
,


* Department of Medicine, UCLA School of Medicine, and
|| Molecular Biology Institute, UCLA, Los Angeles, California;
A. A. Ukhtomsky Physiology Research Institute, St. Petersburg State University, and
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
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) defensin previously described by Tang et al.
(Science, 286, 498502, 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
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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
-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. |
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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.
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defensins. Primer P1
(5'-GTGACCCCAGCCATGAGGACCCTCGCCATC-3') corresponded to nucleotides
3868 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
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. |
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) 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
-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 160 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.
![]() View larger version (40K): [in a new window] |
Figure 1. Deduced amino acid sequences of demidefensins, Also shown are the
sequences of the mature circular ( ) minidefensins RTD-1, -2, and
-3.
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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
monkeys 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. |
View this table: [in a new window] |
Table 1. Relative Abundance of Demidefensin Transcripts in Individual Rhesus
Macaques
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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
-defensins, we tested the
fractions against E. coli in underlay gels containing 10 mM
phosphate buffer ± 100 mM NaCl. Whereas
-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
-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.
![]() View larger version (28K): [in a new window] |
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 -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).
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6 mass units larger
than its unmodified native counterpart.
![]() View larger version (52K): [in a new window] |
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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View this table: [in a new window] |
Table 2. Mass Spectrometric Analysis of Minidefensins
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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.
Received April 23, 2001; revised June 11, 2001; accepted June 18, 2001.
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