Cloning, Characterization, and Phylogenetic Analysis of Siglec-9, a New Member of the CD33-related Group of Siglecs
2000; Elsevier BV; Volume: 275; Issue: 29 Linguagem: Inglês
10.1074/jbc.m002775200
ISSN1083-351X
Autores Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoThe Siglecs are a subfamily of I-type lectins (immunoglobulin superfamily proteins that bind sugars) that specifically recognize sialic acids. We report the cloning and characterization of human Siglec-9. The cDNA encodes a type 1 transmembrane protein with three extracellular immunoglobulin-like domains and a cytosolic tail containing two tyrosines, one within a typical immunoreceptor tyrosine-based inhibitory motif (ITIM). The N-terminal V-set Ig domain has most amino acid residues typical of Siglecs. Siglec-9 is expressed on granulocytes and monocytes. Expression of the full-length cDNA in COS cells induces sialic-acid dependent erythrocyte binding. A recombinant soluble form of the extracellular domain binds to α2–3 and α2–6-linked sialic acids. Typical of Siglecs, the carboxyl group and side chain of sialic acid are essential for recognition, and mutation of a critical arginine residue in domain 1 abrogates binding. The underlying glycan structure also affects binding, with Galβ1–4Glc[NAc] being preferred. Siglec-9 shows closest homology to Siglec-7 and both belong to a Siglec-3/CD33-related subset of Siglecs (with Siglecs-5, -6, and -8). The Siglec-9 gene is on chromosome 19q13.3–13.4, in a cluster with all Siglec-3/CD33-related Siglec genes, suggesting their origin by gene duplications. A homology search of the Drosophila melanogaster and Caenorhabditis elegans genomes suggests that Siglec expression may be limited to animals of deuterostome lineage, coincident with the appearance of the genes of the sialic acid biosynthetic pathway. The Siglecs are a subfamily of I-type lectins (immunoglobulin superfamily proteins that bind sugars) that specifically recognize sialic acids. We report the cloning and characterization of human Siglec-9. The cDNA encodes a type 1 transmembrane protein with three extracellular immunoglobulin-like domains and a cytosolic tail containing two tyrosines, one within a typical immunoreceptor tyrosine-based inhibitory motif (ITIM). The N-terminal V-set Ig domain has most amino acid residues typical of Siglecs. Siglec-9 is expressed on granulocytes and monocytes. Expression of the full-length cDNA in COS cells induces sialic-acid dependent erythrocyte binding. A recombinant soluble form of the extracellular domain binds to α2–3 and α2–6-linked sialic acids. Typical of Siglecs, the carboxyl group and side chain of sialic acid are essential for recognition, and mutation of a critical arginine residue in domain 1 abrogates binding. The underlying glycan structure also affects binding, with Galβ1–4Glc[NAc] being preferred. Siglec-9 shows closest homology to Siglec-7 and both belong to a Siglec-3/CD33-related subset of Siglecs (with Siglecs-5, -6, and -8). The Siglec-9 gene is on chromosome 19q13.3–13.4, in a cluster with all Siglec-3/CD33-related Siglec genes, suggesting their origin by gene duplications. A homology search of the Drosophila melanogaster and Caenorhabditis elegans genomes suggests that Siglec expression may be limited to animals of deuterostome lineage, coincident with the appearance of the genes of the sialic acid biosynthetic pathway. biotinylated polyacrylamide signaling lymphocyte activation molecule SLAM-associated protein neural cell adhesion molecule bacterial artificial chromosome expressed sequence tag polymerase chain reaction enzyme-linked immunosorbent assay Sialic acids are a family of α-keto acids with 9-carbon backbones that are expressed abundantly in animals of the deuterostome lineage (1.Schauer R. Sialic Acids: Chemistry, Metabolism and Function, Cell Biology Monographs. Volume 10. Springer-Verlag, New York1982Crossref Google Scholar, 2.Varki A. Glycobiology. 1992; 2: 25-40Crossref PubMed Scopus (479) Google Scholar, 3.Kelm S. Schauer R. Int. Rev. Cytol. 1997; 175: 137-240Crossref PubMed Google Scholar). They are found mostly at distal positions of oligosaccharide chains of glycoproteins and glycolipids and are thus exposed to the extracellular environment, allowing them to be recognized during the initial contact of cells with various pathogenic agents such as viruses, bacteria, protozoa, and toxins (4.Varki A. FASEB J. 1997; 11: 248-255Crossref PubMed Scopus (488) Google Scholar, 5.Karlsson K.A. Mol. Microbiol. 1998; 29: 1-11Crossref PubMed Scopus (147) Google Scholar). The marked structural complexity of sialic acids can thus be interpreted as a result of evolutionary arms race between the hosts and the pathogens (6.Gagneux P. Varki A. Glycobiology. 1999; 9: 747-755Crossref PubMed Scopus (421) Google Scholar). Recent studies have revealed another prominent role of sialic acids, namely their functions in generating ligands for endogenous lectins (4.Varki A. FASEB J. 1997; 11: 248-255Crossref PubMed Scopus (488) Google Scholar). Siglecs (sialic acid-binding Igsuperfamily lectins) are a family of such sialic acid-recognizing lectins that have been recently defined (7.Crocker P.R. Clark E.A. Filbin M. Gordon S. Jones Y. Kehrl J.H. Kelm S. Le Douarin N. Powell L. Roder J. Schnaar R.L. Sgroi D.C. Stamenkovic K. Schauer R. Schachner M. Van den Berg T.K. Van der Merwe P.A. Watt S.M. Varki A. Glycobiology. 1998; 8: vCrossref PubMed Google Scholar). These proteins are all single-pass type 1 transmembrane polypeptides, with an N-terminal Ig V-set domain, followed by variable numbers of Ig C2-set domains, a transmembrane domain, and a cytoplasmic tail. The first V-set Ig-like domain is the most important in carbohydrate recognition, and the second Ig-like domain may also contribute to the binding (8.Law C.L. Aruffo A. Chandran K.A. Doty R.T. Clark E.A. J. Immunol. 1995; 155: 3368-3376PubMed Google Scholar, 9.Nath D. Van der Merwe P.A. Kelm S. Bradfield P. Crocker P.R. J. Biol. Chem. 1995; 270: 26184-26191Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 10.Vinson M. Van der Merwe P.A. Kelm S. May A. Jones E.Y. Crocker P.R. J. Biol. Chem. 1996; 271: 9267-9272Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11.Van der Merwe P.A. Crocker P.R. Vinson M. Barclay A.N. Schauer R. Kelm S. J. Biol. Chem. 1996; 271: 9273-9280Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Eight members of the family have been described so far in humans, and each shows highly cell type-specific expression: Siglec-1/sialoadhesin (expressed on macrophages) (12.Crocker P.R. Mucklow S. Bouckson V. McWilliam A. Willis A.C. Gordon S. Milon G. Kelm S. Bradfield P. EMBO J. 1994; 13: 4490-4503Crossref PubMed Scopus (224) Google Scholar); Siglec-2/CD22 (on B lymphocytes) (13.Sgroi D. Varki A. Braesch-Andersen S. Stamenkovic I. J. Biol. Chem. 1993; 268: 7011-7018Abstract Full Text PDF PubMed Google Scholar); Siglec-3/CD33 (on myeloid precursors and monocytes) (14.Freeman S.D. Kelm S. Barber E.K. Crocker P.R. Blood. 1995; 85: 2005-2012Crossref PubMed Google Scholar); Siglec-4a/myelin-associated glycoprotein (on oligodendroglia and Schwann cells) (15.Kelm S. Pelz A. Schauer R. Filbin M.T. Tang S. De Bellard M.-E. Schnaar R.L. Mahoney J.A. Hartnell A. Bradfield P. Crocker P.R. Curr. Biol. 1994; 4: 965-972Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar); Siglec-5 (on neutrophils and monocytes) (16.Cornish A.L. Freeman S. Forbes G. Ni J. Zhang M. Cepeda M. Gentz R. Augustus M. Carter K.C. Crocker P.R. Blood. 1998; 92: 2123-2132Crossref PubMed Google Scholar); Siglec-6/OBBP-1 (on B lymphocytes and placental trophoblasts) (17.Patel N. Brinkman-Van der Linden E.C.M. Altmann S.W. Gish K. Balasubramanian S. Timans J.C. Peterson D. Bell M.P. Bazan J.F. Varki A. Kastelein R.A. J. Biol. Chem. 1999; 274: 22729-22738Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar); Siglec-7/AIRM1 (on natural killer cells and monocytes) (18.Falco M. Biassoni R. Bottino C. Vitale M. Sivori S. Augugliaro R. Moretta L. Moretta A. J. Exp. Med. 1999; 190: 793-801Crossref PubMed Scopus (196) Google Scholar, 19.Nicoll G. Ni J. Liu D. Klenerman P. Munday J. Dubock S. Mattei M.G. Crocker P.R. J. Biol. Chem. 1999; 274: 34089-34095Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 20.Angata T. Varki A. Glycobiology. 2000; 10: 431-438Crossref PubMed Scopus (99) Google Scholar); and Siglec-8 (on eosinophils) (21.Floyd H. Ni J. Cornish A.L. Zeng Z.Z. Liu D. Carter K.C. Steel J. Crocker P.R. J. Biol. Chem. 2000; 275: 861-866Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar).Many of the Siglecs have potential tyrosine phosphorylation sites in the context of an immunoreceptor tyrosine-based inhibitory motif in their cytoplasmic tails, suggesting their involvement in intracellular signaling pathways. In fact, Siglecs-3 and -7 have been shown to be capable of transmitting negative regulatory signals upon cross-linking by specific antibodies (18.Falco M. Biassoni R. Bottino C. Vitale M. Sivori S. Augugliaro R. Moretta L. Moretta A. J. Exp. Med. 1999; 190: 793-801Crossref PubMed Scopus (196) Google Scholar, 22.Taylor V.C. Buckley C.D. Douglas M. Cody A.J. Simmons D.L. Freeman S.D. J. Biol. Chem. 1999; 274: 11505-11512Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 23.Ulyanova T. Blasioli J. Woodford-Thomas T.A. Thomas M.L. Eur. J. Immunol. 1999; 29: 3440-3449Crossref PubMed Scopus (115) Google Scholar). In case of CD22/Siglec-2, a negative regulatory role was further proven by the studies using genetically engineered mice (24.O'Keefe T.L. Williams G.T. Davies S.L. Neuberger M.S. Science. 1996; 274: 798-801Crossref PubMed Scopus (468) Google Scholar, 25.Otipoby K.L. Andersson K.B. Draves K.E. Klaus S.J. Farr A.G. Kerner J.D. Perlmutter R.M. Law C.L. Clark E.A. Nature. 1996; 384: 634-637Crossref PubMed Scopus (359) Google Scholar, 26.Sato S. Miller A.S. Inaoki M. Bock C.B. Jansen P.J. Tang M.L.K. Tedder T.F. Immunity. 1996; 5: 551-562Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 27.Nitschke L. Carsetti R. Ocker B. Köhler G. Lamers M.C. Curr. Biol. 1997; 7: 133-143Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). On the other hand, the precise functional importance of sialic acid-binding property of Siglecs is not well understood, although the phenotypic similarity between Siglec-2 null mice and ST6Gal-I (Galβ1–4GlcNAc α2–6 sialyltransferase) null mice (28.Hennet T. Chui D. Paulson J.C. Marth J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4504-4509Crossref PubMed Scopus (306) Google Scholar) suggests that sialic acid binding does affect the signaling activity of this Siglec.Expression of sialic acids is well documented in animals of the deuterostome lineage (primarily in echinoderms and vertebrates), but their expression in another major group of animals, the protostomes (including nematodes, arthropods, and mollusks), is inconspicuous (1.Schauer R. Sialic Acids: Chemistry, Metabolism and Function, Cell Biology Monographs. Volume 10. Springer-Verlag, New York1982Crossref Google Scholar,2.Varki A. Glycobiology. 1992; 2: 25-40Crossref PubMed Scopus (479) Google Scholar). From evolutionary point of view, it is also an open question whether there are any Siglec homologs in the protostome lineage. However, given occasional reports of sialic acids in insects (29.Roth J. Kempf A. Reuter G. Schauer R. Gehring W.J. Science. 1992; 256: 673-675Crossref PubMed Scopus (144) Google Scholar, 30.Davis T.R. Wood H.A. In Vitro Cell. Dev. Biol. Anim. 1995; 31: 659-663Crossref PubMed Scopus (58) Google Scholar, 31.Schauer R. Krisch B. Lapina E.B. Shaw L. Gerardy-Schahn R. Malykh Y.N. Glycoconj. J. 1999; 16: S32Google Scholar), it is possible that the expression of sialic acids and sialic acid binding lectins is simply under more strict spatio-temporal regulation that has diminished possibilities for cDNA cloning of the relevant genes.Here we report the molecular cloning and characterization of a new member of Siglec family in humans, Siglec-9, and show that it is closely related to a Siglec-3/CD33-related subgroup that have arisen by gene duplications. We describe the expression pattern and glycan binding specificity of the molecule. The possible co-evolution of sialic acids and Siglecs is also explored and discussed, taking advantage of the recent near-completion of the genomic DNA sequencing of a fruit fly (Drosophila melanogaster) (32.Adams M.D. et al.Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4770) Google Scholar) and a nematode (Caenorhabditis elegans) (33.The C. elegans Sequencing ConsortiumScience. 1998; 282: 2012-2018Crossref PubMed Scopus (3570) Google Scholar).DISCUSSIONThe Siglec-9 cDNA encodes a protein with many typical features of previously described Siglecs. The first Ig-like domain contains two amino acid residues critical in sialic acid recognition (41.May A.P. Robinson R.C. Vinson M. Crocker P.R. Jones E.Y. Mol. Cell. 1998; 1: 719-728Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar): arginine in β-strand F (Arg120 in Siglec-9) and aromatic amino acid in β-strand G (Trp128). Crystallographic study of Siglec-1/sialoadhesin showed that these residues are directly interacting with the carboxyl group and glycerol-like side chain of sialic acid, respectively (41.May A.P. Robinson R.C. Vinson M. Crocker P.R. Jones E.Y. Mol. Cell. 1998; 1: 719-728Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). A notable exception in Siglec-9 was the lack of aromatic amino acid near its putative N terminus (typically the second residue), which in Siglec-1 is in contact with sialic acid 5-acetamido group. The fact that Siglec-9 shows robust binding to ligands, whereas mutation of this residue results in the complete loss of sialic acid recognition in Siglec-1/sialoadhesin (41.May A.P. Robinson R.C. Vinson M. Crocker P.R. Jones E.Y. Mol. Cell. 1998; 1: 719-728Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), clearly shows that the importance of this aromatic side chain in ligand binding is variable among Siglecs. On the other hand, almost complete loss of ligand binding in R120K mutant indicates that the stable salt bridge between the arginine and carboxyl group of sialic acid is indispensable in ligand binding of Siglec-9, as is the case with all other Siglecs examined so far (10.Vinson M. Van der Merwe P.A. Kelm S. May A. Jones E.Y. Crocker P.R. J. Biol. Chem. 1996; 271: 9267-9272Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11.Van der Merwe P.A. Crocker P.R. Vinson M. Barclay A.N. Schauer R. Kelm S. J. Biol. Chem. 1996; 271: 9273-9280Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 20.Angata T. Varki A. Glycobiology. 2000; 10: 431-438Crossref PubMed Scopus (99) Google Scholar, 22.Taylor V.C. Buckley C.D. Douglas M. Cody A.J. Simmons D.L. Freeman S.D. J. Biol. Chem. 1999; 274: 11505-11512Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 42.Tang S. Shen Y.J. DeBellard M.E. Mukhopadhyay G. Salzer J.L. Crocker P.R. Filbin M.T. J. Cell Biol. 1997; 138: 1355-1366Crossref PubMed Scopus (129) Google Scholar).The cytosolic tail of Siglec-9 contains two tyrosine residues. The amino acid sequence around the first tyrosine (LQY433ASL) is found in the context of an immunoreceptor tyrosine-based inhibitory motif ((S/I/L/V)XYXX(L/V)), which is the docking site for the phosphotyrosine phosphatases, SHP-1 and SHP-2 (55.Vely F. Vivier E. J. Immunol. 1997; 159: 2075-2077PubMed Google Scholar, 56.Newman P.J. J. Clin. Invest. 1999; 103: 5-9Crossref PubMed Scopus (230) Google Scholar). The actual functionality of this motif in Siglec-9 remains to be determined, but this motif is likely to be involved in the signal transduction, judging from the high sequence identity of the motif with those in Siglec-3/CD33 and Siglec-7/AIRM1, which have been shown to interact with SHP-1 (18.Falco M. Biassoni R. Bottino C. Vitale M. Sivori S. Augugliaro R. Moretta L. Moretta A. J. Exp. Med. 1999; 190: 793-801Crossref PubMed Scopus (196) Google Scholar, 22.Taylor V.C. Buckley C.D. Douglas M. Cody A.J. Simmons D.L. Freeman S.D. J. Biol. Chem. 1999; 274: 11505-11512Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 23.Ulyanova T. Blasioli J. Woodford-Thomas T.A. Thomas M.L. Eur. J. Immunol. 1999; 29: 3440-3449Crossref PubMed Scopus (115) Google Scholar). Interestingly, the sequence around the second tyrosine (TEY456SEI) does not strictly conform with but is similar to the proposed SAP-docking site (TIYXX(V/I)) on SLAM/CDw150 (43.Poy F. Yaffe M.B. Sayos J. Saxena K. Morra M. Sumegi J,. Cantley L.C. Terhorst C. Eck M.J. Mol. Cell. 1999; 4: 555-561Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). It is known that SAP interacts with this motif in SLAM and 2B4 (Ig superfamily molecules expressed on T/B lymphocytes and natural killer cells, respectively) to prevent docking of SHP-2 (57.Sayos J. Wu C. Morra M. Wang N. Zhang X. Allen D. van Schaik S. Notarangelo L. Geha R. Roncarolo M.G. Oettgen H. de Vries J.E. Aversa G. Terhorst C. Nature. 1998; 395: 462-469Crossref PubMed Scopus (799) Google Scholar, 58.Tangye S.G. Lazetic S. Woollatt E. Sutherland G.R. Lanier L.L. Phillips J.H. J. Immunol. 1999; 162: 6981-6985PubMed Google Scholar). A mutation in SAP causes human X-linked lymphoproliferative disease (57.Sayos J. Wu C. Morra M. Wang N. Zhang X. Allen D. van Schaik S. Notarangelo L. Geha R. Roncarolo M.G. Oettgen H. de Vries J.E. Aversa G. Terhorst C. Nature. 1998; 395: 462-469Crossref PubMed Scopus (799) Google Scholar). Whether this motif in Siglec-9 actually interacts with SAP and the similar protein EAT-2 (43.Poy F. Yaffe M.B. Sayos J. Saxena K. Morra M. Sumegi J,. Cantley L.C. Terhorst C. Eck M.J. Mol. Cell. 1999; 4: 555-561Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) remains to be determined.Despite the relatively low abundance of mRNA in peripheral blood leukocytes as analyzed by Northern blots, flow cytometry revealed that Siglec-9 is actually expressed on granulocytes and monocytes. In view of expression of Siglec-9 on monocytes, the mRNA expression found in the liver and spleen may be due to Kupfer cells and splenic tissue macrophages, respectively, although this remains to be proven by a direct histochemical approach. The wide distribution of Siglec-9 among cells that can be elicited in the innate immune response, along with its potential as negative regulator of signal transduction, raises the intriguing question whether Siglec-9 functions as general negative regulator of "rapidly responding" cells. Experiments to analyze this hypothetical role of Siglec-9, as well as that of another similarly distributed Siglec, Siglec-5 (on neutrophils and monocytes), is now underway in our laboratory.Siglec-9 recognizes both α2–3- and α2–6-linked sialic acids. Such promiscuous recognition of sialic acid linkages is seen primarily among Siglecs expressed on monocytes (Siglecs-3, -5, -7, and -9) and granulocytes (Siglecs-5, -8, and -9) (16.Cornish A.L. Freeman S. Forbes G. Ni J. Zhang M. Cepeda M. Gentz R. Augustus M. Carter K.C. Crocker P.R. Blood. 1998; 92: 2123-2132Crossref PubMed Google Scholar, 19.Nicoll G. Ni J. Liu D. Klenerman P. Munday J. Dubock S. Mattei M.G. Crocker P.R. J. Biol. Chem. 1999; 274: 34089-34095Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 21.Floyd H. Ni J. Cornish A.L. Zeng Z.Z. Liu D. Carter K.C. Steel J. Crocker P.R. J. Biol. Chem. 2000; 275: 861-866Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 34.Brinkman-Van der Linden E.C.M. Varki A. J. Biol. Chem. 2000; 275: 8625-8632Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Other Siglecs,i.e. Siglecs-1, -2, -4, and -6, show much more strict linkage specificity (12.Crocker P.R. Mucklow S. Bouckson V. McWilliam A. Willis A.C. Gordon S. Milon G. Kelm S. Bradfield P. EMBO J. 1994; 13: 4490-4503Crossref PubMed Scopus (224) Google Scholar, 13.Sgroi D. Varki A. Braesch-Andersen S. Stamenkovic I. J. Biol. Chem. 1993; 268: 7011-7018Abstract Full Text PDF PubMed Google Scholar, 15.Kelm S. Pelz A. Schauer R. Filbin M.T. Tang S. De Bellard M.-E. Schnaar R.L. Mahoney J.A. Hartnell A. Bradfield P. Crocker P.R. Curr. Biol. 1994; 4: 965-972Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 17.Patel N. Brinkman-Van der Linden E.C.M. Altmann S.W. Gish K. Balasubramanian S. Timans J.C. Peterson D. Bell M.P. Bazan J.F. 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Promiscuous as its binding is with regard to sialyl linkage, Siglec-9 still distinguishes between Neu5Acα2–3Galβ1–4GlcNAc (typically found in N-linked glycans) and Neu5Acα2–3Galβ1–3GalNAc (typically found in O-linked glycans and glycosphingolipids). The relevance of this promiscuity and selectivity with regard to Siglec functions is unclear, but it appears to support our hypothesis stated above: that engagement of the Siglecs by polyvalent ligands may be able to elicit negative intracellular signals, thus silencing the cells that are inappropriately activated. Alternatively, Siglecs may be functioning in a manner similar to that of killer cell Ig-like receptors expressed on natural killer cells, which send negative intracellular signals if engaged by major histocompatibility complex class I molecules expressed on target cells. Viral infection or malignant transformation result in down-regulation of major histocompatibility complex class I molecules on cell surface, thus rendering the cell vulnerable to attack by natural killer cells (61.Long E.O. Annu. Rev. Immunol. 1999; 17: 875-904Crossref PubMed Scopus (836) Google Scholar). Likewise, if viral infection or malignant transformation results in drastic change in surface sialic acid expression (as occurs with infection by neuraminidase-producing viruses like influenza virus), this may elicit activation of the Siglec-carrying cells in similar manner. Interestingly, the killer cell Ig-like inhibitory receptor genes are clustered on chromosome 19q13.4 (61.Long E.O. Annu. Rev. Immunol. 1999; 17: 875-904Crossref PubMed Scopus (836) Google Scholar), near the Siglec-3-related genes.Six of the nine known Siglecs (Siglec-3, -5, -6, -7, -8, and -9) are closely related to each other both in primary sequence (see "Results") and chromosomal localization (16.Cornish A.L. Freeman S. Forbes G. Ni J. Zhang M. Cepeda M. Gentz R. Augustus M. Carter K.C. Crocker P.R. Blood. 1998; 92: 2123-2132Crossref PubMed Google Scholar, 19.Nicoll G. Ni J. Liu D. Klenerman P. Munday J. Dubock S. Mattei M.G. Crocker P.R. J. Biol. Chem. 1999; 274: 34089-34095Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 20.Angata T. Varki A. Glycobiology. 2000; 10: 431-438Crossref PubMed Scopus (99) Google Scholar, 21.Floyd H. Ni J. Cornish A.L. Zeng Z.Z. Liu D. Carter K.C. Steel J. Crocker P.R. J. Biol. Chem. 2000; 275: 861-866Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 45.Peiper S.C. Ashmun R.A. Look A.T. Blood. 1988; 72: 314-321Crossref PubMed Google Scholar, 46.Takei Y. Sasaki S. Fujiwara T. Takahashi E. Muto T. Nakamura Y. Cytogenet. Cell Genet. 1997; 78: 295-300Crossref PubMed Scopus (38) Google Scholar) and are hence grouped as "Siglec-3/CD33-related." The genes for these are all localized on chromosome 19q13.3–13.4. The presence of this cluster suggests that these genes emerged by repeated gene duplication at some time during vertebrate evolution. We show here that the previously reported mouse Siglec-3/CD33 does not show a clear relationship to the established human counterpart (Fig.6 B). One explanation is that the true mouse ortholog has not yet been isolated. Another possibility is that some of the gene duplications in this cluster took place after separation of the ancestors of primates and rodents. Notably, there are other clustered gene families in close vicinity of the Siglec-3 subfamily, such as α1–2 fucosyltransferases (19q13.3) (62.Reguigne-Arnould I. Couillin P. Mollicone R. Faure S. Fletcher A. Kelly R.J. Lowe J.B. Oriol R. Cytogenet. Cell Genet. 1995; 71: 158-162Crossref PubMed Scopus (70) Google Scholar), the kallikrein gene cluster (19q13.3–13.4) (63.Yousef G.M. Luo L.Y. Diamandis E.P. Anticancer. Res. 1999; 19: 2843-2852PubMed Google Scholar), and the killer cell Ig-like receptor family (19q13.4) (see above), suggesting that there have been frequent gene duplications in this chromosomal region. In this regard, it is interesting that there are chromosome-specific minisatellites in 19q13.3-qter region (64.Das H.K. Jackson C.L. Miller D.A. Leff T. Breslow J.L. J. Biol. Chem. 1987; 262: 4787-4793Abstract Full Text PDF PubMed Google Scholar) that could have facilitated duplication of these genes by unequal crossing over of sister chromatids in meiotic recombination (65.Fitch D.H. Bailey W.J. Tagle D.A. Goodman M. Sieu L. Slightom J.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7396-7400Crossref PubMed Scopus (113) Google Scholar). Regardless of the similarity in sequences of the Siglec-3/CD33-related group, it is striking that each is expressed in different pattern of cell type specificity and shows distinct patterns of sialic acid recognition. Thus, the gene duplication events may have been selected for by specific new functions in different cell types. In this regard, final definition of the true mouse orthologs may require not only their cloning but also the antibody-based exploration of their expression patterns in different cell types.Although some Ig superfamily genes are highly conserved from nematode to humans (e.g. NCAM and related molecules), there seems to be no distinct Siglec homologs in C. elegans or D. melanogaster, two of the most extensively studied protostome lineage animals. This result is consistent with the fact that this lineage is also thought to lack constitutive expression of sialic acids. Although not conclusive, the results of our homology search on the newly available comprehensive genomic data also support the notion that protostome lineage animals generally do not synthesize sialic acids. We found that both Drosophila andCaenorhabditis genomes apparently lack the enzyme genes required for sialic acid biosynthesis, whereas other genes involved in sugar nucleotide biosynthesis are present and well conserved. Thus, the reported capability of insect cells to express sialic acids under certain circumstances (29.Roth J. Kempf A. Reuter G. Schauer R. Gehring W.J. Science. 1992; 256: 673-675Crossref PubMed Scopus (144) Google Scholar, 31.Schauer R. Krisch B. Lapina E.B. Shaw L. Gerardy-Schahn R. Malykh Y.N. Glycoconj. J. 1999; 16: S32Google Scholar, 66.Castellino F.J. Davidson D.J. Rosen E. McLinden J. Methods Enzymol. 1993; 223: 168-185Crossref PubMed Scopus (7) Google Scholar) should be addressed in other ways, such as the possibility of alternate pathways of biosynthesis and/or uptake of sialic acids or the biosynthetic precursors from the environment. Regardless, our data suggest that the emergence of Siglecs during evolution was dependent on the constitutive expression of sialic acids in deuterostome lineage animals. In this respect, it is particularly interesting to see whether there are any Siglec homologs in echinoderms such as sea urchins and starfishes, which are known to express large amounts of sialic acids. It should be mentioned that when we used full-length amino acid sequences of Siglecs in the homology search of Drosophila genome, we did find some gene products that showed significant homology and similar overall molecular structure, such as irregular optic chiasma C/roughest,neuromusculin, and faint sausage. Although the overall homology was significant between these gene products and human Siglec proteins (especially Siglec-2/CD22), it was limited to the Ig domains 2 and later. Because Siglecs are defined by sialic acid binding, molecules lacking homology in the unique first Ig domain were not considered as Siglec homologs. The same consideration applies tofurrowed, which is considered as fruit fly homolog of selectins (67.Leshko-Lindsay L. Corces V.G. Development. 1997; 124: 169-180Crossref PubMed Google Scholar) but shows low similarity to mammalian selectins in the C-type lectin domain. These phenomena suggest a split of fates between two genes of common ancestry under different biochemical environment characteristic of deuterostome and protostome lineages (presence or absence of sialic acids). The fact that Siglec-2/CD22 shows highest homology with fly proteins also implies that Siglec-2/CD22 may be the closest to the ancestor of mammalian Siglec family; it is unlikely that Siglec-2/CD22 was generated by gene duplication from another Siglec gene and later evolved to acquire similarity to aforementioned fly proteins under different environmental constraints. However, further analysis of the Siglec family in other animals will be needed to address this issue, as well as the issue of the relevant mouse orthologs of Siglec-9 and the other Siglec-3/CD33-related group.Note Added in ProofThe results reported here are in good agreement with another paper on Sigl
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