ABSTRACT
The discovery of Ribonuclease k6 (RNase k6) was an unexpected result of our
ongoing efforts to trace the evolutionary history of the ribonuclease gene
family. The open reading frame of RNase k6, amplified from human genomic DNA,
encodes a 150 amino acid polypeptide with eight cysteines and histidine and
lysine residues corresponding to those found in the active site of the
prototype, ribonuclease A. The single-copy gene encoding RNase k6 maps to human chromosome 14 and orthologous
sequences were detected in both primate and non-primate mammalian species. A single mRNA transcript (1.5 kb) was detected
in all human tissues tested, with lung representing the most abundant source.
At the cellular level, transcripts encoding RNase k6 were detected in normal
human monocytes and neutrophils (but not in eosinophils) suggesting a role for
this ribonuclease in host defense. Of the five previously identified human
ribonucleases of this group, RNase k6 is most closely related to eosinophil-derived neurotoxin (EDN), with 47% amino acid sequence identity; slight
cross-reactivity between RNase k6 and EDN was observed on Western blots probed
with polyclonal anti-EDN antiserum. The catalytic constants determined,
K
m = 5.0
[mu]
M and
k
cat = 0.13 s
-1
, indicate that recombinant RNase k6 has
~40-fold less ribonuclease activity than recombinant EDN. The identification
and characterization of RNase k6 has extended the ribonuclease gene family and
suggests the possibility that there are others awaiting discovery.
The ribonuclease family is composed of proteins with similar primary structure
and enzymatic activity that have diverged to support other, seemingly unrelated
physiologic activities. The prototype of this family is ribonuclease A [bovine
pancreatic ribonuclease (
1
,
2
)]. A human pancreatic ribonuclease with similar sequence has been identified (
3
-
5
) and pancreatic ribonucleases have been isolated from an extensive array of
other mammalian species (
6
,
7
). Two related ribonucleases are the eosinophil-derived neurotoxin (EDN), also known as Rnase Us (
8
) and the eosinophil cationic protein (ECP) (reviewed in
9
,
10
). ECP has been characterized as a cytotoxin, a neurotoxin and as an anti-parasitic and anti-bacterial agent; EDN, despite its sequence homology to ECP (67%) and
100-fold greater ribonuclease activity, shares only ECP's neurotoxicity.
Angiogenin, a structurally atypical member of this gene family, is a tRNA-specific ribonuclease (
11
) that binds to actin on the surface of endothelial cells (
12
); bound angiogenin is endocytosed and translocated to the nucleus, thereby
promoting the endothelial invasiveness necessary for blood vessel formation (
13
). Ribonuclease 4 (RNase 4) also has a unique substrate preference (
14
), although no specific physiologic functions of this protein have been
identified. Others included in this family are bovine brain (
15
,
16
) and bovine seminal ribonucleases (
17
) and several ribonucleases isolated from species of the frog genus,
Rana
(
18
-
20
); no orthologous human sequences corresponding to these bovine or frog
ribonucleases have been identified.
In our previous work, we traced the evolutionary history of the eosinophil
ribonucleases, ECP and EDN (
21
). We found that the EDN/ECP gene pair arose from a gene duplication event that
occurred relatively recently, sometime after the divergence of the Old World
from the New World monkeys and that the genes encoding EDN and ECP are the most
rapidly evolving functional coding sequences known among primate species. We
have since determined that the unusual evolutionary constraints on these two
proteins have promoted both enhanced toxicity (ECP) and increased ribonuclease
activity (EDN) when the properties of these two proteins are compared with
those of a representative single-sequence predecessor (
22
).
In hopes of tracing the molecular evolution of the single ECP/EDN gene through
the non-primate mammalian Orders, we focussed on bovine kidney ribonuclease k2
(RNase k2), a protein originally isolated and described by Irie
et al.
(
23
). Specifically, we were interested in determining whether bovine RNase k2,
whose amino acid sequence was more closely related to human EDN than it was to
any of the other human ribonucleases, represented a direct evolutionary
predecessor of the EDN/ECP genes, or instead represented the bovine ortholog of
a unique human gene that had yet to be discovered. We found the latter to be
the case. In this work, we have isolated unique genomic clones encoding both
bovine Rnase k2 and a novel human ribonuclease, which we have named
Ribonuclease k6 (RNase k6). We have provided a molecular characterization of
RNase k6 and have examined the immunoreactivity and ribonucleolytic activity of
the recombinant protein. Together, bovine RNase k2 and human RNase k6 represent
a heretofore unrecognized arm of the apparently enlarging ribonuclease gene
family and are likely to possess unique physiologic functions.
Bovine genomic DNA was isolated from cells of the MDBK cell line (ATCC-CCL22). Degenerate oligonucleotide primers were designed from amino acids
10 to 15 (5' to 3' primer) and 98 to 102 (3' to 5' primer) of the published amino acid sequence of
bovine RNase k2 (
23
), with sequences as follows: 5' to 3' primer: 5'-TGG TT(CT) GA(AG) AT(ACT) CA(AG) CA-3 and 3' to 5' primer: 5'-AT(GA) AA(GA) AA(TC)
TT(GA) TA(TC) TG-3'. PCR reactions proceeded in a 100 [mu]l volume with 10 [mu]M of each primer, 1 [mu]g template, 0.2 mM dNTPs and 2.5 U
Taq
polymerase and buffer (Boehringer Mannheim, Indianapolis, IN) in a 9600
thermocycler (Perkin-Elmer, Norwalk, CT) with the following parameters: 95oC for 2 min, followed by 35 cycles of 95oC for 30 s, 50oC for 30 s and 72oC for 30 s and completed with 72oC for 5 min. The product was identified by its
mobility on a 6% TBE-acrylamide gel; the product remaining was then subjected to TBE-agarose gel electrophoresis and the band was excised, purified and
ligated into a TA cloning vector (PCRII, Invitrogen, San Diego, CA) as
described previously (
24
). The fragment was identified as encoding bovine RNase k2 by dideoxy sequencing
(US Biochemical, Cleveland, OH). All sequence analyses (including homology and
isoelectric point determinations) were performed with the assistance of the
Wisconsin Genetics Computer Group software on-line at the National Institutes of Health.
The degenerate oligonucleotides described above were used to isolate a human
genomic fragment by polymerase chain reaction as described above using purified
human genomic DNA as a template. An ~300 bp fragment was isolated and identified as encoding a novel
ribonuclease by dideoxy-sequencing. The complete ORF of this intronless gene was obtained by
extension in both the 5' and 3' directions with uni-directional PCR method with reagents included in the
Promoter Finder kit (Clontech, Palo Alto, CA). The nested gene-specific primer pair designed to amplify genomic sequence located 5' of the original isolate were as follows: 1a: (nt 364-338) 5'-ACT GGG CAG CAG CAC TAT AGC GGC ACT-3'; 2a: (nt 333-303) 5'-ATA CTT TCC
TGA AGT GAG TCT GCA GTC-3'. The nested gene-specific primers for amplification of sequence 3' of the original isolate were as follows: 1b: (nt 291-317) 5'-GCC TGT CAA CAT GAC TGA CTG CAG
ACT-3'; 2b: (nt 325-351) 5'-GGA AAG TAT CCC CAG TGC CGC TAT AGT-3'; 1c: (nt 301-336) 5'-ATG ACT GAC
TGC AGA CTC ACT TCA GGA AAG TAT CCC-3'; 2c: (nt 322-360) 5'-TCA GGA AAG TAT CCC CAG TGC CGC TAT AGT GCT GCT
GCC-3'. The first round of amplification in a single direction included
proceeded in a 25 [mu]l reaction volume with 1* Tth buffer, 1.1 mM magnesium acetate, 0.2 mM dNTPs, 0.2 [mu]M adaptor primer 1, 0.2 [mu]M gene-specific primer 1 (a, b or c) and 0.5 [mu]l Advantage Tth polymerase mix. The reaction
parameters included 7 cycles at 94oC for 2 s followed by 68oC for 3 min, then 37 cycles at 94oC for 2 s and 63oC for 3 min, with a completion step of 67oC for 4 min. One [mu]l of each primary amplification was reamplified in
a 25 [mu]l volume including buffer, magnesium acetate, dNTPs as described and
including 0.2 [mu]M adaptor primer 2, 0.2 [mu]M gene-specific primer 2 (a, b or c) and 0.5 [mu]l Advantage Tth polymerase mix, with parameters including 5
cycles of 94oC for 2 s and 68oC for 3 min, followed by 25 cycles at 94oC for 2 s and 63oC for 3 min, with the completion step as described above.
Amplification products obtained were isolated, subcloned and sequenced (see
Results). As a control for potential sequence mutations, the complete coding
sequence was then re-amplified directly from another source of human genomic DNA using flanking
primers; the sequence obtained from this isolate was identical to the original,
suggesting the absence of PCR-induced mutations.
Nitrocellulose membranes with 20 [mu]g restriction digested human or bovine genomic DNA were prepared,
crosslinked and hybridized as described previously (
24
).
Chromosomal localization was performed using a PCR based method as described
(BIOS DNA, New Haven, CT) (
24
,
25
). Briefly, two oligonucleotide primers were tested in order to confirm that
they amplified a specific fragment from only human (and not hamster) genomic
DNA templates. The oligonucleotide primers selected (Fig.
2
A, primers x and y, numbering as per Fig.
2
B) 5' to 3': (nt 121-136) 5'-CCA AGT CCT CTC CAA T-3' and 3' to 5': (nt 360-344) 5'-GGC AGC AGC ACT ATA GC-3'
amplified a single 240 bp fragment from human genomic DNA. Amplification of DNA
isolated from the 25 characterized human:hamster somatic cell hybrids proceeded
as described previously (
24
); products of each reaction were evaluated on TBE-agarose gels stained with ethidium bromide.
A human multi-tissue Northern membrane was obtained from Clontech; the multi-leukocyte Northern membrane was a gift of Dr Philip Murphy. The
membrane was pre-hybridized and hybridized as per manufacturer's instructions; the
hybridization was performed with the radiolabelled human ribonuclease k6 probe
described above. The membrane was then washed with 2* SSPE with 0.1% SDS for 1 h at 42oC followed by 0.2* SSPE with 0.1% SDS for 1 h at 50oC; autoradiograms were developed after 24 h exposure at -80oC. The hybridization with the
oligonucleotide encoding human [beta]-actin was as previously described (
24
).
The portion of the ORF encoding the mature RNase k6 peptide (base pairs 70-450; GenBank accession no. U64998) was re-amplified from human genomic DNA and ligated in-frame into the
Hin
dIII and
Eco
RI sites of the pFCTS bacterial expression vector (International
Biotechnologies, Inc., New Haven, CT) to create hK6#81 (Fig.
4
A). The fidelity of the expression construct was confirmed by dideoxy
sequencing.
Production and isolation of recombinant RNase k6 from bacterial transfectants
was as described previously for recombinant EDN (
22
). The concentration of recombinant protein was determined by comparison to
serial dilutions of a known concentration of a FLAG-conjugated protein standard on Western blots.
Protein samples were subjected to gel electrophoresis in 14% Tris-glycine gels (Novel Experimental Technologies, San Diego, CA). Proteins
were transferred to nitrocellulose membranes and probed with antibodies as per
published procedures (
26
).
The assay used was adapted from the procedure described by Slifman
et al
. (
27
) as described previously (
22
,
28
). Calculations included the following approximations: the average molecular
weight (
M
r
) of tRNA as 28 100 (75-90 ribonucleotides/tRNA molecule *
M
r
341/ribonucleotide), with A
260
of 1.0 corresponding to 40 [mu]g of RNA (
29
). All time points represented averages of triplicate samples. Equivalent
volumes of sham isolations (M2-resin equilibration and glycine elution from equivalent volumes of pFCTS
vector alone bacterial transfectants) had levels of ribonuclease activity that were insignificant compared with that of the recombinant RNase k6 (<0.1%).
The nucleotide sequence of a 281 bp genomic fragment encoding bovine
ribonuclease k2 has been deposited in the GenBank database, accession number
U64997. The fragment was amplified by PCR using degenerate oligonucleotide
primers and bovine genomic DNA as template. The encoded amino acid sequence
matches that determined from peptide fragments from the purified RNase k2
protein described by Irie
et al.
(
23
); included in this partial sequence are seven of the eight characteristic
cysteines, the lysine and one of the histidines comprising the ribonuclease
active site (
2
) and two potential sites for asparagine-linked glycosylation.
The bovine RNase k2 fragment was used to probe a Southern blot of restriction-digested bovine genomic DNA (Fig.
1
A). Whereas single hybridizing bands were detected in the lanes containing DNA
digested with
Eco
RI,
Bam
HI and
Hin
dIII, two bands were evident in the lane containing DNA digested with
Pst
I. As the RNase k2 probe contains no internal
Pst
I sites, these results suggest the existence of an as yet unidentified sequence
similar to RNase k2 (either a novel gene or a polymorphism) within the bovine
genome.
Using the degenerate primers designed to amplify the bovine RNase k2 genomic
fragment (primers p and q, Fig.
2
A), we were successful in isolating a 281 bp fragment encoding a novel human
ribonuclease. This novel isolate shared 77% nucleotide sequence identity and
70% amino acid sequence identity with the previously isolated bovine fragment.
The 5'- and 3'-ends of the complete coding sequence were isolated
using a uni-directional PCR method with adaptor ligated `genomic libraries' and
adaptor primers supplied in the Promoter Finder kit from Clontech Laboratories,
Inc., Palo Alto, CA. The locations of the nested gene-specific primers directed to the 5' (1a and 2a) and 3' (1b and 2b, 1c and 2c) ends of the gene are shown in Figure
2
A (for sequences, see Materials and Methods). For extension in the 3' direction, a 130 bp fragment was amplified from the
Eco
RV-restricted library using nested gene-specific primers 1b and 1c, which provided ~25 bp overlap with the original isolate and an additional 60 bp
of ORF at the 3'-end. The 3'-end of the ORF was completed using primers 1c and 2c
which amplified an 800 bp fragment from the
Dra
I-restricted library. Similarly, 5'-end extension resulted in the amplification of a 500 bp
fragment from the
Sca
I-restricted library which provided an ~200 bp overlap with the original isolate and an additional 90 bp
completing the ORF at the 5'-end. The complete ORF was then re-amplified using flanking primers from a separate source of
human genomic DNA to confirm the original sequence.
The complete coding sequence has been deposited in the GenBank database,
accession number U64998. Highlights of this 14.7 kDa protein (calculated
molecular weight), which we have named RNase k6 (see Discussion), include eight
characteristic cysteines, the ribonuclease active site residues [histidines and
a lysine residue within a conserved sequence motif (
30
)], two potential sites for asparagine-linked glycosylation and a 23 amino acid hydrophobic leader sequence.
Overall, the amino acid sequence of human RNase k6 is 72% identical to the
complete amino acid sequence of its bovine RNase k2 ortholog (
23
). Interestingly, the calculated isoelectric point (pI) of human RNase k6 (pI =
9.49) is significantly higher than that of the bovine protein (pI = 7.65); a
similar increase in pI was noted for human ECP (pI = 11.4) and its evolutionary
predecessor from the New World monkey, mEDN (pI = 8.25) (
21
).
The human RNase k6 gene fragment was used to probe a Southern blot of
restriction-digested human DNA (Fig.
2
B). Single hybridizing bands were detected in the lanes containing DNA digested
with
Eco
RI,
Bam
HI and
Hin
dIII and two bands were detected in the lane containing DNA digested with
Pst
I, as anticipated from the internal
Pst
I site shown in the map in Figure
2
A. In contrast to our findings with the bovine RNase k2 probe, we were unable to
detect any additional, related sequences with these restriction digestions.
As shown in Figure
2
C, we detected orthologs of RNase k6 in a wide variety of primate species,
including chimpanzee (
P
.
troglodytes
), gorilla (
G
.
gorilla
), orangutan (
P
.
pygmaeus
), Old World monkey (macaque,
M
.
fascicularis
) and New World monkey (marmoset,
S
.
oedipus
). Orthologous sequences were also detected in non-primate mammals, including (in addition to the bovine sequence) pig (
S
.
scrofula
) and cat (
F
.
domesticus
).
In Figure
1
D, we present an alignment of the amino acid sequences of human RNase k6 and the
five previously identified human members of the ribonuclease gene family, which
include pancreatic ribonuclease, EDN, ECP, RNase 4 and angiogenin.
Interestingly, the amino acid sequence homologies suggest that RNase k6 is most
closely related to the eosinophil ribonucleases, EDN and ECP.
The chromosomal mapping of RNase k6 was achieved using a PCR-based technique (
24
). Genomic DNAs derived from a characterized panel of human:hamster somatic cell
lines served as templates and a pair of gene-specific primers (Fig.
2
A, arrows x and y) were used to amplify a specific fragment of human RNase k6.
The anticipated 240 bp fragment was amplified from DNA templates from hybrids
numbered 423, 507, 750, 756, 867, 909 and 937 (see reference
24
), consistent with assignment to human chromosome 14.
As shown in Figure
3
A, mRNA encoding human RNase k6 was detected in all human tissues tested, with
lung representing most abundant source. A single transcript of ~1.5 kb was detected. In Figure
3
C, the 1.5 kb mRNA transcript encoding RNase k6 was detected in total RNA
isolated from normal human monocytes and neutrophils, but could not be detected
in total RNA isolated from eosinophils.
The sequence encoding the mature protein (without leader sequence) was used to create plasmid construct hK6#81 (Fig.
4
A). This construct was prepared using the prokaryotic expression vector pFCTS,
which we have used previously to prepare and isolate recombinant ribonucleases
directly from the bacterial periplasm in a biologically active state (
22
,
28
). Features of this expression system include the inducible
tac
promoter, a bacterial secretion piece and a C-terminal sequence tag (FLAG), which does not interfere with proper folding
or activity of recombinant ribonucleases (
22
,
28
). Shown in Figure
4
B are Western blots containing extracts of bacteria transfected with either
hEDNS#1 [induced to produce human EDN, (
22
)] or hK6#81 (producing human RNase k6) probed with the M2 anti-FLAG monoclonal antibody or with polyclonal anti-EDN, respectively. Slight cross-reactivity is detected between human EDN and RNase k6. In
Figure
4
C, the ribonuclease activities of these two proteins are compared. As determined
from double reciprocal plots of initial rates, the Michaelis constant (
K
m
) of RNase k6 is 5.0 [mu]M and the catalytic constant,
k
cat
is 0.13 s
-1
. A comparison to the constants determined for human EDN (
K
m
= 0.70 [mu]M,
k
cat
= 0.91 s
-1
) (
22
) indicates that RNase k6 is ~40-fold less active than EDN when tested against a standard substrate
(yeast tRNA).
We have isolated a genomic fragment encoding a previously unrecognized member of
the ribonuclease gene family, which is perhaps not the last of these
structurally related proteins to be discovered. We have named this protein
`Ribonuclease k6' (RNase k6): `6' indicates that this protein is the sixth
human ribonuclease of this family to be characterized and `k' reflects its
orthologous relationship with bovine RNase k2 (
23
). RNase k6 has features that are fairly typical for this gene family (Fig.
2
D). As a group, the members of this family maintain a set of distinctly spaced
cysteine residues which facilitate formation of specific intrachain disulfide
bonds, as well as conserved lysine and histidine residues analogous to those
originally found in the catalytic site of Ribonuclease A (
2
). All members of this family maintain some degree of ribonuclease activity,
although at different levels of efficiency and with some degree of substrate
preference. There are several amino acids other than those mentioned above that
appear to be conserved in all the human members of this family; some of these
are also conserved in the other mammalian and amphibian ribonucleases whose
sequences have been reported (
6
,
21
). Although the significance of these additional conserved residues has not been
determined, it is interesting to note that interaction between human EDN and
the chaperonin, groEL includes the conserved residue M
36
, suggesting a role for this residue in the folding and subcellular sorting of nascent
ribonucleases within the cells of origin (
31
).
There are several additional features of the gene encoding RNase k6 that are
shared with other members of the ribonuclease gene family, including the
intronless coding sequence (
24
,
32
-
34
) and localization to human chromosome 14 (
24
,
32
-
35
). The mRNA transcript encoding RNase k6 has a near ubiquitous distribution,
similar to that determined for angiogenin (
36
,
37
), human RNase 4 (
24
) and for human pancreatic ribonuclease (Handen and Rosenberg, unpublished
data). The 1.5 kb transcript is slightly larger than the mRNAs encoding EDN,
ECP or angiogenin (
36
,
38
,
39
), but much smaller and less complex than those encoding RNase 4 (
24
). As anticipated from amino acid sequence homology, we demonstrated slight
cross-reactivity between RNase k6 and EDN when both recombinant proteins were
probed with a polyclonal anti-EDN antiserum. In addition, we determined that recombinant RNase k6 was
somewhat less enzymatically active than recombinant EDN (~40 fold), but had more activity than recombinant mEDN, the representative
single-sequence predecessor of the EDN/ECP gene pair isolated from a New World
monkey (
22
).
From an evolutionary perspective, it would seem unlikely that so many distinct
and divergent versions of ribonuclease would have evolved to support the simple
enzymatic activity alone. Indeed, certain members of this family were
originally identified on the basis of their ability to support other biologic
activities. For example, angiogenin was originally identified on the basis of
its ability to promote blood vessel formation (
40
) and ECP and EDN were known to be toxins (
9
,
10
). Since its discovery, the anti-neoplastic activity of bovine seminal ribonuclease has also become appreciated (
41
-
43
). Similarly, other, related ribonucleases from the frog genus
Rana
have displayed both anti-viral and anti-neoplastic activity (
44
-
46
).
Evolutionary principles would also suggest that these proteins have maintained
ribonuclease structure and activity for some crucial reason; the relationship
between ribonuclease activity and biologic function still remains unclear.
Angiogenin is a most interesting example of this point, with many experiments
clearly documenting a direct relationship between angiogenic and ribonuclease
activities (
47
,
48
). Yet, most recent evidence suggests that angiogenin binds to an actin monomer
on the surface of endothelial cells, where it is endocytosed and then
translocated to the nucleus (
12
,
13
,
49
); Moroianu and Riordan (
13
) have established that the nuclear translocation step is crucial for
angiogenesis and that enzymatically inactive angiogenins are capable of
translocation. The relationship between these two apparently crucial phenomena-ribonuclease activity and nuclear translocation-is not immediately clear. Similarly, the tumoricidal and
immunosuppressive activities of bovine seminal ribonuclease have been shown to be directly dependent on its ribonuclease
activity (
50
) and, after internalization, bovine seminal ribonuclease appears to function by
degrading rRNA and thus inhibiting protein synthesis (
51
). It is not yet clear how one achieves specificity, so that somatic cells, including those producing this ribonuclease, are not damaged excessively in the process. The two eosinophil ribonucleases, EDN and ECP, likewise present their own series
of questions. Sorrentino
et al.
(
52
) and Newton
et al.
(
53
) presented evidence suggesting that the (non-physiologic) neurotoxic activity of EDN required active ribonuclease activity. However, both
Molina and colleagues (54) and Rosenberg (
28
) showed that the anti-parasitic and anti-bacterial activities of ECP, respectively, did not require active
ribonuclease activity. ECP is the only ribonuclease identified with an apparent
separation of enzymatic and physiologic activities; the possibility that there
are physiologic functions of ECP and EDN that have not yet been identified
remains an intriguing possibility.
Thus, RNase k6 is currently a protein with enzymatic activity in search of a
physiologic function (as are RNase 4 and EDN). The detection of mRNA encoding
this protein in both neutrophils and monocytes suggests a role for this
ribonuclease in human host defense. We anticipate that the further elucidation
of the biology of this newly discovered ribonuclease will provide more information on the range and scope of activities promoted by the enlarging ribonuclease
gene family.
The authors thank Dr Philip M. Murphy for providing the human leukocyte Northern
blot and Dr Steven Ackerman for the polyclonal anti-EDN antiserum. We would also like to thank Dr John I.Gallin for his
continued support of our work.
REFERENCES
Return