Differential phospholipid binding by site 3 and site 4 toxins. Implications for structural variability between voltage-sensitive sodium channel domains

It has been shown recently that polypeptide toxins that modulate the gating properties of voltage-sensitive cation channels are able to bind to phospholipid membranes, leading to the suggestion that these toxins are able to access a channel-binding site that remains membrane-restricted (Lee, S.-Y., and MacKinnon, R. (2004) Nature 430, 232-235). We therefore examined the ability of anthopleurin B (ApB), a sea anemone toxin that selectively modifies inactivation kinetics of Na(V)1.x channels, and ProTx-II, a spider toxin that modifies activation kinetics of the same channels, to bind to liposomes. Whereas ProTx-II can be quantitatively depleted from solution upon incubation with phosphatidylcholine/phosphatidylserine liposomes, ApB displays no discernible phospholipid binding activity. We therefore examined the activities of structurally unrelated site 3 and site 4 toxins derived from Leiurus and Centruroides venoms, respectively, in the same assay. Like ApB, the site 3 toxin LqqV shows no lipid binding activity, whereas the site 4 toxin Centruroides toxin II, like ProTx-II, is completely bound. We conclude that toxins that modify inactivation kinetics via binding to Na(V)1.x site 3 lack the ability to bind phospholipids, whereas site 4 toxins, which modify activation, have this activity. This inherent difference suggests that the conformation of domain II more closely resembles that of the K(V)AP channel than does the conformation of domain IV.     

Effects of the eukaryotic pore-forming cytolysin Equinatoxin II on lipid membranes and the role of sphingomyelin

Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by (31)P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20-40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state (31)P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The (2)H and (31)P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.     

Do cardiotoxins possess a functional site? Structural and chemical modification studies reveal the functional site of the cardiotoxin from Naja nigricollis

Examination of the literature has revealed that regarding the amino acid sequences, cardiotoxins constitute a family of homogeneous compounds. In contrast, cardiotoxins appear heterogeneous as far as their biological and spectroscopic properties are concerned. As a result, comparison between these molecules with a view to establishing structure-activity correlations is complicated. We have therefore reviewed recent works aiming at identifying the functional site of a defined cardiotoxin, ie toxin gamma from the venom of the spitting cobra Naja nigricollis. The biological and structural properties of toxin gamma are first described. In particular, a model depicting the 3-dimensional structure of the toxin studied by NMR spectroscopy is proposed. The toxin polypeptide chain is folded into 3 adjacent loops rich in beta-sheet structure connected to a small globular core containing the 4 disulfide bonds. A number of derivatives chemically modified at a single aromatic or amino group have been prepared. The structure of each derivative was probed by emission fluorescence, circular dichroism and NMR spectroscopy. Also tested was the ability of the derivatives to kill mice, depolarize excitable cell membranes and lyse epithelial cells. Modification of some residues in the first loop, in particular Lys-12 and at the base of the second loop substantially affected biological properties, with no sign of concomitant structural modifications other than local changes. Modifications in other regions much less affected the biological properties of the toxin. A plausible functional site for toxin gamma involving loop I and the base of loop II is presented. It is stressed that the functional site of other cardiotoxins may be different.     

Elevation of thiobarbituric acid values in the rat liver intoxicated by T-2 toxin

In the present study we first demonstrated that T-2 toxin markedly stimulated lipid peroxidation specifically in the liver of rats. The amount of lipid peroxides in the liver, estimated by the thiobarbituric acid (TBA) method, increased dose dependently, being proportional to the extent of its acute toxicity measured by various parameters in rats fed a commercial diet. Further, to elucidate the mechanism of lipid peroxidation and its role in hepatic injury caused by T-2 toxin, time-course studies on the correlation between lipid peroxide content and some biological and histopathological data were undertaken in rats given 4 mg of the toxin/kg perorally. The TBA reactive substances in the liver began to increase after 6 hr. However, much earlier than this there were some other alterations, which included decreases in the amount of cytochrome P-450 in the liver, of GPT (thereafter an increase) and phospholipids in the plasma, and of basophilic masses in the hepatocytes (arrayed as a rough endoplasmic reticulum in the electron micrograph). The vitamin E-deficient study showed that vitamin E markedly inhibited the stimulative effect of T-2 toxin on lipid peroxidation, but not diminish any other measured parameters of the injury. The toxin-induced stimulation of lipid peroxidation does not appear to be caused by activation of microsomal NADPH-cytochrome c reductase nor by a decrease in the level of cytosolic glutathione peroxidase. These results suggest that T-2 toxin might induce some alteration of the membrane structure and consequently might stimulate lipid peroxidation in situ.     

Studies of the ligand binding to cholera toxin, I. The lipophilic moiety of sialoglycolipids.

The fixation of cholera toxin by ganglioside GGtet1 is dependent on the nature of the carbohydrate as well as the lipid moiety of the glycolipid. The role of the lipid in binding to the toxin investigated with synthetic ganglioside analogues (gangliosidoides). The interaction between glycolipid and toxin was followed by precipitate formation, by inhibition of toxicity and in polyacrylamide gel electrophoresis. For specific precipitation, an aliphatic hydrocarbon chain at least 14 C-atoms in length is required. Some of the gangliosidoides form high molecular weight complexes with cholera toxin at lower molar ratios of ligand to protein than the natural compound. None of the synthetic gangliosidoides equalled natural ganglioside in its ability to inhibit the effects of the toxin in vivo, but some did show considerable inhibitory activity ih monosialo-gangliotetraose or corresponding sialo-glycolipids prevents the easy degradation of the B-protein of cholera toxin into protein subunits by sodium dodecylsulfate.     

Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles.

We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol-containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin.     

Effect of late endosomal DOBMP lipid and traditional model lipids of electrophysiology on the anthrax toxin channel activity

Anthrax toxin action requires triggering of natural endocytic transport mechanisms whereby the binding component of the toxin forms channels (PA₆₃) within endosomal limiting and intraluminal vesicle membranes to deliver the toxin's enzymatic components into the cytosol. Membrane lipid composition varies at different stages of anthrax toxin internalization, with intraluminal vesicle membranes containing ~70% of anionic bis(monoacylglycero)phosphate lipid. Using model bilayer measurements, we show that membrane lipids can have a strong effect on the anthrax toxin channel properties, including the channel-forming activity, voltage-gating, conductance, selectivity, and enzymatic factor binding. Interestingly, the highest PA₆₃ insertion rate was observed in bis(monoacylglycero)phosphate membranes. The molecular dynamics simulation data show that the conformational properties of the channel are different in bis(monoacylglycero)phosphate compared to PC, PE, and PS lipids. The anthrax toxin protein/lipid bilayer system can be advanced as a novel robust model to directly investigate lipid influence on membrane protein properties and protein/protein interactions.     

A 50-kDa fragment from the NH2-terminus of the heavy subunit of Clostridium botulinum type A neurotoxin forms channels in lipid vesicles

1. A 50-kDa fragment representing the NH2-terminus of the heavy subunit of botulinum type A neurotoxin was found, at low pH, to evoke the release of K+ from lipid vesicles loaded with potassium phosphate. Similar K+ release was also observed with the intact neurotoxin, its heavy chain and a fragment consisting of the light subunit linked the 50-kDa NH2-terminal heavy chain fragment. The light subunit alone, however, was inactive. 2. In addition to K+, the channels formed in lipid bilayers by botulinum neurotoxin type A or the NH2-terminal heavy chain fragment were found to be large enough to permit the release of NAD (Mr 665). 3. The optimum pH for the release of K+ was found to be 4.5. Above this value K+ release rapidly decreased and was undetectable above pH 6.0. 4. The binding of radiolabelled botulinum toxin to a variety of phospholipids was assessed. High levels of toxin binding were only observed to lipid vesicles with an overall negative charge; much weaker binding occurred to lipid vesicles composed of electrically neutral phospholipids. 5. A positive correlation between the efficiency of toxin-binding and the efficiency of K+ release from lipid vesicles was not observed. Whereas lipid vesicles containing the lipids cardiolipin or dicetyl phosphate bound the highest levels of neurotoxin, the toxin-evoked release of K+ was low compared to vesicles containing either phosphatidyl glycerol, phosphatidyl serine or phosphatidyl inositol. 6. The implications of these observations to the mechanism by which the toxin molecule is translocated into the nerve ending are discussed.     

A functional domain on the alpha-latrotoxin molecule, distinct from the binding site, involved in catecholamine secretion from PC12 cells: identification with monoclonal antibodies

Seven monoclonal antibodies (mAbs) have been produced against alpha-latrotoxin (alpha-Latx), the toxin component of black widow spider venom that stimulates release of neurotransmitters from PC12 cells. These mAbs were characterized by an enzyme-linked immunosorbent assay and by neutralization analysis of the secretagogue properties of the toxin. The production of a panel of mAbs, possibly directed against different epitopes of alpha-Latx, provides a useful set of reagents to dissect the molecular regions of the toxin having different functions and to describe steps of its mode of action in responsive cells. Attention was focused on one of these mAbs (4C4.1), which inhibits in a dose-dependent fashion both toxin-stimulated and crude venom stimulated dopamine release from PC12 cells, prevents toxin-induced 45Ca2+ accumulation in PC12, alters toxin-dependent phosphoinositide breakdown, and prevents toxin-induced channel formation in artificial lipid bilayers. Since, within certain experimental conditions, mAb 4C4.1 is able to recognize the toxin bound to cells, we conclude that its effects were not a consequence of a direct interference with binding. On the basis of kinetic analysis of mAb interference on toxin action, expressed as accumulation of inositol phosphates and transmitter secretion, we suggest that the described effects result primarily from the blockade of an event immediately successive to binding and central for the full expression of toxin action. The availability of mAb 4C4.1 now makes possible the molecular characterization of the toxin moiety responsible for such an event.     

Vstx1, a modifier of Kv channel gating, localizes to the interfacial region of lipid bilayers

VSTx1 is a tarantula venom toxin which binds to the archaebacterial voltage-gated potassium channel KvAP. VSTx1 is thought to access the voltage sensor domain of the channel via the lipid bilayer phase. In order to understand its mode of action and implications for the mechanism of channel activation, it is important to characterize the interactions of VSTx1 with lipid bilayers. Molecular dynamics (MD) simulations (for a total simulation time in excess of 0.2 micros) have been used to explore VSTx1 localization and interactions with zwitterionic (POPC) and with anionic (POPE/POPG) lipid bilayers. In particular, three series of MD simulations have been used to explore the net drift of VSTx1 relative to the center of a bilayer, starting from different locations of the toxin. The preferred location of the toxin is at the membrane/water interface. Although there are differences between POPC and POPE/POPG bilayers, in both cases the toxin forms favorable interactions at the interface, maximizing H-bonding to lipid headgroups and to water molecules while retaining interactions with the hydrophobic core of the bilayer. A 30 ns unrestrained simulation reveals dynamic partitioning of VSTx1 into the interface of a POPC bilayer. The preferential location of VSTx1 at the interface is discussed in the context of Kv channel gating models and provides support for a mode of action in which the toxin interacts with the Kv voltage sensor "paddle" formed by the S3 and S4 helices.     

Lipid phase coexistence favors membrane insertion of equinatoxin-II, a pore-forming toxin from Actinia equina

Equinatoxin-II is a eukaryotic pore-forming toxin belonging to the family of actinoporins. Its interaction with model membranes is largely modulated by the presence of sphingomyelin. We have used large unilamellar vesicles and lipid monolayers to gain further information about this interaction. The coexistence of gel and liquid-crystal lipid phases in sphingomyelin/phosphatidylcholine mixtures and the coexistence of liquid-ordered and liquid-disordered lipid phases in phosphatidylcholine/cholesterol or sphingomyelin/phosphatidylcholine/cholesterol mixtures favor membrane insertion of equinatoxin-II. Phosphatidylcholine vesicles are not permeabilized by equinatoxin-II. However, the localized accumulation of phospholipase C-generated diacylglycerol creates conditions for toxin activity. By using epifluorescence microscopy of transferred monolayers, it seems that lipid packing defects arising at the interfaces between coexisting lipid phases may function as preferential binding sites for the toxin. The possible implications of such a mechanism in the assembly of a toroidal pore are discussed.     

Translocation of diphtheria toxin A-fragment to the cytosol. Role of the site of interfragment cleavage

Diphtheria toxin contains a trypsin-sensitive region with 3 closely spaced arginines in the sequence (Asn189, Arg190, Val191, Arg192, Arg193, Ser194). Cleavage of the toxin to yield A- and B-fragments ("nicking") appears to occur in a stochastic manner after either of these arginine residues. Isoelectric focusing of A-fragment prepared in vitro showed four bands of varying intensity with pI between 4.5 and 5.0, three of which could be accounted for by the three different cleavage sites. Exposure of cells with surface-bound toxin to pH less than 5.3 induces translocation of A-fragment to a position where it is shielded from external Pronase, presumably in the cytosol. A-fragment translocated in this manner had the same pI as the most acidic A-fragments, indicating that only A-fragments lacking both Arg192 and Arg193 are translocation-competent. This was confirmed by amino acid sequencing. Treatment of A-fragment with carboxypeptidase B eliminated the two bands with the highest pI while there was a concomitant increase in the bands corresponding to the two most acidic A-fragments. Such treatment of nicked diphtheria toxin increased the amount of translocated A-fragment and the ability of toxin to form cation-selective pores in the cell membrane. The site of trypsin cleavage therefore appears to be one of the factors limiting toxin entry to the cytosol.     

Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins

Computer analysis of the three-dimensional structure of ADP-ribosylating toxins showed that in all toxins the NAD-binding site is located in a cavity. This cavity consists of 16 contiguous amino acids that form an a-helix bent over β-strand. The tertiary folding of this structure is strictly conserved despite the differences in the amino acid sequence. Catalysis is supported by two spatially conserved amino acids, each flanking the NAD-binding site. These are: a glutamic acid that is conserved in all toxins, and a nucleophillc residue, which is a histidine in the diphtheria toxin and Pseudomonas exotoxin A, and an arginine in the cholera toxin, the Escherichia coli heat-labile enterotoxins, the pertussis toxin and the mosquitocidal toxin of Bacillus sphaericus. The latter group of toxins presents an additional histidine that appears important for catalysis. This structure suggests a general mechanism of ADP-ribosylation evolved to work on different target proteins.     

Solution structure and lipid membrane partitioning of VSTx1, an inhibitor of the KvAP potassium channel

VSTx1 is a voltage sensor toxin from the spider Grammostola spatulata that inhibits KvAP, an archeabacterial voltage-activated K(+) channel whose X-ray structure has been reported. Although the receptor for VSTx1 and the mechanism of inhibition are unknown, the sequence of the toxin is related to hanatoxin (HaTx) and SGTx, two toxins that inhibit eukaryotic voltage-activated K(+) channels by binding to voltage sensors. VSTx1 has been recently shown to interact equally well with lipid membranes that contain zwitterionic or acidic phospholipids, and it has been proposed that the toxin receptor is located within a region of the channel that is submerged in the membrane. As a first step toward understanding the inhibitory mechanism of VSTx1, we determined the three-dimensional solution structure of the toxin using NMR. Although the structure of VSTx1 is similar to HaTx and SGTx in terms of molecular fold and amphipathic character, the detailed positions of hydrophobic and surrounding charged residues in VSTx1 are very different than what is seen in the other toxins. The amphipathic character of VSTx1, notably the close apposition of basic and hydrophobic residues on one face of the toxin, raises the possibility that the toxin interacts with interfacial regions of the membrane. We reinvestigated the partitioning of VSTx1 into lipid membranes and find that VSTx1 partitioning requires negatively charged phospholipids. Intrinsic tryptophan fluorescence and acrylamide quenching experiments suggest that tryptophan residues on the hydrophobic surface of VSTx1 have a diminished exposure to water when the toxin interacts with membranes. The present results suggest that if membrane partitioning is involved in the mechanism by which VSTx1 inhibits voltage-activated K(+) channels, then binding of the toxin to the channel would likely occur at the interface between the polar headgroups and the hydrophobic phase of the membrane.     

Tetanus toxin induces fusion and aggregation of lipid vesicles containing phosphatidylinositol at low pH

We report here on the ability of tetanus toxin to induce, at low pH, fusion and aggregation of lipid vesicles containing phosphatidylinositol. It has been shown that diphtheria toxin is internalized in acidic vacuoles (endosomes) and that the low endosomal pH could induce a protein conformational change responsible for the interaction with the endosomal membranes and the toxin translocation into the cytoplasm. The data here reported indicate that tetanus toxin might interact with lipid membrane in a similar way as diphtheria toxin suggesting for the two proteins an identical mechanism of entry into cells.     

On the role of polysialoglycosphingolipids as tetanus toxin receptors. A study with lipid monolayers

Lipid monolayers of different compositions were used to study the interaction of tetanus toxin with membrane lipids and to evaluate the role of polysialoglycosphingolipids as membrane receptors. At neutral pH, the toxin binds to dioleoylglycerophosphocholine monolayers and inserts into the phospholipid layer. This effect is potentiated by acidic phospholipids without an apparent preference for a single class of phospholipids. Polysialoglycosphingolipids further increase the fixation and penetration of tetanus toxin in lipid monolayers, but no specific requirement for a particular ganglioside was identified. The ganglioside effect is abolished in the presence of other nervous tissue lipids: cerebrosides and glycosphingolipid sulfates are partially responsible for this effect. The penetration of tetanus toxin in the lipid monolayer is pH dependent. It increases with lowering pH, it is facilitated by acidic phospholipids and by glycosphingolipid sulfates and it is mediated both by hydrophobic and electrostatic interactions as deduced from an analysis of the effect of ionic strength. Fragment B of tetanus toxin the low-pH-driven lipid interaction of the toxin. On the basis of the present findings, the possible role of polysialoglycosphingolipids in the neurospecific binding of tetanus toxin is discussed.     

Prostaglandin and thromboxane in liposomes: suppression of the primary immune response to liposomal antigens

Liposomes containing lipid A as adjuvant and also containing prostaglandin E2 or thromboxane B2 were examined for the ability to influence induction of humoral immunity against liposomal protein or lipid antigens in rabbits. The protein antigen consisted of cholera toxin that was bound to ganglioside GM1 on the surface of the liposomes. High titers of anti-cholera toxin antibodies were produced and IgM and IgG responses were detected. When the immunizing liposomes contained either prostaglandin E2 or thromboxane B2 as part of the lipid bilayer, the primary immune response, involving both IgM and IgG antibodies, was greatly reduced. The secondary immune response observed after a boosting immunization was not suppressed by liposomal eicosanoids. A similar inhibitory effect on the primary response was observed when liposomal lipid antigens were examined instead of cholera toxin. An inhibitory effect of liposomal prostaglandin E2 on the phagocytic uptake of opsonized liposomes by cultured macrophages was also observed, suggesting that liposomal eicosanoids can have direct local effects on macrophages that might influence the immune response to liposomal antigens.     

Raft-targeting and oligomerization of Parasporin-2, a bacillus thuringiensis crystal protein with anti-tumour activity

Parasporin-2 is a newly classified Bacillus thuringiensis crystal toxin with strong cytocidal activities toward human liver and colon cancer cells. Similar to other insecticidal B. thuringiensis crystal toxins, parasporin-2 shows target specificity and damages the cellular membrane. However, the mode of parasporin-2 actions toward the cell membrane remains unknown. Here, we show that this anti-tumour crystal toxin targets lipid rafts and assembles into oligomeric complexes in the membrane of human hepatocyte cancer (HepG2) cells. Upon incubation with HepG2 cells, peripheral membrane-bound toxins, which were recovered in a low-density detergent-resistant membrane fraction, i.e. with lipid rafts, were transformed into heat-stable SDS-resistant membrane-embedded oligomers (approximately 200 kDa). The toxin oligomerization was dependent on temperature and coupled with cell lysis. The toxin oligomerization also occurred in a cell-free membrane system and was required for binding to membrane proteins, the lipid bilayer and cholesterols. These results indicate that parasporin-2 is an oligomerizing and pore-forming toxin that accumulates in lipid rafts.     

Clostridium botulinum C2 toxin. Identification of the binding site for chloroquine and related compounds and influence of the binding site on properties of the C2II channel

Clostridium botulinum C2 toxin belongs to the family of binary AB type toxins that are structurally organized into distinct enzyme (A, C2I) and binding (B, C2II) components. The proteolytically activated 60-kDa C2II binding component is essential for C2I transport into target cells. It oligomerizes into heptamers and forms channels in lipid bilayer membranes. The C2II channel is cation-selective and can be blocked by chloroquine and related compounds. Residues 303-330 of C2II contain a conserved pattern of alternating hydrophobic and hydrophilic residues, which has been implicated in the formation of two amphipathic beta-strands involved in membrane insertion and channel formation. In the present study, C2II mutants created by substitution of different negatively charged amino acids by alanine-scanning mutagenesis were analyzed in artificial lipid bilayer membranes. The results suggested that most of the C2II mutants formed SDS-resistant oligomers (heptamers) similar to wild type. The mutated negatively charged amino acids did not influence channel properties with the exception of Glu(399) and Asp(426), which are probably localized in the vestibule near the channel entrance. These mutants show a dramatic decrease in their affinity for binding of chloroquine and its analogues. Similarly, F428A, which represents the Phi-clamp in anthrax protective antigen, was mutated in C2II in several other amino acids. The C2II mutants F428A, F428D, F428Y, and F428W not only showed altered chloroquine binding but also had drastically changed single channel properties. The results suggest that amino acids Glu(399), Asp(426), and Phe(428) have a major impact on the function of C2II as a binding protein for C2I delivery into target cells.     

Morphological abnormalities, neonatal mortality, and reproductive abnormalities in mice transgenic for diphtheria toxin genes that are driven by the promoter for adipocyte lipid binding protein.

Transgenic mice were used in an experiment that was designed to serve as a model of a possible approach to reducing the amount of carcass fat in meat animals. The objective was to reduce the number of adipocytes in transgenic mice thereby restricting the capacity to accumulate lipid. Our approach employed the technique of genetic ablation. The promoter for the adipocyte lipid binding protein gene was used in an attempt to direct expression of diphtheria toxin genes specifically to adipocytes. Three diphtheria toxin genes were used; they encode, respectively, an extremely cytotoxic wild type toxin, a less toxic attenuated toxin, and a nonfunctional toxin. While it was not possible to accurately assess effects of the transgenes on lipid accumulation, several informative observations were noted. A large percentage of transgenic founder mice that harbor either wild type or attenuated toxin genes are morphologically abnormal, die as neonates, or exhibit reproductive abnormalities including sterility or failure to transmit the transgene to offspring. In contrast, mice that harbor the nonfunctional toxin gene or are nontransgenic rarely have these same abnormalities. These results suggest that the transgenic mice are expressing the transgenes in cells other than adipocytes and that the aberrant production of functional toxin is responsible for the congenital abnormalities. The production of morphological and reproductive abnormalities in transgenic animals should be useful for investigating normal developmental processes.