GBE Multiple Genomic Events Altering Hominin SIGLEC Biology and Innate Immunity Predated the Common Ancestor of Humans and Archaic Hominins Naazneen Khan1,2, Marc de Manuel3, Stephane Peyregne4, Raymond Do1,2, Kay Prufer4, Tomas Marques-Bonet3,5,6,7, Nissi Varki1,2, Pascal Gagneux1,2, and Ajit Varki 1,2 ,* 1 Glycobiology Research and Training Center, Department of Medicine, University of California San Diego 2 Center for Academic Research and Training in Anthropogeny (CARTA),University of California San Diego 3 Institute of Evolutionary Biology (UPF-CSIC), PRBB, Barcelona, Spain 4 Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany 5 Catalan Institution of Research and Advanced Studies (ICREA), Barcelona, Spain 6 CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain 7 Institut Catala de Paleontologia Miquel Crusafont, Universitat Auto noma de Barcelona, Edifici ICTA-ICP, Barcelona, Spain *Corresponding author: E-mail: a1varki@ucsd.edu. Accepted: 12 June 2020 Abstract Human-specific pseudogenization of the CMAH gene eliminated the mammalian sialic acid (Sia) Neu5Gc (generating an excess of its precursor Neu5Ac), thus changing ubiquitous cell surface “self-associated molecular patterns” that modulate innate immunity via engagement of CD33-related-Siglec receptors. The Alu-fusion-mediated loss-of-function of CMAH fixed 2–3 Ma, possibly con- tributing to the origins of the genus Homo. The mutation likely altered human self-associated molecular patterns, triggering multiple events, including emergence of human-adapted pathogens with strong preference for Neu5Ac recognition and/or presenting Neu5Ac-containing molecular mimics of human glycans, which can suppress immune responses via CD33-related-Siglec engage- ment. Human-specific alterations reported in some gene-encoding Sia-sensing proteins suggested a “hotspot” in hominin evolution. The availability of more hominid genomes including those of two extinct hominins now allows full reanalysis and evolutionary timing. Functional changes occur in 8/13 members of the human genomic cluster encoding CD33-related Siglecs, all predating the human common ancestor. Comparisons with great ape genomes indicate that these changes are unique to hominins. We found no evidence for strong selection after the Human–Neanderthal/Denisovan common ancestor, and these extinct hominin genomes include almost all major changes found in humans, indicating that these changes in hominin sialobiology predate the Neanderthal–human diver- gence 0.6 Ma. Multiple changes in this genomic cluster may also explain human-specific expression of CD33rSiglecs in unexpected locations such as amnion, placental trophoblast, pancreatic islets, ovarian fibroblasts, microglia, Natural Killer(NK) cells, and epithelia. Taken together, our data suggest that innate immune interactions with pathogens markedly altered hominin Siglec biology between 0.6 and 2 Ma, potentially affecting human evolution. Key words: sialic acid, hominin, evolution, CD33rSiglecs, common ancestor, Neanderthal/Denisovan, great apes, archaic hominin. Introduction a fixed loss-of-function genomic mutation unique to the hu- Prior to the mid-1990s, the extreme similarity of human and man lineage, in the gene-encoding CMP-Neu5Ac hydroxylase chimpanzee protein sequences suggested that phenotypic (CMAH) (Chou et al. 1998), an event mediated by an Alu–Alu differences were primarily due to differences in gene regula- fusion (Hayakawa et al. 2001). This mutation eliminated bio- tion (King and Wilson 1975). The first definitive exception was synthesis of the common mammalian sialic acid (Sia) ß The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1040 Genome Biol. Evol. 12(7):1040–1050. doi:10.1093/gbe/evaa125 Advance Access publication 18 June 2020 Hominin SIGLEC Biology and Innate Immunity GBE N-glycolylneuraminic acid (Neu5Gc) and caused accumulation Early discovery of some additional human-specific changes of its precursor N-acetylneuraminic acid (Neu5Ac), radically affecting sialic acid biology (Brinkman-Van der Linden et al. changing cell surface extracellular glycosylation throughout 2000; Angata, Varki, et al. 2001; Gagneux et al. 2003; the body in the hominin lineage. The mutation was dated Sonnenburg et al. 2004; Hayakawa et al. 2005; Nguyen to >2 Ma by multiple methods (Chou et al. 2002; et al. 2006) and system-wide genomic and biochemical com- Hayakawa et al. 2006). Additional examples emerged a few parisons of sialic acid biology among primates and rodents years later, including the forkhead box protein P2 (FOXP2) (Altheide et al. 2006) suggested a possible “hotspot” in hu- (Enard et al. 2002, 2009), a myosin heavy chain family mem- man sialic acid evolution (Varki 2009). With the availability of ber (MHY16) (Currie 2004; Stedman et al. 2004), and exam- more human genomes (1000 Genomes Project Consortium ples of gene duplication followed by adaptive evolution et al. 2015), a recent study showed the patterns of genetic (Johnson et al. 2001). Along with biomedical considerations variation of 55 sialic acid biology-related genes in modern (Varki 2000), such discoveries lead to sequencing of the chim- human populations did not significantly deviate from neutral panzee genome (Chimpanzee Sequencing and Analysis expectations and were in fact not significantly different Consortium 2005). There are now many more defined ge- among genes belonging to different functional categories netic differences between humans and our closest evolution- (Moon et al. 2018). ary cousins, involving not only gene expression (Enard et al. Sialic acid binding Ig-like lectin (Siglecs) are type I trans- 2002; Fujiyama et al. 2002; Watanabe et al. 2004; Calarco membrane proteins with an N-terminal immunoglobulin et al. 2007; Kehrer-Sawatzki and Cooper 2007; Cruz-Gordillo (Ig)-like-V-set domain that mediates sialic acid recognition, et al. 2010; Otto et al. 2014; Atkinson et al. 2018) but also and a variable number of Ig-like-C-2 type domains (Angata, other distinct genomic changes ranging from massive (mega- Hingorani, et al. 2001). Siglecs often have a cytoplasmic tail base) structural variation to differences in gene copy numbers, with one or more immunoreceptor tyrosine-based inhibitory de novo genes, pseudogenization (O’Bleness et al. 2012; Ruiz- motifs that can suppress immune cell activation. Alternatively, Orera et al. 2015), human-accelerated regions in noncoding they can recruit adaptor proteins with immunoreceptor regions, and microRNA genes (Eddy 2001; Mello and Conte tyrosine-based activating motifs. Although Siglecs likely have 2004; Kim 2005; Ruiz-Orera et al. 2015). multiple functions, one prominent role appears to be recog- This study focuses on human genes involved in the biology nition of endogenous sialylated glycans as self-associated mo- of sialic acids. All living cells are covered with complex arrays lecular patterns, suppressing reactions of innate immune cells of glycoconjugates, with sialic acids occupying the majority of against self (Varki 2011). Activating Siglecs have a positively terminal positions on such glycan chains in animals of the charged arginine or lysine in their transmembrane domains Deuterostome lineage (including echinoderms and verte- that can recruit DAP12 and activate cellular immune brates) (Gagneux et al. 2015). These acidic, nine-carbon responses against pathogens mimicking endogenous sialic backbone amino-sugars play important roles in cell–cell and acids (Schwarz et al. 2017). CD33-related Siglecs are rapidly cell–matrix interactions, as well as in host–pathogen interac- evolving and among this family, nine inhibitory (hSiglec-3 and tions (Varki et al. 2015). The hominin-specific loss-of-function hSiglec-5 to hSiglec-12) and two activating members (hSiglec- mutation in CMAH mentioned above eliminated biosynthesis 14 and hSiglec-16) have been characterized in humans. of Neu5Gc, which was a target for selective cell recognition by Recently, an article published from our group showed variable various nonhuman pathogens and toxins (Kyogashima et al. presence or absence of functional changes in the 1989; Martin et al. 2005; Campanero-Rhodes et al. 2007; CD33rSIGLEC cluster in 26 mammalian species including great Deng et al. 2014; Alisson-Silva et al. 2018). It is reasonable apes (Khan et al. 2020). to speculate that exogenous pathogen recognition could have With the availability of genomes from both living great initially driven the CMAH loss-of-function, generating a poly- apes (Prado-Martinez et al. 2013; Xue et al. 2015; morphism as the recessive loss-of-function mutant allele rose Kronenberg et al. 2018) and extinct archaic hominins (Reich in frequency. However, this deletion was then fixed in an et al. 2010; Meyer et al. 2012; Prufer et al. 2014), we now ancestral hominin population 2–3 Ma (Chou et al. 2002), systematically reassess the previous discoveries from multiple possibly by directional selection against Neu5Gc via female groups and also report on several new findings. Overall, we immunity during reproduction, a mechanism demonstrated find that multiple complex changes in genes involving sialic in vivo using transgenic mice that carry the same mutation acid biology beyond the initial CMAH mutation did indeed in their Cmah gene as humans (Ghaderi et al. 2011). Given occur but are mostly confined to the CD33rSIGLEC gene clus- the likely timing of these events, we speculated that anti- ter on chromosome 19 (Angata, Hingorani, et al. 2001), Neu5Gc immunity of hominin females immunized against encoding CD33rSiglecs, prominent on innate immune cells Neu5Gc by increased contact with Neu5Gc-rich vertebrate (Varki and Angata 2006). animal prey might have contributed to the origins of the ge- Overall, we found that multiple changes in the nus Homo 2 Ma (Wood and Boyle 2016; Bergfeld et al. CD33rSIGLEC gene cluster are common in all human popula- 2017). tions, postdate the common ancestor with the chimpanzee/ Genome Biol. Evol. 12(7):1040–1050 doi:10.1093/gbe/evaa125 Advance Access publication 18 June 2020 1041 Khan et al. GBE bonobo lineage, but predate the common ancestor with (Peyregne et al. 2017). We also note that signals of positive Neanderthals and Denisovans. Such multiple complex selection are not detectable if the selection coefficient is changes in this gene cluster appear to be associated with smaller than 0.1%. The Ensembl database (release 82) altered expression of these genes, not just confined to innate (Aken et al. 2016) was used to annotate each gene with immune cells, but also in other unexpected human cell types, hg19 coordinates from transcription start to transcription some associated with diseases that appear to be uniquely end. Furthermore, L1CAM, PECAM, CMAH, SIGLEC13, human. SIGLEC16, and SIGLEC17 were excluded because they fall ei- ther in filtered regions not considered for the ancient sweep screen or could not be mapped to hg19 coordinates. As reg- Materials and Methods ulatory regions may also have been the target of positive se- Genome Assembly and Identification of Variants across lection, we extended the start and end coordinates 1 Mb Humans and Great Apes upstream and downstream, respectively. If a neighboring The great ape genome data (total number 147) for CMAH gene was within 1 Mb, we only extended the coordinates and CD33rSIGLECs were derived from three publications until 5 or 1 kb from the transcription start or end of this (Prado-Martinez et al. 2013; Xue et al. 2015; de Manuel neighboring gene. In order to test whether the lack of signa- et al. 2016; Kronenberg et al. 2018). These great ape tures of ancient selection is statistically significant, the candi- genomes were mapped to human reference genome date regions of ancient selection were randomly placed in the (GRCh37/hg19) retrieved from UCSC genome browser, using genome and the random placements of all regions were iter- Burrows–Wheeler aligner and further processed with Picard ated 1,000 times, counting how often no overlap with genes tool to remove duplicates and variant were called using involved in sialic acid biology was detected. The depletion in Genome-analysis toolkit. Moreover, the variants in variant selection is not significant (356 sets never overlap; P value ¼ call format of chimpanzee, bonobos, gorilla, and orangutan 0.356). were visualized in integrative genome viewer with their re- spective reference genome (Pantro4, Gorgor3, and PonAbe2) Immunohistochemistry of Siglecs and annotation file. All archaic hominins raw and processed Paraffin sections were deparaffinized in xylene and rehy- files were obtained from the Max Planck Institute for drated in decreasing concentrations of ethanol and Tris- Evolutionary Anthropology (Reich et al. 2010; Castellano buffered saline–Tween (TBST). Following this, endogenous et al. 2014; Prufer et al. 2014; Slon et al. 2018) (http://cdna. binding sites and peroxidases were blocked with 1% bovine eva.mpg.de/neandertal). Bed coordinates of SIGLECs and serum albumin/TBST and 0.3% H2O2/TBST. This was followed CMAH genes were provided for each human and ape lineage by blocking of endogenous biotin and then heat-induced an- as supplemental (chimpanzee, gorilla, and orangutan) (sup- tigen retrieval was performed in citrate buffer pH 6. Primary plementary file 1, Supplementary Material online). antibodies (mouse anti-Siglec-7 and Rabbit anti-Siglec-13) or Additionally, bed coordinates of additional polymorphism pre- mouse IgG were then overlaid at optimal dilutions, and slides sent in CD33rSIGLECs with allele frequency in great ape pop- were incubated overnight at 4  C in a humid chamber. ulation were also provided as supplemental (supplementary Specific binding was detected using a biotinylated anti- file 2, Supplementary Material online). mouse or anti-rabbit (for Siglec-13), followed by Horseradish Peroxidase (HRP) streptavidin and then biotinyl tyramide en- Inference of Strong Archaic Natural Selection hancement and then HRP streptavidin. Substrate color was The genes involved in sialic acid biology (67 genes) were over- developed, and nuclei were counterstained with hematoxylin lapped with regions displaying signatures of ancient selective and the slides were aqueous mounted for viewing and digital sweeps (Peyregne et al. 2017). The method used to identify photomicrography using an Olympus BH2 microscope with these signatures of archaic selection relies on a hidden an Olympus magnafire camera. Markov model to detect extended regions in the genome where the Neanderthal and Denisovan lineages fall outside Results the human variation (Meyer et al. 2012; Prufer et al. 2014). This method can only detect events that occurred between Multiple Derived, Fixed, or Polymorphic Genomic SIGLEC the split of modern and archaic humans around 0.5 Ma Variants Are Present in All Modern Human Populations (Prufer et al. 2017) and the split of modern human popula- Table 1 summarizes events and frequencies in human popu- tions from each other around 0.2 Ma (Schiffels and Durbin lations based on the 1000 Genomes Project data and com- 2014). For events with a selective advantage of 0.5% or pares these with archaic hominin and great ape genomes. All larger, with an origin of the beneficial mutation as old as human genomic changes in CD33-related SIGLECs (affecting 600,000 years ago, the false positive rate of the method is 8 out of 13 members of this class of genes) were present lower than 0.1% and its true positive rate is larger than 65% across modern human populations. Genome-wide 1042 Genome Biol. Evol. 12(7):1040–1050 doi:10.1093/gbe/evaa125 Advance Access publication 18 June 2020 Hominin SIGLEC Biology and Innate Immunity GBE association studies identified a derived CD33-linked allele NOTE.—All the genomic changes were found in all existing human populations. CD33m (Siglec-3):allele increasing alternate splice form protective against Alzheimer’s disease; ND:not determined; Siglec-5: sialic acid binding arginine residue; Siglec-12: mutation of sialic acid binding arginine residue; Siglec-12: 1-bp insertion resulted in pseudogene (12P); Siglec-12: inactivation polymorphism at position 29 amino acid; Siglec-13: complete deletion by Alu–Alu fusion event; Siglec-14: sialic acid binding arginine residue; Siglec-14: 16-kb deletion/fusion, difficult to read from standard genomic data; Siglec-16:, pseudogene (16P) and gene (16) forms; Siglec-17: mutation of sialic acid binding Orangutan Low call/Y Ancestral Ancestral Ancestral Low call Present (rs3865444(A), rs12459419(T)) that is protective against late ND — Q G R R 0 onset Alzheimer’s disease (Schwarz et al. 2016). The linked single-nucleotide polymorphism was found to be variable Ancestral Not well annotated Present Not well annotated across human populations with highest frequency in native American populations (48%) and lowest in African popula- Ancestral Ancestral Gorilla R/H R/H ND tions (5%). The previously reported Alu-mediated human — Q G R R 0 Great Apes SIGLEC13 deletion (Wang, Mitra, Secundino, et al. 2012) and SIGLEC17 (Siglec-P3) pseudogenization due to Open reading frame disruption and mutation of a critical arginine Ancestral Ancestral Bonobo residue in its V-set domain (Wang, Mitra, Secundino, et al. R/H ND — Q G R R R 0 2012) appear to be fixed in all humans. SIGLEC12, SIGLEC14, and SIGLEC16 show polymorphic pseudogenization in vari- Africa America E. Asia Europe S. Asia Neanderthal Denisovan Chimpanzee able frequencies across human populations, as detailed in ta- Ancestral Ancestral Ancestral Present ble 1. SIGLEC12 also harbors a human-universal mutation of R/H R/H ND — Q G R R 0 the critical arginine residues in the V-set domain (Arg -> Cys, first V-set domain) abrogating its ability to recognize sialic Ancestral acid. An additional SIGLEC12 inactivation mutation Archaic Genomes (%) 100 100 100 100 100 100 100 100 ND ND 50 50 (rs16982743) was found at an overall 18.6% frequency, high- est in Africans (37%) and lowest in East Asians (5%). Another SIGLEC12 polymorphism, that is, frameshift (rs66949844) Detailed Description of Genomic Variants and Their Percentage in Each Lineage Based on CD33rSIGLECs and CMAH Genes Ancestral Ancestral arginine residue; Siglec-17: 1-bp deletion that disrupted the Open reading frame (ORF); CMAH: pseudogene due to 92-bp exon 6 deletion. averages 59% (highest in native Americans and lowest in 100 100 100 100 100 100 100 ND 50 60 67 East Asians) in human population. Additionally, based on the two linked alleles present in SIGLEC16 (rs12611411(T) and rs12984584(C)), the pseudogene variant (SIGLEC16P) is 100 100 100 100 100 100 100 16 63 11 33 87 16 present in higher frequency than the functional allele (SIGLEC16) in human populations (Wang, Mitra, Cruz, et al. 1000 Genomes Data (%) 2012). Because these SIGLEC clusters are undergoing multiple 100 100 100 100 100 100 100 31 67 20 22 82 31 gene conversions, some regions are highly prone to low map- ping quality and hence some variants are not well defined, for 100 100 100 100 100 100 100 19 37 73 67 19 example, SIGLEC14 fusion-deletion, which is highly prevalent 4 in East Asian populations (Yamanaka et al. 2009; Ali et al. 2014). Due to the lack of high coverage sequence data, these 100 100 100 100 100 100 100 48 74 13 21 85 48 regions have not been well genotyped in nonhuman genomes. The CMAH exon deletion is confirmed to be uni- 100 100 100 100 100 100 100 versal to humans. 60 37 30 83 5 5 Fusion/deletion Disrupted ORF Great Ape Genomes Do Not Show a High Frequency of Changes CMAHP SIGLEC Gene Mutations 16P Del AA TT W R C C R * Our previous human–ape comparisons involved the incom- plete draft of a single chimpanzee genome, a few additional incomplete great ape sequences and a small number of hu- rs12611411 rs12984584 Genomic Changes man genomes (Chimpanzee Sequencing and Analysis Consortium 2005). Thus, it remained possible that the geno- /C(rs66949844) Q/*(rs16982743) Exon 6 deletion CD33m (SIGLEC3) rs12459419(T) rs3865444(A) mic changes were actually a common feature of such genes Arg (R)/W Arg (R)/H across all these taxa. The availability of newly annotated ver- Arg (R)/C Deletion Deletion Arg (R) sions of great ape genomes (Prado-Martinez et al. 2013; Xue G/- et al. 2015; de Manuel et al. 2016; Kronenberg et al. 2018) now allows for much better control against ascertainment bias, by comparing human-specific polymorphisms with mul- tiple different genomes for each ape species, capturing po- SIGLEC12 SIGLEC13 SIGLEC14 SIGLEC14 SIGLEC16 SIGLEC17 SIGLEC5 Table 1 tential polymorphisms in the latter as well (Sullivan et al. CMAH Gene 2017). We observed that great ape genomes share none of Genome Biol. Evol. 12(7):1040–1050 doi:10.1093/gbe/evaa125 Advance Access publication 18 June 2020 1043 Khan et al. GBE FIG. 1—Evolutionary changes in hominid SIGLEC genes. The tree represents phylogenetic relatedness among hominids and different types of genomic events are depicted according to the color codes. Siglec expression differences are based on more limited comparisons (see table 1 and fig. 3 and see text for discussion). the mutations observed in human populations. Instead, we as expected, given that both types of sialic acids (Neu5Ac and only observed independent changes in critical arginine resi- Neu5Gc) are present in all these species (Muchmore et al. dues in V-set domain of SIGLEC5/14, rendering them unable 1998). Overall, human genomes harbored a far greater num- to recognize sialic acids (table 1). Although the essential argi- ber of structural/functional changes in CD33rSIGLEC genes nine change (Arg -> His) in SIGLEC5 was found to be poly- (see fig. 1 and supplementary file 2, Supplementary morphic in chimpanzee and gorilla, sequences in orangutan Material online). were not recognized due to low mappability. The SIGLEC14 essential arginine change (Arg -> His) was found to be poly- morphic in chimpanzee, bonobo, and gorilla. However, Most Human Lineage-Specific Mutations Are Also Found sequences of SIGLEC14 in orangutan were not determined in Archaic Hominin Genomes in most of the species due to low coverage, except for To further understand the timing of these human genomic one that harbors an essential arginine change to tyrosine changes of these human genomic variants, we looked for (Arg -> Tyr). The CMAH gene is functional in all great apes their presence in archaic human genomes, that is, 1044 Genome Biol. Evol. 12(7):1040–1050 doi:10.1093/gbe/evaa125 Advance Access publication 18 June 2020 Hominin SIGLEC Biology and Innate Immunity GBE FIG. 2—Description of the ELS scan. (A) Illustration of the selection signature. Patterns of shared mutations (dots) were used to reconstruct the genealogy that relate an archaic human Altai Neanderthal (Prufer et al. 2014); or Denisovan (Meyer et al. 2012); top rectangle to modern humans (bottom rectangles) at any given position along their genomes. There are two types of genealogies: the archaic falls either outside the modern human variation (external genealogy) or inside (internal genealogy). The internal genealogy is the most frequent in the genome. However, if a mutation spread among the ancestors of modern humans (blue dot) more recently than the population split with the ancestors of archaic humans, the archaic will be expected to fall outside the modern human variation in the genomic region around the mutation that has not been unlinked by recombination (blue rectangles). This region is expected to be large if the mutation was positively selected and spread quickly in the population, not leaving enough time for recombination to break its linkage with other mutations (black dots). (B) Time scales for the signatures of selection in humans. The tree represents the simplified population history of Neanderthals, Denisovans, and modern humans (Prufer et al. 2017). The colors indicate the time scales investigated by the ELS scan (red) and other methods (blue) used to detect events of positive selection on the modern human lineage. Neanderthal and Denisovan. The exon deletion leading to (Neanderthal–Denisovan) split from each other around loss-of-function of the CMAH gene is shared by these two 500,000 years ago. As selection may also target neighboring archaic genomes, as expected based on the existing estimates regulatory regions, we defined regulatory domains around of fixation of the mutation over 2 Ma (Hayakawa et al. 2001). each gene and looked for overlaps with candidate sweep Notably, almost all other human CD33rSIGLECs variants (ex- regions. Using these extended gene coordinates, we again cept for CD33-linked variants protective against late onset found no overlap with candidate regions for selective sweeps, Alzheimer’s disease) were also present in these archaic hom- suggesting that none of the genes involved in sialic acid biol- inin genomes in variable frequency (listed in table 1 and fig. 1) ogy exhibits strong signatures of selection more recently than placing their origin before the human–Neanderthal common 0.5 Myr in the common ancestral population of all modern ancestor (0.6 Ma) (Prufer et al. 2017; Hajdinjak et al. 2018). humans. The description of extended lineage sorting (ELS) is Due to unavailability of well annotated sequences, the provided in figure 2. SIGLEC14 fusion/deletion could not be determined in archaic hominins. Human-Specific Expression of CD33rSiglecs in Nonimmune Tissues Lack of Evidence for Strong Selection up to 500,000 Years In addition to these multiple, polymorphic complex genomic Ago changes in the human CD33rSIGLEC gene cluster, there ap- The presence of all genomic SIGLEC changes in human pop- pear to be unusual (derived) human-specific expression pat- ulations indicates that they predate the common ancestor of terns of CD33rSiglecs (in nonhemopoietic cells) in locations modern humans 0.2–0.3 Ma (Hublin et al. 2017). such as placental trophoblast (SIGLEC6) (Kang et al. 2011), Consistent with this observation, the recent article by Moon ovarian fibroblasts (SIGLEC11/16) (Wang et al. 2011), amni- et al. (2018) found that the patterns of genetic variation of otic epithelium (SIGLEC5/14) (Ali et al. 2014), and microglia most CD33rSIGLEC genes did not significantly deviate from (SIGLEC11/16) (Hayakawa et al. 2005). In each of the above neutral expectations, and the few that did significantly deviate instances, we have previously reported human-specific ex- from neutrality experienced either soft sweeps or population- pression and lack of expression in chimpanzee tissue samples specific hard sweeps (Moon et al. 2018). Using a method that (in some instances also other available great ape tissues). With allows detection of selection in deeper time (0.5 Ma) regard to the recent report of SIGLEC7 expression in human (Peyregne et al. 2017), we also found no evidence of strong pancreatic islets (Yamaguchi et al. 2017), we now show that selection after the ancestors of modern and archaic humans such expression is missing in chimpanzee pancreatic islets Genome Biol. Evol. 12(7):1040–1050 doi:10.1093/gbe/evaa125 Advance Access publication 18 June 2020 1045 Khan et al. GBE FIG. 3—Examples of immunohistochemistry using antibodies against Siglec-7 and Siglec-13 on human and chimpanzee tissues (see Materials and Methods for detail). (A) No difference in expression of Siglec-7 in splenic blood cells. There is markedly increased expression of Siglec-7 in human pancreatic islets. (B) Siglec-13 expression in chimpanzee skin and prostatic epithelium. The gene-encoding Siglec-13 is completely deleted in humans. Scale bars ¼ 100 lm. (fig. 3). We also found that Siglec-13, which is deleted in do not show many changes in this cluster, almost all the human hominins, is highly expressed in chimpanzee intestinal epithe- changes are also found in archaic genomes of Neanderthals lium and skin (fig. 3). Current and previously published data and Denisovans (Reich et al. 2010; Castellano et al. 2014; are summarized in table 2. Although we obviously cannot Prufer et al. 2014; Slon et al. 2018; Bokelmann et al. 2019). detect tissue-specific expression in Neanderthals or In keeping with this overall conclusion, there was no evidence Denisovans, it is reasonable to consider the possibility that for strong selective pressure in this cluster after 0.6 Ma, when these unusual human-specific expression patterns arose dur- the human lineage diverged from Neanderthal–Denisovan ing the course of the large-scale changes affecting the common ancestor. CD33rSIGLECs gene cluster during the proposed “hotspot” With regard to timing and order of changes, the most likely and would thus have already existed in both of those extinct possibility is that the CMAH mutation first dramatically altered relatives. sialic acids throughout the body, possibly initiating a series of Discussion events that resulted in SIGLEC cluster changes. The complex Recent availability of sequences of many modern human and dynamic relationships between host and pathogens is genomes (1000 Genomes Project Consortium et al. 2015), ar- often characterized by the Red Queen hypothesis, whereby chaic hominins (Green et al. 2010; Reich et al. 2010, 2011; slowly evolving hosts have to keep changing to keep pace Meyer et al. 2012, 2016; Castellano et al. 2014; Prufer et al. with the pressure exerted by more rapidly evolving pathogens. 2014, 2017; Sawyer et al. 2015; Kuhlwilm et al. 2016; Slon Sialic acids form one primary interface, “the molecular fron- et al. 2018), and great apes (Prado-Martinez et al. 2013; Xue tier” in this evolutionary arms race. The loss-of-function of et al. 2015; de Manuel et al. 2016) provides a tool for under- CMAH and the changes in ST6GAL1 expression undoubtedly standing the role of evolution in shaping genetic architecture led to multiple alterations of human–pathogen interactions. and allowing us to identify the genetic basis of phenotypic traits The direct consequence was the change in human cell surfa- of various species (Haffter et al. 1996; Bryk and Tautz 2014; ces, leading to overexpression of Neu5Ac, escaping Neu5Gc- Valenzano et al. 2015; Castellano and Munch 2020). Our ear- specific pathogens, and favoring human pathogens that in- lier studies noted multiple genomic changes affecting sialic acid teract with Neu5Ac. There are many other known and possi- biology, and we suggested the possibility of a hotspot in ble biological consequences of human Neu5Gc loss (Varki humans affecting these pathways compared with our closest 2009; Okerblom and Varki 2017), including protection from evolutionary relatives (Altheide et al. 2006). However, the lim- various Neu5Gc-recognizing pathogens such as malaria ited availability of annotated and high coverage genomes at caused by Plasmodium reichenowi in African great apes the time left the question unresolved, and our suggestion has (Martin et al. 2005), Escherichia coli K99 gastroenteritis been recently tested by others (Moon et al. 2018), who could (Kyogashima et al. 1989), transmissible gastroenteritis coro- not find evidence for strong selective sweeps in current human navirus (Schwegmann-Wessels and Herrler 2006), and simian populations. We herewith show that apart from the fixed virus 40 (Campanero-Rhodes et al. 2007). Conversely, CMAH CMAH null mutation and increased expression of ST6GAL1 loss likely made humans susceptible to Neu5Ac-preferring (Gagneux et al. 2003), most of the human-specific changes pathogens such as Vibrio cholerae (causative agent of the affecting sialic acid biology are found in the SIGLEC gene clus- human-specific disease cholera) (Alisson-Silva et al. 2018) ter on chromosome 19, and that although great ape genomes and typhoid fever caused by a secreted bacterial toxin that 1046 Genome Biol. Evol. 12(7):1040–1050 doi:10.1093/gbe/evaa125 Advance Access publication 18 June 2020 Table 2 Differential Tissue and Cell Type Expression Profile of SIGLEC Genes among Humans and Great Apes Siglecs Humans “Great Apes” Current Data and Literature Reference(s) Potential Relevance to Diseases That Appear Unique to Humans Hominin SIGLEC Biology and Innate Immunity CD33 High expression in human brain microglia Low expression in chimpanzee brain Schwarz et al. (2016) Alzheimer’s disease microglia Siglec-5 Selectively expressed in amniotic Not expressed in chimpanzee amniotic Ali et al. (2014) Intrauterine infections with group B epithelium epithelium Streptococcus Siglec-6 Selectively expressed in placental Not expressed in placental trophoblast Brinkman-Van der Linden et al. (2007) Preeclampsia trophoblast chimpanzee, gorilla, and orangutan Siglec-7 Selectively expressed in pancreatic islet Not expressed in chimpanzee Present study (fig. 3A) Type I diabetes cells Siglec-11 Selectively expressed in brain microglia Less expression in chimpanzee, no expres- Hayakawa et al. (2005) Escherichia coli K1 meningitis in sion in orangutan brain neonates and infants Siglec-11 Decreased expression in ovarian fibroblasts Strong expression in chimpanzee ovarian Wang et al. (2011) Unknown fibroblasts Siglec-13 Deleted in humans Expressed in chimpanzee monocytes Wang, Mitra, Secundino, et al. (2012) Unknown Siglec-13 Deleted in humans Expressed in chimpanzee epithelium Present study (fig. 3B) Unknown Siglec-14 Polymorphic expression in amniotic Not expressed in chimpanzee amniotic Ali et al. (2014) Intrauterine infections with Group B epithelium epithelium Streptococcus Siglec-16 Polymorphic expression in brain microglia Not expressed in microglia of chimpanzee Hayakawa et al. (2005) Escherichia coli K1 meningitis in and gorilla neonates and infants Siglec-17 No protein expression Expressed in natural killer cells of chim- Wang, Mitra, Secundino, et al. (2012) Unknown panzee, gorilla, and orangutan Genome Biol. Evol. 12(7):1040–1050 doi:10.1093/gbe/evaa125 Advance Access publication 18 June 2020 1047 GBE Khan et al. GBE preferentially recognizes Neu5Ac (Deng et al. 2014). Another secondary consequences of multiple genomic changes that human-specific pathogen Streptococcus pneumoniae recog- occurred in this gene cluster earlier in the hominin lineage. nizes free Neu5Ac released from human cells by its secreted Overall, it is likely that the selective pressures driving all these sialidase (Hentrich et al. 2016). Yet another secondary conse- changes were most prominent sometime after the split from quence appears to have been the evolution of pathogens that the common ancestor of human and chimpanzee, but before express Neu5Ac on their surfaces as a way to engage CD33- the split from the Human–Neanderthal–Denisovan common related Siglecs and downregulate innate immune responses in ancestor. the human host. Again, the existence of many human-specific Taken together, our data suggest that innate immune pathogens that display Neu5Ac supports this scenario encounters with pathogens markedly altered hominin Siglec (Angata 2018). For example, Group B Streptococcus type III, biology between 0.6 and 2 Ma, potentially affecting human interacts with Siglec-9 through sialylated CPS and inhibits in- evolution. Notably, this is the time period when a variety of flammatory response by neutrophils (Carlin et al. 2009). changes were occurring in genus Homo, including exploration Likewise, nontypeable Haemophilus influenzae expressing si- of new habitats (White et al. 1993; Semaw et al. 1997; alic acid on its lipooligosaccharide binds to Siglec-5 and deMenocal 2011), striding bipedalism and running (Bramble reduces cytokine production by myeloid cells (Angata et al. and Lieberman 2004; Lieberman 2015), and scavenging and 2013), and the E. coli K1 strain that causes meningitis in neo- hunting, involving butchery of animal carcasses with stone nates and infection in urinary tract, engages Siglec-11, and tools (Semaw et al. 1997; O’Connell et al. 2002; McPherron escapes killing (Hayakawa et al. 2017). Also, as expected, et al. 2010; Sayers and Lovejoy 2014; Harmand et al. 2015; nonhuman primate Siglec-9 prefers binding to Neu5Gc, Baird et al. 2016), activities that may have increased risk of whereas human Siglec-9 prefers binding to Neu5Ac injury, novel infections, and use of fire (Smith et al. 2015). In (Sonnenburg et al. 2004). Likewise, human Siglec-5 and this regard, it is also interesting that human-like elimination of CD33 prefer binding to Neu5Ac, compared with the baboon Cmah in mice enhances running ability (Okerblom et al. 2018) CD33, which strongly prefers Neu5Gc (Padler-Karavani et al. as well as macrophage activation (Okerblom et al. 2017). 2014). These findings imply that Siglecs underwent adapta- tion in new environments to “catch up” with the changes in human sialome caused by the hominin CMAH mutation. Supplementary Material Some human Siglecs still prefer binding to Neu5Gc (Angata Supplementary data are available at Genome Biology and 2018), perhaps due to incomplete adaptation to the derived, Evolution online. human Neu5Ac-dominant sialome. Although these genomic changes are ancient, evidence also suggests that existing human polymorphisms are associ- Acknowledgments ated with several diseases such as Chronic obstructive pulmo- We thank members of the Varki and Gagneux labs for valu- nary disease, asthma, Alzheimer’s disease, and meningitis able comments on this work. This research was supported by (Yamanaka et al. 2009; Gao et al. 2010; Ali et al. 2014; NIH grant R01GM32373 to A.V.. T.M.-B. is supported by Schwarz et al. 2016). In addition, human Siglec-XII and chim- BFU2017-86471-P (MINECO/FEDER, UE), U01 MH106874 panzee Siglec12 are expressed on macrophages and luminal grant, Howard Hughes International Early Career, Obra epithelia (Mitra et al. 2011). However, human Siglec-XII har- Social “La Caixa” and Secretaria d’Universitats i Recerca, bors a universal mutation (R122C) that makes the protein and CERCA Programme del Departament d’Economia i unable to recognize sialic acid. Interestingly, chimpanzee Coneixement de la Generalitat de Catalunya (GRC 2017 Siglec12 and human Siglec-XII with its arginine experimentally SGR 880). restored strongly prefer Neu5Gc (Mitra et al. 2011). These results suggest a scenario where Siglec-12 lost an endoge- nous ligand and is thus being eliminated from the population. Literature Cited Prior studies have reported human-specific Siglec expres- 1000 Genomes Project Consortium, et al. 2015. A global reference for sion changes in placenta (Siglec-6) (Brinkman-Van der Linden human genetic variation. Nature 526(7571):68–74. Aken BL, et al. 2016. The Ensembl gene annotation system. 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