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Individual protochordates have unique immune-type receptor repertoires
Magazine R465
Correspondence
Individual protochordates have unique immune-type receptor repertoires John P. Cannon1,2, Robert N. Haire3, Natasha Schnitker3, M. Gail Mueller1 and Gary W. Litman1–3* Innate immunity is mediated by a variety of different, nonrearranging receptors and soluble molecules that recognize and facilitate the elimination of a wide range of pathogens [1]. Immunoglobulin (Ig)-type variable (V) region-containing chitinbinding proteins (VCBPs) found in the protochordate amphioxus, which diverged from the vertebrate lineage before the emergence of adaptive immunity, show structural characteristics of innate immune receptors [2]. Here we describe a very high degree of regionalized hypervariability in one family of VCBPs at the level of the individual germline, producing a unique repertoire of proteins in every animal so far analyzed. In species with large populations, such as amphioxus, extensive polymorphisms may compensate for the absence of somatic modification in the maintenance of immune receptor diversity. The diversified VCBPs in the amphioxus Branchiostoma floridae, a cephalochordate, are soluble proteins consisting of two Ig-type V regions joined to a carboxy-terminal chitin-binding domain [2]. Although their ligands are unknown, VCBPs are likely candidates for innate immune receptors and represent the only example of an innate receptor in which the functional unit is a hyperdiversified Ig-type V region. VCBPs are distributed in at least five families and are expressed specifically and abundantly in the gut. Regionalized peptide
sequence hypervariability was noted in the V regions of pooled amphioxus VCBP2 cDNAs [2]. The hypervariable region is centered ~18 residues amino-terminal to the first intradomain cysteine and does not correspond to any of the known complementaritydetermining regions of the rearranging antigen-binding receptors found in jawed vertebrates. However, the basis for the hypervariation is not clear. The hypervariable region of the amino-terminal V region of VCBP2 has been characterized in genomic DNA from individual animals collected in the same local geographical area. The germline of every animal encodes a unique VCBP2 receptor repertoire. In a parallel investigation, a 17-fold representative bacterial artificial chromosome library (CHORI-302) constructed from a single reference animal was screened and six VCBP2 family genes were identified, consistent with previous Southern blot analyses [2]. In sum, a total of 43 different peptides are encoded across the amplified region of VCBP2 by only 13 different animals. The most pronounced differences occur across a segment of ~12–15 residues within a 23–30 residueencoding amplicon (Figure 1). In the majority of cases, any two animals share no more than two specific VCBP2 hypervariable sequences; however, four pairs of animals share three specific hypervariable sequences. Amplicons encoding more conserved regions of the aminoterminal V region of VCBP2 and defensin, an unrelated innate immunity gene, lack extensive polymorphic differences. Nonsystematic studies carried out with additional animals revealed further VCBP2 diversity, implying a still larger gene pool. The localized differences in both length and peptide sequence in the hypervariable region of soluble VCBP2 molecules resemble the junctional variation seen in antigen-binding receptors after combinatorial rearrangement; however, comparisons of cDNA sequences to germline genes amplified from 2 of the 13
specimens indicate that the hyperdiversity in VCBP2 is not derived somatically. High levels of polymorphism have been described previously for other Ig gene superfamily transmembrane molecules. However, in the killer cell Ig-like receptors (KIRs), variation tends to occur throughout the molecule, and in the major histocompatibility complex (MHC) class I and II molecules, variation in the peptide-binding domains is pronounced but is not generally associated with length polymorphism [3,4]. Innate immunity provides an immediate response to pathogenic invasion, a significant and shifting challenge within the marine microbial environment [5] and a likely factor in the survival of the filter-feeding amphioxus. But recognition of molecular patterns on pathogens can be compromised by immune evasion. The ability of amphioxus, which comprises 70% of the total biomass in some ecosystems [6], to maintain a very high degree of receptor diversity by genetic hypervariation within its large population could provide an effective alternative to somatic diversity, a later acquisition of the vertebrate lineage. Increasing evidence points to innate immune receptor diversification as a means of maintaining resistance to pathogens. Maintenance of extensive variation in nonadaptive immune molecules among individuals, well characterized in the leucine-rich repeat plant R genes [7] in which variation is regionalized, and in several Drosophila genes [8], can serve as an effective protective mechanism in the absence of adaptive immunity [9]. In this light, various strategies for diversifying innate receptors may have evolved independently in invertebrates, protochordates and jawless vertebrates before the acquisition of receptor gene rearrangement. Although the particular mechanism that gives rise to the observed genetic polymorphism in VCBP2 genes is unknown, hyperdiversification of the Ig superfamily and other
Current Biology Vol 14 No 12 R466
Figure 1. Regionalized hypervariability of VCBP2 genes from 12 individual amphioxus and a single-animal BAC genomic library (CHORI302). Consensus VCBP2 oligonucleotides were used in PCR employing either genomic DNA or first-strand cDNA as template. At least 30 amplicons from each animal were cloned, sequenced and aligned by ClustalW (with manual correction). Vertical columns of dots represent sets of amplicons recovered from each animal. Identical peptide sequences observed in different animals are displayed at the same horizontal position. Subfamilies (~70% amino acid identity) of VCBP2 sequences are indicated by dot color. At least 30 mRNA (cDNA) amplicons were recovered from animals 11 and 12 and are identical to corresponding genomic DNA. Variation at each amino acid position is indicated by successive changes in shading (no shading, blue, green, yellow, red, magenta, purple). (+) indicates presence of a corresponding peptide-encoding sequence in pooled cDNAs. In a dataset of this size, it is not unreasonable to assume that a modest number of observed changes can be accounted for by either PCR or cloning differences.
receptors in chordates, either at the germline or somatic level, could have provided an advantageous bridge between innate and adaptive immune function until mechanisms emerged in the vertebrates that could generate and select for diversity of antigen-binding receptors. Supplemental data Supplemental data are available at http://www.currentbiology.com/cgi/content/full/14/12 /R465/DC1/ Acknowledgments Thanks to R. Litman, K. Krumeich, B. Pryor, C. Amemiya, and T. Ota. Supported by NIH grant AI23338.
References 1. Medzhitov, R., and Janeway, C.A., Jr. (2002). Decoding the patterns of self and nonself by the innate immune system. Science 296, 298–300. 2. Cannon, J.P., Haire, R.N., and Litman, G.W. (2002). Identification of diversified immunoglobulin-like variable region-containing genes in a protochordate. Nat. Immunol. 3, 1200–1207. 3. Trowsdale, J., and Parham, P. (2004). Mini-review: defense strategies and immunity-related genes. Eur. J. Immunol. 34, 7–17. 4. Uhrberg, M., Valiante, N.M., Shum, B.P., Shilling, H.G., LienertWeidenbach, K., Corliss, B., Tyan, D., Lanier, L.L., and Parham, P. (1997). Human diversity in killer cell inhibitory receptor genes. Immunity 7, 753–763. 5. Fuhrman, J. (1999). Marine viruses and their biogeochemical and ecological effects. Nature 399, 541–548.
6. Bloom, S.A., Simon, J.L., and Hunter, V.D. (1972). Animalsediment relations and community analysis of a Florida estuary. Mar. Biol. 13, 43–56. 7. Bergelson, J., Kreitman, M., Stahl, E.A., and Tian, D. (2001). Evolutionary dynamics of plant Rgenes. Science 292, 2281–2285. 8. Lazzaro, B.P., Sceurman, B.K., and Clark, A.G. (2004). Genetic basis of natural variation in D. melanogaster antibacterial immunity. Science 303, 1873–1876. 9. Hamilton, W.D., Axelrod, R., and Tanese, R. (1990). Sexual reproduction as an adaptation to resist parasites (A review). Proc. Natl. Acad. Sci. USA 87, 3566–3573. 1All
Children’s Hospital, St. Petersburg, Florida 33701, USA. 2H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33620, USA. 3University of South Florida, Children’s Research Institute, St. Petersburg, Florida 33701, USA. *E-mail: [email protected]
Correspondence
Individual protochordates have unique immune-type receptor repertoires John P. Cannon1,2, Robert N. Haire3, Natasha Schnitker3, M. Gail Mueller1 and Gary W. Litman1–3* Innate immunity is mediated by a variety of different, nonrearranging receptors and soluble molecules that recognize and facilitate the elimination of a wide range of pathogens [1]. Immunoglobulin (Ig)-type variable (V) region-containing chitinbinding proteins (VCBPs) found in the protochordate amphioxus, which diverged from the vertebrate lineage before the emergence of adaptive immunity, show structural characteristics of innate immune receptors [2]. Here we describe a very high degree of regionalized hypervariability in one family of VCBPs at the level of the individual germline, producing a unique repertoire of proteins in every animal so far analyzed. In species with large populations, such as amphioxus, extensive polymorphisms may compensate for the absence of somatic modification in the maintenance of immune receptor diversity. The diversified VCBPs in the amphioxus Branchiostoma floridae, a cephalochordate, are soluble proteins consisting of two Ig-type V regions joined to a carboxy-terminal chitin-binding domain [2]. Although their ligands are unknown, VCBPs are likely candidates for innate immune receptors and represent the only example of an innate receptor in which the functional unit is a hyperdiversified Ig-type V region. VCBPs are distributed in at least five families and are expressed specifically and abundantly in the gut. Regionalized peptide
sequence hypervariability was noted in the V regions of pooled amphioxus VCBP2 cDNAs [2]. The hypervariable region is centered ~18 residues amino-terminal to the first intradomain cysteine and does not correspond to any of the known complementaritydetermining regions of the rearranging antigen-binding receptors found in jawed vertebrates. However, the basis for the hypervariation is not clear. The hypervariable region of the amino-terminal V region of VCBP2 has been characterized in genomic DNA from individual animals collected in the same local geographical area. The germline of every animal encodes a unique VCBP2 receptor repertoire. In a parallel investigation, a 17-fold representative bacterial artificial chromosome library (CHORI-302) constructed from a single reference animal was screened and six VCBP2 family genes were identified, consistent with previous Southern blot analyses [2]. In sum, a total of 43 different peptides are encoded across the amplified region of VCBP2 by only 13 different animals. The most pronounced differences occur across a segment of ~12–15 residues within a 23–30 residueencoding amplicon (Figure 1). In the majority of cases, any two animals share no more than two specific VCBP2 hypervariable sequences; however, four pairs of animals share three specific hypervariable sequences. Amplicons encoding more conserved regions of the aminoterminal V region of VCBP2 and defensin, an unrelated innate immunity gene, lack extensive polymorphic differences. Nonsystematic studies carried out with additional animals revealed further VCBP2 diversity, implying a still larger gene pool. The localized differences in both length and peptide sequence in the hypervariable region of soluble VCBP2 molecules resemble the junctional variation seen in antigen-binding receptors after combinatorial rearrangement; however, comparisons of cDNA sequences to germline genes amplified from 2 of the 13
specimens indicate that the hyperdiversity in VCBP2 is not derived somatically. High levels of polymorphism have been described previously for other Ig gene superfamily transmembrane molecules. However, in the killer cell Ig-like receptors (KIRs), variation tends to occur throughout the molecule, and in the major histocompatibility complex (MHC) class I and II molecules, variation in the peptide-binding domains is pronounced but is not generally associated with length polymorphism [3,4]. Innate immunity provides an immediate response to pathogenic invasion, a significant and shifting challenge within the marine microbial environment [5] and a likely factor in the survival of the filter-feeding amphioxus. But recognition of molecular patterns on pathogens can be compromised by immune evasion. The ability of amphioxus, which comprises 70% of the total biomass in some ecosystems [6], to maintain a very high degree of receptor diversity by genetic hypervariation within its large population could provide an effective alternative to somatic diversity, a later acquisition of the vertebrate lineage. Increasing evidence points to innate immune receptor diversification as a means of maintaining resistance to pathogens. Maintenance of extensive variation in nonadaptive immune molecules among individuals, well characterized in the leucine-rich repeat plant R genes [7] in which variation is regionalized, and in several Drosophila genes [8], can serve as an effective protective mechanism in the absence of adaptive immunity [9]. In this light, various strategies for diversifying innate receptors may have evolved independently in invertebrates, protochordates and jawless vertebrates before the acquisition of receptor gene rearrangement. Although the particular mechanism that gives rise to the observed genetic polymorphism in VCBP2 genes is unknown, hyperdiversification of the Ig superfamily and other
Current Biology Vol 14 No 12 R466
Figure 1. Regionalized hypervariability of VCBP2 genes from 12 individual amphioxus and a single-animal BAC genomic library (CHORI302). Consensus VCBP2 oligonucleotides were used in PCR employing either genomic DNA or first-strand cDNA as template. At least 30 amplicons from each animal were cloned, sequenced and aligned by ClustalW (with manual correction). Vertical columns of dots represent sets of amplicons recovered from each animal. Identical peptide sequences observed in different animals are displayed at the same horizontal position. Subfamilies (~70% amino acid identity) of VCBP2 sequences are indicated by dot color. At least 30 mRNA (cDNA) amplicons were recovered from animals 11 and 12 and are identical to corresponding genomic DNA. Variation at each amino acid position is indicated by successive changes in shading (no shading, blue, green, yellow, red, magenta, purple). (+) indicates presence of a corresponding peptide-encoding sequence in pooled cDNAs. In a dataset of this size, it is not unreasonable to assume that a modest number of observed changes can be accounted for by either PCR or cloning differences.
receptors in chordates, either at the germline or somatic level, could have provided an advantageous bridge between innate and adaptive immune function until mechanisms emerged in the vertebrates that could generate and select for diversity of antigen-binding receptors. Supplemental data Supplemental data are available at http://www.currentbiology.com/cgi/content/full/14/12 /R465/DC1/ Acknowledgments Thanks to R. Litman, K. Krumeich, B. Pryor, C. Amemiya, and T. Ota. Supported by NIH grant AI23338.
References 1. Medzhitov, R., and Janeway, C.A., Jr. (2002). Decoding the patterns of self and nonself by the innate immune system. Science 296, 298–300. 2. Cannon, J.P., Haire, R.N., and Litman, G.W. (2002). Identification of diversified immunoglobulin-like variable region-containing genes in a protochordate. Nat. Immunol. 3, 1200–1207. 3. Trowsdale, J., and Parham, P. (2004). Mini-review: defense strategies and immunity-related genes. Eur. J. Immunol. 34, 7–17. 4. Uhrberg, M., Valiante, N.M., Shum, B.P., Shilling, H.G., LienertWeidenbach, K., Corliss, B., Tyan, D., Lanier, L.L., and Parham, P. (1997). Human diversity in killer cell inhibitory receptor genes. Immunity 7, 753–763. 5. Fuhrman, J. (1999). Marine viruses and their biogeochemical and ecological effects. Nature 399, 541–548.
6. Bloom, S.A., Simon, J.L., and Hunter, V.D. (1972). Animalsediment relations and community analysis of a Florida estuary. Mar. Biol. 13, 43–56. 7. Bergelson, J., Kreitman, M., Stahl, E.A., and Tian, D. (2001). Evolutionary dynamics of plant Rgenes. Science 292, 2281–2285. 8. Lazzaro, B.P., Sceurman, B.K., and Clark, A.G. (2004). Genetic basis of natural variation in D. melanogaster antibacterial immunity. Science 303, 1873–1876. 9. Hamilton, W.D., Axelrod, R., and Tanese, R. (1990). Sexual reproduction as an adaptation to resist parasites (A review). Proc. Natl. Acad. Sci. USA 87, 3566–3573. 1All
Children’s Hospital, St. Petersburg, Florida 33701, USA. 2H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33620, USA. 3University of South Florida, Children’s Research Institute, St. Petersburg, Florida 33701, USA. *E-mail: [email protected]