.! , ...... 1.. .., ., ------..ll, - .:U, . 1 4. M-1 W1411.'AN.4"lW*v,1 or,-Al11 m-, R a...... ' the refolding of the short-time denatured 3. R. J. Ellis and S. M. van der Vies, Annu. Rev. 19. H. R. B. Pelham, Cell 46, 959 (1986). Biochem. 60, 321 (1991). 20. G. C. Flynn, J. Pohl, M. T. Flocco, J. E. Rothman, enzyme, but that a fraction of the prolines 4. F. X. Schmid, Curr. Opin. Struct. Biol. 1, 36 (1991). 353, 726 (1991). must be very rapidly buried during the 5. G. Fischer and F. X. Schmid, Biochemistry 29, 21. P.-0. FreskgArd, U. Carlsson, L. G. MArtensson, initiation of the refolding process (pB in 2205 (1990). B.-H. Jonsson, FEBS Lett. 289, 117 (1991). 6. A. E. Eriksson, T. A. Jones, A. Uljas, Proteins 22. R. G. Khalifah, D. J. Strader, S. H. Bryant, S. M. Fig. 3) and thus become inaccessible to Struct. Funct. Genet. 4, 274 (1988). Gibson, Biochemistry 16, 2241 (1977). PPIase. It has been reported (10-12) that 7. C. Fransson et al., FEBS Lett. 296, 90 (1992). 23. L. E. Henderson and D. Henriksson, Anal. Bio- an initiation structure of carbonic anhy- 8. J. L. Cleland and D. I. C. Wang, Biochemistry 29, chem. 51, 288 (1973). 11072 (1990). 24. G. Fischer, H. Bang, C. Mech, Biomed. Biochim. drase is rapidly formed during refolding. 9. J. Buchner et al., ibid. 30, 1586 (1991). Acta 43, 1101 (1984). When the prolines in this core of the 10. U. Carlsson, R. Aasa, L. E. Henderson, B.-H. 25. N. Takahashi, T. Hayano, M. Suzuki, Nature 337, enzyme structure (PB in Fig. 3) are cis-trans Jonsson, S. Lindskog, Eur. J. Biochem. 52, 25 473 (1989). by 1 hour of denaturation, the (1975). 26. G. Fischer, B. Wittmann-Liebold, K. Lang, T. Kief- equilibrated 11. N. Bergenhem, U. Carlsson, J.-A. Karlsson, nt. J. haber, F. X. Schmid, ibid., p. 476. isomerization of these residues to the native Pept. Protein Res. 33, 140 (1989). 27. Supported by grants from the Swedish National conformers limits the rate of reactivation 12. 0. B. Ptitsyn, FEBS Lett. 285, 176 (1991). Board for Technical Development (732-88-04392) = 4 min) in the presence of PPIase. 13. T. Kiefhaber, R. Quaas, U. Hahn, F. Schmid, (U.C. and B.-H.J.), the Swedish Natural Science (ti/2 Biochemistry 29, 3061 (1990). Research Council (K-Ku 9426-300) (B.-H.J.), Mag- However, when the prolines in this core are 14. R. K. Harrison and R. L. Stein, ibid., p. 1684. nus Bergvalls Stiftelse (B.-H.J.), and Carl Tryggers in correct conformation from the beginning 15. R. Wolfenden and A. Radzicka, Chemtracts Bio- Stiftelse (U.C.). Fellowships to P.-OF. and M.S. from of refolding and all the other prolines are chem. Mol. Biol. 2, 52 (1991). Stiftelsen Bengt Lundqvists Minne are also gratefully 16. T. Drakenberg, K.-. Dahlqvist, S. J. Fors6n, J. acknlaedged. We are indebted to Sandoz Ltd. for cis-trans equilibrated by the action of PPIase Phys. Chem. 76, 2178 (1972). their generous gift of CsA. only during the first 3 s of refolding, uncat- 17. R. Wolfenden, Science 222, 1087 (1983). alyzed isomerization of the PPIase-accessi- 18. J. Kallen et al., Nature 353, 276 (1991). 16 April 1992; accepted 27 July 1992 ble prolines (PA; Fig. 3) is rate determining (t1/2 = 5 min) in the reactivation process. Downloaded from Thus, the action of PPIase on the refold- ing HCA II reveals the presence of two Spontaneous and Arterial classes of prolines (accessible and buried), Lesions in Mice Lacking as evidenced by the impact of PPIase on the refolding kinetics. For ribonuclease T1, the Sunny H. Zhang, Robert L. Reddick, Jorge A. Piedrahita, structural context of prolines is important Nobuyo Maeda* http://science.sciencemag.org/ for the efficiency of catalysis (13). That PPIase acts as a classical chaperone is clear- Apolipoprotein E (apoE) is a ligand for receptors that clear remnants of chylomicrons and ly demonstrated also by its independent very low density . Lack of apoE is, therefore, expected to cause accumulation effect on the kinetics and the yield of the in plasma of cholesterol-rich remnants whose prolonged circulation should be atherogenic. refolding reaction. This effect is exemplified ApoE-deficient mice generated by were used to test this hypothesis and to in Fig. 1: after 10 s of refolding, inhibition make a mouse model for spontaneous . The mutant mice had five times of PPIase still gives rise to a 100% yield and normal plasma cholesterol, and developed foam cell-rich depositions in their proximal a relatively slow tl/2 (9 min; chaperone), aortas by age 3 months. These spontaneous lesions progressed and caused severe whereas addition of PPIase after 10 s of occlusion of the coronary artery ostium by 8 months. The severe yet viable phenotype of refolding does not increase the yield but the mutants should make them valuable for investigating genetic and environmental factors on April 28, 2020 does result in a shorter t1,2 (4 min; that modify the atherogenic process. isomerase). Two mechanisms proposed for the ac- tion of PPIase are catalysis by distortion (14) and catalysis by desolvation (15-17). Atherosclerotic cardiovascular disease, the its most important functions is to serve as a The latter mechanism was suggested be- major cause of death in Western society, high affinity ligand for the apoB and cause binding of a peptide segment into a results from complex interactions among apoE(LDL) receptor and for the chylomi- hydrophobic environment, as in the bind- multiple genetic and environmental factors cron-remnant receptor, thereby allowing ing site of PPIase (18), promotes cis-trans (1). Among the factors that have been the specific uptake of apoE-containing par- isomerization by decreasing the charge sep- identified to date are changes in the genes ticles by the liver. aration of the peptide bond and thus creat- involved in lipid metabolism, including the A frequent genetic variant of human ing a peptide that has a more single-bond gene encoding apolipoprotein E (apoE) (2). apoE, apoE-2, differs from the most com- character. Hence, if this in fact is the ApoE is a glycoprotein with a molecular size mon form, apoE-3, by having cysteine at mechanism for PPIase activity, then other of approximately 34 kD that is synthesized position 158 in place of arginine. This chaperones that bind peptide chains to in the liver, brain, and other tissues in both amino acid substitution in the LDL receptor apolar sites (19, 20) might also possess humans and mice (3); it is a structural binding region reduces the binding ability isomerase activity. Because PPIase (which component of all particles other of apoE-2 to less than 2% relative to that of is also cyclophilin) is known to be involved than low density lipoprotein (LDL). One of apoE-3. Homozygosity for the gene Apoe2 is in T cell activation, it is possible that the associated with type III hyperlipoproteine- chaperone function of this protein is impor- S. H. Zhang and R. L. Reddick, Department of Pathol- mia, which is characterized by increased ogy, University of North Carolina at Chapel Hill, Chap- plasma triglyceride and cholesterol levels, tant for essential protein-folding processes el Hill, NC 27599-7525. in the immune response. N. Maeda, Department of Pathology and Curriculum in yellow lipid-laden xanthomatous skin nod- Genetics and of the Program in Molecular Biology and ules, and the early development of athero- Biotechnology, University of North Carolina at Chapel sclerosis (4). The complexities of the REFERENCES AND NOTES Hill, Chapel Hill, NC 27599-7525. J. A. Piedrahita, Department of Veterinary Anatomy pathogenesis of this disease are, however, 1. R. Jaenicke, Prog. Biophys. Mol. Biol. 49, 117 and Public Health, Texas A&M University, College illustrated by the fact that only about 2% of (1987). TX 77840-4458. 2. M.-J. Gething and J. Sambrook, Nature 355, 33 Station, Apoe2/Apoe2 individuals develop hyperlip- (1992). *To whom correspondence should be addressed. oproteinemia. The majority of individuals 468 SCIENCE * VOL. 258 * 16 OCTOBER 1992 with this genotype have, in fact, lower than tion of these results was obtained by agarose pre-P positions were markedly increased. normal plasma cholesterol levels (5). Thus, gel electrophoresis of whole plasma (8). In Thus, the distribution of lipids in the plas- other factors must be necessary for the normal mouse plasma, HDL, which is the ma of the mutants was shifted toward the expression of type III hyperlipoproteinemia major lipoprotein, migrates in the same region where lipoproteins of lower density associated with apoE-2. position as a globulins. VLDL and LDL, normally migrate. Three human kindreds with inherited which migrate more slowly at the pre-P and To better characterize the molecular ba- apoE deficiency have been reported (6). P positions, respectively, are minor compo- sis for the changes associated with the Individuals deficient in apoE, have tubero- nents (9). In the mutant plasma, particles absence of apoE, we determined the apo- eruptive xanthomatosis and type III hyper- that migrate at the at position were reduced lipoprotein compositions of plasma lipopro- lipoproteinemia with elevated cholesterol while those migrating between the P and teins. The lipoprotein particles were first and near normal triglyceride levels. ApoE-deficient mice were generated by inactivating the Apoe gene by targeting (7). Table 1. Plasma cholesterol and triglyceride levels in apoE-deficient mice. Mice of both sexes, 2 to Mice homozygous or heterozygous for the 5 months of age, were kept on regular mouse chow (ProLab formula 3000, 4.5% crude fat). After an disrupted Apoe gene appeared healthy. No overnight fast, blood (about 300 p1) was drawn by retro-orbital bleeding and EDTA, gentamicin sulfate, and aprotinin (Sigma) were added to final concentrations of 2 mM, 50 mg/mI (w/v) and 0.046 difference in their body weights compared trypsin inhibitor unit per milliliter, respectively. Plasma was separated by centrifugation at 14,000g to normal mice was observed. Matings be- for 10 min. at 40C. Cholesterol and triglyceride levels were determined by enzymatic reaction tween homozygotes, between heterozy- followed by spectrophotometric quantitation with 1 gi of plasma and 100 p1 of commercially gotes, and between homozygotes and het- available reagents (cholesterol 20; triglyceride 10; Sigma). HDL cholesterol was measured after erozygotes all yield litters of normal size. No removing the apoB-containing lipoproteins by MgCI2-dextran sulfate precipitation (18). For sex-versus-genotype bias was observed in hyperlipidemic plasma, a 1:2 dilution was made with phosphate-buffered saline prior to precipita- tion for the HDL cholesterol test. When multiple measurements were done on a single animal, Downloaded from the litters, indicating that the mutants had average values were used to derive the overall mean value. The numbers of animals tested are no apparent reproductive problems. How- given in parentheses. ever, significant phenotypic differences be- tween normal animals and the homozygous Animals Total cholesterol HDL cholesterol Triglyceride in mutants were observed in their lipid and in mg/dl + SD (n) in mg/dl + SD (n) mg/dl ± SD (n) lipoprotein profiles (Table 1). The apoE- deficient mice had markedly increased total Normal 86 ± 20 (46) 73 ± 28 (34) 73 ± 36 (32) http://science.sciencemag.org/ plasma cholesterol levels (434 ± 129 mg/ Heterozygous 88 ± 22 (47) 75 ± 18 (29) 102 ± 40 (41) dl), which were five times those of normal Homozygous 434 ± 129 (40) 33 ± 15 (28) 123 ± 51 (42) litter mates (86 ± 20 mg/dl). These levels were unaffected by the age or sex of the animals. Although the total plasma choles- Fig. 1. Cholesterol concentrations in lipoprotein 0.2C terol levels were greatly elevated in the fractions of normal and apoE-deficient mouse VLDL IDL LDL HDL mutants, the HDL (high density lipopro- after Superose 6 column chromatography of 0.15- tein) cholesterol levels, measured after re- total lipoprotein. Total lipoproteins (density E moving apolipoprotein B-containing parti- <1.21 g/ml) from 50 gi of fasting plasma were

isolated by ultracentrifugation (10), applied to a 0.10 were 45% the normal level < on April 28, 2020 cles, only [P Superose 6 (Sigma) column (1 cm by 30 cm), 0 0.0005 (8) by Student's t test for unpaired and eluted at a constant flow rate of 0.4 mI/min 0.05- observations]. The triglyceride levels were with 10 mM tris-HCI buffer, pH 7.4, containing 68% higher than those of normal animals 0.15 M NaCI, 0.01% (wh) EDTA, and 0.02% o.oc0 (P < 0.0005). Marked increase in total (w/v) NaN3. Fractions of 0.32 ml were collected. 0 10 20 30 40 cholesterol accompanied by near normal A portion of each fraction (0.05 ml) was dried Fraction number triglyceride levels was also observed in hu- completely, dissolved in 100 ,u of cholesterol-testing reagent (Sigma), and incubated at 370C for 5 man patients with apoE deficiency (6). In min. Absorbance at 500 nm provides a measure of the relative amounts of cholesterol in each fraction. mice heterozygous for the mutant allele, Open circles, normal mouse; closed circles, homozygous mutant. The approximate positions where cholesterol levels were the same as normal the major human lipoprotein classes normally elute are indicated. animals, but triglyceride levels were in- creased by about 39% (P < 0.005). Differences in the lipoprotein profiles of Fig. 2. SDS-polyacrylamide gel electrophore- VLDL IDL mutants from those of nor- sis of VLDL and IDL fractions from fasting the homozygous plasma from heterozygous mutants (+/-), ho- mal and heterozygous animals were revealed mozygous mutants (-I-), and normal mice+/ ++ +1 1 ++ by fractionating total lipoproteins from (+/+). Plasma (100 p1 each) from six mice B-100_ plasma by gel filtration chromatography was pooled and lipoprotein fractions were B-48~~ (Fig. 1). HDL was the major cholesterol- separated by sequential flotation (10) in a carrying lipoprotein in the plasma of nor- TL-100 bench-top ultracentrifuge with a TLA mal mice; lipoproteins with sizes that cor- 100.3 rotor (Beckman), at 100,000 rpm for 2 respond to VLDL (very low density lipopro- hours for VLDL, IDL, and LDL, and for 4 hours tein), IDL (intermediate density lipopro- for HDL. Each fraction (500 p1) was concen- A- I- and LDL were found in trace trated and desalted by means of a Centricon- tein), only 10 filter apparatus (Amicon). Samples equiv- amounts. In the mutants lacking apoE, the alent to 200, 20, and 100 ,u of original plasma - particles with sizes in the range of HDL from heterozygotes, homozygotes, and nor- were reduced, and nearly 80% of the total mal animals, respectively, were denatured and separated by electrophoresis on a gradient cholesterol was carried in particles having polyacrylamide gel (4 to 20%, Bio-Rad) at 150 V for 45 min. Proteins were stained with Coomassie sizes that are similar to the lower density brilliant blue. Apolipoprotein Cs and apolipoprotein All were not retained in this gel, but there lipoproteins of normal humans. Confirma- were no notable differences in these proteins in other gels. SCIENCE * VOL. 258 * 16 OCTOBER 1992 469 and low levels ofLDL, in contrast to humans who are high in LDL and low in HDL. In addition, mice apparently lack the cho- lesteryl ester transfer protein, an enzyme that transfers cholesterol ester from HDL to VLDL and LDL (13). Despite these differ- ences, apoE-deficient mice have phenotypes remarkably similar to those of apoE-deficient humans. ApoE-deficient individuals in both species have hypercholesterolemia, near normal triglyceride levels, decreased HDL, a large accumulation of particles in the VLDL and IDL size ranges, and a typical broad i c band on agarose gel electrophoresis. They both have an elevated percentage of apoB- 48 in apoB-containing particles, and apoA-I go .*-; and apoA-IV is found in particles in the VLDL density range. lAY Evidence of fatty streaks in the proximal aorta was found in a homozygous mutant mouse that was killed at 3 months of age. .c Downloaded from No pathological changes were noted in the distal aorta and proximal iliac vessels. Lipid - # deposition was found in the liver but not in -if, kidney, lung, or spleen. To further docu- ment the development of the arterial le- Fig. 3. Transverse frozen sections of the proximal aorta stained with Sudan IVB and countterstained sions, a histological study of the proximal with Harris hematoxylin. (A) A section of the aortic sinus of a 5-month-old male hete,rozygote, aortic region (14) of six sets of age-matched http://science.sciencemag.org/ showing the valve attachment sites as outgrowths of smooth muscle cells from the vessel \wall. Final normal, heterozygous and homozygous ani- magnification, x26. (B) An equivalent section of a 5-month-old male homozygote, x,26. Small mals was performed. These animals were 3 arrowheads indicate some of the multilayered intimal foam cell deposits. The entrance to a coronary artery can be seen as an evagination of the aortic wall. (C) A longitudinal section of the to 8 months of age and had been fed normal artery of a 5-month-old normal male, x260, showing normal architecture of the ostiunnco(D) Anry mouse chow (4.5% crude fat). Examination equivalent section of an 8-month-old female homozygote, x260, showing near total occlusLion at the of sections of the proximal aorta showed no ostium of a coronary artery. Abbreviations: ao, aorta; co, coronary artery. abnormalities in the normal and heterozy- gous animals. The luminal surface of the aorta in normal mice was smooth, and separated by sequential density flotation mans, produce B-48 in the liver (1 !1). smooth muscle cells were orderly arranged into the four density ranges that would The significance of the presencie of con- (Fig. 3A). The coronary ostium was free of on April 28, 2020 normally include VLDL, IDL, LDL, and siderable amounts ofapoA-I and apeoA-IV in obstructions (Fig. 3C). In contrast, exami- HDL particles (10). The apolipoprotein the particles with VLDL and IDIL density nation of the same region in all the female compositions of these fractions were then ranges in the mutants remains to 1be deter- and male homozygous mutant animals re- investigated by SDS-polyacrylamide gel elec- mined; in normal humans these ap(olipopro- vealed fatty streak formation. Intimal foam electrophoresis (Fig. 2). Substantial changes teins are present in newly secreted chylomi- cell deposits were present diffusely through- were seen in the apolipoprotein contents cron but are negligible in VLDL atid chylo- out the area of the aortic sinus as illustrated of the VLDL and IDL fractions, while micron remnants (3). Our observattion that in a section of a 5-month-old male (Fig. changes in the LDL and HDL fractions the mutants have about half norrnal HDL 3B). These deposits varied from small col- were minor. As expected, apoE was pre- cholesterol levels also requires c(omment. lections oflipid-filled cells to raised deposits sent in normal and heterozygous mice, but We note that the total amount of apoA-I in within the intima. The small foam cell was absent in the homozygous mutants. the plasma of mutant and normal1 mice is deposits were most often found adjacent to The very low density fraction in the mu- about the same (8) when evaltuated by valve attachment sites, and frequently cov- tants contained substantial amounts of nonreducing SDS-polyacrylamide gel elec- ered this area. Multilayered foam cell de- apoA-I and apoA-IV, both of which were trophoresis (12). Additionally, apoA-I posits were also present between the valve low in this fraction from normal and mRNA levels in the liver and inttestine of attachment sites, and were often in the heterozygous animals. In addition, this, the mutants were approximately thee same as form of continuous deposits. Foam cells fraction had a much greater ratio (about in normal mice (8). Thus, it appearS that the were not found on the valve leaflets. Lipid 20:1) of apoB-48 to apoB-100 than in decreased level of HDL in the honnozygotes was occasionally present in the superficial normal animals (about 1:1); heterozygotes is neither because of reduced synt:hesis nor media. In some areas, the surface of the had essentially normal ratios of apoB-48 to because of accelerated catabolism of apoA-I. lesion was flattened, suggesting an early apoB-100. These results suggest that the Perhaps the apoA-I is redistributedA to rem- stage of fibrous cap formation. The lesions particles accumulated in the very low den- nant particles as a structural comp)onent in progressed with age, and near total occlu- sity fraction of the mutants (Fig. 1) were not the absence of apoE, thereby limLiting the sion at the entrance of a coronary artery was normal VLDL but are likely to have been amount of apoA-I available for HE)L forma- seen in the section of an 8-month-old remnants of chylomicron or VLDL. Rem- tion in the mutants. The reduc;ed HDL female homozygote (Fig. 3D). At this stage, nants of these types in mice are expected to levels may also reflect the absence of apoE- the lesions appeared to contain less lipid be rich in cholesterol and to have apoB-48 as containing HDL particles. but more elongated cells, which represents a major component since mice, unlike hu- Mice naturally have high level

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The presence of these pre-atherosclerot- 10. V. N. Schumaker and D. L. Puppione, Methods perature. Sections were stained to detect lipid Enzymol. 128,155 (1986); R. J. Havel, H. A. Eder, with Sudan IVB followed by counter-staining with ic lesions in the proximal aorta demon- J. H. Bragdon, J. Clin. Invest. 55, 1345 (1955). Harris hematoxylin. strates that a simple lack of apoE is suffi- 11. A. J. Lusis, B. A. Taylor, D. Quon, S. Zollman, R. C. 15. M.-C. Camus, M. J. Chapman, P. Forgez, P. M. cient to initiate atherogenesis. Although LeBoeuf, J. Biol. Chem. 262, 7594 (1987). Laplaud, J. Lipid Res. 24, 1210 (1983); R. S. 12. D. S. France et al., J. Lipid Res. 30, 1997 (1989). Weibust, Genetics 73, 303 (1973). different strains of mice exhibit heritable 13. S. Jiao, T. G. Cole, R. T. Kitchens, B. Pfieger, G. 16. B. Paigen, A. Morrow, C. Brandon, D. Mitchell, P. differences in the levels of their circulating Schonfeld, Metabolism 39, 155 (1990). Holmes, Atherosclerosis 57, 65 (1985); J. D. Mor- lipids (15), no inbred strains are known to 14. After overnight fast, mice were killed with an risett, H.-S. Kim, J. R. Patsch, S. K. Datta, J. J. overdose of avertin (2,2,2-tribromoethanol) and Trentin, Arteriosclerosis 2, 312 (1982); A. Roberts develop lesions spontaneously on a normal the vascular tree was perfused via the heart with and J. S. Thompson, Adv. Exp. Med. Biol. 67, 313 diet. However, they do show significant 4% (wNV) paraformaldehyde in 0.12 M phosphate (1976). differences in their tendencies to form ath- buffer, pH 7.4. The heart and entire aorta, includ- 17. The embryonic stem (ES) cells we used for intro- erosclerotic when on a ing the iliacs, were removed intact and immersed ducing a modification into Apoe were derived plaques placed high in fresh paraformaldehyde. Prior to cryostat sec- from strain 129/Ola. Chimeras made from these fat diet (16). Strain C57BL/6 is more sus- tioning, the heart and an approximately 0.5-cm ES cells were crossed with strain C57BL/6J mice, ceptible to atherogenic diets than others length of ascending aorta was removed from the and F1 offspring that were heterozygous for the that have been tested and strain 129/J is remainder of the aorta. The heart was divided into modified gene were obtained. F2 mice homozy- two halves by cutting transversely along a line gous for the mutation were then generated by moderately susceptible (16). The apoE-de- parallel to the tip of the atria. The half with the crossing two F1 heterozygotes. The genetic back- ficient mice we studied here are mainly F2 attached aortic segment was then placed in OCT ground of an F2 animal is therefore a discrete animals that have a combination of the compound (lissue-Tex), gently compressed to blending of the two strains that is unique to each remove bubbles, positioned in semi-frozen OCT particular animal. genetic backgrounds of both C57BL/6 and to allow cross sectioning of the aorta, and then 18. G. R. Wamick, J. Benderson, J. J. Albers, Clin. 129/a; the precise composition is unique for frozen in OCT compound on a metal pedestal Chem. 28,1379 (1982). each individual animal (17). This genetic prior to sectioning. Eight micrometer-thick frozen 19. We are grateful toO. Smithies for discussions and sections at 24-pm intervals were made beginning critical reading of the manuscript. We thank C. M. heterogeneity may cause some variability with the ascending aorta and proceeding through Sandlin, B. Gibbs, and J. Reynolds for their help in Downloaded from among different individuals in their plasma the entire aortic sinus until the ventricular cham- maintaining and genotyping mice. Supported by lipoprotein phenotypes and in the likeli- ber was reached. Adequacy of the frozen section- an NIH grant HL42630 to N.M. and by a Depart- ing process was monitored by light microscopic ment of Pathology grant to R.L.R. hood of their developing arterial lesions. evaluation of unstained sections. The frozen sec- However, all 40 mice lacking apoE had tions were allowed to dry overnight at room tem- 16 July 1992; accepted 14 September 1992 elevated total plasma cholesterol levels,

and all six that we have studied histologi- http://science.sciencemag.org/ cally had lesions in their proximal aorta. None of these changes were observed in a a normal and heterozygous litter mates. Formation of Gated Channel by Ligand-Specific The early development of lesions in Transport Protein in the Bacterial Outer Membrane mice lacking apoE makes them of great practical value. The combined phenotype Jeanette M. Rutz, Jun Liu, Jeri Ann Lyons, Joanne Goranson, of the homozygotes (high cholesterol and Sandra K. Armstrong, Mark A. Mcintosh, Jimmy B. Feix, early development of non-lethal lesions) Phillip E. Klebba* provides a baseline against which either detrimental or protective genetic and envi- The ferric enterobactin receptor (FepA) is a high-affinity ligand-specific transport protein ronmental factors can be investigated. in the outer membrane of Gram-negative bacteria. Deletion of the cell-surface ligand- on April 28, 2020 These mice should also be of value for use binding peptides of FepA generated mutant proteins that were incapable of high-affinity as an in vivo test system for studies of uptake but that instead formed nonspecific, passive channels in the outer membrane. pharmacological or genetic treatments of Unlike native FepA, these pores acted independently ofthe accessory protein TonB, which hyperlipidemia. suggests that FepA is a gated porin and that TonB acts as its gatekeeper by facilitating the entry of ligands into the FepA channel. The sequence homology among TonB-de- REFERENCES AND NOTES pendent proteins suggests that all ligand-specific outer membrane receptors may function 1. K. Berg, in Progress in Medical Genetics, A. G. by this gated-porin mechanism. Steinberg, A. G. Bearn, A. G. Motulsky, B. Childs, Eds. (Saunders, Philadelphia, 1983), vol. 5, pp. 35-90. 2. J. L. Breslow, Annu. Rev. Med. 42, 357 (1991); S. M. Grundy, Arterioscier. Thromb. 11, 1619 (1991); The cell envelope of Gram-negative bac- ligands (1)-and multicomponent, requir- A. J. Lusis, J. Lipid Res. 29, 397 (1988): G. S. teria contains high-affinity, ligand-specific ing the participation of periplasmic and Getz, T. Mazzone, P. Soltys, S. R. Bates, Arch. outer membrane proteins that translocate cytoplasmic membrane proteins (2). TonB Pathol. Lab. Med. 112,1048 (1988). 3. R. W. Mahley, T. L Innerarity, S. C. Rall, Jr., K. H. substrates into the periplasm. Such trans- is the most notable of these accessory pro- Weisgraber, J. Lipid Res. 25, 1277 (1984). port systems are multifunctional-a single teins; it resides in the cytoplasmic mem- 4. R. W. Mahley and S. C. Rail, Jr., in The Metabolic outer membrane protein usually serves as brane but is thought to project across the Basis of Inherited Disease, C. R. Scriver, A. L. Beaudet, W. S. Sly, D. Valle, Eds. (McGraw-Hill, the surface receptor for several dissimilar periplasmic space and facilitate the trans- New York, ed. 6, 1989), pp. 1195-1213. port function of outer membrane receptors 5. G. Uterrnann, Adv. Exp. Med. Bid. 201, 261 (1986). J. M. Rutz, J. A. Lyons, J. Goranson, P. E. Klebba, by direct, protein-to-protein interactions 6. G. Ghiselli, E. J. Schaefer, P. Gascon, H. B. Department of Microbiology, Medical College of Wis- (3). Brewer, Jr., Science 214, 1239 (1981); D. Kuro- consin, Milwaukee, WI 53226. Ligand-specific receptor proteins have saka et al., Atherosclerosis 88, 15 (1991); H. J. Liu and J. B. Feix, Department of Biophysics, Mabuchi et al., Metabolism 38, 115 (1989). Medical College of Wisconsin, Milwaukee, WI 53226. been considered to be distinct from porins, 7. J. A. Piedrahita, S. H. Zhang, J. R. Hagaman, P. S. K. Armstrong, Department of Microbiology and which form nonspecific, hydrophilic chan- M. Oliver, N. Maeda, Proc. NatI. Acad. Sci. U.S.A. Immunology, East Carolina University School of Med- nels in the outer membrane (4). Porins 89, 4471 (1992). icine, Greenville, NC 27858. 8. S. Zhang et al., unpublished data. M. A. Mcintosh, Department of Molecular Microbiol- contain amphiphilic E strands assembled in 9. R. C. LeBoeuf, D. L. Puppione, V. N. Schumaker, ogy and Immunology, University of Missouri Medical the form of a 13 barrel (5), which acts as a A. J. Lusis, J. Biol. Chem. 258, 5063 (1983); P. N. MO 65212. M. Demacker, H. E. Vos-Janssen, A. van't Laar, A. School, Columbia, molecular sieve that equilibrates small mol- P. Jansen, Clin. Chem. 24, 1439 (1978). *To whom correspondence should be addressed. ecules (<600 daltons) across the bilayer SCIENCE * VOL. 258 * 16 OCTOBER 1992 471 Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E SH Zhang, RL Reddick, JA Piedrahita and N Maeda

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