Download The Nucleotide and Derived Amino Acid

Val. 261. No. 5. Issue of February 15. pp. 1998-2002,1986
Printed in U.S.A.
THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
0 1986 by The American Society of Biological Chemists, Inc
The Nucleotide and Derived Amino Acid Sequence of Human
Apolipoprotein A-IV mRNA and theClose Linkage of Its Gene to the
Genes of Apolipoproteins A-I and C-111”
(Received for publication, August 14, 1985)
Nab11 A. Elshourbagyss, David W.Walkers, Mark S . Boguskil, Jeffrey I. Gordon$, and
John M. TaylorSPII
From $The Gladstone Foundation Laboratories for CardiovascularDisease, the §CardiovascularResearch Institute, and the
Department of Physiology, University of California, Sun Francisco, California 94140-0608 and the YDepartments of Biological
Chemistry and Medicine, Washington University Schoolof Medicine, St. Louis, Missouri 63130
Human apolipoprotein (apo-’) A-IV is a major component
of newly synthesized chylomicrons, but it is not found in
significant amounts in other lipoproteins, including chylomicron remnants (reviewed in Ref. 1).In the rat, apo-A-IV is
also a major component of high density lipoproteins (2). In
all species examined, about half of the circulating apo-A-IV
is found in the lipoprotein-free fraction of plasma (3), but it
may be redistributed to lipoproteins according to therequirements of extracellular lipid metabolism (4). While the overall
* The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked ‘‘advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
The abbreviations used are: apo-, apolipoprotein; kb, kilobases.
clease fragment of the rat cDNA (6) that was 32P-labeledby random
priming (15). Plaque hybridizations were carried out at 42 “C in a
buffer containing 20% deionized formamide, 0.9 M NaCl, 50 mM
sodium phosphate at pH 7.0, 5 mM EDTA, 0.1% sodium dodecyl
sulfate, and 200 pg/ml denatured herring sperm DNA. The cDNA
inserts from positive recombinants were subcloned into bacteriophages M13mp18 and M13mp19 for nucleotide sequence determinations by the dideoxynucleotide chain termination method (16). Human apo-A-IV sequence-specific oligodeoxynucleotide primers were
synthesized with an Applied Biosystems (Foster City, CA)Model
380A synthesizer.
Human apo-A-IV genomic clones were selected from a cosmid (17)
genomic DNA library (provided by Dr. Chris Lau, University of
California at SanFrancisco) by screening with the 32P-labeledinsert
prepared from a cloned human apo-A-IV cDNA that had been characterized as indicated above. Positive recombinants were selected,
and their inserts were subcloned for partial nucleotide sequence
determinations as described above.
Total cellular RNA was prepared from adult rat, marmoset, and
human tissues (18) and examined by dot blot and Northern blot
1998
Downloaded from www.jbc.org by on February 28, 2007
metabolic role of apo-A-1V is unknown, Steinmetz and UterBoth cDNA and genomic clones encoding human apolipoprotein (apo-) A-IV have been isolated and char- mann (5) have demonstrated that human apo-A-IV can be a
acterized. Southern blot analyses of apo-A-IV gene- significant activator of 1ecithin:cholesterol acyltransferase.
containing cosmids revealed that theapo-A-IV gene is This finding is consistent with the structure of rat apo-A-IV
linked to the apo-’A-Iand apo-C-I11 geneswithin a 20- that we determined previously (6).
kilobase span of chromosome 11 DNA. The apo-A-IV
Rat plasma apo-A-IV, a single chain protein of 371 amino
gene is located about 14 kilobases downstream from acids (Mr = 44,465), contains closelyhomologous, tandem
the apo-A-I genein thesame orientation, with the apo- repetitions of a 22-residue segment with amphipathic a-heliC-I11 gene located between them in the opposite ori- cal potential (6). The amphipathic characterof these repeated
entation. The nucleotide sequence
of the corresponding units is probably responsible for the 1ecithin:cholesterol acylhuman apo-A-IV mRNA was determined, and the de- transferase-activating capability of apo-A-IV (7, 8). The serived amino acid sequence showed
that matureplasma
apo-A-IV contained 376 residues. Throughout most of quence and organization of the docosapeptide units are reits length, human apo-A-IVwas found to contain mul- markably homologous to the tandemly repeated segments
tiple tandem 22-residue repeated segments having am- found in apo-A-I (7,9),which suggests that thecorresponding
phipathic, a-helical potential. Amino acid substitutions genes might have arisen from an unequal gene duplication
within these homologous segments were generally con- event and that they may be closelylinked (6).
This study reports the complete structure of human plasma
servative in nature. A comparison of the sequences of
apo-A-IV
and thefinding of an extensive domain of repeated
human and rat apo-A-IV revealed a 79% identity of
amino acid positions
in the amino-terminal 60 residues amphipathic segments within the protein. In addition, the
and a 58% identity in theremainder of the sequences, gene that encodes human apo-A-IV is linked closely to the
with the human protein containing 5 extra residues apo-A-I gene, with the apo-C-I11 geneinterposed. Our results
near the carboxyl terminus. An examination of the expand the previously reported linkage of the apo-A-I and
distribution of apo-A-IV mRNA in different tissues of apo-C-I11genes found on chromosome 11(10-12), raising the
the rat, marmoset, and manshowed that apo-A-IV possibility of a coordinate control of the expression of all
mRNA was abundant in both the liver and small intes- three genes.
tine of the rat, but abundant in only the small intestine
EXPERIMENTALPROCEDURES
of the marmoset and man. It was expressed in only
trace amounts in all other tissues that were examined.
Human apo-A-IV cDNA clones were selected from a XgtlO (13)
These findings on the structureand expression of apo- liver cDNA library (provided by Dr. Beatriz Levy-Wilson, Gladstone
A-IV and the close linkage of its gene to those of apo- Foundation Laboratories) by screening (14) at a reduced stringency
A-I and apo-(3-111 suggest a regulatory relationship with a previously characterized rat apo-A-IV cDNA (6). The hybridization probe was a 1227-base pair XmnI-BstXI restriction endonubetween the threegenes.
Human Apolipoprotein
mRNA A-IV
1999
analyses as described previously (18).Animals were maintained and
tissues were collected as described (18).Tissues from four rats, four
marmosets, and two human trauma victims were examined.
Pstl
Pstl
Pstl
EcoRl
-
c
*
RESULTS ANDDISCUSSION
A
c-m
A-I
-
’ hHA4C202
-
*”””c
hHA4C181
AHA4C201
hHA4C151
-
. ....
*-
”“””
I
l
l
l
- -- --
pHA4G51
““”c
4
2
t
-.
D\u
8
6
l
l
l
l
10
I
l
12
l
I
14
l
l
1
Nucleotides x
FIG. 2. Nucleotide sequence strategy for apo-A-IV mRNA.
The solid bar indicates the nucleotide sequence that was determined
from the cDNA inserts that were cloned in XgtlO. The stippled and
hatched bars and the open bar indicate the sequence that was determined from two exons of a cosmid clone. In this cosmid gene clone,
the regions indicated by the stippled and hatched bars were separated
by an intron, which is illustrated by the line connecting the bars in
the inset. The sites of restriction endonucleases that were used for
the subcloning of DNA fragments into bacteriophage M13 for sequence analysis are indicated. The arrows indicate the direction and
length of the sequence determination, with the solid arrows indicating
the use of a universal primer (16) and the dashed arrows indicating
the use of oligonucleotideprimers that were synthesized to correspond
to sequences that were determined in this study. The X cDNA clones
and the cosmid gene clone employed are identified by their clone
numbers.
A-IP, 3
A-W, 5’
A-I (13) and apo-C-I11 (20) cDNAs. Fig. lA shows that all of
the probes hybridized to each of the cosmids and that the
probes bound to a single HindIII fragment of about 19kb that
23.1.
9.4.
was common to each cosmid insert. Therefore, the apo-A-IV
6.6.
gene was linked closely to theapo-A-I and apo-C-I11 genes.
4.4.
The orientation of the apo-A-IV gene with respect to the
apo-A-I and apo-C-I11 genes was investigated by Southern
blot hybridization. To identify the apo-A-IV gene, two probes
were prepared from a cloned cDNA cleavage of an EcoRI site
within this cDNA yielded a 5”terminal fragment of about
400 base pairs and a 3”terminal fragment of about 700 base
pairs. The cDNA probes for both the apo-A-I and apo-C-I11
I
l
l
1
I
l
l
1
genes had been characterized previously by restriction endo51 52 2121
51 52 21
21
51 52
21
21
52
51
21
21
nuclease mapping and nucleotide sequencing (13, 20) and
corresponded to about three-fourths of the lengths of their
corresponding mRNAs (data not shown). A single Southern
blot of the threeunique cosmid DNAs that hadbeen digested
with EcoRI was prepared and hybridized to each probe sequentially (Fig. lA).The EcoRI enzyme was chosen because
the cosmid vector (17) contained unique sitesfor this enzyme
on either side of the genomic DNA insertion site (BamHI).
The apo-A-I probe hybridized to adifferent size EcoRI
fragment in each cosmid DNA, indicating that the apo-A-I
gene was located at one end of the human genomic DNA
insert. The apo-C-I11 probe bound to this same fragment in
L
‘
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
each cosmid, consistent with the previously described finding
4
18 2
16
146
12
8
10
that it was about 2.6 kb downstream from the apo-A-I gene
Nucleotides x
FIG. 1. Linkage of the human apo-A-I, apo-C-111, and apo- and in theopposite orientation (10, 11).The apo-C-I11probe
A-IV genes. A, the DNAs from cosmid clones pHA4G21, pHA4G51, also bound to a 3.0-kb fragment that was identical in size in
and pHA4G52 weredigested with EcoRI or HindIII and examined by each of the cosmids, indicating that itcontained the 5’ portion
Southern blot hybridization to 32P-labeledcDNA probes specific for of the apo-C-I11 gene (according to the previously described
apo-A-I, apo-C-111, and the 5’ and 3’ portions of apo-A-IV. After ( 1 0 ,l l ) linkage orientation) and that was
it contained within
each hybridization, the probe was removed from the filter (14), and
the same filter was rehybridized to another probe. B, the linkage of the interior of the genomic DNA insert. The 5”terminal apoA-IV probe bound to a 1.2-kb fragment that was the same
the apo-A-I, apo-C-111, and apo-A-IV genes is shown with a fewof
the known restriction endonuclease sites in this gene locus. The size in each cosmid, indicating that it also was contained
arrows indicate the direction of transcription of these genes.
within the interior of the cloned human DNA. The 3”termi0
I
I
I
I
I
I
I
I
I
Downloaded from www.jbc.org by on February 28, 2007
The screening of about 500,000 recombinants yielded 10
clones that were candidates for containing human apo-A-IV
cDNA inserts. DNA was prepared from each of them for
analysis, and examination by agarose gel electrophoresis (14)
indicated that the insertsizes ranged from -300 to 1100 base
pairs in length. The DNAs from six of these candidates were
examined further by hybridization selection and translation
(19) of human intestine mRNA. In each case, the hybridization-selected mRNA directed the synthesis of a M , = 46,000
protein that was immunoprecipitated by an antibody specific
for human apo-A-IV (data not shown). The identity of the
cloned inserts was subsequently demonstrated by nucleotide
sequence analysis as described below.
Linkage of the Apolipoprotein A-I, C-III, and A-IVGenesTo select the corresponding gene, a human genomic cosmid
library was screened with the longest cloned apo-A-IV cDNA
insert. Three positive recombinants were identified that had
an average insert length of 34 kb and some, but not all,
restriction endonuclease fragments in common. The cloned
DNAs were digested with EcoRI and HindIII, resolved by
agarose gel electrophoresis, and blotted to a nitrocellulose
filter (14). The filter was probed with a cloned human apo-AIV cDNA as well as with previously characterized humanapo-
I
3
Human Apolipoprotein A-IV mRNA
2000
G l u V a l Ser A l a A s p G l n V a l A l a
Th,r V a l Met Trp A s p Tyr Phe Ser G l n L e u Ser A s n A s n A l a L y s G l u A l a V a l G l u
GAG GTC AGT GCTGAC
CAG GTG GCC ACA GTG ATG TGGGAC
TACTTC
AGC CAG CTGAGC
AAC AAT G C C AAG GAG GCCGTG
1
30
60
His L e u G l n
GAACAT
CTC CAG
~ y Ser
s
G l u L e u Thr G l n G l n Leu A s n A l a Leu Phe G l n ASP ~ y Lse u G l y G l U V a l A s n
Thr Tyr A l a G l y A s p L e u G l n L y s L y S L e u
AAA T C T GAACTCACC
CAG CAA C T C AATGCCCTCTTC
CAG GAC AAA CTT GGAGAA
GTG AAC ACTTAC
GCA GGTGAC
CTG CAG AAG
AAG
120
V a l Pro Phe A l a Thr G l u L e u
90
CTG
180
150
H i s G l u A r g Leu A l a - L y s A s p Ser G l u L y s L e u L y s G l u G l u
I l e G l y L y S G l U Leu G l U G l u Leu A r g
AAG GACTCG
GAG AAA CTG AAG GAG GAG ATT GGG AAG GAG CTG GAG GAG CTG AGG
210
240
2 10
~ n CCC
; TTT GCC ACC GAG CTGCATGAACGCCTGGCC
Leu Pro His A l a A s n G l u V a l
Ser G l n Lys I l e G l y A s p A s n L e u A r g G l u
Leu G l n G l n A r g L e u G l u
Pro "Yr A l a A S P
A l aA r gL e u
GCC CGG CTG
CTG
CCCCATGCC
AAT GAG GTG AGC CAG
AAG
ATC GGG GAC AAC CTG
CGA
GAG CTT CAG
CAG
CGC CTG GAG CCCTACGffiGAC
300
330
360
G l n Leu A r g Thr G l n V a l A s n Thr G l n A l a G l u G l n Leu A r g A r g G l n Leu Thr Pro Tyr Ala G l n A r g net G l U A r g V a l
Leu A r g G l U
CAG CTG
CGC
ACC
CAG GTC AAC ACG CAG GCC GAG CAG CTG CGG CGC CAG CTG ACCCCCTAC
GCA CAG CGC ATG GAG AGA GTG
CTG
CGG GAG
390
420
450
A s nA l aA s p
AAC GCCGAC
Ser L e u G l n A l a Ser L e u A r g
AGCCTG
CAG GCCTCG
Cl'G
AGG
Pro His A l a ASP G l U Leu LYS A l a LYS I l e ASP G l n A s n V a l G l u G l u L e u L y s G l y A r g
CCCCACGCCGAC
GAG CTC AAG GCC AAG ATCGAC
CAG AAC GTG GAG GAG CTC AAG GGA CGC
480
510
540
ser L e u A l a
GAG GAG CTGCGCCGCAGCCTGGCTCCCTATGCT
600
Leu Thr Pro Tyr A l a ASP G l U Phe LYS V a l LYS I l e ASP G l n Thr V a l G l U G l U L e u A r g A r g
CTT ACG CCCTACGCTGACGAATTC
AAA GTC AAG A I TG A C
CAG ACCGTG
570
Pro Tyr A l a G l n ASP T h r
CAG GAC ACG
630
G l n G l u Lys L e u A s n His G l n L e u G l u G l y Leu Thr Phe G l n Met LYS LYS A s n A l a G l u G l u L e u
LYS A l a A r g I l e Ser A l a Ser A l a
CAG GAG AAG CTC AAC CAC CAG C T T GAG GGCCTGACC
T T C CAG ATG AAG
AAG
AAC GCC GAG GAG CTC AAG GCC AGG <ATCTCGGCCAGTGCC
660
690
720
G l U G l U Leu A r g G l n A r g L e u A l a
Pro Leu A l a G i u A s p V a l A r g G l y A s n
GAG GAG CTG CGG
CAG
AGG
CTG GCG CCCTTGGCC
GAG GACGTGCGTGGCAACCTG
Ser Leu A l a
Leu A r g G l y A s n
Thr G l u G l y L e u G l n L y s
AGG GGC AAC ACC GAG GGG CTG CAG
AAG
TCA
CTG
GCA
7 80
750
810
G l n Met G l u G l n L e u A r g
Thr L y s Leu G l y Pro His A l a G l y A s p V a l G l u G l y
His L e u Sec Phe Leu G l u L y s A s p Leu- A r g A s p L Y S
CAT GCG GGG GACGTG GAA GGC CAC TTG AGC T T C .CTG GAG AAG GAC CTG AGG GAC AAG
CAG ATG GAA CAG CTC AGG ACG AAA CTGGGCCCC
930
96 0
990
V a lA s n
Ser Phe Phe Ser Thr Phe L y s G l u L y s G l u Ser G l n A s p L y s Thr Leu Ser L e u Pro G l u Leu G l u G l n G l n G l n G l u G l n
His
G T C AAC TCC TTCTTC
AGCACC
TCCCTCCCT
GAG CTG GAG CAA CAG
CAG
GAA CAG CAT
T T C AAG GAG AAA GAG AGC CAG GAC AAG ACTCTC
1020
1050
1080
***
Gln Glu Gln Gln Gln Glu Gln Val
G l n Met Leu A l a Pro Leu G l u Ser
CAG GAG CAG CAG
CAG
GAG CAG GTG CAG ATG CTGGCC
CCT TTG GAG AGC TGAGCTGCCCCTGGTGCACTGGCCCCACCCTCGTGGACACCTG
1110
1141
CCCTGCCCTG CCACCTGTCT
AGCTGCTGAG
AATCTAGCCT
C
1291
1171
GTCTGTCCCA AAGAAGTTCT GGTATGAACT TGAGGACACA TGTCCAGTGG GAGGTGAGAC CACCTCTCAA TATTCAATAA
1201
1231
1261
- POly(A)
FIG. 3. Nucleotide and amino acid sequence of human apo-A-IV. The numbers indicate nucleotide
sequence positions; The sequence begins with the first amino acid of the mature plasma protein. The translation
termination codon is indicated by asterisks.
nal apo-A-IV probe bound to a fragment that varied in size
in each cosmid, but 'was different from the fragments that
bound the apo-A-I probe, indicating that the 3'4erminal
portion of the apo-A-IV gene was contained within an EcoRI
fragment located at the opposite end of the insert from the
apo-A-I gene. Our results, together with the previously determined linkage and orientation of the apo-A-I and apo-C-I11
genes (10, ll),also indicated that the apo-A-IV gene had the
same transcriptional orientation as the apo-A-I gene. Additional Southern blot analyses with other restriction endonucleases were consistent with these conclusions (datanot
shown). The linkage of the three genes is illustrated in Fig.
1B. The apo-A-IV gene is located about 8 kb away from the
apo-(3-111 gene and about 14 kb downstream from the apo-AI gene.
Structure of H u m a n Plasma Apolipoprotein A-IV-The nucleotide sequence of human liver apo-A-IV mRNA was determined according to the strategy shown in Fig.'2. Since none
of the cDNAs were full-length copies of the mRNA, the
sequence of the coding portion that contained amino acids 155 of plasma apo-A-IV was determined from the gene, and
the remaining portion was determined from the cloned
cDNAs. The amino-terminal boundary of the mature plasma
protein coding region was identified by comparison to the
previously determined partial amino acid sequence of this
region (21).' In addition, the amino-terminal sequence of rat
apo-A-IV (6) was found to be particularly homologous to the
human sequence. Furthermore, the carboxyl-terminal amino
acid of the 20-residue signal peptide sequence of human apoA-IV (21) was identified as alanine (data not shown), which
is consistent with the position of the mature amino terminus
of human plasma apo-A-IV. This residue was apparently
adjacent to an upstream intron similar to theintron location
of other apolipoprotein genes (22).
The nucleotide sequence of the apo-A-IV plasma protein
coding region of the gene and its corresponding mRNA,
together with the 3"terminal noncoding region, is shown in
Fig. 3. The derived amino acid sequence is also shown. Human
The amino acid sequence of 39 residues beginning at the amino
terminus of human plasma apo-A-IV has been determined by Drs.
Stanley C. Rall, Jr., and Karl H. Weisgraber of the Gladstone Foundation Laboratories (unpublished observations). It is in complete
agreement with the amino acid sequence that was derived from the
nucleotide sequence.
Downloaded from www.jbc.org by on February 28, 2007
G l U Leo G l y G l y His Leu A s p G l n G l n V a l G l u G l u
Phe R r g A r g A r g V a l G l u
Pro Tyr G l y G l u A s n Phe A s n Lys A l a Leu V a l G l n
GAG CTG GGT GGG CAC CTG
GAC
CAG CAG GTG GAG GAG T T C CGA
CGC
CGG GTG GAG CCCTAC
GGG GAA AAC T T C AAC AAA GCC CTG
GTG
CAG
840
870
900
Human Apolipoprotein A-IV mRNA
.........
.........
EVSAWVATVMW
EVTSWVANVMW
human
Rat
Man
A
Liver-
rat
.................................
.................................
DYFTQLSNNAKEAVEQLQKTDVTQQLN TLFQDKLCNINTYADDLQNKLV
0
Large Intestine
human
Testes
rat
Spleen
:::
5.::
Marmoset
@
-
Intestine
Small
40
DYFSQLSNNAKEAVEHLQKSELTQQLN ALFQDKLCEVNTYACDLQKKLV
13
2001
Pancreas
62
PFATELHERLAKDSEKLKEEIC
84
KELEELRARLL
human
PFAVQLSCHLTKETERVREEIQ
KELEDLRANMM
rat
.......
..........
Kidney
Lung
Stomach
Brain
117
95
PHANEVSQKICDNLRELQQRLE
.............
PYADQLRTQVNTQAEQLRRQLT
human
PHANKVSQMFCDNVQKLQEHLR
PYATDLQAQINAQTQDMKRQLT
rat
139
PYAQRMERVLRENADSLQASLR
161
PHADELKAKIDQNVEELKCRLT
human
PFANELKEKFNQNMECLKCQLT
rat
PYADEFKVKIWTVEELRRSLA
205
PYAQDTQEKLNHQLECLTFQMK
human
PRANELKATIDQNLEDLRSRLA
PLAECVQEKLNHQMECLAFQMK
rat
227
KNAEELKARISASAEELRQRLA
249
PLAEDVRCNLRCNTECLQ
human
PLVEDVQSKLKCNTECLQ
rat
.......
....
....
Adrenal
Heart
3 2 1 0.5
.....
.....
. .. .. ..
PYIQRMQTTIQDNVENLQSSMV
..............
w
B
183
. . . . . .. .. .. .. .. ....
.............
Human
-Origin
-1169
- 1101
289
PYCENFNKALVQQMEQLRTKLC
human
KSLEDLNKQLDQQVEVFRRAVE
PLCDKFNMALVQQMEKFRQQLC
rat
..............
1 0.5
-ma
267
KSLAELGCHLDQQVEEFRRRVE
. . . . . . . . . . . ........
Rat
3 2
RNA
-526
-447
-215
31 1
PHACDVECHLSFLEKDLRDKVN
human
SDSCDVESHLSFLEKNLREKVS
rat
SFFSTFKEKESQDKTLSLPELEQQQEQHQEQQQEQVQMLAPLES
human
SFMSTLQKKCSPWPLALPLPEQVQEQVQEQVQPK-----PLES
rat
...............
...............
...................
....
FIG. 4. Alignment of the amino acid sequences of human
and rat apo-A-IV. The optimal alignment of the human and rat
amino acid sequences was made with the use of the PRTALN program
(ktuple = 1, window = 20, gap penalty = 1)obtained from Dr. David
Lipman, National Institutes of Health. The methods used for defining
the boundaries of the repeated sequence units have been described
previously (6, 24). Amino acid identities are indicated by a colon. The
numbers indicate the first residue in each repeat unit.
plasma apo-A-IV contains 376 amino acids, witha M, =
45,150. There are no cysteine residues. Analysis of the sequence by the correlogram algorithm of Kubota et al. (23) as
well as the comparison matrix algorithm of McLachlan (9)
indicated the presence of multiple repeated segments within
human apo-A-IV. As observed inrat apo-A-IV (6),these
segments are not exact duplications, but have conservative
amino acid substitutions thatgenerally preserve their physical
and chemical characteristics (Fig. 4). The repeated units
correlated approximately with the positions of proline residues, which are located with unusual regularity throughout
the sequence. Accordingly, these analysesindicated that
nearly the entire human apo-A-IV protein consisted of 14.5
tandem repetitions of a 22-residue element that is itself the
product of an 11-merduplication (datanot shown). The
optimal alignment of these repeat units is illustrated in Fig.
4, which indicates a limited amount of length polymorphism.
One repeat unit contained a deletion of 4 residues, equivalent
to one turn of an a-helix, which should not disrupt the
surface
properties of the overall alignment of repeat units.
Liver'
'Small
Intestine
J
Small
Liver
L
Intestine
FIG. 5. Distribution and size of apo-A-IV mRNA in the tissues of rats, marmosets, and man. A , aliquots containingdifferent
amounts of total cellular RNA were supplemented with yeast RNA,
denatured, applied to nitrocellulose filters, and examined by hybridization and autoradiogram densitometry as described previously (18).
Rat RNA dot blots were hybridized to a previously characterized rat
apo-A-IV cDNA (6), and the marmoset and human RNA dot blots
were hybridized to a human apo-A-IV cDNA that was characterized
in the present study. Autoradiograms of the blots are shown here. B,
samples containing 30 pg of total cellular RNA were denatured with
glyoxal, electrophoresed in 1.1%agarose gels, blotted to nitrocellulose,
and hybridized to homologous 32P-labeledcDNA probes (18). Autoradiograms of the blots are shown here. The positions of HindIIIdigested SV40 DNA fragments are indicated as molecular size standards.
The human apo-A-IV amino acid sequence was compared
to thatof the rat (Fig. 4), and they
were found to be identical
in 61% of their overall positions. The most highly conserved
portion of the two proteins was in theamino-terminal region,
where there was a 79% identity in the first 60 residues. The
significance of this domain is unknown. The remainder of
apo-A-IV was found to have a 58% homology between the
human and rat proteins. The repeating units in the human
protein are more highly conserved with respect to each other
than those in the rat. This difference may reflect either the
finding that rodent genes evolved considerably faster than
those of man(25) or that there were particular selection
pressures on these proteins.
To achieve an optimal alignment in the carboxyl-terminal
domain, a 5-residue deletion in the rat sequence was postu-
Downloaded from www.jbc.org by on February 28, 2007
...
KNAEELHTKVSTNIDQLQKNLA
.......
................
3 2 1 0.5
2002
Human Apolipoprotein A-IV mRNA
Acknowledgments-We thank Dr. Karl H. Weisgraber for providing antibodies to human apo-A-IV and Dr. Seatriz Levy-Wilson for
providing a human apo-C-I11 cDNA.We thank Drs. Stanley C. Rall,
Jr., and Karl H. Weisgraber for their determination of the amino acid
sequence of the amino-terminal 39 residues of human plasma apo-AIV. Gratitude is expressed to Dr. Robert W. Mahley for his interest
and support of these studies and to Drs. Brian McCarthy, Beatriz
Levy-Wilson, and Stanley C. Rall, Jr., for their helpful discussions.
We thank James X. Warger and Norma Jean Gargasz for graphics
assistance and Barbara Allen and Sally Gullatt Seehafer for editorial
assistance.
REFERENCES
1. Mahley, R. W., Innerarity, T. L., Rall, S. C., Jr., and Weisgraber,
K. H. (1984) J. Lipid Res. 2 5 , 1277-1294
2. Swaney, J. B., Braithwaite, F., and Eder, H. A. (1977) Biochemistry 16,271-278
3. Fidge, N. H. (1980) Biochim. Biophys. Acta 6 1 9 , 129-141
4. DeLamatre, J. G., Hoffmeier, C.A., Lacko, A. G., and Roheim,
P. S. (1983) J. Lipid Res. 2 4 , 1578-1585
5. Steinmetz, A., and Utermann, G. (1985) J. Biol. Chem. 2 6 0 ,
2258-2264
6. Boguski, M. S., Elshourbagy, N. A., Taylor, J. M., and Gordon,
J. I. (1984) Proc. Natl. Acad. Sei. U. S. A . 81,5021-5025
7. Segrest, J. P., Jackson, R. L., Morrisett, J. D., and Gotto, A. M.,
Jr. (1974) FEBS Lett. 38,247-253
8. Kaiser, E. T., and Kezdy, F. J. (1983) Proc.Natl.Acad.Sci.
U. S. A . 8 0 , 1137-1143
9. McLachlan, A.D. (1977) Nature 267,465-466
10. Karathanasis, S. K., McPherson, J., Zannis, V. I., and Breslow,
J. L. (1983) Nature 3 0 4 , 371-373
11. Protter, A. A., Levy-Wilson, B., Miller, J., Bencen, G., White, T.,
and Seilhamer, J. J. (1984) DNA (N. Y.) 3,449-456
12. Bruns, G.A.P., Karathanasis, S. K., and Breslow, J. L. (1984)
Arteriosclerosis 4 , 97-102
13. Seilhamer, J. L., Protter, A. A., Froward, P., and Levy-Wilson,
B. (1984) DNA (N. Y.) 3,309-317
14. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular
CEorting:ALaboratory M u n u t , Cold Spring HarborLaboratory,
Cold Spring Harbor, NY
15. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132,
6-13
16. Messing, J. (1983) Methods Enzymol. 1 0 1 , 20-78
17. Lau, Y.-F., and Kan, Y. W. (1983) Proc. Nutl. Acad. Sci. U. S. A .
8 0 , 5225-5229
18. Elshourbagy, N. A., Liao, W. S., Mahley, R. W., and Taylor, J.
M. (1985) Proc. Natl: Acad. Sci. U. S. A . 82,203-207
19. Ricca, G.A., Hamilton, R. W., McLean, J. W., Conn, A., Kalinyak, J. E., and Taylor, J. M. (1981) J. Biol. Chem. 2 5 6 ,
10362-10368
20. Levy-Wilson, B., Appleby, V., Protter, A,, Auperin, D., and Seilhamer, J. J. (1984) DNA (N. Y.) 3,359-364
21. Gordon, J. I., Bisgaier, C.L.,
Sims, H. F., Sachdev, 0. P.,
Glickman, R.M., and Strauss, A. W. (1984) J. Biol. Chem.
259,468-474
22. Paik, Y.-K., Chang, D. J., Reardon, C. A., Davies, G. E., Mahley,
R. W., and Taylor, J. M. (1985) Proc. Natl. Acad. Sci. U. S. A.
82,3445-3449
23. Kubota, Y., Takahashi, S., Nishikawa, K., and Ooi, T. (1981) J.
Theor. Biol. 91, 347-361
24. Boguski, M. S., Elshourbagy, N. A., Taylor, J. M., and Gordon,
J. I. (1985) Proc. Nutl. Acad. Sci. U. S. A. 82, 992-996
25. Wu., C.-I., and Li, W.-H. (1985) Proc. Natl. Acad. Sci. U. 5'. A .
82,1741-1745
26. Weinberg, R. B., and Spector, M. S.(1985) J. Biol. Chem. 2 6 0 ,
4914-4921
Downloaded from www.jbc.org by on February 28, 2007
lated. In thisregion, the sequence E-Q-X-Q was repeated four
times inthe human proteinand threetimes in the ratprotein,
which may have significance with respect to the ratdeletion.
Analysis of the carboxyl-terminal 44 residues of apo-A-IV did
not reveal a significant sequence homology with the preceding
repeated segments.
Tissue Distribution of Apolipoprotein A-IV mRNA-The
comparison of rat and human apo-A-IV was extended to an
examination of the expression of the corresponding mRNAs
in various tissues. Total cellular RNAs from different tissues
of the rat, the marmoset (a new-world primate), and man
were examined by dot blot and Northernblot hybridizations,
followed by quantitative scanning densitometry (18). Fig. 5A
shows that apo-A-IV mRNA is most abundant in the small
intestine in each animal species. In the adult rat, apo-A-IV
was present in the liver at a level that was 12% of that
observed in the small intestine, with no observable difference
in size between the two tissues (Fig. 5B). In contrast, apo-AIV mRNA in the marmoset liver or human liver was <2% of
that observed in the small intestine. The significance of this
difference is unclear, but it may reflect differences in lipid
metabolism among these species. No other tissue in any of
these three species contained significant amounts of apo-AIV mRNA. These findings indicate that theintestine may be
the only significant source of apo-A-IV in humans and that
the liver may contribute only minor amounts of this apolipoprotein to theplasma.
The precise function of apo-A-IV is unclear. The multiple
repeated amphipathic segments of this protein suggest that it
can be a significant activator of 1ecithin:cholesterolacyltransferase (6); this cofactor activity has been demonstrated recently for human apo-A-IV ( 5 ) .However, the close association
of apo-A-IV with triglyceride-rich lipoproteins further suggests a potential role in the metabolism or structure of these
particles. In thisregard, a recent examination of the properties
of human plasma apo-A-IV has suggested that itslipid binding
properties are especially sensitive to microenvironmental factors (26). The amphipathic structure of apo-A-IV reported in
this study suggests that the association of apo-A-IV with
lipoproteins may be particularly sensitive to their surface
characteristics. Thus, the distribution of apo-A-IV between
lipoproteins and the lipoprotein-free fraction of plasma may
be a function of potential changes in thesurface properties of
lipoproteins asa consequence of their metabolism during
circulation.