C2-set Domains Research Articles

Optimizing Siglec-8-Directed Immunotherapy for Eosinophilic and Mast Cell Disorders

Background/Objective: Current treatments for eosinophilic and mast cell disorders are often ineffective. One promising target to improve outcomes is sialic acid-binding immunoglobulin-like lectin-8 (Siglec-8). As limitations, there are few Siglec-8 monoclonal antibodies (mAbs) available to date, and Siglec-8-directed treatments have so far primarily focused on unconjugated mAbs, which may be inadequate, especially against mast cells. Methods: Here, we used transgenic mice to raise a diverse panel of fully human mAbs that either recognize the V-set domain, membrane-distal C2-set domain, or membrane-proximal C2-set domain of full-length Siglec-8 as a basis for novel therapeutics. Results: All mAbs were efficiently internalized into Siglec-8-expressing cells, suggesting their potential to deliver cytotoxic payloads. Tool T cell-engaging bispecific antibodies (BiAbs) and chimeric antigen receptor (CAR)-modified natural killer (NK) cells using single-chain variable fragments from Siglec-8 mAbs showed highly potent cytolytic activity against Siglec-8-positive cells even in cases of very low target antigen abundance, whereas they elicited no cytolytic activity against Siglec-8-negative target cells. Siglec-8V-set-directed T cell-engaging BiAbs and Siglec-8V-set-directed CAR-modified NK cells induced substantially greater cytotoxicity against cells expressing an artificial smaller Siglec-8 variant containing only the V-set domain than cells expressing full-length Siglec-8, consistent with the notion that targeting membrane-proximal epitopes enhances effector functions of Siglec-8 antibody-based therapeutics. Indeed, unconjugated Siglec-8C2-set mAbs, Siglec-8C2-set-directed T cell-engaging BiAbs, and Siglec-8C2-set-directed CAR-modified NK cells showed high antigen-specific cytolytic activity against Siglec-8-positive human cell lines and primary patient eosinophils. Conclusions: Together, these data demonstrate Siglec-8-directed immunotherapies can be highly potent, supporting their further development for eosinophilic and mast cell disorders.

A bispecific antibody targeting the Ig domains of Siglec-E displays enhanced antitumor effects

Siglec-9 is a promising immune checkpoint molecule, and therapeutics targeting Siglec-9 have the potential to augment anti-tumor immunity. Here, we generated a bispecific antibody, named as aSE4-1-Fc, by fusing two distinct alpaca derived nanobodies, which can simultaneously target the extracellular Ig variable (V)-set domain and C2-set domains of murine Siglec-9 (also known as Siglec-E) with high affinity. In vivo studies showed that aSE4-1-Fc was better than its component antibodies in inhibiting tumor growth/metastasis, and Siglec-E blockade using aSE4-1-Fc generated protective anti-tumor T cell memory. Furthermore, the combination of aSE4-1-Fc with anti-PD-L1 therapy greatly improved the antitumor effects by augmenting both T and NK cells. Taken together, this study emphasizes the importance of Siglec-9 as a potential cancer therapeutic target, demonstrates the synergistic effect of co-inhibition of Siglec-9 and PD-L1, and may have implications for developing engineered antibodies targeting Siglec-9 with enhanced therapeutic efficacy.

Targeting the membrane-proximal C2-set domain of CD33 for improved CAR T cell therapy

Current CD33-targeted immunotherapies typically recognize the membrane-distal V-set domain of CD33. Here, we show that decreasing the distance between Tcell and leukemia cell membrane increases the efficacy of CD33 chimeric antigen receptor (CAR) Tcells. We therefore generated and optimized second-generation CAR constructs containing single-chain variable fragments from antibodies raised against the membrane-proximal C2-set domain, which bind CD33 regardless of whether the V-set domain is present (CD33PAN antibodies). CD33PAN CAR Tcells resulted in efficient tumor clearance and improved survival of immunodeficient mice bearing human AML cell xenografts and, in an AML model with limited CD33 expression, forced escape of CD33neg leukemia. Compared to CD33V-set CAR Tcells, CD33PAN CAR Tcells showed greater invitro and invivo efficacy against several human AML cell lines withdiffering levels of CD33 without increased expression of exhaustion markers. CD33PAN moieties were detected at a higher frequency on human leukemic stem cells, and CD33PAN CAR Tcells had greater invitro efficacy against primary human AML cells. Together, our studies demonstrate improved efficacy with CAR Tcells binding CD33 close to the cell membrane, providing the rationale to investigate CD33PAN CAR Tcells further toward possible clinical application.

Optimizing Siglec-8-Directed Immunotherapy for Eosinophilic and Mast Cell Disorders

Current treatments for diseases in which eosinophils and/or mast cells play an important pathogenic role are often insufficiently effective. One promising target for these disorders is sialic acid immunoglobulin (Ig)-like lectin 8 (Siglec-8), but there are only few monoclonal antibodies (mAbs) against Siglec-8 available, and, so far, Siglec-8-directed therapies have largely focused on unconjugated mAbs, which may lack efficacy. To raise new human mAbs as basis for novel therapeutics, we immunized transgenic (“humanized”) mice with 3T3 cells expressing human full-length Siglec-8 (Siglec-8 FL) or a chimeric mouse/human protein consisting of the V-set domain of mouse CD33 fused with the membrane-proximal C2-set domain of human Siglec-8 or the V-set domain of mouse Siglec-F fused to the membrane-proximal C2-set domain of human Siglec-8. Among 9 unique mAbs we identified and recombinantly produced, 6 (1B6, 1H4, 1B4, 1E1, 1E4, 1E2) recognized Siglec-8 via binding of the V-set domain, 1 (2A3) via binding of the membrane-distal C2-set domain, and 2 (2A7, 2F10) via binding to the membrane-proximal C2-set domain. All mAbs tested were efficiently internalized into Siglec-8+ cells (up to 75% within 4 hours), suggesting their potential to deliver cytotoxic payloads. To assess the value of Siglec-8-directed immunotherapies engaging T or NK cells, we generated tool T cell-engaging Siglec-8/CD3 bispecific antibodies (BiAbs) in the scFv-scFv format as well as Siglec-8 CAR NK cells, using binding sequences from Siglec-8 V-set mAbs. Very low concentrations (10-25 pM) of a Siglec-8/CD3 BiAb built on 1B6 elicited dose-dependent cytolytic activity against EOL-1, HL-60 as well as LUVA cells transduced with Siglec-8 FL. This BiAb also exerted Siglec-8-specific cytotoxicity against HMC 1.2 cells that were enriched for Siglec-8 expression as well as parental LUVA cells, which both display very few Siglec-8 molecules. Similarly, KHYG-1 cells transduced with a Siglec-8 V-set-directed 41BB co-stimulatory domain-containing CAR generated from scFv sequences from 1B6 or 1H4 induced significant cytotoxicity against EOL-1 and RS4;11 cells transduced with Siglec-8 FL. To examine the relationship between the efficacy of T cell and NK cell-engaging therapies and the distance from the target epitope on Siglec-8 to the cell membrane, we engineered human AML and ALL cell line pairs expressing comparable levels of either Siglec-8 FL or an artificial variant of Siglec-8 containing only the V-set domain on the extracellular portion of the protein (Siglec-8 V-set) to bring the V-set domain closer to the cell membrane, and used these cell line pairs as targets for Siglec-8 V-set-directed BiAbs and CAR-modified immune effector cells. Siglec-8 V-set/CD3 BiAbs exerted greater cytotoxicity against AML and ALL cells expressing Siglec-8 V-set than cells expressing Siglec-8 FL. Likewise, Siglec-8 V-set-directed CAR NK cells exerted greater cytotoxicity against AML and ALL cells expressing Siglec-8 V-set than cells expressing Siglec-8 FL, demonstrating that altering the position of the Siglec-8 mAb binding epitope changes the effector functions of Siglec-8 mAb-derived therapies and suggesting membrane-proximal targeting via one of the C2-set domains could enhance Siglec-8-targeted therapies that engage T or NK cells as immune effector cells. To test whether mAbs recognizing one of the C2-set domains could indeed serve as basis for potent therapeutics, we generated a Siglec-8 C2-set/CD3 BiAb in the scFv-scFv format using binding sequences from 2A3 and found it effectively killed EOL-1, ML-1, RS4;11, and LUVA cells transduced with human Siglec-8 FL at very low concentrations. This BiAb also exerted Siglec-8-specific cytotoxicity against Siglec-8-enriched HMC 1.2 cells and parental LUVA cells, which both display Siglec-8 at very low copy number. Likewise, KHYG-1 cells transduced with a Siglec-8 C2-set CAR generated from 2A3 or 2F10 induced significant cytotoxicity against EOL-1 and RS4;11 cells transduced with Siglec-8 FL as well as against Siglec-8-enriched HMC 1.2 cells, albeit to a very modest extent. Together, our data demonstrate Siglec-8-directed immunotherapies can be highly potent, even against target cells expressing very limited copy numbers of Siglec-8 molecules, supporting their further development for use in patients with difficult-to-treat eosinophilic and mast cell disorders.

Sialoadhesin (CD169/Siglec-1) is an extended molecule that escapes inhibitory cis-interactions and synergizes with other macrophage receptors to promote phagocytosis

Sialoadhesin (CD169/Siglec-1, Sn) is a macrophage receptor that interacts with sialic acids on both host cells and pathogens. It is a type 1 membrane protein with an unusually large number of 17 extracellular immunoglobulin (Ig)-like domains, made up of an N-terminal V-set domain that binds sialic acid and 16 adjacent C2-set domains. The potential importance of 17 Ig domains in Sn for mediating cellular interactions has not been investigated experimentally. In the present study, Chinese Hamster Ovary (CHO) cells were stably transfected with full-length or truncated forms of Sn. Using human red blood cells (RBC) as a model system, CHO cells expressing truncated forms of Sn with 4 or less Ig domains were unable to bind RBC in comparison to the full-length protein. Immunoelectron microscopy of the CHO cells indicated that full-length Sn extends ~ 33 nm from the plasma membrane compared with ~ 14 nm for a truncated form with 6 N-terminal Ig domains. Co-expresssion of Sn-expressing CHO cells with heavily glycosylated membrane proteins of differing predicted lengths resulted in selective modulation of Sn-dependent binding to RBC and supported the hypothesis that Sn has evolved 17 Ig domains to escape inhibitory cis-interactions. The functional significance of the extended length of Sn was demonstrated in experiments with macrophages showing that Sn synergizes with phagocytic receptors FcR and TIM-4 to strongly promote uptake of IgG-opsonized and eryptotic RBC respectively.

Targeting the Membrane-Proximal C2-Set Domain of CD33 for Improved CD33-Directed CAR T Cell Therapy

Background: There is increasing interest in targeting CD33 in malignant and non-malignant disorders, but available drugs are ineffective in many patients. As one limitation, therapeutic CD33 antibodies typically recognize the membrane-distal V-set domain. Likewise, currently tested CD33-directed chimeric antigen receptor (CAR) T cells likewise target the V-set domain and have thus far shown limited clinical activity. We have recently demonstrated that binding closer to the cell membrane enhances the effector functions of CD33 antibodies. We therefore raised antibodies against the membrane-proximal C2-set domain of CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain (“CD33 PAN antibodies”). Here, we tested their properties as targeting moiety in CD33 PAN CAR T cell constructs, using a clinically validated lentiviral backbone.Methods: To generate CAR T cells, negatively selected CD8 + T cells were transduced with an epHIV7 lentivirus encoding the scFv from a CD33 PAN antibody (clone 1H7 or 9G2) linked to either a short (IgG 4 hinge only), intermediate (hinge plus IgG 4 CH3 domain), or long (hinge plus IgG 4 CH3 domain plus IgG 4 CH2 domain) spacer, the CD28-transmembrane domain, CD3zeta and 4-1BB intracellular signaling domains, and non-functional truncated CD19 (tCD19) as transduction marker. Similar constructs using scFvs from 2 different V-set domain-targeting CD33 antibodies, including hP67.6 (My96; used in gemtuzumab ozogamicin), were generated for comparison. CAR-T cells were sorted, expanded in IL-7 and IL-15, and used in vitro or in vivo against human AML cell lines endogenously expressing CD33 and cell lines engineered to lack CD33 (via CRISPR/Cas9) with/or without forced expression of different CD33 variants.Results: CD33 V-set-directed CAR T cells exerted significantly more cytolytic activity against AML cells expressing an artificial CD33 variant lacking the C2-set domain (CD33 ΔE3-4) than cells expressing full-length CD33 at similar or higher levels, consistent with the notion that CD33 CAR T cell efficacy is enhanced when targeting an epitope that is located closer to the cell membrane. CD33 PAN CAR T cells were highly potent against human AML cells in a strictly CD33-dependent fashion, with constructs containing the short and intermediate-length spacer demonstrating robust cytokine secretion, cell proliferation, and in vitro cytolytic activity, as determined by 51Cr release cytotoxicity assays. When compared to optimized CD33 V-set CAR T cells, optimized CD33 PAN CAR T cells were significantly more potent in cytotoxicity, proliferation, and cytokine production without appreciably increased acquisition of exhaustion markers. In vivo, CD33 PAN CAR T cells extended survival in immunodeficient NOD.SCID. IL2rg -/- (NSG) mice bearing significant leukemic burdens from various cell line-derived xenografts (HL-60, KG1α and MOLM14) with efficient tumor clearance demonstrated in a dose-dependent fashion.Conclusion: Targeting the membrane proximal domain of CD33 enhances the anti-leukemic potency of CAR T cells. Our data provide the rationale for the further development of CD33 PAN CAR T cells toward clinical testing. DisclosuresFiorenza: Link Immunotherapeutics: Consultancy; Bristol Myers Squibb: Research Funding. Godwin: Pfizer: Research Funding; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Turtle: Allogene: Consultancy; Amgen: Consultancy; Arsenal Bio: Consultancy; Asher bio: Consultancy; Astrazeneca: Consultancy, Research Funding; Caribou Biosciences: Consultancy, Current holder of individual stocks in a privately-held company; Century Therapeutics: Consultancy, Other; Eureka therapeutics: Current holder of individual stocks in a privately-held company, Other; Juno therapeutics/BMS: Patents & Royalties, Research Funding; Myeloid Therapeutics: Current holder of individual stocks in a privately-held company, Other; Nektar therapeutics: Consultancy, Research Funding; PACT Pharma: Consultancy; Precision Biosciences: Current holder of individual stocks in a privately-held company, Other; T-CURX: Other; TCR2 Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

Evolutionary conservation of intrinsically unstructured regions in slit-diaphragm proteins.

Vertebrate kidneys contribute to homeostasis by regulating electrolyte, acid-base balance, removing toxic metabolites from blood, and preventing protein loss into the urine. Glomerular podocytes constitute the blood-urine barrier, and podocyte slit-diaphragm (SD), a modified tight junction, contributes to the glomerular permselectivity. Nephrin, KIRREL1, podocin, CD2AP, and TRPC6 are crucial members of the SD that interact with each other and contribute to the SD’s structural and functional integrity. This study analyzed the distribution of these five essential SD proteins across the organisms for which the genome sequence is available. We found a diverse distribution of nephrin and KIRREL1 ranging from nematodes to higher vertebrates, whereas podocin, CD2AP, and TRPC6 are restricted to the vertebrates. Among invertebrates, nephrin and its orthologs consist of more immunoglobulin-3 domains, whereas in the vertebrates, CD80-like C2-set domains are predominant. In the case of KIRREL1 and its orthologs, more Ig domains were observed in invertebrates than vertebrates. Src Homology-3 (SH3) domain of CD2AP and SPFH domain of podocin are highly conserved among vertebrates. TRPC6 and its orthologs had conserved ankyrin repeats, TRP, and ion transport domains, except Chondrichthyes and Echinodermata, which do not possess the ankyrin repeats. Intrinsically unstructured regions (IURs) are conserved across the SD orthologs, suggesting IURs importance in the protein complexes that constitute the slit-diaphragm. For the first time, a study reports the evolutionary insights of vertebrate SD proteins and their invertebrate orthologs.

Targeting the membrane-proximal C2-set domain of CD33 for improved CD33-directed immunotherapy.

There is increasing interest in targeting CD33 in malignant and non-malignant disorders. In acute myeloid leukemia, longer survival with the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) validates this strategy. Still, GO benefits only some patients, prompting efforts to develop more potent CD33-directed therapeutics. As one limitation, CD33 antibodies typically recognize the membrane-distal V-set domain. Using various artificial CD33 proteins, in which this domain was differentially positioned within the extracellular portion of the molecule, we tested whether targeting membrane-proximal targeting epitopes enhances the effector functions of CD33 antibody-based therapeutics. Consistent with this idea, a CD33V-set/CD3 bispecific antibody (BsAb) and CD33V-set-directed chimeric antigen receptor (CAR)-modified T cells elicited substantially greater cytotoxicity against cells expressing a CD33 variant lacking the entire C2-set domain than cells expressing full-length CD33, whereas cytotoxic effects induced by GO were independent of the position of the V-set domain. We therefore raised murine and human antibodies against the C2-set domain of human CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain (“CD33PAN antibodies”). These antibodies internalized when bound to CD33 and, as CD33PAN/CD3 BsAb, had potent cytolytic effects against CD33+ cells. Together, our data provide rationale for further development of CD33PAN antibody-based therapeutics.

Human-specific microglial Siglec-11 transcript variant has the potential to affect polysialic acid-mediated brain functions at a distance.

CD33-related Siglecs are often found on innate immune cells and modulate their reactivity by recognition of sialic acid-based "self-associated molecular patterns" and signaling via intracellular tyrosine-based cytosolic motifs. Previous studies have shown that Siglec-11 specifically binds to the brain-enriched polysialic acid (polySia/PSA) and that its microglial expression in the brain is unique to humans. Furthermore, human microglial Siglec-11 exists as an alternate splice form missing the exon encoding the last (fifth) Ig-like C2-set domain of the extracellular portion of the protein, but little is known about the functional consequences of this variation. Here, we report that the recombinant soluble human microglial form of Siglec-11 (hSiglec-11(4D)-Fc) binds endogenous and immobilized polySia better than the tissue macrophage form (hSiglec-11(5D)-Fc) or the chimpanzee form (cSiglec-11(5D)-Fc). The Siglec-11 protein is also prone to aggregation, potentially influencing its ligand-binding ability. Additionally, Siglec-11 protein can be secreted in both intact and proteolytically cleaved forms. The microglial splice variant has reduced proteolytic release and enhanced incorporation into exosomes, a process that appears to be regulated by palmitoylation of cysteines in the cytosolic tail. Taken together, these data demonstrate that human brain specific microglial hSiglec-11(4D) has different molecular properties and can be released on exosomes and/or as proteolytic products, with the potential to affect polySia-mediated brain functions at a distance.

Unusual Properties of a Human‐specific and Microglia‐specific Siglec‐11 Transcript Variant

CD33‐related sialic acid‐binding immunoglobulin‐type lectins (Siglecs) are often found on innate immune cells and can modulate their reactivity. Previous studies showed that expression of the inhibitory Siglec‐11 in microglia is unique to humans and exists as an alternate splice form missing the exon encoding the last (5th) Ig‐like C2 set domain of the extracellular portion of the protein. Moreover, the brain‐enriched glycan polysialic acid (polySia/PSA) has been considered as a ligand of Siglec‐11. Here, we show that the human recombinant soluble microglial form hSiglec‐11(4D)‐Fc binds better to immobilized polySia than the tissue macrophage form hSiglec‐11(5D)‐Fc or chimpanzee cSiglec‐11(5D)‐Fc. We found that both intact and cleaved Siglec‐11 are in the culture medium, suggesting their ability to be secreted. Specifically, intact Siglec‐11 is found in exosomes and the microglial splice variant has reduced proteolytic cleavage and enhanced incorporation into exosomes. The exosomes bind to human neuroblastoma in a polySia‐dependent fashion. Mechanistically, cysteine palmitoylation is observed in the intracellular region and may regulate Siglec‐11 localization on lipid rafts and secretion on exosomes. Taken together, we demonstrate that hSiglec‐11(4D) is specifically expressed in human brain microglia and is released on the exosomes and/or proteolytic products, which have the potential to affect brain functions at a distance.Support or Funding InformationNational Institute of General Medical Sciences (NIGMS) R01GM32373

Siglec-7 mediates nonapoptotic cell death independently of its immunoreceptor tyrosine-based inhibitory motifs in monocytic cell line U937

Siglec-7, a sialic acid binding immunoglobulin-like lectin, predominantly transduces inhibitory signals through cytosolic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Here, we report that clustering of Siglec-7 with a specific F(ab')(2) elicited cell death. Interestingly, a truncated Siglec-7 lacking the cytosolic ITIM domain still induced the cell death, suggesting that the ITIMs are not essential for the death signaling. Further analyses of the death signaling revealed that an oxygen radical scavenger, N-acetyl cysteine, completely inhibited the cell death, whereas a pancaspase inhibitor did not. In addition, caspase-3 activation, DNA ladder formation, and nuclear condensation were not detected during the death process, suggesting that the cell death is nonapoptotic. To identify the critical region for the death signaling, we prepared a series of shuffling chimeras between Siglec-7 and Siglec-9, the latter of which did not transduce a death signal. The critical region was mapped to the middle of the membrane-proximal C2-set domain, which contained only six amino acid differences between Siglec-7 and Siglec-9. Point mutation analyses of each of these six amino acids revealed that four of the six amino acids were critical for the death signal. A computer-assisted 3D modeling revealed that these four amino acids were proximally located on the surface of the C2-set domain. In conclusion, Siglec-7 induces nonapoptotic cell death, the signal for which is transduced by an extracellular C2-set domain.

Impact of Salt Bridges on the Equilibrium Binding and Adhesion of Human CD2 and CD58

This study describes quantitative investigations of the impact of single charge mutations on equilibrium binding, kinetics, and the adhesion strength of the CD2-CD58 interaction. Previously steered molecular dynamics simulations guided the selection of the charge mutants investigated, which include the CD2 mutants D31A, K41A, K51A, and K91A. This set includes mutations in which the previous cell aggregation and binding data either agreed or disagreed with the steered molecular dynamics predictions. Surface plasmon resonance measurements quantified the solution binding properties. Adhesion was quantified with the surface force apparatus, which was used previously to study the closely related CD2-CD48 interaction. The results reveal roles that these salt bridges play in equilibrium binding and adhesion. We discuss both the molecular basis of this behavior and its implications for cell adhesion.

Evolution of the human killer cell inhibitory receptor family

Phylogenetic analysis of different domains of human natural killer cell inhibitory receptors (KIR) implicated both intragenic duplication and deletion of exons and interlocus recombination in the evolution of these receptors. In phylogenies of the extracellular immunoglobulin (Ig) superfamily C2-set domains and of the pre-membrane (PM) domain, KIR receptors having two C2-set domains and those having three such domains tended to form separate clusters. However, the phylogenies of the transmembrane (TM) and cytoplasmic (CYT) domains showed quite different topologies, suggesting that major sites of interlocus recombination have been between exon 6 (encoding PM) and exon 7 (encoding TM) and between exon 7 and exons 8–9 (encoding CYT). Examination of the pattern of nucleotide substitution in the exons encoding Ig C2-set domains supported the hypothesis that positive Darwinian selection has acted to diversify the residues within these domains that are involved in contact with class I MHC molecules.

Genomic organization of the siglec gene locus on chromosome 19q13.4 and cloning of two new siglec pseudogenes

The sialic acid binding immunoglobulin-like lectin (Siglec) family of genes is a recently described member of the immunoglobulin superfamily. Within this Siglec family there is a subgroup of genes which bear a high degree of homology with Siglec-3 (CD33), thus designated the Siglec-3-like subgroup of Siglecs. While their mRNA structure has been reported, the full genomic organization of these genes, is not known. Genes of this subgroup have been mapped to chromosome 19q13.4, primarily through in situ hybridization. Through analysis of several bacterial artificial chromosome (BAC) clones, we studied an approximate 700 kb region that encompasses the putative Siglec gene locus on chromosome 19q13.4. We established the first detailed map of the locus, which contains 8 Siglec and Siglec-like genes. Our map shows the relative position of all genes and the precise distances between them, along with the direction of transcription of each gene. To our knowledge, this is the first report that describes the full genomic organization of all members of the CD33-like subgroup of Siglecs, including the promoter sequences of all genes. Members of this subfamily exhibit two patterns of organization of the signal peptide, which is followed by one V-set domain (except for the long form of the siglecL1 gene). Exons containing the C2-set domains are all comparable in size and are separated by linker exons. The transmembrane domain is encoded for by a separate exon of almost the same size in all genes. The total number of exons differs according to the number of C2-set Ig domains, but intron phases are identical. The cytoplasmic domain is always encoded by two exons. We further identified two new Siglec pseudogenes in this locus, and analyzed their tissue expression pattern and their structural features.

New functions for the sialic acid-binding adhesion molecule CD22, a member of the growing family of Siglecs.

New functions for the sialic acid-binding adhesion molecule CD22, a member of the growing family of Siglecs.

Siglec-9, a Novel Sialic Acid Binding Member of the Immunoglobulin Superfamily Expressed Broadly on Human Blood Leukocytes

Here we characterize the properties and expression pattern of Siglec-9 (sialic acid-binding Ig-like lectin-9), a new member of the Siglec subgroup of the immunoglobulin superfamily. A full-length cDNA encoding Siglec-9 was isolated from a dibutyryl cAMP-treated HL-60 cell cDNA library. Siglec-9 is predicted to contain three extracellular immunoglobulin-like domains that comprise an N-terminal V-set domain and two C2-set domains, a transmembrane region and a cytoplasmic tail containing two putative tyrosine-based signaling motifs. Overall, Siglec-9 is approximately 80% identical in amino acid sequence to Siglec-7, suggesting that the genes encoding these two proteins arose relatively recently by gene duplication. Binding assays showed that, similar to Siglec-7, Siglec-9 recognized sialic acid in either the alpha2,3- or alpha2, 6-glycosidic linkage to galactose. Using a specific mAb, Siglec-9 was found to be expressed at high or intermediate levels by monocytes, neutrophils, and a minor population of CD16(+), CD56(-) cells. Weaker expression was observed on approximately 50% of B cells and NK cells and minor subsets of CD8(+) T cells and CD4(+) T cells. These results show that despite their high degree of sequence similarity, Siglec-7 and Siglec-9 have distinct expression profiles.

Identification and Characterization of a Novel Siglec, Siglec-7, Expressed by Human Natural Killer Cells and Monocytes

We describe the characterization of sialic acid-binding Ig-like lectin-7 (siglec-7), a novel member of the siglec subgroup of the immunoglobulin superfamily. A full-length cDNA encoding siglec-7 was isolated from a human primary dendritic cell cDNA library. Siglec-7 is predicted to contain three extracellular immunoglobulin-like domains that comprise an N-terminal V-set domain and two C2-set domains, a transmembrane region and a cytoplasmic tail containing two tyrosine residues embodied in immunoreceptor tyrosine-based inhibition motif-like motifs. Overall, siglec-7 exhibited a high degree of sequence similarity to genes encoding CD33 (siglec-3), siglec-5, OBBP1/siglec-6, and OBBP-like protein and mapped to the same region on chromosome 19q13.3. When siglec-7 was expressed on COS or Chinese hamster ovary cells, it was able to mediate high levels of sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates, suggesting that it may be involved in cell-cell interactions. Among human peripheral blood leukocytes, siglec-7 was found to be present at low levels on granulocytes, intermediate levels on monocytes, and relatively high levels on a major subset of natural killer cells and a minor subset of CD8(+) T cells. Immunoprecipitation experiments indicated that siglec-7 is expressed as a monomer of approximately 65 kDa.

Gene duplication and recombination in the evolution of mammalian Fc receptors.

The immunoglobulin-related chains of cell-surface receptors for the Fc region of immunoglobulins (FcepsilonRIalpha, FcgammaRI, FcgammaRII, and FcgammaRIIIalpha) are encoded by members of a gene family. Phylogenetic analysis of representative members of this family from mammals revealed that FcgammaRIIIalpha genes of human, mouse, and rat are not orthologous to one another in the region of the gene encoding the immunoglobulin C2-set domains. In phylogenetic trees of this region, FcgammaRIIIalpha and FcgammaRII clustered together. However, in trees based on both coding and noncoding regions 5' and 3' to the C2 domains, FcgammaRIIIalpha genes of human, mouse, and rat clustered together. This pattern of relationship is most easily explained as a result of two independent recombinational events occurring in the mouse and rat after these two species diverged, in each of which the exons encoding the C2 domains were donated to an FcgammaRIIIalpha gene by an FcgammaRII gene.

The Amino-terminal Immunoglobulin-like Domain of Sialoadhesin Contains the Sialic Acid Binding Site: COMPARISON WITH CD22

Sialoadhesin and CD22 are members of a recently characterized family of sialic acid-dependent adhesion molecules belonging to the immunoglobulin superfamily. Sialoadhesin is a macrophage-restricted receptor containing 17 extracellular Ig-like domains which recognizes oligosaccharides terminating in NeuAc alpha 2-3Gal in N- and O-linked glycans. CD22 is a B cell-restricted receptor with seven Ig-like domains which selectively recognizes oligosaccharides terminating in NeuAc alpha 2-6Gal in N-glycans. Sequence similarity between these proteins is highest within their first four amino-terminal Ig-like domains. Here we identify the domain(s) containing the binding sites of both molecules by generating a series of extracellular domain deletion mutants fused to the Fc portion of human IgG1. Binding activity was analyzed by solid phase cell adhesion assays and also by surface plasmon resonance using purified glycophorin and CD45 as ligands for sialoadhesin and CD22, respectively. For sialoadhesin, the amino-terminal V-set Ig-like domain was both necessary and sufficient to mediate sialic acid-dependent adhesion of the correct specificity. In contrast, for murine CD22, only constructs containing both the V-set domain and the adjacent C2-set domain were able to mediate sialic acid-dependent binding. These results are consistent with the sialic acid binding site for both proteins residing in the membrane distal V-set domain, but for CD22 a direct contribution in binding from the neighboring C2-set domain cannot be excluded.