A study on the regulation of N ‐glycoloylneuraminic acid biosynthesis and utilization in rat and mouse liver

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N-Glycolylneuraminic acid (Neu5Gc) is a widely expressed sialic acid in mammalian cells. Although humans are genetically deficient in producing Neu5Gc, small amounts are present in human cells in vivo. A dietary origin was suggested by human volunteer studies and by observing that free Neu5Gc is metabolically incorporated into cultured human carcinoma cells by unknown mechanisms. We now show that free Neu5Gc uptake also occurs in other human and mammalian cells. Inhibitors of certain non-clathrin-mediated endocytic pathways reduce Neu5Gc accumulation. Studies with human mutant cells show that the lysosomal sialic acid transporter is required for metabolic incorporation of free Neu5Gc. Incorporation of glycosidically bound Neu5Gc from exogenous glycoconjugates (relevant to human gut epithelial exposure to dietary Neu5Gc) requires the transporter as well as the lysosomal sialidase, which presumably acts to release free Neu5Gc. Thus, exogenous Neu5Gc reaches lysosomes via pinocytic/endocytic pathways and is exported in free form into the cytosol, becoming available for activation and transfer to glycoconjugates. In contrast, N-glycolylmannosamine (ManNGc) apparently traverses the plasma membrane by passive diffusion and becomes available for conversion to Neu5Gc in the cytosol. This mechanism can also explain the metabolic incorporation of chemically synthesized unnatural sialic acids, as reported by others. Finally, to our knowledge, this is the first example of delivery to the cytosol of an extracellular small molecule that cannot cross the plasma membrane, utilizing fluid pinocytosis and a specific lysosomal transporter. The approach could, thus, potentially be generalized to any small molecule that has a specific lysosomal transporter but not a plasma membrane transporter.Sialic acid (Sia) 1 is a generic name for a family of acidic nine carbon sugars typically found as the outermost units of glycan chains on the vertebrate cellular glycocalyx and on secreted glycoproteins (1, 2). Their location and widespread occurrence on all vertebrate cells allow them to be involved in processes such as pathogen binding, inflammation, immune response, and tumor metastasis (3-8).There are more than 50 kinds of Sias known in nature (2, 9, 10). Most are derived via biosynthetic modification of a Sia called N-acetylneuraminic acid (Neu5Ac) (3,4,9). The addition of a single oxygen atom to the N-acetyl group of Neu5Ac gives a very common variation called N-glycolylneuraminic acid (Neu5Gc). The surfaces of most primate cell types studied to date are dominated by two major Sias, which are Neu5Ac and Neu5Gc (11,12).For a Sia molecule to get attached to glycoconjugates it must first be activated by conversion to the sugar nucleotide derivative cytidine monophosphate-Sia (CMP-Sia). Thus, Sias are converted to CMP-Sias in the nucleus, which then return to the cytosol to be transported into the Golgi apparatus, where they serve as high energy donors for attaching Sias to newly synthesized glycoconjugates on their way to the cell surfac...

Section: Fig 3 Uptake Of Free Neu5gc Is Not Specific For Human Cellssupporting

confidence: 90%

Mechanism of Uptake and Incorporation of the Non-human Sialic Acid N-Glycolylneuraminic Acid into Human Cells*

Bardor¹,

Nguyen

Díaz

et al. 2005

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321

“…NeuGc is one of the abundant derivatives of sialic acid and is expressed in most mammals. For example, NeuGc comprises 95 and 11% of total sialic acids expressed in mouse and rat liver glycoconjugates, respectively (16). Almost all sialic acid in rat thymus CD45 and Thy-1 is NeuGc (25).…”

Section: Resultsmentioning

confidence: 99%

“…Previous studies showed that the major mechanism for biosynthesis of NeuGc is hydroxylation of CMP-NeuAc (13)(14)(15)(16). Therefore, the activity of CMP-NeuAc hydroxylase plays a key role in the regulation of NeuGc expression in glycoconjugates.…”

mentioning

confidence: 99%

The Molecular Basis for the Absence ofN-Glycolylneuraminic Acid in Humans

Irie

Koyama²,

Kozutsumi³

et al. 1998

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N-Glycolylneuraminic acid (NeuGc) is abundantly expressed in most mammals, but it is not detectable in humans. The expression of NeuGc is controlled by cytidine monophospho-N-acetylneuraminic acid (CMPNeuAc) hydroxylase activity. We previously cloned a cDNA for mouse CMP-NeuAc hydroxylase and found that the human genome contains a homologue. We report here the molecular basis for the absence of NeuGc in humans. We cloned a cDNA for human CMP-NeuAc hydroxylase from a HeLa cell cDNA library. The cDNA encodes a 486-amino acid protein, and its deduced amino acid sequence lacks a domain corresponding to the N-terminal 104 amino acids of the mouse CMPNeuAc hydroxylase protein, although the human protein is highly identical (93%) to the rest of the mouse hydroxylase protein. The N-terminal truncation of the human hydroxylase is caused by deletion of a 92-base pair-long exon in human genomic DNA. The human hydroxylase expressed in COS-7 cells exhibited no enzymatic activity, and a mouse hydroxylase mutant, which lacks the N-terminal domain, was also inactive. A chimera composed of the human hydroxylase and the Nterminal domain of the mouse hydroxylase displayed the enzyme activity. These results indicate that the human homologue of CMP-NeuAc hydroxylase is inactive because it lacks an N-terminal domain that is essential for enzyme activity. The absence of NeuGc in human glycoconjugates is due to a partial deletion in the gene that encodes CMP-NeuAc hydroxylase.Sialic acids are components of the carbohydrate chains of glycoconjugates and are involved in cell-cell recognition (1, 2) and cell-pathogen interactions (3, 4). Sialic acid is a generic designation used for N-acylneuraminic acids and their derivatives (5, 6). N-Acetylneuraminic acid (NeuAc) 1 and N-glycolylneuraminic acid (NeuGc) are two of the most abundant derivatives. Previous studies suggest that some adhesion molecules recognize glycoconjugates containing NeuGc and NeuAc with different affinities. Sialoadhesin, a cell adhesion molecule of marginal zone macrophages, recognizes NeuAc␣2-3Gal but not NeuGc␣2-3Gal (7). On the other hand, mouse CD22, a B cellrestricted adhesion molecule, binds more strongly to NeuGc␣2-6Gal than to NeuAc␣2-6Gal (7). Influenza hemagglutinins recognize glycoconjugates that contain NeuAc and NeuGc with different affinities (8). These studies suggest that the diversity of sialic acids is biologically important in recognition events mediated by glycoconjugates. Although NeuGc-containing glycoconjugates are found in most mammals (5, 6), NeuGc is not detectable in normal human tissues. Glycoconjugates containing NeuGc are immunogenic in human, and an antibody against NeuGc, which is known as Hanganutziu-Deicher antibody, is produced by patients who receive therapeutic injections of animal antisera (9, 10).NeuGc is assumed to be produced from NeuAc through enzymatic hydroxylation of the N-acetyl residue of free NeuAc, CMP-NeuAc, or glycoconjugate-linked NeuAc (11,12). Previous studies showed that the major mechanism for biosynthesis of N...

“…This conversion from CMP-Neu5Ac to CMP-Neu5Gc in the cytosol is catalyzed by CMP-Neu5Ac hydroxylase (Cmah) (25,26). The ratio of the cytosolic CMP-Neu5Ac and CMP-Neu5Gc pool likely determines the Neu5Ac/Neu5Gc ratio in cell surface sialoglycans, because the Golgi CMP-sialic acid transporter and sialyltransferases can utilize both CMP-Neu5Ac and CMPNeu5Gc as substrates without strong preferences (27)(28)(29). Because of the differential regulation of Cmah expression, the ratio of these two major sialic acids varies markedly among different tissues in different animal species except for certain species like humans that have an inactive CMAH gene (CMAHP) and lack endogenous Neu5Gc expression throughout the body.…”

mentioning

confidence: 99%

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

Naito-Matsui

Davies

Takematsu

et al. 2017

Edited by Amanda J. FosangAll 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.