Morpholinos for splice modificatio

Morpholinos for splice modification



Polymerization of members of the serpin superfamily underlies diseases as diverse as cirrhosis, angioedema, thrombosis and dementia. The Drosophila serpin Necrotic controls the innate immune response and is homologous to human α1-antitrypsin. We show that necrotic mutations that are identical to the Z-deficiency variant of α 1-antitrypsin form urea-stable polymers in vivo. These necrotic mutations are temperature sensitive, which is in keeping with the temperature-dependent polymerization of serpins in vitro and the role of childhood fevers in exacerbating liver disease in Z α-antitrypsin deficiency. In addition, we identify two nec mutations homologous to an antithrombin point mutation that is responsible for neonatal thrombosis. Transgenic flies carrying an S>F amino-acid substitution equivalent to that found in Siiyama-variant antitrypsin (necS>F.UAS) fail to complement nec-null mutations and demonstrate a dominant temperature-dependent inactivation of the wild-type nec allele. Taken together, these data establish Drosophila as a powerful system to study serpin polymerization in vivo.


The serpin (serine proteinase inhibitor) superfamily includes antithrombin, α 1-antitrypsin and PAI-1, which control the coagulation, inflammation and fibrinolytic pathways, respectively. Serpins have a unique method of inhibition that involves a conformational change of the protein (Huntington et al., 2000). This transition is essential for the mechanism of serpin inhibition but it renders serpins susceptible to mutations that affect conformational stability (Carrell and Lomas, 2002) and lead to polymer formation (Lomas et al., 1992; Dafforn et al., 1999; Huntington et al., 1999; Sivasothy et al., 2000). The most well characterized of these conditions is deficiency of the human serpin α1-antitrypsin.

α1Antitrypsin 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 β-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 α 1-antitrypsin predisposes to early-onset emphysema, by failing to protect the lungs against proteolytic attack (Eriksson, 1965). Thus, α 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 α 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 α1-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.

Fig. 1.

Dorsal thoracic region of nec null mutant fly (nec2/nec19). Within a few hours of eclosion, adult flies develop black cuticular patches that are associated with cellular necrosis of the underlying epithelial cells (Green et al., 2000). Necrotic patches are randomly distributed over most of the body surface, but occur preferentially at the proximal leg joints (arrows). Distal leg segments have been dissected away.

We characterize necrotic mutations with amino acid substitutions equivalent to that found in the human Z-variant α-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 α-antitrypsin carriers (Lomas et al., 1992). To test the similarities in the functional constraints between the Nec and α 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 α1-antitrypsin variant, α 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.


Fly strains

The nec alleles used in this study were generated by EMS and X-rays. Transgenic nec mutations were constructed using a nec cDNA (Green et al., 2000) and PCR-based site-directed mutagenesis. Both wild-type (necUAS) and mutant (nec1.UAS, nec9.UAS and necS>F.UAS) strains were expressed using the Gal4/UAS system (Brand and Perrimon, 1993). The Gal4-Act5c driver strain gives constitutive high-level expression of transgenic UAS strains, whereas Gal4-Yp drives high levels of UAS gene expression that are restricted to the fat body of adult female flies [Yolk protein driver P{Yp-Gal4.G} (Georgel et al., 2001)].

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 α1-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).


nec mutations mirror human disease-associated serpin variants

Fourteen mutant strains carrying point mutations in nec were isolated in Cambridge and Strasbourg (by D.G. and J.-M.R.), in addition to mutants isolated by P. Heitzler (Heitzler et al., 1993). Sequencing of these nec alleles identified a range of mutations, including both stop codons and single amino acid substitutions (Table 1). Of particular interest was a mutation that occurred on two occasions (nec9 and nec20) within our collection of alleles. This mutation (Glu421→Lys) is the same as that found in the Z allele of human α1-antitrypsin (Glu342→Lys), as shown on the molecular model in Fig. 2. The Glu342→Lys substitution in Z α1-antitrypsin is at the hinge region of the serpin (Stein and Carrell, 1995), at the junction of β-sheet A and the base of the reactive center loop (Fig. 2A). This mutation perturbs the structure of the serpin molecule such that the reactive center loop of one molecule inserts into β-sheet A of another to initiate polymerization (Fig. 2B) (Lomas et al., 1992). It is these Z-α1-antitrypsin polymers that accumulate in hepatocytes thereby causing `neomorphic function' liver disease and `hypomorphic function' lung disease.

View this table:
Table 1.

Molecular lesions in nec alleles

Fig. 2.

Models of serpin structure. (A) Position of nec mutations mapped onto the scaffold of monomeric α1-antitrypsin (Elliott et al., 2000). The Glu→Lys substitution found in Z-α1-antitrypsin, nec9 and nec20, maps at the hinge region between the reactive center loop (red) and β-sheet A (green). β -sheet B is colored yellow and α-helix A is blue. NecS>F carries the Ser131→Phe substitution homologous to that of α1-antitrypsin-Siiyama, Ser53→Phe. (B) Loop-sheet polymer of Z-α1-antitrypsin and Nec9.

Similarly, NecE421K protein gives a fly mutant phenotype characteristic of lack of Nec activity in the hemolymph. Remarkably, a second amino acid substitution, Gly466→Ser (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 α-helix A of the serpin. The internal face of the α-helix A forms part of the protein core, with the residues from this face interdigitating with those at the back of the β-sheet A. Any perturbation of these residues will lead to a destabilization of the β-sheet A and is likely to lead to polymerization. A number of clinically relevant mutations have been found in the α-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 α -helix A disruption.

nec mutants form urea-stable serpin polymers

The properties of nec9 (Glu421→Lys) and nec1 [an extreme hypomorphic mutation (Δ 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 α 1-antitrypsin (Lomas et al., 1995b) (Δ 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-α1-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 α 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.

Fig. 3.

Conformational stability of mutant serpins. (A) 7.5-15.0% (w/v) non-denaturing PAGE of cell extracts from flies carrying the E421K (nec9) mutation show reduced levels of native-like protein, N, and an increase in levels of higher molecular mass species, H. The more extreme phenotype of nec1 is associated with a further reduction in native protein. (B) 7.5% (w/v) TUG PAGE demonstrates that the higher molecular mass species (arrows) observed in both nec9 and nec1 are insensitive to denaturation in 8M urea. This behavior is characteristic of loop-sheet polymers such as those observed in the livers of individuals with Z-α1-antitrypsin deficiency. These stabilized polymers are not observed in the wild-type flies. (C) The profile of monomeric α 1-antitrypsin and polymerized Z-α1-antitrypsin are shown for comparison, using purified proteins stained with Coomassie. The left of each gel represents 0 M urea and the right 8 M urea.

The rate of polymerization of α1-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 α1-antitrypsin (Lomas et al., 1992), although the secretion of Z α1-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.

Fig. 4.

(A-C) Viability of nec9, nec1 and wild-type (WT) flies at 18°C (A), 25°C (B) and 29°C (C). The survival of nec9 flies, homologous to Z-variant α 1-antitrypsin, is greatly reduced at 25°C and 29°C compared with that of wild-type flies. nec1 flies have shorter survival times than nec9, but survive about twice as long as complete null alleles (nec2/nec19, data not shown). (D-F) Viability of transgenic flies overexpressing wild-type or mutant Nec in a nec+ genetic background (Gal4-Act5c/+; necUAS/+) at 18°C (D), 25°C (E) and 29°C (F). (G) Viability of transgenic females overexpressing mutant Nec at 29°C in the fat body (Gal4-Yp/+; necUAS/+). Sibling males for these genotypes, in which the Yp promotor is inactive, were all healthy and the combined data for all three strains has been plotted as a control.

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 α1-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).


Taken together, our findings identify a class of mutants in the Drosophila Nec serpin that undergo temperature-dependent polymerization and demonstrate, for the first time, an associated temperature-dependent mortality. In human serpins, clinically important mutations cluster in regions critical for the conformational changes essential to the normal function of the protein. We have found that nec mutations in Drosophila map to these same critical regions, underlining their significance in protein function. Furthermore, some nec mutations cause amino acid substitutions identical to those responsible for disease in humans. In particular, both NecE421K and NecG466S have arisen twice independently, proving these residues to be critical for conformational stability. In addition, engineering an amino acid substitution in Nec homologous to that found in the extreme polymeric Siiyama variant of α1-antitrypsin produces an inactive serpin that becomes a dominant-negative-mutant form at higher temperatures. It seems likely that the mechanism of inactivation of the wild-type Nec serpin in flies overexpressing Nec1 or NecS>F is that the polymeric serpin recruits sufficient wild-type Nec to give a deficiency of Nec in the hemolymph. Such heteropolymerization has been observed for the S, I, and Z variants of α1-antitrypsin (Mahadeva et al., 1999).

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.


This worked was funded by the Medical Research Council (UK) and Wellcome Trust. We thank A. Meunier and M. Pal, for help with transgenic constructs and injections, and Dr Linda Sharples of the MRC Biostatistics Unit, University of Cambridge, for help with the statistical analysis.


    • Accepted December 18, 2002.


View Abstract