Institute of Molecular Evolutionary Genetics and Department of Biology, Pennsylvania State University
In vertebrates, there are many secreted regulatory proteins that participate in host defense. Interleukin-2 (IL-2) is one of these proteins, and it is secreted primarily by activated T lymphocytes. Interaction between IL-2 and its receptor on the T cell membrane triggers several signal transduction pathways, resulting in clonal expansion of T cells. While this proliferation-promoting activity is believed to be its main function, IL-2 can stimulate the functional differentiation of T cells as well. IL-2 is also known to be a proliferation and differentiation factor for a variety of cell types, such as B cells, natural killer cells, and myeloid cells (reviewed in Goldsmith and Greene 1994
; Gaulton and Williamson 1994
). Due to its ability to upregulate the immune system, IL-2 has been widely used in immunotherapy for a number of diseases, including cancers and AIDS (e.g., Macey and Johnston 1990
; Zou et al. 1999
). IL-2 has also been of interest to evolutionists for testing the molecular-clock hypothesis (Gillespie 1989
; Ohta 1995
). Here, we analyze the sequences available in GenBank and report detection of positive Darwinian selection in ancestral IL-2 genes of artiodactyls and discuss its implications.
The IL-2 gene sequences of 15 mammalian species were obtained from GenBank (see fig. 1A
for species names) and were aligned using CLUSTAL V (Higgins, Bleasby, and Fuchs 1992
) with some visual adjustments. The number of codons in the alignment was 152 after the gaps were removed. (GenBank accession numbers and the alignment are available on request). We assumed that the phylogenetic tree of the species used was as that shown in figure 1A,
which is generally accepted by molecular evolutionists (Hayasaka, Fujii, and Horai 1996
and references therein; de Jong 1998
) and is also supported by the IL-2 gene.
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In the bNbS test, we examined positive selection for the branches in which high bN/bS ratios were observed. Strictly speaking, this statistical test is inappropriate because it is equivalent to conducting multiple tests. In such a situation, the test is likely to be too liberal. Nevertheless, this will not be a serious problem if additional evidence of positive selection is found, as is shown below.
Comparing the ancestral sequences at the nodes , ß,
, and
(fig. 1A
) and the extant sequence from the deer, we noticed that there were many amino acid substitutions that involved charge changes (fig. 1B
). Let us call the nonsynonymous nucleotide substitutions resulting in charge changes "radical substitutions" and the rest of the nonsynonymous substitutions "conservative substitutions" (Hughes, Ota, and Nei 1990
). The total numbers of conservative (c) and radical (r) nonsynonymous substitutions for branches x, y, z, and w are 42.3 and 36.0, respectively. The potential numbers of conservative (C) and radical (R) sites of the sequences are computed to be 209 and 120, respectively. These numbers were computed by the method of Zhang (2000), which is an extension of the method of Hughes, Ota, and Nei (1990)
and takes into account the transition/transversion bias. When there is no difference in selection intensity on conservative and radical substitutions, r/c is expected to be equal to R/C. In most genes, r/c < R/C, apparently because of a strong purifying selection against radical substitutions in comparison with conservative substitutions (Zhang 2000
). In the present case, however, r/c (0.85) is higher than R/C (0.57), and their difference is statistically significant (P < 0.05; Fisher's exact test). This result, together with the observation of a significantly higher rate of nonsynonymous than synonymous substitution, strongly suggests that positive selection has promoted amino acid substitutions that result in charge changes in the evolution of IL-2 in branches x, y, z, and w. In no other branches of the tree was r/c significantly greater than R/C. In fact, the overall r/c ratio (0.58) for all of the branches excluding x, y, z, and w was almost identical to R/C (0.57), suggesting that for these evolutionary lineages, the selective pressure was nearly the same for conservative and radical amino acid substitutions.
It is worth pointing out that although the rate of charge change substitution was accelerated by positive selection in the four branches examined, the net charge of the IL-2 protein did not change much. This occurred apparently because there were substitutions involving both positively charged and negatively charged amino acids. For example, in branch z, there were 16 amino acid substitutions involving charge changes, and 9 of them increased the net charge of the protein, whereas the remaining 7 decreased it. Interestingly, at some sites, amino acid charges changed back and forth. For instance, at site 30, a substitution from the noncharged glutamine to the positively charged lysine occurred in branch y, but a subsequent substitution in branch z changed it back to a noncharged amino acid (methionine). Similar patterns of charge changes were observed at sites 45, 46, 76, and 98. These observations suggest that positive selection in branches x, y, z, and w did not increase or decrease the net charge of IL-2, but, rather, changed the amino acid charges at individual sites. This pattern is similar to that of the antigen-binding cleft of major histocompatibility complex molecules, in which positive selection promotes charge profile diversity (Hughes, Ota, and Nei 1990
). It is, however, in striking contrast to that of primate eosinophil-cationic protein (ECP), in which the net charge dramatically increased under directional selection during a short period of evolutionary time after a gene duplication event and a novel function subsequently evolved (Zhang, Rosenberg, and Nei 1998
).
A number of host defense genes have been shown to evolve rapidly by positive selection in response to ever-changing pathogens (e.g., Tanaka and Nei 1989
; Hughes, Ota, and Nei 1990
). These genes are all known to interact directly with foreign antigens. In the present case, however, there is no evidence that IL-2 or its receptor directly contacts pathogens. Nevertheless, there is a possibility that they do contact pathogens, as a number of T cell membrane receptors that function in physiological processes are used by pathogens to enter host cells (Cairns and D'Souza 1998
). For instance, the chemokine receptor CCR5, which normally binds to macrophage inflammatory proteins 1
, 1ß, and RANTES to mediate chemotaxis, is used by the human immunodeficiency virus (HIV) as a coreceptor to enter human T cells (reviewed in Cairns and D'Souza 1998
). To examine whether pathogens have been driving the rapid evolution of IL-2, an evolutionary analysis of the IL-2 receptor will be necessary. Interestingly, our preliminary sequence analysis of the alpha chain of the receptor did indicate an elevated rate of evolution in an ancestral lineage of artiodactyls (data not shown) similar to the lineage where IL-2 was found to evolve rapidly. However, because the number of sequences is limited, that result remains inconclusive. Thus, the selective agent on IL-2 is unclear, but the intriguing possibility of direct contact of IL-2 or its receptor with pathogens is worth pursuing in the future, particularly in the context of frequent use of IL-2 clinically.
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1 Present address: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.
1 Keywords: interleukin-2
positive selection
artiodactyls
substitution pattern
2 Address for correspondence and reprints: Jianzhi Zhang, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11N104, 9000 Rockville Pike, Bethesda, Maryland 20892. E-mail: jzhang{at}niaid.nih.gov
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