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(Received for publication, May 19, 1995) From the
Human eosinophil-derived neurotoxin (EDN) and eosinophil
cationic protein (ECP) are members of a unique subfamily of rapidly
evolving primate ribonuclease genes that emerged via a gene duplication
event occurring after the divergence of Old World from New World
monkeys (Rosenberg, H. F., Dyer, K. D., Tiffany, H. L., and Gonzalez,
M.(1995) Nature Genet. 10, 219-223). In this work, we
studied the activity of the protein encoded by the EDN/ECP homolog of
the New World monkey, Saguinus oedipus (marmoset), a
representative of the ``ancestral'' single sequences.
Although the nucleotide sequence of the single marmoset gene (mEDN) was
equally homologous (82%) to both human genes, the encoded amino acid
sequence, calculated isoelectric point, and immunoreactivity all
suggested a closer relationship with EDN. Furthermore, mEDN (at
0.3-1.0 µM concentrations) had no measurable
anti-staphylococcal activity, suggesting functional as well as
structural similarity to EDN. However, with yeast tRNA as substrate,
mEDN had significantly less ribonuclease activity than EDN; Michaelis
constants were nearly identical (K
Eosinophil-derived neurotoxin (EDN) ( We have recently traced the rapid molecular
evolution of the EDN/ECP gene family(14) . While separate genes
encoding EDN and ECP were found in Great Apes and Old World monkeys,
only a single ECP/EDN sequence was detected in the more distant New
World monkeys. These results suggested that ECP and EDN emerged as
distinct sequences as a results of a gene duplication event that
occurred after the divergence of the New World and Old World monkeys (Fig. 1). A representative of this single EDN/ECP sequence was
isolated from the marmoset, Saguinus oedipus (New World
monkey); its nucleotide sequence was found to be equally homologous to
both human EDN and ECP (82% identity), and it encoded a polypeptide
with the structural and catalytic residues analogous to those of other
mammalian ribonucleases.
Figure 1:
A,
estimated evolutionary distances between human and non-human
primates(32, 33) . B, calculated amino acid
sequence divergences and isoelectric points of the individual EDN and
ECP isolates are as described in Rosenberg et
al.(14) ; filled circles designate the sequences
evaluated in this work.
From calculations based on the sequences of
these and other primate homologs, we demonstrated that the genes
encoding EDN and ECP have accumulated non-silent mutations at rates
exceeding those of all other functional coding sequences studied in
primates(14) . The results of this analysis suggested that EDN
and ECP may be responding to unusual evolutionary constraints. In the
work presented here, we have used the single marmoset EDN/ECP as the
basis for the study of the functional evolution of EDN and ECP, in
hopes of learning more about the nature of these constraints.
Figure 2:
A, schematic depicting the human EDN
coding sequence in the prokaryotic expression vector, pFCTS. Features
include the tac promoter, bacterial secretion piece (BSP), and
the FLAG octapeptide (DYKDDDDK) detected by the M2 mAb. Conserved
ribonuclease catalytic residues are highlighted in boxes with
position noted above. B, Coomassie Blue-stained gel containing
total bacterial contents before (lane 1) and 30 min after (lane 2) addition of IPTG. Periplasmic proteins isolated by
heparin-Sepharose chromatography are shown in lane 3. C,
Western blot of lanes 1-3 described in Panel B,
probed with the M2 mAb. Lane 4 contains the periplasmic
isolate shown in lane 3, probed with polyclonal rabbit
anti-EDN antiserum. Amino-terminal sequence of this protein (at arrow) is as indicated. The first serine residue (S)
remains after cleavage of the secretion piece; the remaining residues
are those encoded by the EDN cDNA (KPPQFTWA. .
.)(9, 11) .
Figure 3:
A, Coomassie Blue-stained gel and B, Western blot probed with the M2 mAb containing total cell
extracts (lanes 1 and 3) and periplasmic isolates (lanes 2 and 4) of IPTG-induced bacteria
transfectants. Lanes 1 and 2 contain wild type
recombinant EDN (before and after removal of the secretion piece,
respectively); lanes 3 and 4, recombinant EDN with a
single base pair mutation (A
Determination of
ribonuclease activities of recombinant proteins isolated by M2
anti-FLAG affinity chromatography proceeded as follows. Reactions as
described above were carried out with varying concentrations
(0.89-7.1 µM) of yeast tRNA added in separate
reactions to 0.8 ml of 40 mM sodium phosphate, pH 7.0,
containing either 0.3 pmol of recombinant EDN or 3.6 pmol of
recombinant mEDN isolated on the M2 affinity resin as described.
Equivalent volumes of sham isolations (M2-resin equilibration and
glycine elution of periplasmic proteins isolated from equivalent
volumes of pFCTS vector-alone bacterial transfectants) had levels of
ribonuclease activity that were insignificant compared to human EDN and
represented no more than 15-20% of the experimentally determined
initial rates for mEDN; data presented were determined for
appropriately corrected initial rates. Assay and calculations were as
described above. Michaelis constants (K
In Panel D, the effects of addition of human placental
ribonuclease inhibitor (RNasin) on EDN-catalyzed RNA hydrolysis are
examined. The results demonstrate a reduction in initial rate of
reaction in proportion to the concentration of RNasin added, from 2.9
to 2.5 OD/min (0.067-0.058 nmol/min) with 0.0625 unit/ml RNasin,
and to 1.1 OD/min (0.025 nmol/min) with 0.625 unit/ml RNasin.
Figure 4:
A,
amino acid sequence comparisons of human EDN, mEDN, and human ECP. Lightly shaded boxes enclose the conserved catalytic residues
(His
In Panels B, C,
and D, the cross-reactivity of recombinant mEDN is examined. Panel B shows a Western blot containing total cell extracts
from IPTG-induced transfectants probed with the M2 mAb which detects
all three recombinant proteins (EDN, ECP, and mEDN in lanes 1 through 3, respectively). Although the calculated
molecular mass of ECP (15.6 kDa) is not significantly higher than those
of EDN and mEDN, its cationicity results in the observed reduced
mobility by SDS-PAGE. Panel C shows the identical blot probed
with polyclonal anti-EDN antiserum, and Panel D, the identical
blot probed with anti-ECP antiserum. The anti-EDN antiserum readily
detects both EDN and mEDN, but not ECP; the anti-ECP antiserum detects
ECP and (trace) mEDN, but not EDN. Thus, as predicted by amino acid
sequence homology, mEDN shows cross-reactivity with antisera directed
against both human EDN and human ECP. The toxicity of mEDN for Staphylococcus aureus was examined in Fig. 4, Panel
E. We have shown previously that micromolar concentrations of both
granule-derived and recombinant human ECP were toxic to S. aureus (strain 502A); in contrast, neither granule-derived nor
recombinant human EDN had any measurable antibacterial
activity(4) . The results presented here with purified
recombinant ECP and EDN replicate these findings. The identical
experiments were performed with purified recombinant mEDN (0.3-1
µM); no antibacterial activity was observed.
Figure 5:
Lineweaver-Burk plots (1/v versus 1/[S]) derived from initial rates of reactions
containing A, 0.3 pmol of recombinant EDN and B, 3.6
pmol of recombinant mEDN isolated by M2 affinity chromotography with
varying concentrations of a yeast tRNA substrate (0.89-7.1
µM). Values for K
When expressed with an amino-terminal secretion piece,
recombinant EDN could be isolated in biologically active form directly
from the bacterial periplasm. A similar approach was used previously to
prepare recombinant ECP(4) . Newton et al. (20) reported purification and resolubilization of recombinant
EDN from bacterial inclusion bodies; the approach presented here
results in the production of biologically active protein requiring no
chemical refolding. As was found to be the case for recombinant
ECP(4) , the carboxyl-terminal FLAG peptide (DYKDDDK) aided in
the detection of small amounts of protein and did not interfere with
its transport, folding, or function. We showed that Lys Using this expression system, we have prepared recombinant protein
from a representative ``ancestral'' version of EDN. As shown
in Fig. 1(see also (14) ), the ECP/EDN gene pair
originated from a gene duplication event that occurred after the
divergence of the New World from the Old World monkeys. Although the
single nucleotide sequence isolated from the marmoset (New World
monkey) was found to be equally homologous to both human ECP and EDN
(82%), the encoded amino acid sequence suggested a closer relationship
with the latter protein. The immunoreactivity of the recombinant
protein (mEDN) echoes this observation; while mEDN was detected readily
by anti-EDN antiserum, it was only marginally detectable with anti-ECP.
As such, we anticipated the functionality of mEDN to be more in line
with that of EDN than of ECP. As predicted, mEDN was similar to EDN
in lacking antistaphylococcal activity at low micromolar
concentrations. In an antibacterial assay initially described by Lehrer et al.(24) , micromolar concentrations of
granule-derived ECP were shown to be toxic to stationary phase cultures
of staphylococci. Rosenberg (4) extended these findings,
showing that granule-derived EDN was without effect at the same and at
higher concentrations, and replicated the results with recombinant
proteins. Although the mechanism by which ECP exerts its antibacterial
(and other) toxicity has not been clarified, it has been proposed that
its cationic residues might disrupt membrane phospholipid bilayers via
a mechanism similar to that proposed for the cationic bee venom toxin,
mellitin(10, 25, 26) ; similarly, Young et al. (27) have provided evidence suggesting pore
formation. The benign natures of mEDN and EDN, both proteins with more
neutral physiologic charges (pIs = 8.3 and 8.9, respectively)
are consistent with these hypotheses. From an evolutionary perspective,
these results, along with the structural data, suggest that the
duplication and subsequent mutational events occurred under pressure to
create a more cationic, more toxic protein, such as ECP. With this
in mind, we were quite surprised to find that mEDN was significantly
less effective than EDN as a generalized ribonuclease. With yeast tRNA
as substrate, the Michaelis constants (K Thus, not only did the
duplication and mutational events generating the EDN/ECP gene family
yield a protein that was more cationic, and with greater antibacterial
activity, they also yielded a protein with enhanced ribonuclease
activity; evolutionary constraints appear to have favored the
generation of not one, but two novel functions. Of the two, the latter
function, enhanced ribonuclease activity, is more difficult to
interpret. The physiologic function of EDN is not known; the role of
ribonuclease activity is equally obscure. It is interesting to note,
however, that the neurotoxicity of EDN (the induction of cerebellar
dysfunction and Purkinje cell loss by introduction of EDN into the
cerebrospinal fluid of rabbits, also known as the Gordon phenomenon) (7, 8) was shown to be blocked by ribonuclease
inhibitors(30, 31) . Although itself a nonphysiologic
phenomenon, the observed dependence on ribonuclease activity suggests
that a more careful analysis of the actions of EDN in the central
nervous system may provide clues toward identifying the true
physiologic function of this protein.
Volume 270,
Number 37,
Issue of September 15, pp. 21539-21544, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
EVOLUTION OF NOVEL FUNCTION IN A PRIMATE RIBONUCLEASE GENE FAMILY (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(mEDN)
= 0.67 µM; K
(EDN)
= 0.70 µM), while turnover numbers differed by a
factor of 100 (k
(mEDN) = 0.91
s
; k
(EDN) = 0.64
10 s
). Thus, evolutionary
constraints appear to have promoted two novel functions: increased
cationicity/toxicity (ECP) and enhanced ribonuclease activity (EDN).
The latter result is particularly intriguing, as it suggests a crucial
role for ribonuclease activity in the (as yet to be determined)
physiologic function of EDN.
)and eosinophil
cationic protein (ECP) are small (15-16 kDa) cationic proteins
found in the large specific granules of human eosinophilic
leukocytes(1, 2) . ECP has been characterized as a
cytotoxin, helminthotoxin, and bacterial toxin with ribonuclease
activity that appears to be unrelated to
toxicity(3, 4) . In contrast, EDN has ribonuclease (5, 6) and neurotoxic
activity(7, 8) , but no known physiologic function.
The genes encoding EDN and ECP are striking in their similarity to one
another. The two coding sequences are 85% identical, encoding
polypeptides with structural and catalytic residues that are analogous
to those of other members of the mammalian ribonuclease gene family (9, 10, 11, 12) . The genomic
organization of EDN is likewise identical to that of ECP, and both
genes have been mapped to indistinguishable loci on chromosome 14
(14q24q31)(13) .
Preparation of Plasmid Constructs
The portion of
the open reading frame encoding the mature EDN polypeptide (base pairs
127-528) (9) was PCR-amplified from cDNA and ligated in
frame into the HindIII and EcoRI sites of the pFCTS
bacterial expression vector (International Biotechnologies, Inc., New
Haven, CT) to create hEDNS#1 (Fig. 2, panel A).
Features of this vector include a tac promoter, amino-terminal
bacterial secretion piece, and carboxyl-terminal FLAG polypeptide
(DYKDDDK) detected by the M2 murine monoclonal antibody (IBI). The open
reading frame encoding the mature ECP polypeptide (base pairs
136-534) (10) was amplified and subcloned as described
above to create hECPS#12. The complete coding sequence of marmoset EDN
was isolated directly from genomic DNA purified from an Epstein-Barr
virus-transformed leukocyte cell line from the marmoset S. oedipus (ATCC no. CRL-1612, Rockville, MD) using primers as described
elsewhere (14) (sequence identical to the marmoset isolate
described in Rosenberg et al.(14) ; GenBank accession no. U24099); the original isolate was reamplified with
compatible primers to facilitate insertion into the pFCTS vector,
creating mEDNS#7. The mutant EDN construct encodes a single base pair
mutation (A
G) introduced by overlapping PCR
mutagenesis(15) ; this results in the conversion of
nucleophilic lysine (Lys
) to arginine (Arg)
(hEDNS
LYS#6). All four constructs were confirmed by dideoxy DNA
sequencing (U. S. Biochemical Corp., Cleveland, OH).
SDS-PAGE and Western Blotting
The samples to be
described were subjected to gel electrophoresis in 14% Tris glycine
gels (Novel Experimental Technologies, San Diego, CA). Proteins were
transferred to nitrocellulose membranes (Schleicher & Schuell) and
probed with antibodies as per published procedures(16) .
Briefly, nonspecific binding was blocked with 5% non-fat dry milk
(Safeway, Oakland, CA) in T-TBS (50 mM Tris, pH 8.0, 150
mM sodium chloride, 0.05% Tween 20). The M2 anti-FLAG
monoclonal antibody was used at a 1:200 dilution (14.5 µg/ml) in
T-TBS with 1% gelatin. Polyclonal anti-EDN and anti-ECP antisera
(preadsorbed to eliminate cross-reactivity) (17) were used at
1:300 dilutions in T-TBS with gelatin. Secondary antibodies included
1:1000 dilutions of alkaline phosphatase-conjugated goat anti-mouse and
goat anti-rabbit IgGs (Bio-Rad). Blots were developed in TM buffer (200
mM Tris, pH 9.5, with 10 mM MgSO
) with
300 µg/ml nitro blue tetrazolium and 100 µg/ml
50-bromo-4-chloro-3-indolyl-phosphate (Bio-Rad).Protein Preparations
One-ml overnight cultures of
bacterial transfectants grown in LB broth with 50-100 µg/ml
ampicillin were used to inoculate into 500-1500 ml of Super Broth
(Biofluids, Inc., Rockville, MD) with 50-100 µg/ml ampicillin
which were then grown overnight at 37 °C. Overnight cultures were
diluted 1:1 with fresh Super Broth + ampicillin, and permitted to
grow for an additional 30 min. Isopropyl-1-thio-
-galactopyranoside
(IPTG) (Boehringer Mannheim) was added to a final concentration of
0.5-1.0 mM, and bacteria were harvested by
centrifugation after a 30 min (ECP) or 2 h (EDN,
EDNLys, and mEDN) induction period. SDS-PAGE and
Western blot analysis of proteins were performed on bacteria harvested
from 0.5-ml cultures resuspended directly in 2
reducing sample
buffer(16) .Periplasmic Isolates
The bacteria harvested as
described above were washed twice with 10 mM Tris, pH 8.0, at
room temperature, and once with 0.5 M sucrose in 30 mM Tris, pH 8.0 and 1 mM EDTA. The sucrose-washed pellet was
resuspended in ice-cold distilled water (20 ml/liter bacteria), and the
cellular debris removed by high speed centrifugation. Tris, pH 8.0, and
sodium azide were added to the supernatants to final concentrations of
10 mM and 0.1%, respectively. Ten mg of heparin-Sepharose
CL-6B (Pharmacia Biotech, Inc.) were added to the buffered isolate,
followed by equilibration end-over-end overnight at 4 °C. The
equilibrated resin was washed with 300 volumes of 10 mM Tris,
pH 8.0, and the bound proteins were eluted either directly into 2
reducing sample buffer for SDS-PAGE analysis, or into 0.3 ml of
10 mM sodium phosphate, pH 7.5, with 500 mM sodium
chloride for ribonuclease assay. The concentration of periplasmic
proteins eluted into the phosphate buffer was determined by BCA protein
assay (Pierce, Rockford, IL) with spectrophotometric comparison (562
nm) to bovine serum albumin standards. Quantitation was confirmed by
SDS-PAGE followed by Coomassie Blue staining. Quantitation of
recombinant protein within an individual isolate was performed by
comparison of serial dilutions of the periplasmic isolates to serial
dilutions of a FLAG-containing protein standard of known molarity
(recombinant FLAG-conjugated bacterial alkaline phosphatase) (IBI) on
Western blots probed with the M2 anti-FLAG mAb. Control isolates
(containing no recombinant protein) were prepared from IPTG-induced
cultures of bacteria transfected with the pFCTS vector alone.Isolation of Recombinant Protein on M2 Anti-FLAG Resin
and Amino-terminal Sequencing
Phosphate-buffered saline and
sodium azide were added to the supernatant recovered after resuspension
in ice-cold distilled water and centrifugation as described above, to
final concentrations of 1 and 0.1%, respectively. M2
anti-FLAG-conjugated resin suspension (0.3 ml; IBI) was added to the
supernatant, which was equilibrated end-over-end overnight at 4 °C.
Resin was washed in 300 volumes of phosphate-buffered saline, and bound
protein was eluted directly into 0.4 ml of 100 mM glycine, pH
3.0, and neutralized immediately with 50 µl of 2 M Tris,
pH 7.5. For functional assays (ribonuclease, toxicity), the
concentration of recombinant protein was determined by comparison to
serial dilutions of a known concentration of FLAG-conjugated standard
as described above; for amino-terminal sequencing, the sample was
concentrated, subjected to SDS-PAGE, and transferred to an Immobilon P
membrane as per the manufacturer's instructions (Millipore
Corporation, Bedford, MA). The 15.5-kDa band located by Coomassie
Blue staining was cut from the membrane for determination of the
amino-terminal sequence (performed at National Biological Resources
Branch, NIAID, National Institutes of Health).
Ribonuclease Activity
The assay used was adapted
from the procedure described by Slifman et al. (5) as
described previously(4) . Determination of ribonuclease
activities of recombinant proteins in periplasmic isolates (Fig. 3) proceeded as follows: 5 µl (20 µg) of a 4 mg/ml
solution of yeast tRNA (catalog no. R-9001; Sigma) was added to 0.8 ml
of 40 mM sodium phosphate, pH 7.0, containing 500 ng of
periplasmic proteins eluted from heparin-Sepharose as described. At the
given time points, the reaction was stopped by addition of 0.5 ml of an
ice-cold fresh solution of 20 mM lanthanum nitrate with 3%
perchloric acid. The t = 0 control was prepared by
addition of stop solution to the phosphate and protein-containing
reaction mixture prior to the addition of yeast tRNA. Stopped reactions
were held on ice for 15 min, and insoluble tRNA was removed by
centrifugation for 5-10 min at 12,000 g. The
amount of solubilized RNA was determined from the ultraviolet
absorbance at 260 nm (A) of the supernatant
fraction, with the t = 0 control used as the A
= 0.00 standard (blank). Calculations
included the following approximations: the average molecular weight (M
) of tRNA as M 28,100
(75-90 ribonucleotides/tRNA molecule M 341/ribonucleotide), with A of 1.0
corresponding to 40 µg of RNA (18) .
G) converting
Lys
Arg (before and after removal of the secretion
piece, respectively). C, ribonuclease activity of 500 ng of
periplasmic proteins containing recombinant EDN (open
circles), containing mutant EDN
K (filled
circles), and without recombinant protein (open squares).
Initial rates (OD/min) are shown in the inset. D, ribonuclease
activity of recombinant EDN preparation shown in A in the
presence of 0.5 unit (0.0625 unit/ml; open circles) and 5
units (0.625 unit/ml; filled circles) of human placental
ribonuclease inhibitor (RNasin). Initial rates (OD/min) are shown in the inset. Each time point
represents the average of duplicate
samples.
(M)) and turnover numbers (k (s
)) were determined from the appropriate
intercepts of double reciprocal (Lineweaver-Burk) plots as shown.
Production and Secretion of Recombinant Human
EDN
The coding sequence of the mature protein (without signal
sequence) in the pFCTS bacterial expression vector is shown in Fig. 2, panel A, inserted in-frame with the
vector-encoded amino-terminal OmpA bacterial secretion piece and
carboxyl-terminal octapeptide (FLAG) recognized by the M2 murine mAb.
In Panel C, a Western blot probed with the anti-FLAG M2 mAb
demonstrates the absence of immunoreactive protein in total bacterial
extracts prepared before the addition of IPTG (lane 1), and
its presence 30 min after the addition of IPTG (lane 2). The
predominant immunoreactive band in lane 2 migrates with a
molecular mass 18 kDa, which is consistent with the size of EDN
(15.5 kDa) with an intact secretion piece (2.5 kDa). Immunoreactive
protein was also detected in the periplasmic protein isolate (prepared
by osmotic shock followed by binding to heparin-Sepharose at pH 8.0, as
described under ``Experimental Procedures'') shown in lane 3. Immunoreactive protein detected in the periplasm
migrated with a molecular mass of
15.5 kDa, suggesting cleavage of
the 2.5-kDa bacterial secretion piece in conjunction with translocation
of the recombinant protein to the periplasm. The 15.5-kDa protein was
also detected by polyclonal anti-EDN antiserum (lane 4);
amino-terminal sequencing confirmed the identity of this protein as
recombinant EDN.
Ribonuclease Activity of Recombinant EDN
The
Western blot probed with the M2 mAb (Fig. 3, Panel B)
demonstrates that the production and secretion of the Lys
Arg mutant form of EDN (EDN
Lys, lanes 3 and 4) proceeds in a manner identical to that
of the wild type (lanes 1 and 2). The data in Panel C indicate that periplasmic isolate (500 ng) containing
recombinant EDN has significant ribonuclease activity (100-fold) over
the control (500 ng of periplasmic proteins isolated from an
IPTG-induced vector-alone transfected culture). The conversion of the
conserved Lys
Arg reduces the ribonuclease activity
of recombinant EDN to control level, indicating that this residue is
crucial for ribonuclease activity. The analogous lysine, Lys
is the active-site nucleophile in RNA hydrolysis catalyzed by the
prototype of this gene family, bovine ribonuclease A(19) .
Characterization of mEDN
In Fig. 4, Panel A, the amino acid sequence encoded by the EDN/ECP
homolog of S. oedipus (marmoset) is compared to the sequences
of human EDN and ECP. Although the nucleotide sequence of the marmoset
gene is equally homologous (82.3%) to both human genes, the homologies
of the encoded amino acid sequence (73.2% to EDN, 68.7% to ECP) and the
calculated isoelectric point (pI = 8.3) suggest greater
similarity to human EDN. As such, this EDN/ECP homolog has been
designated marmoset EDN (mEDN).
, Lys
,
His
His
); deeply shaded boxes enclose the conserved cysteines. Open rectangles denote
residues shared by EDN and mEDN but not by ECP and residues shared by
ECP and mEDN but not by EDN; the open squares enclose those
residues unique to mEDN. The percentage similarity of mEDN to both EDN
and ECP, as well as the calculated isoelectric point of each sequence
are listed in the final columns. B, Western blot containing
total cell extracts of IPTG-induced bacterial transfectants probed with
M2 mAb. Lane 1, recombinant ECP; lane 2, recombinant
EDN; lane 3, recombinant mEDN. C and D are
identical blots probed with polyclonal anti-EDN and anti-ECP antisera,
respectively. E, percentage of colony-forming units of S.
aureus surviving after 4 h incubation at 37 °C with
recombinant EDN (open circles), recombinant mEDN (filled
circles), and recombinant ECP (open squares). Each point
represents the average of triplicate samples; error bars as
indicated.
Comparative Ribonuclease Activity
Ribonuclease
activities of EDN and mEDN were determined by evaluating the generation
of acid-soluble ribonucleotide per unit time from varying
concentrations (0.89-7.1 µM) of yeast tRNA
substrate. The Lineweaver-Burk double reciprocal plots (1/v versus 1/[S]) derived from initial rates of these reactions is
shown in Fig. 5; the Michaelis constants (K), turnover numbers (k),
and specificity constants (k
/K
) calculated from these
data are shown in the insets. As anticipated from the observed
conservation of both structural and catalytic residues, mEDN has
measurable ribonuclease activity. Interestingly, the Michaelis
constants (K) for these two ribonucleases are
nearly identical (0.70 µM for EDN; 0.67 µM for mEDN). In contrast, the turnover numbers (k), and therefore, the specificity constants (k
/K
), differ dramatically,
with EDN (k/K
= 1.3
10
M s
) nearly 100-fold more effective at
catalyzing the hydrolysis of yeast tRNA than the marmoset homolog, mEDN (k
/K
= 0.9
10M s
).
(µM), k
(s
), and K
/k
(M
s
) for each
enzyme are listed in the insets.
of recombinant EDN was functionally as well as structurally
homologous to the active site nucleophile (Lys
) of bovine
RNase A, the prototype of the mammalian ribonuclease gene family. The
conversion of Lys
Arg eliminated the ribonuclease
activity of recombinant EDN; similar results were obtained previously
with a Lys
Arg mutant of recombinant
ECP(4) . We also determined that the activity of recombinant
EDN is reduced in a dose-dependent fashion in the presence of human
placental ribonuclease inhibitor, analogous to results obtained with
RNase A (19) as well as with other members of the mammalian
ribonuclease gene family(21, 22, 23) .
) of the
two proteins were about equal. The turnover numbers (k), however, differed by a factor of 100,
indicating that, mEDN is 100-fold less effective than EDN at converting
a molecule of substrate present in the active site into product. From a
structural point of view, it is not immediately clear why this should
be the case, as both mEDN and EDN have the eight cysteine residues
spaced appropriately for the formation of the four characteristic
disulfide bonds, as well as the histidines and lysine analogous to
those in the active site crevice of ribonuclease A(19) ;
neither is mEDN excessively cationic, a feature which has been proposed
as potentially damaging to the catalytic activity of ECP(10) .
A stepwise comparison of ribonuclease activity along evolutionarily
informative pathways (28) is likely to provide information on
the way in which this increase in observed ribonuclease activity was
attained (gradually or at a result of a single transition), and will
identify additional residue(s) crucial to this aspect of EDN's
function. Interestingly, a similar study, focusing on pancreatic
ribonucleases from the order Artiodactyla (cows, sheep, camels) was
recently described by Jermann et al.(29) ; in this
study, the conversion of a single residue (Asp
Gly)
that occurred in conjuction with the evolutionary emergence of the
``true ruminants'' resulted in a 5-fold enhancement of the
hydrolysis of double-stranded RNA.
)-galactopyranoside; mEDN, marmoset EDN; PCR,
polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis;
mAb, monoclonal antibody.
We thank Dr. John Coligan, NBRB, NIAID, for the
amino-terminal sequence data reported in this work, and Dr. Steven
Ackerman for providing the polyclonal antisera. We also thank Dr. John
I. Gallin for his continuing support of our work.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 Y.-C. Huang, Y.-M. Lin, T.-W. Chang, S.-J. Wu, Y.-S. Lee, M. D.-T. Chang, C. Chen, S.-H. Wu, and Y.-D. Liao The Flexible and Clustered Lysine Residues of Human Ribonuclease 7 Are Critical for Membrane Permeability and Antimicrobial Activity J. Biol. Chem., February 16, 2007; 282(7): 4626 - 4633. [Abstract] [Full Text] [PDF]
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 J. Zhang Disulfide-Bond Reshuffling in the Evolution of an Ape Placental Ribonuclease Mol. Biol. Evol., February 1, 2007; 24(2): 505 - 512. [Abstract] [Full Text] [PDF]
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T. Nitto, K. D. Dyer, M. Czapiga, and H. F. Rosenberg Evolution and Function of Leukocyte RNase A Ribonucleases of the Avian Species, Gallus gallus J. Biol. Chem., September 1, 2006; 281(35): 25622 - 25634. [Abstract] [Full Text] [PDF]
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 I. Dupanloup and H. Kaessmann Evolutionary simulations to detect functional lineage-specific genes Bioinformatics, August 1, 2006; 22(15): 1815 - 1822. [Abstract] [Full Text] [PDF]
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J. Zhang Parallel Functional Changes in the Digestive RNases of Ruminants and Colobines by Divergent Amino Acid Substitutions Mol. Biol. Evol., August 1, 2003; 20(8): 1310 - 1317. [Abstract] [Full Text] [PDF]
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 L. O'Bryan, P. Pinkston, V. Kumaraswami, V. Vijayan, G. Yenokida, H. F. Rosenberg, R. Crystal, E. A. Ottesen, and T. B. Nutman Localized Eosinophil Degranulation Mediates Disease in Tropical Pulmonary Eosinophilia Infect. Immun., March 1, 2003; 71(3): 1337 - 1342. [Abstract] [Full Text] [PDF]
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 J. Zhang, K. D. Dyer, and H. F. Rosenberg Human RNase 7: a new cationic ribonuclease of the RNase A superfamily Nucleic Acids Res., January 15, 2003; 31(2): 602 - 607. [Abstract] [Full Text] [PDF]
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 J. Zhang and H. F. Rosenberg From the Cover: Complementary advantageous substitutions in the evolution of an antiviral RNase of higher primates PNAS, April 16, 2002; 99(8): 5486 - 5491. [Abstract] [Full Text] [PDF]
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J. Zhang, K. D. Dyer, and H. F. Rosenberg RNase 8, a novel RNase A superfamily ribonuclease expressed uniquely in placenta Nucleic Acids Res., March 1, 2002; 30(5): 1169 - 1175. [Abstract] [Full Text] [PDF]
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 J. Zhang and H. F. Rosenberg Sequence Variation at Two Eosinophil-Associated Ribonuclease Loci in Humans Genetics, December 1, 2000; 156(4): 1949 - 1958. [Abstract] [Full Text]
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 A. M. HARRISON, C. A. BONVILLE, H. F. ROSENBERG, and J. B. DOMACHOWSKE Respiratory Syncytical Virus-induced Chemokine Expression in the Lower Airways . Eosinophil Recruitment and Degranulation Am. J. Respir. Crit. Care Med., June 1, 1999; 159(6): 1918 - 1924. [Abstract] [Full Text]
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 M. S. Deming, K. D. Dyer, A. T. Bankier, M. B. Piper, P. H. Dear, and H. F. Rosenberg Ribonuclease k6: Chromosomal Mapping and Divergent Rates of Evolution within the RNase A Gene Superfamily Genome Res., June 1, 1998; 8(6): 599 - 607. [Abstract] [Full Text]
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J. Zhang, H. F. Rosenberg, and M. Nei Positive Darwinian selection after gene duplication in primate ribonuclease genes PNAS, March 31, 1998; 95(7): 3708 - 3713. [Abstract] [Full Text] [PDF]
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J. Zhang and H. F. Rosenberg From the Cover: Complementary advantageous substitutions in the evolution of an antiviral RNase of higher primates PNAS, April 16, 2002; 99(8): 5486 - 5491. [Abstract] [Full Text] [PDF]
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J. Zhang, K. D. Dyer, and H. F. Rosenberg Evolution of the rodent eosinophil-associated RNase gene family by rapid gene sorting and positive selection PNAS, April 25, 2000; 97(9): 4701 - 4706. [Abstract] [Full Text] [PDF]
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