the structure of the human apolipoprotein genes

0270-9139/87/0701-056S$02.00/0 HEPATOLOGY Copyright 0 1987 by the American Association for the Study of Liver Diseases

Vol. 7. No. 1. pp. 56S-6OS, 1987 Printed in U. S. A.

The Structure of the Human Apolipoprotein Genes

LAWRENCE CHAN Departments of Cell Biology and Medicine, Baylor College of Medicine, Houston, Texas 77030

The plasma lipoproteins are water-soluble lipid-protein complexes which transport lipids in the vascular and extravascular compartments. The lipoproteins have been classified according to their flotation densities in the ultracentrifuge. By this technique, the major classes of lipoproteins include the chylomicrons, very low density lipoproteins, intermediate density lipoproteins, low density lipoproteins and high density lipoproteins (HDLs). Of the HDL, the two best-defined subclasses are HDL, and HDL3. The major function of the protein components of the lipoproteins, or apolipoproteins, is lipid transport. However, many apolipoproteins serve other additional functions which have been reviewed in detail (1-3).

STRUCTURE OF APOLIPOPROTEINS AND THEIR MRNAS

The cDNAs for a number of apolipoproteins have been isolated. These include those for apo A-I, A-11, A-IV, C-I, C-11, C-I11 and E (4-12). Recently, partial cDNAs for the largest of the apolipoproteins, apo B, have also been identified (13-16). In uitro translation as well as DNA sequence analyses have indicated that the apolipoproteins are similar to other secretory proteins in that they contain a signal peptide as part of the initial nascent polypeptide chain on ribosomes. These are cleaved co- translationally. Furthermore, two of the apolipoproteins, apo A-I and apo A-11, also contain a pro-segment. Apo A-I has a 6-amino acid pro-peptide with the sequence Arg-His-Phe-Trp-Gln-Gln. The Gln-Gln right next to the proteolytic cleavage site is a rather unusual occur- rence (17). Apo A-11, in contrast, has a 5-amino acid pro- peptide with the sequence Ala-Leu-Val-Arg-Arg (18). The two basic amino acids next to the cleavage site is typical of peptides that undergo posttranslational proteolytic processing. The major sites of synthesis of the major apolipoproteins are the liver and the intestine. Apo E appears to be unique in that it is also synthesized in many other organs (19-24). Apo B is the largest of the apolipoproteins. There are two different forms of apo B, a hepatic form (apo B-100) which appears to be slightly more than twice as large as the intestinal form (apo B- 48) (25). Apo B-48 appears to share common sequences

This study was supported by a grant (HL-16512) from the National Institutes of Health.

Address reprint requests to: Lawrence Chan, M.D., Departments of Cell Biology and Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.

with the NH2-terminal portion of apo B-100. Recent studies using monoclonal antibodies indicate that apo B- 100 and apo B-48 are two distinct products of the same gene (26). The exact structural relationship between these two proteins is presently unknown.

GENOMIC STRUCTURES OF HUMAN APOLIPOPROTEINS

Recently, the genomic structures of a number of apolipoproteins have been reported including those of apo A-I (5, 6), apo E (27, 28), apo C-I11 (29), apo A-I1 (30) and apo C-II(31). It is apparent that these apolipoprotein genes have remarkably similar structures, each consist- ing of 4 exons and 3 intervening sequences (IVS). The exon/IVS structures of these genes have been examined (30).

In eukaryotes, exon/IVS junctional structures are well- conserved. IVS are bounded at the 5’ end by a conserved sequence of nine nucleotides with the consensus sequence ,SAGGTeAGT. They are bounded at the 3’ end by another sequence which is pyrimidine-rich with the consensus sequence, PyPyPyPyPyPy XCAGG (32). On examination of all of the exon/IVS junctions in all the apolipoprotein genes published to date (apo A-I, apo E, apo A-11, apo C-111 and apo C-II), they all conform to these 5’ and 3’ splice signals (data not shown). Recently, Keller and Noon (33) examined the IVS further away from the splice junction. They found an internal conserved sequence TACTAAC that is located 20 to 55 nucleotides from the 3’ IVS boundary which might serve as internal splice signals. They analyzed genes from Drosophila, sea urchin, chicken, duck, mouse, rat and man. The analysis of human genes was confined to the globin family. In this gene family, they found the internal 3‘ IVS signal to have the consensus sequence CTGAC. We have searched for potential internal 3‘ IVS splice signals in all of the apolipoprotein genes with known structure (Table 1). We found that, for the apolipoprotein gene family, the consensus sequence CTgAN is present between 8 and 78 nucleotides from the 3’ splice junction of all the 15 IVS for the five genes examined. Recent studies indicate that in IVS processing, splicing events involve an intermediate in the form of a lariat structure (34). In this intermediate, the 5‘ end of the IVS has been released from the preceding exon, and the 5’ terminal G is joined in a branched structure to a point in the IVS near the 3’ splice site. Ruskin et al. (34) report that the branch occurs a t an A and that the

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Vol. 7. No. 1, Suppl. 1987 STRlI(’T1JRK O F T H E H I ’ M A N APOLIPOPROTEIN GENES 57s

sequence around this A is CTGAC, CTAAT, CTGAT, CTAAC and CTCAC in the five cases examined. The consensus sequence we have identified, CTEAN, would then be recognized, and possibly utilized in uiuo for the branch structure. This suggests that this internal 3’ splice signal is operative in the apolipoprotein gene family. We note that the sequence is highly homologous to that in the globin family (see above), the only other human gene family thus examined to date.

Since the apolipoproteins all function as lipid-binding proteins, there has been considerable speculation that they might be evolutionarily related (35). Indeed, amino acid and DNA sequence comparisons have shown that apo A-I, A-11, A-IV, C-I, C-11, C-I11 and E all contain common modules of 1 1-codon repeating units. A detailed analysis of the homologies between apolipoproteins and the individual modules has been recently published by Luo et al. (36). The study strongly suggests that the 11- codon module is a basic building block for the apolipoproteins and forms the basic structure of the putative ancester for all of them.

5’ FLANKING SEQUENCE OF APOLIPOPROTEIN GENES

Since apolipoprotein genes are probably evolutionarily related, we have compared the 5’ flanking sequences of all of the apolipoprotein genes published to date. As shown in Table 2, all of these genes exhibit a GC-rich region of high (>70%) homology within 500 nucleotides

TABLE 1. Internal 3’ splice signal sequences in apolipoprotein IVS

Signal Distance

A- I IVS-I C C A C T C A G C C A (; 22 IVS-I1 G G C C T G A T C T (; (; 24 IVS-111 C, C C C T C A A C C C T 21

IVS-I (; (; (’ T T C A G T G T C :i2 IVS-I1 C A G C T G A A A A G A 48 IVS-111 A C T C T A A T C C C C 16

IVS-I ( . C A G T C A G C C T G 3 1 IVS-I1 T A C C T G A C A C A (; 17 IVS-I11 C C T C T A A C C A T C ‘Lo

IVS-I C C C C T C A C A (; G A 17 IVS-I1 C C A C T C A G C C C T 17 IVS-111 C C C C T G A <’ T (; A T 8

IVS-I G T C C T C A C T G G C :1‘L IVS-I1 A C C C T G A C C C G A 2s IVS-111 T G G C T C A T C C C C 78

A-11

C-I1

C-111

E

upstream from the respective CAP sites. The homologous regions are found in nucleotides -234 to -213 of apo C-11, -497 to -471 of apo A-11, -196 to -179 of apo A- I, -409 to -391 of apo E and -116 to -103 of apo C-111. (The positions for apo A-I and apo C-111 are approximate, since the CAP sites have not been identified in these genes). The lengths of homologous sequences between the apo C-I1 gene and the regions identified for apo E, apo A-I1 and apo A-I are quite extensive, involving 19, 27 and 18 nucleotides, respectively. The homologous region between apo C-I1 and apo C-111 was shorter and involved 14 nucleotides (Table 2). However, since only -189 nucleotides of the 5‘ flanking region of the latter gene have been published, it is possible that more extensive homology might be located further upstream from the CAP site, as is the case with other apolipoprotein genes. One possible explanation for the conservation of 5’ flanking regions in the apolipoprotein genes might be that they are conserved through evolution from genes that have diverged from an ancestral primordial gene. Another possible role of such sequences is that they might be of functional significance, e.g., they might be regulatory sequences which render the respective genes responsive to changes in cellular lipids, or some lipid- protein complexes or other common intermediates in apolipoprotein gene expression. An additional possibility is that these sequences are homologous by random chance.

GENOMIC STRUCTURE OF HUMAN APOLIPOPROTEIN: RELATIONSHIP TO PROTEIN

STRUCTURE The general structural organization of all the apoli-

poprotein genes published to date is shown in Figure 1. It is evident that these genes share very similar structures. There are 4 exons and 3 IVS to each gene. The IVS appear to have very similar locations: IVS-I interrupts the 5’ untranslated region of the gene; IVS-I1 interrupts the translated part of the gene very close to the signal peptidase cleavage site, and IVS-I11 interrupts the part of the gene encoding the mature peptide. The lengths of the first three exons are very similar, and the difference in total length of the mRNAs is accounted for mainly by differences in length of exon 4 (Figure 1). This striking similarity in the gene structures of apo A-I, apo A-11, apo C-11, apo C-I11 and apo E supports the hypoth- esis that these genes have arisen from a common ancester (35, 36), and the individual apolipoprotein genes have evolved through partial and complete gene duplications (36).

Gilbert (37) suggested that the exon/IVS structure of eukaryotic genes might be a record of their evolutionary history: these genes evolved by exploiting RNA splicing to recruit and combine small sements of coding se-

Consensus Structure 0 1 Signals with Flanking Regions quence. Thus, in many instances [e.g., collagen -(38), albumin (39), immunoglobulins (40), ovomucoid (41), rhodopsin (42), 3’-hydroxy-3-methylglutaryl coenzyme A 2 ‘ L : l - 2 1s 2 3 :1

x - 6 9 . 5 reductase (43) and pyruvate kinase (44) genes], the cod- C 7 9 x 13 G 4 3 ‘ L l I 4 - 4 T 2 1 2 1 1 . 5 ~ :{ :{ :{ ing portions of the genes are broken up into pieces

encoding distinct functional domains. We examined the Distance is the number of nucleotides between the 3’ end o f the locations Of IVs-11 and IVs-111 in the apolipoprotein . - -

signal a n d the 3‘ splice point. genes with respect to the secondary structures of the

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TABLE 2. Homologous sequences in the 5' flanking sequence of apolipoprotein genes

Ending nucleotide Reference 6' sequence Beginning

nucleotide

c-I11 -116 A C C C T G C C T C A G G C -103 41 * * * * * * * * * * *

E -409 C T C C C A T C C C A - C T T C T G T C -391 42 * * * * * * * * * * * * * * * *

c-I1 -234 C C C T C C A C A G C C C C C A A C C C A G C C T C T G T C -213 32

A-I1 -497 C C C C C A T A G C C C T C A A C C C T G T C C C T G -471 31 * * * * * * * * * * * * * * * * * * * * *

* * * * * * * * * * * * 1 . 1 .

A-I -196 C C C T G C A C - G C C C C G - - - - C A G C -179 43.44

The asterisks indicate homology to the apo C-I1 sequence only. Numbers indicate distance 5' to the CAP site. For apo A-I and apo (2-111, these are only approximate distances since the CAP sites have not been identified in these genes.

FIG. 1. Structural organization of apolipoprotein genes. Transcrip- tion is from left to right. The heavy bars represent, from left to right, exons 1, 2 ,3 and 4. The thin line represents 5' flanking region, IVS-I, 11, and I11 and 3' flanking region of the respective genes. The numbers h o e the exons indicate the length in nucleotides of the exons. The exact lengths of exon 1 in apo C-I11 and apo A-I are in doubt since the 5' end of the gene has not been determined in these cases. The numbers in parentheses above exon 2 indicate the position of the adenine in the initiation codon, ATG, from the 5' end of exon 2.

individual proteins as predicted by the method of Chou and Fasman (45) (data not shown). Without exception, IVS-I1 interrupts the gene close to the signal peptidase cleavage site, either at regions of random structure (apo C-11, apo C-111), @-turn (apo A-I, apo E) or at the junction between two conformations (i.e., transition between short, probably unstable @-sheet and a-helical region, apo A-11). Similarly, IVS-I11 interrupts the mature peptide region of the different genes at regions of random structure (apo C-11) or @-turn (apo A-11, apo C-111). In these instances, therefore, not only do IVS-I1 and IVS- I11 separate the peptides into distinct functional domains as predicted by Gilbert (37), but they also seem to have preference for the surface of proteins as in the case of many other proteins (46). However, by Chou-Fasman analysis, IVS-I11 appears to interrupt a-helical structures in apo A-I and apo E. To obtain a better definition of the functional domains of the various apolipoproteins, we analyzed the conformation of the mature plasma proteins by the mean helical hydrophobic moment, ( p ~ ) , of Eisenberg et al. (47). This parameter not only gives a measure of helicity, but also takes into consideration the amphiphilicity of a helix. Inspection of Figure 2 reveals

Residue Number FIG. 2. Secondary structure analysis of human apolipoproteins by

the method of Eisenberg et al. (47) and IVS locations. The helical hydrophobic moment, ( p ~ ) , was determined as previously described. Each apolipoprotein was analyzed in 11-residue fragments and the ( p ~ ) values were plotted at the midpoint of each fragment. The bar on the scale on the oertical axis represents 1 kcal. The arrows indicate the positions of the IVS. Each arrow is placed at the residue which marks the end of exon 2 (or the first domain in this figure), e.g., for apo A-11, IVS-111 is located at amino acid 39, and the arrow is placed at the midpoint of the 11-residue peptide which marks the end of the domain encompassed by residues 29-39 or residue 34.

that IVS-I11 consistently interrupts the mature peptide at, or very close to, a minimum value of ( p H ) . It should be noted that the arrows in this figure indicate the IVS position at a site which marks the end of the first domain, e.g., for apo A-11, the arrow marks the domain which ends at residue 39 of the mature peptide (i.e., at the midpoint of the 11-residue peptide which defines the conformation of residues 29-39 or residue 34). However, the domain demarcation is relatively poor for apo C-11. For the two longest apolipoproteins, apo A-I and apo E, analyzed by this method, in addition to the minimum values of ( p H ) marked by IVS-111, other minima seem to appear periodically toward the carboxyl-terminal end of the protein. These subsequent domains are not inter- rupted by IVS and appear to define internally repeated sequences in these proteins (36).

FUTURE DIRECTIONS The application of molecular biological techniques to

lipoprotein research is a relatively recent event. It was only in 1980 that the first eukaryotic apolipoprotein cDNA was cloned (48). Since then, the cDNAs for all

Vol. 7, No. 1, Suppl. 1987 S'rRl1CTlJRE OF THE HITMAN APO1,IPOPROTEIN GENES 59s

the major human apolipoproteins have been cloned and sequenced, and the genomic structures for most of them have been published. In the future, a number of areas will be explored.

The cDNA for the most important of the apolipoproteins, apo B, has been cloned. It is a large cDNA of over 14 kb in length. Its sequence has just been completed (49). The DNA-deduced amino acid sequence of apo B will be the basis for a number of experiments on the structural and functional organization of this unusual protein. Specifically, the low density lipoprotein receptor binding domain and the lipid-binding regions of apo B will be defined, and the relationship between the hepatic apo B (B-100) and intestinal apo B (B-48) will be clari- fied (25).

Another interesting area for exploration is the detailed evolutionary relationship between the individual apolipoproteins. Luo et al. (36) have presented a detailed analysis of the repeating units in the various apolipoproteins. Future experiments will further define the time of divergence of each member of the apolipoprotein multigene family. The possible evolutionary origin of apo B will also be defined.

Finally, restriction fragment length polymorphisms have been described for a number of apolipoproteins (50- 52). Detailed restriction fragment length polymorphisms and haplotype analysis will prove useful in our under- standing of the genetic background for the various dys- lipoproteinemias.

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the structure of the human apolipoprotein genes

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