- Email: [email protected]
CAP 37, A 37 kD human neutrophil granule cationic protein shares homology with inflammatory proteinases
Life Sciences, Vol. 46, pp. 189-196 Printed in the U.S.A.
Pergamon Press
CAP 37, A 37 KD HUMAN NEUTROPHIL GRANULE CATIONIC PROTEIN SHARES HOMOLOGY WITH INFLAMMATORY PROTEINASES H. Anne Pereira1, John K. Spitznagel I, Jan Pohl 2, Douglas E. Wilson z, John Morgan 3, Ilona Palings3, and James W. Larrick 3 1Dept. of Microbiology and Immunology and 2protein Sequencing Facility, Emory University School of Medicine, Atlanta GA 30322 and 3Genelabs Inc., 505 Penobscot Drive, Redwood City, CA 94063. (Received in final form November 17, 1989)
Summary We have previously shown that a major granule-associated cationic protein CAP 37 (Mr = 37 kD) derived from human PMN is a monocyte-specific chemoattractant. The N-terminal amino acid sequence of this novel chemotactic protein shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, a critical substitution of a serine for a histidine at position 41, results in its lack of serine protease activity. The process of inflammation is orchestrated by the sequential release of specific molecules that mediate the activation and chemotaxis of various cell types (1). Early inflammatory lesions are comprised primarily of polymorphonuclear leukocytes (PMN) followed by infiltration of mononuclear phagocytes. Evidence suggests that one or more factors derived from the PMN may modulate the subsequent chemotaxis of monocytes,(2,3). In fact an early study described the absolute requirement of PMNs for the subsequent infiltration of mononuclear phagocytes (4). Previous work from this laboratory demonstrated the antimicrobial activity of a major PMN granule protein CAP 37 [designated Cationic Antimicrobial Protein of Mr = 37kd] (5,6). We have recently demonstrated that this protein is a potent mediator of monocyte specific chemotaxis, and thus may act as a mediator of the second wave of inflammation (Pereira et al., submitted for publication). Stimulation of PMN with opsonized Staphylococcus aureus resulted in a rapid release of approximately 89% of CAP37 into the extracellular milieu. This finding was particularly noteworthy since, if a substance is to act as a chemotactic factor in vivo it would necessarily have to be released extracellularly to effect its function. Immunocytochemical studies performed on peripheral blood cells indicated that CAP37 was present in PMN alone. Mononuclear cells, eosinophils, and red blood cells failed to stain positive with the monospecific mouse antibody to CAP37 used in these studies (Pereira et al., submitted for publication). The present report describes the final purification and Nterminal amino acid sequence of human CAP37. This protein is related to the serine class of proteinases and shares significant homology with several other proteases derived from the granules of inflammatory leukocytes that are thought to mediate tissue damage during the course of inflammatory diseases. 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
190
CAP 37 and Inflammatory Proteinases
Vol. 46, No. 3, 1990
Materials and methods Chemotaxis assay Chemotaxis was measured using the modified Boyden chamber technique (7). Purification of CAP37 CAP37 was purified as previously described (Pereira et al., submitted). Briefly, CAP37 was extracted and purified from PMN crude granule extracts (CGE) through a series of chromatographic steps. The first step of the purification was performed on a Carboxymethyl Sephadex (CMS - C50, Pharmacia) ion exchange column, followed by further separation on a molecular sieve Sephadex G-75 column (Superfine, Pharmacia) (5, 6). The third step of the purification was by hydrophobic interaction HPLC (HIC - HPLC, Biogel TSK phenyl 5PW column, 7.5 mm x 0.75 mm,BioRad Laboratories). Chemotactic activity was followed during the purification process. Prior to sequencing the HIC-HPLC purified material was subjected RP-HPLC. The material was applied to a Dynamax 300A (12 micron, 4.6 x 250 mm) C8 column equilibrated with 0.1% TFA/H20. Purified protein was eluted with a 0-80% gradient of 0.1% TFA/acetonitrile over 30 minutes (flow rate = 1 ml/min). Material in the peak at 16.14 min was concentrated under vacuum (Speed Vac, Savant Instruments). The RP-HPLC purified material was reduced with 2-mercaptoethanol and the reduced protein was alkylated with 4-vinyl pyridine. Alkylated CAP37 was desalted and finally purified by RP-HPLC (Model 130A Micro Separation System, Applied Biosystems, Foster City, CA) on an Aquapore RP-300 (Applied Biosytems, 7 micron, 2.1 x 30 mm) C8 column equilibrated in 0.1% TFA/H20 and eluted with a 0-100% linear gradient of 80% acetonitrile/20% H20/0.085% TFA, over 100 minutes (flow rate = 0.05 ml/min). The eluate containing modified CAP37 was concentrated under vacuum (Speed Vac, Savant Instruments) and sequenced as described below. Microsequencin.q of CAP37 N-terminal protein sequence analys~s was performed using an Applied Biosystems Model 477A Protein/Peptide Sequencer with an on-line Applied Biosystems 120A PTH-Amino Acid Analyzer. Reagents and solvents were from Applied Biosystems, Foster City, CA. Phenylthiohydantoin (PTH)-derivatized amino acids formed sequentially by Edman degradation were separated using an Applied Biosystems PTH C-18 HPLC reverse phase microbore column (2.1 mm ID x 220 mm) by gradient elution. Cysteine was determined as PTH-S-beta-(4-pyridylethyl)-Cys. The sample was applied to an acid-etched glass-fiber filter which had been treated with 3 mg Biobrene (polybrene) and precycled. Peak identification and yield quantitation was based on a standard PTH-amino acid profile. Assays for serine protease activity A.
1-3JH DFP binding studies
Our standard method for the preparation of CAP37, elastase, and cathepsin G involves the treatment of CGE with 5 x 10.3 M diisopropylfluorophosphate (DFP, Sigma), 5 x 10.5 M phenylmethylsulfonylfluoride (PMSF, Sigma), 5 x 10~ M trisodium EDTA (Sigma) and 10" M pepstatin A (Sigma). For these studies, in addition to the
Vol. 46, No. 3, 1990
CAP 37 and Inflammatory Proteinases
191
normal purification protocol, CAP37, elastase, and cathepsin G were prepared from normal CGE that had not been inhibited with DFP or other protease inhibitors. An aliquot of each of these samples was incubated with 1-3-3H DFP (specific activity 3.5 Ci/mmol, Amersham) at a final concentration of 10SM at 37°C overnight. The reaction was stopped with Tris HCI (pH 8.1, final concentration 15 mM) (8). The samples were run on a 15% SDS gel, and following fixation, the gel was treated with Amplify (Amersham) for 30 min, and dried at 80°C. The autoradiograph was developed after 10 and 20 day exposures. B.
Azocasein degradation
Azocasein degradation was measured by the method of Starkey and Barrett (9). In brief, DFP-treated CAP37 (1 ug), elastase (1 ug), cathepsin G (1 ug) and CGE (10 ug) and non DFP-treated CAP37 (1 ug), elastase (1 ug), cathepsin G (1 ug) and CGE (10 ug) in 0.05% Brij 35 were added to 6% azocasein (w/v in 1.25 M Tris-HCL buffer, pH 7.5 containing 2.5 M KCI) and incubated at 50°C for 30 min. Five percent tri chloroacetic acid (TCA) was added to the tubes and the precipitate removed by filtration. The absorbance of the filtrate was measured at 366 nm. One unit of proteolytic activity is the amount which will give a reading of 1.0 at 366 nm. C.
Hemoglobin degradation
Hemoglobin degradation was measured by a modification of the method of Anson (10). Hemoglobin (2% w/v) was denatured in 0.16 M citric acid for 1 h at 37°C and the pH adjusted to 7.2. To the reaction mixture which consisted of 0.4M sodium phosphate buffer pH 7.2 (1 ml), 2% denatured hemoglobin (lml), 0.07 M cysteine (0.1 ml), and 0.1% Triton X-100 (0.1 ml) was added 1 ug of CAP37, elastase or cathepsin G, or 10 ug of CGE. The reaction was stopped with the addition of 3 ml 10% TCA after 18 h at 37°C. The precipitate was filtered and the absorbance of the filtrate measured at 280 nm. D.
BLT esterase activity
Na-Benzyloxycarbonyl L-lysine-thiobenzylesterhydrochloride (BLT, Calbiochem) esterase activity was measured according to the method of Pasternak and Eisen (8). Briefly, to 700 ul of 0.2M Tris - HCI buffer pH 8.1, 100 ul 5'-5'-dithiobis(2-nitrobenzoic acid) (final concentration 2.2 x 10.4 M, Sigma) and 100 ul BLT (final concentration 2 x 104M) was added the sample in 100 ul of the buffer. The mixture was incubated at room temperature for 30 min, and the absorbance read at 412 nm. E.
Cleavage of Suc-Ala.3-pNA and Su.c-Ala2-Pro-Phe-pNA
A modification of the methods according to Starkey and Barrett (9) and Kao et al., (11) was employed. To 1.5 ml of 0.1 M Tris-HCI buffer, pH 7.5 was added the sample in 500 ul of 0.05 % Brij 35. Twenty ul of the substrate (5mg/ml DMSO) was added and the reaction allowed to proceed at 50°C for 10 min. The absorbance at 410 nm was measured. F.
C!eavage of tosyl-arginine-methyl-ester
The hydrolysis of TAME was determined as described in the Worthington Enzyme manual (12).
192
CAP 37 and Inflammatory Proteinases
Vol. 46, No. 3, 1990
Results and Discussion The final purification step on a C8 reverse phase column yielded a major peak at 16.14 minutes (see figure 1). Microsequencing of this peak yielded the N-terminal sequence shown in figure 2. A search of the protein sequence data base using the program FASTAMAIL through BIONET (13) revealed a novel sequence. This sequence has substantial homology with the N-termini of a number of serine proteinases known to participate in the inflammatory response (see figure 3). Highest homology (57.5%) was with human elastase (14), also known as medullasin (15,16). CAP37 shares 45% homology with cathepsin G (17), 45% homology with bovine plasminogen, and 42.5% with human complement factor D. Other proteinases sharing significant homology include the rat mast cell proteases I and II and proteinases derived from cytotoxic T cells.
80~
SOd 58d
40~ E
2~
.
.
.
.
,
5
.
.
.
.
,
.
.
.
18 T|me
.
,
.
.
15 (min.)
.
.
.
-
-
-
20
-
1
.
.
.
.
25
FIG 1. Final Purification Step of CAP 37 on C8 reverse phase HPLC. Purified human CAP37 elutes at 16.14 minutes of a 0-->80% gradient of acetonitrile. The first 20 residues of CAP37 were similar to the first 20 amino acid residues of the recently published sequence of an antibacterial substance named azurocidin (18). Azurocidin was purified from the membranes of azurophil granules of human PMN, and was reported to have a molecular weight of 29 kD. Despite the 1 5 10 15 I-V-G-G-R-K-A-R-P-R-Q-F-P-F-L-A-S-
20 I-Q-N-Q
25 30 35 40 G-R-H-F-C-G-G-A-L- I-H-A-R-F-V-M-T-A-A-S
FIG 2. NH 2 - terminal sequence of CAP37.
Vol. 46, No. 3, 1990
CAP37 ,LAST FACTD PLASM CATG RMCPI RMCPII CCPI HF
CAP 37 and Inflammatory Proteinases
193
1 I0 20 I-.-,-,-,-,-.-,-,-,-,-,-,-,-,-.-,-,-,-,-,-,-,-,-,--I-.-,-,-,-,-.-,-,-,-.-.-,-,-,-.-,-,-,-,-,-,-,-,-,--I-L-G-G-R-E-A-E-A-H-A-R-P-Y-M-A-S-V-Q-L-N-G-A-E-L--I-V-G-G-C-V-S-,-,-,-,-.-,-.-,-.-'-,-,-,-,-'-,-,-'--I- I - G - G - R - E - S - R - P - H - S - R - P - Y - M - A - Y - L - Q I-Q-S-P-A-G-QI- I - G - G - V - E - S - R - P - H - S - R - P - Y - M - A - H - L - E I-,-T-E-R-G-YI- I - G - G - V - , - S - I - P - H - S - R - P - Y - M - A - H - L - D - I - V - T - E - K - G - L I-,-'-,-,-,-,-,-,-,-,-,-,-,-,-.-,-,-,-,-,-,-,-,-,-,I- I - G - G - D - T - V - V - P - H - S - R - P - Y - M - A - L - L - K - L - S - S - N - T ......
CAP37 ,LAST FACTD PLASM CATG RMCPI RMCPII CCPI HF
30 . --.-.-.-.-.-,-.-.-.-.-.-.-.-.-.-.---
40
+.
--,-,-.-,-,-,-.-,-,-,-.-.-,-,-.-,-,--,-,-,-,-,-.-.-,-,-.-.-,-,-.-.-,-,--,-,-,-,-,-,-,-,-,-.-,-,-,-.-.-,-,-,-,---,-,-,-,-,-.-,-,-,-,-.-,-,-,-.-,-,-,-.-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-.-,-,-,-.-,-,-,-,-,-,-,-,-,-,-,-.-,-,-.-.-,-,-.---,-,-,-,-,-,-,-,-,-,-,-.-,-,-,-.-,-,I-C-A-G-A-L-I-E-K-N-W-V-L-T-A-A-H-C-
61 42 42 42 45 46 46 45 42
FIG 3. Composite alignment of the amino acid sequences of CAP37 with several inflammatory cell proteases. ELAST (elastase), FACTD (complement factor, D), PLASM (bovine plasminogen), CATG (cathepsin G), RMCPI (rat mast cell, protease I), RMCP II (rat mast cell protease II), CCPI (cytotoxic T cell protease I), HF (cytotoxic T cell protease, H factor); * indicates position of disulfide bonds. + indicates expected location of 'his' residue of the serine protease cataly'dc triad. disparity in publications association composition
the molecular weights, CAP37 and azurocidin appear similar. Previous from our laboratory in 1984 and 1986 had described the PMN-granule of CAP37, its physical and chemical properties, including its amino acid and its antibacterial activity (5, 6).
The serine endopeptidases are the largest class of mammalian proteinases. The sequence of the CAP37 protein expands the number of serine endopeptidase-homologues associated with inflammatory cells. CAP37 shares highest homology with granulocyte elastase. The elastase/medullasin cDNA encodes a protein of 237 amino acid residues. PMN elastase is a proteolytic enzyme with broad substrate specificity that resides in the azurophil granules of PMNs and monocytes. It is a 28 kd protein that removes the hyaluronic acid-binding region of cartilage proteoglycan and then fragments the glycosaminoglycan attachment region (19). Although elastin is generally resistant to proteolytic attack, it is degraded by elastase (20). PMN elastase also degrades fibronectin and laminin (21), and type I, type II and type IV collagens (22). The activity of elastase is modulated in vivo by alpha-1proteinase inhibitor and alpha-2-macroglobulin. Two clinically important antiinflammatory drugs, gold sodium thiomalate and pentosan polysulfate act in part by inhibiting leukocyte elastase (23).
* One proteolytic N.D. = not done.
CGE (10ug) CGE + DFP Elastase (lug) Elastase + DFP Cathepsin G (lug) Cathepsin G + DFP CAP37 (lug) CAP37 + DFP Trypsin (lug)
(A366nm)
Azocasein
unit
will
give
a reading
0.3356 0.0343 0.1926 0.0056 0.0867 0.0070 0.0001 0 N.D. of
1.0
at
2.1979 0.0727 1.9246 0.0135 2.4738 0.0519 0.0444 0.0356 2.4326
(412nm)
BLT
activity*
of CAP37
absorbance
0.1712 0.0100 0.0258 0.0212 0.2395 0.0100 0.0069 0 N.D.
S u c - A l a 2Pro-PhepNA (412nm)
respective
0.0479 0 N.D. N.D. N.D. N.D. 0.0179 0.0189 0.4691
(280nm)
TAME
the
of proteolytic
Hemoglobin (280nm)
Units
TABLE 1 of ~ - D F P B i n d i n g and S e r i n e P r o t e o l y t i c A c t i v i t y with other Known Serine Proteases.
+++ 0.1386 0.0232 ++ 0.0676 0 + 0.1017 0.0018 0.0026 0.0020 N.D.N.D.
3H-DFP
Comparison
reading.
0.1789 0.0016 0.0407 0.0089 0.0010 0.0026 0.0013 0.009 N.D
Suc-Ala3pNA (412nm)
~D o
Z o
o~
o
o m ~° m m
o
m
m
H
m
c~
Vol. 46, No. 3, 1990
CAP 37 and Inflammatory Protelnases
195
Cathepsin G, also present in the azurophil granules is structurally and catalytically related to the pancreatic chymotrypsins and to the chymases of mast cell granules (17). Although cathepsin G has limited action on elastin or type I collagen, it can solubilize collagen from cartilage and may generate physiologically active components from the complement proteins (24). Plasma alpha-1 antichymotrypsin and alpha-1 proteinase inhibitor and alpha 2- macroglobulin are the physiological inhibitors of cathepsin G (25,26). As shown in figure 3 the cys-26 residue of CAP37 which corresponds to the cys-42 residue of the first di-sulfide bond (cys-42 to cys-58 in chymotrypsin numbering) of serine proteases appears to be conserved. Whether the other two disulfide bonds are conserved will be ascertained when the amino acid sequence of CAP37 is completed. In addition to the conserved disulfide bonds, another important feature of the serine proteases is the residues forming the 'charge relay system' of the active catalytic site which occurs at his-57, asp-102 and ser-195 (chymotrypsin numbering). In the sequence of CAP37 the site of the conserved histidine is replaced by a serine residue. Because of the strong homologies between CAP37 and the other serine proteases it was important to determine if CAP37 would bind the serine protease inhibitor DFP, and whether it was capable of degrading various substrates specific for serine proteases. Table 1 shows that uninhibited CAP37, unlike uninhibited CGE, elastase and cathepsin G was unable to bind 3H-DFP. The intensity of the reaction obtained on the autoradiograph was strongest with CGE, and slightly less intense with elastase. Uninhibited cathepsin G also bound DFP but to a lesser extent than CGE and elastase. Azocasein and hemoglobin were degraded by uninhibited CGE, elastase and cathepsin G. However as indicated in Table 1, CAP37 failed to cleave either of these substrates. BLT esterase activity, a trypsin-like esterase activity, which has been associated with the cytolytic T lymphocyte/perforin system (8) could not be demonstrated with CAP37. The values obtained with uninhibited CGE, elastase and cathepsin G were comparable to the values obtained with 1 ug of trypsin. Further assessment of the trypsin-like activity of CAP37 using TAME was once again negative when compared to the trypsin control. Both CAP37 and cathepsin G unlike CGE and elastase were unable to cleave the synthetic peptide Suc-AlafpNA, a substrate specific for elastase-like activity. Cleavage of the Suc-Ala2-Pro-Phe-pNA synthetic substrate, which is specific for chymotrypsin-like esterases, was obtained with CGE and cathepsin G, but not with elastase and CAP37. These results suggest that the replacement of the his-41 by a serine residue destroys the ability of CAP37 to bind 3H-DFP and to cleave various substrates specific for serine esterase activity. It is conceivable that through evolution the catalytic site of CAP37 was changed without affecting those domains involved in its chemotactic and bactericidal activities. Studies of the cloned cDNA for CAP37 (Morgan et al, in preparation.) will help to elucidate the role of this molecule in the inflammatory response. Expression of this molecule combined with purification of substantial quantities of recombinant protein will be required to determine the substrate specificity (if any) of the CAP37 molecule. This will also permit study of its regulation during inflammation and the relevance of its binding to endotoxin (27) to its antimicrobial action.
196
CAP 37 and Inflammatory Proteinases
Vol. 46, No. 3, 1990
Acknowledgements This work was supported by Public Health Service grant AI17662 (JKS) from the National Institute of Allergy and Infectious Disease. References 1. 2. 3. 4. 5. 6.
P.O. WILKINSON, Chemotaxis and Inflammation. 2nd Ed. Edinburgh: Churchill Livingstone, 119-142, (1982). P.A. WARD, J. Exp. Med. 128, 1201-1221 (1968). J.I. GALLIN, Ann. Rev. Med. 36, 263-274 (1985). A.R. PAGE, and GOOD, R.A. Amer. J. Pathol. 34, 645-669 (1958). W.M. SHAFER., MARTIN, L.E., and SPITZNAGEL, J.K. Infect. Immun. 45, 29 35 (1984). W.M. SHAFER, MARTIN, L.E., SPITZNAGEL, J.K. Infect. Immun. 53, 651-655 (1986).
7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
R. SNYDERMAN, and PIKE, M.C. Leukocyte Chemotaxis J.l. Gallin and P.G. Quie, (eds) New York: Raven Press. pp. 73-84, (1978). M.S. PASTERNAK, and EISEN, H.N. Nature, 315, 743 - 745 (1985). P . M . STARKEY, and BARRET, A.J. Biochem. J. 155, 255 - 263 (1976). M.L ANSON. J. Gen. Physiol. 23, 79 - 89 (1940). R.C. KAO, WEHNER, N.G., SKUBITZ, K.M., GRAY, B.H., and HOIDAL, J.R.J. Clin. Invest. 82, 1963 - 1 9 7 3 ( 1 9 8 8 ) . Worthington Enzyme Manual. Ed. LA. Decker, New Jersey, pp 221 - 224 (1977). W.R. PEARSON, and LIPMAN, D.J. Proc. Natl. Acad. Sci. 85, 2444-2448 (1988). S. SINHA, WATOREK. W., KARR. S., et al. Proc. Natl. Acad. Sci. 84, 22282232, (1987). K. OKANO, AOKI, Y., SAKURAI, T. et al. J. Biol. Chem. 102,,13-16 (1987). FARLEY, D., SALVESEN, G., TRAVIS, J. Biol. Chem. Hoppe.Seyler. 369, 3-7 (suppl) (1988). SALVESEN, G., FARLEY, D., SHUMAN, J., et al. Biochem. 26, 2289-2293 (1987). J.E. GABAY, SCOTT, R.W., CAMPANELLI, D., GRIFFITH, J., WILDE, C., MARRA, M.N., SEEGER, M., and NATHAN, C.F. Proc. Nat. Acad. Sci. USA. 86, 5610 - 5614 (1989). KEISER, H., GREENWALD, R.A., FEINSTEIN, G. and JANOFF, A. J. Clin. Invest. 57, 625-632 (1976). BARRETT,A.J., Methods Enzymol 80, 581-588 (1981). CAMPBELL, E.J., SENIOR, R.M., McDONALD, J.A., and COX, D.L. J. Clin. Invest. 70, 845-852 (1982). PIPOLY, D.J. and CROUCH, E.C. Biochem. 26, 5748-5755 (1987). BAICI, A., SALGAM, P., FEHR, K. and BONI, A. Biochem. Pharmacol. 30, 703-710 (1981). STARKEY, P.M., BARRETT, A.J., BURLEIGH, M.C. Biochem. Biophys. Acta. 483, 386-392 (1977). OHLSSON, K. and OLSSON, I. Scand. J. Clin. Lab. Invest. 34, 349-357 (1974). BARRETT, A.J. and STARKEY, P.M. Biochem. J. 133, 709-715 (1973). MODRZAKOWSKI, M.C. and SPITZNAGEL, J.K. Infect. Immun. 25, 597-602 (1979).
Pergamon Press
CAP 37, A 37 KD HUMAN NEUTROPHIL GRANULE CATIONIC PROTEIN SHARES HOMOLOGY WITH INFLAMMATORY PROTEINASES H. Anne Pereira1, John K. Spitznagel I, Jan Pohl 2, Douglas E. Wilson z, John Morgan 3, Ilona Palings3, and James W. Larrick 3 1Dept. of Microbiology and Immunology and 2protein Sequencing Facility, Emory University School of Medicine, Atlanta GA 30322 and 3Genelabs Inc., 505 Penobscot Drive, Redwood City, CA 94063. (Received in final form November 17, 1989)
Summary We have previously shown that a major granule-associated cationic protein CAP 37 (Mr = 37 kD) derived from human PMN is a monocyte-specific chemoattractant. The N-terminal amino acid sequence of this novel chemotactic protein shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, a critical substitution of a serine for a histidine at position 41, results in its lack of serine protease activity. The process of inflammation is orchestrated by the sequential release of specific molecules that mediate the activation and chemotaxis of various cell types (1). Early inflammatory lesions are comprised primarily of polymorphonuclear leukocytes (PMN) followed by infiltration of mononuclear phagocytes. Evidence suggests that one or more factors derived from the PMN may modulate the subsequent chemotaxis of monocytes,(2,3). In fact an early study described the absolute requirement of PMNs for the subsequent infiltration of mononuclear phagocytes (4). Previous work from this laboratory demonstrated the antimicrobial activity of a major PMN granule protein CAP 37 [designated Cationic Antimicrobial Protein of Mr = 37kd] (5,6). We have recently demonstrated that this protein is a potent mediator of monocyte specific chemotaxis, and thus may act as a mediator of the second wave of inflammation (Pereira et al., submitted for publication). Stimulation of PMN with opsonized Staphylococcus aureus resulted in a rapid release of approximately 89% of CAP37 into the extracellular milieu. This finding was particularly noteworthy since, if a substance is to act as a chemotactic factor in vivo it would necessarily have to be released extracellularly to effect its function. Immunocytochemical studies performed on peripheral blood cells indicated that CAP37 was present in PMN alone. Mononuclear cells, eosinophils, and red blood cells failed to stain positive with the monospecific mouse antibody to CAP37 used in these studies (Pereira et al., submitted for publication). The present report describes the final purification and Nterminal amino acid sequence of human CAP37. This protein is related to the serine class of proteinases and shares significant homology with several other proteases derived from the granules of inflammatory leukocytes that are thought to mediate tissue damage during the course of inflammatory diseases. 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
190
CAP 37 and Inflammatory Proteinases
Vol. 46, No. 3, 1990
Materials and methods Chemotaxis assay Chemotaxis was measured using the modified Boyden chamber technique (7). Purification of CAP37 CAP37 was purified as previously described (Pereira et al., submitted). Briefly, CAP37 was extracted and purified from PMN crude granule extracts (CGE) through a series of chromatographic steps. The first step of the purification was performed on a Carboxymethyl Sephadex (CMS - C50, Pharmacia) ion exchange column, followed by further separation on a molecular sieve Sephadex G-75 column (Superfine, Pharmacia) (5, 6). The third step of the purification was by hydrophobic interaction HPLC (HIC - HPLC, Biogel TSK phenyl 5PW column, 7.5 mm x 0.75 mm,BioRad Laboratories). Chemotactic activity was followed during the purification process. Prior to sequencing the HIC-HPLC purified material was subjected RP-HPLC. The material was applied to a Dynamax 300A (12 micron, 4.6 x 250 mm) C8 column equilibrated with 0.1% TFA/H20. Purified protein was eluted with a 0-80% gradient of 0.1% TFA/acetonitrile over 30 minutes (flow rate = 1 ml/min). Material in the peak at 16.14 min was concentrated under vacuum (Speed Vac, Savant Instruments). The RP-HPLC purified material was reduced with 2-mercaptoethanol and the reduced protein was alkylated with 4-vinyl pyridine. Alkylated CAP37 was desalted and finally purified by RP-HPLC (Model 130A Micro Separation System, Applied Biosystems, Foster City, CA) on an Aquapore RP-300 (Applied Biosytems, 7 micron, 2.1 x 30 mm) C8 column equilibrated in 0.1% TFA/H20 and eluted with a 0-100% linear gradient of 80% acetonitrile/20% H20/0.085% TFA, over 100 minutes (flow rate = 0.05 ml/min). The eluate containing modified CAP37 was concentrated under vacuum (Speed Vac, Savant Instruments) and sequenced as described below. Microsequencin.q of CAP37 N-terminal protein sequence analys~s was performed using an Applied Biosystems Model 477A Protein/Peptide Sequencer with an on-line Applied Biosystems 120A PTH-Amino Acid Analyzer. Reagents and solvents were from Applied Biosystems, Foster City, CA. Phenylthiohydantoin (PTH)-derivatized amino acids formed sequentially by Edman degradation were separated using an Applied Biosystems PTH C-18 HPLC reverse phase microbore column (2.1 mm ID x 220 mm) by gradient elution. Cysteine was determined as PTH-S-beta-(4-pyridylethyl)-Cys. The sample was applied to an acid-etched glass-fiber filter which had been treated with 3 mg Biobrene (polybrene) and precycled. Peak identification and yield quantitation was based on a standard PTH-amino acid profile. Assays for serine protease activity A.
1-3JH DFP binding studies
Our standard method for the preparation of CAP37, elastase, and cathepsin G involves the treatment of CGE with 5 x 10.3 M diisopropylfluorophosphate (DFP, Sigma), 5 x 10.5 M phenylmethylsulfonylfluoride (PMSF, Sigma), 5 x 10~ M trisodium EDTA (Sigma) and 10" M pepstatin A (Sigma). For these studies, in addition to the
Vol. 46, No. 3, 1990
CAP 37 and Inflammatory Proteinases
191
normal purification protocol, CAP37, elastase, and cathepsin G were prepared from normal CGE that had not been inhibited with DFP or other protease inhibitors. An aliquot of each of these samples was incubated with 1-3-3H DFP (specific activity 3.5 Ci/mmol, Amersham) at a final concentration of 10SM at 37°C overnight. The reaction was stopped with Tris HCI (pH 8.1, final concentration 15 mM) (8). The samples were run on a 15% SDS gel, and following fixation, the gel was treated with Amplify (Amersham) for 30 min, and dried at 80°C. The autoradiograph was developed after 10 and 20 day exposures. B.
Azocasein degradation
Azocasein degradation was measured by the method of Starkey and Barrett (9). In brief, DFP-treated CAP37 (1 ug), elastase (1 ug), cathepsin G (1 ug) and CGE (10 ug) and non DFP-treated CAP37 (1 ug), elastase (1 ug), cathepsin G (1 ug) and CGE (10 ug) in 0.05% Brij 35 were added to 6% azocasein (w/v in 1.25 M Tris-HCL buffer, pH 7.5 containing 2.5 M KCI) and incubated at 50°C for 30 min. Five percent tri chloroacetic acid (TCA) was added to the tubes and the precipitate removed by filtration. The absorbance of the filtrate was measured at 366 nm. One unit of proteolytic activity is the amount which will give a reading of 1.0 at 366 nm. C.
Hemoglobin degradation
Hemoglobin degradation was measured by a modification of the method of Anson (10). Hemoglobin (2% w/v) was denatured in 0.16 M citric acid for 1 h at 37°C and the pH adjusted to 7.2. To the reaction mixture which consisted of 0.4M sodium phosphate buffer pH 7.2 (1 ml), 2% denatured hemoglobin (lml), 0.07 M cysteine (0.1 ml), and 0.1% Triton X-100 (0.1 ml) was added 1 ug of CAP37, elastase or cathepsin G, or 10 ug of CGE. The reaction was stopped with the addition of 3 ml 10% TCA after 18 h at 37°C. The precipitate was filtered and the absorbance of the filtrate measured at 280 nm. D.
BLT esterase activity
Na-Benzyloxycarbonyl L-lysine-thiobenzylesterhydrochloride (BLT, Calbiochem) esterase activity was measured according to the method of Pasternak and Eisen (8). Briefly, to 700 ul of 0.2M Tris - HCI buffer pH 8.1, 100 ul 5'-5'-dithiobis(2-nitrobenzoic acid) (final concentration 2.2 x 10.4 M, Sigma) and 100 ul BLT (final concentration 2 x 104M) was added the sample in 100 ul of the buffer. The mixture was incubated at room temperature for 30 min, and the absorbance read at 412 nm. E.
Cleavage of Suc-Ala.3-pNA and Su.c-Ala2-Pro-Phe-pNA
A modification of the methods according to Starkey and Barrett (9) and Kao et al., (11) was employed. To 1.5 ml of 0.1 M Tris-HCI buffer, pH 7.5 was added the sample in 500 ul of 0.05 % Brij 35. Twenty ul of the substrate (5mg/ml DMSO) was added and the reaction allowed to proceed at 50°C for 10 min. The absorbance at 410 nm was measured. F.
C!eavage of tosyl-arginine-methyl-ester
The hydrolysis of TAME was determined as described in the Worthington Enzyme manual (12).
192
CAP 37 and Inflammatory Proteinases
Vol. 46, No. 3, 1990
Results and Discussion The final purification step on a C8 reverse phase column yielded a major peak at 16.14 minutes (see figure 1). Microsequencing of this peak yielded the N-terminal sequence shown in figure 2. A search of the protein sequence data base using the program FASTAMAIL through BIONET (13) revealed a novel sequence. This sequence has substantial homology with the N-termini of a number of serine proteinases known to participate in the inflammatory response (see figure 3). Highest homology (57.5%) was with human elastase (14), also known as medullasin (15,16). CAP37 shares 45% homology with cathepsin G (17), 45% homology with bovine plasminogen, and 42.5% with human complement factor D. Other proteinases sharing significant homology include the rat mast cell proteases I and II and proteinases derived from cytotoxic T cells.
80~
SOd 58d
40~ E
2~
.
.
.
.
,
5
.
.
.
.
,
.
.
.
18 T|me
.
,
.
.
15 (min.)
.
.
.
-
-
-
20
-
1
.
.
.
.
25
FIG 1. Final Purification Step of CAP 37 on C8 reverse phase HPLC. Purified human CAP37 elutes at 16.14 minutes of a 0-->80% gradient of acetonitrile. The first 20 residues of CAP37 were similar to the first 20 amino acid residues of the recently published sequence of an antibacterial substance named azurocidin (18). Azurocidin was purified from the membranes of azurophil granules of human PMN, and was reported to have a molecular weight of 29 kD. Despite the 1 5 10 15 I-V-G-G-R-K-A-R-P-R-Q-F-P-F-L-A-S-
20 I-Q-N-Q
25 30 35 40 G-R-H-F-C-G-G-A-L- I-H-A-R-F-V-M-T-A-A-S
FIG 2. NH 2 - terminal sequence of CAP37.
Vol. 46, No. 3, 1990
CAP37 ,LAST FACTD PLASM CATG RMCPI RMCPII CCPI HF
CAP 37 and Inflammatory Proteinases
193
1 I0 20 I-.-,-,-,-,-.-,-,-,-,-,-,-,-,-.-,-,-,-,-,-,-,-,-,--I-.-,-,-,-,-.-,-,-,-.-.-,-,-,-.-,-,-,-,-,-,-,-,-,--I-L-G-G-R-E-A-E-A-H-A-R-P-Y-M-A-S-V-Q-L-N-G-A-E-L--I-V-G-G-C-V-S-,-,-,-,-.-,-.-,-.-'-,-,-,-,-'-,-,-'--I- I - G - G - R - E - S - R - P - H - S - R - P - Y - M - A - Y - L - Q I-Q-S-P-A-G-QI- I - G - G - V - E - S - R - P - H - S - R - P - Y - M - A - H - L - E I-,-T-E-R-G-YI- I - G - G - V - , - S - I - P - H - S - R - P - Y - M - A - H - L - D - I - V - T - E - K - G - L I-,-'-,-,-,-,-,-,-,-,-,-,-,-,-.-,-,-,-,-,-,-,-,-,-,I- I - G - G - D - T - V - V - P - H - S - R - P - Y - M - A - L - L - K - L - S - S - N - T ......
CAP37 ,LAST FACTD PLASM CATG RMCPI RMCPII CCPI HF
30 . --.-.-.-.-.-,-.-.-.-.-.-.-.-.-.-.---
40
+.
--,-,-.-,-,-,-.-,-,-,-.-.-,-,-.-,-,--,-,-,-,-,-.-.-,-,-.-.-,-,-.-.-,-,--,-,-,-,-,-,-,-,-,-.-,-,-,-.-.-,-,-,-,---,-,-,-,-,-.-,-,-,-,-.-,-,-,-.-,-,-,-.-,-,-,-,-,-,-,-,-,-,-,-,-,-,-,-.-,-,-,-.-,-,-,-,-,-,-,-,-,-,-,-.-,-,-.-.-,-,-.---,-,-,-,-,-,-,-,-,-,-,-.-,-,-,-.-,-,I-C-A-G-A-L-I-E-K-N-W-V-L-T-A-A-H-C-
61 42 42 42 45 46 46 45 42
FIG 3. Composite alignment of the amino acid sequences of CAP37 with several inflammatory cell proteases. ELAST (elastase), FACTD (complement factor, D), PLASM (bovine plasminogen), CATG (cathepsin G), RMCPI (rat mast cell, protease I), RMCP II (rat mast cell protease II), CCPI (cytotoxic T cell protease I), HF (cytotoxic T cell protease, H factor); * indicates position of disulfide bonds. + indicates expected location of 'his' residue of the serine protease cataly'dc triad. disparity in publications association composition
the molecular weights, CAP37 and azurocidin appear similar. Previous from our laboratory in 1984 and 1986 had described the PMN-granule of CAP37, its physical and chemical properties, including its amino acid and its antibacterial activity (5, 6).
The serine endopeptidases are the largest class of mammalian proteinases. The sequence of the CAP37 protein expands the number of serine endopeptidase-homologues associated with inflammatory cells. CAP37 shares highest homology with granulocyte elastase. The elastase/medullasin cDNA encodes a protein of 237 amino acid residues. PMN elastase is a proteolytic enzyme with broad substrate specificity that resides in the azurophil granules of PMNs and monocytes. It is a 28 kd protein that removes the hyaluronic acid-binding region of cartilage proteoglycan and then fragments the glycosaminoglycan attachment region (19). Although elastin is generally resistant to proteolytic attack, it is degraded by elastase (20). PMN elastase also degrades fibronectin and laminin (21), and type I, type II and type IV collagens (22). The activity of elastase is modulated in vivo by alpha-1proteinase inhibitor and alpha-2-macroglobulin. Two clinically important antiinflammatory drugs, gold sodium thiomalate and pentosan polysulfate act in part by inhibiting leukocyte elastase (23).
* One proteolytic N.D. = not done.
CGE (10ug) CGE + DFP Elastase (lug) Elastase + DFP Cathepsin G (lug) Cathepsin G + DFP CAP37 (lug) CAP37 + DFP Trypsin (lug)
(A366nm)
Azocasein
unit
will
give
a reading
0.3356 0.0343 0.1926 0.0056 0.0867 0.0070 0.0001 0 N.D. of
1.0
at
2.1979 0.0727 1.9246 0.0135 2.4738 0.0519 0.0444 0.0356 2.4326
(412nm)
BLT
activity*
of CAP37
absorbance
0.1712 0.0100 0.0258 0.0212 0.2395 0.0100 0.0069 0 N.D.
S u c - A l a 2Pro-PhepNA (412nm)
respective
0.0479 0 N.D. N.D. N.D. N.D. 0.0179 0.0189 0.4691
(280nm)
TAME
the
of proteolytic
Hemoglobin (280nm)
Units
TABLE 1 of ~ - D F P B i n d i n g and S e r i n e P r o t e o l y t i c A c t i v i t y with other Known Serine Proteases.
+++ 0.1386 0.0232 ++ 0.0676 0 + 0.1017 0.0018 0.0026 0.0020 N.D.N.D.
3H-DFP
Comparison
reading.
0.1789 0.0016 0.0407 0.0089 0.0010 0.0026 0.0013 0.009 N.D
Suc-Ala3pNA (412nm)
~D o
Z o
o~
o
o m ~° m m
o
m
m
H
m
c~
Vol. 46, No. 3, 1990
CAP 37 and Inflammatory Protelnases
195
Cathepsin G, also present in the azurophil granules is structurally and catalytically related to the pancreatic chymotrypsins and to the chymases of mast cell granules (17). Although cathepsin G has limited action on elastin or type I collagen, it can solubilize collagen from cartilage and may generate physiologically active components from the complement proteins (24). Plasma alpha-1 antichymotrypsin and alpha-1 proteinase inhibitor and alpha 2- macroglobulin are the physiological inhibitors of cathepsin G (25,26). As shown in figure 3 the cys-26 residue of CAP37 which corresponds to the cys-42 residue of the first di-sulfide bond (cys-42 to cys-58 in chymotrypsin numbering) of serine proteases appears to be conserved. Whether the other two disulfide bonds are conserved will be ascertained when the amino acid sequence of CAP37 is completed. In addition to the conserved disulfide bonds, another important feature of the serine proteases is the residues forming the 'charge relay system' of the active catalytic site which occurs at his-57, asp-102 and ser-195 (chymotrypsin numbering). In the sequence of CAP37 the site of the conserved histidine is replaced by a serine residue. Because of the strong homologies between CAP37 and the other serine proteases it was important to determine if CAP37 would bind the serine protease inhibitor DFP, and whether it was capable of degrading various substrates specific for serine proteases. Table 1 shows that uninhibited CAP37, unlike uninhibited CGE, elastase and cathepsin G was unable to bind 3H-DFP. The intensity of the reaction obtained on the autoradiograph was strongest with CGE, and slightly less intense with elastase. Uninhibited cathepsin G also bound DFP but to a lesser extent than CGE and elastase. Azocasein and hemoglobin were degraded by uninhibited CGE, elastase and cathepsin G. However as indicated in Table 1, CAP37 failed to cleave either of these substrates. BLT esterase activity, a trypsin-like esterase activity, which has been associated with the cytolytic T lymphocyte/perforin system (8) could not be demonstrated with CAP37. The values obtained with uninhibited CGE, elastase and cathepsin G were comparable to the values obtained with 1 ug of trypsin. Further assessment of the trypsin-like activity of CAP37 using TAME was once again negative when compared to the trypsin control. Both CAP37 and cathepsin G unlike CGE and elastase were unable to cleave the synthetic peptide Suc-AlafpNA, a substrate specific for elastase-like activity. Cleavage of the Suc-Ala2-Pro-Phe-pNA synthetic substrate, which is specific for chymotrypsin-like esterases, was obtained with CGE and cathepsin G, but not with elastase and CAP37. These results suggest that the replacement of the his-41 by a serine residue destroys the ability of CAP37 to bind 3H-DFP and to cleave various substrates specific for serine esterase activity. It is conceivable that through evolution the catalytic site of CAP37 was changed without affecting those domains involved in its chemotactic and bactericidal activities. Studies of the cloned cDNA for CAP37 (Morgan et al, in preparation.) will help to elucidate the role of this molecule in the inflammatory response. Expression of this molecule combined with purification of substantial quantities of recombinant protein will be required to determine the substrate specificity (if any) of the CAP37 molecule. This will also permit study of its regulation during inflammation and the relevance of its binding to endotoxin (27) to its antimicrobial action.
196
CAP 37 and Inflammatory Proteinases
Vol. 46, No. 3, 1990
Acknowledgements This work was supported by Public Health Service grant AI17662 (JKS) from the National Institute of Allergy and Infectious Disease. References 1. 2. 3. 4. 5. 6.
P.O. WILKINSON, Chemotaxis and Inflammation. 2nd Ed. Edinburgh: Churchill Livingstone, 119-142, (1982). P.A. WARD, J. Exp. Med. 128, 1201-1221 (1968). J.I. GALLIN, Ann. Rev. Med. 36, 263-274 (1985). A.R. PAGE, and GOOD, R.A. Amer. J. Pathol. 34, 645-669 (1958). W.M. SHAFER., MARTIN, L.E., and SPITZNAGEL, J.K. Infect. Immun. 45, 29 35 (1984). W.M. SHAFER, MARTIN, L.E., SPITZNAGEL, J.K. Infect. Immun. 53, 651-655 (1986).
7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
R. SNYDERMAN, and PIKE, M.C. Leukocyte Chemotaxis J.l. Gallin and P.G. Quie, (eds) New York: Raven Press. pp. 73-84, (1978). M.S. PASTERNAK, and EISEN, H.N. Nature, 315, 743 - 745 (1985). P . M . STARKEY, and BARRET, A.J. Biochem. J. 155, 255 - 263 (1976). M.L ANSON. J. Gen. Physiol. 23, 79 - 89 (1940). R.C. KAO, WEHNER, N.G., SKUBITZ, K.M., GRAY, B.H., and HOIDAL, J.R.J. Clin. Invest. 82, 1963 - 1 9 7 3 ( 1 9 8 8 ) . Worthington Enzyme Manual. Ed. LA. Decker, New Jersey, pp 221 - 224 (1977). W.R. PEARSON, and LIPMAN, D.J. Proc. Natl. Acad. Sci. 85, 2444-2448 (1988). S. SINHA, WATOREK. W., KARR. S., et al. Proc. Natl. Acad. Sci. 84, 22282232, (1987). K. OKANO, AOKI, Y., SAKURAI, T. et al. J. Biol. Chem. 102,,13-16 (1987). FARLEY, D., SALVESEN, G., TRAVIS, J. Biol. Chem. Hoppe.Seyler. 369, 3-7 (suppl) (1988). SALVESEN, G., FARLEY, D., SHUMAN, J., et al. Biochem. 26, 2289-2293 (1987). J.E. GABAY, SCOTT, R.W., CAMPANELLI, D., GRIFFITH, J., WILDE, C., MARRA, M.N., SEEGER, M., and NATHAN, C.F. Proc. Nat. Acad. Sci. USA. 86, 5610 - 5614 (1989). KEISER, H., GREENWALD, R.A., FEINSTEIN, G. and JANOFF, A. J. Clin. Invest. 57, 625-632 (1976). BARRETT,A.J., Methods Enzymol 80, 581-588 (1981). CAMPBELL, E.J., SENIOR, R.M., McDONALD, J.A., and COX, D.L. J. Clin. Invest. 70, 845-852 (1982). PIPOLY, D.J. and CROUCH, E.C. Biochem. 26, 5748-5755 (1987). BAICI, A., SALGAM, P., FEHR, K. and BONI, A. Biochem. Pharmacol. 30, 703-710 (1981). STARKEY, P.M., BARRETT, A.J., BURLEIGH, M.C. Biochem. Biophys. Acta. 483, 386-392 (1977). OHLSSON, K. and OLSSON, I. Scand. J. Clin. Lab. Invest. 34, 349-357 (1974). BARRETT, A.J. and STARKEY, P.M. Biochem. J. 133, 709-715 (1973). MODRZAKOWSKI, M.C. and SPITZNAGEL, J.K. Infect. Immun. 25, 597-602 (1979).