Potential small effector molecules restoring cellular defects due to sialic acid biosynthetic enzyme deficiency: Pathological relevance to GNE myopathy.

GNEM (GNE Myopathy) is a rare neuromuscular disease caused due to biallelic mutations in sialic acid biosynthetic GNE enzyme (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine Kinase). Recently direct or indirect role of GNE in other cellular functions have been elucidated. Hyposialylation of IGF-1R leads to apoptosis due to mitochondrial dysfunction while hyposialylation of ß1 integrin receptor leads to altered F-actin assembly, disrupted cytoskeletal organization and slow cell migration. Other cellular defects in presence of GNE mutation include altered ER redox state and chaperone expression such as HSP70 or PrdxIV. Currently, there is no cure to treat GNEM. Possible therapeutic trials focus on supplementation with sialic acid, ManNAc, sialyllactose and gene therapy that slows the disease progression. In the present study, we analyzed the effect of small molecules like BGP-15 (HSP70 modulator), IGF-1 (IGF-1R ligand) and CGA (cofilin activator) on cellular phenotypes of GNE heterozygous knock out L6 rat skeletal muscle cell line (SKM­GNEHz). Treatment with BGP-15 improved GNE epimerase activity by 40 % and reduced ER stress by 45 % for SKM­GNEHz. Treatment with IGF-1 improved epimerase activity by 37.5 %, F-actin assembly by 100 %, cell migration upto 36 % (36 h) and atrophy by 0.44-fold for SKM­GNEHz. Treatment with CGA recovered epimerase activity by 49 %, F-actin assembly by 132 % and cell migration upto 41 % (24 h) in SKM­GNEHz. Our study shows that treatment with these small effector molecules reduces the detrimental phenotype observed in SKM­GNEHz, thereby, providing insights into potential therapeutic targets for GNEM.

Evaluation of N-Acetylmannosamine Administration to Restore Sialylation in GNE-Deficient Human Embryonal Kidney Cells.

BACKGROUND: A key mechanism in the neuromuscular disease GNE myopathy (GNEM) is believed to be that point mutations in the GNE gene impair sialic acid synthesis - maybe due to UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) activity restrictions - and resulting in muscle tissue loss. N-acetylmannosamine (ManNAc) is the first product of the bifunctional GNE enzyme and can therefore be regarded as a precursor of sialic acids. This study investigates whether this is also a suitable substance for restoring the sialic acid content in GNE-deficient cells. METHODS: A HEK-293 GNE-knockout cell line was generated using CRISPR-Cas9 and analyzed for its ability to synthesize sialic acids. The cells were then supplemented with ManNAc to compensate for possible GNE inactivity and thereby restore sialic acid synthesis. Sialic acid levels were monitored by immunoblot and high performance liquid chromatography (HPLC). RESULTS: The HEK-293 GNE-knockout cells showed almost no polysialylation signal (immunoblot) and a reduced overall (-71%) N-acetylneuraminic acid (Neu5Ac) level (HPLC) relative to total protein and normalized to wild type level. Supplementation of GNE-deficient HEK-293 cells with 2 mM ManNAc can restore polysialylation and free intracellular sialic acid levels to wild type levels. The addition of 1 mM ManNAc is sufficient to restore the membrane-bound sialic acid level. CONCLUSIONS: Although the mechanism behind this needs further investigation and although it remains unclear why adding ManNAc to GNE-deficient cells is sufficient to elevate polysialylation back to wild type levels - since this substance is also converted by the GNE, all of this might yet prove helpful in the development of an appropriate therapy for GNEM.

Elucidation of ER stress and UPR pathway in sialic acid-deficient cells: Pathological relevance to GNEM.

Accumulation of misfolded proteins in endoplasmic reticulum (ER) generates a stress condition in the cell. The cell combats ER stress by activating unfolded protein response (UPR) and ERAD (ER stress-associated degradation) pathway. Failure to restore favorable folding environment results in cell dysfunction and apoptosis. Various neurodegenerative disorders are characterized by the accumulation of misfolded protein, protein aggregates, and ER stress. GNE myopathy (GNEM) is a neuromuscular disorder pathologically characterized by rimmed vacuole formation due to the accumulation of protein aggregates. More than 200 mutations in key sialic acid biosynthetic enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) have been identified worldwide in the muscle biopsies of GNE myopathy patients. However, the cellular and molecular pathomechanism leading to the disease ar poorly understood. In the present study, the phenomenon of ER stress has been elucidated in GNE mutant cells overexpressing GNE mutations of Indian origin. The effect of GNE mutations on activation of UPR signaling via inositol-requiring transmembrane kinase/endoribonuclease 1 (IRE-1), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor-6 (ATF6) were deciphered to understand the effect of GNE mutations on these proteins. GRP78 was upregulated with increased X-box-binding protein-1 (XBP-1) splicing and CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) upregulation leading to increased apoptosis of GNE mutant cells. Insulin-like growth factor 1 (IGF-1) ligand rescued the cells from apoptotic phenotype by supporting cell survival mechanism. Our study indicates a balance of cell death and survival that decides cell fate and offers potential therapeutic targets to combat ER stress in diseases associated with dysfunctional UPR pathway.

Free sialic acid storage disorder: Progress and promise.

Lysosomal free sialic acid storage disorder (FSASD) is an extremely rare, autosomal recessive, neurodegenerative, multisystemic disorder caused by defects in the lysosomal sialic acid membrane exporter SLC17A5 (sialin). SLC17A5 defects cause free sialic acid and some other acidic hexoses to accumulate in lysosomes, resulting in enlarged lysosomes in some cell types and 10-100-fold increased urinary excretion of free sialic acid. Clinical features of FSASD include coarse facial features, organomegaly, and progressive neurodegenerative symptoms with cognitive impairment, cerebellar ataxia and muscular hypotonia. Central hypomyelination with cerebellar atrophy and thinning of the corpus callosum are also prominent disease features. Around 200 FSASD cases are reported worldwide, with the clinical spectrum ranging from a severe infantile onset form, often lethal in early childhood, to a mild, less severe form with subjects living into adulthood, also called Salla disease. The pathobiology of FSASD remains poorly understood and FSASD is likely underdiagnosed. Known patients have experienced a diagnostic delay due to the rarity of the disorder, absence of routine urine sialic acid testing, and non-specific clinical symptoms, including developmental delay, ataxia and infantile hypomyelination. There is no approved therapy for FSASD. We initiated a multidisciplinary collaborative effort involving worldwide academic clinical and scientific FSASD experts, the National Institutes of Health (USA), and the FSASD patient advocacy group (Salla Treatment and Research [S.T.A.R.] Foundation) to overcome the scientific, clinical and financial challenges facing the development of new treatments for FSASD. We aim to collect data that incentivize industry to further develop, obtain approval for, and commercialize FSASD treatments. This review summarizes current aspects of FSASD diagnosis, prevalence, etiology, and disease models, as well as challenges on the path to therapeutic approaches for FSASD.

Identification of an N-acetylneuraminic acid-presenting bacteria isolated from a human microbiome.

N-Acetylneuraminic acid is the most abundant sialic acid (SA) in humans and is expressed as the terminal sugar on intestinal mucus glycans. Several pathogenic bacteria harvest and display host SA on their own surfaces to evade Siglec-mediated host immunity. While previous studies have identified bacterial enzymes associated with SA catabolism, no reported methods permit the selective labeling, tracking, and quantitation of SA-presenting microbes within complex multi-microbial systems. We combined metabolic labeling, click chemistry, 16S rRNA gene, and whole-genome sequencing to track and identify SA-presenting microbes from a cultured human fecal microbiome. We isolated a new strain of Escherichia coli that incorporates SA onto its own surface and encodes for the nanT, neuA, and neuS genes necessary for harvesting and presenting SA. Our method is applicable to the identification of SA-presenting bacteria from human, animal, and environmental microbiomes, as well as providing an entry point for the investigation of surface-expressed SA-associated structures.

Evolutionary conservation of human ketodeoxynonulosonic acid production is independent of sialoglycan biosynthesis.

Human metabolic incorporation of nonhuman sialic acid (Sia) N-glycolylneuraminic acid into endogenous glycans generates inflammation via preexisting antibodies, which likely contributes to red meat-induced atherosclerosis acceleration. Exploring whether this mechanism affects atherosclerosis in end-stage renal disease (ESRD), we instead found serum accumulation of 2-keto-3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (Kdn), a Sia prominently expressed in cold-blooded vertebrates. In patients with ESRD, levels of the Kdn precursor mannose also increased, but within a normal range. Mannose ingestion by healthy volunteers raised the levels of urinary mannose and Kdn. Kdn production pathways remained conserved in mammals but were diminished by an M42T substitution in a key biosynthetic enzyme, N-acetylneuraminate synthase. Remarkably, reversion to the ancestral methionine then occurred independently in 2 lineages, including humans. However, mammalian glycan databases contain no Kdn-glycans. We hypothesize that the potential toxicity of excess mannose in mammals is partly buffered by conversion to free Kdn. Thus, mammals probably conserve Kdn biosynthesis and modulate it in a lineage-specific manner, not for glycosylation, but to control physiological mannose intermediates and metabolites. However, human cells can be forced to express Kdn-glycans via genetic mutations enhancing Kdn utilization, or by transfection with fish enzymes producing cytidine monophosphate-Kdn (CMP-Kdn). Antibodies against Kdn-glycans occur in pooled human immunoglobulins. Pathological conditions that elevate Kdn levels could therefore result in antibody-mediated inflammatory pathologies.

Second sialic acid-binding site of influenza A virus neuraminidase: binding receptors for efficient release.

Influenza A viruses (IAVs) are a major cause of human respiratory tract infections and cause significant disease and mortality. Human IAVs originate from animal viruses that breached the host species barrier. IAV particles contain sialoglycan receptor-binding hemagglutinin (HA) and receptor-destroying neuraminidase (NA) in their envelope. When IAV crosses the species barrier, the functional balance between HA and NA needs to be adjusted to the sialoglycan repertoire of the novel host species. Relatively little is known about the role of NA in host adaptation in contrast to the extensively studied HA. NA prevents virion aggregation and facilitates release of (newly assembled) virions from cell surfaces and from decoy receptors abundantly present in mucus and cell glycocalyx. In addition to a highly conserved catalytic site, NA carries a second sialic acid-binding site (2SBS). The 2SBS preferentially binds α2,3-linked sialic acids and enhances activity of the neighboring catalytic site by bringing/keeping multivalent substrates in close contact with this site. In this way, the 2SBS contributes to the HA-NA balance of virus particles and affects virus replication. The 2SBS is highly conserved in all NA subtypes of avian IAVs, with some notable exceptions associated with changes in the receptor-binding specificity of HA and host tropism. Conservation of the 2SBS is invariably lost in human (pandemic) viruses and in several other viruses adapted to mammalian host species. Preservation or loss of the 2SBS is likely to be an important factor of the viral host range.

A novel glycosidase plate-based assay for the quantification of galactosylation and sialylation on human IgG.

Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLD-MSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson's r correlation coefficient of 0.8208 between the two methods.

Structure of Merkel Cell Polyomavirus Capsid and Interaction with Its Glycosaminoglycan Attachment Receptor.

Merkel cell polyomavirus (MCPyV) is a human double-stranded DNA tumor virus. MCPyV cell entry is unique among members of the polyomavirus family as it requires the engagement of two types of glycans, sialylated oligosaccharides and sulfated glycosaminoglycans (GAGs). Here, we present crystallographic and cryo-electron microscopic structures of the icosahedral MCPyV capsid and analysis of its glycan interactions via nuclear magnetic resonance (NMR) spectroscopy. While sialic acid binding is specific for α2-3-linked sialic acid and mediated by the exposed apical loops of the major capsid protein VP1, a broad range of GAG oligosaccharides bind to recessed regions between VP1 capsomers. Individual VP1 capsomers are tethered to one another by an extensive disulfide network that differs in architecture from previously described interactions for other PyVs. An unusual C-terminal extension in MCPyV VP1 projects from the recessed capsid regions. Mutagenesis experiments show that this extension is dispensable for receptor interactions.IMPORTANCE The MCPyV genome was found to be clonally integrated in 80% of cases of Merkel cell carcinoma (MCC), a rare but aggressive form of human skin cancer, strongly suggesting that this virus is tumorigenic. In the metastasizing state, the course of the disease is often fatal, especially in immunocompromised individuals, as reflected by the high mortality rate of 33 to 46% and the low 5-year survival rate (<45%). The high seroprevalence of about 60% makes MCPyV a serious health care burden and illustrates the need for targeted treatments. In this study, we present the first high-resolution structural data for this human tumor virus and demonstrate that the full capsid is required for the essential interaction with its GAG receptor(s). Together, these data can be used as a basis for future strategies in drug development.

Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria.

The human gut symbiont Ruminococcus gnavus scavenges host-derived N-acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D NMR, we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-2-deoxy-2,3-dehydro-N-acetylneuraminic acid intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram-negative and Gram-positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionization spray MS that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolize 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli depended on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.

GBA3: a polymorphic pseudogene in humans that experienced repeated gene loss during mammalian evolution.

The gene encoding the cytosolic ß-glucosidase GBA3 shows pseudogenization due to a truncated allele (rs358231) that is polymorphic in humans. Since this enzyme is involved in the transformation of many plant ß-glycosides, this particular case of gene loss may have been influenced by dietary adaptations during evolution. In humans, apart from the inactivating allele, we found that GBA3 accumulated additional damaging mutations, implying an extensive GBA3 loss. The allelic distribution of loss-of-function alleles revealed significant differences between human populations which can be partially related with their staple diet. The analysis of mammalian orthologs disclosed that GBA3 underwent at least nine pseudogenization events. Most events of pseudogenization occurred in carnivorous lineages, suggesting a possible link to a ß-glycoside poor diet. However, GBA3 was also lost in omnivorous and herbivorous species, hinting that the physiological role of GBA3 is not fully understood and other unknown causes may underlie GBA3 pseudogenization. Such possibility relies upon a putative role in sialic acid biology, where GBA3 participates in a cellular network involving NEU2 and CMAH. Overall, our data shows that the recurrent loss of GBA3 in mammals is likely to represent an evolutionary endpoint of the relaxation of selective constraints triggered by diet-related factors.

α2,3 linkage of sialic acid to a GPI anchor and an unpredicted GPI attachment site in human prion protein.

Prion diseases are transmissible, lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of the prion protein (PrPSc) after conversion of the cellular prion protein (PrPC). The glycosylphosphatidylinositol (GPI) anchor of PrPC is involved in prion disease pathogenesis, and especially sialic acid in a GPI side chain reportedly affects PrPC conversion. Thus, it is important to define the location and structure of the GPI anchor in human PrPC Moreover, the sialic acid linkage type in the GPI side chain has not been determined for any GPI-anchored protein. Here we report GPI glycan structures of human PrPC isolated from human brains and from brains of a knock-in mouse model in which the mouse prion protein (Prnp) gene was replaced with the human PRNP gene. LC-electrospray ionization-MS analysis of human PrPC from both biological sources indicated that Gly229 is the ω site in PrPC to which GPI is attached. Gly229 in human PrPC does not correspond to Ser231, the previously reported ω site of Syrian hamster PrPC We found that ∼41% and 28% of GPI anchors in human PrPCs from human and knock-in mouse brains, respectively, have N-acetylneuraminic acid in the side chain. Using a sialic acid linkage-specific alkylamidation method to discriminate α2,3 linkage from α2,6 linkage, we found that N-acetylneuraminic acid in PrPC's GPI side chain is linked to galactose through an α2,3 linkage. In summary, we report the GPI glycan structure of human PrPC, including the ω-site amino acid for GPI attachment and the sialic acid linkage type.

Sialic Acid-Siglec-E Interactions During Pseudomonas aeruginosa Infection of Macrophages Interferes With Phagosome Maturation by Altering Intracellular Calcium Concentrations.

Pseudomonas aeruginosa (PA) is commonly associated with nosocomial and chronic infections of lungs. We have earlier demonstrated that an acidic sugar, sialic acid, is present in PA which is recognized and bound by sialic acid binding immunoglobulin type lectins (siglecs) expressed on neutrophils. Here, we have tried to gain a detailed insight into the immunosuppressive role of sialic acid-siglec interactions in macrophage-mediated clearance of sialylated PA (PA+Sia). We have demonstrated that PA+Sia shows enhanced binding (~1.5-fold) to macrophages due to additional interactions between sialic acids and siglec-E and exhibited more phagocytosis. However, internalization of PA+Sia is associated with a reduction in respiratory burst and increase in anti-inflammatory cytokines secretion which is reversed upon desialylation of the bacteria. Phagocytosis of PA+Sia is also associated with reduced intracellular calcium ion concentrations and altered calcium-dependent signaling which negatively affects phagosome maturation. Consequently, although more PA+Sia was localized in early phagosomes (Rab5 compartment), only fewer bacteria reach into the late phagosomal compartment (Rab7). Possibly, this leads to reduced phagosome lysosome fusion where reduced numbers of PA+Sia are trafficked into lysosomes, compared to PA-Sia. Thus, internalized PA+Sia remain viable and replicates intracellularly in macrophages. We have also demonstrated that such siglec-E-sialic acid interaction recruited SHP-1/SHP-2 phosphatases which modulate MAPK and NF-κB signaling pathways. Disrupting sialic acid-siglec-E interaction by silencing siglec-E in macrophages results in improved bactericidal response against PA+Sia characterized by robust respiratory burst, enhanced intracellular calcium levels and nuclear translocation of p65 component of NF-κB complex leading to increased pro-inflammatory cytokine secretion. Taken together, we have identified that sialic acid-siglec-E interactions is another pathway utilized by PA in order to suppress macrophage antimicrobial responses and inhibit phagosome maturation, thereby persisting as an intracellular pathogen in macrophages.

A Sialic Acid-Binding Protein SABP1 of Toxoplasma gondii Mediates Host Cell Attachment and Invasion.

Many obligate intracellular apicomplexan parasites have adapted a distinct invasion mechanism involving a close interaction between the parasite ligands and the sialic acid (SA) receptor. We found that sialic acid binding protein-1 (SABP1), localized on the outer membrane of the zoonotic parasite Toxoplasma gondii, readily binds to sialic acid on the host cell surface. The binding was sensitive to neuraminidase treatment. Cells preincubated with recombinant SABP1 protein resisted parasite invasion in vitro. The parasite lost its invasion capacity and animal infectivity after the SABP1 gene was deleted, whereas complementation of the SABP1 gene restored the virulence of the knockout strain. These data establish the critical role of SABP1 in the invasion process of T. gondii. The previously uncharacterized protein, SABP1, facilitated T. gondii attachment and invasion via sialic acid receptors.

In vivo evolutionary engineering of riboswitch with high-threshold for N-acetylneuraminic acid production.

Riboswitches with desired properties, such as sensitivity, threshold, dynamic range, is important for its application. However, the property change of a natural riboswitch is difficult due to the lack of the understanding of aptamer ligand binding properties and a proper screening method for both rational and irrational design. In this study, an effective method to change the threshold of riboswitch was established in vivo based on growth coupled screening by combining both positive and negative selections. The feasibility of the method was verified by the model library. Using this method, an N-acetylneuraminic acid (NeuAc) riboswitch was evolved and modified riboswitches with high threshold and large dynamic range were obtained. Then, using a new NeuAc riboswitch, both ribosome binding sites and key gene in NeuAc biosynthesis pathway were optimized. The highest NeuAc production of 14.32 g/l that has been reported using glucose as sole carbon source was obtained.

The 2nd sialic acid-binding site of influenza A virus neuraminidase is an important determinant of the hemagglutinin-neuraminidase-receptor balance.

Influenza A virus (IAV) neuraminidase (NA) receptor-destroying activity and hemagglutinin (HA) receptor-binding affinity need to be balanced with the host receptor repertoire for optimal viral fitness. NAs of avian, but not human viruses, contain a functional 2nd sialic acid (SIA)-binding site (2SBS) adjacent to the catalytic site, which contributes to sialidase activity against multivalent substrates. The receptor-binding specificity and potentially crucial contribution of the 2SBS to the HA-NA balance of virus particles is, however, poorly characterized. Here, we elucidated the receptor-binding specificity of the 2SBS of N2 NA and established an important role for this site in the virion HA-NA-receptor balance. NAs of H2N2/1957 pandemic virus with or without a functional 2SBS and viruses containing this NA were analysed. Avian-like N2, with a restored 2SBS due to an amino acid substitution at position 367, was more active than human N2 on multivalent substrates containing α2,3-linked SIAs, corresponding with the pronounced binding-specificity of avian-like N2 for these receptors. When introduced into human viruses, avian-like N2 gave rise to altered plaque morphology and decreased replication compared to human N2. An opposite replication phenotype was observed when N2 was combined with avian-like HA. Specific bio-layer interferometry assays revealed a clear effect of the 2SBS on the dynamic interaction of virus particles with receptors. The absence or presence of a functional 2SBS affected virion-receptor binding and receptor cleavage required for particle movement on a receptor-coated surface and subsequent NA-dependent self-elution. The contribution of the 2SBS to virus-receptor interactions depended on the receptor-binding properties of HA and the identity of the receptors used. We conclude that the 2SBS is an important and underappreciated determinant of the HA-NA-receptor balance. The rapid loss of a functional 2SBS in pandemic viruses may have served to balance the novel host receptor-repertoire and altered receptor-binding properties of the corresponding HA protein.

From "Serum Sickness" to "Xenosialitis": Past, Present, and Future Significance of the Non-human Sialic Acid Neu5Gc.

The description of "serum sickness" more than a century ago in humans transfused with animal sera eventually led to identification of a class of human antibodies directed against glycans terminating in the common mammalian sialic acid N-Glycolylneuraminic acid (Neu5Gc), hereafter called "Neu5Gc-glycans." The detection of such glycans in malignant and fetal human tissues initially raised the possibility that it was an oncofetal antigen. However, "serum sickness" antibodies were also noted in various human disease states. These findings spurred further research on Neu5Gc, and the discovery that it is not synthesized in the human body due to a human-lineage specific genetic mutation in the enzyme CMAH. However, with more sensitive techniques Neu5Gc-glycans were detected in smaller quantities on certain human cell types, particularly epithelia and endothelia. The likely explanation is metabolic incorporation of Neu5Gc from dietary sources, especially red meat of mammalian origin. This incorporated Neu5Gc on glycans appears to be the first example of a "xeno-autoantigen," against which varying levels of "xeno-autoantibodies" are present in all humans. The resulting chronic inflammation or "xenosialitis" may have important implications in human health and disease, especially in conditions known to be aggravated by consumption of red meat. In this review, we will cover the early history of the discovery of "serum sickness" antibodies, the subsequent recognition that they were partly directed against Neu5Gc-glycans, the discovery of the genetic defect eliminating Neu5Gc production in humans, and the later recognition that this was not an oncofetal antigen but the first example of a "xeno-autoantigen." Further, we will present comments about implications for disease risks associated with red meat consumption such as cancer and atherosclerosis. We will also mention the potential utility of these anti-Neu5Gc-glycan antibodies in cancer immunotherapy and provide some suggestions and perspectives for the future. Other reviews in this special issue cover many other aspects of this unusual pathological process, for which there appears to be no other described precedent.

Hollow S-nitrosothiols nanoparticle with polymeric brushes for nitric oxide (NO)-releasing as tumor targeted chemotherapy.

A kind of tumor targeting nitric oxide donor nanoparticle with brushes is described in this paper. The poly(4-vinylphenylboronic acid) polymeric brush, which shows glucose and pH dual responsiveness, endows the ability of hollow S-nitrosothiols nanoparticle to accurate recognition and binding with the sialic acid over-expressed type tumor cells, such as HepG2 and MCF-7 cells. In vitro experiments, including cells capture and release experiments, confocal fluorescence microscope characterization, cytotoxicity assay with different cells, demonstrate the selective recognition and the controlled NO release to kill tumor cells for these S-nitrosothiols nanoparticles. Low concentration of the released NO from the S-nitrosothiols nanoparticles in the transmission would participate physiological activity and avoid serious side effects because the endogenous nature and the physiological necessity to regulate normal biological functions. To the best of our knowledge, this is the first report about polymer nanoparticles as NO donors with functional brushes to selectively identify tumor cells and release NO in a controlled manner.

Sialic acid is a critical fetal defense against maternal complement attack.

The negatively charged sugar sialic acid (Sia) occupies the outermost position in the bulk of cell surface glycans. Lack of sialylated glycans due to genetic ablation of the Sia-activating enzyme CMP-sialic acid synthase (CMAS) resulted in embryonic lethality around day 9.5 post coitum (E9.5) in mice. Developmental failure was caused by complement activation on trophoblasts in Cmas-/- implants and was accompanied by infiltration of maternal neutrophils at the fetal-maternal interface, intrauterine growth restriction, impaired placental development, and a thickened Reichert's membrane. This phenotype, which shared features with complement receptor 1-related protein Y (Crry) depletion, was rescued in E8.5 Cmas-/- mice upon injection of cobra venom factor, resulting in exhaustion of the maternal complement component C3. Here we show that Sia is dispensable for early development of the embryo proper but pivotal for fetal-maternal immune homeostasis during pregnancy, i.e., for protecting the allograft implant against attack by the maternal innate immune system. Finally, embryos devoid of cell surface sialylation suffered from malnutrition due to inadequate placentation as a secondary effect.