Gene 383 (2006) 64 – 70
www.elsevier.com/locate/gene

Origin and evolution of gene for prolactin-induced protein
Takashi Kitano a,⁎, Wei Tian a , Kazuo Umetsu a , Isao Yuasa b ,Kentaro Yamazaki a , Naruya Saitou c , Motoki Osawa daDepartment of Experimental and Forensic Pathology, Yamagata University School of Medicine, 2-2-2 Iidanishi, Yamagata 990-9585, Japanb
Division of Legal Medicine, Faculty of Medicine, Tottori University, Yonago 683-8503, Japanc
Division of Population Genetics, National Institute of Genetics, Mishima 411-8540, Japand
Department of Forensic Medicine, Tokai University School of Medicine, Isehara, 259-1193, Japan
Received 29 March 2006; received in revised form 10 July 2006; accepted 14 July 2006
Available online 28 July 2006
Received by T. Gojobori

Abstract
Prolactin-induced protein (PIP) is a small protein secreted into the fluid in several glands. We determined the PIP coding sequences of 5
hominoid species and estimated the numbers of synonymous and nonsynonymous substitutions for each branch of the mammalian PIP gene tree.
The branch connecting hominoids and Old World monkeys showed significantly higher nonsynonymous than synonymous substitutions. Thesechanges tended to be accumulated in the fibronectin-binding domain. Many other primate branches also showed higher nonsynonymous thansynonymous substitutions, thus suggesting that the PIP genes of primates have experienced some kind of positive selection. We also considered
the phylogenetic relationship of the PIP gene with the alpha-2-macroglobulin gene family. The results indicate that the PIP gene arose by partial
gene duplication from a member of the alpha-2-macroglobulin gene family after the divergence between amphibians and other tetrapods.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Phylogenetic tree; Nonsynonymous substitution; Positive selection; Mammal; Gene duplication

1. Introduction
The hormonally responsive prolactin-induced protein (PIP) isexpressed in various normal exocrine organs such as sweat,
salivary, and lacrimal glands, as well as in seminal plasma(Haagensen and Mazoujian, 1986). Although its functions insecretory fluids are not well understood, the protein is reported to
be a potent inhibitor of T lymphocyte programmed cell death
induced by cross-linking of CD4 and T cell receptor (Gaubin et al.,1999). It was also reported that the protein is an aspartyl proteinasewith specific fibronectin-degrading ability (Caputo et al., 2000).
The CD4-binding domains and fibronectin-binding domains on
the protein have been identified (Basmaciogullari et al., 2000;
Caputo et al., 2003).
Abbreviations: PIP, prolactin-induced protein; A2M, alpha-2-macroglobulin; FN, fibronectin.
⁎ Corresponding author. Tel.: +81 23 628 5271; fax: +81 23 628 5273.
E-mail address: tkitano@med.id.yamagata-u.ac.jp (T. Kitano).
0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2006.07.014

The human PIP gene is located on chromosome 7q32–36(Myal et al., 1989), and the entire gene is about 7 kb long. The
gene has 4 exons (Myal et al., 1991) comprising 146 aminoacids (Murphy et al., 1987). PIP shares homology with anothersecretory protein, the seminal vesicle autoantigen (SVA). SVA,a secretory protein present in mouse seminal plasma, exerts aninhibitory effect on the motility of spermatozoa by interactingwith the cell membrane phospholipid (Huang et al., 1999).
Evolutionary studies on the PIP genes have yielded limited
data (Osawa et al., 2004; Clark and Swanson, 2005), with theresults suggesting the operation of positive selection on thegene. Thus, in this study, we sequenced the PIP coding
sequences (CDS) of 5 primate species, and carried out a moredetailed evolutionary analysis of this gene. We also examined
the phylogenetic relationship of the PIP gene with other genes
having homologous sequences, such as those of alpha-2macroglobulin (A2M), pregnancy-zone protein (PZP), alpha-2macroglobulin-like 1 (A2ML1), and ovostatin 2 (OVOS2), andconsidered the origin of the mammalian PIP gene.

T. Kitano et al. / Gene 383 (2006) 64–70

2. Materials and methods
2.1. DNA sequencing of primate PIP genes
Genomic DNAs from 2 common chimpanzees (Pantroglodytes), 3 gorillas (Gorilla gorilla), 1 orangutan (Pongo
pygmaeus), 1 agile gibbon (Hylobates agilis), and 1 siamang(Symphalangus syndactylus) were used for DNA sequencing.
PCR amplification was performed in a volume of 20 μl
containing 50 ng of DNA, 1× PCR buffer, 0.2 mM concentration of each dNTP, 0.5 μM concentration of each oligonucleotide primer, 1.5 mM MgCl2, and 1 U of AmpliTaq Goldpolymerase (Applied Biosystems). All PCR primers were
designed based on intron regions to amplify full CDSs, basedon the human (NC_000007) and chimpanzee (AB098482)
sequences in the DDBJ/EMBL/GenBank international nucleotide sequence database. A list of primers used in this study isavailable from one of the authors (TK). The typical PCR
conditions used in this study consisted of 40 cycles of 30-s
denaturation at 95 °C, followed by 15-s primer annealing at
55 °C and 1-min extension at 72 °C. Immediately proceedingthese cycles, a 10-min hot-start step at 95 °C was included. The
PCR products were confirmed by 1.5% agarose gel electrophoresis and purified by using a QIAquick PCR purification kit(Qiagen). The purified PCR products were sequenced with a
BigDye terminator cycle sequencing kit version 1.1 and an ABI
Prism 3100 genetic analyzer (Applied Biosystems). Bothstrands were sequenced, and 2 overlapping peaks on a sitewere regarded to indicate heterozygosity, and the site wasdesignated by following Nomenclature Committee of theInternational Union of Biochemistry (NC-IUB).
2.2. Analysis of PIP coding sequences
The CDSs of the PIP gene for the following 8 mammals
were retrieved from the nucleotide sequence database: human(Homo sapiens, J03460), chimpanzee (AB098482), Japanesemacaque (Macaca fuscata, AB098481), cow (Bos taurus,
AB098480), rabbit (Oryctolagus cuniculus, AB098478), guinea
pig (Cavia porcellus, AB098479), mouse (Mus musculus,
S73282), and rat (Rattus norvegicus, AF054270). The CLUSTALW program (Thompson et al., 1994) was used for themultiple alignment. Aligned regions without gaps were used for
subsequent analyses. A phylogenetic tree was constructed byusing the neighbor-joining method (Saitou and Nei, 1987) alongwith the two-parameter method (Kimura, 1980). MEGA3
software (Kumar et al., 2004) was used for the tree construction.
The ancestral sequences on the tree were reconstructed by the
likelihood-based Bayesian method (Yang et al., 1995) using the
BASEML program in the PAML package (Yang, 1997). The
numbers of synonymous and nonsynonymous substitutions foreach branch were estimated by using the modified Nei and
Gojobori's method (Nei and Gojobori, 1986; Zhang et al.,1998). The ratio (R) of transitional changes to transversionalchanges for the data set was obtained by counting the total
numbers of transitional and transversional changes that wereobserved in the entire phylogenetic tree. This method yielded65

R = 296 / 254 = 1.17; therefore, we assumed R = 1 in subsequent
computations. Fisher's exact tests for synonymous andnonsynonymous substitutions were performed according toZhang et al. (1998).
2.3. Polymorphism data for the human PIP geneWe used genomic DNAs from 30 Japanese and 30 Germans
to sequence 2 regions (I and II) of the human PIP gene. The
regions I (615 bp) and II (664 bp) contained exon 2 and exons
3–4, respectively (Supplementary Fig. 1). PCR and sequencing
were done as described above. Chimpanzee (NW_109346) andJapanese macaque (AB098481) sequences were used in order todetermine ancestral states for each SNP (single nucleotide
polymorphism). Two measures of nucleotide variability, π (Nei,
1987) and θW (Watterson, 1975), were calculated. Tajima's(1989) D and Fay and Wu's (2000) H were calculated to test for
deviations from the standard neutral model by using the allele
frequency spectrum. A program for performing these statistics,
kindly provided by J.C. Fay, was used.
2.4. Analysis of the relationship between PIP and its relatedgenes
We performed PSI-BLAST (Altschul et al., 1997) with 10
passes against the NCBI non-redundant protein database. The
human PIP (NP_002643) was used as a query for the homology
search. From the PSI-BLAST result, we collected amino acidsequences that gave hit length of more than 100 amino acidresidues and higher than 30% positive (meaning identical orsimilar) residues without alignment gaps. Human, cow, dog, andmouse were used as representative mammals, with the chicken(Gallus gallus) for birds, African clawed frog (Xenopus laevis) for
amphibians, and pufferfish (Tetraodon nigroviridis) and zebrafish(Danio rerio) for fish. We also obtained homologous sequencesfrom the Japanese lamprey (Lethenteron japonicum) and 7
invertebrate species, i.e., purple sea urchin (Strongylocentrotus
purpuratus), sea scallop (Chlamys farreri), horseshoe crab
(Limulus sp.), red flour beetle (Tribolium castaneum), honeybee (Apis mellifera), soft tick (Ornithodoros moubata), andkuruma shrimp (Marsupenaeus japonicus). The list of thesesequences is given in Supplementary Table 1. Redundantsequences and sequences that contained an alignment gap of
more than 5 contiguous amino acid residues were not used for the
analysis (see Supplementary Table 1). In total, 92 amino acidsequences were used for the tree reconstruction of the PIP genes
and their related genes. Furthermore we carried out tBLASTn
searches against gss (genome survey sequence), wgs (wholegenome shotgun sequence), and htgs (unfinished high throughput
genomic sequences) databases for chicken, African clawed frog,
zebrafish, and pufferfish (Takifugu rubripes) by using the human
PIP (NP_002643) as a query. However, any other additionalsequences were not observed. The T-Coffee program (Notredame
et al., 2000) was used for the multiple alignment. For maximum
likelihood phylogenetic inference, first, a neighbor-joining tree
was constructed with the JTT model (Jones et al., 1992), followedby the branch swapping (nearest neighbor interchange) method,

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T. Kitano et al. / Gene 383 (2006) 64–70

with the neighbor-joining tree used as the initial tree. The
PROTML program in the MOLPHY package (Adachi and
Hasegawa, 1996) was used.
3. Results and discussion
3.1. Synonymous and nonsynonymous substitutions in mammalian PIP genes
We sequenced the PIP genes from 2 common chimpanzees,
3 gorillas, 1 orangutan, 1 siamang and 1 agile gibbon (DDBJ/
EMBL/GenBank International Nucleotide Sequence Databaseaccession numbers are AB251899–AB251906). Fig. 1 shows amultiple alignment of translated amino acid sequences of the
mammalian PIP proteins. The chimpanzee sequences determined in this study were identical to the sequence encoded by
the chimpanzee PIP gene deposited in the database except for 1
synonymous change (T342C). In the gorillas, 2 polymorphic
sites were observed (G343A and A359G). Both substitutions
could change the type of amino acid and were located in theneighborhood of each other (Fig. 1). We used 343 = G and
359 = A for the following analyses as a representative ofgorillas.
Fig. 2 shows the phylogenetic relationship among mammalian PIP genes and the numbers of synonymous andnonsynonymous substitutions on each branch. The dN/dS
ratio of the number of nonsynonymous substitutions per

nonsynonymous site (dN) to the number of synonymous
substitutions per synonymous site (dS) was estimated for eachbranch. Nine branches showed a dN/dS value higher than 1, and
8 of them were in the primate lineages. Application of Fisher's
exact test showed that the number of nonsynonymous
substitutions was significantly higher than that of the synonymous ones (p = 0.02) in the branch that connected hominoids
and an Old World monkey, where 18 nonsynonymous
substitutions (n), 1 synonymous substitutions (s), 112.7
potential synonymous sites (S), and 310.3 potential nonsynonymous sites (N) were estimated. Table 1 shows the numbers ofamino acid changes for each domain among the primate PIP
proteins. On the branch of Node b–Node c, 5 out of 18 aminoacid changes were clustered in the FN-1 domain. The rate ofamino acid changes in this domain was 2 to 3 times higher thanthat for the other regions. This domain also accumulated aminoacid changes in the branch that connected to an Old World
monkey (Node b–Macaque). In contrast, amino acid changes inhominoids tended to accumulate in the FN-2 domain. The NH2terminal region, which is the first CD4-binding domain, wasconserved among the primate PIP proteins. Furthermore, 4cysteine residues for 2 pairs of disulfide bonds (Schaller et al.,1991) were conserved among the mammals examined (Fig. 1).
By using site-specific mutagenesis, Caputo et al. (2000)
demonstrated that an aspartate residue (they called it Asp22)
was required for proteinase activity. This aspartate residue islocated in the FN-1 domain (Fig. 1), and is shared by human and

Fig. 1. Amino acid sequences for present-day and ancestral PIP proteins. Dots show the same amino acids as those of the sequence at node a. Four cysteine residues (67,
91, 93, and 125) for 2 pairs of disulfide bonds are shown on a grey background. CD4-1 and CD4-2 indicate CD4-binding domains; and FN-1 and FN-2, fibronectinbinding domains. The 2 underlined residues were polymorphic among 3 gorillas (V115I and Q120R). The aspartate residue (Asp22) is indicated by the asterisk.

T. Kitano et al. / Gene 383 (2006) 64–70

67

Fig. 2. A phylogenetic tree of mammalian PIP genes. dN/dS are indicated above each branch, and a branch that has a dN/dS value higher than 1 is shown by a thick line.
The numbers of nonsynonymous (n) and synonymous (s) substitutions are indicated as n/s below each branch. Three ancestral nodes are shown by a (common
ancestor of primates, rodents, and rabbit), b (common ancestor of primates), and c (common ancestor of hominoids). The statistical significance was tested by Fisher's
exact test (⁎, less than 5%).

great apes. If so, the proteinase activity of PIP was acquired
during the evolution of the great apes. Further study is needed toelucidate the time of acquisition of proteinase activity by PIP
proteins.
Although the function of the PIP protein is not wellunderstood, it has been suggested that PIP is a binding factor
playing a role in host defense against infections (Schenkels et al.,
1997; Gaubin et al., 1999). The majority of genes are considered
to be evolving under neutral mutation pressure. However, some
genes are evolving through positive selection. For example,
several genes are considered to be positively selected byinteraction between host defense systems and pathogens(Hughes and Nei, 1988, 1989; Ina and Gojobori, 1994; Kitano
et al., 1998; Kitano and Saitou, 1999; Schaner et al., 2001; Baumet al., 2002; Osada et al., 2002; Sumiyama et al., 2002; Filip andMundy, 2004; etc.). The high rate of nonsynonymous substitutions for the primate PIP genes also suggests the existence of

positive selection on this gene, which selection might be causedby some kind of interaction between the PIP protein andpathogens.
To clarify whether the higher number of nonsynonymous
substitutions of the PIP gene in the primate lineages was a resultof positive selection or relaxation of selection, we sequenced
and analyzed polymorphisms in the genome sequence of thehuman PIP gene (DDBJ/EMBL/GenBank International Nucleotide Sequence Database accession numbers are AB267097–
AB267216). In total, 3 polymorphic sites in Japanese and 5 inGermans were observed in the 2 sequenced regions (I and II).
Table 2 summarizes the statistics on the polymorphisms of thehuman PIP gene (see also Supplementary Fig. 1). The
nucleotide diversity (π) determined by the SNP Consortium(Sachidanandam et al., 2001) for the entire human genome is
0.075%, and the corresponding values for the human PIP genes
reported here (Region I + II) were 0.022% for Japanese andTable 1
Number of amino acid changes for each domain of primate PIP proteins

No. of residues
Node b–Node c
Node b–Macaque
In hominoids

CD4-1

IT-1

FN-1

IT-2

CD4-2

IT-3

FN-2

C-terminal

Total

35
2 (0.06)
2 (0.06)
2 (0.06)6
1 (0.17)2 (0.33)
2 (0.33)

16
5 (0.31)5 (0.31)2 (0.13)20
2 (0.10)5 (0.25)
3 (0.15)28
4 (0.14)3 (0.11)7 (0.25)

30 (0.00)0 (0.00)0 (0.00)10
1 (0.10)0 (0.00)
5 (0.50)28
3 (0.11)5 (0.18)
6 (0.21)

146
18
22
27

The number of amino acid changes per residue for each domain is shown in parentheses. CD4-1 and CD4-2: 2 CD4-binding domains, FN-1 and FN-2: 2 fibronectinbinding domains, IT-1, IT-2, and IT-3: 3 interval regions, C-terminal: C-terminal region.

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T. Kitano et al. / Gene 383 (2006) 64–70

Table 2Polymorphisms in humans at the PIP locus
Region I (613 bp)

Region II (664 bp)

JapaneseJapanese

Germans

Region I + II
(1277 bp)

Germans Japanese

S
2
1
1
4
π (%)
0.026
0.034
0.019
0.123
θW (%)
0.070
0.035
0.032
0.129
−1.072−0.029−0.526−0.105
DTajima
HFay & Wu −1.612⁎⁎ −1.377⁎⁎ −1.645⁎⁎ −0.232

Germans

3
5
0.022
0.080
0.050
0.084
− 1.101− 0.101− 3.258⁎⁎⁎ − 1.609⁎

S: number of segregating sites; π: average number of nucleotide differences persite between 2 sequences; θW: nucleotide diversity based on the proportion ofsegregating sites in a sample; DTajima: Tajima's D (Tajima, 1989); HFay & Wu:
Fay and Wu's H (Fay and Wu, 2000). Asterisks indicate the significance level(⁎10%, ⁎⁎5%, ⁎⁎⁎1%).

0.080% for Germans. Although the π in region II of Germans
was slightly high, these values are comparable with that value
for the entire human genome. Tajima's D, which examines thedifference (D) between π and θW, was negative for all estimates.
However, none of the D values differed significantly from theneutral expectation of zero. In contrast, Fay and Wu's H-test
showed significance values except for the region II of Germans.
The results revealed an excess of derived variants at highfrequency that were likely to have been influenced byhitchhiking of the derived changes under positive selection on
the human PIP gene. Thus, our data suggest that the higher
number of nonsynonymous substitutions of the PIP gene in the
primate lineages was caused by positive selection rather than byrelaxation of selection.
3.2. Phylogenetic relationship between the PIP gene and theA2M gene family, and origin of the PIP geneWe searched the NCBI protein database by PSI-BLAST
using the human PIP amino acid sequence as a query, andobserved weak homologies with the C-terminal region ofmembers of the A2M gene families including PZP, OVOS2, andA2ML1 (Supplementary Table 1). The average percent of aminoacid identities between PIP and A2M, PZP, A2ML1, OVOS2 inmammalians were 16.1%, 16.3%, 17.1%, and 16.7%, respectively. Fig. 3A shows a phylogenetic tree among the A2M genefamily with the PIP and SVA genes. The amino acid length

Fig. 3. A phylogenetic tree of the A2M gene family constructed by the maximum likelihood method (A). Bootstrap values greater than 80% are shown on each branch.
White squares, black circles, and white circles indicate internal nodes connecting fish, frog, and chicken, respectively. A phylogenetic tree of the A2ML1 cluster
excluding the PIP and SVA genes constructed by the maximum likelihood method (B). Bootstrap values greater than 80% are shown on each branch. Arrows withnumbers indicate candidate branches connecting the PIP–SVA cluster. A phylogenetic tree of the A2ML1–PIP–SVA cluster constructed by the maximum likelihood
method (C). Black circles and white circles indicate internal nodes connecting fish, frog, and chicken, respectively.

T. Kitano et al. / Gene 383 (2006) 64–70

compared was 92. The phylogenetic tree was constructed byusing the region of exon 2 to exon 4 for the human PIP genewhich corresponds to a part of exon 19 to exon 21 for the human
A2M gene. In fact, A2M gene family members shared thelocation of exon–intron boundaries (Supplementary Fig. 2)
excluding invertebrate sequences. All invertebrate sequences
formed a cluster, and a lamprey sequence was located on the
middle between the vertebrate and invertebrate sequences.
Thus, the lamprey and invertebrates sequences can be used as
out-groups of the A2M gene family for vertebrates. Two large
clusters were observed in vertebrates; the A2M cluster(including A2M, PZP, OVOS2, and Murinoglobulin) and theA2ML1–PIP–SVA cluster. Although the branching position of
the pufferfish sequence (CAG06475) did not form a cluster withother fish sequences, the fish cluster formed a cluster with theA2M cluster. There are 2 possibilities for this result; one isthat fish genes homologous to the A2ML1–PIP–SVA cluster
genes were deleted or undetected, and the other is that thebranching point of the fish homologous genes is not correct. It isalso possible to assume that the gene duplication between theA2M cluster and the A2ML1–PIP–SVA cluster occurred after
the divergence between fish and other vertebrates. In any case,our data suggest that the A2ML1–PIP–SVA cluster emerged
after the divergence between fish and other vertebrates. Frog
homologous genes were observed in the both clusters.
To obtain more reliable phylogenetic relationships in theA2ML1–PIP–SVA cluster, we used A2M domain sequences andconstructed a phylogenetic tree excluding the PIP and SVA genes(B). The amino acid length compared was 339, and the pufferfish(CAG06475) was used as an out-group. Three frog sequences
formed a cluster. It is not clear whether these 3 genes were allelicor represented different loci. Three chicken sequences did notform a cluster and their genes were located at different
chromosomal positions. By using this topology (Fig. 3B), we
added the PIP–SVA cluster for each branch, indicated by thearrow, with number and estimated likelihood values for each tree.
Fig. 3C shows the maximum likelihood tree for the A2ML1–
PIP–SVA cluster. The amino acid length compared was 104.
Although the likelihood values of 8 trees excluding tree number 7
were not significantly different, the PIP–SVA genes formed acluster with the predicted chicken gene (XP_416480), which is
not probably an orthologue of the PIP–SVA genes. This predicted
chicken gene was located on chromosome 1 (chr.1) in the
68,370K–68,416K region. In contrast, human and mouse synteny
around the PIP gene was conserved; and the corresponding
region of the chicken gene was located on chr.1 in the 71,750K–
72,130K region, although the location of the probable chicken
PIP orthologous gene was in the genome sequence gap. Thus it
is not clear whether a chicken PIP orthologous gene exists or not.
We also BLAST-searched against the chicken EST database using
human PIP gene as a query, but obtained no PIP homologous
sequence.
Although the exact branching point of the PIP gene is notclear, it is possible to predict that after the divergence between
amphibians and other tetrapods, the PIP gene have emerged by apartial gene duplication, since mammalian PIP genes are shorter
than other A2M gene members. After the gene duplication, the69

ancestral PIP gene might have acquired a new specific function
and might have become expressed in sweat, salivary, andlacrimal glands, and in the seminal plasma in the mammalianlineage. The gene duplications that produced SVA genes
occurred in the mammalian lineage as described elsewhere(Yoshida et al., 2001; Osawa et al., 2004). Based on analysis of
the chicken genome sequence (International Chicken Genome
Sequencing Consortium, 2004), some genes such as those forcasein milk proteins and salivary-associated proteins (statherin
and histatins) arose in the mammalian lineage after thedivergence between mammalians and birds. It is likely that the
PIP gene is also a mammalian-specific gene and appeared
during mammalian evolution.
Acknowledgements
We are grateful to the Great Ape Information Network(GAIN) for providing us chimpanzee, gorilla, and orangutan
samples, to Ueno Zoo, Tokyo, for providing us with the gorilla
samples, and to Dr. Takenaka (deceased) for providing us the
gibbon DNA samples.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.gene.2006.07.014.
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