1 Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2
3EH, UK
2 Department of Haematology, University of Cambridge, Cambridge Institute for
Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY,
UK
3 UPR 9022 CNRS, Institut de Biologie Moléculaire et Cellulaire,
Strasbourg, France
4 Department of Medicine, University of Cambridge, Cambridge Institute for
Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY,
UK
* Present address: Department of Genome Sciences, University of Washington,
Seattle, WA 98195, USA
Present address: Biological Sciences, University of Manchester, 2.205 Stopford
Building, Oxford Road, Manchester M13 9PT, UK
Author for correspondence (e-mail:
d.gubb{at}gen.cam.ac.uk)
Accepted 18 December 2002
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SUMMARY |
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Key words: Necrotic, Serpin, Polymer, Z-variant 1-antitrypsin, Conformational disease, Drosophila
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INTRODUCTION |
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1Antitrypsin is synthesized in the liver and is the most
abundant circulating proteinase inhibitor in humans. Most individuals carry
the normal M allele but 1 in 25 northern Europeans are heterozygous for the Z
variant (Blanco et al., 2001
).
The Z mutation (Glu342
Lys) favors the spontaneous formation
of polymers between the reactive center loop of one molecule and ß-sheet
A of another (Lomas et al.,
1992
; Dafforn et al.,
1999
; Sivasothy et al.,
2000
). These polymers are retained within hepatocytes to form
inclusion bodies that are associated with neonatal hepatitis
(Sveger, 1976
), cirrhosis
(Sveger, 1988
) and
hepatocellular carcinoma (Eriksson et al.,
1986
). In addition, the lack of circulating
1-antitrypsin predisposes to early-onset emphysema, by
failing to protect the lungs against proteolytic attack
(Eriksson, 1965
). Thus,
1-antitrypsin deficiency results in two clear phenotypes.
The toxic effect of polymer accumulation causes cirrhosis while the consequent
lack of inhibitory activity results in emphysema.
Polymerization also underlies deficiency of antithrombin, C1-inhibitor and
1-antichymotrypsin, which are associated with thrombosis
(Bruce et al., 1994
),
angio-edema (Aulak et al.,
1993
; Eldering et al.,
1995
) and emphysema (Gooptu et
al., 2000
), respectively, and the accumulation of mutant
neuroserpin protein within the brain, which causes an inclusion body dementia
(Davis et al., 1999
;
Belorgey et al., 2002
).
The necrotic (nec) gene in Drosophila
melanogaster is one of a cluster of serpin transcripts at 43A on the
second chromosome (Green et al.,
2000). Loss-of-function nec mutants hatch as weak adults
that develop black melanized spots on the body and leg joints
(Fig. 1) and die within a few
days of eclosion. In addition to the visible phenotype, the Toll-mediated
immune response to fungal infections is constitutively activated in
nec mutants (Levashina et al.,
1999
). The Nec protein consists of a serpin core, which has
sequence homology with
1-antitrypsin, and a
polyglutamine-rich N-terminal extension of 79 amino acids that is not found in
other serpins (Green et al.,
2000
). Nec protein is synthesized in the fat body, the insect
equivalent of the liver, and secreted into the hemolymph.
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We characterize necrotic mutations with amino acid substitutions
equivalent to that found in the human Z-variant -antitrypsin. These
mutants survive longer than necrotic null flies and retain inactive
polymers of Nec. Moreover, the mutants are sensitive to increases in
temperature: this is in keeping with the temperature-dependent formation of
serpin polymers in vitro and the role of childhood fevers in exacerbating
liver disease in Z-variant
-antitrypsin carriers
(Lomas et al., 1992
). To test
the similarities in the functional constraints between the Nec and
1-antitrypsin serpins further, we engineered the
Ser131
Phe transition in the Nec protein
(NecS>F) to be equivalent to that responsible for the most
extreme polymerogenic
1-antitrypsin variant,
1-antitrypsin-Siiyama [Ser53
Phe
(Lomas et al.,
1993
,Lomas et al.,
1993
)]. We found that overexpression of NecS>F
failed to complement the genetic lesion in nec-null mutant flies and,
furthermore, produced a temperature-dependent dominant phenotype in a
nec+ genetic background. The striking parallels between
the behavior of Nec and human serpins establishes Drosophila as a
powerful in vivo system with which to both study polymerization and test
therapeutic agents for human disease.
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MATERIALS AND METHODS |
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DNA sequencing
PCR fragments containing the whole of the nec transcript were
isolated from the genomic DNA of each mutant using the primers
3'-TGTGATCGACACGGAATCCCA-5' and
3'-CTCTTCCAATCGCCGTATAGC-5'. Both strands of each fragment were
sequenced using oligonucleotide primers (Sigma-Genosys) and the ABI Bigdye
Terminator Cycle Sequencing Kit (Perlin Elmer).
Survival times
Flies (n=50) of each mutant or wild-type (Oregon-R) strain,
heterozygous with the null mutation nec2, were scored for
survival at 18°C, 25°C and 29°C. Survival of 50 transgenic flies
overexpressing mutant or wild-type Nec protein, in a nec+
genetic background, were scored at 18°C, 25°C and 29°C. The log
rank test was used for statistical analysis of results.
Protein analysis
Protein was extracted by homogenizing whole flies in 100 mM Tris (pH 8), 5
mM EDTA, 50 mM NaCl and treating with general use protease and phosphatase
inhibitor cocktail (Sigma). Following centrifugation, the supernatant was
removed and loaded on a polyacrylamide gel. Protein was detected by western
blotting using a rabbit anti-nec antibody and a goat anti-rabbit
horseradish-peroxidase-conjugated secondary antibody. Protein bands were
detected by chemiluminescence. M and Z 1-antitrypsins were
purified from the plasma of homozygotes as described previously
(Lomas et al.,
1993
,Lomas et al.,
1993
) and detected directly by Coomassie staining of gels.
Native and transverse-urea-gradient polyacrylamide gel
electrophoresis
Native and transverse-urea-gradient (TUG) PAGE were carried out using 10%
and 8% (w/v) polyacrylamide gels, respectively
(Lomas et al., 1995a).
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RESULTS |
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Similarly, NecE421K protein gives a fly mutant phenotype
characteristic of lack of Nec activity in the hemolymph. Remarkably, a second
amino acid substitution, Gly466Ser
(NecG466S), also occurred twice within the 14 nec
point mutations (nec7 and nec22). This
lesion is analogous to a mutation of antithrombin that is associated with
polymer formation, loss of inhibitory function and thrombosis [equivalent to
the Gly424
Arg substitution in antithrombin
(Jochmans et al., 1994
)].
The nec1 mutation results in the deletion of two
isoleucine residues at positions 118 and 119 in the -helix A of the
serpin. The internal face of the
-helix A forms part of the protein
core, with the residues from this face interdigitating with those at the back
of the ß-sheet A. Any perturbation of these residues will lead to a
destabilization of the ß-sheet A and is likely to lead to polymerization.
A number of clinically relevant mutations have been found in the
-helix
A (Stein and Carrell, 1995
),
and it is likely that the deletion of the two residues observed in the
nec1 mutation would represent an extreme case of
-helix A disruption.
nec mutants form urea-stable serpin polymers
The properties of nec9 (Glu421Lys) and
nec1 [an extreme hypomorphic mutation (
Ile118, Ile119) with slight residual Nec activity] were
analyzed alongside the wild-type protein using non-denaturing and
transverse-urea-gradient (TUG) gels. A progressive reduction in the native
protein was seen from wild type through nec9 to
nec1 (Fig.
3A). A corresponding progressive increase in the higher molecular
mass species was seen in these mutants. These higher molecular mass bands were
resistant to unfolding in 8 M urea (Fig.
3B), which is characteristic of serpin polymers. The resolution of
only a single higher molecular mass band in samples from
nec9 and nec1 flies suggests that
polymer formation is halted at a low-order oligomer stage. Similar behavior is
shown by human polymeric variants, such as the Mmalton variant of
1-antitrypsin (Lomas et
al., 1995b
) (
Phe52) and the Rouen VI variant
(Asn187
Asp) of antithrombin
(Bruce et al., 1994
), as well
as the trimeric form of Hsp47 (Dafforn et
al., 2001
). Note, however, that the clear
Z-
1-antitrypsin polymer ladder represents Coomassie-stained
purified protein, whereas the Nec data are from immunoblotted TUG gels of
whole-fly protein extracts. Under these conditions, the failure to detect
high-order Nec polymer ladders may reflect the increased background staining
or post-translational modification (e.g. glycosylation) of Nec. Despite these
caveats, the critical feature that the Nec9, Nec1 and Z
1-antitrypsin TUG gels have in common is the lack of a
S
R serpin transition, which is clearly shown by the sigmoidal form in
the wild-type Nec and antitrypsin TUG gels.
|
The rate of polymerization of 1-antitrypsin variants is
temperature dependent in vitro (Lomas et
al., 1992
; Lomas et al.,
1993
,Lomas et al.,
1993
). It has thus been suggested that childhood fevers might
exacerbate liver disease in individuals with Z
1-antitrypsin
(Lomas et al., 1992
), although
the secretion of Z
1-antitrypsin from cultured cell lines
does not support this hypothesis (Burrows
et al., 2000
). The temperature dependence of serpin polymerization
was assessed in vivo by the survival of nec mutant fly strains. Null
mutations of nec cause adult flies to die rapidly after eclosion
(Green et al., 2000
) but
hypomorphic nec mutants survive for several days to a week at
25°C (Fig. 4B). We tested
survival of each nec allele, heterozygous with a deletion of the
nec chromosomal region, at a range of defined temperatures. When the
culture temperature was reduced from 25°C to 18°C, the relative
survival rate of different alleles was not affected. An increase to 29°C,
however, had a significant effect: nec9 flies had a
shorter survival time relative to control flies than that seen at 25°C
(P=0.0068). Moreover, nec1 flies had a shorter
survival time than nec9 flies
(Fig. 4A-C). The more severe
phenotype of the nec1 flies correlates with the thicker
polymer band and the reduced monomer band seen in these mutants. These results
provide clear evidence that higher temperatures reduce survival times, and
that the probable mechanism is by increasing the rate of polymer
formation.
|
To test directly the toxicity of mutant Nec proteins, we overexpressed
putative polymeric mutant proteins in a nec+ genetic
background (Fig. 4D-G). In
addition to nec9.UAS and nec1.UAS, we
recovered a necS>F.UAS strain (carrying the amino acid
substitution homologous to that found in the extreme polymeric Siiyama variant
of 1-antitrypsin, Ser53
Phe). Consistent
with our previous results, Nec1 overexpression strongly reduced
viability, whereas Nec9 overexpression resulted in a moderate
reduction. Overexpressing wild-type Nec protein also weakly reduced viability
compared with +/nec2 flies
(Fig. 4D-F). An unexpected
result, however, is that, at all three culture temperatures,
Gal4-Act5c/+; nec1.UAS/+ flies show a moderate
nec phenotype, despite carrying a wild-type nec allele. The
NecS>F protein appears to be inactive at 18°C and 25°C,
with the viability of necS>F.UAS flies being comparable
with +/nec2 (compare
Fig. 4D,E with 4A,B) and
greater than necUAS controls. The NecS>F
protein fails to complement lack of wild-type Nec in a nec-null
background and nec2/nec19;
necS>F.UAS/Gal4-Act5c flies retain an extreme Nec
phenotype. However, NecS>F behaves like Nec1 at
29°C in a nec+ background, and Gal4-Act5c/+;
necS>F.UAS/+ flies develop a moderate Nec phenotype
that is associated with strongly reduced viability. The major site of toxicity
of the mutant Nec proteins is probably in the fat body, the normal site of Nec
synthesis. The female-specific fat body driver Gal4-Yp reduces
viability of transgenic nec9.UAS, nec1.UAS and
necS>F.UAS females compared with sibling males
(Fig. 4G).
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DISCUSSION |
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The genetic analysis of serpins in model organisms has been hampered by the broad substrate specificity of most serpin molecules. As a consequence, loss-of-function serpin mutations rarely produce mutant phenotypes in mice and the target proteases remain inhibited by the activity of related serpins. The tight specificity of Nec for its substrate protease and the lack of functionally redundant Nec-like serpin activities in Drosophila provides us with a unique opportunity for genetic analysis of serpin function. The Nec mutant phenotype will enable the development of a model to facilitate the study of serpin polymerization in vivo and to test therapeutic agents for human disease.
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ACKNOWLEDGMENTS |
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