Proinflammatory Macrophage Activation by the Polysialic Acid-Siglec-16 Axis Is Linked to Increased Survival of Patients with Glioblastoma.

PURPOSE: Interactions with tumor-associated microglia and macrophages (TAM) are critical for glioblastoma progression. Polysialic acid (polySia) is a tumor-associated glycan, but its frequency of occurrence and its prognostic value in glioblastoma are disputed. Through interactions with the opposing immune receptors Siglec-11 and Siglec-16, polySia is implicated in the regulation of microglia and macrophage activity. However, due to a nonfunctional SIGLEC16P allele, SIGLEC16 penetrance is less than 40%. Here, we explored possible consequences of SIGLEC16 status and tumor cell-associated polySia on glioblastoma outcome. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded specimens of two independent cohorts with 70 and 100 patients with newly diagnosed glioblastoma were retrospectively analyzed for SIGLEC16 and polySia status in relation to overall survival. Inflammatory TAM activation was assessed in tumors, in heterotypic tumor spheroids consisting of polySia-positive glioblastoma cells and Siglec-16-positive or Siglec-16-negative macrophages, and by exposing Siglec-16-positive or Siglec-16-negative macrophages to glioblastoma cell-derived membrane fractions. RESULTS: Overall survival of SIGLEC16 carriers with polySia-positive tumors was increased. Consistent with proinflammatory Siglec-16 signaling, levels of TAM positive for the M2 marker CD163 were reduced, whereas the M1 marker CD74 and TNF expression were increased, and CD8+ T cells enhanced in SIGLEC16/polySia double-positive tumors. Correspondingly, TNF production was elevated in heterotypic spheroid cultures with Siglec-16-expressing macrophages. Furthermore, a higher, mainly M1-like cytokine release and activating immune signaling was observed in SIGLEC16-positive as compared with SIGLEC16-negative macrophages confronted with glioblastoma cell-derived membranes. CONCLUSIONS: Collectively, these results strongly suggest that proinflammatory TAM activation causes the better outcome in patients with glioblastoma with a functional polySia-Siglec-16 axis.

Simple and practical sialoglycan encoding system reveals vast diversity in nature and identifies a universal sialoglycan-recognizing probe derived from AB5 toxin B subunits.

Vertebrate sialic acids (Sias) display much diversity in modifications, linkages, and underlying glycans. Slide microarrays allow high-throughput explorations of sialoglycan-protein interactions. A microarray presenting ~150 structurally defined sialyltrisaccharides with various Sias linkages and modifications still poses challenges in planning, data sorting, visualization, and analysis. To address these issues, we devised a simple 9-digit code for sialyltrisaccharides with terminal Sias and underlying two monosaccharides assigned from the nonreducing end, with 3 digits assigning a monosaccharide, its modifications, and linkage. Calculations based on the encoding system reveal >113,000 likely linear sialyltrisaccharides in nature. Notably, a biantennary N-glycan with 2 terminal sialyltrisaccharides could thus have >1010 potential combinations and a triantennary N-glycan with 3 terminal sequences, >1015 potential combinations. While all possibilities likely do not exist in nature, sialoglycans encode enormous diversity. While glycomic approaches are used to probe such diverse sialomes, naturally occurring bacterial AB5 toxin B subunits are simpler tools to track the dynamic sialome in biological systems. Sialoglycan microarray was utilized to compare sialoglycan-recognizing bacterial toxin B subunits. Unlike the poor correlation between B subunits and species phylogeny, there is stronger correlation with Sia-epitope preferences. Further supporting this pattern, we report a B subunit (YenB) from Yersinia enterocolitica (broad host range) recognizing almost all sialoglycans in the microarray, including 4-O-acetylated-Sias not recognized by a Yersinia pestis orthologue (YpeB). Differential Sia-binding patterns were also observed with phylogenetically related B subunits from Escherichia coli (SubB), Salmonella Typhi (PltB), Salmonella Typhimurium (ArtB), extra-intestinal E.coli (EcPltB), Vibrio cholera (CtxB), and cholera family homologue of E. coli (EcxB).

Development and applications of sialoglycan-recognizing probes (SGRPs) with defined specificities: exploring the dynamic mammalian sialoglycome.

Glycans that are abundantly displayed on vertebrate cell surface and secreted molecules are often capped with terminal sialic acids (Sias). These diverse 9-carbon-backbone monosaccharides are involved in numerous intrinsic biological processes. They also interact with commensals and pathogens, while undergoing dynamic changes in time and space, often influenced by environmental conditions. However, most of this sialoglycan complexity and variation remains poorly characterized by conventional techniques, which often tend to destroy or overlook crucial aspects of Sia diversity and/or fail to elucidate native structures in biological systems, i.e. in the intact sialome. To date, in situ detection and analysis of sialoglycans has largely relied on the use of plant lectins, sialidases, or antibodies, whose preferences (with certain exceptions) are limited and/or uncertain. We took advantage of naturally evolved microbial molecules (bacterial adhesins, toxin subunits, and viral hemagglutinin-esterases) that recognize sialoglycans with defined specificity to delineate 9 classes of sialoglycan recognizing probes (SGRPs: SGRP1-SGRP9) that can be used to explore mammalian sialome changes in a simple and systematic manner, using techniques common in most laboratories. SGRP candidates with specificity defined by sialoglycan microarray studies were engineered as tagged probes, each with a corresponding nonbinding mutant probe as a simple and reliable negative control. The optimized panel of SGRPs can be used in methods commonly available in most bioscience labs, such as ELISA, western blot, flow cytometry, and histochemistry. To demonstrate the utility of this approach, we provide examples of sialoglycome differences in tissues from C57BL/6 wild-type mice and human-like Cmah-/- mice.

Evolution of Human-Specific Alleles Protecting Cognitive Function of Grandmothers.

The myelomonocytic receptor CD33 (Siglec-3) inhibits innate immune reactivity by extracellular V-set domain recognition of sialic acid (Sia)-containing "self-associated molecular patterns" (SAMPs). We earlier showed that V-set domain-deficient CD33-variant allele, protective against late-onset Alzheimer's Disease (LOAD), is derived and specific to the hominin lineage. We now report multiple hominin-specific CD33 V-set domain mutations. Due to hominin-specific, fixed loss-of-function mutation in the CMAH gene, humans lack N-glycolylneuraminic acid (Neu5Gc), the preferred Sia-ligand of ancestral CD33. Mutational analysis and molecular dynamics (MD)-simulations indicate that fixed change in amino acid 21 of hominin V-set domain and conformational changes related to His45 corrected for Neu5Gc-loss by switching to N-acetylneuraminic acid (Neu5Ac)-recognition. We show that human-specific pathogens Neisseria gonorrhoeae and Group B Streptococcus selectively bind human CD33 (huCD33) as part of immune-evasive molecular mimicry of host SAMPs and that this binding is significantly impacted by amino acid 21 modification. In addition to LOAD-protective CD33 alleles, humans harbor derived, population-universal, cognition-protective variants at several other loci. Interestingly, 11 of 13 SNPs in these human genes (including CD33) are not shared by genomes of archaic hominins: Neanderthals and Denisovans. We present a plausible evolutionary scenario to compile, correlate, and comprehend existing knowledge about huCD33-evolution and suggest that grandmothering emerged in humans.

Sialoglycan-binding patterns of bacterial AB5 toxin B subunits correlate with host range and toxicity, indicating evolution independent of A subunits.

Many pathogenic bacteria secrete AB5 toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y. pestis' adaptability to numerous hosts. We previously showed selective binding of other B5 pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Here, we report that A subunit phylogeny evolved independently of B subunits and suggest a future B subunit nomenclature based on bacterial species names. We also found via phylogenetic analysis of B subunits, which bind Sias, that homologous molecules show poor correlation with species phylogeny. These data indicate ongoing lateral gene transfers between species, including mixing of A and B subunits. Consistent with much broader host range of Y. pestis, we show that YpeB recognizes all mammalian Sia types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, which is abolished by a mutation (Y77F) eliminating Sia recognition, suggesting that cell proliferation and death are promoted via lectin-like crosslinking of cell surface sialoglycoconjugates. These findings help explain the host range of Y. pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.

Human-specific polymorphic pseudogenization of SIGLEC12 protects against advanced cancer progression.

Compared with our closest living evolutionary cousins, humans appear unusually prone to develop carcinomas (cancers arising from epithelia). The SIGLEC12 gene, which encodes the Siglec-XII protein expressed on epithelial cells, has several uniquely human features: a fixed homozygous missense mutation inactivating its natural ligand recognition property; a polymorphic frameshift mutation eliminating full-length protein expression in ~60%-70% of worldwide human populations; and, genomic features suggesting a negative selective sweep favoring the pseudogene state. Despite the loss of canonical sialic acid binding, Siglec-XII still recruits Shp2 and accelerates tumor growth in a mouse model. We hypothesized that dysfunctional Siglec-XII facilitates human carcinoma progression, correlating with known tumorigenic signatures of Shp2-dependent cancers. Immunohistochemistry was used to detect Siglec-XII expression on tissue microarrays. PC-3 prostate cancer cells were transfected with Siglec-XII and transcription of genes enriched with Siglec-XII was determined. Genomic SIGLEC12 status was determined for four different cancer cohorts. Finally, a dot blot analysis of human urinary epithelial cells was established to determine the Siglec-XII expressors versus non-expressors. Forced expression in a SIGLEC12 null carcinoma cell line enriched transcription of genes associated with cancer progression. While Siglec-XII was detected as expected in ~30%-40% of normal epithelia, ~80% of advanced carcinomas showed strong expression. Notably, >80% of late-stage colorectal cancers had a functional SIGLEC12 allele, correlating with overall increased mortality. Thus, advanced carcinomas are much more likely to occur in individuals whose genomes have an intact SIGLEC12 gene, likely because the encoded Siglec-XII protein recruits Shp2-related oncogenic pathways. The finding has prognostic, diagnostic, and therapeutic implications.

The Alzheimer's disease-protective CD33 splice variant mediates adaptive loss of function via diversion to an intracellular pool.

The immunomodulatory receptor Siglec-3/CD33 influences risk for late-onset Alzheimer's disease (LOAD), an apparently human-specific post-reproductive disease. CD33 generates two splice variants: a full-length CD33M transcript produced primarily by the "LOAD-risk" allele and a shorter CD33m isoform lacking the sialic acid-binding domain produced primarily from the "LOAD-protective" allele. An SNP that modulates CD33 splicing to favor CD33m is associated with enhanced microglial activity. Individuals expressing more protective isoform accumulate less brain ß-amyloid and have a lower LOAD risk. How the CD33m isoform increases ß-amyloid clearance remains unknown. We report that the protection by the CD33m isoform may not be conferred by what it does but, rather, from what it cannot do. Analysis of blood neutrophils and monocytes and a microglial cell line revealed that unlike CD33M, the CD33m isoform does not localize to cell surfaces; instead, it accumulates in peroxisomes. Cell stimulation and activation did not mobilize CD33m to the surface. Thus, the CD33m isoform may neither interact directly with amyloid plaques nor engage in cell-surface signaling. Rather, production and localization of CD33m in peroxisomes is a way of diminishing the amount of CD33M and enhancing ß-amyloid clearance. We confirmed intracellular localization by generating a CD33m-specific monoclonal antibody. Of note, CD33 is the only Siglec with a peroxisome-targeting sequence, and this motif emerged by convergent evolution in toothed whales, the only other mammals with a prolonged post-reproductive lifespan. The CD33 allele that protects post-reproductive individuals from LOAD may have evolved by adaptive loss-of-function, an example of the less-is-more hypothesis.

Physiological Exploration of the Long Term Evolutionary Selection against Expression of N-Glycolylneuraminic Acid in the Brain.

All vertebrate cell surfaces display a dense glycan layer often terminated with sialic acids, which have multiple functions due to their location and diverse modifications. The major sialic acids in most mammalian tissues are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), the latter being derived from Neu5Ac via addition of one oxygen atom at the sugar nucleotide level by CMP-Neu5Ac hydroxylase (Cmah). Contrasting with other organs that express various ratios of Neu5Ac and Neu5Gc depending on the variable expression of Cmah, Neu5Gc expression in the brain is extremely low in all vertebrates studied to date, suggesting that neural expression is detrimental to animals. However, physiological exploration of the reasons for this long term evolutionary selection has been lacking. To explore the consequences of forced expression of Neu5Gc in the brain, we have established brain-specific Cmah transgenic mice. Such Neu5Gc overexpression in the brain resulted in abnormal locomotor activity, impaired object recognition memory, and abnormal axon myelination. Brain-specific Cmah transgenic mice were also lethally sensitive to a Neu5Gc-preferring bacterial toxin, even though Neu5Gc was overexpressed only in the brain and other organs maintained endogenous Neu5Gc expression, as in wild-type mice. Therefore, the unusually strict evolutionary suppression of Neu5Gc expression in the vertebrate brain may be explained by evasion of negative effects on neural functions and by selection against pathogens.

Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E.

CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer.

Lectin galactoside-binding soluble 3 binding protein (LGALS3BP) is a tumor-associated immunomodulatory ligand for CD33-related Siglecs.

Lectin galactoside-binding soluble 3 binding protein (LGALS3BP, also called Mac-2 binding protein) is a heavily glycosylated secreted molecule that has been shown previously to be up-regulated in many cancers and has been implicated in tumor metastatic processes, as well as in other cell adhesion and immune functions. The CD33-related subset of sialic acid-binding immunoglobulin-like lectins (Siglecs) consists of immunomodulatory molecules that have recently been associated with the modulation of immune responses to cancer. Because up-regulation of Siglec ligands in cancer tissue has been observed, the characterization of these cancer-associated ligands that bind to inhibitory CD33-related Siglecs could provide novel targets for cancer immunomodulatory therapy. Here we used affinity chromatography of tumor cell extracts to identify LGALS3BP as a novel sialic acid-dependent ligand for human Siglec-9 and for other immunomodulatory Siglecs, such as Siglec-5 and Siglec-10. In contrast, the mouse homolog Siglec-E binds to murine LGALS3BP with lower affinity. LGALS3BP has been observed to be up-regulated in human colorectal and prostate cancer specimens, particularly in the extracellular matrix. Finally, LGALS3BP was able to inhibit neutrophil activation in a sialic acid- and Siglec-dependent manner. These findings suggest a novel immunoinhibitory function for LGALS3BP that might be important for immune evasion of tumor cells during cancer progression.

Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer.

Certain pathogenic bacteria are known to modulate the innate immune response by decorating themselves with sialic acids, which can engage the myelomonocytic lineage inhibitory receptor Siglec-9, thereby evading immunosurveillance. We hypothesized that the well-known up-regulation of sialoglycoconjugates by tumors might similarly modulate interactions with innate immune cells. Supporting this hypothesis, Siglec-9-expressing myelomonocytic cells found in human tumor samples were accompanied by a strong up-regulation of Siglec-9 ligands. Blockade of Siglec-9 enhanced neutrophil activity against tumor cells in vitro. To investigate the function of inhibitory myelomonocytic Siglecs in vivo we studied mouse Siglec-E, the murine functional equivalent of Siglec-9. Siglec-E-deficient mice showed increased in vivo killing of tumor cells, and this effect was reversed by transgenic Siglec-9 expression in myelomonocytic cells. Siglec-E-deficient mice also showed enhanced immunosurveillance of autologous tumors. However, once tumors were established, they grew faster in Siglec-E-deficient mice. In keeping with this, Siglec-E-deficient macrophages showed a propensity toward a tumor-promoting M2 polarization, indicating a secondary role of CD33-related Siglecs in limiting cancer-promoting inflammation and tumor growth. Thus, we define a previously unidentified impact of inhibitory myelomonocytic Siglecs in cancer biology, with distinct roles that reflect the dual function of myelomonocytic cells in cancer progression. In keeping with this, a human polymorphism that reduced Siglec-9 binding to carcinomas was associated with improved early survival in non-small-cell lung cancer patients, which suggests that Siglec-9 might be therapeutically targeted within the right time frame and stage of disease.

Inverse hormesis of cancer growth mediated by narrow ranges of tumor-directed antibodies.

Compelling evidence for naturally occurring immunosurveillance against malignancies informs and justifies some current approaches toward cancer immunotherapy. However, some types of immune reactions have also been shown to facilitate tumor progression. For example, our previous studies showed that although experimental tumor growth is enhanced by low levels of circulating antibodies directed against the nonhuman sialic acid N-glycolyl-neuraminic acid (Neu5Gc), which accumulates in human tumors, growth could be inhibited by anti-Neu5Gc antibodies from a different source, in a different model. However, it remains generally unclear whether the immune responses that mediate cancer immunosurveillance vs. those responsible for inflammatory facilitation are qualitatively and/or quantitatively distinct. Here, we address this question using multiple murine tumor growth models in which polyclonal antibodies against tumor antigens, such as Neu5Gc, can alter tumor progression. We found that although growth was stimulated at low antibody doses, it was inhibited by high doses, over a linear and remarkably narrow range, defining an immune response curve (IRC; i.e., inverse hormesis). Moreover, modulation of immune responses against the tumor by altering antibody avidity or by enhancing innate immunity shifted the IRC in the appropriate direction. Thus, the dualistic role of immunosurveillance vs. inflammation in modulating tumor progression can be quantitatively distinguished in multiple model systems, and can occur over a remarkably narrow range. Similar findings were made in a human tumor xenograft model using a narrow range of doses of a monoclonal antibody currently in clinical use. These findings may have implications for the etiology, prevention, and treatment of cancer.

Rapid evolution of binding specificities and expression patterns of inhibitory CD33-related Siglecs in primates.

Siglecs are sialic acid-binding Ig-like lectins that recognize sialoglycans via amino-terminal V-set domains. CD33-related Siglecs (CD33rSiglecs) on innate immune cells recognize endogenous sialoglycans as "self-associated molecular patterns" (SAMPs), dampening immune responses via cytosolic immunoreceptor tyrosine-based inhibition motifs that recruit tyrosine phosphatases. However, sialic acid-expressing pathogens subvert this mechanism through molecular mimicry. Meanwhile, endogenous host SAMPs must continually evolve to evade other pathogens that exploit sialic acids as invasion targets. We hypothesized that these opposing selection forces have accelerated CD33rSiglec evolution. We address this by comparative analysis of major CD33rSiglec (Siglec-3, Siglec-5, and Siglec-9) orthologs in humans, chimpanzees, and baboons. Recombinant soluble molecules displaying ligand-binding domains show marked quantitative and qualitative interspecies differences in interactions with strains of the sialylated pathogen, group B Streptococcus, and with sialoglycans presented as gangliosides or in the form of sialoglycan microarrays, including variations such as N-glycolyl and O-acetyl groups. Primate Siglecs also show quantitative and qualitative intra- and interspecies variations in expression patterns on leukocytes, both in circulation and in tissues. Taken together our data explain why the CD33rSiglec-encoding gene cluster is undergoing rapid evolution via multiple mechanisms, driven by the need to maintain self-recognition by innate immune cells, while escaping 2 distinct mechanisms of pathogen subversion.

Quantum dot nanometal surface energy transfer based biosensing of sialic acid compositions and linkages in biological samples.

Current methods for analyzing sialic acid diversity in modifications and linkages require multistep processing, derivatization, and chromatographic analyses. We here report a single-step optical method for identification and quantification of different compositions of sialoglycans on glycoproteins and in serum. This was achieved by measuring and quantifying nanometal surface energy transfer (NSET) signals between quantum dots and gold nanoparticles bound to specific sialic acid binding proteins (SBPs) and sialic acid moieties, respectively. The biosensing process is based on the NSET turn-on by external sialic acid species that compete for binding to the SBPs. Selectivity of the biosensor toward sialoglycans can be designed to detect the total amount, glycosylation linkages (α2-6 vs α2-3), and modifications (9-O-acetyl and N-glycolyl groups) in the samples. This nanobiosensor is a prototype expected to achieve limits of the detection down to the micromolar range for high-throughput quantification and analysis of different compositions of sialoglycans present in biological or biomedical samples.

Specific inactivation of two immunomodulatory SIGLEC genes during human evolution.

Sialic acid-recognizing Ig-like lectins (Siglecs) are signaling receptors that modulate immune responses, and are targeted for interactions by certain pathogens. We describe two primate Siglecs that were rendered nonfunctional by single genetic events during hominin evolution after our common ancestor with the chimpanzee. SIGLEC13 was deleted by an Alu-mediated recombination event, and a single base pair deletion disrupted the ORF of SIGLEC17. Siglec-13 is expressed on chimpanzee monocytes, innate immune cells that react to bacteria. The human SIGLEC17P pseudogene mRNA is still expressed at high levels in human natural killer cells, which bridge innate and adaptive immune responses. As both resulting pseudogenes are homozygous in all human populations, we resurrected the originally encoded proteins and examined their functions. Chimpanzee Siglec-13 and the resurrected human Siglec-17 recruit a signaling adapter and bind sialic acids. Expression of either Siglec in innate immune cells alters inflammatory cytokine secretion in response to Toll-like receptor-4 stimulation. Both Siglecs can also be engaged by two potentially lethal sialylated bacterial pathogens of newborns and infants, agents with a potential impact on reproductive fitness. Neanderthal and Denisovan genomes show human-like sequences at both loci, corroborating estimates that the initial pseudogenization events occurred in the common ancestral population of these hominins. Both loci also show limited polymorphic diversity, suggesting selection forces predating the origin of modern humans. Taken together, these data suggest that genetic elimination of Siglec-13 and/or Siglec-17 represents signatures of infectious and/or other inflammatory selective processes contributing to population restrictions during hominin origins.

Cross-comparison of protein recognition of sialic acid diversity on two novel sialoglycan microarrays.

DNA and protein arrays are commonly accepted as powerful exploratory tools in research. This has mainly been achieved by the establishment of proper guidelines for quality control, allowing cross-comparison between different array platforms. As a natural extension, glycan microarrays were subsequently developed, and recent advances using such arrays have greatly enhanced our understanding of protein-glycan recognition in nature. However, although it is assumed that biologically significant protein-glycan binding is robustly detected by glycan microarrays, there are wide variations in the methods used to produce, present, couple, and detect glycans, and systematic cross-comparisons are lacking. We address these issues by comparing two arrays that together represent the marked diversity of sialic acid modifications, linkages, and underlying glycans in nature, including some identical motifs. We compare and contrast binding interactions with various known and novel plant, vertebrate, and viral sialic acid-recognizing proteins and present a technical advance for assessing specificity using mild periodate oxidation of the sialic acid chain. These data demonstrate both the diversity of sialic acids and the analytical power of glycan arrays, showing that different presentations in different formats provide useful and complementary interpretations of glycan-binding protein specificity. They also highlight important challenges and questions for the future of glycan array technology and suggest that glycan arrays with similar glycan structures cannot be simply assumed to give similar results.

Human cholesterol 7alpha-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype.

Bile acid synthesis plays a critical role in the maintenance of mammalian cholesterol homeostasis. The CYP7A1 gene encodes the enzyme cholesterol 7alpha-hydroxylase, which catalyzes the initial step in cholesterol catabolism and bile acid synthesis. We report here a new metabolic disorder presenting with hyperlipidemia caused by a homozygous deletion mutation in CYP7A1. The mutation leads to a frameshift (L413fsX414) that results in loss of the active site and enzyme function. High levels of LDL cholesterol were seen in three homozygous subjects. Analysis of a liver biopsy and stool from one of these subjects revealed double the normal hepatic cholesterol content, a markedly deficient rate of bile acid excretion, and evidence for upregulation of the alternative bile acid pathway. Two male subjects studied had hypertriglyceridemia and premature gallstone disease, and their LDL cholesterol levels were noticeably resistant to 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. One subject also had premature coronary and peripheral vascular disease. Study of the kindred, which is of English and Celtic background, revealed that individuals heterozygous for the mutation are also hyperlipidemic, indicating that this is a codominant disorder.