Ganglioside expression in tissues of mice lacking the tumor necrosis factor receptor 1

This study presents a comparative analysis of gangliosides from lymphoid (spleen and thymus) and other tissues (brain, liver, lung, muscle) of C57BL/6 mice homozygous (-/-) and heterozygous (+/-) for the tumor necrosis factor receptor 1 (TNFRp55). Quantitative and qualitative differences in the expression of the lipid-bound N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) and of various ganglioside biosynthesis pathways were detected between the tissues of the TNFRp55 -/- and the control TNFRp55 +/- mice. Sialic acid profiles showed a strong decrease in the absolute amount of sialic acids (Neu5Ac + Neu5Gc) in the lungs and thymus of homozygous (1.41 and 0.3 ng/mg wet weight, respectively) compared with control heterozygous animals (7.18 and 2.05 ng/mg wet weight, respectively). Considerable differences of Neu5Ac/Neu5Gc ratios in the lungs, muscle, spleen, and thymus were also detected. The gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) were the dominant gangliosides in the lungs of the control animals, whereas the knockout mice almost completely lacked these structures in this organ. Reduced expression of GM1b-type gangliosides (GM1b and GalNAc-GM1b) was also found in the lungs, spleen, and thymus of the TNFRp55 knockout mice. On the other hand, neolacto-series gangliosides were more abundant in the lungs, brain, and muscle of the knockout mice, whereas their expression in the liver, spleen, and thymus was similar in both groups of animals. This study provides in vivo evidence that TNF signaling via the TNFRp55 is involved in the acquisition of a distinct ganglioside assembly in different mouse organs. TNFRp55 signaling seems to be especially important for the activation of the GM1b-type ganglioside biosynthetic pathway that is a unique characteristic of the mouse lymphoid tissues.     

Developmental sialic acid modifications in rat organs.

The changes in expression of sialic acids in Sprague-Dawley rats in the prenatal and early postnatal time period have been examined in multiple organs, both visceral and non-visceral. In all organs examined, there is a dramatic increase in both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) shortly after birth. The bulk of the sialic acid is present in the ganglioside fraction in all tissues examined. As total amounts of sialic acid present in gangliosides decrease, the proportion present in the low molecular weight cytosolic fraction increases. A curious observation is that Neu5Ac hydroxylase activity is present at the time of the increase in sialic acid, but its activity does not correlate with Neu5Gc expression after the early postnatal period. This implies that Neu5Gc expression has another level of regulation besides CMP-Neu5Ac hydroxylase activity.     

Implication of N-glycolylneuraminic acid in regulation of cell adhesiveness of C2C12 myoblast cells during differentiation into myotube cells

Glycoconjugate Journal - A transition of sialic acid (Sia) species on GM3 ganglioside from N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc) takes place in mouse C2C12 myoblast...     

Biosynthesis of N-glycolyneuraminic acid. The primary site of hydroxylation of N-acetylneuraminic acid is the cytosolic sugar nucleotide pool

N-Glycolylneuraminic acid (Neu5Gc) is an oncofetal antigen in humans and is developmentally regulated in rodents. We have explored the biology of N-acetylneuraminic acid hydroxylase, the enzyme responsible for conversion of the parent sialic acid, N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. We show that the major sialic acid in all compartments of murine myeloma cell lines is Neu5Gc. Pulse-chase analysis in these cells with the sialic acid precursor [6-3H]N-acetylmannosamine demonstrates that most of the newly synthesized Neu5Gc appears initially in the cytosolic low-molecular weight pool bound to CMP. The percentage of Neu5Gc on membrane-bound sialic acids closely parallels that in the CMP-bound pool at various times of chase, whereas that in the free sialic acid pool is very low initially, and rises only later during the chase. This implies that conversion from Neu5Ac to Neu5Gc occurs primarily while Neu5Ac is in its sugar nucleotide form. In support of this, the hydroxylase enzyme from a variety of tissues and cells converted CMP-Neu5Ac to CMP-Neu5Gc, but showed no activity towards free or alpha-glycosidically bound Neu5Ac. Furthermore, the majority of the enzyme activity is found in the cytosol. Studies with isolated intact Golgi vesicles indicate that CMP-Neu5Gc can be transported and utilized for transfer of Neu5Gc to glycoconjugates. The general properties of the enzyme have also been investigated. The Km for CMP-Neu5Ac is in the range of 0.6-2.5 microM. No activity can be detected against the beta-methylglycoside of Neu5Ac. On the other hand, inhibition studies suggest that the enzyme recognizes both the 5'-phosphate group and the pyrimidine base of the substrate. Taken together, the data allow us to propose pathways for the biosynthesis and reutilization of Neu5Gc, with initial conversion from Neu5Ac occurring primarily at the level of the sugar nucleotide. Subsequent release and reutilization of Neu5Gc could then account for the higher steady-state level of Neu5Gc found in all of the sialic acid pools of the cell.     

Preferential Accumulation of 14C-N-Glycolylneuraminic Acid over 14C-N-Acetylneuraminic Acid in the Rat Brain after Tail Vein Injection

The two main molecular species of sialic acid existing in nature are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Neu5Ac is abundant in mammalian brains and plays crucial roles in many neural functions. In contrast, Neu5Gc is present only at a trace level in vertebrate brains. The brain-specific suppression of Neu5Gc synthesis, which is a common feature in mammals, suggests that Neu5Gc has toxicity against brain functions. However, in vivo kinetics of Neu5Gc in the whole body, especially in the brain, has not been studied in sufficient detail. To determine the in vivo kinetics of Neu5Gc, ¹⁴C-Neu5Gc was enzymatically synthesized and injected into rat tail veins. Although most of ¹⁴C-Neu5Gc was excreted in urine, a small amount of ¹⁴C-Neu5Gc was detected in the brain. Brain autoradiography indicated that ¹⁴C-Neu5Gc was accumulated predominantly in the hippocampus. ¹⁴C-Neu5Gc transferred into the brain was incorporated into gangliosides including GM1, GD1a, GD1b, GT1b and GQ1b. Reduction of ¹⁴C-Neu5Gc after intracerebroventricular infusion was slower than that of ¹⁴C-Neu5Ac in the brain and hippocampus. The results suggest that Neu5Gc is transferred from blood into the brain across the blood brain barrier and accumulates in the brain more preferentially than does Neu5Ac.     

Solution conformations of GM3 gangliosides containing different sialic acid residues as revealed by NOE-based distance mapping, molecular mechanics, and molecular dynamics calculations.

The conformation of the GM3 ganglioside, Neu5Ac alpha 2-3Gal beta 1-4Glc beta 1-1 Cer, and its analogs containing the Neu5Gc or Neu4Ac5Gc residues (Gc = glycolyl, CH2OHCO) was investigated in Me2SO-d6 solution with the aid of a distance-mapping procedure based on rotating-frame NOE contacts, with hydroxyl protons being used as long-range sensors defining the distance constraints. A pronounced flexibility found for both the Neu-Gal and Gal-Glc linkages was confirmed by 1000-ps molecular dynamics simulations. Similar results, although based on a smaller number of NOE constraints, were obtained for GM3 gangliosides anchored in mixed D2O/dodecylphosphocholine-d38 micelles and for the Neu5Ac-, Neu5Gc-, and Neu5,9Ac2-sialyllactoses dissolved in D2O. No noteworthy differences in conformational behavior of the glycan chains of the three gangliosides or sialyllactoses were observed in either of the media.     

Determination of sialic acids in immune system cells (coelomocytes) of sea urchin,Paracentrotus lividus,using capillary LC-ESI-MS/MS

Coelomocytes are considered to be immune effectors of sea urchins. Coelomocytes are the freely circulating cells in the body fluid contained in echinoderm coelom and mediate the cellular defence responses to immune challenges by phagocytosis, encapsulation, cytotoxicity and the production of antimicrobial agents. Coelomocytes have the ability to recognize self from non-self. Considering that sialic acids play important roles in immunity, we determined the presence of sialic acid types in coelomocytes of Paracentrotus lividus. Homogenized coelomocytes were kept in 2 M aqueous acetic acid at 80 °C for 3 h to liberate sialic acids. Sialic acids were determined by derivatization with 1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride (DMB) followed by capillary liquid-chromatography-electrospray ionization/tandem mass spectrometry (CapLC-ESI-MS/MS). Standard sialic acids; Neu5Ac, Neu5Gc, KDN and bovine submaxillary mucin showing a variety of sialic acids were used to confirm sialic acids types. We found ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac₂, Neu5,7Ac₂, Neu5,8Ac₂, Neu5,7,9Ac₃, Neu5Gc7,9Ac₂, Neu5Gc7Ac) isolated in limited amounts from total coelomocyte population. Neu5Gc type of sialic acids in coelomocytes was the most abundant type sialic acid when compared with other types. This is the first report on the presence of sialic acid types in coelomocytes of P. lividus using CapLC-ESI-MS/MS-Ion Trap system (Capillary Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry).     

A structural difference between the cell surfaces of humans and the great apes

The sialic acids are major components of the cell surfaces of animals of the deuterostome lineage. Earlier studies suggested that humans may not express N-glycolyl-neuraminic acid (Neu5Gc), a hydroxylated form of the common sialic acid N-acetyl-neuraminic acid (Neu5Ac). We find that while Neu5Gc is essentially undetectable on human plasma proteins and erythrocytes, it is a major component in all the four extant great apes (chimpanzee, bonobo, gorilla and orangutan) as well as in many other mammals. This marked difference is also seen amongst cultured lymphoblastoid cells from humans and great apes, as well as in a variety of other tissues compared between humans and chimpanzees, including the cerebral cortex and the cerebrospinal fluid. Biosynthetically, Neu5Gc arises from the action of a hydroxylase that converts the nucleotide donor CMP-Neu5Ac to CMP-Neu5Gc. This enzymatic activity is present in chimpanzee cells, but not in human cells. However, traces of Neu5Gc occur in some human tissues, and others have reported expression of Neu5Gc in human cancers and fetal tissues. Thus, the enzymatic capacity to express Neu5Gc appears to have been suppressed sometime after the great ape-hominid divergence. As terminal structures on cell surfaces, sialic acids are involved in intercellular cross-talk involving specific vertebrate lectins, as well as in microbe-host recognition involving a wide variety of pathogens. The level of sialic acid hydroxylation (level of Neu5Ac versus Neu5Gc) is known to positively or negatively affect several of these endogenous and exogenous interactions. Thus, there are potential functional consequences of this widespread structural change in humans affecting the surfaces of cells throughout the body. Am J Phys Anthropol 107:187-198, 1998. © 1998 Wiley-Liss, Inc.     

A shift from N-glycolyl- to N-acetyl-sialic acid in the GM3 ganglioside impairs tumor development in mouse lymphocytic leukemia cells

Humans, in contrast to other mammals, do not synthesize N-glycolyl-neuraminic acid (Neu5Gc) due to a deletion in the gene (cmah) encoding the enzyme responsible for this conversion, the cytidine monophospho-N-acetyl-neuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase). The detection of considerable amounts of Neu5Gc-sialoconjugates, in particular gangliosides, in human malignancies makes these antigens attractive targets for immunotherapy, in particular with monoclonal antibodies (mAbs). We have previously described a GM3(Neu5Gc) ganglioside-specific mAb, named 14F7, with the ability to kill tumor cells in a complement-independent manner. Silencing the cmah gene in GM3(Neu5Gc)-expressing L1210 mouse lymphocytic leukemia B cells caused the abrogation of this cytotoxic effect. We now show that cmah-silenced L1210 cells (cmah-kd) express a high level of GM3(Neu5Ac) and have an impaired ability for anchorage-independent cell growth and tumor development in vivo. No evidences of increased immunogenicity of the cmah-kd cell line were found. These results provide new evidences on the role of GM3(Neu5Gc), or Neu5Gc-sialoconjugates in general, in tumor biology. As an important tool in this study, we used the humanized version (here referred to as 7C1 mAb) of a recently described, rationally-designed mutant of 14F7 mAb that is able to bind to both GM3(Neu5Gc) and GM3(Neu5Ac). In contrast to its parental antibody, the humanized 14F7 (14F7hT) mAb, 7C1 mAb was able to kill not only GM3(Neu5Gc)-expressing L1210 wild type cells, but also GM3(Neu5Ac)-expressing cmah-kd cells, which endorses this antibody as a potential agent for cancer immunotherapy.     

Isolation and characterization of gangliosides from pig lymphocytes

Two major gangliosides from pig spleen lymphocytes, accounting for 57% of the total lipid-bound sialic acids, were isolated and purified to homogeneity by column chromatography on DEAE-Sephadex and silica gel. They were identified as GM3 (II3Neu5GcLacCer), and GD3 (II3(Neu5Gc)2LacCer), by thin-layer chromatography in comparison with standards and by analysis of the constituent sugars. The major fatty acids of these gangliosides were stearic acid and myristic acid, respectively. In addition to these gangliosides, GD2 and bands comigrating on thin-layer chromatography with authentic GM2, GM1, GD1a and GD1b were found. These compounds also occur in pig peripheral blood lymphocytes, where, however, GD3 represents about 70% of the total lipid-bound sialic acid.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INCORPORATION OF N-GLYCOLYLHEXOSAMINES INTO MAMMALIAN GLYCANS BY FEEDING N-GLYCOLYLGALACTOSAMINE

The outermost positions of mammalian cell-surface glycans are predominantly occupied by the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). To date, hydroxylation of CMP-Neu5Ac resulting in the conversion into CMP-Neu5Gc is the only known enzymatic reaction in mammals to synthesize a monosaccharide carrying an N-glycolyl group. In our accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, jbc.M112.363549), we report a metabolic pathway for degradation of Neu5Gc, demonstrating that N-acetylhexosamine pathways are tolerant toward the N-glycolyl substituent of Neu5Gc breakdown products. In this study, we show that exogenously added N-glycolylgalactosamine (GalNGc) serves as a precursor for Neu5Gc de novo biosynthesis, potentially involving seven distinct mammalian enzymes. Following the GalNAc salvage pathway, UDP-GalNGc is epimerized to UDP-GlcNGc, which might compete with the endogenous UDP-GlcNAc for the sialic acid biosynthetic pathway. Using UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase-deficient cells, we confirm that conversion of GalNGc into Neu5Gc depends on this key enzyme of sialic acid biosynthesis. Furthermore, we demonstrate by mass spectrometry that the metabolic intermediates UDP-GalNGc and UDP-GlcNGc serve as substrates for assembly of most major classes of cellular glycans. We show for the first time incorporation of GalNGc and GlcNGc into chondroitin/dermatan sulfates and heparan sulfates, respectively. As demonstrated by structural analysis, N-glycolylated hexosamines were found in cellular gangliosides and incorporated into Chinese hamster ovary cell O-glycans. Remarkably, GalNAc derivatives altered the overall O-glycosylation pattern as indicated by the occurrence of novel O-glycan structures. This study demonstrates that mammalian N-acetylhexosamine pathways and glycan assembly are surprisingly tolerant toward the N-glycolyl substituent.     

Comparison of theN-glycoloylneuraminic andN-acetylneuraminic acid content of platelets and their precursors using high performance anion exchange chromatography

N-Acetylneuraminic acid (Neu5Ac) andN-glycoloylneuraminic acid (Neu5Gc) are distributed widely in nature. Using a Carbopac PA-1 anion exchange column, we have determined the ratios of Neu5Ac and Neu5Gc in hydrolysates of platelets and their precursors: a rat promegakaryoblastic (RPM) cell line and a human megakaryoblastic leukemia cell line (MEG-01). The ratio of Neu5Gc:Neu5Ac in cultured RPM cells is 16:1, whereas in platelet rich plasma and cultured MEG-01 cells it is 1:38 and 1:28, respectively. The nature of these sialic acids from RPM cells was verified using thin layer chromatography and liquid secondary ion mass spectrometry. The relevance of increased Neu5Gc levels in early stages of development is discussed.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - RPM rat promegakaryoblast - MEG-01 human megakaryoblastic leukaemia cell line - PAD pulsed amperometric detection - WGA wheat germ agglutinin - FCS foetal calf serum - PPEADF phosphatidylethanolamine dipalmitoyl - LSIMS liquid secondary ion mass spectrometry - HPAEC high performance anion exchange chromatography - TBA thiobarbituric acid     

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

The relative contribution of N-glycoloyl-beta-D-neuraminic acid (Neu5Gc) to total sialic acids expressed in mouse and rat liver glycoconjugates was found to be 95% and 11%, respectively. This considerable difference in sialic acid composition made these two tissues suitable models for a comparative investigation into the regulation of Neu5Gc biosynthesis and utilization. An examination of the CMP-glycoside specificity of Golgi-associated sialyltransferases using CMP-N-acetyl-beta-D-neuraminic acid (CMP-Neu5Ac) and CMP-Neu5Gc revealed no significant tissue-dependent differences. The Golgi membrane CMP-sialic acid transport system from rat liver did, however, exhibit a slightly higher internalisation rate for CMP-Neu5Ac, though no preferential affinity for this sugar nucleotide over CMP-Neu5Gc was observed. In experiments, where Golgi membrane preparations were incubated with an equimolar mixture of labelled CMP-Neu5Ac and CMP-Neu5Gc, no significant tissue-dependent differences in [14C]sialic acid composition were observed, either in the luminal soluble sialic acid fraction or in the precipitable sialic acid fraction, results which are consistent with the above observations. From this experiment, evidence was also obtained for the presence of a Golgi-lumen-associated CMP--sialic acid hydrolase which exhibited no apparent specificity for either CMP-Neu5Ac or CMP-Neu5Gc. The specific activity of the CMP-Neu5Ac hydroxylase, the enzyme responsible for the biosynthesis of Neu5Gc, was found to be 28-fold greater in high-speed supernatants of mouse liver than of rat liver. No hydroxylase activity was detected in the Golgi membrane preparations. It is therefore proposed that the cytoplasmic ratio of CMP-Neu5Ac and CMP-Neu5Gc produced by the hydroxylase, remains largely unmodified after CMP-glycoside uptake into the Golgi apparatus and transfer on to growing glycoconjugate glycan chains. The close relationship between the total sialic acid composition and the sialic acid pattern in the CMP-glycoside pools of the tissues lends considerable weight to this hypothesis.     

Loss of N-glycolylneuraminic acid in human evolution. Implications for sialic acid recognition by siglecs

The common sialic acids of mammalian cells are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans are an exception, because of a mutation in CMP-sialic acid hydroxylase, which occurred after our common ancestor with great apes. We asked if the resulting loss of Neu5Gc and increase in Neu5Ac in humans alters the biology of the siglecs, which are Ig superfamily members that recognize sialic acids. Human siglec-1 (sialoadhesin) strongly prefers Neu5Ac over Neu5Gc. Thus, humans have a higher density of siglec-1 ligands than great apes. Siglec-1-positive macrophages in humans are found primarily in the perifollicular zone, whereas in chimpanzees they also occur in the marginal zone and surrounding the periarteriolar lymphocyte sheaths. Although only a subset of chimpanzee macrophages express siglec-1, most human macrophages are positive. A known evolutionary difference is the strong preference of mouse siglec-2 (CD22) for Neu5Gc, contrasting with human siglec-2, which binds Neu5Ac equally well. To ask when the preference for Neu5Gc was adjusted in the human lineage, we cloned the first three extracellular domains of siglec-2 from all of the great apes and examined their preference. In fact, siglec-2 had evolved a higher degree of recognition flexibility before Neu5Gc was lost in humans. Human siglec-3 (CD33) and siglec-6 (obesity-binding protein 1) also recognize both Neu5Ac and Neu5Gc, and siglec-5 may have some preference for Neu5Gc. Others showed that siglec-4a (myelin-associated glycoprotein) prefers Neu5Ac over Neu5Gc. Thus, the human loss of Neu5Gc may alter biological processes involving siglec-1, and possibly, siglec-4a or -5.     

Detection, isolation, and characterization of oligo/poly(sialic acid) and oligo/poly(deaminoneuraminic acid) units in glycoconjugates.

We have evaluated methods for separation, preparation, and characterization of alpha-2----8-linked oligomers of sialic acids (Neu5Ac and Neu5Gc) and deaminated neuraminic acid (KDN; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) recently found as a naturally occurring novel type of sialic acid analogue. (A) We examined preparative anion-exchange chromatography for fractionation and preparation of oligo(Neu5Ac), oligo(Neu5Gc), and oligo(KDN). (B) We also examined the TLC method for separation and differentiation of the partial acid hydrolysates of colominic acid, as well as polysialoglycoproteins (PSGP) and poly(KDN)-glycoproteins (KDN-gp) isolated from rainbow trout eggs, and for discrimination of lower oligomers of Neu5Ac, Neu5Gc, and KDN. (C) We developed the high-performance adsorption-partition chromatographic method for (a) separation of monomers and oligomers of three nonulosonates according to the difference in substituents at C-5 and the presence or absence of 9-O-acetyl groups in oligo(KDN) and (b) separation of three homologous series of lower oligomers according to the degree of polymerization. (D) We examined and compared high-performance anion-exchange chromatographic separation of 3H-labeled oligo(Neu5Ac), oligo(Neu5Gc), and oligo(KDN) alditols by using Mono-Q HR 5/5 resin. (E) We examined a method of selective and quantitative microprecipitation for separation and purification of oligomers and polymers of Neu5Ac by treating them with cetylpyridinium chloride. We also used PSGP and KDN-gp to test both the sensitivity and the selectivity of this method.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: RESISTANCE OF α2-8-LINKED N-GLYCOLYLNEURAMINIC ACID TO ENZYMATIC CLEAVAGE

The sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) and its hydroxylated derivative N-glycolylneuraminic acid (Neu5Gc) differ by one oxygen atom. CMP-Neu5Gc is synthesized from CMP-Neu5Ac, with Neu5Gc representing a highly variable fraction of total Sias in various tissues and among different species. The exception may be the brain, where Neu5Ac is abundant and Neu5Gc is reported to be rare. Here, we confirm this unusual pattern and its evolutionary conservation in additional samples from various species, concluding that brain Neu5Gc expression has been maintained at extremely low levels over hundreds of millions of years of vertebrate evolution. Most explanations for this pattern do not require maintaining neural Neu5Gc at such low levels. We hypothesized that resistance of α2-8-linked Neu5Gc to vertebrate sialidases is the detrimental effect requiring the relative absence of Neu5Gc from brain. This linkage is prominent in polysialic acid (polySia), a molecule with critical roles in vertebrate neural development. We show that Neu5Gc is incorporated into neural polySia and does not cause in vitro toxicity. Synthetic polymers of Neu5Ac and Neu5Gc showed that mammalian and bacterial sialidases are much less able to hydrolyze α2-8-linked Neu5Gc at the nonreducing terminus. Notably, this difference was not seen with acid-catalyzed hydrolysis of polySias. Molecular dynamics modeling indicates that differences in the three-dimensional conformation of terminal saccharides may partly explain reduced enzymatic activity. In keeping with this, polymers of N-propionylneuraminic acid are sensitive to sialidases. Resistance of Neu5Gc-containing polySia to sialidases provides a potential explanation for the rarity of Neu5Gc in the vertebrate brain.     

N-glycolylneuraminic acid conjugates: implications of their absence in mammalian biochemistry

N-glycolylneuraminic acid (Neu5Gc) is one of the two most common forms of sialic acids present in glycoproteins and glycolipids of mammalian tissues. It is synthesized from the most ubiquitous sialic acid, N-acetylneuraminic acid (Neu5Ac) in a hydroxylation reaction catalysed by the enzyme Neu5Ac hydroxylase. Though Neu5Gc conjugates are prevalent in many tissues of mammals, they are absent in glycolipids and only trace amounts are present in glycoproteins of the brain and central nervous system. In humans Neu5Ac is the main sialic acid as Neu5Ac hydroxylase is inactive due to mutation of its gene. The importance of sialic acids in biochemical phenomena and the distinct roles played by specific forms of these amino sugars is adequately reflected in functional studies of selectin and sialoadhesin families of adhesion molecules. The absence of Neu5Gc, therefore, in tissues of humans and brain of mammals has raised interest, especially with regard to its impact on biochemical differences evident between humans and other mammals. It is suggested that though Neu5Gc conjugates are important in cellular interactions, their presence in brain and the central nervous system is deleterious to the latter's normal functions. Their interaction with other cellular components to form supramolecular associations is indicated that may have a bearing on major biochemical differences, a few of which are presently evident between humans and other mammals.     

A sialylated glycan microarray reveals novel interactions of modified sialic acids with proteins and viruses

Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2-3- and α2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2-6- and α2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2-6-linked Neu5Ac9Lt or α2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.     

Evidence of free radical participation in N-glycolylneuraminic acid generation in liver of chicken treated with gallotannic acid

The occurrence of N-glycolylneuraminic acid (Neu5Gc) in cancerous tissue and inflammatory diseases, conditions associated with increased oxidative stress suggests the participation of reactive oxygen radicals in Neu5Gc generation, where an oxygen atom is transferred. To study this possibility, we treated two groups of domesticated birds and rabbits with different dosages of gallotannic acid (GTA), a compound known to cause generation of reactive oxygen species (ROS). The antioxidant status and leukocyte capacity, as well as amount and form of sialic acids were assessed in plasma and liver. Results showed that while lipid peroxides were increased, white blood cell (WBC) count was decreased significantly in all treated groups. The increased sialic acids and low protein contents were observed in plasma, possibly as a result of decreased sialic acid cycling crucial for formation of new glycoconjugates in tissues, caused by decreased protein synthesis due to microsomal degranulation. The activities of antioxidant enzymes were also decreased in treated groups, implying increased oxidative stress. The presence of Neu5Gc and apparent absence of Neu5Ac hydroxylase activity in liver of chicken treated with GTA indicate that free radicals might be involved in the non-enzymatic hydroxylation of N-acetylneuraminic acid (Neu5Ac) to Neu5Gc in liver, which normally does not express this sialic acid.     

A uniquely human consequence of domain-specific functional adaptation in a sialic acid-binding receptor

Most mammalian cell surfaces display two major sialic acids (Sias), N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans lack Neu5Gc due to a mutation in CMP-Neu5Ac hydroxylase, which occurred after evolutionary divergence from great apes. We describe an apparent consequence of human Neu5Gc loss: domain-specific functional adaptation of Siglec-9, a member of the family of sialic acid-binding receptors of innate immune cells designated the CD33-related Siglecs (CD33rSiglecs). Binding studies on recombinant human Siglec-9 show recognition of both Neu5Ac and Neu5Gc. In striking contrast, chimpanzee and gorilla Siglec-9 strongly prefer binding Neu5Gc. Simultaneous probing of multiple endogenous CD33rSiglecs on circulating blood cells of human, chimp, or gorilla suggests that the binding differences observed for Siglec-9 are representative of multiple CD33rSiglecs. We conclude that Neu5Ac-binding ability of at least some human CD33rSiglecs is a derived state selected for following loss of Neu5Gc in the hominid lineage. These data also indicate that endogenous Sias (rather than surface Sias of bacterial pathogens) are the functional ligands of CD33rSiglecs and suggest that the endogenous Sia landscape is the major factor directing evolution of CD33rSiglec binding specificity. Exon-1-encoded Sia-recognizing domains of human and ape Siglec-9 share only approximately 93-95% amino acid identity. In contrast, the immediately adjacent intron and exon 2 have the approximately 98-100% identity typically observed among these species. Together, our findings suggest ongoing adaptive evolution specific to the Sia-binding domain, possibly of an episodic nature. Such domain-specific divergences should also be considered in upcoming comparisons of human and chimpanzee genomes.