Isolation of cationic peptides from rat polymorphonuclear leukocyte granule contents using fast protein liquid chromatography

ANALYTICAL

BIOCHEMISTRY

158,377-38 1 (1986)

Isolation of Cationic Peptides from Rat Polymorphonuclear Leukocyte Granule Contents Using Fast Protein Liquid Chromatography MICHAELJ.LOEFFELHOLZ'ANDMALCOLMC.MODRZAKOWSKI Department of Zoological Osteopathic Medicine,

and Biomedical Ohio University,

Sciences, and College Athens, Ohio 45701

of

Received February 28, 1986 Separation of extracted rat polymorphonuclear leukocyte (PMN) granule contents using fast protein liquid chromatography yielded four major protein fractions. These fractions consisted of myeloperoxidase (peak A), neutral protease (peak B), lysozyme (peak C), and low molecular weight, cationic peptides (peak D). This study represents the first noted purification ofthe cationic IIIC. peptides of rat PMN granules. 0 1986 Academic h KEY WORDS: rat; polymorphonuclear leukocyte; leukocyte granules; cationic proteins; antimicrobial factors; fast protein liquid chromatography.

The nonoxidative bactericidal activity of polymorphonuclear leukocyte (PMN)2 granule contents has been extensively studied in this laboratory and others ( 1,3,6- 11,16,17). Evidence suggests that this bacterial activity is due largely to cationic proteins of a variety of molecular weights which function due to their highly positively charged properties, rather than by any enzymatic activities. Cationic proteins isolated from rabbit and human PMN have been shown to increase the outer membrane permeability of target gram-negative bacteria (9,16,17). Weiss et al. ( 17) have proposed that this increase in permeability is caused by alterations of lipopolysaccharide (LPS) following adsorption of cationic proteins to anionic moieties of the LPS molecule. The granule extract from rat PMN has been shown to contain two cationic peptides with approximate molecular weights of 3600 and 5800 (Richard L. Hodinka, Ph.D. Dissertation, Ohio University, 1983). Sephadex-G- 100 col’ To whom correspondence should be addressed Department of Zoological and Biomedical Sciences, Irvine Hall, Ohio University, Athens, Ohio 4570 1. * Abbreviations used: PMN, polymorphonuclear leukocyte; LPS, lipopolysaccharide; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate. 377

umn chromatography of granule extract produced a fraction containing these peptides which possessed potent bactericidal activity for rough LPS mutants of Salmonella typhimurium (3). This fraction contained lysozyme in addition to cationic peptides, as Sephadex G100 chromatography lacked the resolving power to separate the low molecular weight components of granule extract. It could not therefore be concluded in the report whether the observed killing of S. typhimurium was due to bactericidal activity of the cationic peptides, of lysozyme, or of a synergistic interaction between both components. In the present report, we have utilized fast protein liquid chromatography (FPLC) to isolate the cationic peptides of rat PMN granule extract. The use of FPLC to isolate proteins from crude biological samples has been described (5,15). The high resolving power and recovery rates of FPLC make it particularly suited for protein purification when working with limited quantities of material, as is the case with rat PMN granule extract. MATERIALS

AND METHODS

Isolation of rat PMN granule contents.

Granules and their contents were isolated from 0003-2697/86 $3.00 Copyright 0 1986 by Academic Press. Inc. All rigltts of reproduction in any form resewed.

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rat peritoneal PMN using previously described SDS-PAGE. The molecular weights of peak methods (3,8). The contents of the granules D cationic peptides were estimated with sowere extracted overnight twice with 0.2 M so- dium dodecyl sulfate (SDS)-PAGE using the dium acetate buffer (pH 4.0) containing 10 urea-phosphate gel system of Shapiro (12), mM calcium chloride, then concentrated by modified by Bethesda Research Laboratories ultrafiltration (UM-05 filter, Amicon, Lexing(BRL, Gaithersburg, Md.) for resolving low ton, Mass.) to approximately 30 mg/ml acetate molecular weight proteins. Peak D protein ( 10 buffer prior to chromatography. pg) and marker proteins (60 fig) (BRL) were FPLC. Granule extract (200-500 ~1) was electrophoresed at a constant voltage of 60 in chromatographed at room temperature on a a 15% acrylamide gel containing 0.1% SDS Superose 12 gel filtration column (HR10/30) and 6 M urea. Gels were stained and destained (Pharmacia, Uppsala, Sweden) equilibrated as described for cationic-PAGE. with degassed, filter sterilized (0.45 pm pore Protein determination. Protein content of Superose column eluates was monitored by size) sodium acetate buffer. The flow rate was 0.5 ml/min. Fractions of 1.O ml were collected AZgO. Protein contents of column fractions and kept on ice. Fractions representing each were determined by the method of Lowry et peak were pooled, dialyzed extensively against al. (4) with egg white lysozyme (Sigma) as a phosphate-buffered saline (pH 7.0), and con- standard. centrated by ultrafiltration (UM-05 filter). RESULTS AND DISCUSSION Protein standards (bovine albumin, ovalbumin, trypsinogen, and egg white lysozyme) The elution profile obtained from the FPLC (Sigma Chemical Co., St. Louis, MO.) in so- separation of rat PMN granule contents shows dium acetate buffer were also applied to the three large protein peaks designated A, B, and Superose 12 column under identical condiC, and three smaller peaks eluting after peak tions, and their elution positions versus mo- C designated together as peak D (Fig. 1). The lecular weights were plotted to estimate the recovery rate of granule protein from the Sumolecular weights of native peak D peptides. perose column was approximately 50% as deEnzyme assays. Myeloperoxidase was as- termined by protein assays. Comparison of sayed using the procedure described by Wor- elution positions of peak D peptides with those thington Biochemicals Corporation ( 18). Pro- of molecular weight standards shows the three tease was assayed using the procedure of Star- peptides of peak D to have molecular weights key and Barrett ( 14). Lysozyme was assayed using the procedure of Shugar (13). Further description of assays can be found in the footnotes to Table 1. Cationic-PAGE. Isolated granule extract 10 fractions were examined with cationic polyC 1 acrylamide gel electrophoresis (PAGE) using 0 06 0 the system of Gabriel (2). Isolated fractions 06 (20 pg) and crude granule extract (200 pg) were electrophoresed in 10% acrylamide gels at a constant current of 4.0 mA per gel. In addition, myeloperoxidase and lysozyme standards ( 10 pg each) were electrophoresed for comparison with crude granule extract. Gels were stained FRACTION NUMBER with 0.25% Coomassie blue R250 in 25% isoFIG.1. Superose 12 fast protein liquid chromatography propanol/ 10% acetic acid and destained with of rat PMN granule extract. Conditions of chromatography are described under Materials and Methods. 25% isopropanaol/lO% acetic acid.

14

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GRANULE

TABLE

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379

I

ENZYME ACTIVITIES OF RAT PMN GRANULE EXTRACX AND FRACTIONS FROM SUPEROSE12 FAST PROTEIN LIQUID CHROMATOGRAPHY Units/mg protein Enzyme

Crude granule extract

Peak A

Peak B

Peak C

Peak D

Myeloperoxidase” Neutral proteaseb LysozymeC

21.9 3.2 13,966.2

120.5 0.1 0

0.7 10.3 2153.8

0 8.6 102,325.6

0 1.3 0

u One unit of peroxidase activity is that amount of enzyme decomposing I .Oumole of peroxide/min at 25°C. Activity was expressed as the change in optical density at 460 nm per min per mg protein with o-dianisidine as the hydrogen donor (18). b One unit of proteolytic activity is that amount of enzyme producing a change in optical density at 366 nm of 1.O in 30 min at 50°C with azocasein as the substrate (14). ’ One unit of lvsozvme is that amount of enzvme uroducing a change in optical density at 450 nm of 0.00 I/ml at 25°C with Micro&&s lysodeikticus as the substrate’( 13).

of approximately 4100, 5500, and 7700 (data peptides identified by Hodinka (Ph.D. Dissernot shown). Enzymatic analysis indicates tation, Ohio University, 1983). Peak D protein peaks A, B, and C to consist of myeloperoxi(lane e) appears to contain a faint band which dase, neutral protease, and lysozyme, respec- correlates with a neutral protease of peak B tively. Peak D lacks any substantial activity of the enzymes tested (Table 1). Figure 2 shows the electrophoretic patterns of crude granule extract and isolated peaks in cationic polyacrylamide gels. The migration pattern of crude granule extract components was similar to that observed by Hodinka and Modrzakowski (3) and the migration of peaks A (lane b) and C (lane d) correlated with that of myeloperoxidase and lysozyme standards, respectively (data not shown). Lane e shows peak D to consist of two peptides, in contrast to the three apparent peptides observed in the elution profile of chromatographed granule extract. The consistent presence of these cationic peptides in rat PMN granule extract virtually rules out the possibility that they are the result of random degradation of proteins by granule proteases. It is likely that rat PMN granule extract contains only two cationic peptides, as they have been consistently observed in cationic-PAGE conducted in this laboratory. It is possible that the 7700-Da peptide eluting from the Superose column is FIG. 2. Cationic-PAGE of (a), crude granule extract; actually a dimer form of the 4 lOO-Da peptide. (b), peak A; (c), peak B; (d), peak C; (e), peak D. Conditions The 4 100- and 5500-Da peptides correspond of electrophoresis are described under Materials and closely to the molecular weights of the cationic Methods.

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(lane c). Enzyme analysis shows peak D to contain a small amount of proteolytic activity (Table 1). More careful fraction collection, or further purification steps, such as the reversephase high-pressure liquid chromatography technique described by Selsted et al. (10) for the isolation of rabbit PMN cationic peptides may be required to purify the cationic peptides to complete homogeneity. Peak D peptides were found to have molecular weights of approximately 3500 and 6800 when examined with SDS-PAGE (Fig. 3). While the molecular weight of the 3500Da peptide is close to that determined by gel filtration chromatography conducted in this study and by SDS-PAGE conducted by Hodinka (Ph.D. Dissertation), the molecular weight of the 6800-Da peptide is higher by approximately IOOO-Da. From these results, it can be concluded that rat PMN granule extract probably contains two cationic peptides with

FIG.

ditions

3. SDS-PAGE are described

of isolated peak D peptides. under Materials and Methods.

Con-

MODRZAKOWSKI

molecular weights of between 3500 and 4 100, and 5500 and 6800. There appear to be two classes of antimicrobial cationic proteins based on size: large proteins with molecular weights between 36,000 and 60,000; and small peptides with molecular weights between 3500 and 7000. While only large and small cationic proteins have been isolated from human (16) and rat (3) PMN granules respectively, both large ( 17) and small (10) cationic proteins have been isolated from rabbit PMN granules. Homology studies to determine the relatedness of the various cationic proteins could prove interesting. Cationic, antimicrobial proteins from human and rabbit PMN granules have been characterized. Weiss et al. have described cationic proteins from human ( 16) and rabbit ( 17) PMN with molecular weights between 50,000 and 60,000. The bactericidal activity of these noncatalytic proteins against gram-negative bacteria was accompanied by an increase in outer membrane permeability. ModIzakowski and Spitznagel(8) isolated a 36,000-to 37,000Da cationic protein from human PMN granules which possessed bactericidal activity against S. typhimurium and was adsorbed by isolated LPS. Low molecular weight cationic peptides with potent antimicrobial activity have also been characterized. Selsted et al. ( 10) have utilized HPLC to purify several 4000-Da cationic peptides from rabbit PMN. The antimicrobial activity of cationic, noncatalytic proteins from rat PMN granules has not, until now, been examined. This was due in part, to the difficulty in isolating the low molecular weight proteins. It is possible that rabbit and rat cationic peptides possess similar mechanisms of antimicrobial activity. While standard gel filtration chromatography techniques appear to be incapable of separating the low molecular weight proteins from lysozyme, the resolving power of FPLC produces excellent separation. An initial examination of isolated peak D peptides from rat PMN has shown these granule components to possess potent bactericidal activity (manuscript submitted for publication). Studies to

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characterize this bactericidal activity are currently in progress, and an analysis of the amino acid composition of the cationic peptides is planned.

GRANULE

381

CONTENTS

6. Modrzakowski, M. C., Dosch-Meier, D., and Hodinka, R. L. (1983) Cunad. J. Mcrobiol.

29,

1339-1343.

I. Modtzakowski, M. C., and Paranavitana, C. J. (198 1) Infect. 8.

Immun.

Modrzakowski, Infect.

Immun.

32, 668-674.

M. C., and Spitznagel, J. IS. (1979) 25, 591-602.

Odeberg, H., and Olsson, I. (1965) Infecf. Immun. 14, 1269-1275. 10. Selsted, M. E., Szklarek, D., and Lehrer, R. I. (1984) Infect. Immun. 45, 150- 154. Il. Shafer, W. M., Casey, S. G., and Spitznagel, J. K. , ? ( (1984) Infect. Immun. 43, 834-838. i L. Shapiro, A. L., Vinuela, E., and Maizel, J. V., Jr. ( 1967) Biochem. Biophys. Rex Commun. 28,8 15-820. 13. Shugar, D. (1952) Biochim. Biophys. Acta 8, 3029.

ACKNOWLEDGMENTS The authors thank the laboratory of Dr. Peter Johnson, Department of Chemistry, Ohio University, for assistance with, and the use of the Pharmacia FPLC system. This work was supported by Ohio University Baker Fund Award 84- 12, and by research funds fim the Ohio University College of Osteopathic Medicine.

309.

14. Starkey, P. M., and Barrett, A. J. (1976) Biochem.

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