(Received for publication, October 17, 1995; and in revised form, February 14, 1996)
From the
To gain further insights into the cytokine network of birds, we
used polymerase chain reaction technology to clone a cDNA that codes
for a chicken homolog of the interferon-induced guanylate-binding
proteins (GBPs). In its N-terminal moiety, the 64-kDa chicken GBP
contains two sequence blocks of 100 and 19 amino acids, respectively,
that are about 70% identical to mammalian GBPs. The first region
includes two motifs of the canonical GTP-binding consensus element. The
other parts of chicken GBP are poorly conserved, except for a CAAX motif at the extreme C terminus which might signal isoprenylation.
Like mammalian GBPs, recombinant chicken GBP specifically bound to
agarose-immobilized guanine nucleotides and hydrolyzed GTP to both GDP
and GMP. Regulation by interferons was also conserved: chicken GBP RNA
was barely detectable in uninduced chicken cells. Low GBP RNA levels
were found in cells treated with type I interferon, whereas very high
levels were observed in cells treated with supernatant of a chicken T
cell line that secretes a -interferon-like activity. Together with
recent phylogenetic studies of interferon genes, these results suggest
that in spite of low sequence conservation, the various components of
the avian interferon system are functionally well conserved.
An important role of interferons (IFNs) ()is to
activate the virus defense system of vertebrate hosts(1) .
Phylogenetic analysis of this presumably archetypal antiviral strategy
has been hampered by the lack of information on the molecular nature of
IFNs and their intracellular effector molecules in non-mammalian
species. It was known for almost 40 years that chicken cells can be
induced to secrete IFN(2) , but the molecular analysis of this
activity proved difficult. The recent cloning of the first cDNA for a
chicken IFN (3) showed that its primary sequence is poorly
conserved. This finding raised the possibility that the avian IFN
system has evolved independently and might use fundamentally different
effector molecules(4) .
Among the IFN effector molecules of
mammals, the guanylate-binding proteins (GBPs) are of particular
interest due to their unusual biochemical properties and dramatic
up-regulation in IFN-treated cells. Unlike other proteins with
GTP-binding activity, GBPs bind to agarose-immobilized GTP, GDP, and
GMP with very high
affinity(5, 6, 7, 8) . Furthermore,
GBPs not only hydrolyze GTP to GDP as conventional GTPases do, but they
also degrade a significant fraction of this substrate to
GMP(9) . Unlike most other GTPases, GBPs carry only the first
two motifs of the tripartite GTP-binding consensus
element(10) , while the third (N/T)XKD motif is
missing(7) . All mammalian GBPs carry a CAAX isoprenylation signal motif at their C termini (7, 9) . Human GBP1 was recently shown to be
farnesylated in vitro(9) and in vivo. ()In mouse and human cells, GBPs are strongly induced in
response to IFN-
and to a lesser extent by IFN-
and IFN-
(type I IFNs)(5, 8) . In mouse cells, this induction
process is mediated by the IFN-induced transcription factor
IRF-1(11) . Mouse and human cells contain at least two
functional GBP genes(7) , but the physiological roles of their
encoded products in the IFN response remains unknown.
Here we
demonstrate that GBPs also exist in birds. Analysis of a chicken cDNA
clone showed that sequence conservation of GBPs is restricted to
regions in the N-terminal moiety that harbor the GTP-binding consensus
motifs. Nonetheless, the biochemical properties of recombinant chicken
GBP closely resembled those of mammalian GBPs. Another conserved
feature of GBPs was their regulation: chicken GBP gene was most
strongly induced by an IFN--like activity present in chicken T
cell supernatants.
Figure 1:
Primary sequence of chicken
GBP. Shown is the nucleotide sequence of a cDNA clone that codes for
chicken GBP (GenBank accession no. X92112). Its deduced
amino acid sequence is presented in the single-letter code. The two
regions with high similarity to mammalian GBPs are shaded. Two
motifs of the canonical GTP-binding consensus element (10) are
highlighted by a solid line; the putative third motif is
highlighted by a broken line. Asterisks mark a putative
isoprenylation motif.
Figure 2: Guanine nucleotide binding and GTPase activity of chicken GBP. A, purified recombinant His-ChGBP (lane 1) was allowed to react with CTP agarose (lane 3) or GTP agarose (lane 4), and bound material was analyzed by 10% SDS-PAGE and Coomassie Blue staining. Three times more His-ChGBP was used for the binding experiments than loaded in lane 1. Protein size marker (M). B, purified recombinant His-ChGBP (0.3 µg/µl) was incubated for 1 h at 37 °C in the presence of 200 µM GTP, and the reaction product was analyzed for the presence of GDP and GMP by HPLC. A control reaction lacking His-ChGBP was performed under identical conditions. The chromatograms were monitored at 252 nm. The identity of the nucleotide peaks and their retention times are indicated. The asterisks mark signals of an unknown buffer contaminant.
Since
human GBP1 was recently shown to be an unorthodox GTPase that
hydrolyzes GTP to GDP as well as to GMP(9) , we examined
whether the chicken GBP would exhibit similar biochemical activity.
Incubation of His-ChGBP with 200 µM GTP for 60 min
resulted in partial hydrolysis of this substrate to GDP and GMP (Fig. 2, panel B). The reaction product consisted of
approximately 87% GDP and 13% GMP. ATP was not hydrolyzed by purified
His-ChGBP at a detectable rate under our reaction conditions (data not
shown), indicating that the preparation was virtually free of
contaminating phosphatases. Furthermore, histidine-tagged dehydrofolate
reductase or a variant of human GBP1 with a mutation in the first
element of the GTP-binding motif ()that were both expressed
and purified by similar means failed to hydrolyze GTP at a detectable
rate (data not shown). The fact that GMP was a product of the chicken
GBP-catalyzed GTP hydrolysis reaction demonstrated that, like human
GBP1, the chicken homolog has unusual GTPase properties. However, the
two recombinant proteins differed clearly in their fidelity by which
they synthesized GMP. Human GBP1 preferentially hydrolyzed GTP to GMP (9) , whereas chicken GBP degraded GTP predominantly to GDP.
Kinetic studies showed that the specific GTPase activity of our best
preparations of His-ChGBP were about 3 nmol of GTP/min/mg, a value that
was about 10-fold lower than that determined for human
GBP1(9) .
In a first series of experiments, RNAs from the various cell lines were tested for chicken GBP expression by Northern blot analysis (Fig. 3). Untreated cells showed virtually no GBP RNAs. CEC-32 cells treated with supernatant of the MAF-secreting T cell line contained high levels of three GBP RNAs of 2-3.5-kilobase pair length, which are either the products of multiple GBP genes or transcription variants of a single GBP gene (Fig. 3). Similarly treated HD-11 cells contained high levels of a single GBP RNA of about 2 kilobase pairs. Treatment of CEC-32 and HD-11 cells with supernatant of the control T cell line did not result in the accumulation of detectable levels of GBP RNAs (Fig. 3). Recombinant chicken type I IFN induced the GBP genes of CEC-32 and HD-11 cells only very weakly (signals hardly visible on the short exposure shown in Fig. 3). LMH cells and primary chicken embryo cultures did not contain detectable levels of GBP RNAs under all induction conditions studied (Fig. 3, and data not shown). In all cell lines, the IRF-1 gene was induced very strongly by supernatant of the MAF-secreting T cell line, to a lower extent by recombinant type I IFN, and not detectably by supernatant of the control T cell line (Fig. 3). In LMH and HD-11 cells, the Mx gene was strongly induced by type I IFN but not by the T cell supernatants, whereas in CEC-32 cells the Mx gene seemed inert to induction by the various chicken cytokines (Fig. 3). Taken together, these results suggested a strong conservation of GBP regulation in response to cytokines in mammals and birds.
Figure 3:
Detection of GBP transcripts in
established chicken cell lines under various induction conditions. LMH,
CEC-32 and HD-11 cells were treated for 15 h with either plain culture
medium (), 500 U/ml of recombinant chicken type I IFN (ChIFN1), 50-fold diluted supernatant of a chicken T cell line
that secretes antiviral and MAF activity (MAF), or 50-fold
diluted supernatant of a chicken T cell line that fails to secrete MAF (no MAF). The cells were lysed, the RNAs were extracted, and
samples (20 µg/lane) of total RNA were subjected to Northern blot
analysis. The membrane was sequentially hybridized to radiolabeled
chicken GBP, chicken IRF-1(18) , chicken Mx (22) and
chicken glyceraldehyde-3-phosphate dehydrogenase (23) cDNA
probes.
To
determine whether the induced GBP RNAs were translated into functional
proteins, CEC-32 cells were incubated with either MAF-containing T cell
supernatant or plain medium in the presence of radiolabeled amino
acids, before cell lysates were prepared and samples were analyzed for
proteins capable of binding to agarose-immobilized GTP. Two GTP-binding
proteins of M 65,000 and 70,000 were detected in
the MAF-induced cells that were absent from the uninduced control cells (Fig. 4). These proteins most likely represent bona fide chicken GBPs.
Figure 4:
Induction of endogenous guanine
nucleotide-binding proteins in CEC-32 cells. Cells maintained in the
presence of [S]methionine and
[
S]cysteine were treated for 15 h with either
plain medium (
) or with 50-fold diluted supernatant of a T cell
line that secretes antiviral and MAF activity (MAF). Cell
lysates were prepared and allowed to react with GTP-agarose, before the
bound proteins were eluted and separated by 10% SDS-PAGE. The gel was
soaked in Amplify (Amersham), dried, and exposed to x-ray film. The gel
positions of two induced GTP-binding proteins are
marked.
Our experiments showed that, although the primary sequences of chicken and mammalian GBPs differ greatly, their biochemical properties and their regulation by IFNs are remarkably well conserved. The previously characterized chicken homologs of IRF-1(18) , ICSBP(18) , and Mx protein (22) had revealed a different picture: the coding sequences of these genes are well conserved over most regions. The situation reported here for the GBPs is reminiscent to that reported for type I IFNs of birds and mammals, which show minimal sequence conservation in spite of strong functional similarities(3) .
Sequence conservation between chicken and mammalian GBPs is prominent in two regions (Fig. 1, shaded areas), one of which is 100 amino acids long and harbors two motifs of the tripartite GTP-binding consensus element(10) . The second region is only 19 amino acids long and starts 25 residues downstream. Since GBPs are unique among GTP-binding proteins in that they lack the classical third motif ((N/T)KXD) of the tripartite GTP-binding element(7) , this constellation suggests that the missing motif is hidden in the second conserved region. Considering the chemical nature of the various amino acids, we speculate that the sequence TVRD (amino acid positions 175 to 178 in ChGBP; Fig. 1) is required for guanine nucleotide binding of GBPs. Substitution of the (N/T)KXD motif by TVRD might explain the unorthodox nucleotide binding properties of GBPs. Site-directed mutagenesis and structural studies of the GBP nucleotide binding pocket will be necessary to evaluate this hypothesis.
Although the
characteristic biochemical features of mammalian GBPs are conserved in
the chicken GBP, we still observed differences between the GTPase
activities associated with chicken GBP and human GBP1. The most
important difference was that GMP represented only a minor product of
the chicken GBP-catalyzed hydrolysis reaction, whereas it was the
predominant product of the human GBP1-catalyzed reaction(9) .
In this respect, chicken GBP closely resembles GBP2, the product of a
second human GBP gene(7) : hydrolysis of GTP by GBP2 yielded
about 7-fold more GDP than GMP under standard reaction conditions. ()It thus seems that although all GBPs can form GMP, they do
it to variable extents.
Chicken GBP RNA was not found in uninduced
cells, but it was abundantly present in cells treated with supernatant
of a T cell line that secretes antiviral and MAF activity. Based on
other results (B. Kaspers, unpublished results), we previously
speculated that these two activities might result from the avian
homolog of IFN-(17) . The results described here seem to
support this view and further provide a new method for measuring this
activity with very high sensitivity. Interestingly, the GBP gene(s) of
CEC-32 and HD-11 cells were strongly inducible, whereas those of LMH
and chicken embryo cells were not. By monitoring the induction of IRF-1
RNA we could demonstrate that all these cell lines responded otherwise
equally well to the inducing agents, indicating that the corresponding
cytokine receptors and signal transduction pathways are functional. The
fact that primary embryo cells failed to express the GBP genes, whereas
two of three established cell lines expressed them strongly is a
puzzling finding. It suggests that genetic differences may exist
between the chickens used to establish the various cell lines. Genetic
differences could also explain the fact that the Mx gene could not be
induced in CEC-32 cells, although it was strongly induced in type I
IFN-treated LMH and HD-11 cells. The situation in the chicken may thus
resemble that of mice, where inbred strains with genetic defects of Mx
and GBP genes were described(8, 24) .