851

The Mutation Causing the CommonApolipoprotein A-IV Polymorphism Is a

Glutamine to Histidine Substitutionof Amino Acid 360

Heli Tenkanen, Matti Lukka, Matti Jauhiainen, Jari Metso,

Marc Baumann, Leena Peltonen, and Christian Ehnholm

Apolipoprotein (apo) A-IV is a protein involved in the metabolism of chylomicrons and highdensity lipoproteins. This protein displays genetic polymorphism due to two main codominantalleles, A-IV' and A-IV2. We have identified the mutation that leads to this polymorphism. It iscaused by a single-base substitution of guanine for thymine in the third base of codon 360. Thissubstitution leads to a glutamine to histidine change. Direct sequencing of amplified DNA fromeight subjects in a three-generation pedigree has demonstrated that the guanine to thyminesubstitution can explain the apo A-IV polymorphism. In 32 unrelated individuals, a correspon-dence between apo A-IV phenotype determined by isoelectric focusing and genotype determinedwith Fnu4HI digestion of amplified DNA could be demonstrated. The enzymelecithin: cholesteryl acyltransferase (LCAT) is activated by apo A-IV. Under our in vitroconditions, the isoprotein apo A-IV 1-1 is a better LCAT activator than is the isoprotein apoA-IV 2-2. A knowledge of the molecular mechanism underlying the apo A-IV polymorphism willhelp to elucidate the mechanisms involved in LCAT activation. (Arteriosclerosis and Thrombosis1991;ll:851-856)

Apolipoprotein (apo) A-IV is a major compo-nent of intestinally derived triglyceride-richlipoproteins.1 Apo A-IV is a 46,000-d glyco-

protein synthesized in the small intestine during fatabsorption.2-3 In fasting plasma, about 20% of apoA-IV is found associated with high density lipoproteins(HDLs), while the major portion of plasma apo A-IV ispresent in the lipoprotein-deficient fraction.4-5 Thephysiological function of apo A-IV is, at present, un-known. However, several lines of evidence have impli-cated apo A-IV as an important element in the metab-olism of chylomicrons and HDL.6 It has been shownthat apo A-IV in vitro can function as a cofactor for theenzyme lecithin: cholesteryl acyltransferase (LCAT)7-8

and that apo A-FV may act as an activator of a plasmafactor involved in the conversion of HDL subclasses.9 Itmay also function in "reverse cholesterol transport" bypromoting efflux of cholesterol from cells.10 The apoA-IV gene, located on chromosome 11 in close linkage

From the National Public Health Institute (H.T., M.L., M.J.,J.M., L.P., C.E.) and the Department of Medical Biochemistry(M.B.), University of Helsinki, Helsinki, Finland.

Supported by the Sigrid Juselius Foundation, Helsinki, Finland.Address for correspondence: Christian Ehnholm, National Public

Health Institute, Mannerheimintie 166, 00300 Helsinki, Finland.Received September 3,1990; revision accepted February 4,1991.

with the apo A-I gene,1112 has been isolated andcharacterized,13 and the primary structure of apo A-IVhas been elucidated.14

By use of two-dimensional electrophoresis1115 orisoelectric focusing (IEF),16-18 genetic polymorphism ofapo A-IV has been demonstrated. Genetic studies areconsistent with a single locus with several codominantalleles. The gene frequencies of the two most commonalleles, A-IV' and A-IV2, have been determined inseveral populations.16-18-24 The gene frequencies forthe apo A-IV' allele vary between 0.88 and 0.94 andthose for apo A-IV2 from 0.06 to 0.12. In addition,several rare apo A-IV variants have been de-scribed. 15,16,18,23,25

It has been reported that the two alleles at the apoA-IV locus influence plasma HDL cholesterol andtriglyceride levels.2024 It was therefore of interest tostudy which mutation causes the apo A-IV polymor-phism and how this might influence plasma HDLlevels. We now report on a point mutation causingthe common apo A-IV polymorphism and on theeffect of this mutation on LCAT activation.

MethodsPlasma Samples

Blood was collected from nonfasting subjects intoEDTA-containing tubes. The plasma was separated

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by centrifugation and stored at -20°C until used.Plasma used for purification of apo A-IV was col-lected by plasmapheresis.

Apolipoprotein A-IV PhenotypingPhenotype analysis was performed by immunoblot-

ting after IEF of the serum.18

Isolation of ApolipoproteinsHuman apo A-IV was isolated from lymph ob-

tained from a patient with chylothorax. The lymphwas centrifuged in a Beckman Model 50.2 rotor(Beckman Instruments, Palo Alto, Calif.) for 12hours at 40,000 rpm. After washing and delipidation,the chylomicron apolipoprotein was dissolved in 10mM tris(hydroxymethyl)aminomethane (Tris) HC1(pH 8.0) buffer containing 6 M urea,7 chromato-graphed on a DEAE-Sepharose column (Pharma-cia-LKB Biotechnology, Uppsala, Sweden) equili-brated with the same buffer, and eluted with lineargradient 10-300 mM Tris HC1 (pH 8.0) containing 6M urea. The apo A-IV-containing fractions werepooled, dialyzed against 50 mM NH4HCO3, lyo-philized, and subjected to high-pressure liquid chro-matography (HPLC).26

Human apo A-IV was isolated from plasma asdescribed by Weinberg and Scanu.27 The proteinsabsorbed to intralipid were, upon delipidation,further purified by DEAE-Sepharose chromatog-raphy and HPLC.

Apolipoprotein A-I PreparationHuman apo A-I was isolated from HDLs

(d=1.063-1.210 g/ml) after delipidation by prepara-tive IEF in Ultradex (Pharmacia-LKB Biotechnol-ogy) as described.28

Lecithin '• Cholesteryl Acyltransferase PreparationLCAT was isolated from fresh plasma collected by

plasmapheresis by a modification28-29 of the methoddescribed by Albers et al.30 The following changes inpurification steps were made: the anion exchangerAccell QMA (Millipore Corp., Bedford, Mass.) re-placed DEAE-Sephadex A-50 (Pharmacia-LKBBiotechnology), and the final purification step wasgel filtration on Sephacryl S-200 (high resolution,Pharmacia-LKB Biotechnology). The purified en-zyme was stored at 4°C in 10 mM Tris HC1 (pH 7.0)containing 150 mM NaCl, 1 mM EDTA, and 5 mM)3-mercaptoethanol. The enzyme preparation wasused within 4 weeks.

Preparation of Apolipoprotein A-I-EggPhosphatidylcholine-Cholesterol and ApolipoproteinA-IV-Egg Phosphatidylcholine-CholesterolProteoliposome Substrates

Apo A-I- and apo A-IV 1-1- or apo A-IV 2-2-eggphosphatidylcholine-cholesterol proteoliposomeswere prepared by a slight modification of the sodiumcholate dialysis method described by Chen and Al-bers.31 The substrates consisted of complexes of egg

yolk phosphatidylcholine-cholesterol-(l,2-[3H]cho-lesterol-apo A-I, -apo A-IV 1-1, or -apo A-IV 2-2) inthe molar ratio of 250:12.5:0.8, respectively. Typi-cally, egg phosphatidylcholine, cholesterol, and [3H]cholesterol as a tracer in organic solvents were mixedin a glass vial, and the organic solvents were evapo-rated to dryness under N2. The residual organic phasewas removed by lyophilization. The dried lipid mixturewas then solubilized by vortexing in 10 mM Tris HC1buffer (pH 7.4) containing 150 mM NaCl and 1 mMEDTA. Then homogeneous apo A-I or apo A-IV(isoforms 1-1 or 2-2) and sodium cholate (final con-centration, 55 mM) were added. The mixture wasvortexed for 1 minute and then incubated at 25°C for20 minutes after which it was dialyzed against 10 mMTris HC1 buffer (pH 7.4) containing 150 mM NaCl and1 mM EDTA for approximately 50 hours to removesodium cholate. The molar ratio of the components inthe proteoliposome was determined by cholesterol,phospholipid, and protein quantification. The sub-strates were stored at 4°C under N2 until used.

Lecithin: Cholesteryl Acyltransferase AssayLCAT transacylase activity (phosphatidylcholine

cleavage followed by cholesterol esterification) wasanalyzed in an assay mixture typically consisting of0.2-0.25 ml assay buffer, 10 mM Tris HC1 (pH 7.4),with 150 mM NaCl and 1 mM EDTA, 0.125 ml 2%bovine serum albumin, and 0.05-0.1 ml apo A-I- orapo A-IV-containing proteoliposome substrate. Themixture was preincubated at 37°C for 15 minutes toactivate the substrate, after which 0.025 ml 0.1 M/3-mercaptoethanol and 30-50 fi\ enzyme was added.After vortexing, the reaction mixture was incubated at37°C for 30 minutes. The reaction was terminated bythe addition of 8 ml CHClj/CH3OH (2:1, vol/vol), andthe lipids were extracted by the method of Folch et al32

and analyzed by thin-layer chromatography.28

N-Terminal Sequence AnalysisThe isolated proteins (apo A-IV 1-1 and apo A-IV

2-2) were digested with Endoproteinase Lys-C(Wako Chemicals, Neuss, FRG), and the releasedpeptides were separated by HPLC by use of a VydacC-18 reverse-phase column equilibrated with 0.1%trifluoroacetic acid in H2O eluted with a gradient ofacetonitrile (0-70% in 30 minutes). The sequenceanalysis was performed by automated Edman degra-dation with a modified Applied Biosystems Model477A/120A on-line pulsed liquid-phase/gas-phase se-quencer in the gas-phase mode.

DNA PreparationDNA was isolated from 5 ml of EDTA-augmented

blood33 from subjects belonging to different apoA-IV phenotypes.

Amplification of DNAGenomic DNA (0.5 /xg) isolated from apo A-IV

1-1 and 2-2 subjects was amplified by the polymerasechain reaction (PCR) technique.34 The primers used

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Tenkanen el al Apo A-IV Polymorphism 853

Gin360 His360

ApoA-JZ 1-1A C G T

1 - 2 2 - 2

A C G T A C G T

362/ G/Glu A

361/ G/Gin A

360//GGin A — mmm'

c —59//Gin A

C

58/ A/Glu A

G

G 362/A /G luG

G 361/A /GinC

•«_'""•"—» ' _ _ " " " — T 36o/_ — ~ A /HiHis

G 359/A /GinC

A 358/A / GGlu

FIGURE 1. Autoradiograms of the region of

sequencing gels showing location of the mu-tation that causes polymorphism of the apo-lipoprotein (apo) A-Wgene. Autoradiogramsfrom left to right are those obtained from apoA-W 1-1, A-IV 1-2, and A-W 2-2 subjects.Codon numbers and amino acid substitutionare indicated. Gin, glutamine; His, histidine;A, adenine; C, cytosine; G, guanine; T, thy-mine; Glu, glutamic acid.

were chosen according to the published apo A-IVgene structure13: exon 1, 5'-CACTGCAGCGCAG-GTGAGCTC-3' and 5'-GCCTCCATCCTGCAC-TACTC-3'; exon 2, 5'-GAGCCAGGGCTGAGGT-CAGTGC-3' and 5'-TTGAGTTGCTGGGTGAGT-TC-3'; exon 3, 5'-TGCCCTCTTCCAGGACAAAC-3' and 5'-GCTCTCCAAAGGGGCCAGCATC-3'(Figure 3). The PCR was performed in a reactionmixture containing 1 /iM of primers, deoxyadenosinetriphosphate, deoxycytidine triphosphate, deoxygua-nosine triphosphate, and deoxythymidine triphos-phate, (0.2 mM each), 20 mM Tris HC1 (pH 8.8), 1.5mM MgCl2,0.1% Tween-20,0.1 mg/ml gelatin, and 2.5units Thermus aquaticus (Taq) DNA polymerase (USBiochemical Corp., Cleveland, Ohio) in a final volumeof 100 fil. The cycles of denaturation (1 minute at95°C), annealing (1 minute at 60°C), and elongation (2minutes at 72°C) were repeated 30 times. The elonga-tion time for exon 3 was 3 minutes. The amplifiedproducts were analyzed on 2% agarose gels and iden-tified by staining with ethidium bromide and Southernblotting.35 The apo A-IV gene probe was a kind gift ofJohn Taylor, Gladstone Foundation Laboratories, SanFrancisco, Calif.

CloningFor sequencing, amplified DNA from each exon

was blunt-end ligated36 into the Sma I site in thepGem3 (Promega, Madison, Wis.).

Sequence AnalysisThe cloned fragments were sequenced with the

dideoxy-chain termination reaction37 by use of acommercial kit (Sequenase.2, Cleveland, Ohio) withSP6 and T7 primers (Promega). For sequencing ofthe third exon, two additional primers, located about

250 nucleotides from each end of the exon, wereused.

For direct sequencing of PCR fragments of exon 3,the DNA was subjected to agarose gel electrophore-sis, eluted from the agarose gel with a commercial kit(Gene clean II, La Jolla, Calif.), boiled for 10 min-utes, cooled on ice, and sequenced according to theSequenase.2 kit protocol except that the annealingtime used was 10 minutes. A sequencing primer,5'-CACTTGAGCTTCCTGGAG-3', located 107 nu-cleotides 5' of the mutation point, was used.

Synthesis of PrimersPrimers for PCR amplification and sequencing

were synthesized on an Applied Biosystems Model381A DNA synthesizer.

Statistical MethodsPaired t tests (two-sided p values) were used to

assess differences in LCAT activation properties ofapo A-IV isoforms.

ResultsSequence analysis of amplified DNA correspond-

ing to the three exons of the apo A-IV gene fromsubjects homozygous either for apo A-IV 1-1 or 2-2only revealed one difference, a single-base substitu-tion at position 2,387. An autoradiogram of sequenc-ing gels of DNA from subjects with the phenotypesapo A-IV 1-1, apo A-IV 1-2, and apo A-IV 2-2 areshown in Figure 1. The substitution is a guanine tothymine change in the third base of codon 360,causing a glutamine to histidine substitution. Thisamino acid substitution results in an increase in thepositive charge of the apo A-IV 1-1 isoprotein by oneunit and explains the difference observed betweenthe isoproteins on IEF.

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Apo A-UE 1 - 1-CAG-

Codon -CAG-

-o1 - 2-CAG--CAT-

1 >1-1

-CAG--CAG-

D1 - 2 1 - 2 1 -2

- C A G - -CAG- -CAG--CAT- - C A T - - C A T -

FIGURE 2. Pedigree of a kindred with different electropho-retic apolipoprotein (apo) A-IV phenotypes. Apo A-IV pheno-types and corresponding codons for amino acid 360 in apoA-IV are indicated. Squares denote males, and circles denotefemales. C, cytosine; A, adenine; G, guanine; T, thymine.

To establish that this point mutation is the basis forthe common apo A-IV polymorphism, the inheri-tance of this mutation was studied in a family andcompared with the inheritance of the electrophoreticapo A-IV1 and apo A-IV2 alleles. The family tree of akindred consisting of three generations is illustratedin Figure 2. From these subjects, the portion of theapo A-IV gene that corresponds to exon 3 wasamplified by PCR as illustrated schematically inFigure 3. After amplification, the DNA from thefamily members was analyzed by direct DNA se-quencing (data not shown). It is evident that the apoA-IV1 allele cosegregates with the -CAG- coding forglutamine, while the -CAT- coding for histidine atposition 360 segregates with apo A-IV2.

The guanine to thymine substitution in the apoA-IV2 allele results in the loss of one recognition sitefor the restriction enzyme Fnu4Hl. This enzyme canbe used to demonstrate the substitution after ampli-fication of part of exon 3 with a tailored primer.38

Using this method, we determined the genotypes of32 unrelated subjects, and the results were concor-dant with the phenotypes obtained by IEF andimmunoblotting.

The mutation was further verified by amino acidsequence analysis of apo A-IV protein purified from

one subject homozygous for the apo A-IV' allele andfrom two subjects homozygous for the apo A-IV2

allele. In all cases, the results were in accordancewith those obtained by DNA sequencing.

As apo A-IV is an activator of LCAT, it was ofinterest to study whether the mutation described inter-feres with the activating capacity of apo A-IV. Theisoproteins apo A-FV 1-1 and apo A-IV 2-2 were eachisolated from two different subjects. In three separateexperiments the activation of LCAT by the two isopro-teins was compared with that of apo A-I. In theseexperiments apo A-I always was a better activator thaneither of the two apo A-IV isoforms (Figure 4). Themean percent activations caused by the isoproteins apoA-IV 1-1 and apo A-IV 2-2 compared with that of apoA-I were 43% and 36%, respectively. In all threeexperiments apo A-IV 1-1 was a somewhat betteractivator (/7<0.01) than was apo A-IV 2-2.

DiscussionIn the present study we determined the mutation

that causes the common electrophoretic apo A-IVpolymorphism. This polymorphism, which is identicalwith USP1,11 can easily be determined by IEF fol-lowed by Western blotting.18-20 It is the result of twocommon codominant alleles, apo A-IV1 and apoA-IV2, at a single genetic locus. PCR amplification ofthe apo A-IV alleles followed by cloning into aplasmid and nucleotide sequencing revealed that aguanine to thymine transition in the third position ofcodon 360 was the difference between the two alleles.This single-base substitution leads to a change ofamino acid 360. A glutamine at this position in apoA-IV 1-1 is substituted by histidine in apo A-IV 2-2.Analysis of the apo A-IV gene from a three-genera-tion kindred by use of PCR followed by directsequencing of the products further established thatthis point mutation underlies the common apo A-IVpolymorphism. Also, direct sequencing of the apoA-IV protein purified from subjects homozygous foreither the apo A-IV1 or apo A-IV2 allele were inagreement with the above findings.

In a previous study14 in which the primary struc-ture of apo A-IV was determined by direct proteinsequencing, the amino acid at position 360 wasglutamine, the amino acid corresponding to the more

apo ADZ 1-1

apo ABZ 2-2

162 127

FIGURE 3. Schematic presentationof the region of the apolipoprotein(apo) A-IV gene amplified in thisstudy. Exons are shown as blackboxes, and introns are indicated bylines. Their sizes (in number of nu-cleotides) are given above the bar.Positions of the primers used for thepolymerase chain reaction amplifica-tion are indicated by arrows. Positionwhere the base exchange occurs ismarked by a box. C, cytosine; A,adenine; G, guanine, T, thymine.

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Tenkanen et al Apo A-IV Polymorphism 855

200

E> 150O

c

~ 100

o<0

< 50

T• I

apo A- I

•

-

T i-

ipoA-X: i- £

T_L

poA-n.2- 2

100 O

3"01

o

01TJ

50 OJ»

H

Oo3

Q>O

B

O3

01(0

oo

•o01

a

FIGURE 4. Bar graph showing activation (nmol/ml/hat left; percent at right) of purified lecithin'.cholesteryIacyltransferase (LCAT) by apolipoprotein (apo) A-Iand two isoforms of apo A-IV. Activating capacity ofapo A-I and of the two isoforms of apo A-IV, apo A-IV1-1 and apo A-IV 2-2, are represented by columns.Each column represents the activating capacity ofisoprotein isolated from a separate subject. The assaywas performed with apoprotein-containing proteolipo-some substrates in which the molar ratio of eggphosphatidylcholinelcholesterollapoprotein was250:12.5:0.8. They were prepared by the sodiumcholate detergent dialysis procedure. Each columnrepresents the mean of six assays (±SD) performed inthree separate experiments.

common allele apo A-IV1. However, as this is dif-ferent from the amino acid histidine derived fromliver cDNA analysis by Elshourbagy et al,12 thoseauthors concluded that sequence heterogeneity atthis position could not be excluded. We now demon-strate that this sequence heterogeneity is geneticallydetermined and explains the apo A-IV polymor-phism.

This mutation causes the loss of one FnuAHlrestriction site. The DNA fragments resulting afterdigestion are small, thus making use of this enzymefor apo A-IV typing complicated. However, thesetechnical difficulties can be avoided by using PCRwith a tailored primer.38 In all 32 subjects studied bythis method, the common apo A-IV polymorphismcould be explained by a guanine to thymine substitu-tion of nucleotide 2,387.

The role of apo A-IV in lipoprotein metabolism isat present far from clear. It has been postulated to beinvolved in chylomicron and HDL metabolism.6

Therefore, the observation that in at least somepopulations20-24 the alleles at the apo A-IV locus havesignificant effects on plasma HDL cholesterol andtriglycerides is of interest and may help to unravel thephysiological role of apo A-IV. In this regard, knowl-edge on the variation in the apo A-IV gene, whichgives rise to apo A-IV polymorphism, may be impor-tant for understanding the underlying molecularmechanisms.

The LCAT enzyme is important for normal HDLmetabolism,39 and apo A-IV has been shown to be anactivator of LCAT at least in vitro.7-8 Our observa-tion, that under the assay conditions described apoA-IV is about one third as effective as apo A-I inactivating LCAT, is in line with previous work.7-8 Theuse of different isoforms of apo A-IV for activationindicated that the isoform apo A-IV 1-1 is a some-what better activator than apo A-IV 2-2. This obser-vation is contradictory to data recently reported thatapo A-IV 2-2 is more efficient in activating LCAT

than is apo A-IV l-l.40 As it is known that LCATactivation by apoproteins depends on the composi-tion of the lipid substrate,7 it is possible that thisapparent discrepancy is due to assay conditions.

The activating capacity of apo A-I on LCAT isthought to be due to its amphipathic helixes, whichare similar to those found in apo A-IV. Recently, itwas shown that a major LCAT-activating effect ofapo A-I is associated with two 22-mer tandem am-phipathic helix repeats, one located between residues66-87 and the other between residues 99-120 of apoA-I.41 It is conceivable that amphipathic regions ofthe apo A-IV molecule in a similar way take part inthe activation of LCAT. The amino acid substitutionon which the apo A-IV polymorphism is based (atresidue 360) is located in the center of one such22-mer amphipathic helix, amino acids 351-373.Whether the small changes in the activating capacityof different apo A-IV isoforms that we have observedduring in vitro incubations have any physiologicalrelevance in vivo remains to be established.

Acknowledgments

We thank Liisa Ikavalko for expert technical as-sistance and Liisa Palonen for help in preparing themanuscript.

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KEY WORDS • apolipoprotein polymorphisms • mutations •polymerase chain reaction • lecithin :cholesteryl acyltransferase

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H Tenkanen, M Lukka, M Jauhiainen, J Metso, M Baumann, L Peltonen and C Ehnholmhistidine substitution of amino acid 360.

The mutation causing the common apolipoprotein A-IV polymorphism is a glutamine to

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