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1
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Institute for Genetics, University of Cologne, Cologne, Germany; and
Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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induces a number of cellular programs functional in innate
and adaptive resistance to infectious pathogens. It has recently become
clear that the complete cellular response to IFN- is extraordinarily
complex, with >500 genes (i.e., 
0.5% of the genome) activated. We
made suppression-subtractive hybridization differential libraries from
IFN--stimulated primary mouse embryonic fibroblasts and from a mouse
macrophage cell line, ANA-1, in each case with reference to
unstimulated cells. Of 250 clones sequenced at random from the two
libraries, >35% were representatives of one or the other of two small
unrelated families of GTPases, the 65-kDa and 47-kDa families. These
families dominate the IFN--induced response in both cell types. We
report here the full-length sequences of one new 65-kDa and two new
47-kDa family members. The 65-kDa family members are under
transcriptional control of IRF-1, whereas the 47-kDa family members are
inducible in embryonic fibroblasts from IRF-1-/- mice.
Members of both GTPase families are strongly up-regulated in livers of
wild-type mice infected with the pathogenic bacterium, Listeria
monocytogenes, but not in IFN-R0/0 mice. These
GTPases appear to be dedicated to the IFN- response, since resting
levels are negligible and since neither family shows any significant
relationship to any other described family of GTPases. Understanding
the role of these GTPases in IFN--mediated resistance against
pathogens is the task for the future. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is an immunomodulatory cytokine secreted mainly by activated
thymus-derived (Th1 and TC1) cells and NK cells. IFN-
orchestrates a variety of cellular programs in different target cells
(reviewed in Refs. 1 and 2). Important IFN--stimulated cellular
programs are the quantitative and qualitative enhancement of Ag
presentation by MHC class I and class II pathways, nitric oxide and
respiratory bursts involved in direct antimicrobial activation of
macrophages, and translational inhibition directed against viral
replication. The cellular response to IFN- is extraordinarily
complex. Recent estimates suggest that >500 genes are inducible (3, 4), of which 300 have been identified (Ref. 5 and T. Klamp,
unpublished observations). Thus, as many as 200 genes active in the
cellular response to IFN- remain to be described.
Partially nonidentical programs are triggered by IFN- in different
cell types. In macrophages, IFN- stimulates numerous pathways which
reflect the specialized role of this cell type in early innate immunity
(e.g., inducible nitric oxide synthase, IL-12, phox, complement
components, FcR, etc.), whereas in fibroblasts a more limited range
of functions mainly focused on Ag presentation and direct antiviral
action is induced. In the present study, two independent analyses of
the complexity of IFN--regulated gene induction in macrophages and
fibroblasts have been conducted. To this end, the
suppression-subtractive hybridization
(SSH)3 technique (6) was used
to identify cytokine-inducible genes in the mouse macrophage cell line
ANA-1 and in mouse primary embryonic fibroblasts (MEF). The most
abundant classes of differential cDNAs recovered from both cell types
were members of two cytokine-inducible guanylate-binding protein (GBP)
families, the 65-kDa and 47-kDa families. In the mouse, the 65-kDa GBP
family consists of two previously known genes cloned and described
full-length, mGBP1/mag-1 (7, 8) and mag-2 (8). Previously known members
of the 47-kDa family are the proteins encoded by the IRG-47 (9), TGTP
(10)/Mg21 (11), LRG-47 (12), and IGTP (13) cDNAs.
New members of each family were identified in both cell types and are
cloned and characterized in this study. We show that all members of the
two families are strongly induced by IFN- in the livers of mice
infected with the facultative intracellular bacterium Listeria
monocytogenes. Furthermore, we present additional evidence that a
significant portion of the IFN- response in fibroblasts as well as
in macrophages remains to be described, since at least 15 fragments of
putative new IFN--inducible genes were recovered from our screens.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6J mice were obtained from the animal house at the
Institute for Genetics, University of Cologne, Cologne, Germany. Mice
deficient for IRF-1 (IRF-1-/-), or the IFN- receptor
(IFN-R0/0) have been described elsewhere (14, 15) and
were kindly provided by M. Aguet and C. Weissmann. Mice lacking TNFRp55
(TNFRp55-/-) were described by Pfeffer et al. (16). All
mice were maintained in a conventional animal facility.
L. monocytogenes infection
C57BL/6J, IRF-1-/-, IFN-R0/0, and
TNFRp55-/- mice were infected i.p. with one-tenth of the
LD50 of Listeria monocytogenes (strain EGD).
Twenty-four hours after infection, the mice were killed, and livers
were removed and snap-frozen in liquid N2.
Cells and tissue culture
Primary embryonic fibroblasts were isolated from C57BL/6J,
IRF-1-/-, and IFN-R0/0 mice at day 14 post
coitum as described (17) and were grown in DMEM (Life Technologies,
Gaithersburg, MD) supplemented with 10% heat-inactivated, low
endotoxin FCS (HyClone, Logan, UT), 2 mM L-glutamine (Life
Technologies), 1 mM sodium pyruvate (ICN, Aurora, OH), 100 U/ml
penicillin (Life Technologies), 100 µg/ml streptomycin (Life
Technologies), and 1x nonessential amino acids (Life
Technologies).
Given that macrophage lines differ in their differentiation stages and
responsiveness to IFN-, the inducibility of the well-characterized
IFN--induced inducible nitric oxide synthase mRNA was compared by
RT-PCR in various macrophage cell lines. The C57BL/6J-derived
macrophage cell line ANA-1 (18) was the strongest responder among those
tested (data not shown). ANA-1 was grown in low endotoxin RPMI
(Biochrom, Berlin, Germany), supplemented with 10%
heat-inactivated, low endotoxin FCS (HyClone) and 50 µM 2-ME (Life
Technologies).
For the preparation of SSH cDNA libraries (see below), MEFs were
cultured as indicated for 3 or 24 h in medium containing 1000 U/ml
recombinant mouse IFN- (Genzyme Diagnostics, Cambridge, MA). ANA-1
cells were stimulated with 2000 U/ml recombinant human TNF-
(Genzyme) and 55 U/ml recombinant mouse IFN- (Genzyme) for
16 h. For Northern blot analysis, both MEFs and ANA-1 were
stimulated with 1000 U/ml IFN- for 24 h.
RNA isolation
Total RNA was extracted from cell lines and tissues using the
RNeasy mini kit (Qiagen, Hilden, Germany), the DNA/RNA isolation
kit (Amersham, Little Chalfont, U.K.) or the acid guanidinium
thiocyanate-phenol-chloroform extraction method (19).
Poly(A)+ RNA was isolated from total RNA with the Oligotex
mRNA kit (Qiagen) for the generation of the PCR-Select libraries and
the poly(A)+ RNA isolation kit (Amersham) for the
generation of the 
-cDNA library according to the manufacturers’
instructions. The amount of RNA was quantified spectrophotometrically.
Northern blot analysis
Ten micrograms of total RNA were electrophoresed in 1%
denaturing agarose-formaldehyde gels (20) and then transferred onto
Hybond-N nylon membranes (Amersham Life Sciences) according to standard
protocols. Equal loading, transfer, and quality of the RNA was examined
by staining the membranes with methylene blue (21, 22).
[-32P]dCTP (Amersham)-labeled probes were generated by
the random priming method (23) using the Rediprime DNA labeling system
(Amersham Life Sciences). Hybridizations were performed overnight at
42°C in a buffer containing 50% formamide, 5x Denhardt’s solution,
5x standard saline-phosphate-EDTA (SSPE), 1% SDS, and 10% dextran
sulfate. Membranes were washed under stringent conditions. The
hybridization signal was detected by autoradiography, using Kodak
X-OMAT AR films. Labeled cDNA of mouse glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as a control probe to reveal the amount
of loaded total RNA, and the RNA Millenium Marker (Ambion) was used as
the RNA size standard.
PCR amplification of mGBP-2 and mGBP-4
mRNA prepared from C57BL/6J embryonic fibroblasts stimulated for
24 h with 1000 U/ml recombinant IFN- was reverse-transcribed
with Moloney murine leukemia virus reverse transcriptase (Boehringer
Mannheim) in the presence of an oligo(dT)-anchor primer (Boehringer
Mannheim). After degradation of the mRNA template with RNase H
(Boehringer Mannheim), this library was used to amplify mGBP-2 with
primers mGBP-2 5' (5'-GAAAAGCTGCTTCTTCTTCTTCTCT-3') and mGBP-2 3'
(5'-TCAAGACATGTTGTCACAGTGG-3') (nucleotides 100–124 and
2405–2384, respectively) and mGBP-4 with primers MM44 5'
(5'-GGAAGCTCAGGGAAAGAACTAA-3') and MM44 3'
(5'-CATACATTCTCTTATGAAAACCCAC-3') (nucleotides 23–44 and
2443–2419, respectively).
Suppression-subtractive hybridization/PCR-Select
SSH to generate subtracted cDNA libraries was performed essentially as described (6), using reagents and procedures provided by Clontech (Palo Alto, CA; PCR-Select cDNA Subtraction Kit). Briefly, double-stranded cDNA (tester cDNA) was synthesized from 2 µg of mRNA isolated from cytokine-stimulated MEF and ANA-1 cells (see above). Driver cDNA was prepared from unstimulated MEF and ANA-1 cells. The differential PCR products obtained after two rounds of subtraction were cloned without further purification into pGEM-T Easy (Promega, Madison, WI) or pCR 2.1 (Invitrogen, San Diego, CA) vectors. The SSH protocol generates clonal fragments of differential cDNAs bounded at both ends by RsaI restriction sites.
Construction of size-selected cDNA libraries
from IFN--stimulated fibroblasts
C57BL/6J embryonic fibroblasts were stimulated with 1000 U/ml
IFN- for 24 h. After mRNA isolation (U.S. Biochemical,
Cleveland, OH), cDNA was synthesized from 5 µg of mRNA (ZAP-cDNA
synthesis protocol; Stratagene, Heidelberg, Germany), and the libraries
were constructed using the Uni-Zap XR vector kit from Stratagene
according to the manufacturer’s instructions with the following
alteration. Before ligation of the cDNA into the Uni-Zap XR vector
arms, the cDNA was electrophoresed in a 1% agarose gel and three size
fractions (0.4–1.4 kb, 1.4–4 kb, and >4 kb) were eluted according to
standard procedures (22) and ligated into the vector arms to generate
three independent libraries. For the screening of the new members of
the two GBP-families, the medium size library was used (size range,
1.4–4 kb).
The library was screened with [-32P]dCTP-labeled
probes (Rediprime; Amersham). Filters were hybridized overnight at
65°C in HYB-9 DNA hybridization solution (Biozym, Gwent, U.K.). After
rescreening, cDNA inserts were isolated by in vivo excision of the
pBluescript phagemid from the Uni-Zap XR vector, according to the
manufacturer’s instructions.
All solutions for molecular biology were made with ultrapure water
(0.055 µS/cm) derived from a combined reverse osmosis/ultrapure water
system (ß 75/
UV/UF; USF Seral Reinstwassersysteme Gmbh,
Ransbach-Baumbach, Germany) equipped with UV (185/254 nm) and
ultrafiltration (5000-kDa cutoff).
Nucleotide sequencing, sequence alignment, and analysis
Sequencing was performed in our sequencing facility on an ABI sequencer (Applied Biosystems). Sequences were aligned manually using SeqPup version 0.6 by Don Gilbert (available at ftp://iubio.bio.indiana.edu/molbio/seqpup/). Dot plot analyses and hydrophobicity plots were performed using the Wisconsin Package version 9.1, Genetics Computer Group, Madison, WI. Phylogenetic trees were constructed using PHYLIP version 3.572c (24). The EMBL Data Library accession numbers are mGBP-1/mag-1 (M55544), mGBP-2 (AJ007970), mag-2 (M81128), mGBP-4 (MM44731), hGBP-1 (M55542), hGBP-2 (M55543), rat GBP (M80367), chicken GBP (X92112), pig GBP (F14838), IGTP (U53219), LRG-47 (U19119), IRG-47 (M63630), Mg21/TGTP (L38444), GTPI (AJ007972), IIGP (AJ007971).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-stimulated MEFs and from
IFN- and TNF--activated ANA-1 macrophages
Differential libraries of cytokine-induced MEFs and ANA-1
macrophages were generated using the SSH protocol as described
(Materials and Methods). MEFs were stimulated with 1000 U of
IFN- per ml of medium for 3 and 24 h to generate SSH libraries
representing an early and a late time point in the IFN- response. A
total of 182 clones from the 2 MEF libraries were sequenced. Cells of
the C57BL/6J macrophage cell line ANA-1 (18) were stimulated with a
mixture of 55 U of IFN- and 2000 U of TNF- per ml of medium for
16 h, and a third SSH library was generated from which 70 clones
were sequenced.
Members of the 65-kDa and 47-kDa families of IFN-inducible guanylate-binding proteins are the most abundant messages recovered
The identities of cloned cDNA fragments recovered from MEF and
ANA-1 macrophage-differential libraries are summarized in Table I
. Fragments belonging to the 65-kDa and
47-kDa GBP families were strikingly overrepresented in both libraries
(Table IB), with the new 65-kDa family mGBP-2 sequence (see below) with
36 fragments recovered being by far the most abundantly represented
single gene. Of the other known IFN--inducible genes recovered from
the SSH libraries, the transcription factor IRF-1 was represented five
times in total and the chemokine IP-10 nine times. Fragments of new
members of the 47-kDa family, IIGP and GTPI (see below), were recovered
from both MEF libraries. The two GBP families were abundant in both MEF
and ANA-1 libraries. There was a possibly significant bias in the MEF
libraries toward a higher abundance of GBP family members relative to
other induced sequences.
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A), all of
which were strongly induced by IFN- in the respective cell type over
low or undetectable resting levels (data not shown). Ongoing
experiments will determine whether the yield of MEF and ANA-1-specific
clones is a statistical accident or due to differential regulation. mGBP-2 and mGBP-3 are new members of the 65-kDa family
The abundant cDNA named mGBP-2 in Table IB was contained in
fragments spanning a full-length sequence similar to but distinct from
the published mGBP-1/mag-1 (7, 8). Since C57BL/6J mice do not express
mGBP-1 (25), it was possible that these fragments represented a
homologous gene of which the promoter and 5'-end of the first exon has
been described from BALB/c mice and named mGBP-2 (26), but of which no
complete sequence has yet been described. We therefore performed PCR on
mRNA from IFN--stimulated MEFs using primers based on our fragment
sequences representing the putative 5'- and 3'-ends of mGBP-2 (see
Materials and Methods). The single 2.5-kb product yields an
apparently full-length coding sequence confirmed in full-length clones
from a MEF -cDNA library (data not shown). The 5'-end of the new
sequence is identical with the published short segment of mGBP-2 (26);
this new cDNA thus represents the first full-length mGBP-2 sequence.
The high yield of mGBP-2 fragments from the subtractive libraries is
consistent with the high frequency of mGBP-2 in the 1.4–4 kb
size-selected primary MEF -cDNA library, in which mGBP-2 clones were
represented at a frequency of 1.8%. The open reading frame (ORF) is
1767 nucleotides long, encoding a putative protein of 589 amino acids
with a predicted molecular mass of 66.7 kDa and an isoelectric point of
5.52. Fig. 1 shows the new mGBP-2
sequence aligned with family members mGBP-1/mag-1 and mag-2 and a
sequence (MM44731; deposited by B. H. Han, D. J. Park, R. W. Lim, and
H. D. Kim) of another member of the 65-kDa family for which we
propose the name mGBP-3. This sequence, extracted from a BLAST search
of the GenBank database, arises from a cDNA isolated from erythroid
progenitor cells infected with Friend virus. It contains an ORF of 1863
nucleotides and codes for a putative protein of 620 residues with a
predicted molecular mass of 70.8 kDa and an isoelectric point of 6.6.
No cDNA fragments representing mGBP-3 were recovered from MEF or ANA-1
SSH libraries (Table I), but a 2.4-kb product identical with the
database sequence was identified from RT-PCR on mRNA isolated from MEFs
stimulated with IFN- (see Materials and Methods). All
members of the 65-kDa GBP family possess the canonical
G(X4)GKS and D(X2)G
motifs conserved in most GBPs (27), while all lack the
(N/T)(K/Q)XD motif, as previously noted for mGBP-1 (7). A
stretch of six amino acids (VVVAIV) immediately preceding the
G(X4)GKS motif is perfectly conserved in all
members of the family including all known 65-kDa homologues from human,
rat, pig, and chicken (7, 28, 29, 30). The sequences of the 65-kDa GBP
family are further analyzed below (Table II, Figs. 6 and 7).
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Fragments representing all known members of the 47-kDa GBP family
(LRG-47, IRG-47, Mg21/TGTP, and IGTP) were recovered from the SSH
libraries. Two adjacent segments of an apparently new 47-kDa family
member (labeled IIGP in Table IB) were represented by 5 SSH fragments.
Multiple apparently full-length clones containing both sequence
segments were recovered from the -cDNA library following screening
with one SSH fragment. The ORF is 1239 nucleotides in length,
generating a protein of 413 residues, predicted molecular mass of 47.5
kDa, and isoelectric point of 6.23. Three adjacent segments of a second
apparently new 47-kDa member (labeled GTPI in Table IB) were
represented by 4 fragments. GTPI -cDNA library clones obtained by
screening with one SSH fragment contain a 1185-nucleotide ORF,
generating a putative protein of 395 residues with a predicted
molecular mass of 45.2 kDa and an isoelectric point of 6.95. The IIGP
and GTPI putative amino acid sequences are aligned to known members of
the 47-kDa GBP family in Fig. 2. The
sequence of IIGP is closer to Mg21/TGTP and IRG-47, whereas the
sequence of GTPI is closer to LRG-47 and IGTP (Table III). All
canonical GBP motifs are present in all members of the 47-kDa family,
although LRG-47, IGTP, and GTPI have an unusual methionine in the first
motif, turning the canonical GKS sequence into GMS.
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on IRF-1
Members of the 65-kDa and 47-kDa GBP families were shown by
Northern blot analysis of MEF mRNA to be massively induced from
undetectable constitutive levels by IFN- alone after 24 h of
stimulation (Fig. 3, A and
B, tracks 1 and 2). No induction
could be detected in MEFs from IFN-R0/0-mice (Fig. 3, A and B, track 3). Of the 47-kDa
members, all except GTPI were also strongly induced in
IFN--stimulated ANA-1 macrophages. Of the 65-kDa members, we were
unable to detect induction of mag-2 in ANA-1 even though the original
cDNA was cloned from IFN--induced RAW 264.7, another macrophage cell
line (8). Furthermore, the mag-2 Northern blot shows two bands at 2.8
and 4.1 kb, as originally described by Wynn et al. (8), while the mag-2
sequence deposited in GenBank is 3.2 kb long. These discrepancies
remain to be resolved. The inducibility of mGBP-3 is still under
investigation.
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in MEFs lacking a functional IRF-1 gene. No
detectable induction of 65-kDa family member mRNA was found after
24 h of IFN- induction in MEFs from IRF-1-/- mice
(Fig. 3A, track 4). All 47-kDa mRNAs were
strongly induced in IRF-1-/- MEFs, although IGTP and
IRG-47 showed some dependence on IRF-1 (Fig. 3B, track
4). Induction of GBPs in the livers of L. monocytogenes-infected mice
Twenty-four hours after i.p. infection of normal C57BL/6J mice
with Listeria, mRNAs of mGBP-2, mag-2, and all 47-kDa GBPs
were strongly induced in the liver (Fig. 4, A and B, tracks 1and 2). In IFN-R0/0 mice, no induction
of any transcript was detectable (Fig. 4, A and
B, tracks 3 and 4), showing the
absolute dependence of this induction on IFN-. All GBP genes were
also strongly induced in Listeria-infected
TNFRp55-/- mice, suggesting that induced TNF- does not
markedly contribute to the induction of these genes in vivo via the
TNF- p55 receptor, which mediates the TNF--dependent protective
response to Listeria (31). Additional experiments are needed
to reveal whether the small reduction in signal observed for mGBP-2,
mag-2, and possibly LRG-47 and IIGP in the TNFRp55-/-
track is a consistent and significant effect.
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Nucleotide and amino acid sequence comparisons (Tables II and III)
show the 47-kDa family to be more divergent among its members and
therefore either older or more rapidly evolving than the 65-kDa family.
This observation is consistent with the lack of known members of this
family recovered thus far from human or indeed any other material. A
more extended analysis would be required to distinguish between these
two possibilities. Both families can also be seen from Tables II and
III to break down into two subfamilies, and this is confirmed by the
unrooted trees of protein sequences shown in Fig. 5. Thus, mGBP-1 and -2 cluster closely
together near human GBP-1 and 2, and distant from the mag-2/mGBP-3
cluster among the 65-kDa proteins, while IGTP, GTPI, and LRG-47 cluster
together and distant from IIGP/IRG-47/Mg21/TGTP among the 47-kDa
proteins. Dot plot comparisons between and within subfamily members
(Fig. 6, A and B)
demonstrate the complete colinearity of the 65-kDa family at both high
and low stringency: the only characteristic distinction between the
subfamilies is at the extreme C terminus. Structural divergence between
the subfamilies in the C-terminal half of the 65-kDa proteins, however,
is clearly apparent from the hydrophobicity plots (Fig. 7). Among the 47-kDa GTPases, sequence
divergence at the C terminus is more marked than elsewhere both within
and between subfamilies. The hydrophobicity plots show distinct
structural features at individual, subfamily, and family levels. The
dot plot shown in Fig. 6C compares a 65-kDa (mGBP-2) and a
47-kDa (IIGP) family member, documenting the absence of any sequence
relatedness between the two families.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in primary MEF and to a
synergistic mixture of IFN- and TNF- in a mouse macrophage cell
line, ANA-1. The SSH technique of Diatchenko et al. (6) was used in
preference to differential display (32), firstly because of the built
in focus on the recovery of cDNAs representing mRNAs with large ratios
of induced to noninduced levels and secondly because of the relative
ease of processing the differentially expressed material.
From the analysis of a total of 182 MEF clones and 70 ANA-1 clones, our
results (Table I) show the striking and unexpectedly high yield of
GTPases of the 65-kDa and 47-kDa families in both cell types.
Altogether nine different GTPases of these two families were
identified, including new members of each family, and members of these
families were individually the most abundant cDNA fragments recovered.
Subtractive hybridization differential cloning, in which high molar
ratios of driver (i.e., noninduced in our case) to tester (i.e.,
induced in our case) cDNA are used to remove sequences found in both
pools, will reduce, if not eliminate, sequences that may be
differential but have nevertheless a significant constitutive level of
expression. The results of our analysis are consistent with this
expectation. Thus no component of the classical class I Ag presentation
pathway (including the structural proteins H-2Kb, Db,
ß2-microglobulin; the endoplasmic reticulum components
TAP1, TAP2, gp96, and tapasin; and the proteasome components LMP2,
LMP7, PA28, and PA28ß) was recovered from either cell type.
Although all these components are inducible by IFN-, the pathway is
constitutively active at a certain level in both cell types. The
recovery of GTPase fragments in such abundance thus reflects the high
abundance of these messages in the induced mRNA pool and their
remarkably low level of expression in noninduced cells (Fig. 3). In
confirmation of this view, human GBP-1 was identified in early
experiments in human fibroblasts as one of the most abundantly induced
proteins, with about 3 x 105 protein molecules per
cell 24 h after IFN- stimulation (33, 34), while, as noted
above, the newly identified homologue mGBP-2 represented 1.8% of a
primary cDNA library constructed from IFN--stimulated MEFs.
In the two cell types studied, therefore, these GTPases are extreme
specialists of the IFN--induced state in the sense that they are
virtually absent in resting cells and highly abundant after induction.
That the same is also true for liver in vivo is suggested by the
Northern blots shown in Fig. 4, where the low signal apparent in some
tracks from noninfected mice is consistent with a low level of
induction by endogenous cytokine production induced by background
pathogens in these conventionally reared animals. The 47-kDa GTPase
IGTP has been reported to show high constitutive levels of mRNA in
tissues rich in lymphoid cells including the thymus and small intestine
(13). Whether this expression can be generalized for other
IFN-inducible GTPases and whether it reflects constitutive
transcription rather than transcription induced by local production of
IFN- should be determined by further analysis of expression of these
gene families in tissues of IFN-- or IFN- receptor-deficient
mice.
As noted in Results, cDNA fragments representing members of
both the 47-kDa and 65-kDa GTPase families were recovered from both the
3-h and 24-h SSH libraries, showing that mRNAs from both families are
significantly elevated both relatively early and relatively late in the
cellular response to IFN-. Nevertheless, the induction of the two
families was radically different in MEFs from IRF-1-/-
mice (Fig. 3). Both the 65-kDa family members, mGBP-2 and mag-2, were
absolutely IRF-1 dependent, as documented elsewhere also for the
promoters of mGBP-1 and mGBP-2 (26, 35), while the 47-kDa family
members showed only more or less trivial reductions in signal in the
IRF-1-deficient fibroblasts. Promoter sequences of both mGBP-1 and
mGBP-2 show an apparently inactive STAT1-binding IFN- activation
site (26), and two functional IFN-stimulated regulatory elements
expected to act as IRF-1 binding sites. It is therefore likely that all
65-kDa GTPases are classical secondary response genes, relying for
their induction on prior synthesis of the main secondary transcription
factor IRF-1 induced by IFN-. Transcription of IGTP as a
representative of the 47-kDa family, on the other hand, has been shown
to be significant within 1 h of induction, and this early
transcription is independent of de novo protein synthesis (13),
consistent with our demonstration of significant independence from
IRF-1. Thus, IGTP, and by implication the other 47-kDa family members,
is at least partly a primary response gene with induction of mRNA
presumably by activated STAT1 within minutes after IFN- stimulation.
Transcription of the 47-kDa GTPases is presumably sustained by
subsequent induction through IRF-1 or other secondary transcription
factors, explaining the significant but incomplete dependence of 24-h
mRNA levels on IRF-1 in our experiments, and the significant
sensitivity of IGTP mRNA levels at all except the earliest times to
cycloheximide (13). Although all the 47-kDa GTPase family members show
a degree of independence of IRF-1, IRG-47 appears to be the most
sensitive, perhaps consistent with an earlier study of the IRG-47
promoter in which an IFN-stimulated regulatory element was described
and which concluded that IRF-1 was an essential transcription factor
for this gene (36).
Both the MEFs and ANA-1 macrophages used to generate the SSH libraries
in our study are derived from C57BL/6J mice. Neither library yielded
any copies of the 65-kDa GTPase prototype, mGBP-1, while the most
abundant fragments from both cell types proved to represent the close
homologue, mGBP-2. This result is consistent with early studies from
Staeheli et al. (25, 37), who showed that most inbred strains of mouse,
including C57BL/6J, made no detectable mGBP-1 protein. Although a short
5'-cDNA fragment corresponding to a mGBP-1 homologue was subsequently
identified, sequenced, and named mGBP-2 from BALB/c material (26), no
full-length sequence of mGBP-2 has been available. The full-length
sequence of mGBP-2 presented here shows it to be the closest known
relative to mGBP-1 with 80.5% amino acid identity (Table II). Among
the 65-kDa GTPases, mag-2 presents an anomaly in that despite strong
induction in MEFs (Fig. 3) and in the livers of
Listeria-infected mice (Fig. 4), no induction was detected
in ANA-1 macrophages. Since mag-2 was first identified as an
IFN--inducible gene in a macrophage cell line, RAW264.7, this
failure cannot be attributed to the difference in cell type alone.
However, RAW264.7 is derived from the BALB/c strain, unlike the
C57BL/6J-derived ANA-1. Polymorphism in expression of members of the
65-kDa GTPase family is reminiscent of that observed in mice in another
IFN-inducible GTPase, namely the Mx protein, which confers resistance
to influenza virus. Polymorphism among mouse strains in expression or
function of members of the 47-kDa GTPase family has not been reported.
The existence of polymorphism between members of the 65-kDa GTPase family suggests the existence of some undisclosed but persistent selection pressure on these proteins. A similar conclusion may also be reached from the fine structure of the two GTPase families which are both divided into distinct subfamilies. That these subdivisions imply functional divergence is suggested in both families of GTPases. In the case of the 65-kDa family, the mGBP-1 and -2 proteins share a C-terminal motif CTIL which functions as a modification signal for isoprenylation (38) and presumably membrane attachment, while this motif is absent from mag-2 and mGBP-4. For the 47-kDa proteins, IGTP, GTPI, and LRG-47 share the unusual amino acid, methionine, in the first canonical GTP-binding motif, while this residue is replaced by the typical lysine in the members of the second subfamily. Although IGTP has been shown to be a GTPase (13), no GTPase activity of other 47-kDa GTPases has been documented. Divergence in the products of GTP hydrolysis has recently been demonstrated between the human GBP-1 (favoring GMP) and GBP-2 (favoring GDP) proteins of the 65-kDa family (39), suggesting that GTPase activity per se may be a target for evolutionary modification.
The ubiquitous and abundant presence of these two families of GTPases
in the cellular response to IFN-, and their structural and
biochemical diversification all point to their playing well-defined and
specific roles in pathogen resistance. The present experiments provide
a hint that if either family of proteins is involved in activation of
cellular defense against intracellular pathogens such as L.
monocytogenes, it is more likely to be the IRF-1-independent,
largely primary response 47-kDa family, rather than the
IRF-1-dependent, exclusively secondary response 65-kDa family. Although
the molecular mechanisms by which IFN-R and TNFRp55 trigger such
defenses are not yet clear (15, 16, 40), gene products induced by STAT1
and interferon consensus sequence binding protein may be more
significant than those induced by IRF-1 in defense against
Listeria (41, 42, 43). A distinct perspective has been offered
recently by Taylor et al. (44) who proposed, on the basis of finding
the IGTP 47-kDa GTPase associated with the endoplasmic reticulum
membrane, that this GTPase might function in protein processing for Ag
presentation. Perhaps the strongest clue to the function of these
enigmatic proteins is from the Mx protein, a large GTPase inducible by
type I IFN, which is known to interfere directly with the replication
of a number of RNA viruses, especially influenza (45). Very recent
experiments have suggested specific antiviral activity in the Mg21/TGTP
47-kDa GTPase (46). Mg21/TGTP-transfected L-cells
showed resistance to viral plaque formation by an RNA virus, vesicular
stomatitis virus, but not to the DNA virus for herpes simplex.
Note added in proof:
While our manuscript was in review, Han
et al. (47) published a characterization of mGBP-3, including the
demonstration that it is induced by IFN-.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Ulrich Boehm, Institute for Genetics, University of Cologne, Zülpicher Strasse 47, D-50674 Köln, Germany. E-mail address:
3 Abbreviations used in this paper: SSH, suppression subtractive hybridization; GBP, guanylate-binding protein; MEF, mouse embryonic fibroblasts; IRF-1, IFN-regulatory factor 1; IFN-R, IFN- receptor; TNFRp55, 55-kDa TNF receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ORF, open reading frame.
Received for publication May 6, 1998. Accepted for publication August 26, 1998.
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