-Phe-4-nitroanilide (AEPF-4NA) was from ton X-100) and in enzyme-spiked serum during8hofincubation at 37°C. Bachem; succinyl-L-Val-Pro-Phe-4-nitroanilide (VPF-4NA) was provided Aliquots were withdrawn at intervals to measure residual chymase and by Dr. John Burnier of Genentech. Angiotensin I was purchased from cathepsin G activity using AAPF-4NA and VPF-4NA, respectively, in ␣ ␣ Peninsula Laboratories (now Bachem). Antipeptidases 2M and 1ACT 1-ml cuvettes. In additional experiments, stability to five cycles of freezing were from EMD Biosciences/Calbiochem, and human serum used in assay and thawing was examined in serum spiked with 10 ng/ml active chymase. development was from Sigma-Aldrich. Design and synthesis of a colorimetric substrate of chymase Size exclusion chromatography and immunoblotting of Screening of recombinant human chymase with a combinatorial library of chymase-spiked serum tetrapeptide substrates (21) identified Arg-Glu-Thr-Tyr or Arg-Glu-Thr- Normal human serum (100 ␮l) spiked with 340 ng of active human chy- by guest on September 25, 2021 Phe as being highly favored in the P4–P1 positions on the N-terminal side ␣ mase or prochymase was chromatographed using an AKTA purifier system of the site of hydrolysis. A synthetic inhibitor, N -benzoxycarbonyl-L-Arg- (GE Healthcare) on a Superose 6 GL 10/300 size exclusion column equil- P Glu-Thr-Phe -phosphonate, which was synthesized based on these se- ibrated with PBS. Outflow was monitored for absorbance at 280 nm. Ali- quences, inhibited chymase selectively in comparison with cathepsin G quots of column fractions were assayed for amidolytic activity with RETF- (21), which encouraged us to design an assay substrate, acetyl-L-Arg-Glu- 4NA in a 96-well format as noted for chymase-spiked serum. Aliquots Thr-Phe-4-nitroanilide (RETF-4NA), which was custom-synthesized by from each fraction were divided into six pools covering distinct molecular AnaSpec. mass regions. Portions of each pool were electrophoresed, electroblotted to a polyvinylidene difluoride membrane, and probed with anti-human ␣ M Assessment of concentration of active enzyme 2 mAb 2D9 (Abcam) and anti-human chymase (CC-1; Abcam). The column Chymase activity was measured by addition of enzyme to buffer containing was calibrated with thyroglobulin (669 kDa), apoferritin (460 kDa), and 1 mM AAPF-4NA, 45 mM Tris-HCl (pH 8.0), 1.8 M NaCl, and 9% BSA (66 kDa). Human chymase was also chromatographed in PBS to ␣ DMSO. Cathepsin G activity was measured by addition of enzyme to assay establish elution behavior in the absence of 2M and other serum proteins. buffer containing 1 mM VPF-4NA, 0.1 M HEPES (pH 7.5), 0.5 M NaCl, and 10% DMSO. Chymotrypsin activity was measured in buffer containing Recruitment and pathological stratification of subjects with 1 mM AAPF-4NA, 0.1 M HEPES (pH 7.5), 0.5 M NaCl, and 10% DMSO. mastocytosis Change in A410 nm was monitored at 25°C. The concentration of active enzyme in each preparation was determined by referencing observed ac- Study participants were evaluated at the National Institutes of Health (Be- tivity under these conditions to reported specific activity, which is 2.1 ϫ thesda, MD) as part of Institutional Review Board-approved research pro- 6 ϫ 7 ϫ 7 10 , 2.4 10 , and 1.7 10 A410 nm/min/M for human cathepsin G (22), tocols exploring the pathogenesis of mastocytosis. Twenty-five patients human chymase (6), and bovine chymotrypsin (23), respectively. who met World Health Organization criteria for mastocytosis between 2003 and 2008 were included (24). The 15 adult subjects were classified as Kinetic comparisons of peptide-based colorimetric substrates follows: 13 with indolent systemic mastocytosis and 2 with aggressive Hydrolysis of substrates was compared using recombinant human chy- systemic mastocytosis. Of the 10 pediatric subjects, 7 were classified as mase, human cathepsin G, and bovine chymotrypsin in the presence and indolent systemic mastocytosis and 3 as cutaneous mastocytosis. ␣ ␣ absence of 2M. For experiments involving 2M, each enzyme was incu- ␣ bated with 1000-fold molar excess of 2M in PBS (pH 7.4) at 25°C for 30 Measurement of immunoreactive tryptase in subjects with ␣ min, followed by incubation for 30 min with 2-fold molar excess of 1ACT mastocytosis to inactivate any residual free enzyme. To assess relative sensitivity and ␣ specificity for chymase free in solution and when bound to 2M, we com- As part of establishing World Health Organization diagnostic criteria for pared kinetic attributes of the novel substrate RETF-4NA with those of systemic mastocytosis, a total tryptase level was obtained for all partici- AAPF-4NA, AEPF-4NA, and VPF-4NA. Substrates were dissolved in PBS pants. Serum was collected at the National Institutes of Health, frozen to (pH 7.4) containing 0.05% DMSO and 0.01% Triton X-100. Reactions Ϫ20°C, and then shipped to the Mayo Medical Laboratories, where serum ␣ were initiated by addition of free or 2M-bound enzyme. The reaction total tryptase was measured via fluorescence enzyme immunoassay, with mixture was pipetted in triplicate in 180-␮l aliquots into wells of a Costar normal level of Ͻ11.5 ng/ml, according to the laboratory. Serum for the 3320 flat bottom 96-well plate (Corning Life Sciences), which then was chymase experiments was handled and mailed to the San Francisco Vet- sealed with TempPlate RT optical film (USA Scientific) to minimize evap- erans Affairs Medical Center in a similar manner. ␣ 5772 CHYMASE CAPTURE BY 2-MACROGLOBULIN

FIGURE 1. Screening of peptidyl nitroanilide substrates against chymase and related chymotryptic peptidases. A, Performance of chymotrypsin, ca- thepsin G, and chymase vs standard substrates AAPF-4NA, VPF-4NA, and AEPF-4NA in comparison with novel substrate RETF-4NA, which was synthesized based on results of combinatorial screening of chymase with peptide substrates. To facilitate comparison between enzymes, results are normalized to substrate turnover (molecules of substrate hydrolyzed per second by each molecule of active enzyme). B, Performance of the chymotryptic peptidases vs novel substrate RETF-4NA. The dashed vertical line indicates the concentration of RETF-4NA used in this work in assays of chymotryptic activity in serum. Downloaded from

Measurement of chymase activity in subjects with mastocytosis strate has been used by investigators to assay all three peptidases. Serum from subjects with mastocytosis was assayed in duplicate for RETF- For cathepsin G, the best substrate by far was VPF-4NA, although 4NA-hydrolyzing activity in sealed microtiter plates as described for as- this peptidase is weak overall compared with chymotrypsin and says of chymase-spiked serum. RETF-4NA-hydrolyzing activity was mea- chymase (as revealed by kcat/Km specificity constants in Table I).

sured in duplicate in separate aliquots of the same serum samples after Consequently, VPF-4NA is more efficiently hydrolyzed by chy- http://www.jimmunol.org/ ␮ preincubation with 100 M chymostatin, a chymase inhibitor. Activity motrypsin and chymase than by cathepsin G, and it has compar- observed in the presence of chymostatin was considered background. The ⌬ atively little ability to discriminate among these enzymes. For chy- difference in A410 nm measured with and without chymostatin was con- sidered to be chymase-like activity. Concentration of active chymase in mase, VPF-4NA and RETF-4NA are the best of the substrates native samples was determined by extrapolation from standard curves gen- examined and yield similar specificity constants. However, as re- erated using serum spiked with known concentrations of recombinant ac- vealed in Fig. 1B and as hypothesized from results of combinato- tive chymase. rial screening, our novel substrate RETF-4NA is substantially Assessment of angiotensin II-generating activity in chymase more selective than the other substrates for chymase in comparison

bound to inhibitors with cathepsin G and chymotrypsin. When kcat/Km is compared for enzymes incubated in PBS, the ratios for chymase, chymotrypsin, by guest on September 25, 2021 Active chymase (1 pmol) was incubated in 7 ␮l of PBS at 37°C for 15 min ␣ ␣ and cathepsin G are 15:8.5:1 for AEPF-4NA and a much more with 5 pmol of human 2M or 5 pmol of human 1ACT. To verify the ␣ reaction of chymase with 2M, 1 pmol of chymase was first incubated in selective 55:8.0:1 for RETF-4NA. A selectivity advantage is also ␣ PBS at 37°C for 15 min with 5 pmol of 2M and then for 15 min with 5 noted for AEPF-4NA incubated with ␣ M, as shown in Table I. ␣ ␮ 2 pmol of 1ACT. Following these incubations, 1 l of the resulting mix- Based on these sensitivity and selectivity profiles, AEPF-4NA and tures (containing 170 fmol of chymase) was incubated with 1 nmol of angiotensin I in 50 ␮l of PBS for 30 min at 37°C. Reactions were termi- RETF-4NA were tested as candidate substrates with which to con- nated by addition of 1 ␮l of 12 N HCl, diluted with 60 ␮l of an aqueous struct a serum-based, chymase-selective assay. solution of 10% acetonitrile/0.1% trifluoroacetic acid, and injected onto a 2.1 ϫ 250-mm BioBasic C-18 column (Thermo Scientific) equilibrated in 10% acetonitrile/0.1% trifluoroacetic acid on the AKTA purifier system Chymase measured with high selectivity and sensitivity in (GE Healthcare). Angiotensin I and cleavage products were eluted with a chymase-spiked serum linear gradient of 10–40% acetonitrile over 2.7 ml (three column vol- In pilot experiments (not shown), background activity in serum umes). Outflow was monitored for A280 nm. Chromatograms were analyzed using Unicorn 5.0 software (GE Healthcare). was higher when using AEPF-4NA than when using RETF-4NA. We tested AEPF-4NA because it is commercially available and Angiotensin generation by chymase in native human serum because our laboratory previously identified a preference by chy- One microliter of native serum or chymase-spiked serum was incubated mase for peptidic substrates with Glu in the P3 position, that is, with 20 nmol of angiotensin I in PBS for 16 h at 37°C with or without 2 three residues on the N-terminal side of the site of hydrolysis (21). mM captopril, 0.4 mM chymostatin, or both inhibitors. Products were ex- Indeed, as shown in Fig. 1 and Table I, AEPF-4NA is more readily tracted on PepClean C-18 spin columns (Thermo Scientific/Pierce), eluted with 50% acetonitrile/0.1% trifluoroacetic acid, and dried by vacuum cen- hydrolyzed by chymase than by the other peptidases. However, trifugation. Pellets were resuspended in 110 ␮l of 10% acetonitrile/0.1% AEPF-4NA is not as selective as custom-synthesized RETF-4NA, trifluoroacetic acid and subjected to reverse-phase HPLC as described for which is fully optimized based on preferences identified by com- angiotensin hydrolyzed in the presence of purified inhibitors. binatorial substrate screening. To test RETF-4NA selectivity in serum, 10 pM active chymase, chymotrypsin, and cathepsin G Results were added separately to aliquots of low-background serum con- RETF-4NA is a sensitive and selective substrate for chymase taining 1.4 mM RETF-4NA. At this concentration, which is well ␣ when free or bound to 2M above the predicted Km of chymase and chymotrypsin but likely As revealed in Fig. 1 and Table I, the kinetic performances of the well below that of cathepsin G, chymotrypsin activity was 10% colorimetric substrates compared in this study differ markedly. For that of chymase, and cathepsin G activity was 0. This was as pre- chymotrypsin, the best substrate in terms of maximum hydrolytic dicted by screening of enzyme-substrate combinations in PBS and ␣ rate is AAPF-4NA, which is much less rapidly hydrolyzed by ca- 2M (Table I), which revealed that hydrolytic rates at substrate thepsin G and chymase, although this commercially available sub- concentrations well above Km are much higher for chymase than The Journal of Immunology 5773

Table I. Kinetic comparisons of peptidyl nitroanilide hydrolysis by chymotryptic peptidases

AAPF-4NA AEPF-4NA RETF-4NA VPF-4NA

Chymase a Ϯ Ϯ Ϯ Ϯ kcat 9.3 0.8 8.8 0.2 19.7 0.1 17.2 0.4 a Ϯ Ϯ Ϯ Ϯ Km 0.31 0.07 0.15 0.01 0.64 0.05 0.29 0.04 a kcat/Km 30 59 31 59 ϩ ␣ Chymase 2M Ϯ Ϯ Ϯ Ϯ kcat 49 2511871 8.9 0.6 Ϯ Ϯ Ϯ Ϯ Km 0.28 0.02 0.08 0.08 0.32 0.01 0.16 0.03 kcat/Km 170 640 270 55 Cathepsin G Ϯ Ϯ Ϯ Ϯ kcat 1.4 0.1 3.7 0.8 7.3 11.9 13.8 0.4 Ϯ Ϯ Ϯ Ϯ Km 0.97 0.06 0.93 0.08 13 36 0.69 0.07 kcat/Km 1.4 3.9 0.56 20 ϩ ␣ Cathepsin G 2M Ϯ Ϯ Ϯ Ϯ kcat 0 0 0.71 0.26 0 0 2.4 2.1 Ϯ Ϯ Km 0.12 0.15 0.8 1.4 kcat/Km 5.9 3.0 Chymotrypsin Ϯ Ϯ Ϯ Ϯ kcat 68 8 12.8 0.5 5.0 0.4 11.8 0.4 Ϯ Ϯ Ϯ Ϯ Downloaded from Km 0.52 0.14 0.39 0.04 1.1 0.2 0.07 0.01 kcat/Km 130 33 4.5 170 ϩ ␣ Chymotrypsin 2M Ϯ Ϯ Ϯ Ϯ kcat 48 1311 3.9 0.4 22 1 Ϯ Ϯ Ϯ Ϯ Km 0.035 0.003 0.17 0.02 0.057 0.026 0.037 0.007 kcat/Km 1400 180 68 59

a Ϫ1 Ϫ1 Ϫ1 Units of kcat, Km, and kcat/Km are s , mM, and s mM , respectively. http://www.jimmunol.org/ for chymotrypsin, and that hydrolysis of RETF-4NA is nearly un- chymase. The assay also is sensitive, being capable of detecting ␣ ϳ detectable for cathepsin G preincubated with 2M. active chymase in serum at or above 1 pM. More detailed kinetic evaluation of RETF-4NA hydrolysis by Ϯ Ϫ1 chymase in spiked serum revealed kcat of 9.6 0.3 s and Km of Serum stabilizes chymase and cathepsin G activity 0.48 Ϯ 0.03 mM (yielding nominal k /K of 20 sϪ1mMϪ1). The cat m As shown in Fig. 3, chymase activity in serum is remarkably stable k and k /K estimates in this case are minimum values because cat cat m to assay over time in serum, as compared with PBS. This stability they assume that all chymase added to serum remains active, contributes to the enhancement of assay sensitivity achieved by by guest on September 25, 2021 which is likely not to be the case. The net effect is that chymase prolonged incubation. added to serum behaves similarly to the same concentration of chymase added to PBS, in terms of overall kinetic performance. ␣ The better performance of 2M-bound chymase in serum is prob- ably offset by inhibition of a portion of the pool of active enzyme ␣ by antipeptidases other than 2M. As shown in Fig. 2, chymase activity can be measured over a large range of enzyme concentra- tions without evidence of a plateau effect or deviation from lin- earity. This is evidence that the assay has a wide dynamic range, ␣ likely reflecting the large molar excess of 2M available to engage

FIGURE 3. Stability of chymase and cathepsin G activity in serum. Left

panel, Examples of repeated measurements of A410 nm in single wells of a 96-well plate in a kinetic spectrophotometer at 37°C. Individual wells con- tained RETF-4NA substrate (0.5 mM) and spiked chymase concentrations as follows: F, 2.1 ng/ml; E, 0.68 ng/ml; f, 0.23 ng/ml; and Ⅺ, 0.08 ng/ml. FIGURE 2. RETF-4NA-hydrolyzing activity in chymase-spiked se- Right panel, Results of long-term incubations of human cathepsin G and rum. Human serum was spiked with human chymase over the range of chymase in serum (E, cathepsin G; F, chymase) and PBS (Ⅺ, cathepsin G; concentrations of enzyme indicated by the x-axis. There is a strong f, chymase). Enzyme-spiked serum and PBS were incubated as stocks for linear correlation between concentrations of chymase in spiked serum 8 h. Aliquots were withdrawn at the indicated intervals and subjected to and observed rates of substrate hydrolysis, as reflected by change in cuvette-based spectrophotometric assay of chymotryptic activity using ⌬ milliabsorbance ( mA410 nm) per minute. Standard curves based on this AAPF-4NA for chymase and VPF-4NA for cathepsin G. Results are ex- relationship allow determination of levels of chymase-like activity in pressed as percentage of activity relative to activity measured at the start of native serum, as in Fig. 4. incubation. ␣ 5774 CHYMASE CAPTURE BY 2-MACROGLOBULIN

␣ FIGURE 4. Coelution of chymase activity with 2M in serum. The chromatogram represented by the solid line shows absorbance of Superose 6 fractions generated by serum subjected to gel filtration chromatography in PBS. Downward arrows indicate elution positions of standard proteins of known size applied separately to the column in PBS. The asterisk indicates chymase (ϳ30 kDa) applied to the column in PBS. The dashed line indi- Downloaded from cates levels of chymase-like (RETF-4NA-hydrolyzing) activity assessed in individual fractions of eluate generated by serum premixed with active chymase. Fractions were also collected in six larger pools, as indicated, then concentrated and subjected to reducing SDS-PAGE and immunoblot- ␣ ting using Abs recognizing 2M. Results, as revealed by the immunoblot, ␣ reveal strongest 2M immunoreactivity in pool 2, which also contains most FIGURE 6. ␣ http://www.jimmunol.org/ of the chymase activity. Generation of angiotensin II by 2M-captured chymase. These HPLC chromatograms reveal products resulting from incubation of ␣ angiotensin I with chymase alone, chymase plus 1ACT, chymase plus ␣ M, or chymase plus the combination of ␣ ACT and ␣ M. Absorbance of Chymase activity in serum co-elutes with ␣ M 2 1 2 2 the eluate was monitored continuously at 260 nm. Incubation time and As shown in Fig. 4, the vast majority of activity of chymase, when chymase concentration were the same in each reaction and were selected so the enzyme is spiked into serum and size-fractionated by gel chro- that digestion would allow visualization of parent as well as product pep- tides. Elution positions of angiotensin I and the bioactive product angio- matography, elutes at an apparent Mr consistent with capture by ␣ ␣ ␣ tensin II are as noted. 2M. Furthermore, 2M immunoreactivity of 2M in column frac- tions coelutes with the peak of chymase activity, providing further by guest on September 25, 2021 evidence that chymase, when mixed with the complex mixture of peptidase inhibitors and other proteins in serum, binds mainly to with serum levels of immunoreactive total tryptase. Levels of ac- ␣ 2M, which preserves its activity. tive chymase (in ng/ml) are always lower than those of immuno- reactive tryptase in paired samples. The highest chymase activity Detection of chymase activity in serum of subjects with is in a subject with aggressive systemic mastocytosis. Nearly all mastocytosis subjects with indolent systemic mastocytosis have readily detect- As shown in Fig. 5, most subjects with systemic mastocytosis have able chymase-like activity. However, all three of the subjects with detectable serum chymase-like activity, which correlates weakly cutaneous mastocytosis have low levels at or near the threshold for detection. ␣ Chymase captured and protected from serpins by 2M generates angiotensin II As revealed in the chromatograms in Fig. 6, chymase generates bioactive angiotensin II from angiotensin I when preincubated ␣ with 2M, but it has no detectable activity when preincubated with ␣ ␣ the 1ACT. However, when preincubated with 2M and ␣ 1ACT together, chymase’s angiotensin II-generating capacity is preserved, consistent with chymase reacting more slowly with ␣ ␣ ␣ 1ACT than with 2M, and gaining protection within the 2M “cage” from inhibition by the serpin. This finding also reveals that ␣ the size of the cage in chymase-bound 2M is sufficiently large to admit the decapeptide angiotensin I, which is more than twice the FIGURE 5. Serum chymase activity in mastocytosis. Chymase activity length of the tri- and tetrapeptide nitroanilide substrates used in and immunoreactive tryptase was measured in serum from subjects with var- this work to develop the serum chymase assay. The results shown ious types of mastocytosis. Chymase activity was measured using the RETF- in Fig. 6 also suggest that rates of hydrolysis by ␣ M-bound chy- 4NA microtiter plate assay. Total immunoreactive ␣ plus ␤ serum tryptase 2 mase are, if anything, greater than for chymase free in solution. (protryptase plus mature tryptase) was measured by ELISA. Each symbol rep- resents data from a single subject and sample. The dashed line indicates the Chymase generates angiotensin II in serum active chymase detection threshold (ϳ0.03 ng/ml) of the assay. Mastocytosis abbreviations are as follows: ASM, aggressive systemic mastocytosis; ISM, As shown in Fig. 7, the combination of chymase and captopril indolent systemic mastocytosis; CM, cutaneous mastocytosis. almost fully ablates angiotensin-generating capacity of native and The Journal of Immunology 5775

with the fate of other secreted immune peptidases. For example, and chymase’s closest relative, cathepsin G, are inactivated in plasma by serpin-class inhibitors in a small fraction of 1 s (half life of ϳ0.4 ms (25)). The half-life of the chymase- ␣ ␣ 2M in blood, if typical of other serine peptidase- 2M complexes, is Ͼ106-fold longer than that of neutrophil elastase. Under the optimized conditions of our assay, the activity of ␣ chymase bound to 2M in serum ex vivo is remarkably stable, especially compared with stability of pure chymase in PBS or of chymase combined with serpins. We exploited this stability, which allows prolonged incubations with chymase substrates, to increase the sensitivity of the serum assay. The assay we report in this work is an alternative to the development and application of immuno- assay-based assays. Although Abs raised against human chymase FIGURE 7. Generation of angiotensin II by serum chymase. The graph work well in immunohistochemical applications and in blotting of shows results of measurement of angiotensin II-generating capacity of na- purified chymase, they are less successful as components of im- tive serum and of the same sample spiked with recombinant human chy- munoassays for detecting chymase in complex biological fluids, mase (50 or 100 pM). Some samples were preincubated with an ACE including serum (26). This may be because most chymase released Downloaded from inhibitor (captopril), a chymase inhibitor (chymostatin), or with both in- ␣ from mast cells becomes covalently linked to (and caged by) 2M, hibitors, as indicated. in which form its major Ab-binding epitopes may be shielded from interacting productively with Abs. To our knowledge, the only chymase-spiked serum alike. The sample of serum used in the reported successful use of an immunoassay to detect chymase in studies in Fig. 7 was chosen for its low baseline chymase-like (i.e., human serum was in postmortem specimens in cases of anaphy-

chymostatin-sensitive) activity to allow exploration of concentra- laxis (27). However, in the vast majority of cases the level of http://www.jimmunol.org/ tion-responsiveness to chymase in spiking experiments. Addition chymase determined by immunoassay is below the level of detec- of chymase to serum increases chymostatin-sensitive angiotensin tion (26, 27). Unlike activity-based assays, immunoassays have the II-generating activity in proportion to the concentration of added potential to detect prochymase, denatured chymase, and other pro- chymase. These findings establish that chymase can generate an- teolytically inactive forms, which are not expected to be captured ␣ giotensin II in serum. In the sample of serum used in the Fig. 7 by 2M because they are unable to cleave the bait region. studies, native ACE-like (captopril-sensitive) activity is at least Investigations in mice suggest that little if any prochymase is ϳ4-fold greater than that of native chymase-like (chymostatin- stored by mast cells, except in the case of animals lacking the sensitive) activity, as reflected by relative angiotensin II-generat- intracellular chymase-activating enzyme dipeptidylpeptidase I ing capacity revealed in the first four bars of the graph. In other (28). In humans, it is not known whether there is constitutive re- by guest on September 25, 2021 samples (not shown), native chymase-like angiotensin II-generat- lease of prochymase from mast cells in tissues. However, if a ma- ing activity exceeds ACE-like activity. These findings suggest that jor portion of chymase were released in the proenzyme form, one chymase can generate angiotensin II in native serum and that its would expect greater ease in developing immunoassays, since contribution can be similar to or even greater than that of ␣ prochymase is not captured by 2M. In contrast, the great majority soluble ACE. of circulating immunoreactive ␤ , which are produced by most human mast cells, is thought to be immature, inactive proen- Discussion zyme (29, 30). This is also true of ␣ tryptases in humans who This work reveals that an active form of human chymase can be possess ␣ genes (31). However, levels of mature ␤ tryptases can ␣ captured by 2M, in which form it can cleave small peptide sub- rise substantially in some subjects shortly after anaphylaxis (32), strates, including angiotensin I, and is protected from irreversible presumably because the active tryptase tetramer, which is much inactivation by serpins and other antipeptidases in biological flu- larger than monomeric chymase, is too big to be engulfed by the ids. We exploited ␣ M binding to develop a sensitive and specific 2 ␣ M cage (33). The weak correlation of active chymase levels with assay for chymase activity in the serum of subjects with mastocy- 2 tryptase levels in our mastocytosis samples, as well as the major tosis. These studies reveal that chymase, after secretion by mast difference between tryptase and chymase in the range of measured cells and capture by ␣ M, can cleave small peptides for longer than 2 concentrations, may relate to major disparity between tryptases once thought possible. Extravascular chymase captured and pro- ␣ and chymase in the extent to which mast cells release the two tected by 2M may be an important source of non-ACE-generated angiotensin II near tissue sites of mast cell degranulation. The peptidase types as proenzymes. If human chymase, unlike trypta- ␣ ses, is released mainly from granules via a regulated pathway, then portion of 2M-caged chymase making its way to the bloodstream provides the basis of our serum assay and may be a mobile source there is the potential that chymases are released acutely in larger of angiotensin II-generating chymase in blood and tissues remote amounts in settings of anaphylaxis, which is a possibility that we from original sites of mast cell degranulation. The half-life of pep- are exploring. Nonetheless, it seems likely that chymase activity in ␣ serum is influenced by total body burden of mast cells, in that in tidase-bound 2M in blood in vivo is 9–12 min, with activity of labeled complex peaking at 30–40 min in liver (24), which ap- vitro assays of mast cells suggest that chymase “leaks” from mast pears to be a major site of uptake and destruction of the complex. cell granules in the absence of specific stimulation at a low but Thus, chymase activity in blood will reflect a balance between steady rate (34). This hypothesis is consistent with our assay re- ␣ rates of production and removal of the chymase- 2M complex. sults in subjects with mastocytosis, in that chymase levels are ␣ The half-life of peptidase- 2M complexes before entering the much higher in systemic mastocytosis than in more localized cu- bloodstream is not known. Nonetheless, the duration of chymase taneous mastocytosis. Nonetheless, a few subjects with the indo- ␣ activity following capture by 2M is profoundly longer compared lent subtype of systemic mastocytosis had levels at or below the ␣ 5776 CHYMASE CAPTURE BY 2-MACROGLOBULIN level of detection, which may reflect low mast cell burden or pos- Disclosures sibly variations in peptidase phenotype, because mastocytosis cells The authors have no financial conflicts of interest. can vary in relative expression of tryptases and chymase (35). The present studies underscore the value of combinatorial pep- References tide substrate screening in developing selective substrates for spe- 1. Reilly, C. F., D. A. Tewksbury, N. B. Schechter, and J. Travis. 1982. Rapid cific peptidases. Our custom substrate RETF-4NA was synthesized conversion of angiotensin I to angiotensin II by neutrophil and mast cell pro- ϳ teinases. J. Biol. Chem. 257: 8619–8622. for the present studies based on results of a screen of 160,000 2. Wintroub, B. U., N. B. Schechter, G. S. 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