(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 (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.
Eosinophil-derived neurotoxin (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) .
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
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.
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 (M)) and turnover numbers (k
(s
)) were determined from the appropriate
intercepts of double reciprocal (Lineweaver-Burk) plots as shown.
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, 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.
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(µM), k
(s
), and K
/k
(M
s
) for each
enzyme are listed in the insets.
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 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) .
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) 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.
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.