(Received for publication, July 31, 1995; and in revised form, August 11, 1995)
From the
The lipoprotein Lp(a), a major inherited risk factor for atherosclerosis, consists of a low density lipoprotein-like particle containing apolipoprotein B-100 plus the distinguishing component apolipoprotein(a) (apo(a)). Human apo(a) contains highly repeated domains related to plasminogen kringle four plus single kringle five and protease-like domains. Apo(a) is virtually confined to primates, and the gene may have arisen during primate evolution. One exception is the occurrence of an Lp(a)-like particle in the hedgehog. Cloning of the hedgehog apo(a)-like gene shows that it is distinctive in form and evolutionary history from human apo(a), but that it has acquired several common features. It appears that the primate and hedgehog apo(a) genes evolved independently by duplication and modification of different domains of the plasminogen gene, providing a novel type of ``convergent'' molecular evolution.
The lipoprotein Lp(a) ()has gained increasing
attention due to its role as a novel major risk factor for
atherosclerosis and for the unusual nature of its distinguishing
protein component, apolipoprotein(a) (apo(a)) (see (1) and
references therein). DNA cloning and sequencing revealed the unexpected
homology of apo(a) to plasminogen and the presence of 37 domains that
are most closely related to the fourth kringle of
plasminogen(2) . It has since been found that individual
alleles contain from
12 to 40 similar or identical kringles,
encoding proteins that range in apparent molecular mass from
250,000 to 800,000(3) . The apo(a) gene likely arose from a
duplicated version of the plasminogen gene followed by exon deletions,
multiplications, and single base substitutions(2) .
Although its precise function remains unclear, Lp(a) is concentrated in the artery wall by virtue of binding to fibrin, plasminogen receptors, matrix, and other targets(4, 5, 6, 7) . As an inactive homolog of plasminogen, apo(a) competes for the binding and activation of plasminogen and interferes with clot lysis (for reviews, see (8, 9, 10, 11) ). It has been speculated that a selective advantage of apo(a) may be its ability to deliver cholesterol to wound sites for cell biosynthesis(12) . These properties becomes pathogenic in the face of elevated plasma concentration, exposure to high fat diet, and increased life span. For example, the inhibition of plasminogen activation and the prolonged presence of thrombus on the vessel wall may promote the growth and migration of smooth muscle cells and the development of atherosclerotic lesions through several intermediate pathways(13, 14, 15) .
Of evolutionary as
well as practical interest is the observation that the existence of the
Lp(a) particle and the apo(a) protein are restricted to Old World
monkeys, apes, and
humans(16, 17, 18, 19, 20) ,
with one intriguing exception: the insectivore, hedgehog(21) .
Although some members (e.g. tree shrews and elephant shrews)
of the original order Insectivora have now been reclassified, hedgehogs
are still considered to be ``primitive'' extant mammals whose
ancestors diverged from other orders of placental mammals about 90
million years ago(22, 23) . The analysis of mammalian
hemoglobin sequences(24) , and our phylogenetic tree based on
plasminogen sequences (see Fig. 5A) support the
anatomical classification and clearly place hedgehogs on the most
distant mammalian branch, far removed from primates. Although it is
difficult to document unpublished negative findings, we know of no
other nonprimate in which the presence of Lp(a) has been unequivocally
demonstrated. ()While confinement of apo(a) to a subset of
primates is consistent with the recent advent of the apo(a) gene, its
existence in hedgehogs would require other evolutionary scenarios.
Perhaps the apo(a) gene arose before the major divergence of mammalian
orders, became disabled in most mammalian species, and retained
exceptional sequence similarity to the plasminogen gene in primates
through extreme functional constraints and gene conversion (some
domains of the two human genes are virtually identical; (2) ).
Alternatively, apo(a)-like genes might have arisen more than once
during mammalian evolution. To evaluate these possibilities, we have
isolated and sequenced cDNA and genomic clones of hedgehog apo(a) and
plasminogen. The results point to the independent creation of an
apo(a)-like gene in the hedgehog lineage by remodeling a duplicated
plasminogen gene in a way that is distinct from the evolution of
primate apo(a).
Figure 5: Relationship of plasminogen and apo(a) protein sequences. A, dendrogram of plasminogen protein sequences of human, rhesus monkey (Macacamulatta), cow (Bostaurus), pig (Susscrofa), mouse (Musmusculus), and hedgehog (E. europaeus) show hedgehog to be on the most distant branch. B, dendrogram of hedgehog apo(a) kringles and plasminogen kringle three from several species. Identical kringle sequences are represented only once. The branching pattern is consistent with the possibility that the ancestor of hedgehog apo(a) diverged from plasminogen before the speciation included in the analysis. More recent rounds of kringle duplication followed the initial split to form the multiple domains within the present hedgehog apo(a). Length of branches indicate percentage of sequence identity corrected for gap penalties.
Figure 1: Blot analysis of hedgehog apo(a) protein and mRNA. A, hedgehog lipoprotein fraction contains an apolipoprotein(a)-immunoreactive component. The density <1.215 g/ml fraction of hedgehog plasma was electrophoresed in 5% polyacrylamide under nonreducing (lane1) and reducing (lane2) conditions, transferred to nitrocellulose, and exposed to antiserum raised against human Lp(a). Lane3 is a reduced sample of human plasma with two apo(a) isoforms migrating at 0.92 and 0.96 the rate of apo B-100, whose position, at 550 kDa, is indicated. Hedgehog and human apo(a) share regions of 8 consecutive and 14 of 16 identical amino acids, which could be sites accounting for immunological cross-reactivity. B, hedgehog liver plasminogen and apo(a) mRNA. A fragment of plasminogen cDNA detects the 3-kb hedgehog plasminogen mRNA (lane1), while a 45-base oligonucleotide derived from hedgehog apo(a) genomic sequence hybridizes to a major band at 10 kb and a weaker a band at 12 kb by Northern blotting. By analogy to the human case, the bands are likely to represent the two alleles of the apo(a) gene in this individual animal, which differ in expression level as well as size (due to differences in number of tandemly repeated kringle domains). It should be noted that, due to import restrictions, A and B represent samples from different animals, A being derived from plasma of an African hedgehog and B from liver RNA of a European hedgehog.
Figure 2: Alignment of hedgehog apo(a) tandem kringle repeats and comparison to hedgehog plasminogen kringle three. Nucleotide sequence of two overlapping cDNA clones comprising 9209 bp (which overlap within the region of kringle 20) was translated and aligned in rows of its 93-amino acid kringle domains (GenBank U33170). Partial sequencing of additional clones confirmed the overlap. The derived consensus sequence of the repeated domains is shown below, with the two most common amino acids listed at some positions. The bottom line gives the sequence of the corresponding region of hedgehog plasminogen kringle three, with asterisks indicating identity to apo(a) consensus residues. The six canonical kringle cysteines are boxed, while the seventh cysteine, occurring only in kringle 31 is highlighted. (Human apo(a) has one kringle with a seventh cysteine, located in a different position.) The seven residues of the potential lysine binding pocket are underlined in the toprow. The underlinesbelow the consensus sequence show where direct peptide sequence was obtained, with only one discrepancy from cDNA predicted sequence. Arrowsabove the topline point to the location of introns in the hedgehog apo(a) and plasminogen genes. While the second two introns are in identical locations in the two genes, the first intron is located in the codon immediately preceding position one (Q) of hedgehog apo(a) but is 10 codons further upstream in the hedgehog plasminogen gene. The phase of the introns between the two genes is conserved. The initial residues of the mRNA were not contained in cDNA clones. As occurs in the case of human apo(a), we have noted individual variation in the size of hedgehog apo(a) isoforms by Western blot analysis of seven animals (data not shown).
Failure to detect hedgehog apo(a) mRNA or cDNA clones could have been due to a nonhepatic site of synthesis, low expression in some animals or, low homology to probes used. We also considered the possibility that a distinct apo(a) did not exist in hedgehogs but that its plasminogen could bind to low density lipoprotein and produce the type of particle that had been observed. The strategy of screening genomic, rather than cDNA, libraries circumvented some of these potential pitfalls. Genomic DNA libraries were constructed and screened with a kringle 2-4 fragment of hedgehog plasminogen cDNA at reduced stringency. Two types of clones were isolated, those containing DNA sequence that precisely matched that of hedgehog plasminogen cDNA and clones with related, but distinct sequence. A 45-base oligonucleotide designed to be specific to the latter gene detected bands of 10 and 12 kb on a Northern blot containing hedgehog liver RNA (Fig. 1B; lane2) This pattern resembles that found with human liver RNA samples that show up to two distinct apo(a) transcripts, ranging from about 7-12 kb, due to differing expression levels and size of apo(a) alleles, respectively.
Use of this oligonucleotide probe allowed isolation of hedgehog liver cDNA clones that encode an apo(a)-like protein (Fig. 2). (Although not an exact replica of human apo(a), we shall heretofore refer to the hedgehog analog as ``apo(a),'' rather than ``apo(a)-like,'' for convenience.) Sequence analysis of two large overlapping clones revealed the presence of 31 tandem ``kringle'' domains that contained pairwise nucleotide identity ranging from 74 to 100%. Unlike human apo(a), hedgehog apo(a) clones lack a protease-like domain. Comparison of nucleotide and translated amino acid sequences reveal that all of the hedgehog apo(a) domains far more closely resemble kringle three of hedgehog plasminogen than any other of its other plasminogen kringles. The translated amino acid sequences of hedgehog apo(a) kringles (defined as the region bounded by its six cysteines) have 69-78% amino acid identity to hedgehog plasminogen kringle three; no gaps are required in the alignment. This compares to amino acid identity of only 51-54% with the other kringles of hedgehog plasminogen. The similarity to non-plasminogen kringle domains in the data base is also significantly lower.
The identity of cloned cDNA and the plasma protein was confirmed by limited amino acid sequencing. After isolation by density centrifugation followed by gel electrophoresis, the protein was cleaved with trypsin, and three fragments were isolated by HPLC and sequenced. Peptide sequence of 16, 15, and 9 residues matched the predicted cDNA sequence with only one apparent discrepancy (See Fig. 2and ``Experimental Procedures'').
Figure 3: Scenario for the independent evolution of human and hedgehog apo(a) genes. The sequences of plasminogen and hedgehog and human apo(a) cDNA are compared. The sequences are divided into domains, and the percentage of nucleotide identity is shown between the corresponding multiple kringle domains of hedgehog apo(a) and hedgehog plasminogen and between human apo(a) and human plasminogen. The data suggest that hedgehog apo(a) derived from a plasminogen gene by elimination of all but the kringle three-like domain, followed by extensive multiplication of this 279-base domain and limited base substitutions, including loss of the ``seventh cysteine'' of kringle three in all but the last copy. In contrast, human apo(a) has retained the kringle five and protease domains of plasminogen and multiplied the 342-base domain of kringle four, while acquiring a seventh cysteine in only the next to last of the repeated domains. This one unpaired cysteine (indicated by asterisk) is responsible for the binding of human apo(a) to apoB-100 and is postulated to be the case for the hedgehog protein. The human sequence data is from (2) .
To
our knowledge, this represents the only example of the independent,
parallel evolution of a similar protein more than once. Although the
human and hedgehog apo(a)-like proteins are not identical, they share
several common attributes. Each contain numerous copies of kringle
domains and forms a covalent complex with apoB-100 in a circulating
Lp(a) lipoprotein particle of density 1.07 g/ml. Since virtually
all apo(a) in humans is linked to apoB-100 in lipoprotein particles,
this linkage is presumed to be a key to its function. Interestingly, a
scenario can be proposed by which this linkage arose in complementary
fashion in the human and hedgehog versions of the gene. Human apo(a),
which is not in itself lipophilic, forms a disulfide bond to the
lipid-associated apoB-100 using the unpaired cysteine of its only
kringle, which contains seven rather than six cysteines. Human apo(a)
consists of multiple kringles derived from plasminogen kringle four,
which contains six cysteine residues. It acquired a single unpaired
seventh cysteine in only its penultimate kringle four homologue, which
fulfills the cross-linking function. In contrast, hedgehog apo(a)
derived from repeated plasminogen kringle three domains, which contain
seven cysteine residues. Subsequently, this extra cysteine was
apparently lost from all but one of its multiple kringle domains (Fig. 2). Since the cysteine residue at this location in
plasminogen kringle three forms a disulfide bond with an unpaired
cysteine in kringle two, this cysteine in the last kringle domain of
hedgehog apo(a) is likely to be at a surface and available for covalent
bonding. We can speculate that the retention of the unpaired cysteine
in one kringle proved useful for binding to a target protein, but there
was selective pressure to eliminate the seventh cysteine from the other
kringle copies as they multiplied to avoid the formation of internal
cross-linking and interprotein polymerization.
Another salient property of human apo(a) is its ability to bind to substrates shared by its plasminogen homologue, yet not to function as an activable plasmin-like protease itself. Rouy et al.(32) demonstrated that hedgehog Lp(a) binds to lysine and to immobilized fibrin surfaces and in the presence of tissue plasminogen activator, inhibits the binding and activation of plasminogen. The fibrin binding of human plasminogen kringle four and human apo(a) occurs via a lysine binding site comprising a trough lined by three aromatic residues flanked at one end by two cationic and at the other end by two anionic residues (33, 34, 35, 36) (Fig. 4). (These sites may also be involved in binding of human apo(a) to other extracellular and cell surface targets.) The reported binding of hedgehog Lp(a) to lysine (32) could be accounted for by the conserved residues located at corresponding positions in the kringles of hedgehog apo(a). Perhaps significantly, these residues are conserved in hedgehog apo(a) and hedgehog plasminogen kringle three (with one Phe to Tyr substitution), while in human plasminogen kringle three, two nonconservative differences from the binding site motif (Phe to His and Asp to Lys) exist (Fig. 4). Hence it could be speculated that while hedgehog plasminogen kringle three could readily serve as a precursor for the evolution of fibrin-binding apo(a) kringles, it would have been more difficult to convert human plasminogen kringle three to such an activity.
Figure 4: Lysine binding pockets. The toptworows show the amino acids that comprise the known lysine binding pocket of human plasminogen kringle four and the 37th kringle four-like repeat of human apo(a), its major lysine binding kringle(33, 34, 35, 36) . They are grouped into the two cationic, three neutral aromatic, and two anionic amino acids that form the binding site. Numbers below indicate amino acid positions as numbered in Fig. 2. At corresponding positions in sequence alignment, numerous hedgehog apo(a) kringles and hedgehog plasminogen kringle three have the conservatively altered sequences shown in rowsthree and four. In contrast, human plasminogen kringle three (bottomrow) contains differently charged histidine and lysine residues at corresponding locations. We speculate that the hedgehog plasminogen gene offered potential lysine binding kringles for apo(a) from its third kringle domain, while during evolution of the human type of apo(a), plasminogen kringles one or four were more amenable substrates than kringle three.
Perhaps the most notable feature of apo(a) kringles is their sheer multiplicity, occurring in both hedgehog and human proteins. The adaptive value of multiple kringle domains for apo(a) is unknown, but the fact that human apo(a) isoforms appear to have 12 or more kringles suggests that there might be a minimum functional requirement. Electron microscopy and velocity centrifugation indicate that human apo(a) can extend in a loose coiled fashion away from the spherical lipoprotein particle(37, 38) . This may allow for noncovalent capture of extended target sites such as other apoB-containing particles (39) or multiple sites on fibrin or other matrix macromolecular complexes. This could serve to increase the concentration of lipids at target sites or to increase binding avidity.
Proteolytic activity is not a vital part of the function of this member of the protease superfamily, and its very absence may have been adaptive. Examination of the original sequence of human apo(a) cDNA suggested lack of such activity. Key differences from plasminogen sequence were noted, and the recombinant protein was shown to lack plasmin-like catalytic activity (2) (although non-plasmin-like catalytic activity has been proposed by one group, (40) and (41) ). Sequence analysis of rhesus monkey apo(a) showed that it has eliminated the catalytic triad of this domain(16) , while comparison of the rhesus and human sequences showed that the protease domain has accumulated changes at all codon positions at the same rate, as would an inactive pseudogene(42) . Now we find that apo(a) appears to function in hedgehogs with no trace of a catalytic domain.
To recapitulate, human and hedgehog apo(a) have apparently evolved independently by remodeling different parts of a copied plasminogen gene. No piece of plasminogen is shared by the two forms of apo(a). Convergent evolution has endowed both incarnations of apo(a) with many tandem copies of kringle domains, the ability to form a complex with apoB-containing lipoproteins, lysine and fibrin binding capability, competitive inhibition of plasminogen activation, and lack of plasmin-like proteolytic activity. Several types of analysis indicate that the original duplication of genes, which led the present plasminogen and apo(a) genes of hedgehog, may have occurred in the distant past. A relationship tree of kringle three domains reveals one major branch for the plasminogens of cow, pig, human, rhesus, mouse, and hedgehog and a separate branch for the apo(a) kringles (Fig. 5B). If the two genes have evolved at similar rates (as shown in the primate case(42) ), this implies that the gene duplication giving rise to the ancestor of the hedgehog apo(a)-like protein preceded the divergence of these mammalian species. Consistent with ancient divergence is the observation that the high degree of synonymous nucleotide substitutions, which are roughly similar (and nearly saturated) when comparing hedgehog plasminogen to hedgehog apo(a) (54%) or hedgehog plasminogen kringle three to human plasminogen kringle three (55%). Finally, we analyzed intron sequences of hedgehog apo(a) and plasminogen genes obtained by polymerase chain reaction and found that beyond the immediate vicinity of splice junctions they are so divergent as to prevent meaningful sequence alignment. In contrast, some genes retain intron sequence conservation between mouse and humans(49) . Hence the ``hedgehog type'' of apo(a) may have a long history and not be restricted to this species. A systematic search among other related animals now appears appropriate.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33171 [GenBank]and U33170[GenBank].