Aspartic Proteinase Phylogeny and the Origin of Pregnancy-Associated Glycoproteins

Austin L. Hughes*,, Jonathan A. Green{dagger}, Helen Piontkivska* and R. Michael Roberts{dagger}

* Department of Biological Sciences, University of South Carolina
{dagger} Department of Animal Science, University of Missouri-Columbia

Correspondence: E-mail: austin{at}biol.sc.edu.


    Abstract
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The phylogenetic relationships of eukaryotic aspartic proteinases were reconstructed in order to understand the origin of pregnancy-associated glycoproteins (PAGs), which constitute a large gene family expressed in the trophoblast and placenta of mammals in the order Artiodactyla. The phylogeny supported the hypothesis that PAGs originated in mammals, being most closely related to a group of PAG-like molecules (including rodent pepsin F) found in other mammalian orders. These two groups in turn form a sister group to a group of digestive enzymes from birds and mammals, which includes pepsin A. Sequence similarity in the promoter region of artiodactyl PAGs and mouse pepsin F also supported a close relationship between these genes. Ancestral sequence reconstruction revealed that, at the residues corresponding to positions 148–150 of pepsin A, in the ancestor of artiodactyl PAGs the motif QNL was replaced by EPV; and EPV (or occasionally EPI) is conserved at these sites in known PAGs. The conservation of this ancestral change suggests that it may be important to PAG function, particularly the fact that PAGs lack proteinase activity in spite of the conservation of active site residues in most PAGs.

Key Words: aspartic proteinase • PAGs • pregnancy-associated glycoproteins


    Introduction
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The majority of the proteinases, the enzymes that hydrolyze peptide bonds, have been placed in four major categories on the basis on the amino acid residues or other chemical groups directly involved in the catalytic mechanism: serine proteinases, cysteine proteinases, metalloproteinases, and aspartic proteinases (Barrett 1992). Within each of these categories is contained numerous molecules, some of which share no sequence homology outside of the active site residues; thus, it is possible that the same type of proteolytic mechanism has evolved independently more than once (Barrett 1992).

Aspartic proteinases have been described in animals, plants, fungi, and retroviruses (Davies 1990). The eukaryotic aspartic proteinases are characterized by a bilobed structure. Within each lobe resides an aspartic acid residue in the context of an invariant D-T/S-G motif. The aspartic acids within each lobe are brought into close proximity with one another and, together, in conjunction with a water molecule, are directly involved in the catalytic mechanism (Blundell et al. 1998). All eukaryotic aspartic proteinases show evidence of sequence homology outside the active site, which suggests that they share a common ancestry. Aspartic proteinases from vertebrates include a number of enzymes whose three-dimensional structures are known, such as the digestive enzymes pepsin (Sielecki et al. 1990) and chymosin (Newman et al. 1991); the lysosomal enzyme cathepsin D (Baldwin et al. 1993); and renin, which is involved in control of blood pressure through conversion of angiotensinogen to angiotensin I (Dhanaraj et al. 1992). Among the most interesting members of the vertebrate aspartic proteinase family are the pregnancy-associated glycoproteins (PAGs) of mammals in the order Artiodactyla, the even-toed hoofed mammals. Pregnancy-associated glycoproteins have been cloned or purified from placenta of mammals of sheep, goat, bovine, moose, and pig (Szafranska et al. 1995; Xie et al. 1997; Huang et al. 1999; Garbayo et al. 2000; Green et al. 2000). The PAGs are encoded by a large multi-gene family (Xie et al. 1997; Hughes et al. 2000). Many members of this family have been predicted to lack protease function, although the ability to bind peptides remains unaltered (Xie et al. 1997). Furthermore, there is evidence that natural selection has acted to diversify the members of the PAG gene family at amino acid residues in solvent-accessible positions; such changes have been hypothesized to modulate peptide binding (Xie et al. 1997; Hughes et al. 2000).

Additional PAG-like molecules have been reported from other mammals outside the Artiodactyla (Chen et al. 2001). These PAG-like molecules (sometimes known as pepsin F) differ from PAGs in that the PAG-like molecules are encoded by only one or two genes, whereas the PAGs are encoded by a large multi-gene family. Furthermore, whereas PAG expression is restricted to the placenta, PAG-like molecules are expressed both in extra-embryonic membranes and in the neonatal stomach. In addition, PAG-like genes have been found in the genomes of mammals belonging to a number of different orders (Carnivora, Lagomorpha, Perissodactyla, and Rodentia), but not in the human genome.

Because both PAGs and PAG-like molecules are known only from eutherian (placental) mammals, it seems a plausible hypothesis that these molecules originated within the placental mammals. A preliminary phylogenetic analysis by Chen et al. (2001) placed PAGs close to a group of PAG-like molecules from non-artiodactyl mammals, including a mouse proteinase expressed in the yolk sac and neonatal stomach. However, because this phylogenetic analysis included only mammalian aspartic proteinases, it was not possible to test the hypothesis that PAGs originated within the mammals. The phylogenetic tree lacked any statistical estimation of the confidence of branches, and thus it was not possible to assess the reliability of clustering patterns. In the present study, a comprehensive phylogenetic analysis of eukaryotic aspartic proteinases is used to reconstruct the relationships of major mammalian classes of aspartic proteinases and the evolutionary origin of PAGs.

We used sequence comparisons of promoter regions to obtain additional information regarding the relationships of PAGs; and we used reconstruction of amino acid changes in the phylogeny to identify key features of primary structure unique to the PAGs. Coupled with information on the three-dimensional structure of other aspartic proteinases, these features highlighted structural changes that may be important to the function of the PAGs, which is still poorly understood.


    Methods
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Sequences Analyzed
A phylogenetic analysis of the eukaryotic aspartic proteinases was based on 66 representative amino acid sequences (fig. 1). These sequences were chosen to provide representatives from major eukaryotic taxa, including animals, plants, fungi, and protists (table 1). Within animals, representative vertebrate and invertebrate sequences were chosen; and within vertebrates, representative sequences from different classes were chosen. In preliminary analyses with a larger number of sequences, the results were essentially the same as those presented here (data not shown). Among the 66 sequences were six representative PAGs from Artiodactyla (fig. 1).



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FIG. 1. Minimum evolution tree of aspartic proteinases, based on the gamma-corrected amino acid distance at 287 aligned sites. Numbers on the branches are confidence levels for the significance test of the interior branch; only values >=95% are shown

 

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Table 1 Organisms Represented in Phylogenetic Analyses.

 
In additional analyses, a set of 41 complete PAG sequences from pig, bovine, sheep, and goat were used; the accession numbers of these additional sequences were as follows: U30251, AF020513, Q28057, AF020514, AF191331, AF191326, AF192331, U94794, AF191330, AF020506, AF020508, Q29432, AF192337, AF020509, AF192333, U94790, T10264, U94789, AF191328, AF191329, U94795, AF191335, AF192332, AF192336, A41545, M73961, U94792, AF191332, AF191336, AF191334, AF192335, AF020511, AF192334, AF020507, U94791, AF020512, AF272735, I46617, Q29078, Q29079, and AF315377. In preliminary phylogenetic analyses, the vertebrate aspartic proteinases known as memapsins (Yan et al. 1999) were found to be very distant from all other eukaryotic aspartic proteinases; thus they were not included in the present analyses.

In analyses of promoter regions, we used promoter sequences for porcine PAG2 (U39198), bovine PAG1 (L237833), bovine PAG2 (AY212886), and mouse pepsin F (AY212887).

Statistical Methods
Amino acid sequences and noncoding DNA sequences were aligned using the CLUSTAL W program (Thompson, Higgins, and Gibson 1994). In the case of coding DNA sequences, the amino acid sequence alignment was imposed on the DNA. In phylogenetic analyses and pairwise comparisons with a set of sequences, all sites at which the alignment postulated a gap in any sequence were excluded from the analysis. All alignments are available from the authors by request.

Phylogenetic trees were reconstructed from amino acid sequence alignments by the maximum parsimony (MP) method (Swofford 2000); by the quartet maximum likelihood (QML) method (Strimmer and von Haeseler 1996) implemented in the program TreePuzzle 5.0; by the minimum evolution (ME) method (Rzhetsky and Nei 1992). In the QML method, we used the JTT model (Jones, Taylor, and Thornton 1992) of amino acid evolution and the assumption that rate variation among sites followed a gamma distribution. In the ME analysis, amino acid sequence distances were estimated by the gamma model (Ota and Nei 1994). The parameter a of the gamma distribution was estimated by the method of Gu and Zhang (1997) and by the TreePuzzle 5.0 program; both methods yielded identical estimates (a = 1.3). All methods produced essentially identical results, and only the ME tree is shown here. The reliability of clustering patterns in the ME analysis was tested by the interior branch length test, using bootstrap estimation of the standard error of branch lengths as implemented in the MEGA2 program (Kumar et al. 2001).

Ancestral amino acid sequences and amino acid replacements were reconstructed by the MP method and by the maximum likelihood (ML) method of Yang, Kumar, and Nei (1995). Ancestral reconstruction was applied only to a sub-tree of closely related sequences. When sequences are closely related, both the MP and likelihood methods are predicted to give reliable inference of ancestral sequences (Zhang and Nei 1997). Ancestral DNA sequences for selected sites were reconstructed by the MP method. Because of long evolutionary distances, the ML method was not able to reconstruct DNA sequences in this case.

To test the hypothesis of sequence conservation in promoter regions, we compared the pattern of nucleotide substitution in available PAG promoter sequences with that at fourfold degenerate sites in the coding region. The single-invariant test of Rzhetsky and Nei (1992) rejected the applicability of the Jukes-Cantor model of sequence evolution to the PAG promoter sequences, but it did not reject the Kimura two-parameter (K2P) model (Kimura 1980). Therefore we used the K2P model to estimate the numbers of nucleotide substitutions per site in promoter regions. So that a comparable model was applied to both coding and noncoding regions in these comparisons, we estimated the numbers of substitutions per site at fourfold degenerate sites in coding regions using the method of Li, Wu, and Luo (1985). This method is equivalent to the K2P method at fourfold degenerate sites.


    Results
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Figure 1 shows the ME tree of aspartic proteinases. The tree was rooted with sequences from malaria parasites. In this phylogeny, support was weak for most of the deepest branches. However, a large cluster including vertebrate cathepsin E, vertebrate pepsins, artiodactyl PAGs, and PAG-like sequences from other mammals received highly significant support (fig. 1). Within this cluster, a subfamily of vertebrate digestive enzymes including pepsin A and chymosin (designated the pepsin A subfamily in figure 1) formed a sister group to the PAGs and PAG-like sequences. The PAG-like sequences from mammals other than artiodactyls formed a separate cluster which received highly significant support (fig. 1). This cluster of PAG-like molecules included proteins from the orders Rodentia (rat and mouse), Perissodactyla (horse and zebra), and Carnivora (cat); and it clustered as a sister group to the true PAGs from Artiodactyla (fig. 1). Because both of these clusters included only mammalian sequences, the phylogeny supported the hypothesis that PAGs originated within the mammals.

In addition to the cluster including pepsin A, PAG, and PAG-like molecules, three other clusters of vertebrate aspartic proteinases received strong support. Cathepsin C from mammals, chicken, and the frog Xenopus clustered with brook trout gastricin; and this cluster was supported by a highly significant internal branch (fig. 1). Similarly, cathepsin D from mammals, chicken, and zebrafish formed a cluster supported by a significant internal branch (fig. 1). Also, mammalian cathepsin E clustered with zebrafish nothepsin, and this cluster was supported by a significant internal branch (fig. 1). Thus, the phylogenetic analysis strongly supported the hypothesis that cathepsin C/gastricin, cathepsin D, and cathepsin E/nothepsin represent distinct subfamilies of aspartic proteinases that arose by gene duplications prior to the divergence of bony fishes from tetrapods. Nevertheless, there were no significantly supported clusters including molecules from both vertebrates and invertebrates (fig. 1). Thus, the phylogenetic analysis did not provide any evidence that vertebrate genomes include any aspartic proteinases that arose by gene duplication prior to the origin of the vertebrates.

To identify amino acid changes that might be crucial to the evolution of new function in the ancestor of PAGs, amino acid changes reconstructed by the MP and ML methods were examined in the sub-tree of PAGs and PAG-like molecules, rooted with the pepsin A subfamily (fig. 1). Both methods of reconstruction revealed three changes occurring at adjacent amino acid sites along the branch leading to PAGs (fig. 2). The sites involved correspond to residues 148–150 of the mature mammalian pepsin molecule (Sielecki et al. 1990) and to residues 150–152 of mature bovine PAG1 (accession number Q29432). These reconstructions indicated that the ancestor of PAG and PAG-like molecules had the amino acid sequence QNL at these sites, and that this was changed to EPV in the ancestor of PAGs. The posterior probabilities estimated by the ML method for the EPV motif in the PAG ancestor were all quite high, particularly in the case of the first two residues (fig. 2). All available artiodactyl PAG sequences retain EPV or EPI at these positions (fig. 2).



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FIG. 2. Reconstructed amino acid changes at the positions corresponding to positions 148–150 of pepsin in the ancestor of mammalian PAG-like molecules and the ancestor of true PAG molecules of Artiodactyla. Numbers in brackets below each reconstructed amino acid are the posterior probabilities of the reconstruction given the phylogenetic tree and the substitution model. Numbers in parentheses correspond to the percentages of each amino acid found in available sequences

 
To test whether positive Darwinian selection may have played a role in fixation of the EPV motif in the PAG ancestor, we examined DNA sequence changes along the branch leading to the PAG ancestor using sequences reconstructed by the MP method. The reconstructed DNA sequence for these three codons changed from CAG AAT GTC to GAG CCT GTC. Thus, there were four nonsynonymous changes but no synonymous changes. Although the number of sites is small, the preponderance of nonsynonymous changes is consistent with the hypothesis that natural selection acted to favor these amino acid changes (Hughes and Nei 1988; Hughes 1999).

The promoter regions of artiodactyl PAG-encoding genes showed homology to the mouse gene encoding pepsin F (table 2). However, there was little evidence of homology between PAG promoters and published promoter sequences from mammalian pepsin A or renin genes (data not shown). In pairwise comparisons among PAG promoters, there was a region proximal to the start site that showed significantly lower sequence divergence than synonymous sites in the coding region (table 2). In comparisons between PAG and pepsin F, the same region likewise showed significantly lower sequence divergence than synonymous sites in the coding region (table 2). The reduced rate of nucleotide substitution in the proximal promoter region is evidence that this region is subject to purifying selection and thus to functional constraint. The fact that this pattern is seen both within the PAGs and in comparisons between PAGs and pepsin F is evidence that these two classes of genes are subject to similar functional constraints in this portion of their promoter regions.


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Table 2 Mean Numbers of Nucleotide Substitutions per 100 Sites Among Three Artiodactyl PAGs and Mouse Pepsin F in Promoter and Coding Regions.

 

    Discussion
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Phylogenetic analysis of aspartic proteinases from a wide variety of eukaryotes supported the hypothesis that PAGs of Artiodactyla arose by gene duplication within the mammals. The closest relatives of the PAGs were shown to be a group of PAG-like molecules from various mammals, whereas PAGs and PAG-like molecules were shown to be related to a group of digestive enzymes from birds and mammals here designated the pepsin A subfamily (fig. 1). This group of PAG-like molecules includes mouse pepsin F; and a close relationship between pepsin F and PAGs was supported by evidence that these two groups of genes share functional constraints in a portion of the promoter region (table 2).

Reconstruction of amino acid changes in the phylogeny of aspartic proteinases revealed that, in the ancestral PAG, the motif QNL was replaced by EPV at the residues corresponding to residues 148–150 of pepsin. The fact that the EPV motif (or occasionally EPI) is conserved in all known PAGs suggests that this sequence change may have been important in the evolution of a function unique to PAGs. A preponderance of nonsynonymous nucleotide changes in the EPV codons on the branch leading to the PAGs supports the hypothesis that natural selection favored changes in these residues, which is consistent with their having played an important role in the origin of a functionally novel class of proteins.

The aspartic proteinases have an internally duplicated structure, with N-terminal and C-terminal domains that show evidence of homology to each other (Sielecki et al. 1990). This structure appears to have arisen by an ancient internal gene duplication (Tang et al. 1978). Each of these two domains includes a short conserved sequence containing an aspartate residue (residues 32 and 215, respectively, in pepsin) that contributes to the active site (Sielecki et al. 1990). In the three-dimensional structure of the molecule, a six-stranded anti-parallel ß-sheet creates a hydrophobic core on the side of the protein opposite to the substrate-binding cleft that contains Asp 32 and Asp 215. In porcine pepsin A, residues 149 and 150 are located at the beginning of one of the strands of this ß-sheet and are involved in hydrogen bonding with other strands (Sielecki et al. 1990) (fig. 3). The corresponding residues play an identical role in bovine chymosin (Newman et al. 1991). A similar structure is seen in rennin (Dealwis et al. 1994), which is less closely related to PAGs than are pepsin A and chymosin (fig. 1). Because of the potential importance of residues 148–150 in maintaining the connection between the N-terminal and C-terminal domains of aspartic proteinases, changes in these residues in the ancestor of PAGs may have served to alter the three-dimensional structure of the molecule. Such a change in structure may explain the observation that PAGs lack aspartic proteinase activity in spite of the fact that the active site residues are conserved in most PAGs (Xie et al. 1997).



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FIG. 3. Model of porcine pepsin A with the QDL motif (residues 148–150) colored yellow (at arrow). The two catalytic amino acids are also colored yellow. The QDL residues are at the beginning of one of the strands forming the six-stranded ß-pleated sheet that creates the hydrophobic core of the molecule

 


    Acknowledgements
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
This research was supported by National Institutes of Health grants GM34940 to A.L.H. and HD21896 to R.M.R.


    Footnotes
 
Claudia Schmidt-Dannert, Associate Editor Back


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 Methods
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 Acknowledgements
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Accepted for publication June 27, 2003.





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