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.     

Regulation of sialic acid O-acetylation in human colon mucosa

The expression of O-acetylated sialic acids in human colonic mucins is developmentally regulated, and a reduction of O-acetylation has been found to be associated with the early stages of colorectal cancer. Despite this, however, little is known about the enzymatic process of sialic acid O-acetylation in human colonic mucosa. Recently, we have reported on a human colon sialate-7(9)-O-acetyltransferase capable of incorporating acetyl groups into sialic acids at the nucleotide-sugar level [Shen et al., Biol. Chem. 383 (2002), 307-317]. In this report, we show that the CMP-N-acetyl-neuraminic acid (CMP-Neu5Ac) and acetyl-CoA (AcCoA) transporters are critical components for the O-acetylation of CMP-Neu5Ac in Golgi lumen, with specific inhibition of either transporter leading to a reduction in the formation of CMP-5-N-acetyl-9-O-acetyl-neuraminic acid (CMP-Neu5,9Ac2). Moreover, the finding that 5-N-acetyl-9-O-acetyl-neuraminic acid (Neu5,9Ac2 could be transferred from neo-synthesised CMP-Neu5,9Ac2 to endogenous glycoproteins in the same Golgi vesicles, together with the observation that asialofetuin and asialo-human colon mucin are much better acceptors for Neu5,9Ac2 than asialo-bovine submandibular gland mucin, suggests that a sialyltransferase exists that preferentially utilises CMP-Neu5,9Ac2 as the donor substrate, transferring Neu5,9Ac2 to terminal Galbeta1,3(4)R- residues.     

Metabolism of sialic acid in regenerating rat liver.

In regenerating rat liver slices 24 h after partial hepatectomy, the incorporation of [1-14C]glucosamine into 'free sialic acid' (N-acetylneuraminic acid + CMP-N-acetylneuraminic acid) decreased to below 50% of the control values and the incorporation into protein-bound sialic acid decreased to the same extent. The incorporation of [14C]glucosamine into 'free sialic acid' decreased during the period from 6 to 47 h after hepatectomy, showing a minimum at 12 h, and recovered to the control value by 96 h. At 12 h, the activities of UDP-N-acetylglucosamine 2-epimerase (UDP-2-acetoamido-2-deoxy-D-glucose 2-epimerase, EC 5.1.3.14) and N-acyl-D-mannosamine kinase (ATP: 2-acylamino-2-deoxy-D-mannose 6-phosphotransferase, EC 2.7.1.60) in the liver were significantly decreased. The amount of protein-bound sialic acid in the liver was not changed after partial hepatectomy, but the amount in plasma was changed, with a similar pattern to that of the incorporation of [14C]glucosamine into slice 'free sialic acid'. These results indicate that the synthesis of sialic acid in the liver much decreases in the early stage of regeneration and that this may be correlated with the decreased synthesis of plasma sialoglycoproteins.     

Histochemical identification of 9-O-acyl sialic acids: studies of bovine submandibular and rat sublingual gland and human colon

Summary Formalin-fixed tissue specimens containing glycoproteins with side chain O-acylated sialic acids were used to re-examine, compare and evaluate the usefulness of three methods based on the periodic acid-borohydride reduction-saponification-periodic acid-Schiff sequence (PA-Bh-KOH-PAS) for the histochemical identification of 9-O-acyl sialic acids (9-O-AcSA). Method I, modified from Vehet al. (1979), involved a comparison of the staining intensely obtained when both oxidation steps of the PA-Bh-KOH-PAS sequence were carried out with the selective oxidation technique of Volzet al. (1987) with that obtained when the initial oxidation step was carried out with 0.5m periodic acid for 4h at room temperature. Methods II and III, modified from Reidet al. (1978), involved an initial PA-Bh step under oxidation conditions that cleaved all the vicinal diols associated with neutral sugars and side chain unsubstituted and 7-O-acyl sialic acids. The Schiff staining obtained following subsequent re-oxidation with either 0.5m (method II) or 1% periodic acid (method III) for 4h at room temperature (PA-Bh-PAS procedure) identifies 9-O-AcSa.The results of this study indicate that (a) bovine submandibular gland acinar cell glycoproteins contain 9-O-AcSA as well as sialic acids which have ester substituents at C7 or C8, or which are di-(C7C8, C7C9, C8C9) or tri-(C7C8C9) substituted, (b) the side chain O-acyl sialic acids of the glycoproteins of Sprague Dawley rat sublingual gland acinar cells are entirely or almost entirely 9-O-AcSA and (c) it is likely that the majority of the human adult and foetal glycoproteins studied contain small quantities of 9-O-AcSA mixed with sialic acids which are substituted at C7 or C8 or which have two or three side chain O-acyl substituents. However, the interpretation of the results are complicated by observations that indicate that (a) treatment with 0.5m periodic acid either extracts or removes sialic acids from bovine submandibular gland glycoproteins, (b) some human colonic epithelial glycoproteins apparently contain a component other than 9-O-AcSA that oxidises slowly with periodic acid and (c) 1% periodic acid for 2h at room temperature oxidises a small but significant quantity of 9-O-AcSA, thus reducing the intensity of staining in methods II and III. It is concluded that when adequately controlled, methods I, II and III are capable of detecting 9-O-AcSA in glycoproteins containing large quantities of the sialic acid. However, these methods may not detect small quantities of 9-O-AcSA in the presence of large quantities of sialic acids which have O-acyl substitutents at positions C7 or C8 or which have two (C7C8, C7C9, C8C9) or three (C7C8C9) side chain O-acyl substituents. Thus, caution should be used when interpreting data that indicates the absence of 9-O-AcSA.     

The role of sialic acid in human polyomavirus infections

JC virus (JCV) and BK virus (BKV) are human polyomaviruses that infect approximately 85% of the population worldwide [1,2]. JCV is the underlying cause of the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML), a condition resulting from JCV induced lytic destruction of myelin producing oligodendrocytes in the brain [3]. BKV infection of kidneys in renal transplant recipients results in a gradual loss of graft function known as polyomavirus associated nephropathy (PVN) [4]. Following the identification of these viruses as the etiological agents of disease, there has been greater interest in understanding the basic biology of these human pathogens [5,6]. Recent advances in the field have shown that viral entry of both JCV and BKV is dependent on the ability to interact with sialic acid. This review focuses on what is known about the human polyomaviruses and the role that sialic acid plays in determining viral tropism.     

The effect of dietary linoleic acid on rat platelet sialic acid

The total sialic acid content of blood platelets from rats raised for 8 weeks or 12 months on a diet containing 1% linoleic acid (1LA) was significantly lower (by over 30%) than that from those raised on an isocalorific diet containing 6% linoleic acid (6LA). The transfer of sialic acid to endogenous glycoprotein acceptor was also significantly lower (up to almost 4-fold) in 1LA platelet and megakaryocyte-rich preparations but the transfer to exogenous glycoprotein acceptor was similar in both 1LA and 6LA platelets. The megakaryocyte-rich fraction of 1LA animals showed a reduced phosphodolichol-sensitive N-acetylglucosaminyl (but not mannosyl) transfer to endogenous glycoprotein compared with 6LA animals. No significant difference was found between the megakaryocytes of 1LA and 6LA animals in the incorporation of radioactive mannose and glucosamine into the glycoprotein of the whole cells. It was concluded that the decreased transfer of sialic acid to glycoproteins of platelets and megakaryocyte of animals on the 1LA diet was due to the decreased availability of sialyl acceptor. The formation of N-linked oligosaccharide was the same in both 1LA and 6LA megakaryocytes, and thus any differences in phosphodolichol-mediated N-glycosylation did not account for this decreased availability of sialyl acceptor.     

Developmental regulation of D-beta-hydroxybutyrate dehydrogenase in rat liver and brain

The activities of ketone-metabolizing enzymes in rat brain increase 3- to 5-fold during the suckling period before decreasing to the adult level after weaning. We have observed that a similar developmental pattern also exists for D-beta-hydroxybutyrate dehydrogenase (BDH) in rat liver. Utilizing antibodies prepared against the purified protein we determined that the changes in BDH activities in both brain and liver are due to changes in the amount of BDH in the mitochondria. In vitro translations of isolated RNA followed by immunoprecipitation revealed that the increase in BDH activity and content was correlated with an increase in the level of functional BDH-mRNA in both liver and brain.     

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.     

Developmental regulation of phospholipid methylation in rat brain synaptosomes

Phospholipid methylation was studied in cortical synaptosomes prepared from 7 and 14 day and adult rat brain. Using varying concentrations of [³H] S-adenosylmethionine, Km and Vmax values were determined for the formation of [³H] phosphatidylmonomethylethanolamine (PME), [³H] phosphatidyl-dimethylethanolamine and [³H] phosphatidylcholine (PC). At 25°C, the Km values for the formation of all three products, significantly decreased with development. Increasing the temperature to 37°C increased the Km values in the 14 day and adult but not the 7 day preparation. The Vmax values at 25°C were highest at 7 and 14 days, depending on the product and then decreased in the adult. At 37°C, the Vmax values were highest in the 14 day preparation. The overall results are discussed in terms of the developmentally regulated decrease in both synaptic membrane PC and membrane fluidity.     

Developmental regulation of amino acid transport in Neurospora crassa

Conidia of Neurospora crassa exhibit an ability to transport various amino acids against a concentration gradient. The conidial transport system has previously been characterized in terms of kinetics, competitions, and genetic control. This study describes the development of a new and highly active transport capability which is elaborated during the early stages of development but prior to evident germination. It has been named “postconidial” transport activity and represents as much as 20-fold greater initial rates as compared to those observed with conidia. Development of the postconidial transport activity requires protein synthesis and can be partially repressed when the substrate amino acid is present during the developmental preincubation period. A mutant has been utilized which exhibits normal conidial but fails to develop normal postconidial transport activity for any amino acid examined. Although temperature optimum and pH dependence are similar in conidial and postconidial systems, there is evidence that the new activity is not a simple amplification of an existing capability. This is reflected as a change in competition patterns between particular amino acids as development proceeds.     

Is the lectin binding pattern of human breast and colon cancer cells influenced by modulators of sialic acid metabolism?

Sialic acid residues are the most abundant terminal carbohydrate residues of mammalian cells. Modification of the sialic acid residues by exposure of cells in culture to sialic acid precursor analogues resulted in a modifed susceptibility to polyoma viruses. In the present study, human breast and colon cancer cell lines were exposed for 65 h to these acid precursor analogues at 5 mM and their lectin binding pattern was analysed. Use of a panel of several different lectins indicated that the pretreatment of these cell lines with the sialic acid analogues did not change their lectin binding profile. The incorporation of these precursors into membrane glycoproteins was assessed by reversed phase high-performance liquid chromatography, which clearly demonstrated that the precursors were incorporated. The results therefore indicate that these analogues are highly specific for sialic acid and do not interfere with other biosynthetic pathways of membrane glycoconjugates.     

Circadian regulation of electrolyte absorption in the rat colon

The intestinal transport of nutrients exhibits distinct diurnal rhythmicity, and the enterocytes harbor a circadian clock. However, temporal regulation of the genes involved in colonic ion transport, i.e., ion transporters and channels operating in absorption and secretion, remains poorly understood. To address this issue, we assessed the 24-h profiles of expression of genes encoding the sodium pump (subunits Atp1a1 and Atp1b1), channels (α-, β-, and γ-subunits of Enac and Cftr), transporters (Dra, Ae1, Nkcc1, Kcc1, and Nhe3), and the Na(+)/H(+) exchanger (NHE) regulatory factor (Nherf1) in rat colonic mucosa. Furthermore, we investigated temporal changes in the spatial localization of the clock genes Per1, Per2, and Bmal1 and the genes encoding ion transporters and channels along the crypt axis. In rats fed ad libitum, the expression of Atp1a1, γEnac, Dra, Ae1, Nhe3, and Nherf1 showed circadian variation with maximal expression at circadian time 12, i.e., at the beginning of the subjective night. The peak γEnac expression coincided with the rise in plasma aldosterone. Restricted feeding phase advanced the expression of Dra, Ae1, Nherf, and γEnac and decreased expression of Atp1a1. The genes Atp1b1, Cftr, αEnac, βEnac, Nkcc1, and Kcc1 did not show any diurnal variations in mRNA levels. A low-salt diet upregulated the expression of βEnac and γEnac during the subjective night but did not affect expression of αEnac. Similarly, colonic electrogenic Na(+) transport was much higher during the subjective night than the subjective day. These findings indicate that the transporters and channels operating in NaCl absorption undergo diurnal regulation and suggest a role of an intestinal clock in the coordination of colonic NaCl absorption.     

Intracellular pH regulation in colonocytes of rat proximal colon

The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.     

Association of sialic acid with microsomal membrane structures in rat liver

The amount of sialic acid on phospholipid basis increases from rough, through smooth II and smooth I microsomes, to Golgi membranes, all of them free from most of the adsorbed and luminal protein. The incorporation rate of glucosamine-³H into sialic acid also follows a similar order. Deoxycholate removes phospholipid and sialic acid to an identical extent, and a significant part of the latter remains after trypsin and neuraminidase treatment. The sialic acid/phospholipid ratio decreases in phenobarbital-induced smooth but not in rough membranes, while the incorporation rate of glycosamine-³H into sialic acid decreases in both subfractions.     

Alteration in sialyltransferase and sialic acid expression accompanies cell differentiation in rat intestine

The subcellular distribution of sialyltransferase and its product of action, sialic acid, was investigated in the undifferentiated cells of the rat intestinal crypts and compared with the pattern observed in the differentiated cells present in the surface epithelium. Sialyltransferase was immunocytochemically detected with an antibody, affinity-purified on a beta-galactosidase/sialyltransferase fusion protein, which recognizes only protein epitopes of the enzyme. A similar pattern and intensity of immunolabeling were observed in the Golgi apparatus, apical and basolateral plasma membranes of both undifferentiated and differentiated absorptive cells. However, in the goblet cells, the mucus was only weakly labeled in cells present in the basal portion of the crypts but increased in intensity through the zone of migration to the surface epithelium. Sialic acid as detected with the Limax flavus lectin was observed in the Golgi apparatus and post-Golgi apparatus structures of both absorptive and goblet cells regardless of their position along the crypt-to-surface epithelium axis. However, a striking difference in the plasma membrane distribution of sialic acid existed between undifferentiated cells of the lower half of the crypts and those of the upper half and the surface epithelium: in the former, label was present in both the apical and basolateral domain, whereas in the latter it became restricted to the apical domain. These results suggest that the presence of sialyltransferase immunoreactivity in the goblet cell mucus and the polarization of sialic acid to the apical plasma membrane of both goblet and absorptive cells may be markers for the differentiated state.