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Ajit Varki

Publications and source records attributed to Ajit Varki.

At least 19 recordsLinked to original sources
PubMed2006

Group B streptococcal capsular sialic acids interact with siglecs (immunoglobulin-like lectins) on human leukocytes.

Group B Streptococcus (GBS) is classified into nine serotypes that vary in capsular polysaccharide (CPS) architecture but share in common the presence of a terminal sialic acid (Sia) residue. This position and linkage of GBS Sia closely resembles that of cell surface glycans found abundantly on human cells. CD33-related Siglecs (CD33rSiglecs) are a family of Sia-binding lectins expressed on host leukocytes that engage host Sia-capped glycans and send signals that dampen inflammatory gene activation. We hypothesized that GBS evolved to display CPS Sia as a form of molecular mimicry limiting the activation of an effective innate immune response. In this study, we applied a panel of immunologic and cell-based assays to demonstrate that GBS of several serotypes interacts in a Sia- and serotype-specific manner with certain human CD33rSiglecs, including hSiglec-9 and hSiglec-5 expressed on neutrophils and monocytes. Modification of GBS CPS Sia by O acetylation has recently been recognized, and we further show that the degree of O acetylation can markedly affect the interaction between GBS and hSiglec-5, -7, and -9. Thus, production of Sia-capped bacterial polysaccharide capsules that mimic human cell surface glycans in order to engage CD33rSiglecs may be an example of a previously unrecognized bacterial mechanism of leukocyte manipulation.

Animals↗
PubMed2006

System-wide genomic and biochemical comparisons of sialic acid biology among primates and rodents: Evidence for two modes of rapid evolution.

Numerous vertebrate genes are involved in the biology of the oligosaccharide chains attached to glycoconjugates. These genes fall into diverse groups within the conventional Gene Ontology classification. However, they should be evaluated together from functional and evolutionary perspectives in a "biochemical systems" approach, considering each monosaccharide unit's biosynthesis, activation, transport, modification, transfer, recycling, degradation, and recognition. Sialic acid (Sia) residues are monosaccharides at the outer end of glycans on the cell-surface and secreted molecules of vertebrates, mediating recognition by intrinsic or extrinsic (pathogen) receptors. The availability of multiple genome sequences allows a system-wide comparison among primates and rodents of all genes directly involved in Sia biology. Taking this approach, we present further evidence for accelerated evolution in Sia-binding domains of CD33-related Sia-recognizing Ig-like lectins. Other gene classes are more conserved, including those encoding the sialyltransferases that attach Sia residues to glycans. Despite this conservation, tissue sialylation patterns are shown to differ widely among these species, presumably because of rapid evolution of sialyltransferase expression patterns. Analyses of N- and O-glycans of erythrocyte and plasma glycopeptides from these and other mammalian taxa confirmed this phenomenon. Sia modifications on these glycopeptides also appear to be undergoing rapid evolution. This rapid evolution of the sialome presumably results from the ongoing need of organisms to evade microbial pathogens that use Sia residues as receptors. The rapid evolution of Sia-binding domains of the inhibitory CD33-related Sia-recognizing Ig-like lectins is likely to be a secondary consequence, as these inhibitory receptors presumably need to keep up with recognition of the rapidly evolving "self"-sialome.

Amino Acid Sequence↗
PubMed2006

Loss of Siglec expression on T lymphocytes during human evolution.

We report here that human T cells give much stronger proliferative responses to specific activation via the T cell receptor (TCR) than those from chimpanzees, our closest evolutionary relatives. Nonspecific activation using phytohemagglutinin was robust in chimpanzee T cells, indicating that the much lower response to TCR simulation is not due to any intrinsic inability to respond to an activating stimulus. CD33-related Siglecs are inhibitory signaling molecules expressed on most immune cells and are thought to down-regulate cellular activation pathways via cytosolic immunoreceptor tyrosine-based inhibitory motifs. Among human immune cells, T lymphocytes are a striking exception, expressing little to none of these molecules. In stark contrast, we find that T lymphocytes from chimpanzees as well as the other closely related "great apes" (bonobos, gorillas, and orangutans) express several CD33-related Siglecs on their surfaces. Thus, human-specific loss of T cell Siglec expression occurred after our last common ancestor with great apes, potentially resulting in an evolutionary difference with regard to inhibitory signaling. We confirmed this by studying Siglec-5, which is prominently expressed on chimpanzee lymphocytes, including CD4 T cells. Ab-mediated clearance of Siglec-5 from chimpanzee T cells enhanced TCR-mediated activation. Conversely, primary human T cells and Jurkat cells transfected with Siglec-5 become less responsive; i.e., they behave more like chimpanzee T cells. This human-specific loss of T cell Siglec expression associated with T cell hyperactivity may help explain the strikingly disparate prevalence and severity of T cell-mediated diseases such as AIDS and chronic active hepatitis between humans and chimpanzees.

Animals↗
PubMed2006

The group B streptococcal sialic acid O-acetyltransferase is encoded by neuD, a conserved component of bacterial sialic acid biosynthetic gene clusters.

Nearly two dozen microbial pathogens have surface polysaccharides or lipo-oligosaccharides that contain sialic acid (Sia), and several Sia-dependent virulence mechanisms are known to enhance bacterial survival or result in host tissue injury. Some pathogens are also known to O-acetylate their Sias, although the role of this modification in pathogenesis remains unclear. We report that neuD, a gene located within the Group B Streptococcus (GBS) Sia biosynthetic gene cluster, encodes a Sia O-acetyltransferase that is itself required for capsular polysaccharide (CPS) sialylation. Homology modeling and site-directed mutagenesis identified Lys-123 as a critical residue for Sia O-acetyltransferase activity. Moreover, a single nucleotide polymorphism in neuD can determine whether GBS displays a "high" or "low" Sia O-acetylation phenotype. Complementation analysis revealed that Escherichia coli K1 NeuD also functions as a Sia O-acetyltransferase in GBS. In fact, NeuD homologs are commonly found within Sia biosynthetic gene clusters. A bioinformatic approach identified 18 bacterial species with a Sia biosynthetic gene cluster that included neuD. Included in this list are the sialylated human pathogens Legionella pneumophila, Vibrio parahemeolyticus, Pseudomonas aeruginosa, and Campylobacter jejuni, as well as an additional 12 bacterial species never before analyzed for Sia expression. Phylogenetic analysis shows that NeuD homologs of sialylated pathogens share a common evolutionary lineage distinct from the poly-Sia O-acetyltransferase of E. coli K1. These studies define a molecular genetic approach for the selective elimination of GBS Sia O-acetylation without concurrent loss of sialylation, a key to further studies addressing the role(s) of this modification in bacterial virulence.

Acetyltransferases↗
PubMed2006

Multiple hepatic receptors cooperate to eliminate secretory mucins aberrantly entering the bloodstream: are circulating cancer mucins the "tip of the iceberg"?

Hollow organs lined by columnar epithelial cells normally secrete mucins and their proteolytic fragments vectorially into the lumen. These heterogeneously O-glycosylated molecules are known to aberrantly enter the bloodstream in the setting of epithelial carcinomas and possibly during injury or inflammation. We have recently shown that carcinoma mucin fragments can trigger the rapid formation of platelet-rich microthrombi in vivo. Thus, mechanisms to clear such aberrantly secreted mucins must exist. Indeed, we found that i.v. injected carcinoma mucin fragments had an approximately 1 minute half-life in mice, which was primarily due to rapid clearance by hepatic reticuloendothelial cells. Inhibition of known glycan-recognizing hepatic clearance receptors showed involvement of multiple partially overlapping clearance systems. Studies of genetically deficient mice and incomplete competition between different mucins confirmed this result. Thus, multiple hepatic clearance receptors cooperate to eliminate secretory mucins entering the circulation, limiting potential pathology. This may also explain why mucin-type clustered O-glycosylation is rare on plasma proteins. Notably, small subsets of injected carcinoma mucins remained unrecognized by clearance systems, had a much longer half-life, and carried highly sialylated O-glycans. Similar circulating mucins were found in tumor-bearing mice despite lack of saturation of hepatic clearance mechanisms. Thus, circulating cancer mucins currently used as clinical diagnostic markers likely represent only the clearance-resistant "tip of the iceberg." Such aberrantly circulating mucins could play pathologic roles not only in cancer but also during injury or inflammation of hollow organs and in liver disease.

Adenocarcinoma↗
PubMed2006

L-selectin facilitation of metastasis involves temporal induction of Fut7-dependent ligands at sites of tumor cell arrest.

Hematogenous carcinoma metastasis is supported by aggregated platelets and leukocytes, forming tumor cell emboli. Early tumor cell-platelet interactions can be mediated by P-selectin binding to tumor cell surface ligands and this process is blocked by heparin. We previously showed that L-selectin deficiency also attenuates experimental metastasis. However, the mechanisms and timing of L-selectin action remained unknown. Here, we study how L-selectin facilitates establishment of pulmonary metastatic foci in syngeneic mice by using experimental metastasis to time events following entry of tumor cells into the bloodstream. Although L-selectin deficiency did not affect platelet aggregation or initial tumor cell embolization, the association of leukocytes with tumor cells was reduced and tumor cell survival was diminished 24 hours later. Temporal inhibition of L-selectin by a function-blocking antibody reduced metastasis. Moreover, although selectin blockade by heparin 6 to 18 hours after tumor cell injection was synergistic with P-selectin deficiency in reducing metastasis, there was no further effect in L-selectin-deficient animals. Thus, heparin apparently works at these time points primarily by blocking L-selectin. Endogenous L-selectin ligands were concomitantly induced adjacent to established intravascular tumor cell emboli in a similar time window when leukocytes were also present. Metastasis was attenuated in mice missing these induced endogenous L-selectin ligands due to fucosyltransferase-7 deficiency. Thus, L-selectin facilitation of metastasis progression involves leukocyte-endothelial interactions at sites of intravascular arrest supported by local induction of L-selectin ligands via fucosyltransferase-7. These data provide the first explanation for how leukocyte L-selectin facilitates tumor metastasis.

Animals↗
PubMed2006

9-O-acetylation of exogenously added ganglioside GD3. The GD3 molecule induces its own O-acetylation machinery.

Sialic acids are sometimes 9-O-acetylated in a developmentally regulated and cell-type-specific manner. Cells naturally expressing the disialoganglioside GD3 often O-acetylate the terminal sialic acid residue, giving 9-O-acetyl-GD3 (9AcGD3), a marker of neural differentiation and malignant transformation. We also reported that Chinese hamster ovary cells transfected with GD3 synthase can spontaneously O-acetylate some of the newly synthesized GD3. It is unclear whether such phenomena result from induction of the 9-O-acetylation machinery and whether induction is caused by the GD3 synthase protein or by the GD3 molecule itself. We now show that exogenously added GD3 rapidly incorporates into the plasma membrane of Chinese hamster ovary cells, and 9AcGD3 is detected after approximately 6 h. The incorporated GD3 and newly synthesized 9AcGD3 have a half-life of approximately 24 h. This phenomenon is also seen in other cell types, such as human diploid fibroblasts. Inhibitors of gene transcription, protein translation, or endoplasmic reticulum-to-Golgi transport each prevent induction of 9-O-acetylation, without affecting GD3 incorporation. Inhibition of the initial clathrin-independent internalization of incorporated GD3 also blocks induction of 9-O-acetylation. Thus, new synthesis of one or more components of the 9-O-acetylation machinery is induced by incorporation and internalization of GD3. Prepriming with structurally related gangliosides fails to accelerate the onset of 9-O-acetylation of subsequently added GD3, indicating a requirement for specific recognition of GD3. To our knowledge, this is the first example wherein a newly expressed or exogenously introduced ganglioside induces de novo synthesis of an enzymatic machinery to modify itself, and the first evidence for a mechanism of induction of sialic acid O-acetylation.

Acetylation↗
PubMed2006

Myeloid precursors and acute myeloid leukemia cells express multiple CD33-related Siglecs.

OBJECTIVES: CD33 is a cell surface marker of committed myelomonocytic precursors and circulating monocytes, and is also found on acute myeloid leukemia (AML) cells. CD33 belongs to a family of sialic acid-binding cell surface proteins named Siglecs, among which there are 7 other functional CD33-related Siglecs (CD33rSiglecs). We sought to characterize the spectrum of expression of the other CD33rSiglecs on bone marrow precursors and AML cells and asked if they can potentially serve as targets for therapy. METHODS: Cell surface CD33rSiglecs were analyzed by flow cytometry. The ability of certain anti-Siglec antibodies to target toxin-mediated cell killing of Siglec-expressing cell lines was characterized and compared. RESULTS: We demonstrate that Siglecs-3, -5, -6, -7, and -9 are expressed on subsets of normal bone marrow precursors, including promonocytes and myelocytes. Furthermore, most AML (but not ALL) cells express these Siglecs. There is substantial variability in Siglec type and expression level between cases, with each having a unique "CD33rSiglec fingerprint." Individual anti-Siglec antibodies along with a saporin toxin-conjugated secondary antibody can target myelomonocytic leukemia cells for death, and targeting of multiple Siglecs improves cell killing. Cytotoxicity was further enhanced by sialidase treatment of target cells, which improves antibody binding. We also confirmed that antibody binding induced rapid internalization of Siglecs from the cell surface, which is a requirement for cell killing via saporin. CONCLUSIONS: Multiple CD33rSiglecs are expressed on normal and malignant myelomonoyctic cells. Targeting these Siglecs, possibly in combinations, could improve anti-CD33 antibody therapy or be used as an alternative to anti-CD33.

Antibodies, Monoclonal↗
PubMed2006

Discovery of Siglec-14, a novel sialic acid receptor undergoing concerted evolution with Siglec-5 in primates.

Immune receptors that show high mutual sequence similarity and have antagonizing signaling properties are called paired receptors, and are believed to fine-tune immune responses. Siglecs are sialic acid-recognizing receptors of the immunoglobulin (Ig) superfamily expressed on immune cells. Human Siglec-5, encoded by SIGLEC5 gene, has four extracellular Ig-like domains and a cytosolic inhibitory motif. We discovered human Siglec-14 with three Ig-like domains, encoded by the SIGLEC14 gene, adjacent to SIGLEC5. Human Siglec-14 has almost complete sequence identity with human Siglec-5 at the first two Ig-like domains, shows a glycan binding preference similar to that of human Siglec-5, and associates with the activating adapter protein DAP12. Thus, Siglec-14 and Siglec-5 appear to be the first glycan binding paired receptors. Near-complete sequence identity of the amino-terminal part of human Siglec-14 and Siglec-5 indicates partial gene conversion between SIGLEC14 and SIGLEC5. Remarkably, SIGLEC14 and SIGLEC5 in other primates also show evidence of gene conversions within each lineage. Evidently, balancing the interactions between Siglec-14, Siglec-5 and their common ligand(s) had selective advantage during the course of evolution. The "essential arginine" critical for sialic acid recognition in both Siglec-14 and Siglec-5 is present in humans but mutated in almost all great ape alleles.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Fixation of the human-specific CMP-N-acetylneuraminic acid hydroxylase pseudogene and implications of haplotype diversity for human evolution.

The human CMP-N-acetylneuraminic acid hydroxylase gene (CMAH) suffered deletion of an exon that encodes an active center for the enzyme approximately 3.2 million years ago (MYA). We analyzed a 7.3-kb intronic region of 132 CMAH genes to explore the fixation process of this pseudogene and the demographic implication of its haplotype diversity. Fifty-six variable sites were sorted into 18 different haplotypes with significant linkage disequilibrium. Despite the rather low nucleotide diversity, the most recent common ancestor at CMAH dates to 2.9 MYA. This deep genealogy follows shortly after the original exon deletion, indicating that the deletion has fixed in the population, although whether this fixation was facilitated by natural selection remains to be resolved. Remarkable features are exceptionally long persistence of two lineages and the confinement of one lineage in Africa, implying that some African local populations were in relative isolation while others were directly involved in multiple African exoduses of the genus Homo. Importantly, haplotypes found in Eurasia suggest interbreeding between then-contemporaneous human species. Although population structure within Africa complicates the interpretation of phylogeographic information of haplotypes, the data support a single origin of modern humans, but not with complete replacement of archaic inhabitants by modern humans.

Animals↗
PubMed2005

Differential metastasis inhibition by clinically relevant levels of heparins--correlation with selectin inhibition, not antithrombotic activity.

PURPOSE: Unfractionated heparin reduces metastasis in many murine models. Multiple mechanisms are proposed, particularly anticoagulation and/or inhibition of P-selectin and L-selectin. However, the doses used are not clinically tolerable and other heparins are now commonly used. We studied metastasis inhibition by clinically relevant levels of various heparins and investigated the structural basis for selectin inhibition differences. EXPERIMENTAL DESIGN: Five clinically approved heparins were evaluated for inhibition of P-selectin and L-selectin binding to carcinoma cells. Pharmacokinetic studies determined optimal dosing for clinically relevant anticoagulant levels in mice. Experimental metastasis assays using carcinoma and melanoma cells investigated effects of a single injection of various heparins. Heparins were compared for structural relationships to selectin inhibition. RESULTS: One (Tinzaparin) of three low molecular weight heparins showed increased selectin inhibitory activity, and the synthetic pentasaccharide, Fondaparinux, showed none when normalized to anticoagulant activity. Experimental metastasis models showed attenuation with unfractionated heparin and Tinzaparin, but not Fondaparinux, at clinically relevant anticoagulation levels. Tinzaparin has a small population of high molecular weight fragments not present in other low molecular weight heparins, enriched for selectin inhibitory activity. CONCLUSIONS: Heparin can attenuate metastasis at clinically relevant doses, likely by inhibiting selectins. Equivalent anticoagulation alone with Fondaparinux is ineffective. Clinically approved heparins have differing abilities to inhibit selectins, likely explained by size distribution. It should be possible to size fractionate heparins and inhibit selectins at concentrations that do not have a large effect on coagulation. Caution is also raised about the current preference for smaller heparins. Despite equivalent anticoagulation, hitherto unsuspected benefits of selectin inhibition in various clinical circumstances may be unwittingly discarded.

Animals↗
PubMed2005

A human-specific gene in microglia.

Recent studies have shown multiple differences between humans and apes in sialic acid (Sia) biology, including Siglecs (Sia-recognizing-Ig-superfamily lectins). Comparisons with the chimpanzee genome indicate that human SIGLEC11 emerged through human-specific gene conversion by an adjacent pseudogene. Conversion involved 5 cent untranslated sequences and the Sia-recognition domain. This human protein shows reduced binding relative to the ancestral form but recognizes oligosialic acids, which are enriched in the brain. SIGLEC11 is expressed in human but not in chimpanzee brain microglia. Further studies will determine if this event was related to the evolution of Homo.

Animals↗
PubMed2005

Evolution of human-chimpanzee differences in malaria susceptibility: relationship to human genetic loss of N-glycolylneuraminic acid.

Chimpanzees are the closest evolutionary cousins of humans, sharing >99% identity in most protein sequences. Plasmodium falciparum is the major worldwide cause of malaria mortality. Plasmodium reichenowi, a morphologically identical and genetically very similar parasite, infects chimpanzees but not humans. Conversely, experimental P. falciparum infection causes brief moderate parasitization and no severe infection in chimpanzees. This surprising host specificity remains unexplained. We modified and enhanced traditional methods for measuring sialic acid (Sia)-dependent recognition of glycophorins by merozoite erythrocyte-binding proteins, eliminating interference caused by endogenous Sias on transfected cells, and by using erythroleukemia cells to allow experimental manipulation of Sia content. We present evidence that these remarkable differences among such closely related host-parasite pairs is caused by species-specific erythrocyte-recognition profiles, apparently related to the human-specific loss of the common primate Sia N-glycolylneuraminic acid. The major merozoite-binding protein erythrocyte-binding antigen-175 of P. falciparum apparently evolved to take selective advantage of the excess of the Sia N-acetylneuraminic acid (the precursor of N-glycolylneuraminic acid) on human erythrocytes. The contrasting preference of P. reichenowi erythrocyte-binding antigen-175 for N-glycolylneuraminic acid is likely the ancestral condition. The surprising ability of P. falciparum to cause disease in New World Aotus monkeys (geographically isolated from P. falciparum until arrival of peoples from the Old World) can be explained by parallel evolution of a human-like Sia expression pattern in these distantly related primates. These results also have implications for the prehistory of hominids and for the genetic origins and recent emergence of P. falciparum as a major human pathogen.

Animals↗
PubMed2005

Siglecs--the major subfamily of I-type lectins.

Animal glycan-recognizing proteins can be broadly classified into two groups-lectins (which typically contain an evolutionarily conserved carbohydrate-recognition domain [CRD]) and sulfated glycosaminoglycan (SGAG)-binding proteins (which appear to have evolved by convergent evolution). Proteins other than antibodies and T-cell receptors that mediate glycan recognition via immunoglobulin (Ig)-like domains are called "I-type lectins." The major homologous subfamily of I-type lectins with sialic acid (Sia)-binding properties and characteristic amino-terminal structural features are called the "Siglecs" (Sia-recognizing Ig-superfamily lectins). The Siglecs can be divided into two groups: an evolutionarily conserved subgroup (Siglecs-1, -2, and -4) and a CD33/Siglec-3-related subgroup (Siglecs-3 and -5-13 in primates), which appear to be rapidly evolving. This article provides an overview of historical and current information about the Siglecs.

Animals↗