Adaptive Evolution of the IgA Hinge Region in Primates
Kenta Sumiyama,*† Naruya Saitou,† and Shintaroh Ueda**Department of Biological Sciences, Graduate School of Science, University of Tokyo; †Laboratory of Evolutionary Genetics,
National Institute of Genetics, Mishima 411-8540, Japan
IgA is a major component that prevents the penetration of pathogenic bacteria into mucosal surfaces. The IgA
antibody is cleaved at the IgA hinge region with high specificity by IgA-specific proteases produced by several
pathogenic bacteria. We conducted a genomic sequence analysis of the IgA genes of a wide spectrum of primates,
including the first intron and second exon, which consist of the hinge region and the CH2 domain, to find evidenceof positive selection. Because the hinge region is quite small, we combined the largest collection of sequences that
could be clearly aligned and evaluated the total numbers of synonymous and nonsynonymous substitutions on thephylogenetic tree. The nonsynonymous to synonymous substitution ratio (dN/dS test) showed that hominoids, Old
World monkeys, and New World monkeys have dN/dS ratios of 5.4, 6.3, and 4.2, respectively. Fisher’s exact
probability tests showed statistical significance for the Old World monkey. Because the substitution rates of theflanking sequences are more or less similar to the synonymous rates of the hinge region, these high values of dN/
dS should be the result of positive selection at the hinge region. Combining the high sequence variability in each
population and the highly accelerated nonsynonymous substitution rates in the hinge region, we conclude that thisunusual IgA evolution is a molecular evidence of adaptive evolution possibly caused by the host-parasite
relationship.

Introduction
The IgA antibody is a major component of the secreted immune response and is known to inhibit colonization of pathogenic bacteria on the mucosal surface.
The heavy chain constant region of IgA consists of three
CH domains and a hinge region, which are encoded by
an immunoglobulin Ca gene. The hinge region, locatedin the beginning of the CH2 exon, links Fab, including
the antigen recognition sites, and Fc, including the effector sites, and can vary the distance between two antigen recognition sites with some flexibility. Hominoids
have two Ca loci in their genomes, except for orangutan, whereas Old World monkeys have a single Ca gene
(Kawamura, Omoto, and Ueda 1990 and references
therein). Sequence comparison among the hominoid Ca
genes reveals the hypervariability of the hinge region
that might be caused by frequent remodeling associatedwith their reiterated structure (Kawamura, Omoto, andUeda 1990). Because the IgA is a secreted antibody, thismolecule may be subject to positive selection driven bybacterial proteolysis. It is known that the bacterial IgAspecific proteases inactivate the IgA molecule by cleaving it at the hinge region (Plaut et al. 1975; Kilian, Mestecky, and Schrohenloher 1979; Male 1979; Plaut 1983).
As for IgA, we found by a survey based on PCR–single
strand conformation polymorphism (SSCP) that the Ca
hinge region of Old World monkeys had an unusuallyhigh polymorphism, although the hinge region is locatedwithin the constant region (Sumiyama et al. 1998).
In view of the evolutionary hypervariability among
hominoids and the unusually high polymorphism of the
Ca gene hinge region among Old World monkeys, there
is a possibility that this gene has experienced positive
Key words: immunoglobulin Ca, IgA, hinge, positive selection.
Address for correspondence and reprints: Kenta Sumiyama, Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, Connecticut 06520. E-mail:kenta.sumiyama@yale.edu.
Mol. Biol. Evol. 19(7):1093–1099. 2002
q 2002 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

selection during primate evolution. With this aim, we
obtained 31 new Ca hinge sequences from Old Worldand New World monkeys and evaluated their dN and dSvalues by using a tree-based comparison method.
Materials and Methods
Specimens
Nucleotide sequences of the following species were
newly determined: patas monkey (Erythrocebus patas),
Tibetan macaque (Macaca thibetana), stump-tailed macaque (Macaca arctoides), rhesus macaque (Macaca
mulatta), lion-tailed macaque (Macaca silenus), pigtailed macaque (Macaca nemestrina), Taiwan macaque(Macaca cyclopis), Assamese macaque (Macaca assamensis), crab-eating macaque (Macaca fascicularis), anubis baboon (Papio anubis), hamadryas baboon (Papio
hamadryas), green monkey (Cercopithecus aethiops),
black-and-white colobus (Colobus polykomos), bandedleaf monkey (Presbytis melalophos), silvered leaf monkey (Presbytis cristata), obscurus leaf monkey (Presbytis obscurus), Javan leaf monkey (Presbytis comata),
white-thighed leaf monkey (Presbytis femoralis), nightmonkey (Aotus trivirgatus), cotton-top tamarin (Saginus
oedipus), white-lipped tamarin (Saginus labiatus), Geoffroy’s spider monkey (Ateles geoffroyi), and long-haired
spider monkey (Ateles belzebuth). Their total genomic
DNAs were prepared from peripheral blood by standard
procedure.
PCR Amplification and Fragment Analysis
The following PCR primer pair was used to amplify an approximately 400-bp DNA fragment involving
the intron, hinge, and CH2 regions: 59-CCA AGC TTC
TAC ACG AAT CCC AGC CAG GAT GTG-39 and 59CCG AAT TCT CCC ACT TGA GGG CGT CCA GGT
GAA-39. The PCR program was 35–40 cycles of 958C
for 1 min, 558C for 1 min, and 728C for 2 min. A PCRSSCP analysis was performed to check whether the PCR
products contain single or multiple sequences, where
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Sumiyama et al.

PCR products were run on nondenaturing gel containing
5% acrylamide, 5% glycerol, and 0.53 TBE buffer at
168C constant temperature, as described before (Sumiyama et al. 1998). In the PCR-SSCP analysis, the following internal primers were used to obtain better resolution in gel: 59-TCT GAC CAG TTC AGG CCA TCT
C-39 and 59-GGC CGG CGC AGC GAC AGT-39.
Sequence Determination
Nucleotide sequences of both strands were determined using the ABI Taq DyeDeoxy Terminator Cycle
sequencing kit, according to the manufacturer’s instructions, using an ABI PRISM 377 DNA sequencer. In
some heterozygous cases, their nucleotide sequenceswere determined by using allele-specific sequencing
primers.
Sequence Analyses
Hominoid sequences were taken from Kawamura
et al. (1991). The Neighbor-joining (NJ) method (Saitou
and Nei 1987) was used for tree reconstruction of thehinge-flanking region by using CLUSTAL W (Thompson, Higgins, and Gibson 1994), with ‘‘exclude gap’’
option. Maximum likelihood analysis was done by using
the NucML program of the MOLPHY package (Adachi
and Hasegawa 1996) with the HKY85 model in orderto evaluate the reliability of tree topology. We used the
PAUP 3.1.1. program (Illinois Natural History Survey)
for the construction of the maximum parsimony (MP)tree for the hinge region. For the purpose of estimating
the ancestral sequences on the tree internal nodes, weused the baseml program of the PAML package (Yang
1999) with the HKY85 model. Values of dN and dS were
calculated for each phylogenetic tree branch between thegiven sequences using the computer program package
ODEN (Ina 1994) that employs Nei and Gojobori’s(1986) method. Window analysis was conducted using
the wina program of Endo, Ikeo, and Gojobori (1996).
Statistical tests for positive selection were conducted byusing Fisher’s exact probability test, where we set thenull hypothesis that nonsynonymous substitutions persite and synonymous substitutions per site are equal(strictly neutral condition). Therefore, the ratio of unchanged nonsynonymous sites to unchanged synonymous sites was expected to be equal to that of the nonsynonymous changes to synonymous changes (e.g.,
Zhang, Kumar, and Nei 1997).
Results and Discussion
Window Analysis of Hominoid Genomic Sequences
We performed a window-based analysis on the
published Ca sequences to study the nonsynonymousand synonymous substitution pattern for the entire coding region of the immunoglobulin Ca genes. Kawamura
et al. (1991) previously determined the whole genomic
sequences of the chimpanzee, gorilla, and orangutan Ca
genes. The human genomic sequence (Takahashi et al.
1982; accession number is J00220) was also available.
We first obtained pairwise distances of both synony-

FIG. 1.—Window analysis of the hominoid immunoglobulin Ca1
genes. Vertical and horizontal axes indicate the total distance (DT) value and the window position, respectively. DS (dotted line) and DN(thick line) designate DT of synonymous and nonsynonymous substitutions, respectively. Sliding window is shifted by three bases (onecodon). Window size is 20 codons. Note that the nucleotide positionindicates the start point of each window.

mous (dS) and nonsynonymous (dN) substitutions in a
default 20-codon window of the wina program. The
hinge region of hominoids consists of 18 codons suchthat this default window size seems to be appropriate.
We calculated the total distance (DT) of the phylogenetic tree among species for dS and dN, according
to the established tree topology—(((human, chimpanzee), gorilla), orangutan). Ancestral nucleotide sequences of all interior nodes were reconstructed by using the
PAML program, and then the number of nucleotide substitutions on each branch was calculated. DT is definedas the sum of all branches. DT of each window wasplotted against the first nucleotide position of each window along the entire coding sequence (fig. 1). The DTof the nonsynonymous substitutions clearly exceeds thatof the synonymous substitutions at the hinge region.
This observation supports the idea that the hominoid
hinge region has been evolving under some kind of positive selection. Besides the hinge region, both synonymous and nonsynonymous rates were raised at the endof the CH2 domain. We do not know the reason for thisobservation, however, it is possible that recombinations
or gene conversions occurred and raised overall variation in this region.
Genomic Sequences of the IgA Gene of Nonhominoid
Primates
In our previous report, we showed by PCR-SSCP
DNA fragment analysis that some nonhominoid primates have very high polymorphism and high heterozygosity (Sumiyama et al. 1998). Here, we determined
the hinge and flanking sequences of various nonhominoid primates to perform a more detailed sequence analysis. Newly determined immunoglobulin Ca gene sequences of nonhominoid primates are supplied as supplementary material, and the predicted hinge amino acidsequences are shown in table 1. Sequence data are available in DDBJ/EMBL/GenBank International Nucleotide
Sequence Database (accession numbers AB013752–
AB013790).

Positive Selection in the IgA Hinge

1095

Table 1
Amino Acid Sequences of the Immunoglobulin Ca Hinge Region in PrimatesSpecies

Amino Acid Sequence

Old World monkeys (Cercopithecinae)
Rhesus macaque-2, crab-eating macaque-1 . . . . . . . . . . . . . . . . . . . . .
Lion-tailed macaque, pit-tailed macaque-1, Taiwan
macaque-2,a Assamese macaque-2,a rhesus macaque-4a. . . . . . . . .
Rhesus macaque-3a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tibetan macaque,a stump-tailed macaquea . . . . . . . . . . . . . . . . . . . . . .
Assamese macaque-1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rheses macaque-1, crab-eating macaque-2a . . . . . . . . . . . . . . . . . . . .
Hamadryas baboon-1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Anubis baboon,a hamadryas baboon-2. . . . . . . . . . . . . . . . . . . . . . . . .
Patas monkey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Green monkey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hamadryas baboon-3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rhesus macaque-5, Taiwan macaque-1, pig-tailed macaque-2 . . . . .
Rhesus macaque-6a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Old World monkeys (Colobinae)
Black-and-white colobus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Silvered leaf monkey-1, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Silvered leaf monkey-2, obscurus leaf monkey, bandedleaf monkey-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Banded leaf monkey-2, white-thighed leaf monkey, Javanleaf monkey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

SETKPCLSQTKPCLPQPKPCL
PPPKPCLLPPNSCLPPPNSCLPKPNSCL
SQPKSCL
IPPKPCFASPKCPLPSSKCPSLKPPITPPCPS
IPPTPIPLPCPSIPPRCPPQPPCPPCCS
RPPCPPCCSHPPCYS

Hominoids
Human Ca1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chimpanzee Ca1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gorilla Ca1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Orangutan Ca. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Human Ca2, chimpanzee Ca2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gorilla Ca2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gibbon Ca2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PSTPPTPSPSTPPTPSPSPSTPCPPTPSTPPTPSPSPSTPPTPSPSTPPTPSPPPRPTPTPSTPPCPPPS
PPPPP
PPSPP
PPPHP

New World monkeys
Cotton-top tamarin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Night monkey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Geoffroy’s spider monkey. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Long-haired spider monkey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
White-lipped moustached tamarin . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PPQPLCPCHSPPECPPPCHP
PPLPPCPCHSPPLPPPPCHSPPLPSCPCHSa Only the hinge region is determined.

Our sequences begin at the end of exon 1, corresponding to the CH1 domain, and end at the middle ofexon 2, corresponding to the hinge region and the CH2
domain. There is a gap located just before the exon 1
and intron 1 boundary. This 3-bp gap does not cause a
nonsense mutation. Exon-intron boundary sequences arewell conserved throughout the sequences. We did notobserve any large deletion or insertion in the subsequent
intron region, although there are several single substitutions and minor deletions, such that the multiple alignment of all intron sequences was straightforward. Thisis also the case in exon 2, other than the hinge region,
and most substitutions in this region occurred at thethird positions leading to synonymous substitutions. We
did not find any large gap here. The boundary sequences
between intron 1 and exon 2 are also well conserved.
In contrast to the flanking region, the hinge regionis unusually hypervariable. Because of the complexity
associated with the hinge region (different length, many
substitutions, and partially reiterated structure; see table1), a multiple alignment of all sequences could not be
performed at once. Instead of making an overall multiple alignment in the hinge region, we show the align-

ment within the hinge to be the largest collection ofsequences for which homology can be clearly assessed
within Old World monkeys, New World monkeys, andhominoids (fig. 2). We use these unambiguously aligned
sequences for subsequent analysis of the hinge region.
Phylogenetic Analysis of the Nonhypervariable HingeFlanking Sequences
To elucidate the basic phylogenetic relationshipamong sequences used here, we constructed an NJ treeusing 343 bp hinge-flanking sequences with 1,000 bootstrap replications (fig. 3). It should be noted that thesenonhinge region sequences were unambiguouslyaligned. In this NJ tree, Old World monkeys, hominoids,
and New World monkeys are separated clearly. Interestingly, internal clusters in Old World monkeys do notnecessarily correspond to that of the species relationship. For example, three rhesus macaque sequences (rhesus mac-1, -2, and -5) are located in different clusters.
The same is true for two pig-tailed macaque sequences.
These patterns suggest that an ancestral polymorphism
may have been maintained in the extant Old World mon-

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Sumiyama et al.

FIG. 3.—A phylogenetic tree reconstructed for the Ca hingeflanking sequences by using the NJ method. The bootstrap values outof 1,000 replications are indicated where their values are larger than500. Macaque and baboon are abbreviated as mac and bab,
respectively.

FIG. 2.—The nucleotide sequences and their predicted translationsof the immunoglobulin Ca genes in the hinge region of the largest
collection of unambiguously aligned sequences from (a) Old World
monkeys, (b) hominoids, and (c) New World monkeys. The full-length
sequence alignment from the end of the exon 1 to the middle of theexon 2 region will be shown at the MBE website as supplementarydata. To save space, names for each sequence are shortened, such as
crab (crab-eating macaque), lion (lion-tailed macaque), rhesus (rhesus
macaque), tibetan (Tibetan macaque), assam (Assamese macaque), hamad (hamadryas baboon), patas (patas monkey), green (green monkey), gspider (Geoffroy’s spider monkey), lspider (long-haired spider
monkey), night (night monkey), wtamarin (white-lipped moustachedtamarin), ctamarin (cotton-top tamarin), hsa1 (human Ca1), ptr1
(chimpanzee Ca1), ggo1 (gorilla Ca1), and ppo (orangutan Ca).

keys (trans-species polymorphism). This is indicative of
overdominant selection (Klein et al. 1993).
Because the bootstrap probabilities for the internalbranches were not high in figure 3, it is difficult to conclude this with certainty. Hence, we compared this unusual topology (original tree: ORTR) with the species
tree (SPTR) using the NucML program of the MOLPHY
package. For simplification purpose, we compared only
four sequences: rhesus macaque-1, rhesus macaque-5,
patas monkey, and human as an apparent outgroup.
ORTR (log likelihood 5 2687.6, and RELL-BP 50.93) was clearly favored to SPTR (log likelihood 52698.1, with standard error 7.2, and RELL-BP 5
0.053). We also compared pig-tailed macaque-1, pigtailed macaque-2, African green monkey, and humanand obtained similar results. ORTR (log likelihood 5
2686.8, and RELL-BP 5 0.93) was favored to SPTR
(log likelihood 5 2696.0, with standard error 6.3, andRELL-BP 5 0.033).

We previously reported a very high extent of heterozygosity in the hinge region in macaques (Sumiyama
et al. 1998), which is another indication of overdominantselection. Considering these results together, existenceof overdominant selection is quite probable in the Cagene hinge region for Old World monkeys.
When hominoids are considered, special attention
is necessary because they have two Ca loci (Kawamura,
Saitou, and Ueda 1992). Ca1 and Ca2 are positioned
close to each other in the phylogenetic tree in every
hominoid species. This is the result of increased similarity caused by a gene conversion event between thetwo duplicated Ca loci (Kawamura, Saitou, and Ueda
1992). In other primates, there is only one locus for the
Ca gene; thus, they are free from this gene conversion
problem.
Phylogeny and Substitution Pattern of theHypervariable Hinge Sequences
Because the hinge region itself is small and hypervariable, it was necessary to use methods different fromthose used for the hinge-flanking regions. First, we constructed MP trees for those nucleotide sequences corresponding to 10 Old World monkey hinge regions
shown in figure 2a. Ten equally parsimonious trees wereobtained (fig. 4a). Nine of the ten MP trees contained
only nonsynonymous substitutions. Only one MP tree
(MPTREEp5) contained one synonymous substitution,
as shown with a diamond in figure 4a.
Second, we constructed a phylogenetic networkfrom the same data set (fig. 4b). The phylogenetic network is a summarization of all possible phylogenies andis more informative than the simple consensus tree because it can show the existence of parallel substitutions
or recombinations (or both) with parallelogram. In thephylogenetic network, only one possible synonymous
substitution is observed in a triangle. Parallelograms
shown in this network were probably caused by parallel
substitutions.

Positive Selection in the IgA Hinge

1097

Table 2
Numbers of Nonsynonymous Substitutions andSynonymous Substitutions in all Maximum Parsimony
Trees of Each Group, Their SdN/SdS Values, and Fisher’s
Exact Probabilities
Nonsy- Synnony- onymous mous

FIG. 4.—Phylogenetic relationship and nucleotide substitution patterns among immunoglobulin Ca genes-alleles of Old World monkeys.
a, All MP trees (10) of Old World monkeys (fig. 2a) calculated by PAUP
3.1.1. Operational taxonomic units (OTUs) are as folows—1: rhesus macaque-2, crab-eating macaque-1; 2: lion-tailed macaque, pig-tailed macaque-1, Taiwan macaque-2, Assamese macaque-2, rhesus macaque-4;
3: rhesus macaque-3; 4: Tibetan macaque, stump-tailed macaque; 5: patas monkey; 6: anubis baboon, hamadryas baboon-2; 7: hamadryas baboon-1; 8: rhesus macaque-1, crab-eating macaque-2; 9: Assamese macaque-1; 10: green monkey. A synonymous substitution is indicated bya diamond. b, Phylogenetic network for Old World monkeys. Parallelograms designate existence of incompatible sites. A curve in the network
indicates an incompatible site, which cannot be drawn in three dimensions. All 10 MP trees in figure 4a are included in this network. A
synonymous substitution is designated by a diamond.

To further examine our observations, we computed
Fisher’s exact tests (table 2). Note that the commonlyused t-test is not suitable for very low expected valuesof substitutions. The results are listed in table 2. Notethat the nonsynonymous and synonymous substitution
reconstruction may be biased by the codon usage and
the GC%, it is observed that GC3 5 0.795 and ENc(effective number of codon) 5 39.75 for the entire crabeating macaque Ca-coding sequence (X53705). Nevertheless, the null hypothesis of equal expectation of nonsynonymous and synonymous substitutions was rejected
for all MP trees. We also conducted the same analysis
on hominoids and New World monkeys. They also showhigh nonsynonymous to synonymous ratios, but thesewere not statistically significant (see table 2).
To test whether a high ratio of nonsynonymous
substitution to synonymous substitution is caused by anincreased nonsynonymous substitution rate or the suppression of the synonymous rate, substitutions of theflanking intron were counted and compared with thehinge nonsynoymous substitutions. We reconstructed thetree and ancestral sequence on each node using the
PAML program and then calculated the total substitution
number for both intron and hinge regions, using Assamese mac-1 and -2, crab-eating mac-1 and -2, hamadryas
bab-1 and -2, patas monkey, and African green monkey
sequences. The results reveal 23 nonsynonymous changes and 127 invariant sites in total in the hinge region.
In the intron region, there are 18 site changes and a totalof 2,122 invariant sites. This is highly significant (P ,0.00000001) by Fisher’s exact test. Synonymous chang-

Old World monkeys
MPTREE 1–4, 6–10 . . . Change
No change
MPTREE 5. . . . . . . . . . . Change
No change
Hominoids . . . . . . . . . . . Change
No change
New World monkeys . . . Change
No change

19
157
18
158
10
130
10
76

0
55
1
54
1
69
1
33

P

SdN/
SdS

0.0045

—

0.034

6.3

0.070

5.4

0.125

4.2

NOTE.—Substitution numbers are based on parsimony estimates so that it
could have some error.

es in the hinge region are 1 and 59 for the invariantsites. Fisher’s exact test cannot rule out the null hypothesis of the equal substitution rate (P 5 0.409). If weassume that the intron shows a neutral rate of evolution,these data suggest that only the nonsynonymous substitution rate has increased in the hinge region. Together
with the high ratio of nonsynonymous to synonymous
substitutions in the hinge region, we conclude that positive selection operates in the hinge region.
Other Supports for Positive Selection: IgA Sequence
Variation in Other Mammals and Vast IgA Protease
Variation in Pathogenic Bacteria
We found that the IgA hinge region showed evidence of adaptive evolution in primates. Furthermore,some other mammalian species also show hypervariability in their IgA hinge regions, although nonsynonymous-synonymous substitution ratio tests have not been
conducted because of alignment difficulties (Osborne etal. 1988; Burnett et al. 1989; Brown and Butler 1994).
Our results suggest that adaptive evolution of the IgA
hinge is likely to have occurred in many mammalian
species. IgA is the most prevalent immunoglobulin in
secretions, and it plays a crucial role for the preventionof pathogenic bacterial penetration of the mucosal surface. Because IgA is an indispensable factor for mammalian species, the bacterial IgA protease could be astrong selective pressure on the IgA gene.
There is a possibility of coevolution between host
IgA and proteases of the pathogenic bacteria. IgA proteases of pathogenic bacteria consist of several different
classes. They are found in a wide variety of pathogenic
bacteria, including Haemophilus, Neisseria, and Streptococcus (Plaut et al. 1975; Kilian, Mestecky, andSchrohenloher 1979; Male 1979). These proteases arehighly specific enzymes that cleave certain peptide
bonds of the IgA hinge region. Interestingly, their catalytic mechanisms and gene structures are completely
different among bacteria (Mortensen and Kilian 1984;
Bachovchin et al. 1990; Gilbert, Plaut, and Wright 1991;

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Sumiyama et al.

Lomholt, Poulsen, and Kilian 1995), such as thiol-activated protease from Bacteroides melaninogenicus, serine-type proteases from Haemophilus influenzae andNeisseria gonorrhoeae, and metalloprotease from Streptococcus sanguis. Such wide variations among IgA proteases from different origins with the same substratespecificity strongly suggest functional convergence in
IgA protease evolution. This convergent evolution of the
IgA proteases of pathogenic bacteria indicates the importance of inactivation of the host IgA to retain theirpathogenicity. This fact indicates strong host-parasite interaction between host IgA and parasite protease, which
results in positive selection pressure on both sides.
Conclusions
The dN/dS value is a powerful indicator for positive
selection with which several genes evolving under positive selection have so far been identified (Hughes and Nei
1988, 1989; Tanaka and Nei 1989; Messier and Stewart1997). Here, we used a tree-based multiple sequencecomparative method in order to test the smaller region.
We showed that nonsynonymous substitutions in thehinge were significantly larger than synonymous substitutions in the hinge and flanking intron substitutions. Thisstrongly suggests the existence of positive selection in the
IgA hinge region of primates. This selection could be a
result of the adaptation against pathogenic parasites
where an increasing variety in the IgA hinge sequence isrequisite for the secretory immune system.
Supplementary Materials
The full version of the alignment data will beshown at the MBE website (www.molbiolevol.org).
Acknowledgments
This study was supported by Grants-in-Aids for
Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan, NIG Cooperative Research Program, and the Cooperation Research Programof Primate Research Institute, Kyoto University. We
thank Drs. T. Ishida and O. Takenaka for providing samples of crab-eating macaque and leaf monkeys,
respectively.
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RODNEY HONEYCUTT, reviewing editor
Accepted February 15, 2002