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(Journal of Leukocyte Biology. 2001;70:461-464.)
© 2001 by Society for Leukocyte Biology

Circular minidefensins and posttranslational generation of molecular diversity

Larisa Leonova*,{dagger}, Vladimir N. Kokryakov{dagger},{ddagger}, Galina Aleshina{dagger}, Teresa Hong*, Tung Nguyen*, Chengquan Zhao*,§, Alan J. Waring* and Robert I. Lehrer||

* Department of Medicine, UCLA School of Medicine, and
|| Molecular Biology Institute, UCLA, Los Angeles, California;
{dagger} A. A. Ukhtomsky Physiology Research Institute, St. Petersburg State University, and
{ddagger} Institute of Experimental Medicine, St. Petersburg, Russia; and
§ Department of Pathology and Laboratory Medicine, MCP Hahnemann School of Medicine, Philadelphia, Pennsylvania

Correspondence: Robert I. Lehrer, M.D., Department of Medicine, UCLA Center for the Health Sciences, Los Angeles, CA 90095-1690. E-mail: rlehrer{at}mednet.ucla.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 BONE MARROW PROCUREMENT AND...
 PEPTIDE SYNTHESIS
 OXIDATION AND CYCLIZATION
 RACE ANALYSIS
 IDENTIFICATION OF DEMIDEFENSIN R...
 INDIVIDUAL VARIATION
 IDENTIFICATION OF CIRCULAR...
 NUMBER OF DISULFIDE BONDS
 REFERENCES
 
We purified two new minidefensins (RTD-2 and RTD-3) from the bone marrow of rhesus monkeys. Both were circular octadecapeptides that contained three intramolecular disulfide bonds and were homologous to RTD-1, a circular ({theta}) defensin previously described by Tang et al. (Science, 286, 498–502, 1999). However, whereas the 18 residues of RTD-1 represent spliced nonapeptide fragments derived from two different demidefensin precursors, RTD-2 and -3 comprise tandem nonapeptide repeats derived from only one of the RTD-1 precursors. Thus, circular minidefensins are products of a novel posttranslational system that generates effector molecule diversity without commensurate genome expansion. A system wherein two demidefensin genes can produce three circular minidefensins might allow n such genes to produce (n/2)(n+1) peptides.

Key Words: antimicrobial • peptides • defensins • innate immunity • RTD


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
BONE MARROW PROCUREMENT AND...
 PEPTIDE SYNTHESIS
 OXIDATION AND CYCLIZATION
 RACE ANALYSIS
 IDENTIFICATION OF DEMIDEFENSIN R...
 INDIVIDUAL VARIATION
 IDENTIFICATION OF CIRCULAR...
 NUMBER OF DISULFIDE BONDS
 REFERENCES
 
Vertebrate defensins are arginine-rich antimicrobial peptides with largely ß-sheet structures that are stabilized by three intramolecular disulfide bonds [1 ]. {alpha} and ß defensins of humans, which differ in the placement and connectivity of their cysteine residues, arose from a common ancestral gene that existed before reptilian and avian lineages diverged [2 , 3 ]. RTD-1, a peptide recently isolated from granulocytes of the rhesus monkey (Macaca mulatta) [4 ], is the index member of a third vertebrate defensin subfamily that is called "circular minidefensins" in this report. This study began when we found an unusual protegrin-sized peptide in rhesus monkey leukocytes. Because it resisted conventional sequencing, we undertook a cloning strategy to identify its precursor, expecting it to be a cathelicidin. Instead, we found several {alpha}-defensin transcripts whose sequence contained a premature stop codon after the third cysteine residue. While this work was in progress, Tang et al. described the cyclic nature of RTD-1 [4 ]. Our present report describes two additional rhesus circular minidefensins, RTD-2 and RTD-3, and discusses them with respect to the generation of molecular diversity at the peptide level.


   BONE MARROW PROCUREMENT AND PROCESSING
TOP
 ABSTRACT
 INTRODUCTION
 BONE MARROW PROCUREMENT AND...
PEPTIDE SYNTHESIS
 OXIDATION AND CYCLIZATION
 RACE ANALYSIS
 IDENTIFICATION OF DEMIDEFENSIN R...
 INDIVIDUAL VARIATION
 IDENTIFICATION OF CIRCULAR...
 NUMBER OF DISULFIDE BONDS
 REFERENCES
 
Bone marrow was obtained from seven adult rhesus monkeys (M. mulatta) euthanized at the University of California at Davis Regional Primate Center, Davis, CA, for reasons unrelated to this research. Aliquots of fresh marrow (0.5 mL each) were dispensed into tubes containing 3.75 mL of TRI Reagent BD (Molecular Research Center, Cincinnati, OH) and 350 µL of 5 N acetic acid, mixed vigorously, and rapidly frozen. After transport and thawing, a protein fraction was precipitated by adding isopropanol to the phenol-ethanol supernatant in a 2:1 (v/v) ratio, centrifuging the mixture at 12,000 g for 10 min, and washing the precipitate three times by centrifuging and resuspending it in 0.3 M guanidine hydrochloride in 95% ethanol, with a holding period of 20 min at room temperature preceding each wash step centrifugation. The resulting precipitate was washed with 100% ethanol and air-dried for 10 min.

With vigorous mixing, this precipitate was dissolved in 10% acetic acid-9 M urea and clarified by centrifugation (10,000 g, 20 min, 4°C). The supernatant was subjected to preparative continuous acid-urea polyacrylamide gel electrophoresis (PAGE) as previously described [5 ]. Eluted fractions were collected, examined by analytical acid-urea- and sodium dodecyl sulfate-PAGE, and tested in radial diffusion assays [6 ]. Fractions containing low-molecular-weight components that were active in 100 mM NaCl were purified to apparent homogeneity by reversed-phase (RP)-high-performance liquid chromatography (HPLC), with a 4.6- by 250-mm C18-column (Vydac, The Separations Group, Hesperia, CA) and a 1% min-1 linear gradient of acetonitrile in 0.1% trifluoroacetic acid. The circular minidefensins described in this report eluted at 28.5% (RTD-2), 30.5% (RTD-1), and 32.1% (RTD-3) acetonitrile.


   PEPTIDE SYNTHESIS
TOP
 ABSTRACT
 INTRODUCTION
 BONE MARROW PROCUREMENT AND...
 PEPTIDE SYNTHESIS
OXIDATION AND CYCLIZATION
 RACE ANALYSIS
 IDENTIFICATION OF DEMIDEFENSIN R...
 INDIVIDUAL VARIATION
 IDENTIFICATION OF CIRCULAR...
 NUMBER OF DISULFIDE BONDS
 REFERENCES
 
Because RTD-like peptides are circular, they are inherently refractory to sequencing by conventional Edman degradation. Therefore, we prepared synthetic peptides corresponding to RTD-1, -2, and –3 for comparison with the native peptides. Peptides were synthesized with an ABI 431 A synthesizer, using prederivatized polyethylene glycol-polystyrene arginine resin from PerSeptive Biosystems (Framingham, MA), FastMocTM chemistry [7 ] with double coupling throughout. Crude peptide was reduced with excess dithiothreitol under N2 for 15 h at 50°C in 6 M guanidine-HCI, 0.2 M Tris-HCI, and 0.2 mM EDTA (pH 8.2). After adding glacial acetic acid (final concentration, 5%), the reduced peptide was stored under N2 until purified by RP-HPLC.


   OXIDATION AND CYCLIZATION
TOP
 ABSTRACT
 INTRODUCTION
 BONE MARROW PROCUREMENT AND...
 PEPTIDE SYNTHESIS
 OXIDATION AND CYCLIZATION
RACE ANALYSIS
 IDENTIFICATION OF DEMIDEFENSIN R...
 INDIVIDUAL VARIATION
 IDENTIFICATION OF CIRCULAR...
 NUMBER OF DISULFIDE BONDS
 REFERENCES
 
To form disulfide bonds, reduced peptides were dissolved at 0.1 mg/mL in 0.1% acetic acid. After adjusting the pH to 7.4 with NH4OH, solutions were stirred for 24 h at room temperature in air before acetic acid was added (5%, final concentration). After purification by RP-HPLC, the oxidized acyclic peptides were 6 atomic mass units smaller than their corresponding reduced forms, as determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) (data not shown). To cyclize these oxidized peptides, they were incubated for 18 h at room temperature in a 3:1 mixture of ethylenediaminecarbodiimide and anhydrous N-hydroxybenzotriazole in dimethylsulfoxide. The product was lyophilized and purified by RP-HPLC on a C18 column, using a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. The cyclic peptides had the expected mass by MALDI-TOF analysis and were obtained in a yield of 50–70% (data not shown).


   RACE ANALYSIS
TOP
 ABSTRACT
 INTRODUCTION
 BONE MARROW PROCUREMENT AND...
 PEPTIDE SYNTHESIS
 OXIDATION AND CYCLIZATION
 RACE ANALYSIS
IDENTIFICATION OF DEMIDEFENSIN R...
 INDIVIDUAL VARIATION
 IDENTIFICATION OF CIRCULAR...
 NUMBER OF DISULFIDE BONDS
 REFERENCES
 
Rapid 3' amplification of the cDNA end (3'-RACE) was performed with a kit (Gibco-BRL, Gaithersburg, MD). Total monkey bone marrow RNA (1 µg) and 1 µl of 10 µM adapter primer were used to obtain first-strand cDNA. Primers P1 and P2 were designed according to cDNA sequences of human {alpha} defensins. Primer P1 (5'-GTGACCCCAGCCATGAGGACCCTCGCCATC-3') corresponded to nucleotides 38–68 of human neutrophil peptide (HNP)-1 [8 ]. Primer P2 (5'-GTCTGCCCTCTCTGCTCGCCCTGCC) corresponded to nucleotides 1-25 of HNP-4 [9 ]. PCR (35 cycles) was done in a 50-µL final volume with 10% of the first-strand cDNA, 10 pmol of each Abridged Universal amplification primer (Gibco-BRL, Rockville, MD), the human {alpha} defensin primers, and 1 µL of Advantage cDNA polymerase (Clontech, Palo Alto, CA). A Perkin-Elmer (Foster City, CA) model 2400 GeneAmp PCR system was used, with the following cycle temperatures and times: 94°C, 20 s; 55°C, 20 s; and 72°C, 40 s. After agarose gel electrophoresis, the PCR products were cloned into a PCR II vector with a TA kit (Invitrogen) and sequenced by the fluorescein-labeled dideoxynucleotide terminator method on an Applied Biosystems 373 DNA sequencer.


   IDENTIFICATION OF DEMIDEFENSIN R mRNA
TOP
 ABSTRACT
 INTRODUCTION
 BONE MARROW PROCUREMENT AND...
 PEPTIDE SYNTHESIS
 OXIDATION AND CYCLIZATION
 RACE ANALYSIS
 IDENTIFICATION OF DEMIDEFENSIN R...
INDIVIDUAL VARIATION
 IDENTIFICATION OF CIRCULAR...
 NUMBER OF DISULFIDE BONDS
 REFERENCES
 
We found three rhesus transcripts that encoded demidefensins. Their nucleotide sequences can be found in GenBank, with the following accession numbers: AF184156, AF184157, and AF184158. The deduced amino acid sequences of these demidefensins are shown in Figure 1 , along with those of the mature circular ({theta}) minidefensins RTD-1, -2, and -3. Because a mutation in the ancestral defensin gene introduced a stop codon in mid-sequence of each demidefensin, each monkey demidefensin peptide contains only three cysteine residues instead of the six found in conventional {alpha}-defensins. Demidefensin transcripts have 76-amino-acid residues. Demidefensin 3 (pI, 5.79) was much more acidic than demidefensins 1 (pI, 8.02) or 2 (pI, 7,08). Residues 1–60 of the transcripts correspond to their "prepro" regions and are invariant (60/60 identical residues) in demidefensins 1, 2, and 3. In contrast, their C terminal 16 residues varied. Comparing demidefensins 1 and 2 or 2 and 3 showed 11/16 (68.8%) to be identical, whereas in demidefensins 1 and 3, they were 10/16 (62.5%) identical.



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  		Figure 1. Deduced amino acid sequences of demidefensins, Also shown are the sequences of the mature circular ({theta}) minidefensins RTD-1, -2, and -3.

 

   INDIVIDUAL VARIATION
TOP
 ABSTRACT
 INTRODUCTION
 BONE MARROW PROCUREMENT AND...
 PEPTIDE SYNTHESIS
 OXIDATION AND CYCLIZATION
 RACE ANALYSIS
 IDENTIFICATION OF DEMIDEFENSIN R...
 INDIVIDUAL VARIATION
IDENTIFICATION OF CIRCULAR...
 NUMBER OF DISULFIDE BONDS
 REFERENCES
 
To examine demidefensin expression in different monkeys, we designed PCR primers, P3 (5'-GCCATGCTTCTCCTGGTGGCCC-3') and P4 (5'-CTCTCAAAGTAACTTCTGAGC-3') that were common to demidefensins 1, 2, and 3. We used these primers to amplify bone marrow cDNA from four different monkeys and cloned and sequenced ~100 PCR products (Table 1 ). Clones encoding demidefensin 1 mRNA accounted for about three-fourths of these clones and were recovered 15-fold more often than clones with demidefensin 2 mRNA. Transcripts for demidefensin 3, which was not represented in RTD-1, -2, or -3, were recovered from each monkey’s bone marrow and constituted about 20% of the total recovered mRNA. Although data from only four monkeys were acquired, Table 1 indicates that demidefensin gene polymorphism exists in this species. The peptide products, if any, resulting from demidefensin 3 remain to be identified.


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  		Table 1. Relative Abundance of Demidefensin Transcripts in Individual Rhesus Macaques

 

   IDENTIFICATION OF CIRCULAR MINIDEFENSINS
TOP
 ABSTRACT
 INTRODUCTION
 BONE MARROW PROCUREMENT AND...
 PEPTIDE SYNTHESIS
 OXIDATION AND CYCLIZATION
 RACE ANALYSIS
 IDENTIFICATION OF DEMIDEFENSIN R...
 INDIVIDUAL VARIATION
 IDENTIFICATION OF CIRCULAR...
NUMBER OF DISULFIDE BONDS
 REFERENCES
 
We developed a strategy to identify {approx}2-kDa peptides with three disulfide bonds and salt-insensitive activity against Escherichia coli ML-35P, an "engineered" strain we have described elsewhere [10 ]. Summarized briefly, we began by performing preparative continuous acid-urea-PAGE and identifying fractions of interest by radial diffusion assays. To distinguish circular minidefensins from conventional {alpha}-defensins, we tested the fractions against E. coli in underlay gels containing 10 mM phosphate buffer ± 100 mM NaCl. Whereas {alpha}-defensins were active only under low-salt conditions, fractions containing circular minidefensins were also active in the presence of 100 mM NaCl. In Figure 2 , the peak C fractions were active only under low-salt conditions and contained only {alpha}-defensins (data not shown). Peaks A and B, which were also effective in 100 mM NaCl, were later found to contain RTD-1 and RTD-3, respectively. RTD-2, which we generally recovered only in small amounts, was not identified in this run.



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  		Figure 2. Identification of candidate circular defensins. Fractions obtained by preparative electrophoresis were tested (in radial diffusion assays) for their ability to kill E. coli ML-35p in the presence and absence of 100 mM NaCl. Peak C fractions were active only under low-salt conditions and contained only {alpha}-defensins (data not shown). Peaks A and B, which were also effective in 100 mM NaCl, were later found to contain RTD-1 and RTD-3, respectively. RTD-2, was generally recovered only in small amounts, and was not identified in this run. The insert shows an analytical acid-urea-PAGE gel of the starting material, stained either with Coomassie blue (CB) or with silver (Ag).

 

   NUMBER OF DISULFIDE BONDS
TOP
 ABSTRACT
 INTRODUCTION
 BONE MARROW PROCUREMENT AND...
 PEPTIDE SYNTHESIS
 OXIDATION AND CYCLIZATION
 RACE ANALYSIS
 IDENTIFICATION OF DEMIDEFENSIN R...
 INDIVIDUAL VARIATION
 IDENTIFICATION OF CIRCULAR...
 NUMBER OF DISULFIDE BONDS
REFERENCES
 
Once candidate circular minidefensins were identified in this manner, they were purified by preparative RP-HPLC (Fig. 3 ), and the presence of three intramolecular disulfide bonds was ascertained by performing MALDI-TOF measurements before and after the peptides were reduced with dithiothreitol (Table 2 ). As anticipated, each reduced peptide was ~6 mass units larger than its unmodified native counterpart.



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  		Figure 3. Characteristics of rhesus circular minidefensins. The left panel shows the RP-HPLC elution profiles of purified native RTD-1, -2, and -3. Their synthetic counterparts had identical elution times (data not shown) The right panel is an acid-urea-PAGE gel that shows the similar migration of synthetic and native RTDs. Lanes a and b contain the bone marrow protein fractions from two different rhesus macaques before the preparative electrophoresis step.

 

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  		Table 2. Mass Spectrometric Analysis of Minidefensins

 
To establish the identity of the native peptides, we compared them to their synthetic counterparts. Each corresponding pair had identical HPLC retention times (Fig. 3) and masses. Native and synthetic peptides that were trypsinized and then reduced with dithiothreitol had identical fragmentation patterns by MALDI-TOF analysis, with measured masses that were, as expected, within experimental error (Table 2) .

These experiments show that rhesus monkey bone marrow can produce at least three circular octadecapeptides: the recently described RTD-1 [4 ] and RTD-2 and -3, the peptides described in this report. RTD-2 and -3 both contain duplicated nonapeptide elements in tandem. In RTD-2, these elements are contributed by demidefensin 1. In RTD-3, they come from demidefensin 2. In contrast, the RTD-1 derives one nonapeptide element from each of these demidefensins. Our search strategy did not identify any circular minidefensins containing fragments of demidefensin 3. It is possible that this strategy (which required candidate peptides to show activity against E. coli in 100 mM NaCl) was insensitive to their presence. Alternatively, the protein-rich fraction used to start our peptide isolation might have been depleted of such peptides by the multiple differential extractions used in its preparation. These and other possibilities are being explored and will be reported at a later date.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grant AI-22839 from the National Institutes of Health.

Received April 23, 2001; revised June 11, 2001; accepted June 18, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 BONE MARROW PROCUREMENT AND...
 PEPTIDE SYNTHESIS
 OXIDATION AND CYCLIZATION
 RACE ANALYSIS
 IDENTIFICATION OF DEMIDEFENSIN R...
 INDIVIDUAL VARIATION
 IDENTIFICATION OF CIRCULAR...
 NUMBER OF DISULFIDE BONDS
 REFERENCES
 

  1. Zimmermann, G. R., Legault, P., Selsted, M. E., Pardi, A. (1995) Solution structure of bovine neutrophil beta-defensin-12: the peptide fold of the beta-defensins is identical to that of the classical defensins Biochemistry 34,13,663-13,671
  2. Liu, L., Zhao, C., Heng, H. H., Ganz, T. (1997) The human beta defensin-1 and alpha-defensins are encoded by adjacent genes: two peptide families with differing disulfide topology share a common ancestry Genomics 43,316-320[Medline]
  3. Zhao, C., Nguyen, T., Liu, L., Sacco, R. E., Brogden, K. A., Lehrer, R. I. (2001) Gallinacin-3, an inducible epithelial beta-defensin in the chicken Infect. Immun 69,2684-2691[Abstract/Free Full Text]
  4. Tang, Y. Q., Yuan, J., Osapay, G., Osapay, K., Tran, D., Miller, C. J., Ouellette, A. J., Selsted, M. E. (1999) A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins Science 286,498-502[Abstract/Free Full Text]
  5. Harwig, S. S., Chen, N. P., Park, A. S., Lehrer, R. I. (1993) Purification of cysteine-rich bioactive peptides from leukocytes by continuous acid-urea-polyacrylamide gel electrophoresis Anal. Biochem 208,382-386[Medline]
  6. Lehrer, R. I., Rosenman, M., Harwig, S. S., Jackson, R., Eisenhauer, P. (1991) Ultrasensitive assays for endogenous antimicrobial polypeptides J. Immunol. Methods 137,167-173[Medline]
  7. Fields, C. G., Lloyd, D. H., Macdonald, R. L., Otteson, K. M., Noble, R. L (1991) HBTU activation for automated Fmoc solid-phase peptide synthesis Peptide Res 4,95-101
  8. Daher, K. A., Lehrer, R. I., Ganz, T., Kronenberg, M. (1988) Isolation and characterization of human defensin cDNA clones Proc. Natl. Acad. Sci. USA 85,7327-7331[Abstract/Free Full Text]
  9. Palfree, R. G. E., Sadro, L. C., Solomon, S. (1993) The gene encoding the HNP-4 precursor contains a recent 86-base duplication and is located on chromosome 8 Mol. Endocrinol 7,199-205[Abstract]
  10. Lehrer, R. I., Barton, A., Ganz, T. (1988) Concurrent assessment of inner and outer membrane permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry J. Immunol. Methods 108,153-158[Medline]



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