Purification, Primary Structures, and Antibacterial Activities of B-Defensins, a New Family of Antimicrobial Peptides from Bovine Neutrophils*

A new family of cysteine-rich antimicrobial peptides from bovine neutrophils was isolated and characterized. Thirteen structurally homologous peptides were purified to homogeneity from a granule-rich cyto- plasmic fraction of purified blood neutrophils. The complete sequences of the peptides were determined by a combination of enzymatic digestion, Edman degradation, and additional biochemical characterization of the carboxyl termini. The peptides are characterized by a highly cationic 38-42-residue chain which includes 6 invariantly spaced cysteines which form three disulfides. They share a highly conserved consensus sequence which is also found in a recently described epithelial antimicrobial peptide from bovine trachea. The in vitro antibacterial activities of the 13 neutrophil peptides, determined in assays using Staphylococcus aureus and Escherichia coli as test organisms, dem- onstrated that each peptide possessed antimicrobial activity, and that several were as active as

peptides which equip these cells to inactivate ingested microbial targets by mechanisms considered to be "oxygen-independent" (1-3). These granule proteins constitute an antimicrobial arsenal which includes defensins, a family of broad spectrum antibiotic peptides which are released into the phagosome during phagolysosome fusion (1,4). To date, members of the defensin family have been isolated from neutrophils of human (5), rabbit (6), rat (7,8), and guinea pig origin (9, lo), and most recently from the Paneth cells of mouse small intestine (11,12). The current investigation was initiated to determine whether defensins may contribute to the antimicrobial activity of ruminant neutrophils, leukocytes which possess a class of morphologically unique cytoplasmic granules previously shown to contain potent bactericidal polypeptides (13).
The unique features of ruminant granulocytes were first described by Gennaro and Baggiolini and coworkers (14,15) who demonstrated that neutrophils of cattle, goats, sheep, and ibex are endowed with large numbers of unusually large cytoplasmic granules which are distinct from the classical azurophil and specific granules. Subsequent studies (13) established that the majority of the antibacterial polypeptides of bovine neutrophils are contained in these unique organelles. Romeo and  have demonstrated that the large granules of bovine neutrophils contain potent microbicidal peptides which are structurally distinct from defensins. These include three arginine-rich peptides, termed bactenecins, which efficiently kill several Gram-positive and Gram-negative bacteria in vitro (16,18). Recently, we reported the isolation and characterization of a novel tridecapeptide amide from bovine neutrophils. Termed indolicidin, this cationic peptide was shown to be unusually rich in tryptophan and to have potent bactericidal activity against Escherichia coli and Staphylococcus aureus (19).
In this report, we describe the results of studies which were initiated to determine the presence and biologic role of defensins in bovine neutrophils. The strategy for detection and isolation of bovine defensins was based on the isolation of low molecular weight, cysteine-containing antimicrobial peptides extracted from granule-rich lysates of purified neutrophils. Using gel filtration and reversed-phase HPLC, 13 structurally related peptides were purified to homogeneity, tested for antibacterial activity, and fully sequenced. Though possessing some features of defensins, namely their similar size, cationicity, and the presence of three intramolecular disulfides, the bovine peptides differ significantly in structure from defensins, and thus represent a new class of host defense 6641 New Family of Antimicrobial Peptides peptides. To distinguish them from classical defensins, we propose that this novel peptide family be termed /3-defensins.

MATERIALS AND METHODS
Bovine Neutrophils-Polymorphonuclear leukocytes (PMN) were purified from 1-liter batches of fresh citrated bovine blood. Following sedimentation for 40 min at 700 X g and 37 "C, the erythrocyte column was subjected to 7 s of hypotonic lysis, after which isotonicity was restored using 3 X phosphate-buffered saline. The leukocyte-rich suspension was then sedimented at 120 X g (4 "C, 15 min). Residual erythrocytes were lysed by repeating this procedure one or two times. Aliquots were removed for quantitation by hemocytometry and differential counts. Preparations obtained by this procedure contained an average of 4 X loB cells per liter of whole blood of which 97 f 3% were neutrophils. Half of the preparations were treated with 2 mM diisopropylfluorophosphate (DFP (20)). Neutrophil preparations were then cooled to 4 "C for 20 min and disrupted by nitrogen cavitation in a Parr bomb (21). The cavitate was centrifuged at 800 X g for 10 min at 4 "C, and the granule-containing supernatant was collected. Granules were harvested by centrifugation at 27,000 x g for 40 min and stored at -80 "C.
PMN Granule Extracts-Preparations of frozen granules from 1-5 X 1 0 ' ' PMN were extracted with 5 ml of ice-cold 10% acetic acid per 1 X 1 0 ' cell equivalents. After stirring on ice for 18 h, the suspension was clarified by centrifugation at 27,000 X g for 20 min at 4 "C and the supernatants were lyophilized and stored at -80 "C.
Size Exclusion Chromatography-Lyophilized granule extract was dissolved in 10% acetic acid at a concentration of approximately 1 X 10' cell equivalents per ml, clarified by centrifugation, and loaded onto a 4.8-X 110-cm column of BioGel P-60 equilibrated in 5% acetic acid. The column was run at 8 "C with an elution rate of 2 cm per h, and 15-ml fractions were collected with continuous monitoring at 280 nm.
Reversed-phose HPLC (RP-HPLC)-Low molecular weight components eluting from the size-exclusion column were further resolved by RP-HPLC on a Waters 510 binary system on a 1 X 25-cm Vydac C-18 column. Water and acetonitrile containing 0.1% trifluoracetic acid or 0.13% heptafluorobutyric acid were used for gradient elution. Purified peptides were lyophilized, dissolved in 0.01% acetic acid at 500 pg/ml, and stored at -70 "C.
Polyacrylamide Gel Electrophoresis-SDS (22) and acid-urea (23) gel electrophoresis were used to the estimate molecular mass and/or purity of protein preparations as previously described (24).
Amino Acid Analysis-The amino acid composition of each peptide was determined on 6 N HCl hydrolysates (2 h, 150 "C) of native and performic acid-oxidized or reduced and alkylated samples (25). Tryptophan content was determined by sequence analysis and by spectroscopic measurement on a Beckman DU 60 spectrophotometer by the method of Edelhoch (26).
Sequence Analysis-Samples for sequence analysis were reduced with dithiothreitol and alkylated with vinyl pyridine or iodoacetamide (27) and purified by RP-HPLC. Two cycles of manual Edman d e g radation (28) were performed on all samples to identify aminoterminally blocked peptides. 3 to 4 nmol of each N-blocked peptide was incubated with 2 pg of pyroglutamate aminopeptidase (Boehringer Mannheim) for 5 h at room temperature in 50 r l of 0.1 M sodium phosphate, 0.01 M disodium EDTA, 5 mM dithiothreitol, 5% glycerol, pH 8.0. The deblocked peptide was then purified by RP-HPLC prior to automated Edman sequence analysis. Automated sequence analysis was performed on an Applied Biosystems 475A instrument configured with on-line phenylthiohydantoin-amino acid analysis.
Carboxyl-terminal amino acids were determined by amino acid analysis of residues released by carboxypeptidases A or Y (29).
Approximately 1 nmol of S-alkylated peptide was dissolved in 50 pl of 0.05 M sodium phosphate buffer, pH 8.0, or 0.125 M ammonium bicarbonate, pH 8.0, containing 3-6 pg of carboxypeptidase A (Boehringer Mannheim) for 20-60 min at 37 'C. Released tryptophan was identified after derivitization to the phenylthiocarbamyl derivative as described above under amino acid analysis. For digestion with carboxypeptidase Y (Boehringer Mannheim), 4 nmol of peptide (BNBD-6) was incubated with 1 pg of enzyme in 50 pl of 0.05 M pyridineacetate buffer, pH 5.5, at room temperature for 4 h. The mixture was acidified with acetic acid and subjected to RP-HPLC. Released tryptophan was identified by its coelution with authentic tryptophan on Mass Spectrometry-Native peptide mass was determined by positive ion fast atom bombardment mass spectrometry on a VG 7070E-RP-HPLC. HF instrument. Scans were from 3450-6200 m/z at a scan speed of 300 s/decade with a resolution of 500 for five cumulative scans. Ions were generated by bombardment of the meta-nitrobenyl alcohol matrix by a neutral xenon atom beam accelerated under an 8-kv potential.
Trypsin and Chymotrypsin Treatment-Proteolytic digestion of selected peptides was carried out with a-chymotrypsin (Boehringer Mannheim) or tosylamide-2-phenylethyl chloromethyl ketonetreated trypsin (Worthington). S-Pyridylethylated peptide (approximately 2 nmol) was dissolved in 50 pl of 0.125 M ammonium bicarbonate and incubated with 0.2 pg of enzyme at 37 "C for 1-5 min. Peptide fragments were purified by RP-HPLC, characterized by amino acid analysis, and in some cases sequenced.
Antimicrobial Assay-E. coli ML35 and S. aureus 502A were utilized in a radial diffusion assay recently described by Lehrer et al. (30). Bacteria were grown to mid-log phase in trypticase soy broth (TSB), diluted into 10 ml of warm (43 "C) 1% agarose containing 3 mg of TSB, buffered with 10 mM sodium phosphate, pH 7.4. Five pl of each peptide solution was pipeted into wells formed with a 4-mm cork borer and allowed to incubate at 37 "C for 3-4 h. Plates were then overlaid with 10 ml of sterile 1% agar containing 2 X TSB. Following incubation for 18-24 h, the diameter of the clear zone surrounding each well was measured using a magnified transilluminator.

RESULTS
Purification of Bovine Neutrophil Peptides-Previous electrophoretic analyses of the acid-soluble proteins of bovine PMN granules demonstrated that these preparations contain a complex mixture of proteins varying in size from 1000 to 200,000 Da (19). In order to isolate putative defensins from bovine neutrophil granules, 1-3 x 10" cell equivalents of acidsolubilized granule protein was fractionated on a BioGel P-60 column, and antibacterial activity in pooled eluent fractions was assayed as described in "Materials and Methods." Each of the peaks (A-F in Fig. 1) contained bactericidal activity against S. aureus and E. coli (data not shown). As described in a recent report, peak F was predominantly comprised of indolicidin, a novel 13-residue antibiotic peptide amide (19).
SDS-PAGE of pooled fractions from the P-60 column indicated that most of the proteins eluting in peak E were approximately 5 kDa (data not shown), and amino acid analysis demonstrated that the overall cysteine content of material in this peak was approximately 15%. Since these are features consistent with the size and composition of defensins, peak E fractions were combined and further purified by HPLC. The initial RP-HPLC purification of peak E fractions yielded a complex chromatogram (Fig. 2) in which most peaks contained two or more peptides as determined by acid-urea PAGE. One of the earliest peaks (indicated by the asterisk in Fig. 2) contained an antibacterial peptide of approximately 1500 Da. Automated sequence analysis (data not shown) revealed that this peptide was identical to the cyclic dodecapeptide bactenecin described earlier by Romeo et al. (16). Subsequent steps in the purification of the 13 peptides described here employed modified gradient conditions and/or use of 0.13% heptafluorobutyric acid as the ion-pairing agent. These steps enabled the purification of 13 unique peptides, each of which was determined to be pure by its homogeneous behavior on analytical RP-HPLC (Fig. 3) and acid-urea PAGE (Fig. 4). As described below, the peptides constitute a family of related peptides (bovine neutrophil /3-defensins (BNBDs)) which we have numbered 1-13 based on their increasing retention times on RP-HPLC. (Fig. 3 and Table   I). Peptides eluting in unnumbered peaks in Fig. 2 were characterized by amino acid analysis and SDS-PAGE and were either devoid of cysteine or were much larger than BNBD 1-13, indicating that these peptides were unrelated to /3- neutrophils was chromatographed on a Bio-Gel P-60 column as described under "Materials and Methods." Fractions corresponding to peak E were lyophilized and subjected to further purification as shown in Fig. 2.
FIG. 2. Reversed-phase HPLC of peak E fractions. One-tenth of the pooled fractions frompeak E ( Fig. 1) was loaded on a 1 X 25-cm Vydac C-18 column equilibrated in 0.1% trifluoracetic acid/water (solvent A) at a flow rate of 3.0 ml/min. A linear gradient of acetonitrile (20-45%) containing 0.1% trifluoracetic acid (solvent B) was applied at the rate of 0.33% per min. Fractions were collected using the peak cutting mode of a Pharmacia Frac-200 fraction collector. The identity of the b-defensin(s) eluting in each peak is indicated by numbers 1-13 which corresponds to the nomenclature in Table I  The cellular content of P-defensin peptide was estimated by quantitating the amount of homogeneous BNBD 1-13 recovered and correcting for losses at each step in purification. Using acid-urea and SDS-PAGE to assess recovery, we estimated that approximately 80% of the cellular content of Pdefensins was extracted from granule-enriched fractions and that recovery from the P-60 column was virtually quantitative. Assuming 75% recovery during RP-HPLC, the quantity of the combined 13 8-defensins, averaged from two complete purifications, was approximately 4.9 mg/lO" neutrophils. The most abundant P-defensin was BNBD-3, present at approximately 2.2 mg/lOlo cells, and the quantity of each of the remaining peptides was similarly estimated as summarized in Table I.
Amino Acid Analysis-The composition of each peptide was established by amino acid analysis of native and performic acid-oxidized or S-carboxamidomethylated samples, and each was analyzed at least twice. Absorbance scans of each of the peptides were carried out between 300 and 200 nm, providing an accurate estimate of tyrosine and tryptophan content (26). As summarized in Table I, the 13 peptides contained from 38 to 42 amino acids, 6 of which were half-cystine residues. The native peptides did not react with Ellman's reagent or iodoacetamide, indicating that the cysteines were most likely pres-70 ent as disulfides. In addition to their high cysteine content, the peptides were generally rich in the basic amino acids arginine and lysine, but tyrosine and alanine were relatively uncommon.
Sequence Analyses-Two cycles of manual Edman degradation allowed the identification of amino-terminal residues of six peptides (BNBDs 1, 2, 8, 11, 12, and 13). The amino termini of the remaining seven peptides were deblocked with pyroglutamate aminopeptidase, demonstrating the presence of a pyroglutamyl residue at the amino terminus of each of these peptides. Automated sequence analysis was carried out on 1-5 nmol of each S-alkylated peptide. Repetitive sequencing yields averaged 93-97%, allowing for unambiguous assignment of 511 of 519 amino acids by automated Edman degradation. The 8 residues requiring additional steps for identification included the carboxyl-terminal tryptophans of BNBDs 1, 2, 3, 6, 11, 12, and 13, and the carboxyl-terminal arginine of BNBD-4. With the exceptions of BNBD-4 and -6, the carboxyl terminus of each of the eight above-mentioned peptides was determined by analysis of carboxypeptidase A- Cys (1.59). The overall composition of this fragment and its content of (3) arginine residues are consistent with the presence of the Arg-Arg dipeptide at the carboxyl terminus.
The tryptophan assigned as the carboxyl terminus of BNBD-6 was released poorly by carboxypeptidases A and Y.
To confirm the carboxyl-terminal tryptophan, the mass of BNBDB was determined on a sample of native peptide by fast atom bombardment mass spectrometry. The monoisotopic mass of BNBD-6 was 4814.2 a.m.u., in close agreement with the theoretical mass of 4816, and consistent with the presence of the carboxyl-terminal tryptophan. Furthermore, ultraviolet spectral analysis (26) indicated the presence of a single tryptophan in both the intact peptide and in the carboxyl-terminal chymotryptic fragment containing residues 33-42. The sequences of all 13 peptides were in excellent agreement with their respective amino acid compositions ( Table I).
The complete amino acid sequences of BNBD 1-13, shown in Fig. 5, reveal the high degree of primary structural similarity of this peptide family. Like defensins, each peptide is characterized by 6 invariant cysteine residues, 2 of which are sequential and situated near the peptide carboxyl terminus. However, the spacing of the other cysteine residues in the sequence differs from defensins, and the disulfide connectivities, determined in BNBD-12, differ from those of defensins (31). Given these structural distinctions, we have proposed that this new family of peptides be designated P-defensins.
In addition to the conserved cysteines, the 8-defensin sequences contain several amino acids that are highly if not absolutely conserved (Fig. 5). By aligning highly conservative substitutions (Ser/Thr; Val/Ile/Leu/Phe) and one position where only Pro or Arg appears in the primary structures of 10 or more P-defensins, a common consensus sequence of 27 amino acids is revealed (Fig. 6).
A sequence similarity search using the BLAST alogorithm (32) revealed only a single protein with substantial identity to the 8-defensins, this being tracheal antimicrobial peptide (TAP), a peptide isolated by Diamond et al. (33) from bovine tracheal epithelium. The primary structure of TAP contains the 27-residue 8-defensin consensus sequence, though it is not identical to any of the neutrophil-derived P-defensins described here (Fig. 6).

TABLE I Amino acid compositions, RP-HPLC retention times, electrophoretic mobilities, and cellular quantities of BNBD 1-13
Values determined from vapor phase HCI hydrolysis as described in experimental procedures. Numbers in parentheses indicate residues determined by sequence analysis.
Tryptophanyl residues identified by UV spectrophotometry and release by carboxypeptidase A.

spectrometry.
Relative order of migration on acid urea-PAGE with 1 being the highest Rf and 13 the lowest Rf value (see Fig. 4). Approximate content of each peptide based on recovery as described under "Results."   I 1 1 1 1 1 1 1 1 1 1  I I I I I I I I I I I I I   TAP FIG. 6. Amino acid sequence of bovine tracheal antimicrobial peptide (TAP) and the 8-defensin concensus. The 0-defensin concensus consists of 27 residue positions in which the amino acid is absolutely conserved (11 residues) or where conservative or limited substitutions occur (16 residues): h, hydrophobic (Leu, Ile, Val, or Phe); S/T, Ser or Thr; P/R, Pro or Arg. The disulfide connectivities as determined in BNBD-12 (31) are also shown.

~P~I C P R~~Q I C V P I R C P Q B H~Q I Q T~~
Antimicrobial Activity of &Defensins-The antibacterial activity of each 0-defensin was evaluated using S. aureus 502A and E. coli ML35 as test organisms. Using a sensitive radial diffusion assay, each peptide was tested against the two bacterial organisms with B-defensin concentrations ranging from 10 to 300 pg/ml. The data presented in Fig. 7 reveal the dose-dependent activity of each peptide as measured by the size of the clear zone surrounding the sample well. In most cases, the log of the peptide concentration was linearly related to the diameter of the growth-free zone. Though the relative potencies of peptides differed, all 13 were active against E. coli, and all but BNBD-1 and BNBD-5 were active against S. aureus in the range of concentrations tested. In most cases, the zone of clearing was greater against E. coli than S. aureus.
The antibacterial activities of the 6-defensins were compared with those of three previously characterized antimicrobial peptides: rabbit neutrophil defensin NP-1, the most potent of the classical defensins (24), indolicidin (19), and the cyclic dodecapeptide bactenecin described above (16). Like the @-defensins, the latter two peptides were purified from bovine neutrophil granules. As shown in Fig. 7, the antistaphylococcal activity of rabbit NP-1 was the greatest of any of the peptides tested, though the activity of NP-1 against E. coli was modest when compared to nearly all of the B-defensins. The potency of the dodecapeptide bactenecin was similar to that of several of the 8-defensins against both bacteria, but less so than the most active 8-defensins (e.g. BNBDs 7,9, 12, and 13.) On a mass basis, indolicidin was the most active peptide against E. coli, and it was nearly as active as rabbit NP-1 against S. aureus.

DISCUSSION
Defensins were the first antimicrobial peptides isolated from leukocytes and, until this report, were the only phagocyte-derived molecules known which contain a conserved tridisulfide structural motif. Though we did not detect classical defensins in bovine neutrophils, the search for them led to the discovery of a new class of distinct but related peptide antibiotics. The P-defensins constitute a highly conserved family of at least 13 neutrophil peptides which are characterized by a disulfide motif different from that of the defensin family (31, 34). In the accompanying paper (31), we report the determination of the disulfide structure in BNBD-12 and discuss possible structural relationships between classical and 8-defensins which emerge from a comparison of the respective cysteine connectivities.
Unlike classical defensins which have free amino termini, seven of the 13 B-defensins were found to be blocked at the amino terminus with a pyroglutamyl residue which results from the enzymatic cyclization of amino-terminal glutamine (35). The significance of the pyroglutamyl amino termini is not known, but may confer some aspect of functional specificity (see below), or may provide these particular 8-defensins with increased resistance to endogenous leukocyte proteases.
Three of the 8-defensins appear to be amino-terminal proc-essing variants of corresponding peptides which are slightly longer. The sequences of BNBD-2 and -8 are identical to BNBD-3 and -9 respectively, except that the latter two peptides each have an pyroglutamyl-glycine dipeptide extension.
In addition, BNBD-12 and -13 have identical sequences except that BNBD-13 has a Ser-Gly-Ile-Ser amino-terminal tetrapeptide (Fig. 5). The occurrence of highly related sequences varying in structure at only the amino terminus raises the possibility that the smaller peptides might be generated by proteolytic degradation during purification or, alternatively, that these minor sequence variations reflect genetic polymorphisms expressed in individual animals. The first possibility is unlikely since all steps in the 8-defensin purification up to RP-HPLC were carried out at 0-8 "C at pH < 2.3. In addition, we compared the yield of @-defensins purified from cells which were treated with DFP, a permeant serine protease inhibitor, with those from untreated cells and found no difference in the total and relative yield of the 13 peptides described here. The possibility that the sequence differences reflect genetic polymorphism in the @-defensin locus is excluded since all 13 @-defensins described were isolated from the cells of individual Hereford cows. Therefore, it is most likely that each pair of amino-terminal analogs is produced by differential processing of a common precursor. The proteolytic processing might be a regulated process for producing the observed mixture of P-defensin analogs. Alternatively, the amino-terminal analogs may reflect incomplete or asynchronous post-translational processing which results as granulopoiesis ceases in the bone marrow. The relative ratios of the three @-defensin pairs, estimated from yields obtained during several purifications are approximately 1:8 for BNBD-2:BNBD-3, 1:l for BNBD9:BNBDd, and 1O:l for BNBD-12:BNBD-13. Of the first pair, BNBD-3 has the dipeptidyl extension and is much more abundant. We speculate that the small amount of BNBD-2 may represent the fraction of BNBD-3 in which the amino-terminal glutamine was not converted to pyroglutamate, thereby allowing for additional proteolytic processing to occur. As measured by our in vitro studies, BNBD-2 and -3 have equivalent antibacterial activity against both test organisms. Similarly, the antibacterial activities of BNBD-12 and -13 are approximately equal, though BNBD-12 is by far the more abundant of this pair. This suggests that, like the BNBD-2 and -3 pair, BNBD-13 may be represent an incompletely processed precursor of BNBD-12. In contrast, the BNBD-8 and -9 pair differ in antibacterial potency, with the more abundant BNBD-9 having the greater in vitro activity (Fig. 7). This difference in activity reveals that the amino terminus is a structural determinant of function, much as the amino terminus of human defensins plays a role in antimicrobial potency (5).
The methods used for purifying @-defensins were chosen to minimize processing which might occur if the neutrophils were activated. Frank et al. (17) have shown that the arginine/ proline-rich bactenecins are converted from inactive precur-  (0) were used to assess antibacterial activity of 13 neutrophil 8-defensins in addition to rabbit defensin NP-1, bactenecin dodecapeptide, and indolicidin. Activity is expressed as the diameter of clearing (millimeters) resulting from the application of 5 pl of peptide at the concentrations shown. sors to actively antimicrobial mature peptides when bovine neutrophils are stimulated with various secretagogues. Interestingly, we have found no evidence for such an inducible processing pathway for activation of #?-defensins. Furthermore, the yield of #?-defensin peptides in these studies was unaffected by treatment with DFP or the temperature used for cell extraction, suggesting that these peptides reside in granules as fully processed molecules.
Like the neutrophil #?-defensins, the epithelial peptide TAP is reported to be active in vitro against E. coli and S. aureus as well as Klebsiellapneumoniae and Pseudomonas aeruginosa (33). Because the antibacterial assay method used in this study differed from that employed by Diamond et al. (33), it is not possible to directly compare our antimicrobial potency data with the former study. It is interesting to note that the deduced TAP precursor has a glutamine in the position corresponding to the pyroglutamyl residue in the seven aminoblocked #?-defensins. TAP is the first example of an antimicrobial peptide to be isolated from tracheal epithelium, and its discovery suggests an important role for natural peptide antibiotics in host defense of epithelial surfaces. Recent reports from Ouellette et al. (11) and Selsted et al. (12) have extended this observation in studies which characterized defensins from the epithelium of the small intestine. The presence of #?-defensins in myeloid elements and tracheal epithelium suggests the possibility that members of this peptide family may be expressed and function in other tissues as well.