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     

Low incidence of N-glycolylneuraminic acid in birds and reptiles and its absence in the platypus

The sialic acids of the platypus, birds, and reptiles were investigated with regard to the occurrence of N-glycolylneuraminic (Neu5Gc) acid. They were released from tissues, eggs, or salivary mucin samples by acid hydrolysis, and purified and analyzed by thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry. In muscle and liver of the platypus only N-acetylneuraminic (Neu5Ac) acid was found. The nine bird species studied also did not express N-glycolylneuraminic acid with the exception of an egg, but not tissues, from the budgerigar and traces in poultry. Among nine reptiles, including one turtle, N-glycolylneuraminic acid was only found in the egg and an adult basilisk, but not in a freshly hatched animal. BLAST analysis of the genomes of the platypus, the chicken, and zebra finch against the CMP-N-acetylneuraminic acid hydroxylase did not reveal the existence of a similar protein structure. Apparently monotremes (platypus) and sauropsids (birds and reptiles) cannot synthesize Neu5Gc. The few animals where Neu5Gc was found, especially in eggs, may have acquired this from the diet or by an alternative pathway. Since Neu5Gc is antigenic to man, the observation that this monosaccharide does not or at least only rarely occur in birds and reptiles, may be of nutritional and clinical significance.     

Multiple changes in sialic acid biology during human evolution

Humans are genetically very similar to “great apes”, (chimpanzees, bonobos, gorillas and orangutans), our closest evolutionary relatives. We have discovered multiple genetic and biochemical differences between humans and these other hominids, in relation to sialic acids and in Siglecs (Sia-recognizing Ig superfamily lectins). An inactivating mutation in the CMAH gene eliminated human expression of N-glycolylneuraminic acid (Neu5Gc) a major sialic acid in “great apes”. Additional human-specific changes have been found, affecting at least 10 of the <60 genes known to be involved in the biology of sialic acids. There are potential implications for unique features of humans, as well as for human susceptibility or resistance to disease. Additionally, metabolic incorporation of Neu5Gc from animal-derived materials occurs into biotherapeutic molecules and cellular preparations - and into human tissues from dietary sources, particularly red meat and milk products. As humans also have varying and sometime high levels of circulating anti-Neu5Gc antibodies, there are implications for biotechnology products, and for some human diseases associated with chronic inflammation.     

Functional Evaluation of Activation-dependent Alterations in the Sialoglycan Composition of T Cells

Sialic acids (Sias) are often conjugated to the termini of cellular glycans and are key mediators of cellular recognition. Sias are nine-carbon acidic sugars, and, in vertebrates, the major species are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), differing in structure at the C5 position. Previously, we described a positive feedback loop involving regulation of Neu5Gc expression in mouse B cells. In this context, Neu5Gc negatively regulated B-cell proliferation, and Neu5Gc expression was suppressed upon activation. Similarly, resting mouse T cells expressed principally Neu5Gc, and Neu5Ac was induced upon activation. In the present work, we used various probes to examine sialoglycan expression by activated T cells in terms of the Sia species expressed and the linkages of Sias to glycans. Upon T-cell activation, sialoglycan expression shifted from Neu5Gc to Neu5Ac, and the linkage shifted from α2,6 to α2,3. These changes altered the expression levels of sialic acid-binding immunoglobulin-like lectin (siglec) ligands. Expression of sialoadhesin and Siglec-F ligands increased, and that of CD22 ligands decreased. Neu5Gc exerted a negative effect on T-cell activation, both in terms of the proliferative response and in the context of activation marker expression. Suppression of Neu5Gc expression in mouse T and B cells prevented the development of nonspecific CD22-mediated T cell-B cell interactions. Our results suggest that an activation-dependent shift from Neu5Gc to Neu5Ac and replacement of α2,6 by α2,3 linkages may regulate immune cell interactions at several levels.     

Osmolality as a lever to modulate the N-glycolylneuraminicacid (Neu5Gc) level of a recombinant glycoprotein produced in Chinese hamster ovary cells

Glycoproteins could be highly sialylated, and controlling the sialic acid levels for some therapeutic proteins is critical to ensure product consistency and efficacy. N-acetylneuraminic acid (Neu5Ac, or NANA) and N-glycolylneuraminic acid (Neu5Gc, or NGNA) are the two most common forms of sialic acids produced in mammalian cells. As Neu5Gc is not produced in humans and can elicit immune responses, minimizing Neu5Gc formation is important in controlling this quality attribute for complex glycoproteins. In this study, a sialylated glycoprotein was used as the model molecule to study the effect of culture osmolality on Neu5Gc. A 14-day fed-batch process with osmolality maintained at physiological levels produced high levels of Neu5Gc. Increase of culture osmolality reduced the Neu5Gc level up to 70–80%, and the effect was proportional to the osmolality level. Through evaluating different osmolality conditions (300–450 mOsm/kg) under low or high pCO₂, we demonstrated that osmolality could be an effective process lever to modulate the Neu5Gc level. Potential mechanism of osmolality impact on Neu5Gc is discussed and is hypothesized to be cytosol NADH availability related. Compared with cell line engineering efforts, this simple process lever provides the opportunity to readily modulate the Neu5Gc level in a cell culture environment.     

Chemo-enzymatic synthesis of the carbohydrate antigen N-glycolylneuraminic acid from glucose

N-Glycolylneuraminic acid (Neu5Gc) is a non-human sialic acid, which may play a significant role in human pathologies, such as cancer and vascular disease. Further studies into the role of Neu5Gc in human disease are hindered by limited sources of this carbohydrate. Using a chemo-enzymatic approach, Neu5Gc was accessed in six steps from glucose. The synthesis allows access to gram-scale quantities quickly and economically and produces Neu5Gc in superior quality to commercial sources. Finally, we demonstrate that the synthesized Neu5Gc can be incorporated into the cell glycocalyx of human cells, which do not naturally synthesize this sugar. The synthesis produces Neu5Gc suitable for in vitro or in vivo use.     

Importance of high-performance liquid chromatographic analysis of serum N-acylneuraminic acids in evaluating surgical treatment in patients with early endometrial cancer

The objectives of this study were the quantification of the two major sialic acid (Sia) forms – N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic acids (Neu5Gc) – in serum before and after surgical treatment of early endometrial cancer and the relation of their levels with the progress of surgical therapy. The major Sia forms were liberated from sera glycoconjugates by mild acid hydrolysis, separated as per-O-benzoylated derivatives by a highly sensitive reversed-phase HPLC method and detected at 231 nm. Total Sia content in sera of healthy women was not related to age and body weight. Neu5Ac was identified as the major Sia in sera from both cancer patients, healthy individuals as well as in tissue specimens (≥94% of total Sia). In patients with endometrial cancer the total Sia level before surgical treatment (709.5±306.5 mg/l) was significantly higher (p≤0.0001) than that of the control group (213.5±88.7 mg/l). The elevation in Sia level was exclusively due to Neu5Ac. Following surgical therapy, serum Neu5Ac levels (699.4±305.6 mg/l) were significantly decreased (305.9±114.5 mg/l). In one case, where Neu5Ac level was increased 15 days and eight months after surgery (1.8 and 2.5 times as compared to control, respectively), a metastasis not detected during surgery was recorded. The obtained results suggest that Neu5Ac level in serum may be used as a tumor marker in evaluating the suitability of surgical treatment in early endometrial cancer.     

CMP-N-acetylneuraminic acid hydroxylase activity determines the wheat germ agglutinin-binding phenotype in two mutants of the lymphoma cell line MDAY-D2

The dominant glycosylation mutants of MDAY-D2 mouse lymphoma cells, designated class 2 (D33W25 and D34W25) were selected for their resistance to the toxic effects of wheat germ agglutinin (WGA) and shown to express elevated levels of Neu5Gc. In accordance with this, the activity of CMP-Neu5Ac hydroxylase was found to be substantially higher in the mutant cells. The hydroxylase in the D33W25 mutant cells exhibited kinetic properties identical to those of the same enzyme from mouse liver. Growth rate experimentsin vivo andin vitro, where the mutant cells grew more slowly at low cell densities in serum-free medium and also formed slower growing tumours in syngeneic mice, indicate that CMP-Neu5Ac hydroxylase expression may be associated with altered growth of the mutant cells.Abbreviations WGA wheat germ agglutinin - Neu5Ac N-acetyl--d-neuraminic acid - Neu5Gc N-glycology--d-neuraminic acid - CMP-Neu5Ac cytidine-5-monophospho-N-acetylneuraminic acid - CMP-Neu5Gc cytidine-5-monophospho-N-glycoloylneuraminic acid - FACS fluorescence-activated cell sorting - buffer A triethylamine hydrogen carbonate, pH 7.6 (concentration given at appropriate points in the text) - SFM serum free medium - IMDM Iscove's modified Dulbecco's medium - CMP-Neu5Ac hydroxylase CMP-N-acetylneuraminate: NAD(P)H oxido-reductase (N-acetyl hydroxylating) (EC 1.14.99.18); CMP-sialate hydrolase (EC 3.1.4.40); sialic acid-pyruvate lyase (EC 4.1.3.3)     

Adhesion ofHelicobacter pylori strains toα-2,3-linked sialic acids

Helicobacter pylori is a human pathogen associated with gastritis and peptic ulcer. Adhesion properties ofH. pylori to various structures have been described in the literature, including evidence for sialic acid-binding. To study the specificity and frequency of sialic acid-binding, fourteenH. pylori strains were investigated using haemagglutination with derivatized erythrocytes carrying sialic acids only on defined glycans and using haemagglutination inhibition assays. From these studiesH. pylori strains can be grouped into sialic acid-dependent and sialic acid-independent classes. The sialic acid-dependent strains require -2,3-linked sialic acid for haemagglutination. The potential roles of sialic acid-dependent adhesions forH. pylori-related infections are discussed.Abbreviations Sia sialic acids - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolylneuraminic acid - Neu5Fm N-formylneuraminic acid - Neu5TFA N-trifluoroacetylneuraminic acid - RBC human red blood cells (erythrocytes)     

Coexpression of CMP-sialic acid transporter reduces N-glycolylneuraminic acid levels of recombinant glycoproteins in Chinese hamster ovary cells

Recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells contain two forms of sialic acids; N-acetylneuraminic acid (Neu5Ac) as a major type and N-glycolylneuraminic acid (Neu5Gc) as a minor type. The Neu5Gc glycan moieties in therapeutic glycoproteins can elicit immune responses because they do not exist in human. In the present work, to reduce Neu5Gc levels of recombinant glycoproteins from CHO cell cultures, we coexpressed cytidine-5′-monophosphate-sialic acid transporter (CMP-SAT) that is an antiporter and transports cytosolic CMP-sialic acids (both forms) into Golgi lumen. When human erythropoietin was used as a target human glycoprotein, coexpression of CMP-SAT resulted in a significant decrease of Neu5Gc level by 41.4% and a notable increase of Neu5Ac level by 21.2%. This result could be reasonably explained by our hypothesis that the turnover rate of Neu5Ac to Neu5Gc catalyzed by CMP-Neu5Ac hydroxylase would be reduced through facilitated transportation of Neu5Ac into Golgi apparatus by coexpression of CMP-SAT. We confirmed the effects of CMP-SAT coexpression on the decrease of Neu5Gc level and the increase of Neu5Ac level using another glycoprotein human DNase I. Therefore, CMP-SAT coexpression might be an effective strategy to reduce the levels of undesired Neu5Gc in recombinant therapeutic glycoproteins from CHO cell cultures.     

Binding specificity of influenza C-virus to variablyO-acetylated glycoconjugates and its use for histochemical detection ofN-acetyl-9-O-acetylneuraminic acid in mammalian tissues

The specificity of influenza C-virus binding to sialoglycoconjugates was tested with various naturallyO-acetylated gangliosides or syntheticallyO-acetylated sialic acid thioketosides, which revealed binding to 9-O-acetylatedN-acetylneuraminic acid. Binding was also observed with a sample of Neu5,7Ac₂-GD3, however at a lower degree. Sialic acids with two or threeO-acetyl groups in the side chain of synthetic sialic acid derivatives are not recognized by the virus. In these experiments, bound viruses were detected with esterase substrates. Influenza C-virus was also used for the histological identification of mono-O-acetylated sialic acids in combination with an immunological visualization of the virus bound to thin-sections. The occurrence of these sialic acids was demonstrated in bovine submandibular gland, rat liver, human normal adult and fetal colon and diseased colon, as well as in human sweat gland. Submandibular gland and colon also contain significant amounts of glycoconjugates with two or three acetyl esters in the sialic acid side chain, demonstrating the value of the virus in discriminating between mono- and higherO-acetylation at the same site. The patterns of staining showed differences between healthy persons and patients with colon carcinoma, ulcerative colitis or Crohn's disease. Remarkably, some human colon samples did not showO-acetyl sialic acid-specific staining. The histochemical observations were controlled by chemical analysis of tissue sialic acids.Abbreviations BSA bovine serum albumin - BSM bovine submandibular gland mucin - HAU haemagglutination units - HPLC high-performance liquid chromatography - HPTLC high-performance thin-layer chromatography - Neu5Ac N-acetylneuraminic acid - Neu5,9Ac₂ N-acetyl-9-O-acetylneuraminic acid - Neu5,7,9Ac₃ N-acetyl-7,9-di-O-acetylneuraminic acid - Neu5,7,8,9Ac₄ N-acetyl-7,8,9-tri-O-acetylneuraminic acid - PBS phosphate-buffered saline - TLC thin-layer chromatography Dedicated to Prof. Dr Nathan Sharon on the occasion of his 70th birthday.     

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.     

Preparation ofN-acetylneuraminic acid from delipidated egg yolk

Egg yolk, a large proportion of the egg, was studied for the preparation ofN-acetylneuraminic acid (Neu5Ac). The delipidated hen egg yolk (DEY; 500 kg containing 0.2% w/w, Neu5Ac) was hydrolysed with HCl (pH 1.4) at 80 °C and neutralized with NaOH (pH 6.0). The mixture was filtered and electrodialysed until the conductivity was 240 µS cm^–1. The filtrate was applied on a column of Dowex HCR-W2 (20–50 mesh), followed by a column of Dowex 1-X8 (200–400 mesh). The latter column was washed with water, and then eluted with a linear gradient of HCO₂H (0–2m). The eluates containing Neu5Ac were concentrated using a reverse osmosis membrane and, finally, rotary evaporated at 40 °C. The residue was then lyophilized to yield 500 g Neu5Ac. The purity of Neu5Ac was >98% (TBA method). HPLC, NMR spectroscopy and TLC chromatography of the product obtained from the DEY showed that Neu5Ac was the sole derivative present in egg yolk. The DEY, a byproduct from egg processing plants, was found to be an excellent source for the large-scale preparation of Neu5Ac.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycolylneuraminic acid - DEY delipidated egg yolk - HPLC high performance liquid chromatography - TLC thin layer chromatography - NMR nuclear magnetic resonance - IR infrared spectroscopy Presented at the 11th International Symposium on Glycoconjugates, Toronto, Canada.     

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.     

The presence of N-acetylneuraminic acid in Malpighian tubules of larvae of the cicada Philaenus spumarius

Sialic acid-containing glycoconjugates are generally considered to be unique to the deuterostomes, a lineage of the animal kingdom which includes animals from the echinoderms up to the vertebrates. There are, however, two isolated reports of sialic acid occurring in the insect species Drosophila melanogaster and Galleria mellonella. Since insects are classified as protostomes, these findings call previous assumption on the phylogenetic distribution and thus on the evolution of sialic acids into question. Here, we report the occurrence of N-acetylneuraminic acid (Neu5Ac) in larvae of the cicada Philaenus spumarius. Cytochemical analysis of larval sections with lectins from Sambucus nigra and Limax flavus suggested the presence of sialic acids in the concrement vacuoles of the Malpighian tubules. The monoclonal antibody MAb 735, which is specific for polysialic acid, labelled the same structures. A chemical analysis performed by HPLC of fluorescent derivatives of sialic acids and by GLC-MS provided sound evidence for the presence of Neu5Ac in the Philaenus spumarius larvae. These data suggest that in this cicada Neu5Ac occurs in 2,8-linked polysialic acid structures and in 2,6-linkages. The results provide further evidence for the existence of sialic acids in insects and in linkages known to occur in glycoconjugates of deuterostomate origin.     

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).     

The Sialoadhesins — A family of sialic acid-dependent cellular recognition molecules within the immunoglobulin superfamily

For many years evidence has accumulated that sialic acids function in cellular interactions either by masking or as a recognition site. However, receptors or adhesion molecules mediating such functions between eukaryotic cells were unknown until about 5 years ago, when it was found that the members of the Selectin family mediate adhesion of leukocytes to specific endothelia through binding to sialylated glycans like sialyl Lewis^x. More recently, the Sialoadhesin family of sialic acid-dependent adhesion molecules was defined within the superfamily of immunoglobulin-like molecules. So far, it has been shown that sialoadhesin (Sn), CD22, CD33, the myelin-associated glycoprotein (MAG) and the Schwann cell myelin protein (SMP) belong to this family. In contrast to the Selectins, these proteins are associated with diverse biological processes, i.e. hemopoiesis, neuronal development and immunity. In this review their properties, carbohydrate specificities and potential biological functions are discussed. Finally, we provide perspectives with respect to the nature of ligands, implications of sialic acid modifications and future research.Abbreviations IgSF immunoglobulin superfamily - MAG myelin-associated glycoprotein - Sia sialic acid - SMP Schwann cell myelin protein - Sn sialoadhesin     

Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

CHO cells express glycoproteins containing both the N‐acetylneuraminic acid (Neu5Ac) and minor amounts of the N‐glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein therapeutics. During process development of a glycosylated fusion protein expressed by CHO cells, a number of culture conditions were identified that affected the Neu5Gc content of the recombinant glycoprotein. Sodium butyrate (SB), a well‐known additive reported to enhance recombinant protein productivity in specific cases, minimally affected product titers here, but did decrease Neu5Gc levels by 50–62%. A shift in culture temperature to a lower value after the exponential growth phase was used to extend the culture period. It was found that the Neu5Gc levels were 59% lower when the temperature shift occurred later near the stationary phase of the culture compared to an early‐temperature shift, near the end of the exponential growth phase. Studies on the effects of pCO₂ with this product showed that the Neu5Gc levels were 46% lower at high pCO₂ conditions (140 mmHg) compared to moderate pCO₂ levels (20–80 mmHg). Finally, a comparison of sodium carbonate versus sodium hydroxide as the base used for pH control resulted in a reproducible 33% decrease in Neu5Gc in bioreactors using sodium hydroxide. These results are of practical importance as SB is a commonly tested additive, and the other factors affecting Neu5Gc can conveniently be used to reduce or control Neu5Gc in processes for the manufacture of glycoprotein therapeutics. Biotechnol. Bioeng. 2010;105: 1048–1057. © 2009 Wiley Periodicals, Inc.     

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.     

Structural Determination of Monosialyl Trisaccharides Obtained From Caprine Colostrum

Acidic oligosaccharides were separated by dialysis, ion-exchange, preparative paper and gel chromatography from caprine colostrum. Four sialyl trisaccharides were characterized by ¹H-NMR spectrometry as follows: α-N-acetylneuraminyl-(2,6)-β-d-galactopyranosyl-(1,4)-2-N-acetamido-2-deoxy-d-glucopyranose (Neu5Ac α 2-6Gal β 1-4GlcNAc), α-N-acetylneuraminyl-(2,3)-β-d-galactopyranosyl-(1,4)-d-glucopyranose (Neu5Ac α 2-3Gal β-1-4Glc), α-N-acetylneuraminyl-(2,6)-β-d-galactopyranosyl-(1,4)-d-glucopyranose (Neu5Ac α 2-6Gal β 1-4Glc) and α-N-glycolylneuraminyl-(2,6)-β-d-galactopyranosyl-(1,4)-d-glucopyranose (Neu5Gc α 2-6Gal β 1-4Glc).