Title: SIGLEC12, a Human-specific Segregating (Pseudo)gene, Encodes a Signaling Molecule Expressed in Prostate Carcinomas
Abstract: The primate SIGLEC12 gene encodes one of the CD33-related Siglec family of signaling molecules in immune cells. We had previously reported that this gene harbors a human-specific missense mutation of the codon for an Arg residue required for sialic acid recognition. Here we show that this R122C mutation of the Siglec-XII protein is fixed in the human population, i.e. it occurred prior to the origin of modern humans. Additional mutations have since completely inactivated the SIGLEC12 gene in some but not all humans. The most common inactivating mutation with a global allele frequency of 58% is a single nucleotide frameshift that markedly shortens the open reading frame. Unlike other CD33-related Siglecs that are primarily found on immune cells, we found that Siglec-XII protein is expressed not only on some macrophages but also on various epithelial cell surfaces in humans and chimpanzees. We also found expression on certain human prostate epithelial carcinomas and carcinoma cell lines. This expression correlates with the presence of the nonframeshifted, intact SIGLEC12 allele. Although SIGLEC12 allele status did not predict prostate carcinoma incidence, restoration of expression in a prostate carcinoma cell line homozygous for the frameshift mutation induced altered regulation of several genes associated with carcinoma progression. These stably transfected Siglec-XII-expressing prostate cancer cells also showed enhanced growth in nude mice. Finally, monoclonal antibodies against the protein were internalized by Siglec-XII-expressing prostate carcinoma cells, allowing targeting of a toxin to such cells. Polymorphic expression of Siglec-XII in humans thus has implications for prostate cancer biology and therapeutics. The primate SIGLEC12 gene encodes one of the CD33-related Siglec family of signaling molecules in immune cells. We had previously reported that this gene harbors a human-specific missense mutation of the codon for an Arg residue required for sialic acid recognition. Here we show that this R122C mutation of the Siglec-XII protein is fixed in the human population, i.e. it occurred prior to the origin of modern humans. Additional mutations have since completely inactivated the SIGLEC12 gene in some but not all humans. The most common inactivating mutation with a global allele frequency of 58% is a single nucleotide frameshift that markedly shortens the open reading frame. Unlike other CD33-related Siglecs that are primarily found on immune cells, we found that Siglec-XII protein is expressed not only on some macrophages but also on various epithelial cell surfaces in humans and chimpanzees. We also found expression on certain human prostate epithelial carcinomas and carcinoma cell lines. This expression correlates with the presence of the nonframeshifted, intact SIGLEC12 allele. Although SIGLEC12 allele status did not predict prostate carcinoma incidence, restoration of expression in a prostate carcinoma cell line homozygous for the frameshift mutation induced altered regulation of several genes associated with carcinoma progression. These stably transfected Siglec-XII-expressing prostate cancer cells also showed enhanced growth in nude mice. Finally, monoclonal antibodies against the protein were internalized by Siglec-XII-expressing prostate carcinoma cells, allowing targeting of a toxin to such cells. Polymorphic expression of Siglec-XII in humans thus has implications for prostate cancer biology and therapeutics. IntroductionSialic acids (Sias) 5The abbreviations used are: SiaSialic acidPCaprostate cancerITIMimmunoreceptor tyrosine-based inhibitory motif. are nine-carbon backbone sugar molecules typically found at terminal positions of glycan chains in Deuterostomes (vertebrates and some “higher” invertebrates), making them potentially important in recognition events (1Angata T. Varki A. Chem. Rev. 2002; 102: 439-469Crossref PubMed Scopus (1023) Google Scholar, 2Schauer R. Curr. Opin. Struct. Biol. 2009; 19: 507-514Crossref PubMed Scopus (496) Google Scholar, 3Chen X. Varki A. ACS Chem. Biol. 2010; 5: 163-176Crossref PubMed Scopus (391) Google Scholar). One class of intrinsic Sia recognition proteins in vertebrates are Siglecs (sialic acid-binding immunoglobulin-like lectins). Siglecs are single-pass transmembrane proteins, with a Sia-binding site in the extracellular N-terminal Ig-like V-set domain (4Varki A. Angata T. 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Immunol. 2007; 7: 255-266Crossref PubMed Scopus (1367) Google Scholar, 8Varki A. Crocker P.R. Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Bertozzi C.R. Hart G.W. Etzler M.E. Essentials of Glycobiology. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2009: 459-474Google Scholar, 9Cao H. Crocker P.R. Immunology. 2011; 132: 18-26Crossref PubMed Scopus (125) Google Scholar).CD33-related Siglecs (CD33rSiglecs) are encoded by a subset of SIGLEC genes clustered on chromosome 19 in humans and chimpanzees. They are homologous in sequence and typically expressed on immune cells (10Cornish 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). Analyses of genomic SIGLEC sequences across humans, chimpanzees, baboons, rats, and mice showed that CD33rSiglecs are evolving rapidly (11Angata T. Margulies E.H. Green E.D. Varki A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13251-13256Crossref PubMed Scopus (135) Google Scholar). This is particularly pronounced in the Sia-recognizing V-set domain, suggesting that this domain is under the greatest selection pressure (11Angata T. Margulies E.H. Green E.D. Varki A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13251-13256Crossref PubMed Scopus (135) Google Scholar, 12Altheide T.K. Hayakawa T. Mikkelsen T.S. Diaz S. Varki N. Varki A. J. Biol. Chem. 2006; 281: 25689-25702Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 13Cao H. Lakner U. de Bono B. Traherne J.A. Trowsdale J. Barrow A.D. Eur. J. Immunol. 2008; 38: 2303-2315Crossref PubMed Scopus (79) Google Scholar, 14Cao H. de Bono B. Belov K. Wong E.S. Trowsdale J. Barrow A.D. Immunogenetics. 2009; 61: 401-417Crossref PubMed Scopus (36) Google Scholar).This study focuses on Siglec-12 (formerly Siglec-L1). We have shown previously that human Siglec-12 has an Arg → Cys (R122C) substitution mutation resulting in a protein unable to bind Sias (15Angata T. Varki N.M. Varki A. J. Biol. Chem. 2001; 276: 40282-40287Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). By convention, the protein is referred to as Siglec-XII 6Siglec-XII is the product of the human SIGLEC12 gene that cannot bind sialic acids because of a mutation in the critical arginine residue. in humans, to differentiate it from Siglec-12 in other primates, where the Sia-binding arginine is present. The gene in both cases is referred to as SIGLEC12. Reversing this mutation in vitro restored Sia binding (15Angata T. Varki N.M. Varki A. J. Biol. Chem. 2001; 276: 40282-40287Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Thus, except for the R122C mutation, the Sia-binding domain and reading frame were noted to remain intact.The C-terminal signaling domain in CD33-related Siglecs has an immunoreceptor tyrosine-based inhibitory motif (ITIM) or an immunoreceptor tyrosine-based switch motif. ITIMs typically recruit the protein tyrosine phosphatases SHP-1 and SHP-2 or the lipid phosphatase SHIP-1, generally resulting in inhibitory downstream signaling (16Avril T. Attrill H. Zhang J. Raper A. Crocker P.R. Biochem. Soc Trans. 2006; 34: 1024-1027Crossref PubMed Scopus (38) Google Scholar). The function of the immunoreceptor tyrosine-based switch motif in CD33-related Siglecs is unclear. Siglec-XII has an ITIM motif in its cytoplasmic C terminus.Analysis of chimpanzee, bonobo, gorilla, and orangutan sequences shows that they all have a functional SIGLEC12 gene with the key Arg residue intact (15Angata T. Varki N.M. Varki A. J. Biol. Chem. 2001; 276: 40282-40287Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Because humans and chimpanzees are typically ∼99% identical in protein coding regions (17Varki A. Altheide T.K. Genome Res. 2005; 15: 1746-1758Crossref PubMed Scopus (208) Google Scholar, 18Go Y. Niimura Y. Mol. Biol. Evol. 2008; 25: 1897-1907Crossref PubMed Scopus (76) Google Scholar) but have significant physiological, anatomical, and biomedical differences (such as a lower incidence of carcinomas in the latter) (19Varki N.M. Strobert E. Dick Jr., E.J. Benirschke K. Varki A. Annu. Rev. Pathol. 2011; 6: 365-393Crossref PubMed Scopus (82) Google Scholar), it is important to explore these genetic variations.Here we ask whether the R122C mutation is universal to humans and explore the allele frequency of an additional polymorphic frameshift insertion mutation in the human SIGLEC12 gene, which results in a premature stop codon in some individuals. Using newly generated monoclonal antibodies against Siglec-XII, we also confirm and extended earlier work where we noted unexpected expression in epithelial cells in addition to immune cells (15Angata T. Varki N.M. Varki A. J. Biol. Chem. 2001; 276: 40282-40287Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Finally, we describe the mechanistic and potential therapeutic significance of Siglec-XII expression in human carcinomas, particularly in prostate carcinomas.DISCUSSIONThe transmembrane position of Siglecs, along with their preference for binding sialic acids, the outermost sugars on cell surface glycans, make them an important part of the cell-cell communication system both within an organism and in between organisms such as host and pathogens. Previous work comparing SIGLEC loci in mice, rats, baboons, chimpanzees, and humans showed that CD33-related Siglecs are evolving rapidly, especially in the Sia-binding domain (11Angata T. Margulies E.H. Green E.D. Varki A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13251-13256Crossref PubMed Scopus (135) Google Scholar). This rapid evolution could be in response to slight changes in the Sia molecular structure in the host itself and/or on surface of pathogens. Apart from SIGLEC12, other Siglecs that have undergone human-specific changes in functional gene status, expression, or ligand binding include SIGLEC1, SIGLEC5/14, SIGLEC6, SIGLEC7, SIGLEC9, SIGLEC11, SIGLEC13, and SIGLEC16 (29Varki A. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 8939-8946Crossref PubMed Scopus (200) Google Scholar). Such large scale differences within a single class of gene indicate strong selection pressure on CD33-related SIGLEC loci in humans (29Varki A. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 8939-8946Crossref PubMed Scopus (200) Google Scholar, 30McMillan S.J. Crocker P.R. Carbohydr Res. 2008; 343: 2050-2056Crossref PubMed Scopus (34) Google Scholar).Although Siglec-12 in chimpanzees and the reverse mutated C122R human protein are shown to bind Sias (15Angata T. Varki N.M. Varki A. J. Biol. Chem. 2001; 276: 40282-40287Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), its natural ligands are unknown. In humans, Siglec-XII with a Cys instead of the essential Arg is expressed as a non-Sia binding full-length protein with cytosolic signaling domains intact. It is unclear whether the protein can mediate normal downstream signaling in the absence the essential Arg, but our microarray experiment shows a large scale gene down-regulation upon stable expression. This suggests that Siglec-XII retains some downstream signaling activity. Despite the R122C mutation, it is possible that Siglec-XII weakly recognizes sialic acids that results in the observed signaling effect. As shown in a previous work by others (31Yu Z. Lai C.M. Maoui M. Banville D. Shen S.H. J. Biol. Chem. 2001; 276: 23816-23824Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), Cos-7 cells expressing Siglec-XII bind sialylated red blood cells marginally better (∼4–5-fold) than nonexpressing Cos-7 cells, although this binding was undetectable when compared with a reverse mutated human C122R Siglec-XII (15Angata T. Varki N.M. Varki A. J. Biol. Chem. 2001; 276: 40282-40287Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). This phenomenon might arise from other interactions between sialic acids and Siglecs, such as that between the sialic acid glycerol group and a conserved hydrophobic amino acid as seen in crystal structures of Siglec-5 (Tyr-133), Siglec-7 (Trp-132), and Sialoadhesin (Trp-106) (32Zhuravleva M.A. Trandem K. Sun P.D. J. Mol. Biol. 2008; 375: 437-447Crossref PubMed Scopus (59) Google Scholar, 33Attrill H. Takazawa H. Witt S. Kelm S. Isecke R. Brossmer R. Ando T. Ishida H. Kiso M. Crocker P.R. van Aalten D.M. Biochem. J. 2006; 397: 271-278Crossref PubMed Scopus (64) Google Scholar, 34May 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). This hydrophobic amino acid is conserved in all Siglecs (4Varki A. Angata T. Glycobiology. 2006; 16: 1R-27RCrossref PubMed Scopus (427) Google Scholar). In addition there are other interactions between the sialic acid and the protein backbone (35Alphey M.S. Attrill H. Crocker P.R. van Aalten D.M. J. Biol. Chem. 2003; 278: 3372-3377Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). It has also been shown that certain Siglecs, such as Siglec-11, Siglec-6, and myelin-associated glycoprotein, still retain some Sia binding in the absence of the essential Arg (36Angata T. Kerr S.C. Greaves D.R. Varki N.M. Crocker P.R. Varki A. J. Biol. Chem. 2002; 277: 24466-24474Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 37Brinkman-Van der Linden E.C. Hurtado-Ziola N. Hayakawa T. Wiggleton L. Benirschke K. Varki A. Varki N. Glycobiology. 2007; 17: 922-931Crossref PubMed Scopus (84) Google Scholar, 38Vinson M. Strijbos P.J. Rowles A. Facci L. Moore S.E. Simmons D.L. Walsh F.S. J. Biol. Chem. 2001; 276: 20280-20285Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Siglec-XII might also have some residual sialic acid binding that might be enough to trigger downstream signaling.In addition to the R122C mutation, if the frameshift mutation is also present, there is no Siglec-XII expression, as confirmed by comparing genotype with immunohistochemistry. Thus, individuals homozygous for SIGLEC12P will lack any Siglec-XII-mediated signaling. This suggests a potential biochemical difference between genotypically different individuals and thus a potential target for natural selection.Unlike other Siglecs, Siglec-XII is expressed on human and chimpanzee epithelia, including human breast and prostate carcinomas. Epithelial carcinomas are rare in chimpanzees but occur at high frequencies in humans (19Varki N.M. Strobert E. Dick Jr., E.J. Benirschke K. Varki A. Annu. Rev. Pathol. 2011; 6: 365-393Crossref PubMed Scopus (82) Google Scholar, 29Varki A. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 8939-8946Crossref PubMed Scopus (200) Google Scholar, 39McClure H.M. Am. J. Phys. Anthropol. 1973; 38: 425-429Crossref PubMed Scopus (71) Google Scholar, 40Seibold H.R. Wolf R.H. Lab. Anim. Sci. 1973; 23: 533-539PubMed Google Scholar). One might hypothesize that this difference results in part from the inability of human Siglec-XII to bind sialylated ligands, giving less efficient ITIM signaling. In contrast, chimpanzee Siglec-12 maintains sialic acid binding, thus giving full ITIM signaling and better inhibition. In keeping with this, we found it difficult to stably express chimpanzee Siglec-12 and human C122R Siglec-XII, i.e. the human Siglec reverse mutated to restore Sia binding. 8N. Mitra, T. Angata, and A. Varki, unpublished observations. In contrast, expressing the non-Sia binding full-length human Siglec-XII allows stable expression.Regardless of these speculations, microarray data show that in presence of Siglec-XII, almost all of the observed changes in gene expression (with one exception) are down-regulations. Functional analyses of the affected genes show that many of them are associated with cellular movement and cancer (supplemental Table S1). Some genes known to be up-regulated in prostate cancers such as GDF15, TTF3, RUNX2, ITGB4, MMP1, and S100A9 are down-regulated in Siglec-XII-expressing PC-3 cells. GDF15 (alternatively MIC-1) expressed by PCa cells can disrupt cell adhesion and is associated with bone homeostasis (41Vanhara P. Lincová E. Kozubik A. Jurdic P. Soucek K. Smarda J. Differentiation. 2009; 78: 213-222Crossref PubMed Scopus (32) Google Scholar, 42Bauskin A.R. Brown D.A. Kuffner T. Johnen H. Luo X.W. Hunter M. Breit S.N. Cancer Res. 2006; 66: 4983-4986Crossref PubMed Scopus (197) Google Scholar). TFF3 is elevated in PCa, although its function is not clear (43Vestergaard E.M. Nexø E. Tørring N. Borre M. Ørntoft T.F. Sørensen K.D. Int. J. Cancer. 2010; 127: 1857-1865Crossref PubMed Scopus (49) Google Scholar). RUNX2, a transcription factor, has an anti-apoptotic effect in early cancer but contributes to subsequent bone metastasis (44Pratap J. Lian J.B. Stein G.S. Bone. 2011; 48: 30-36Crossref PubMed Scopus (77) Google Scholar). ITGB4 overexpression is also associated with epithelial cancers and their proliferation (45Bon G. Di Carlo S.E. Folgiero V. Avetrani P. Lazzari C. 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Other studies show strong expression of E-cadherin in metastatis of PCa (49De Marzo A.M. Knudsen B. Chan-Tack K. Epstein J.I. Urology. 1999; 53: 707-713Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 50Rubin M.A. Mucci N.R. Figurski J. Fecko A. Pienta K.J. Day M.L. Hum. Pathol. 2001; 32: 690-697Crossref PubMed Scopus (152) Google Scholar). The authors hypothesize that after a temporary loss of expression required for metastasis, the cancer cells regain E-cadherin expression for tumor establishment. Thus, the overall effect of CDH1 down-regulation is unclear.The presence of the inhibitory ITIM motif in Siglec-XII could be the likely cause of the observed down-regulation. A previous study (31Yu Z. Lai C.M. Maoui M. Banville D. Shen S.H. J. Biol. Chem. 2001; 276: 23816-23824Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) showed that Siglec-XII (called S2V in that paper) binds to the tyrosine phosphatases SHP-1 and SHP-2 in a phosphorylation-dependent manner (co-immunoprecipitation worked only in the presence of pervanadate). Despite the R122C mutation, Siglec-XII was phosphorylated by the kinase c-Src. The authors also showed that SHP-1 interaction with Siglec-XII was mediated by the ITIM domain in Siglec-XII. Since Siglec-XII has been shown to associate with SHP-1 and SHP-2 and because the interaction of SHP-1 is ITIM-dependent, it is reasonable to conclude that the observed gene down-regulation is due to the inhibitory ITIM domain. It is theoretically possible that these observed effects could vary, based on the amount of Siglec-XII expression. However, our two independently stable lines with different levels of Siglec-XII expression (differing by almost one order of magnitude, see supplemental Fig. S1) still show a similar pattern of gene down-regulation.U.S. population data show that PCa is the most prevalent form of cancer diagnosed among men and the second leading cause of cancer-related deaths (51Jemal A. Siegel R. Xu J. Ward E. CA Cancer J. Clin. 2010; 60: 277-300Crossref PubMed Scopus (12308) Google Scholar). Lack of symptoms during the early stages makes periodic screening essential for timely treatment of this disease. The most common screening method is the prostate-specific antigen test (52Sarrats A. Comet J. Tabarés G. Ramírez M. Aleixandre R.N. de Llorens R. Peracaula R. Prostate. 2010; 70: 1-9Crossref PubMed Scopus (36) Google Scholar), which has been shown to have a small effect on the mortality rate while picking up false positives (53Schröder F.H. Hugosson J. Roobol M.J. Tammela T.L. Ciatto S. Nelen V. 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Genes Dev. 2010; 24: 1967-2000Crossref PubMed Scopus (685) Google Scholar).Our own analysis of SIGLEC12 genomic status in PCa (mostly early stage PCa obtained during screening) and normal samples showed a similar allelic distribution in both groups, suggesting that the presence or absence of the frameshift does not predict the incidence of PCa. Indeed, this region of the genome was not picked in a recent large genome-wide association study that reported seven new prostate cancer susceptibility loci (61Eeles R.A. Kote-Jarai Z. Al Olama A.A. Giles G.G. Guy M. Severi G. Muir K. Hopper J.L. Henderson B.E. Haiman C.A. Schleutker J. Hamdy F.C. Neal D.E. Donovan J.L. Stanford J.L. Ostrander E.A. Ingles S.A. John E.M. Thibodeau S.N. Schaid D. Park J.Y. Spurdle A. Clements J. Dickinson J.L. Maier C. Vogel W. Dörk T. Rebbeck T.R. Cooney K.A. Cannon-Albright L. Chappuis P.O. Hutter P. Zeegers M. Kaneva R. Zhang H.W. Lu Y.J. Foulkes W.D. English D.R. Leongamornlert D.A. Tymrakiewicz M. 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However, based on our gene expression and tumor growth data, SIGLEC12 genomic and expression status could still have an effect on the long term progression of the cancer and the eventual severity of the disease. Indeed, although the majority of patients diagnosed with early stage prostate cancer will develop microscopic disease with advancing age, only a minority will have metastatic disease (62Esserman L. Shieh Y. Thompson I. J. Am. Med. Assoc. 2009; 302: 1685-1692Crossref PubMed Scopus (433) Google Scholar).For an initial indication of the role of Siglec-XII in prostate cancer development, we monitored the growth of human prostate cancer cells stably transfected with human SIGLEC12 or empty vector in nude mice. Cells expressing Siglec-XII showed a significant growth advantage over nonexpressing cells. This small growth difference over 70 days (time period of the mouse experiment) could become pertinent over many years, the usual time that it takes for a clinically significant prostate cancer to develop in humans. It is currently unknown whether this result extends to humans, but this is testable by association studies on large cohorts with known outcomes.A recent study on polymorphic nonsense single-nucleotide polymorphisms in the human genome found SIGLEC12 to be one of the 12 outliers, among the 167 genes studied (24Yngvadottir B. Xue Y. Searle S. Hunt S. Delgado M. Morrison J. Whittaker P. Deloukas P. Tyler-Smith C. Am. J. Hum. Genet. 2009; 84: 224-234Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). By calculating a statistical parameter called FST, which is a measure of population differentiation, it was found that a majority of nonsense single-nucleotide polymorphisms have a low FST value (< 0.09), suggesting that these single-nucleotide polymorphisms were at least mildly deleterious. SIGLEC12, on the other hand, had a high FST of 0.221 and a relatively high heterozygosity of 0.317, suggesting a balancing selection or a selective sweep. It is unlikely that PCa, which usually afflicts individuals in the postreproductive period, would affect this selective sweep. Future studies will work to uncover the selective target in or around the SIGLEC12 region on chromosome 19.All of the experiments in this study to understand the possible functions of Siglec-XII including microarray, tumor growth in nude mice, and cell killing experiments, were done in PC-3 cells stably expressing Siglec-XII under antibiotic selection. Although the effects observed in cells overexpressing Siglec-XII may not be relevant under normal physiological conditions in cancer cells, where Siglec-XII expression levels might be low, we would like to point out that the expression levels of the protein in Siglec-XII PC-3 cells and the three cancer cell lines natively expressing Siglec-XII are within range of each other (supplemental Fig. S3). Thus, the studies presented here are still relevant under conditions of native Siglec-XII expression. In the future, we aim to study the effect of Siglec-XII more directly by knocking down its expression in endogenously expressing cell lines.Human Siglec-XII is expressed at low levels in the epithelium. Because 40% of human individuals are homozygous for SIGLEC12P and do not show any obvious phenotype, it can be concluded that this gene is not essential for survival, although its presence might become pertinent under special conditions. This apparently more specialized function makes it a target for in vivo experimentation. In addition, Siglec-XII has a low and very narrow expression profile (it is expressed in only a very few cell types), thus making it a suitable candidate for antibody-mediated targeting for drug delivery to cancer cells expressing it. Because Siglec-XII can be internalized upon antibody binding, a simultaneous delivery of a toxin attached to the antibody could be a viable approach to future cancer therapy. IntroductionSialic acids (Sias) 5The abbreviations used are: SiaSialic acidPCaprostate cancerITIMimmunoreceptor tyrosine-based inhibitory motif. are nine-carbon backbone sugar molecules typically found at terminal positions of glycan chains in Deuterostomes (vertebrates and some “higher” invertebrates), making them potentially important in recognition events (1Angata T. Varki A. Chem. Rev. 2002; 102: 439-469Crossref PubMed Scopus (1023) Google Scholar, 2Schauer R. Curr. Opin. Struct. Biol. 2009; 19: 507-514Crossref PubMed Scopus (496) Google Scholar, 3Chen X. Varki A. ACS Chem. Biol. 2010; 5: 163-176Crossref PubMed Scopus (391) Google Scholar). One class of intrinsic Sia recognition proteins in vertebrates are Siglecs (sialic acid-binding immunoglobulin-like lectins). Siglecs are single-pass transmembrane proteins, with a Sia-binding site in the extracellular N-terminal Ig-like V-set domain (4Varki A. Angata T. Glycobiology. 2006; 16: 1R-27RCrossref PubMed Scopus (427) Google Scholar, 5Crocker P.R. Paulson J.C. Varki A. Nat. Rev. Immunol. 2007; 7: 255-266Crossref PubMed Scopus (1367) Google Scholar, 6von Gunten S. Bochner B.S. Ann. N.Y. Acad. Sci. 2008; 1143: 61-82Crossref PubMed Scopus (134) Google Scholar, 7Lopez P.H. Schnaar R.L. Curr. Opin. Struct. Biol. 2009; 19: 549-557Crossref PubMed Scopus (230) Google Scholar, 8Varki A. Crocker P.R. Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Bertozzi C.R. Hart G.W. Etzler M.E. Essentials of Glycobiology. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2009: 459-474Google Scholar, 9Cao H. Crocker P.R. Immunology. 2011; 132: 18-26Crossref PubMed Scopus (125) Google Scholar). Such V-set domains are followed by one or more C2-set Ig-like domains. Siglecs can signal through one or more tyrosine-based signaling motif(s) in the cytoplasmic tail (5Crocker P.R. Paulson J.C. Varki A. Nat. Rev. Immunol. 2007; 7: 255-266Crossref PubMed Scopus (1367) Google Scholar, 8Varki A. Crocker P.R. Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Bertozzi C.R. Hart G.W. Etzler M.E. Essentials of Glycobiology. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2009: 459-474Google Scholar, 9Cao H. Crocker P.R. Immunology. 2011; 132: 18-26Crossref PubMed Scopus (125) Google Scholar).CD33-related Siglecs (CD33rSiglecs) are encoded by a subset of SIGLEC genes clustered on chromosome 19 in humans and chimpanzees. They are homologous in sequence and typically expressed on immune cells (10Cornish 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). Analyses of genomic SIGLEC sequences across humans, chimpanzees, baboons, rats, and mice showed that CD33rSiglecs are evolving rapidly (11Angata T. Margulies E.H. Green E.D. Varki A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13251-13256Crossref PubMed Scopus (135) Google Scholar). This is particularly pronounced in the Sia-recognizing V-set domain, suggesting that this domain is under the greatest selection pressure (11Angata T. Margulies E.H. Green E.D. Varki A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13251-13256Crossref PubMed Scopus (135) Google Scholar, 12Altheide T.K. Hayakawa T. Mikkelsen T.S. Diaz S. Varki N. Varki A. J. Biol. Chem. 2006; 281: 25689-25702Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 13Cao H. Lakner U. de Bono B. Traherne J.A. Trowsdale J. Barrow A.D. Eur. J. Immunol. 2008; 38: 2303-2315Crossref PubMed Scopus (79) Google Scholar, 14Cao H. de Bono B. Belov K. Wong E.S. Trowsdale J. Barrow A.D. Immunogenetics. 2009; 61: 401-417Crossref PubMed Scopus (36) Google Scholar).This study focuses on Siglec-12 (formerly Siglec-L1). We have shown previously that human Siglec-12 has an Arg → Cys (R122C) substitution mutation resulting in a protein unable to bind Sias (15Angata T. Varki N.M. Varki A. J. Biol. Chem. 2001; 276: 40282-40287Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). By convention, the protein is referred to as Siglec-XII 6Siglec-XII is the product of the human SIGLEC12 gene that cannot bind sialic acids because of a mutation in the critical arginine residue. in humans, to differentiate it from Siglec-12 in other primates, where the Sia-binding arginine is present. The gene in both cases is referred to as SIGLEC12. Reversing this mutation in vitro restored Sia binding (15Angata T. Varki N.M. Varki A. J. Biol. Chem. 2001; 276: 40282-40287Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Thus, except for the R122C mutation, the Sia-binding domain and reading frame were noted to remain intact.The C-terminal signaling domain in CD33-related Siglecs has an immunoreceptor tyrosine-based inhibitory motif (ITIM) or an immunoreceptor tyrosine-based switch motif. ITIMs typically recruit the protein tyrosine phosphatases SHP-1 and SHP-2 or the lipid phosphatase SHIP-1, generally resulting in inhibitory downstream signaling (16Avril T. Attrill H. Zhang J. Raper A. Crocker P.R. Biochem. Soc Trans. 2006; 34: 1024-1027Crossref PubMed Scopus (38) Google Scholar). The function of the immunoreceptor tyrosine-based switch motif in CD33-related Siglecs is unclear. Siglec-XII has an ITIM motif in its cytoplasmic C terminus.Analysis of chimpanzee, bonobo, gorilla, and orangutan sequences shows that they all have a functional SIGLEC12 gene with the key Arg residue intact (15Angata T. Varki N.M. Varki A. J. Biol. Chem. 2001; 276: 40282-40287Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Because humans and chimpanzees are typically ∼99% identical in protein coding regions (17Varki A. Altheide T.K. Genome Res. 2005; 15: 1746-1758Crossref PubMed Scopus (208) Google Scholar, 18Go Y. Niimura Y. Mol. Biol. Evol. 2008; 25: 1897-1907Crossref PubMed Scopus (76) Google Scholar) but have significant physiological, anatomical, and biomedical differences (such as a lower incidence of carcinomas in the latter) (19Varki N.M. Strobert E. Dick Jr., E.J. Benirschke K. Varki A. Annu. Rev. Pathol. 2011; 6: 365-393Crossref PubMed Scopus (82) Google Scholar), it is important to explore these genetic variations.Here we ask whether the R122C mutation is universal to humans and explore the allele frequency of an additional polymorphic frameshift insertion mutation in the human SIGLEC12 gene, which results in a premature stop codon in some individuals. Using newly generated monoclonal antibodies against Siglec-XII, we also confirm and extended earlier work where we noted unexpected expression in epithelial cells in addition to immune cells (15Angata T. Varki N.M. Varki A. J. Biol. Chem. 2001; 276: 40282-40287Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Finally, we describe the mechanistic and potential therapeutic significance of Siglec-XII expression in human carcinomas, particularly in prostate carcinomas.