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◥ for the mutations, we narrowed potential can- RESEARCH ARTICLE didates to seven SNVs, including a heterozy- gous frameshift (fs) deletion in the LIM domain and actin binding 1 (LIMA1)gene(LIMA1: METABOLISM NM_001113546:exon7:c.916_923del:p.K306fs) on chromosome 12 (fig. S1). LIMA1 is also named as epithelial protein lost in neoplasm (EPLIN) or A LIMA1 variant promotes low plasma sterol regulatory element binding protein 3 (SREBP3). LIMA1 shows little sequence sim- LDL cholesterol and decreases ilarity to SREBP-1 or -2 but is a homolog of an unknown gene that fused with the N-terminal intestinal cholesterol absorption domain of SREBP2 in SRD-2 cells (12). The func- tion of LIMA1 in lipid metabolism has not been described to date. Ying-Yu Zhang1,2*, Zhen-Yan Fu3*, Jian Wei2*, Wei Qi4, Gulinaer Baituola3, Jie Luo2, We validated the seven variants by Sanger Ya-Jie Meng3, Shu-Yuan Guo4,5, Huiyong Yin4,5, Shi-You Jiang2, Yun-Feng Li2, sequencing and found LIMA1-K306fs SNV to be Hong-Hua Miao1, Yong Liu2, Yan Wang2, Bo-Liang Li1, the only one that cosegregated with the low LDL-C Yi-Tong Ma3†, Bao-Liang Song2† phenotype within this family (Fig. 1, A and B). Genome-wide linkage analysis on nine members A high concentration of low-density lipoprotein cholesterol (LDL-C) is a major risk factor of Family 1 revealed that LIMA1, but not the for cardiovascular disease. Although LDL-C levels vary among humans and are heritable, other six candidate SNVs, was located in the the genetic factors affecting LDL-C are not fully characterized. We identified a rare chromosomal regions with a logarithm of odds Downloaded from frameshift variant in the LIMA1 (also known as EPLIN or SREBP3) gene from a Chinese (LOD) score > 1.5 (fig. S2). Together, these analyses family of Kazakh ethnicity with inherited low LDL-C and reduced cholesterol absorption. In suggest the LIMA1-K306fs SNV as the responsible a mouse model, LIMA1 was mainly expressed in the small intestine and localized on the variant for low LDL-C. The genomic structure of brush border membrane. LIMA1 bridged NPC1L1, an essential protein for cholesterol LIMA1 isshowninFig.1C. absorption, to a transportation complex containing myosin Vb and facilitated cholesterol Detailed information about Family 1 is listed Lima1 intableS1.Theplasmatotalcholesterol(TC)and uptake. Similar to the human phenotype, -deficient mice displayed reduced http://science.sciencemag.org/ cholesterol absorption and were resistant to diet-induced hypercholesterolemia. Through LDL-C levels of LIMA1-K306fscarriers(+/K306fs) our study of both mice and humans, we identify LIMA1 as a key protein regulating intestinal were significantly lower than those of wild-type cholesterol absorption. (WT) (+/+) individuals (Fig. 1, D and E). However, the plasma levels of triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and glucose were ardiovascular disease (CVD) is the leading attributed to the common single-nucleotide var- similar in both LIMA1-K306fs and WT individuals cause of death worldwide (1), and a high iants (SNVs) identified by genome-wide associ- (Fig. 1, F to H). Given that LDL-C is influenced concentration of plasmalow-densitylipo- ation studies (GWASs). Together, these analyses by both endogenous cholesterol biosynthesis protein cholesterol (LDL-C) is a major risk suggest that the genetic factors influencing LDL-C and intestinal cholesterol absorption, we used C – factor (2). LDL-C concentration is a com- have not been fully characterized. gas chromatography mass spectrometry (GC-MS)

plex trait that is influenced by environmental Because the majority of human genetic var- to measure the campesterol:lathosterol ratio on June 7, 2018 and genetic factors. About 40 to 50% of the iants are rare and vary among populations with (Ca:L ratio) (13) to estimate relative cholesterol phenotypic variances of LDL-C are due to genetic different demographic histories (9), examination absorption in the plasma of Family 1 members. factors (3), including mutations in the LDL re- of a diverged population may help to identify We found that the LIMA1-K306fs mutation was ceptor (LDLR)(4), autosomal recessive hyper- additional susceptible variants. In our search for associated with a significantly lower Ca:L ratio cholesterolemia (ARH)(5), proprotein convertase LDL-C–associated mutations, we focused on than that of the WT members (Fig. 1I), suggest- subtilisin/kexin type 9 (PCSK9)(6), and Niemann- Chinese Kazakhs, one of the major ethnic groups ing that LIMA1-K306fs carriers have reduced Pick C1-like 1 (NPC1L1)(7) genes. However, a re- in western China that has not (to the best of our intestinal cholesterol absorption. cent large-scale analysis showed that only 2.5% of knowledge) been included in lipid GWASs to To our knowledge, the LIMA1-K306fsisapre- subjects with severely high LDL-C harbored the date. The Kazakhs mainly descend from the viously unknown mutation and has not been known genetic variants identified from familial Turkic and medieval Mongol peoples (10)and reported in any published databases, including hypercholesterolemia (8). In addition, only about exhibit marked differences in the SNVs across the 121,370 allele–containing Exome Aggrega- 10 to 20% of the total variances in LDL-C can be their genomes. The Kazakhs live in isolated re- tion Consortium (ExAC) database. We further gions and usually marry within their own ethnic sequenced the coding regions of LIMA1 in 510 group. These features make the Chinese Kazakhs Chinese Kazakh individuals with relatively normal 1The State Key Laboratory of Molecular Biology, Institute of a resourceful population to characterize the LDL-C concentrations (2.7 to 3.36 mmol/liter) and Biochemistry and Cell Biology, University of Chinese ethnic-specific variants associated with LDL-C. 509 Chinese Kazakh individuals with low LDL-C Academy of Sciences, Chinese Academy of Sciences, concentrations (0.29 to 2.42 mmol/liter). No 2 LIMA1 Shanghai 200031, China. Hubei Key Laboratory of Cell Identification of a variant K306fs variants were found for individuals with Homeostasis, College of Life Sciences, Institute for Advanced associated with lower plasma LDL-C Studies, Wuhan University, Wuhan 430072, China. 3State normal LDL-C, but an additional K306fs hetero- Key Laboratory of Pathogenesis, Prevention and Treatment During the Cardiovascular Risk Survey in western zygote was detected in participants with low of High Incidence Diseases in Central Asia, Heart Center, China (11), we found a Chinese Kazakh family LDL-C (fig. S3A). In addition, no K306fs variant First Affiliated Hospital of Xinjiang Medical University, Urumqi (named Family 1) with inherited low levels of was found in a total of ~9400 individuals from 830054, Xinjiang, China. 4School of Life Science and Technology, ShanghaiTech University, Shanghai 200031, LDL-C (Fig. 1A). To identify the causal SNV(s), theDallasHeartStudyandtheBiobankStudy China. 5Institute for Nutritional Sciences, Shanghai Institutes the samples from three subjects exhibiting low (fig. S3A). Together, these results demonstrate for Biological Sciences, Chinese Academy of Sciences, LDL-C and one exhibiting normal LDL-C were that K306fs is a rare mutation in different pop- Shanghai 200031, China. analyzed by whole-exome sequencing. By using ulations, including Chinese Kazakhs (fig. S3A). *These authors contributed equally to this work. †Corresponding author. Email: [email protected] (Y.-T.M.); a dominant model, filtering against common The K306fs created a premature stop codon and [email protected] (B.-L.S.) variants, and considering functional prediction caused a 60% truncation of the LIMA1 protein

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(Fig. 1J). This mutation did not change the To rule out the possibility that dietary choles- with cholesterol. However, the cholesterol fluo- mRNA stability, and the truncated protein could terol might be trapped in the enterocytes fol- rescence was substantially reduced in Lima1- or be detected in humans (fig. S1, C to E). lowing uptake, we performed filipin staining on Npc1l1-deficient mice (Fig. 2H). We also mea- Targeted sequencing of LIMA1 in ~1000 mouse intestine. A robust cholesterol signal was sured the cholesterol contents in intestinal epi- Chinese Kazakhs revealed three additional observed in the enterocytes of WT mice gavaged thelial cells after cholesterol gavage and found families, with low LDL-C levels, carrying the L25I variant (LIMA1:NM_001113546:exon2: c.73C>A:p.L25I) in LIMA1 (fig. S3, A and B) A Family 1 B (see table S1 for details about the additional 12 LIMA1: c.916_923del:p.K306fs families). The LIMA1-L25I (Leu25→Ile) mutation I cosegregated with the low LDL-C phenotype 3.01 2.21 within the families. The L25I carriers (+/L25I) +/+ +/K306fs exhibited lower plasma TC and LDL-C concentra- 1 23 4567 tions and a lower Ca:L ratio than WT individuals II (fig. S3, C to E). When expressed in cells, the L25I +/+ mutation did not affect RNA stability or transla- 2.95 1.01 1.19 3.35 1.20 2.81 2.92 +/+ +/K306fs +/K306fs +/+ +/K306fs +/+ +/+ tional efficiency but resulted in accelerated turn- over rate (fig. S3, G to K). The LDL-C levels of 12 +/K306fs the +/L25I individuals were not as low as those III of the +/K306fs individuals (compare Fig. 1A with fig. S3B), suggesting that L25I may partially im- 1.86 1.34 +/K306fs +/K306fs pair LIMA1 function by destabilizing the protein. Downloaded from C D ** E *** LIMA1 6 4 Lima1-deficient mice display lower c.916_923del:p.K306fs dietary cholesterol absorption 3 4 The human genetic study suggested that LIMA1 2

may have a role in cholesterol metabolism. To 2 (mmol/L) 1234567 8 91011 1 investigate its function, we first examined the Plasma LDL-C http://science.sciencemag.org/ 0 expression pattern of LIMA1 in mice. LIMA1 Plasma TC (mmol/L) 0 +/+ +/K306fs +/+ +/K306fs was highly expressed in the small intestine, in- cluding the duodenum, jejunum, and ileum. It LIMA1 F NS G NS J 1 753 was modestly expressed in the liver and was 2.0 2.5 WT minimally detectable in the heart, spleen, lung, 1.5 2.0 K306fs brain, and pancreas (Fig. 2A). The intestine 1.5 accounts for ~50% of cholesterol input daily (14) 1.0 LIMA1-Flag WT- K306fs 1.0 (mmol/L) (mmol/L) 1 2 3 kDa and is the major tissue for cholesterol absorption, Plasma TG 0.5 0.5 which is mediated by the key transmembrane Plasma HDL-C 150 protein NPC1L1 (15). It is known that NPC1L1 0.0 0.0 IB: Flag +/+ +/K306fs +/+ +/K306fs 100

facilitates intestinal cholesterol uptake through on June 7, 2018 a vesicular transport mechanism (16). Next, we H I generated intestine-specific Lima1–deficient 8 NS 2.0 * (I-Lima1−/−) mice to investigate the function 6 1.5 of LIMA1 (fig. S4A) (17). LIMA1 was specifically depleted from the mouse intestine without af- 4 1.0 37 fecting the level of NPC1L1 (Fig. 2B). Notably, (mmol/L) 2 0.5 LIMA1 mainly localized on the brush border Ca: L Ratio Plasma glucose 0.0 0 IB: CHC membrane of the small intestine (Fig. 2C). The +/+ +/K306fs +/+ +/K306fs 150 I-Lima1−/− mice appeared normal without ob- vious morphological change in the small intestine, Fig. 1. Identification of the K306fs mutation in the LIMA1 gene associated with lower plasma as revealed by immunostaining with antibody to LDL-C in a Chinese Kazakh family. (A) Pedigree of the Kazakh family with lower levels of plasma Villin (fig. S4B). By orally administering radio- LDL-C. Squares and circles indicate males and females, respectively; Roman numerals indicate labeled cholesterol, we observed significantly generations; and Arabic numerals indicate individual family members. Each member’s LDL-C − lower cholesterol uptake in I-Lima1+/ (35.5%) and concentration (millimoles per liter) and LIMA1 genotype are shown below the squares and circles. − − I-Lima1 / (28.6%)micethaninWTlittermates The normal range of LDL-C concentration is 2.7 to 3.1 mmol/liter (37). Half-filled squares and (51.6%) (Fig. 2D). Two hours after administration, circles represent individuals (+/K306fs) with lower LDL-C levels. (B) DNA sequencing data of an the amount of 3H-cholesterol in the liver was unaffected man (II: 4) and an affected man (II: 2) with the heterozygous frameshift deletion in LIMA1. − − − lower in I-Lima1+/ and I-Lima1 / mice (34.0 and WT, wild type; MT, mutant; E, Glu; Q, Gln; K, Lys; N, Asn; V, Val; P, Pro; C, Cys; A, Ala; R, Arg; S, Ser. 59.1%, respectively) than in WT mice (Fig. 2E). The (C) Genomic structure of the human LIMA1 gene. The LIMA1-K306fs mutation identified in Family − amount of plasma 3H-cholesterol in I-Lima1+/ 1 (A) is indicated in red. (D to H) Plasma TC (D), LDL-C (E), TG (F), HDL-C (G), and glucose (H) levels − − and I-Lima1 / micewas29.6and54.5%lessthan of the members of Family 1 (A). Data are expressed as mean ± SD. Statistical analyses, unpaired in WT mice, respectively (Fig. 2F). The plasma t test. **P < 0.01; ***P < 0.001; NS, not statistically significant. (I) Plasma Ca:L ratio from dual-isotope ratio method also showed that family members in (A), measured by GC-MS. Ca, campesterol; L, lathosterol. Data are expressed cholesterol absorption was reduced by ~40% in as mean ± SD. Statistical analyses, unpaired t test. *P < 0.05. (J) Immunoblots (IB) showing Lima1-deficient mice (Fig. 2G) (18). Together, the protein levels of LIMA1 variants. Plasmids encoding FLAG-tagged LIMA1(WT) and LIMA1(K306fs) these results demonstrate that ablation of Lima1 were transfected into CRL1601 cells, and cells were harvested for Western blotting analysis in the small intestine impairs dietary cholesterol 48 hours later. Immunoblots represent at least three independent experiments. Clathrin heavy chain absorption in a gene dosage–dependent manner. (CHC) was used as a loading control.

Zhang et al., Science 360, 1087–1092 (2018) 8 June 2018 2of6 RESEARCH | RESEARCH ARTICLE

Fig. 2. Metabolic characteristics of A B C intestine-specific Lima1 knockout Liver Small intestine LIMA1/Hoechst mice. (A) Expression profile of mouse +/- -/- +/- -/- heartliverspleenlungkidneybrainpancreasduodenumjejunumileum kDa LIMA1 and NPC1L1 proteins. Tissues 130 WT taken from 8-week-old male C57BL/6 WT I-Lima1I-Lima1WT I-Lima1I-Lima1kDa IB: LIMA1 96 150 mice were immediately homogenized IB: LIMA1 and subjected to SDS–polyacrylamide 250 100 IB: NPC1L1 gel electrophoresis followed by immu- 250 IB: NPC1L1 I-Lima1-/- noblotting with anti-LIMA1, anti- 150 IB: CHC NPC1L1, or anti-CHC. Immunoblots 130 are representative of at least three IB: CHC 150 independent experiments. (B) D E F G Expression of LIMA1 and NPC1L1 *** *** *** ** proteins in liver and small intestine ** 100 80 ** 0.5 0.3 * from WT, I-Lima+/−, and I-Lima1−/− 0.4 80 mice. Immunoblots are representative 60 0.3 0.2 60 of at least three independent 40 0.2 40 experiments. CHC was used as a 0.1 20 loading control. (C) Representative 0.1 20 images showing localization of LIMA1 0 0.0 0.0 0 H-chol. in liver (% dose)

+/- -/- 3 +/- -/- +/- -/- -/- H-chol. in plasma (% dose) Cholesterol absorption (%) Cholesterol absorption (%) in the small intestine from 8-week-old WT WT 3 WT WT − − male mice (WT or I-Lima1 / ). Scale Downloaded from I-Lima1 I-Lima1 I-Lima1 I-Lima1 I-Lima1 I-Lima1 I-Lima1 bar, 10 mm. (D) Cholesterol absorption from mice (8 to 12 weeks, n = 6), as HIJ WT I-Lima1-/- Npc1l1-/- WT -/- measured using the fecal dual-isotope I-Lima1 10 Npc1l1-/- *** ratio method after oral gavage with 200 ### 14 3 ### 8 C-cholesterol and H-sitosterol for ## 150 3 days. Data are expressed as mean ± *** 6

*** http://science.sciencemag.org/ Corn oil ns ns * SD. Statistical analyses, one-way 100

(mg/g) 4 ANOVA (analysis of variance). **P < 50 2 0.01; ***P < 0.001. (E and F) Amount intestine (%) Fecal cholesterol 3 of H-cholesterol in the liver (E) and 0 0 Cholesterol in small

n in corn oil Corn oil Cholesterol plasma (F) of mice ( = 6) after oral Cholesterol in corn oil gavage with 3H-cholesterol for 2 hours. Data are mean ± SD. Statistical K L M WT NS 8 I-Lima1-/- analyses, one-way ANOVA. *P < 0.05; WT I-Lima1-/- NS Npc1l1-/- P P 2.0 NS NS ** < 0.01; *** < 0.001. (G) Choles- 6 terol absorption in mice (8 to 12 1.5 n weeks, = 5), as measured using the 4 on June 7, 2018 1.0 plasma dual-isotope ratio method NS NS 3 (% dose) after injection of H-cholesterol intra- (mg/g) 2 0.5 NS NS NS NS venously and oral gavage with H-sitosterol in feces 3 14C-cholesterol for 3 days. Data are Fecal triglyceride 0.0 0 Day1 Day2 Day3 Day4 mean ± SD. Statistical analyses, unpaired t test. **P <0.01.(H and I) N O P VLDL WT Analysis of free cholesterol from intestinal 20 ### 30 -/- − − − − 200 I-Lima1 samples of I-Lima1 / , Npc1l1 / ,and ### 15 HDL WT male mice (8 weeks, n = 6). (H) 150 *** 20 Representative images of filipin staining 100 10 *** LDL from at least three independent *** experiments. Scale bar, 10 mm. (I) The 50 5 *** 10 Liver TC (mg/g) levels of cholesterol and phospholipids Plasma TC (mg/dL) 0 0 in the small intestine were measured Chow HCD Chow HCD Cholesterol (mg/dL) 0 enzymatically. The relative amount 52010 15 3025 403545 of cholesterol was normalized to that of Fraction number phospholipids. Data are expressed as mean ± SD. Statistical analyses, two-way ANOVA. ##P < 0.01 and ###P < (M) Fecal excretion of sitosterol in I-Lima1−/−, Npc1l1−/−, and WT mice. 0.001 as compared with mice of the same genotype gavaged with corn oil; Data show amount of 3H-sitosterol in the feces and are expressed as ***P < 0.001 as compared with WT mice in the same group; ns, not mean ± SD for 8- to 12-week-old mice (n = 6). Statistical analyses, statistically significant as compared with WT mice in the same group. two-way ANOVA. NS, not statistically significant. (N and O)I-Lima1−/−, (J and K) Fecal cholesterol and triglyceride levels in WT, I-Lima1−/−, and Npc1l1−/−, and WT male mice (8 to 12 weeks, n = 6) were fed a chow diet Npc1l1−/− mice. Feces were collected for 3 days to measure fecal or a HCD (1% cholesterol) for 7 days. Plasma TC (N) and liver TC (O) cholesterol (J) and triglycerides (K). Data are expressed as mean ± SD. levels were measured. Data are expressed as mean ± SD. Statistical Statistical analyses, one-way ANOVA. *P < 0.05; ***P < 0.001; NS, not analyses, two-way ANOVA. ###P < 0.001 as compared with WT mice statistically significant. (L) Distribution of ABCG8 protein in the small fed the chow diet; ***P < 0.001 as compared with WT mice fed the HCD. intestine of I-Lima1−/− or WT mice (8-week-old mice). Images are (P) Representative cholesterol concentration in FPLC fractions from representative of at least three independent experiments. Scale bar, 10 mm. the serum of I-Lima1−/− and WT mice fed the HCD for 7 days.

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A C LIMA1 E Myosin Vb NPC1L1-CT67 binding LIMA1-491-541 binding 1266 1332 491 541 WT + WT + + Δ491-511 - NPC1L1 LIMA1 Merge - + LIMA1 Myosin Vb-2-80 binding 2 80 WT + Myosin Vb LIMA1 Merge Δ21-40 - GST-CT67 - + GST + ---- His6-LIMA1-491-541 - ++- + His6-LIMA1+++++ Δ 12 345 kDa His6-LIMA1-491-541- 491-511 - - - + - Myosin Vb NPC1L1 Merge - - - - pellet 35 GST + GST-Myosin Vb-2-80 B (IB: GST) 25 ++- + - GST-Myosin Vb-2-80-Δ21-40 --- - + 130 NPC1L1-T7 - + - + pellet 12345 kDa (IB: His6) 100 Myosin Vb-EGFP + +++ 35 Pull down: His6 siRNA control Lima1 pellet

input 35 Downloaded from 1 2 3 4 kDa (IB: GST) (IB: GST) 25 250 25 pellet IB: EGFP 250 (IP: T7) NPC1L1-CT67 pellet IB: T7 150 D LIMA1 binding (IB: His6) 15 250 1753 IB: EGFP Pull down: His6 input 250 WT + Δ161-187 - http://science.sciencemag.org/ IB: T7 150 35 - input (IB: GST) Fig. 3. LIMA1 interacts with NPC1L1 and myosin Vb. (A) Distribution of NPC1L1, 25

LIMA1,andmyosinVbinmousesmall His6-LIMA1 + + Δ161-187 intestine (8-week-old mice). Images are GST-NPC1L1-CT67 - +++ F Microfilament representative of at least three independent GST + --- kDa 491-511 experiments. Scale bar, 10 mm. (B) Knock- 1234 35 LIMA1 down of Lima1 reduced the interaction pellet 21-40 NPC1L1 between NPC1L1 and myosin Vb. siRNA and (IB: GST) 25 -C164LG-

plasmids were cotransfected into the

130 on June 7, 2018 pellet -Q1277KR- CRL1601 cells. Cell lysates were immuno- (IB: His6) 100 Myosin Vb precipitated with anti-T7–coupled agarose Pull down: His6 and subjected to Western blotting. Immuno- Vesicle 35 blots represent at least three independent input (IB: GST) experiments. (C) Interaction of His6-LIMA1 25 and GST-NPC1L1-CT67 (WT, L1273ALE→AAAA,

Q1277KR→AAA, or E1281E→AA) proteins analyzed by in vitro pull-down assay. by in vitro pull-down assay. (F)SchematicmodelofNPC1L1–LIMA1–myosin (D) Interaction of His6-LIMA1 (WT, D161-187, or C164LG→AAA) with GST-NPC1L1- Vb interaction. The Q1277KR residues of NPC1L1 and the C164LG residues of CT67 proteins analyzed by in vitro pull-down assay. Immunoblots represent at LIMA1 are required for NPC1L1-LIMA1 interaction. The region (residues 491 to least three independent experiments. (E) Interaction of His6-LIMA1(491-541) 511) of LIMA1 and the region (residues 21 to 40) of myosin Vb are required for (WT or D491-511) and GST-myosin Vb(2-80) (WT or D21-40) proteins analyzed LIMA1–myosin Vb association. lower levels in Lima1- or Npc1l1-deficient mice munostaining (Fig. 2L). Similarly, knockdown plasma and liver TC levels, respectively. The than in WT mice (Fig. 2I). In contrast, fecal of Lima1 did not impair the plasma membrane plasma and liver TC contents of I-Lima1−/− were, cholesterol was higher in I-Lima1−/− and Npc1l1−/− localization of the transfected ABCG5-ABCG8 respectively, 28.8 and 58.3% lower than those of mice than in WT mice (Fig. 2J), although the protein complex in cultured cells (fig. S4C). Func- WT mice fed the HCD (Fig. 2, N and O). All mice fecal triglyceride level was similar (Fig. 2K). These tional studies showed that the kinetics of fecal showed similar plasma and liver TG levels (fig. results demonstrate that intestinal cholesterol excretion of sitosterol was comparable in I- S4, D and E). Fast protein liquid chromatography absorption was reduced in I-Lima1−/− mice. Lima1−/− mice and WT littermates (Fig. 2M), (FPLC) analysis showed that cholesterol contents ABCG5 and ABCG8 are known to form an which suggests that Lima1 ablation affects neither in very-low-density lipoprotein (VLDL), LDL, and essential protein complex to excrete cholesterol the function of ABCG5-ABCG8 nor the excretion HDL were all markedly lower in I-Lima1−/− animals and plant sterols into the intestinal lumen (19). of sterols. than in WT mice fed the HCD (Fig. 2P). We examined whether ablation of Lima1 affected We next evaluated the role of LIMA1 in diet- Relative to levels of mice on the chow diet, the localization and function of ABCG5-ABCG8, induced hypercholesterolemia. When fed a chow protein levels of HMG-CoA reductase (HMGCR) which would then alter cholesterol homeostasis. diet, all mice showed similar TC levels in the decreasedinthesmallintestineandliverofWT The brush border membrane localization of en- plasma and liver (Fig. 2, N and O). In com- mice fed the HCD. However, the HMGCR levels dogenous ABCG8 protein appeared normal in parison, mice that consumed a high-cholesterol in I-Lima1−/− and Npc1l1−/− animals were higher I-Lima1−/− mice, as revealed by anti-ABCG8 im- diet (HCD) had 1.63-fold and 4-fold increases in than in WT mice on the HCD (fig. S4F). We

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Fig. 4. LIMA1 regulates the NPC1L1 transpor- A Cholesterol depletion B Lima1 myosin Contrl siRNA tation. (A) Knockdown of or siRNA 0 30 45 60 (min) Vb slows transport of NPC1L1 from the ERC to Lima1 siRNA Myosin Vb siRNA the PM. Control siRNA or siRNA targeting rat 1.0 Lima1 myosin Vb Control or was transfected into 0.8 CRL1601 cells stably expressing NPC1L1-EGFP *** 0.6 individually. Forty-eight hours later, the cells *** *** were depleted of cholesterol for various time 0.4

Lima1 (of total) durations. Scale bar, 10 mm. Confocal micros- 0.2 copy images are representative of at least three 0.0 independent experiments. (B) Quantification Intracellular NPC1L1 0 30 45 60 (min) of the intracellular localization of NPC1L1-EGFP Myosin Vb Cholesterol depletion shown in (A). Data are expressed as mean ± SD of three independent experiments. Statistical analyses, two-way ANOVA. ***P < 0.001. C D Lima1 Cholesterol depletion +/+ (C) WT (+/+) and heterozygous 0.8 frameshift deletion (+/Q303fs) CRL1601 +/Q303fs 0 30 45 60 (min) cells stably expressing NPC1L1-EGFP were 0.6 *** depleted of cholesterol for the indicated times. *** Confocal microscopy images are representative +/+ 0.4 of at least three independent experiments. *** Downloaded from (of total) Scale bar, 10 mm. (D) Quantification of 0.2 the intracellular localization of NPC1L1-EGFP

+/Q303fs Intracellular NPC1L1 shown in (C). Data are expressed as mean ± SD 0.0 of three independent experiments. Statistical 0 30 45 60 (min) analyses, two-way ANOVA. ***P < 0.001. Cholesterol depletion (E) NPC1L1-EGFP(WT) and NPC1L1-EGFP E F (Q1277KR→AAA) variants were expressed in Cholesterol depletion http://science.sciencemag.org/ CRL1601 cells and depleted of cholesterol for WT 0 30 45 60 (min) 1.0 the indicated times. Confocal microscopy images represent at least three independent 0.8 experiments. Scale bar, 10 mm. (F) Quantification WT 0.6 *** of the intracellular localization of NPC1L1-EGFP 0.4 *** *** shown in (E). Data are expressed as mean ± SD (of total) 0.2 of three independent experiments. Statistical 0.0 analyses, two-way ANOVA. ***P < 0.001. Intracellular NPC1L1 (G) Representative immunoelectron microscopy NPC1L1-EGFP 0 30 45 60 (min) images of NPC1L1 in the jejunum of I-Lima1−/− Cholesterol depletion and WT mice from three independent G H on June 7, 2018 -/- ) experiments. Arrows indicate NPC1L1-positive WT I-Lima1 2 25 *** particles. Scale bar, 500 nm. (H) Quantification from (G) of the density of gold particles 20 indicating NPC1L1 protein beneath microvilli. 15 Data are expressed as mean ± SD of three NPC1L1 10 independent experiments. Statistical analyses, 5 unpaired t test. ***P < 0.001. 0 microvilli (per 0.2 µm Gold particles beneath WT I-Lima1-/- observed similar trends in the mRNA levels of (15, 20), we used this drug to pharmacologically complex from WT mouse intestinal epithelial Hmgcr, HMG-CoA synthase 1(Hmgcs1), and Ldlr inactivate NPC1L1 to address whether LIMA1 is cells and identified itsbindingproteinsby (fig. S4, G to J), indicating that I-Lima1 −/− mice epistatic to NPC1L1. After treatment, cholesterol tandem mass spectrometry (fig. S6A). The spec- take up less cholesterol from diet and com- absorption efficiency in WT mice was reduced to ificity of the antibody to LIMA1 (anti-LIMA1) was pensatorily express more cholesterol synthesis– a similar level as in Npc1l1-deficient mice (fig. validated by Western blotting and immuno- and cholesterol uptake–related genes. Similar S5K). Ablation of Lima1 decreased cholesterol staining (Fig. 2, B and C). The anti-LIMA1 im- phenotypes were detected in Npc1l1−/− mice (15). absorption, but to a lesser extent than in Npc1l1 munoprecipitation (IP) from Lima1 −/− intestinal We next generated whole-body Lima1 knock- knockout mice. Ezetimibe treatment in I-Lima1−/− epithelial cells and the anti-EGFP IP from WT out mice (fig. S5A). The heterozygotes appeared mice further decreased cholesterol absorption intestinal epithelial cells were similarly per- normal but displayed less dietary cholesterol to the same level as in Npc1l1-deficient mice formed, and the identified proteins served as absorption and lower plasma TC levels as com- (fig. S5K). These data indicate that LIMA1 func- nonspecific binders (fig. S6A and table S2). The pared with WT mice (fig. S5, B to H), similar tions upstream of NPC1L1 in affecting cholesterol LIMA1-binding candidates specifically identified to human heterozygotes with the LIMA1 loss- absorption. from anti-LIMA1 IP of WT samples are listed in of-function mutation (Fig. 1A). The homozygotes fig. S6B and table S2. Cadherin and b-catenin are also appeared normal and displayed reduced LIMA1 interacts with NPClL1 and myosin Vb known LIMA1-binding proteins (21), thus vali- dietary cholesterol absorption (fig. S5, I and J). To reveal the mechanism by which LIMA1 dating our purification results. Notably, NPC1L1 Because ezetimibe is a potent inhibitor of cho- mediates dietary cholesterol absorption, we and myosin Vb, both required for efficient in- lesterol absorption through targeting of NPC1L1 immunoprecipitated the LIMA1-containing testinal cholesterol absorption (16, 22–30), were

Zhang et al., Science 360, 1087–1092 (2018) 8 June 2018 5of6 RESEARCH | RESEARCH ARTICLE

among the LIMA1-binding candidates, and we The LIMA1-NPC1L1 interaction is REFERENCES AND NOTES found that LIMA1 bound NPC1L1 and myosin required for cholesterol absorption 1. R. Lozano et al., Lancet 380, 2095–2128 (2012). Vb (fig. S7A). LIMA1 was mainly present on the Because NPC1L1-LIMA1 interaction is critical 2. W. B. Kannel, T. R. Dawber, A. Kagan, N. Revotskie, J. Stokes III, – brush border membrane of mouse small intes- for NPC1L1 recycling, ablating this interaction Ann. Intern. Med. 55,33 50 (1961). 3. G. Pilia et al., PLOS Genet. 2, e132 (2006). tine and colocalized with NPC1L1 and myosin by peptide competition would impair NPC1L1 4. H. H. Hobbs, M. S. Brown, J. L. Goldstein, Hum. Mutat. 1, Vb (Fig. 3A). These results suggest that LIMA1 trafficking and therefore decrease cholesterol 445–466 (1992). may work together with NPC1L1 and myosin Vb absorption. To test this hypothesis, we fused the 5. C. K. Garcia et al., Science 292, 1394–1398 (2001). – to facilitate cholesterol absorption. LIMA1(161-187) region with the transferrin re- 6. M. Abifadel et al., Nat. Genet. 34, 154 156 (2003). 7. Myocardial Infarction Genetics Consortium Investigators, The association between NPC1L1 and myosin ceptor (TrfR) for membrane anchoring (fig. S13A) N. Engl. J. Med. 371, 2072–2082 (2014). Vb was reduced by Lima1 depletion (Fig. 3B), in- (31). LIMA1(161-187) outcompeted the LIMA1- 8. N. S. Abul-Husn et al., Science 354, aaf7000 (2016). dicating that LIMA1 may bridge myosin Vb to NPC1L1 interaction, whereas the mutated peptide 9. S. Gravel et al., Proc. Natl. Acad. Sci. U.S.A. 108, 11983–11988 (2011). – NPC1L1. Through a series of truncations and LIMA1(161-187)-C LG→AAA [LIMA1(161-187)-mu] 10. H. B. Paksoy, Z. V. Togan, Centr. Asian Surv. 11,83 100 (1992). 164 11. Y. N. Yang et al., PLOS ONE 7, e35270 (2012). mutations followed by co-IP and in vitro pull- failed to do so (fig. S13B). Consistently, LIMA1 12. J. Yang, M. S. Brown, Y. K. Ho, J. L. Goldstein, J. Biol. Chem. down assays, we identified that the Q1277KR res- (161-187), but not LIMA1(161-187)-mu, substantially 270, 12152–12161 (1995). idues (Q, Gln; K, Lys; R, Arg) of NPC1L1 and the decreased the cellular transport of NPC1L1 to the 13. T. A. Miettinen, R. S. Tilvis, Y. A. Kesäniemi, Am. J. Epidemiol. – C LG residues (C, Cys; G, Gly) of LIMA1 were PM(fig.S13,CandD). 131,20 31 (1990). 164 14. J. J. Repa, D. J. Mangelsdorf, Annu. Rev. Cell Dev. Biol. 16, critical for NPC1L1-LIMA1 interaction (Fig. 3, C We next delivered the fusion protein into mouse 459–481 (2000). and D, and figs. S7 and S8) and that the amino liver using adenovirus and investigated the 15. S. W. Altmann et al., Science 303, 1201–1204 (2004). acid 21 to 40 region of myosin Vb and the amino function of the NPC1L1-LIMA1 complex in vivo. 16. L. Ge et al., Cell Metab. 7, 508–519 (2008). 17. F. 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Lipid Res. 50, 1653–1662 (2009). recruiting myosin Vb to NPC1L1 sults (fig. S13B), LIMA1(161-187)-TrfR fusion, but 24. L. Ge et al., Proc.Natl.Acad.Sci.U.S.A.108, 551–556 (2011). We next tested whether LIMA1 played a role in not LIMA1(161-187)-mu, disrupted the interac- 25. L. J. Wang et al., J. Biol. Chem. 286, 7397–7408 (2011). NPC1L1 translocation. Knockdown of Lima1 or tion between NPC1L1 and LIMA1 in mouse liver 26. C. Xie, N. Li, Z. J. Chen, B. L. Li, B. L. Song, J. Biol. Chem. 286, 35933–35942 (2011). http://science.sciencemag.org/ myosin Vb blocked transportation of NPC1L1 (fig. S13E). Compared with TrfR-expressing mice, 27. J. H. Zhang et al., J. Biol. Chem. 286,25088–25097 (2011). from the endocytic recycling compartment (ERC) an increase in the biliary cholesterol concen- 28. C. Xie et al., J. Lipid Res. 53, 2092–2101 (2012). 29. P. S. Li et al., Nat. Med. 20,80–86 (2014). to the plasma membrane (PM) (Fig. 4, A and B). tration and a decrease in the liver and plasma – – 30. P. Xie et al., Atherosclerosis 237, 609 617 (2014). Expression of small interfering RNA (siRNA) TC levels were detected in mice expressing the 31. D. J. Yamashiro, B. Tycko, S. R. Fluss, F. R. Maxfield, Cell 37, resistant LIMA1 effectively rescued the impaired LIMA1(161-187)-TrfR fusion protein (fig. S13F). 789–800 (1984). NPC1L1 translocation in Lima1-knockdown cells However, compared with the control, expression 32. R. E. Temel et al., J. Clin. Invest. 117, 1968–1978 (2007). 33. J. L. Goldstein, M. S. Brown, Arterioscler. Thromb. Vasc. Biol. (fig. S11, A and B), but expression of the truncated of LIMA1(161-187)-mu had no effect on biliary 29, 431–438 (2009). protein LIMA1(1-306) failed to do so (fig. S11, C cholesterol, liver TC, or plasma TC concentra- 34. E. A. Stein et al., N. Engl. J. Med. 366, 1108–1118 (2012). and D). Overexpression of LIMA1(1-306) out- tions (fig. S13F), indicating that LIMA1(161-187) 35. D. 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S12A) and delayed the transportation of NPC1L1 reabsorption from bile. Plasma levels of aspar- 37. Joint Committee for the Revision of Guideline for the on June 7, 2018 from the ERC to the PM (fig. S12, B and C). We tate aminotransferase (AST) were similarly low Prevention and Treatment of Dyslipidemia in Chinese Adults, also generated a heterozygous Lima1 frameshift among different groups (fig. S13F, bottom panel), Chin. Circ. J. 31, 10 (2016). deletion cell line (+/Q303fs) by using CRISPR- suggesting no obvious liver injury. ACKNOWLEDGMENTS Cas9 to mimic the human LIMA1-K306fs mutation We thank H. Hobbs and J. Cohen at the UT Southwestern (fig. S12D). The +/Q303fs cells showed signifi- Discussion Medical Center for help with human genetic studies, Y.-X. Qu and cantly attenuated NPC1L1 transportation to the Our study identifies that LIMA1 influences plasma J. Xu for technical assistance, and S. Robine (Institut Curie) for PM compared with WT cells (Fig. 4, C and D), cholesterol levels through regulation of intestinal the villin-Cre ERT2 transgenic mice. Funding: This work was supported by grants from the MOST of China (2016YFA0500100), NNSF of China suggesting a dominant inhibitory function. Given cholesterol absorption in both humans and mice. (31690102, 31430044, 81260041, 31771568, 31701030, 91754102, that LIMA1(1-306) was expressed in human A growing body of evidence has indicated that U1403221, and 31600651), Xinjiang Key Research and Development carriers (fig. S1, C to E), the +/K306fs hetero- lower LDL-C levels are associated with reduced Project (2016B03053), Science and Technology Support Project of zygotes may harbor a low Ca:L ratio due to the risk of CVD. LDL-C can be decreased by statin Xinjiang (2017E0269), 111 Project of Ministry of Education of China (B16036), and the Natural Science Foundation of Hubei Province dominant inhibitory effect of the K306fs allele drugs, which inhibit the rate-limiting enzyme (2016CFA012). Author contributions: B.-L.S. conceived of the project. (Fig. 1I), which also contributes to the low HMGCR in the cholesterol biosynthesis pathway Y.-Y.Z., Z.-Y.F., J.W., Y.-T.M., and B.-L.S. designed the experiments. LDL-C concentration in this family (Fig. 1, D (33), or by ezetimibe, which blocks NPC1L1- Y.-Y.Z., Z.-Y.F., J.W., W.Q., G.B., Y.-J.M., S.-Y.G., S.-Y.J., Y.-F.L., and and E). mediated intestinal cholesterol absorption (15). H.-H.M. performed the experiments. Y.-Y.Z., Z.-Y.F., J.W., W.Q., J.L., H.Y., Y.L., Y.W., B.-L.L, Y.-T.M., and B.-L.S. analyzed the data. Y.-Y.Z., Replacement of Q1277KR with AAA (A, Ala) in The PCSK9 inhibitors, which act by elevating W.Q., and B.-L.S. wrote the paper with input from all authors. NPC1L1, which abolished the NPC1L1-LIMA1 in- LDLR levels, are a third class of drugs that de- Competing interests: The authors declare no competing financial teraction, substantially decreased the transporta- crease plasma LDL-C and have demonstrated interests. Data and materials availability: Whole-exome sequencing tion rate of NPC1L1 from the ERC to the PM clinical benefits (34). Despite the availability of datasets were deposited in Sequence Read Archive with accession number SRP139972 (www.ncbi.nlm.nih.gov/sra/). (Fig. 4, E and F). In mouse small intestine, a these therapeutics, the prevalence of CVD con- considerable number of NPC1L1-positive particles tinues to rise, and many individuals are in- SUPPLEMENTARY MATERIALS accumulated in the apical cytoplasm beneath the tolerant to statins and/or ezetimibe or are unable www.sciencemag.org/content/360/6393/1087/suppl/DC1 microvilli of I-Lima1−/− mice compared with WT to reach their target LDL-C levels using these Materials and Methods animals (Fig. 4, G and H). Together, these results strategies (35, 36). Thus, there is still a need to Figs. S1 to S13 Tables S1 and S2 suggest that LIMA1 acts as a scaffold protein identify targets and therapeutics that provide References (38–44) regulating NPC1L1 transportation to the PM by alternative ways to lower LDL-C and treat CVD, – – 13 August 2017; resubmitted 7 February 2018 forming the NPC1L1 LIMA1 myosin Vb triplex and inhibition of LIMA1 may provide a new di- Accepted 17 April 2018 (Fig. 3F). rection for treating hypercholesterolemia. 10.1126/science.aao6575

Zhang et al., Science 360, 1087–1092 (2018) 8 June 2018 6of6 A LIMA1 variant promotes low plasma LDL cholesterol and decreases intestinal cholesterol absorption Ying-Yu Zhang, Zhen-Yan Fu, Jian Wei, Wei Qi, Gulinaer Baituola, Jie Luo, Ya-Jie Meng, Shu-Yuan Guo, Huiyong Yin, Shi-You Jiang, Yun-Feng Li, Hong-Hua Miao, Yong Liu, Yan Wang, Bo-Liang Li, Yi-Tong Ma and Bao-Liang Song

Science 360 (6393), 1087-1092. DOI: 10.1126/science.aao6575

A missing link in cholesterol absorption Cholesterol is important for general health, but too much can build up in artery walls and cause cardiovascular disease. Low-density lipoprotein cholesterol (LDL-C) is often referred to as ''bad cholesterol''; keeping LDL-C within stringent limits is recommended to reduce the risk of heart attack and stroke. Zhang et al. discovered that some Downloaded from individuals have an inherited frameshift mutation in the LIMA1 gene (also known as EPLIN or SREBP3). The gene has not been linked to lipid metabolism before, but altered LIMA1 was found to maintain low plasma LDL-C by reducing the absorption of cholesterol through the intestine. Pharmacological targeting through the LIMA1 pathway might thus provide a strategy to improve heart health. Science, this issue p. 1087 http://science.sciencemag.org/

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