(Received for publication, August 10, 1995; and in revised form, November 6, 1995)
From the
We have identified and characterized a novel proline- and arginine-rich protein component of lipoproteins, present in up to five sialylated isoforms, in rabbit blood plasma. The pI of the desialylated protein is 5.7. Based upon its N-terminal sequence, a complete cDNA sequence of 555 nucleotides was cloned from rabbit liver.The synthesized protein is predicted to contain 124 amino acids, including a typical signal peptide of 27 residues. The mature protein of 97 amino acids, designated apolipoprotein C-IV, is associated with the lipoproteins of blood plasma, primarily very low density and high density lipoproteins. It contains two potential amphipathic helices characteristic of plasma apolipoproteins and forms discoidal micelles with phosphatidylcholine. Northern analysis shows a single 0.6-kilobase apolipoprotein C-IV mRNA, detected only in the liver, and Southern analysis suggests a single copy gene. Sialylated apolipoprotein C-IV is secreted from transfected mammalian cells. Nucleotide sequence comparisons demonstrate a strong homology to portions of the upstream regions of the mouse and human apolipoprotein C2 genes, within each of which a distinct gene has recently been identified. The nucleotide sequences and the predicted amino acid sequences, as well as corresponding cDNA sequences in the rat and monkey, indicate that the apolipoprotein C4 gene has been highly conserved during mammalian evolution.
Lipoproteins in blood plasma of mammals contain a variety of
protein components, most of which are water-soluble proteins, encoded
by a gene family characterized by repeating sequences of amphipathic
helices containing 11 or 22 amino acids(1) . Those of low
molecular mass (approximately 6-12 kDa) are generally called
``C'' apoproteins, of which three are well
recognized(2) . These apoproteins are found in
triglyceride-rich lipoproteins (chylomicrons and very low density
lipoproteins (VLDL)) ()and high density lipoproteins (HDL),
among which they readily transfer according to the relative
concentrations and, presumably, surface areas of the lipoprotein
particles(3) . ApoC-I and apoC-II lack carbohydrate, whereas
apoC-III has an O-linked carbohydrate moiety containing up to
three sialic acid residues(2) . The functions of the C
apoproteins are only partly understood. During postprandial lipemia, C
apoproteins move from HDL to nascent chylomicrons and are returned to
HDL as chylomicrons are metabolized by lipoprotein lipase(4) .
ApoC-II is a specific cofactor for the action of lipoprotein lipase
upon the ester bond of triacylglycerols and glycerophosphatides of
triglyceride-rich lipoproteins(5, 6) . The other C
apoproteins can inhibit this action of apoC-II(5, 6) ,
and all of the C apoproteins can inhibit the clearance of remnants of
triglyceride-rich lipoproteins by the liver(7) .
The C apoproteins are most commonly visualized in triglyceride-rich lipoproteins by isoelectric focusing electrophoresis in polyacrylamide gels. During studies of lipoprotein metabolism in the rabbit, we observed a complex pattern of apoproteins in triglyceride-rich lipoproteins(8) , unlike that found in other mammals. This pattern, which was also seen in the C apoproteins separated from other soluble apoproteins by gel permeation chromatography, was found to reflect the presence of a second sialoglycoprotein, in addition to apoC-III. Based upon the N-terminal amino acid sequence, which was unusually rich in proline, we have now cloned the cDNA and expressed this protein which was found to be homologous to that of a recently identified gene in the apoE-apoC1-apoC2 gene cluster on mouse chromosome 7(9) .
Apolipoproteins were obtained after delipidation of lipoprotein fractions with ethanol:ether (3:1)(13) . C apoproteins were separated from apoVLDL dissolved in 0.015 M Tris, 8 M urea, pH 8.2, by chromatography on Sephadex G-200 (Pharmacia Biotech Inc.)(14) . Individual apolipoproteins were separated by preparative isoelectric focusing electrophoresis as described (15) or by anion exchange chromatography on DEAE-cellulose (DE52, Whatman). For the latter, elution was with a linear gradient of 0.01-0.15 M Tris (pH 8.2) containing 6 M urea. Apolipoproteins and their components were evaluated by one-dimension isoelectric focusing (pH 3.5-7.0)(16) , SDS-polyacrylamide gel electrophoresis(17) , and two-dimensional electrophoresis(18) . The amino acid composition of individual C apoproteins was determined in an amino acid analyzer(19) . Content of cysteine was evaluated after treatment of proteins with performic acid(20) . For amino acid sequencing, samples containing 800 pmol of purified protein were subjected to Edman degradation in an Applied Biosystems 470A gas-phase Sequencer. The phenylthiohydantoin-derivatives were identified and quantified by reverse-phase HPLC with an on-line Applied Biosystems 120A PTH analyzer.
Based upon the N-terminal amino acid sequence of
apoC-IV: EPEGtP(n/t)PLPA(p/a)EESRWS(l/t)V (parentheses represent
ambiguities, and lower case letters indicate weak signals), degenerate
primers were synthesized (Operon Co.) complementary to the first 9
amino acid residues: P1S3,
5`GA(A/G)CCNGA(A/G)GGNACNCCNAA(T/C)CCN(C/T)T3` (N = A, G, C, or T); and the nested primer: P2S2,
5`CCI(C/T)TICCIGCI(C/G)CIGA(A/G)GA(A/G)3`, (I = deoxyinosine) to amino acid residues 8 to 14 as
indicated in Fig. 5. The 3`-portion of the apoC-IV cDNA was
first screened by three successive rounds of PCR. The first round was
asymmetric, with CDS-primed single-stranded cDNA as the template.
Amplification was carried out with P1S3 (4 pmol) as primer in 67 mM Tris (pH 8.3), 12 mM (NH)
SO
, 2 mM MgCl
, 17 µg/ml bovine serum albumin, 0.15 mM concentration each of dATP, dGTP, dCTP, and dTTP, and 1 unit of Taq polymerase (Life Technologies, Inc.) in a thermal cycler
(Perkin-Elmer) for 30 cycles at 94 °C, 45 s; 50 °C, 60 s; 72
°C, 2 min, with a last extension at 72 °C for 7 min. One µl
of the first round PCR product was used in the second round PCR
amplification, probed by 8 pmol each of CDS and P1S3 under the same
conditions for 30 cycles. Two bands (major, 380 bp; minor, 330 bp) were
resolved by electrophoresis in 1.5% agarose gel and purified, and each
was used in the third round PCR amplification with 14 pmol each of CDS
and P2S2 under the same conditions for 30 cycles. The amplification
product from the 380-bp band was
360 bp (about 21 bp shorter as
expected for the nested P2S2 primed product). The 330-bp band yielded
only unrelated products. The PCR product containing the 360-bp cDNA was
treated with 1.5 units of T4-DNA polymerase and 5 units of
polynucleotide kinase (Life Technologies, Inc.) (30) and
purified by ethanol precipitation before cloning into the SmaI
site of Bluescript II KS
vector (Stratagene). The
resultant cDNA inserts were sequenced on both directions by the
dideoxynucleotide chain termination method (31, 32) using sequenase Conversion 2.0 DNA sequencing
kits (U. S. Biochemical Corp.).
Figure 5: Nucleotide and deduced protein sequences of the cDNA for apoC-IV. The cDNA was isolated from rabbit liver by PCR screening. The open reading frame of 124 amino acids, in one-letter code for the predicted amino acids, is indicated below the nucleotide sequence. A translation initiation codon, ATG, is indicated by bold letters. Positions of oligonucleotides used for PCR amplifications and their orientations with respect to the sense strand are indicated by arrows. * represents the stop codon. The polyadenylation signal is underlined. +1 indicates the start of the mature protein determined by protein sequencing.
We used the 5` rapid amplification
of cDNA ends (5`-RACE) method (26) to clone the 5`-portion of
apoC-IV cDNA. The cDNA was synthesized from 2.5 µg of total RNA
with 12.5 units of avian myeloblastosis virus reverse transcriptase
(Boehringer Mannheim) and 4 pmol of a specific primer P5A1
(5`GCCACCTCTCTCTGGTCCTAGTCA3`) at 42 °C for 1 h and 52 °C for
30 min. The RT products were purified on a Centricon 100 column (Amicon
Co.) and tailed with oligo(dA) and 18 units of terminal DNA transferase
(Pharmacia Biotech) at 37 °C for 5 min. The reaction mixtures were
diluted 200-fold and used in 5`-RACE reaction. The amplification was
performed as above except annealing was at 50 °C for 45 s. The
nested PCR was carried out with 100-fold dilution of the first PCR
product and 12 pmol each of CDS and the nested primer P5A2
(5`AGTCAGCAGCGGACCCACCAGCTCCTT3`) as above except annealing was at 62
°C for 30 s. A single band of PCR product (280 bp) was cloned
and sequenced as above.
A full-length apoC-IV cDNA was obtained by sequencing the PCR product primed with the sense primer p8S (5`CCGAATTCGCAGAGAGGGACAGA3`) and the antisense primer p8A (5`CCGAATTCTGGGTGAGCAGGTGA3`) amplified at 94 °C, 45 s; 62 °C, 30 s; 72 °C, 90 s for 40 cycles, with a last extension at 72 °C for 7 min. This set of primers, with an inserted EcoRI site in each, was derived from the 5`- and 3`-ends of the separately cloned nucleotide sequences. Several deletions of the cloned cDNA were made and sequenced in both directions to resolve ambiguities. Each full-length RT-PCR and cloning were repeated three times.
For immunoprecipitation, 2 µl of anti-apoC-IV antiserum was added to 5 ml of the medium collected from each dish of transfected cells and the mixture, supplemented with 2 µg/ml each of apoprotinin, leupeptin, and pepstatin A, and 0.02% sodium azide, was incubated with rotation at 4 °C overnight. Then, 80 µl (0.1 g/ml) of protein A-linked Sepharose CL-4B beads (Pharmacia) were added and incubation was continued for 4 h. Beads were sedimented at low speed for 5 s and washed twice with cold phosphate-buffered saline. For desialylation, immunoisolated protein on the beads was incubated with 7.5 milliunits of neuraminidase as above for 24 h. The protein was eluted with SDS sample buffer at 95 °C for 5 min and subjected to SDS-PAGE and Western blot analysis.
Figure 1: Isoelectric focusing polyacrylamide slab gel electrophoretograms (pH 3.5-7.0) of rabbit apoVLDL. Lanes 1 and 2, 70 µg of apoVLDL from a rabbit injected with turpentine 48 h before blood was sampled; lane 3, 6 µg of purified desialylated apoC-IV; lanes 4 and 5, 70 µg of apoVLDL from a normal rabbit injected with 0.15 M NaCl as above. The first of each pair of apoVLDL samples was treated with neuraminidase. SAA = serum amyloid A. The minor band in lane 3 may represent carbamylated apoC-IV.
Figure 2: Isoelectric focusing polyacrylamide tube gel electrophoretograms (pH 3.5-7.0) of C apolipoproteins of VLDL from cholesterol-fed rabbits. Lane at left shows unfractionated C apoproteins. Individual C apoproteins were separated by preparative isoelectric focusing electrophoresis and identified on the basis of amino acid composition (see text). The group of three gels at the right is from a separate preparation of apoVLDL that was treated with neuraminidase.
The individual proteins were also isolated from the C apoprotein fraction by ion exchange chromatography (Table 1). In addition to the three proteins separated by preparative isoelectric focusing, a major component was found with an amino acid composition that resembled human apoC-I together with a component tentatively identified as the 12-kDa thrombolytic fragment of apoE(42) . The novel proline- and arginine-rich protein (PARP) thus appeared to be a sialoglycoprotein, normally present as isoforms with up to 4 or 5 sialic acid residues. Its apparent molecular mass on SDS-gel electrophoresis was about 14 kDa. This was confirmed by two-dimensional electrophoresis with Coomassie Blue staining and immunoblotting (Fig. 3), which also showed that the apparent molecular mass of the sialylated forms of PARP was greater than that of the desialylated protein. PARP contained approximately 2 cysteine residues (Table 1). Upon immunoblotting apoVLDL separated by SDS gel electrophoresis without addition of reducing agents, bands of higher molecular weight, consistent with homodimers and homotrimers, were observed (data not shown). These components were absent in the presence of reducing agents, consistent with disulfide linkage of the oligomers.
Figure 3: Two-dimensional gel electrophoretic analysis of apoVLDL (A and C) and desialylated apoVLDL (B and D). Gels from one preparation of apoVLDL were stained with Coomassie Blue R-250 (A and B) and those from another preparation of apoVLDL were subjected to Western blotting with apoC-IV antiserum (C and D). Cathode was at left and anode at right of each gel.
PARP was also evident upon
isoelectric focusing of apoHDL (data not shown). By radioimmunoassay,
PARP was distributed mainly between VLDL and HDL of a rabbit fed
regular chow, with undetectable amounts in IDL, LDL, and
lipoprotein-free serum ( > 1.21 g/ml), whereas PARP was
detected only in VLDL from a cholesterol-fed rabbit (Table 2).
PARP was also found in apoVLDL isolated from perfusates of isolated
rabbit livers (data not shown). Thus, PARP appeared to be a bona fide
apolipoprotein that was distributed in a manner resembling that of
other C apoproteins. Based upon these properties, PARP was
provisionally designated as apoC-IV.
The affinity of apoC-IV for lipids was confirmed by its ability to convert small unilamellar vesicles of dimyristoylphosphatidylcholine to discoidal particles. These discs were, however, considerably larger than those produced with rabbit apoE (Fig. 4).
Figure 4:
Negatively stained preparations of
discoidal complexes of apoC-IV with dimyristoyl phosphatidylcholine
with apoC-IV (top) and rabbit apoE (bottom). For each
preparation, unilamellar liposomes of dimyristoylphosphatidylcholine
prepared in a French pressure cell (120-170 µg) were mixed
with one-fifth the mass of apoC-IV or apoE in 0.25 ml of 0.15 M NaCl and incubated at 37 °C for 1 h before negative staining.
180,000.
The amino
acids at positions -3 and -1 upstream of the N terminus of
the mature protein (Val and Cys, respectively) fit well into the
``(-3,-1) rule'' for the signal cleavage site of
eukaryotes(44) . The calculated pI of the mature protein is
5.05. Amphipathic -helix analysis (45, 46, 47) of the predicted amino acid
sequence indicates two possible regions (residues 48-71 and
92-113) of the protein that may be responsible for lipid binding (Fig. 6). The sequence lacks the N-glycosylation
consensus site NX(S/T), but there are several candidate Ser
and Thr residues for O-glycosylation. Two cysteines are
present near the C terminus.
Figure 6: Comparison of helical wheel diagrams (45) of predicted amino acids of homologous regions of rabbit apoC-IV cDNA (A and B), mouse ACL gene (C and D)(9) , and human apoC-IV cDNA (E and F)(54) . Negatively and positively charged amino acids are indicated by - and +, respectively. * indicates proline residue 57 in rabbit and mouse sequences.
DataBank (Swiss-prot, EMBL, and GenBank) searching revealed significant homology between apoC-IV cDNA (146-307 bp) and the 5`-flanking regions of cynomolgus monkey apoC-II (86-247 bp, 77%(48) ) and mouse apoC-II (114-244 bp, 82%(49) ). This 5`-flanking region of the mouse apoC2 gene has been sequenced recently (9) and contains a novel gene (the ``ACL'' gene) within the mouse apoE-apoC1-apoC2 gene cluster on chromosome 7. The complete nucleotide sequences of rabbit apoC-IV and mouse ACL cDNA shared 64% homology and the predicted amino acid sequences shared 62% amino acid identity.
Figure 7:
Northern blot analysis of tissue
expression of the apoC4 gene. A, 5 µg of
poly(A) RNA (lane 1) or 20 µg of total
RNA (lanes 2-7) from various tissues were subjected to
electrophoresis in a denaturing agarose/formaldehyde gel, transferred
to a nylon membrane, and probed with
P-labeled full-length
apoC-IV cDNA. The membrane was exposed to x-ray film for 9 days at
-70 °C. Lanes 1 and 2, liver; 3,
kidney; 4, brain; 5, heart; 6, small
intestine; 7, skeletal muscle. Positions of standard RNA size
markers (G319a, Promega) are indicated on the left. B,
ethidium bromide-stained RNA blotted on a nylon membrane shows the
equivalence of RNA mass after transfer.
Figure 8:
Southern blot analysis of genomic DNA.
Genomic DNA (20 µg) from rabbit liver was digested with restriction
endonucleases: B, BamHI; H, HindIII; P, PstI; and E, EcoRI, subjected to electrophoresis in 0.8% agarose gel,
transferred to a nylon membrane, and probed with P-labeled
full-length apoC-IV cDNA. The membrane was exposed to x-ray film for 6
days at -70 °C. Positions of standard DNA markers (1-kb
ladder, Life Technologies, Inc.) are indicated on the left.
Figure 9: Western blot analysis of rabbit apoC-IV expressed in E. coli (A) and CHO cells (B). A, lanes 1, molecular mass markers; 2, 0.25 µg of desialylated rabbit apoVLDL; 3, cell extract transformed with pET3c vector alone; 4, cell extract transformed with pET3c vector containing the cDNA encoding the 124 amino acids of apoC-IV and an additional 14-amino acid linker sequence; 5, cell extract transformed with pET3b vector alone; 6, cell extract transformed with pET3b vector containing the cDNA encoding only the 97 amino acids of mature apoC-IV; 7, 0.25 µg of sialylated rabbit apoVLDL. B, lanes 1, molecular mass markers; 2, medium from transfected CHO cells with pMT2 vector alone; 3 and 4, media from transfected CHO cells with pMT2 apoC-IV cDNA; 5 and 6, 0.1 µg of rabbit apoVLDL. Expressed protein in lanes 3 and 6 was desialylated with neuraminidase.
In cell extracts from transfected COS-1, COS-7, Chinese hamster ovary (CHO), and HepG2 cells, we observed several nonspecific bands around 10-18 kDa in Western blot analysis with our polyclonal antiserum (data not shown). Since apoC-IV was isolated from blood plasma and should be detected in medium of transfected cells as a secreted protein, we used the medium from cells to assess the expression of apoC-IV cDNA. Fig. 9B shows that the immunoisolated proteins from medium of CHO cells transfected with pMT2 vector harboring apoC-IV cDNA yielded a specific band of about 16 kDa (lane 4). After desialylation, this band shifted to about 14 kDa (lane 3). Thus, the mobility of asialoglycosylated apoC-IV was similar to that of the unglycosylated protein. Furthermore, the apparent molecular mass of the expressed apoC-IV cDNA product before and after desialylation was similar to the sialylated and desialylated forms of apoC-IV from rabbit apoVLDL, respectively, consistent with the presence of a sialylated carbohydrate moiety. Similar results were obtained with transfected COS-7 cells (data not shown).
We have identified a novel protein associated with lipoproteins in blood plasma of rabbits. Other proteins, such as serum amyloid A, which is secreted in large amounts as part of the acute phase response(50) , associate with lipoproteins and many others, such as complement 4B-binding protein(51, 52) , have been found to associate with lipoproteins to a limited extent. The novel protein, which we have designated as apoC-IV, appears to be a bona fide apolipoprotein by several criteria: 1) it is present primarily or exclusively in association with lipoproteins; 2) it binds to phospholipids, converting unilamellar liposomes of dimyristoylphosphatidylcholine to discoidal particles; 3) it is present in measurable quantities at all times and does not appear to be a typical acute phase reactant; 4) it contains two regions predicted to form amphipathic helices of the type found in most soluble apolipoproteins. Based on its molecular weight and distribution mainly in VLDL and HDL, apoC-IV appears to fall into the group of C apolipoproteins. Like apoC-III, it is a sialoglycoprotein, present in several isoforms.
The content of apoC-IV among rabbit C apolipoproteins is appreciable, as judged from Coomassie Blue staining and ion exchange chromatography, the mass of apoC-IV appears to be substantially greater than that of apoC-II and similar to that of apoC-I and apoC-III.
The N-terminal sequence of apoC-IV, with 5 prolines among the first 12 amino acids, gave no clue to its provenance. However, its structure otherwise is typical with respect to its signal peptide and the presence of amphipathic helical sequences downstream. As with other C apolipoproteins, apoC-IV mRNA is expressed mainly (possibly exclusively) in the liver. As predicted, it is evidently secreted from transfected mammalian cells as a sialoglycoprotein. The apparent molecular mass of sialylated apoC-IV is about 15,500 Da; that of asialoC-IV (approximately 14,000 Da) in SDS-PAGE is about 3,000 Da higher than that of the aminoacyl chain (11,020 Da). This anomalous behavior is evidently not related to the presence of one or more O-linked carbohydrate moieties (Fig. 9). The proline-rich N-terminal region may contribute to the reduced mobility.
The protein sequence of apoC-IV showed little homology with that of other apolipoproteins, but, surprisingly, the cDNA sequence showed a striking homology with the 5`-flanking region of monkey and mouse apoC-II(48, 49) . The basis for this homology has become clear from the recent work of van Eck, Hoffer and associates(9) , who initially found two additional exons 5` to the start site of the mouse apoC2 gene(49) . Evidence for a similar gene (the ``ECL'' gene) was found earlier in the rat by Shen and Howlett(53) , in the region of the apoE gene, but with an orientation apparently opposite to that found in the mouse. Van Eck and associates (9) have recently shown that these exons are contained within a novel gene in the apoE, apoC1, apoC2 gene cluster on mouse chromosome 7, the orientation of which is identical with that of the other three genes in this cluster (Fig. 10). This gene, which they designated the apoC2-linked (ACL) gene, is composed of three exons and is expressed in liver as a transcript of 473 bp. It encodes a putative protein of the precise length of apoC-IV (124 amino acids). The mRNA for the putative mouse protein, like apoC-IV, is expressed solely in the liver. From the sequence of the C-terminal portion of the putative signal peptide(9) , we predict that the site of signal peptide cleavage would be the same as for apoC-IV (following residue 27). The overall homology of amino acids 1-27 is 68%. Except for the first 7 amino acids (Fig. 5), the predicted sequence of the mature mouse protein also shows striking homology to apoC-IV, 71% for amino acids 35-124 and, when conservative substitutions are included, 80% (Fig. 10). Essentially the same amphipathic helical regions are predicted for the putative mouse protein encoded by the ACL gene and apoC-IV (Fig. 6).
Figure 10: Comparison of the deduced amino acid sequences of the rabbit apoC-IV cDNA and the mouse ACL gene(9) , partial sequence of the rat ECL gene(53) , the cynomolgus monkey 5`-flanking region of apoC-II cDNA(48) , and the human apoC4 gene (54) . Identical amino acids are indicated by - -. Gaps were introduced to maximize alignments. The stop codon in the rat sequence is indicated by an asterisk. The numbers assigned to the right are based on the methionine at the start of the open reading frame deduced from the nucleotide sequences.
After this work was completed, we became aware of the sequence of the human gene, also present in the same gene cluster just upstream of the apoC2 gene on chromosome 19(54) . The sequence (Fig. 10) includes a 9-nucleotide insert similar to that of the monkey gene, yielding a predicted protein of 127 amino acids. Based upon the hydropathy profile of the predicted protein and the assumption that the N terminus is initiated at the start of exon two, Allan and associates (54) predicted the signal peptide to have 25 amino acids. The amphipathic helices predicted for the human protein also include two regions. A 21-mer (residues 95-116) resembles that found for the rabbit and mouse sequences but differs with respect to the polar face, for which only the predicted human sequence contains a positively charged amino acid (Lys-110) (Fig. 6). A 28-mer amphipathic helix (residues 47-74) differs from the corresponding rabbit and mouse sequences by the presence of a codon for threonine (ACA) at residue 60 (Fig. 10). In all other species (monkey at predicted residue 60 and mouse, rat and rabbit at predicted residue 57), the equivalent codon specifies proline (CC(G/A)) which should produce a kink in the helix(55) . If this were not the case, the sequences for the species other than the human would predict a similar amphipathic helix in this region (Fig. 6).
The structure of
the mouse and human apoC4 genes as well as that of the rabbit ()is similar, with three exons and two introns (two short
exons upstream, the first encoding most of the signal peptide, and a
longer terminal exon encoding the C-terminal region). The second and
third exons each contain an amphipathic helical region. This genomic
organization and the location of the apoC4 gene within the apoE, apoC1,
apoC2 gene cluster provide strong support for inclusion of apoC-IV in
the apolipoprotein gene family, which includes not only the four C
apoproteins and apoE, but also apoA-I, -A-II, and -A-IV(1) .
Conservation of the genomic and protein structure of apoC-IV across
mammalian evolution from mouse to humans strongly suggests that apoC-IV
has an important function. It is thus puzzling that the protein
apparently is not expressed and secreted at a more substantial level in
species other than the rabbit. A low level of secretion in humans has
been predicted by Allan et al.(54) , based upon a
level of expression of apoC-IV mRNA in human liver only about 1% of
that of apoC-II. Clearly, further work is needed to clarify the reasons
for such variable expression of apoC-IV (and in particular the basis
for an important function at some stage of life).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U39356[GenBank].