The mechanism of the periodate-thiobarbituric acid reaction of sialic acids.
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ABSTRACT:
SUBMITTER: Paerels GB
PROVIDER: S-EPMC1207218 | biostudies-other | 1965 Sep
REPOSITORIES: biostudies-other
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Project description:The human glycome is characterized by a high degree of sialylation, affecting, amongst others, cell-cell interactions and protein half-life. An established method for the linkage isomer-specific characterization of N-glycan sialylation is based on the linkage-specific derivatization of sialylated glycoconjugates, inducing ethyl esterification of α2,6-linked sialic acids and lactonization of α2,3-linked sialic acids. While the carboxylic acid activator and nucleophile used in this reaction received extensive investigation, the role of the catalyst was never thoroughly explored. A frequently used catalyst for the linkage-specific esterification of sialic acids is 1-hydroxybenzotriazole (HOBt). Here, a systematic evaluation was performed of five HOBt alternatives in combination with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in ethanol for the linkage-specific derivatization of sialic acids. Derivatized glycans were analyzed by MALDI-TOF-MS and the catalyst performance was evaluated based on the completeness of the reactions and the linkage-specificity obtained. The use of both 6-Cl-HOBt and 6-CF3-HOBt resulted in high linkage-specificity and minimal byproduct formation, similar to the benchmark method using HOBt. Performing the reaction with these catalysts at neutral or acidic pH showed comparable efficiencies on both sialyllactose and complex-type N-glycans. The reported investigations resulted in an expansion of the reaction space for linkage-specific sialic acid derivatization.
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Project description: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.
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Project description:Carbohydrates have long been known to mediate intracellular interactions, whether within one organism or between different organisms. Sialic acids (Sias) are carbohydrates that usually occupy the terminal positions in longer carbohydrate chains, which makes them common recognition targets mediating these interactions. In this review, we summarize the knowledge about animal disease-causing agents such as viruses, bacteria and protozoa (including the malaria parasite Plasmodium falciparum) in which Sias play a role in infection biology. While Sias may promote binding of, e.g., influenza viruses and SV40, they act as decoys for betacoronaviruses. The presence of two common forms of Sias, Neu5Ac and Neu5Gc, is species-specific, and in humans, the enzyme converting Neu5Ac to Neu5Gc (CMAH, CMP-Neu5Ac hydroxylase) is lost, most likely due to adaptation to pathogen regimes; we discuss the research about the influence of malaria on this trait. In addition, we present data suggesting the CMAH gene was probably present in the ancestor of animals, shedding light on its glycobiology. We predict that a better understanding of the role of Sias in disease vectors would lead to more effective clinical interventions.
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Project description:Boronic acids are well known for their ability to reversibly interact with the diol groups found in sugars and glycoproteins. However, they are generally indiscriminate in their binding. Herein we describe the discovery of a group of heterocyclic boronic acids demonstrating unusually high affinity and selectivity for sialic acids (SAs or N-acetylneuraminic acid), which are sugar residues that are intimately linked with tumor growth and cancer progression. Remarkably, these interactions strengthen under the weakly acidic pH conditions associated with a hypoxic tumoral microenvironment. In vitro competitive binding assays uncovered a significantly higher ability of 5-boronopicolinic acid, one of the derivatives identified in this work as a strong SA-binder, to interact with cell surface SA in comparison to a gold-standard structure, 3-propionamidophenylboronic acid, which has proven to be an efficient SA-binder in numerous reports. This structure also proved to be suitable for further chemical conjugation with a well-preserved SA-binding capability. These findings suggest an attractive alternative to other ongoing boronic acid based chemistry techniques aiming to achieve tumor-specific chemotherapies and diagnoses.
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Project description:All living cells are covered with a dense "sugar-coat" of carbohydrate chains (glycans) conjugated to proteins and lipids. The cell surface glycome is determined by a non-template driven process related to the collection of enzymes that assemble glycans in a sequential manner. In mammals, many of these glycans are topped with sialic acids (Sia), a large family of acidic sugars. The "Sialome" is highly diverse owing to various Sia types, linkage to underlying glycans, range of carriers, and complex spatial organization. Presented at the front of cells, Sia play a major role in immunity and recognition of "self" versus "non-self," largely mediated by the siglecs family of Sia-binding host receptors. Albeit many mammalian pathogens have evolved to hijack this recognition system to avoid host immune attack, presenting a fascinating host-pathogen evolutionary arms race. Similarly, cancer cells exploit Sia for their own survival and propagation. As part of this ongoing fitness, humans lost the ability to synthesize the Sia type N-glycolylneuraminic acid (Neu5Gc), in contrast to other mammals. While this loss had provided an advantage against certain pathogens, humans are continuously exposed to Neu5Gc through mammalian-derived diet (eg, red meat), consequently generating a complex immune response against it. Circulating anti-Neu5Gc antibodies together with Neu5Gc on some human tissues mediate chronic inflammation "xenosialitis" that exacerbate various human diseases (eg, cancer and atherosclerosis). Similarly, Neu5Gc-containing xenografts are exposed to human anti-Neu5Gc antibodies with implications to sustainability. This review aimed to provide a glimpse into the evolution of Sia and their implications to xenotransplantation.
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