Mechanism of uptake and incorporation of the non-human sialic acid N-glycolylneuraminic acid into human cells

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.     

N-glycolylneuraminic acid deficiency in mice: implications for human biology and evolution

Humans and chimpanzees share >99% identity in most proteins. One rare difference is a human-specific inactivating deletion in the CMAH gene, which determines biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc). Since Neu5Gc is prominent on most chimpanzee cell surfaces, this mutation could have affected multiple systems. However, Neu5Gc is found in human cancers and fetuses and in trace amounts in normal human tissues, suggesting an alternate biosynthetic pathway. We inactivated the mouse Cmah gene and studied the in vivo consequences. There was no evidence for an alternate pathway in normal, fetal, or malignant tissue. Rather, null fetuses accumulated Neu5Gc from heterozygous mothers and dietary Neu5Gc was incorporated into oncogene-induced tumors. As with humans, there were accumulation of the precursor N-acetylneuraminic acid and increases in sialic acid O acetylation. Null mice showed other abnormalities reminiscent of the human condition. Adult mice showed a diminished acoustic startle response and required higher acoustic stimuli to increase responses above the baseline level. In this regard, histological abnormalities of the inner ear occurred in older mice, which had impaired hearing. Adult animals also showed delayed skin wound healing. Loss of Neu5Gc in hominid ancestors approximately 2 to 3 million years ago likely had immediate and long-term consequences for human biology.     

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.     

N.M.R. spectroscopy and calcium binding of sialic acids: N-glycolylneuraminic acid and periodate-oxidized N-acetylneuraminic acid

N.m.r. spectroscopy (¹H- and ¹³C-) of N-glycolylneuraminic acid, and of its interaction product with Ca²⁺ at pH 7, indicated that a 1:1 complex is formed, with a formation constant of 193 M^?1 [compared to 121 M^?1 for N-acetylneuraminic acid (1)]. From analysis of electric-field shifts, an approximate position of the Ca²⁺ ion in the complex is inferred, with the hydroxyl group of the N-glycolyl group providing the additional binding. Compound 1 was oxidized with sodium periodate, and ¹³C-n.m.r. spectroscopy was applied in an attempt to identify the aldehyde formed, and to demonstrate that the loss of the glycerol-1-yl side-chain (carbon atoms 8 and 9) decreases its Ca²⁺ ion-binding capacity.     

Reduced diversity of the human erythrocyte membrane sialic acids in polycythemia vera. Absence of N-glycolylneuraminic acid and characterisation of N-acetylneuraminic acid 1,7 lactone

Sialic acids from the erythrocyte (RBC) membrane of a patient suffering from polycythemia vera, a malignant orphan disorder of hematopoietic cells, was studied using GC/MS. We found that the sialic acid diversity of these membranes was drastically reduced since only four entities were identified: Neu5Ac (91.5%) and its 1,7 lactone Neu5Ac1,7L (7.5%) which is absent in normal RBC, Neu4,5Ac(2) (0.50%) and Neu4,5Ac(2) 9Lt (0.50%); in normal RBC, Neu5,7Ac(2), Neu5,9Ac(2), Neu5Ac9Lt, Neu5Ac8S and Neu, as well as traces of Kdn, were also present. Neu5Gc and its O-alkylated or O-acetylated derivatives, which are considered by various authors as cancer markers, were not detected.     

Uptake of sialic acid by human erythrocyte. Characterization of a transport system

Upon incubation of human red blood cells (RBC) with [4-9-14C] N-acetylneuraminic acid, the cells incorporated this sugar, as demonstrated by the identification of labelled N-acetylmannosamine in the cytosol, as a result of the action of the sialic acid pyruvate-lyase we discovered previously (Biochimie 84 (2002) 655). The mechanism is saturable and indicates the presence of a limited number of transporter molecules in the RBC membrane. This transport process may have relevance to the desialylation of membrane glycoconjugates which occurs during ageing of erythrocytes.     

Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (N-glycolylneuraminic acid) binding activity.

The hemagglutinating activity of transmissible gastroenteritis virus (TGEV), an enteric porcine coronavirus, was analyzed and found to be dependent on the presence of alpha-2,3-linked sialic acid on the erythrocyte surface. N-Glycolylneuraminic acid was recognized more efficiently by TGEV than was N-acetylneuraminic acid. For an efficient hemagglutination reaction the virions had to be treated with sialidase. This result suggests that the sialic acid binding site is blocked by virus-associated competitive inhibitors. Porcine respiratory coronavirus (PRCV), which is serologically related to TGEV but not enteropathogenic, was found to be unable to agglutinate erythrocytes. Incubation with sialidase did not induce a hemagglutinating activity of PRCV, indicating that the lack of this activity is an intrinsic property of the virus and not due to the presence of competitive inhibitors. Only monoclonal antibodies to an antigenic site that is absent from the S protein of PRCV were able to prevent TGEV from agglutinating erythrocytes. The epitope recognized by these antibodies is located within a stretch of 224 amino acids that is missing in the S protein of PRCV. Our results indicate that the sialic acid binding activity is also located in that portion of the S protein. The presence of a hemagglutinating activity in TGEV and its absence in PRCV open the possibility that the sialic acid binding activity contributes to the enterotropism of TGEV.     

Inhibition of alkaline phosphatase by sialic acid.

The interaction of human organ alkaline phosphatases (orthophosphoric-monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1) with sugars was studied. Hexosamines, N-acetylneuraminic acid (NANA or sialic acid), N-acetylmuramic acid and N-acetylglycolylneuraminic acid inhibited human organ alkaline phosphatase activities. Of these, sialic acid was the most effective inhibitor. The pH profiles for the enzymes in the absence and presence of sialic acid were similar. The sialic acid - enzyme complex was more heat stable than the free enzyme between 20 and 45 degrees C. Lineweaver-Burk plots of 1/v versus 1/S at various concentrations of sialic acid showed intersecting straight lines indicating that the mechanism of inhibition was a mixed type. The Ki value obtained from the plots of 1/v versus the square of sialic acid concentration was 0.07 mM for the hepatic, sialidase-treated hepatic, and intestinal alkaline phosphatases. The respective Hill coefficients varied somewhat with the alkaline phosphatase isoenzyme. Hyperbolic curves were obtained when the percentage of remaining activity was plotted against the substrate concentration at different concentrations of sialic acid. The Hill coefficient was lowered in the presence of sialic acid. The sialidase-treated hepatic enzymes used gave the most effective conversion. Partial denaturation of the enzyme with urea, or pronase digestion had a little if any effect on the sialic acid inhibition with constant time.     

Accelerated uptake of 5-hydroxytryptamine by human blood platelets enriched in a sialic acid.

The enzymically catalysed incorporation of N-acetylneuraminic acid into human platelets, whether suspended in their own citrated plasma or in buffered saline containing 0.17 mM-sucrose, accelerated the uptake of 5-hydroxytryptamine. This acceleration decreased with time. The observations may be explained by assuming that N-acetylneuraminic acid is a component of a transport receptor for 5-hydroxytryptamine.     

Blood clotting factor IX. Loss of activity after cleavage of sialic acid residues

Enzymatic cleavage of sialic acid from human blood clotting factor IX results in a loss of factor IX clotting activity. The loss of clotting activity and the rate of release of sialic acid follow the same time courses. Control experiments have ruled out several explanations for the loss of factor IX activity: proteolytic degradation, inhibitory effects of free sialic acid, and non-specific inhibition of the clotting assays. Furthermore, no inhibition was seen when similar enzymatic cleavage was carried out on factor X and factor VIII. Therefore, we suggest that the loss of factor IX activity is the direct result of cleavage of sialic acid from the protein. Most of the inhibition appeared to be an effect on the activity of factor IXa itself, and thus far, little or no effect has been shown on the activation of factor IX to IXa. The structural basis for this unusual effect of sialic acid on protein function currently is being investigated.     

The sialic acid content and isoelectric point of human kininogen.

The sialic acid content of highly purified human kininogen was found to be about 8.6 mol/mol(mol.wt. 50,000). The isoelectric point (pH 4.9 +/- 0.2) is much higher than that of bovine low-molecular-weight kininogen, but is close to that expected from the amino acid and sialic acid analyses.     

Germinal center marker GL7 probes activation-dependent repression of N-glycolylneuraminic acid, a sialic acid species involved in the negative modulation of B-cell activation

Sialic acid (Sia) is a family of acidic nine-carbon sugars that occupies the nonreducing terminus of glycan chains. Diversity of Sia is achieved by variation in the linkage to the underlying sugar and modification of the Sia molecule. Here we identified Sia-dependent epitope specificity for GL7, a rat monoclonal antibody, to probe germinal centers upon T cell-dependent immunity. GL7 recognizes sialylated glycan(s), the alpha2,6-linked N-acetylneuraminic acid (Neu5Ac) on a lactosamine glycan chain(s), in both Sia modification- and Sia linkage-dependent manners. In mouse germinal center B cells, the expression of the GL7 epitope was upregulated due to the in situ repression of CMP-Neu5Ac hydroxylase (Cmah), the enzyme responsible for Sia modification of Neu5Ac to Neu5Gc. Such Cmah repression caused activation-dependent dynamic reduction of CD22 ligand expression without losing alpha2,6-linked sialylation in germinal centers. The in vivo function of Cmah was analyzed using gene-disrupted mice. Phenotypic analyses showed that Neu5Gc glycan functions as a negative regulator for B-cell activation in assays of T-cell-independent immunization response and splenic B-cell proliferation. Thus, Neu5Gc is required for optimal negative regulation, and the reaction is specifically suppressed in activated B cells, i.e., germinal center B cells.     

Insights into the evolution of sialic acid catabolism among bacteria

Background  

Sialic acids comprise a family of nine-carbon amino sugars that are prevalent in mucus rich environments. Sialic acids from the human host are used by a number of pathogens as an energy source. Here we explore the evolution of the genes involved in the catabolism of sialic acid.     

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.     

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.     

Versatile biosynthetic engineering of sialic acid in living cells using synthetic sialic acid analogues.

Sialic acids are critical components of many glycoconjugates involved in biologically important ligand-receptor interactions. Quantitative and structural variations of sialic acid residues can profoundly affect specific cell-cell, pathogen-cell, or drug-cell interactions, but manipulation of sialic acids in mammalian cells has been technically limited. We describe the finding of a previously unrecognized and efficient uptake and incorporation of sialic acid analogues in mammalian cells. We added 16 synthetic sialic acid analogues carrying distinct C-1, C-5, or C-9 substitutions individually to cell cultures of which 10 were readily taken up and incorporated. Uptake of C-5- and C-9-substituted sialic acids resulted in the structural modification of up to 95% of sialic acids on the cell surface. Functionally, binding of murine sialic acid-binding immunoglobulin-like lectin-2 (Siglec-2, CD22) to cells increased after N-glycolylneuraminic acid treatment, whereas 9-iodo-N-acetylneuraminic acid abolished binding. Furthermore, susceptibility to infection by the B-lymphotropic papovavirus via a sialylated receptor was markedly enhanced following pretreatment of host cells with selected sialic acid analogues including 9-iodo-N-acetylneuraminic acid. This novel experimental strategy allows for an efficient biosynthetic engineering of surface sialylation in living cells. It is versatile, extending the repertoire of modification sites at least to C-9 and enables detailed structure-function studies of sialic acid-dependent ligand-receptor interactions in their native context.     

Amino acid preferences of small proteins. Implications for protein stability and evolution.

The dependence of amino acid frequency on sequence length has been examined for the 20 natural amino acids using a set of 2275 protein sequences with little sequence identity. As expected, the frequency of cysteine increases dramatically for sequences shorter than 100 amino acids with a length-dependence that corresponds to an average of two Cys per sequence independent of length. Surprisingly dramatic changes were also observed for the frequencies of arginine, lysine, aspartic acid, and glutamic acid: Arg and Lys frequencies increase for short sequences whereas Asp and Glu frequencies decrease. These changes do not appear to be due to an over-abundance of DNA- and membrane-binding proteins in the database and may, therefore, be related to protein stability. Possible stabilizing mechanisms include increased hydrogen bonding by Arg and increased hydrophobic stabilization due to the amphiphilic character of Arg and Lys. These observations suggest that amino acid composition played an important role in the evolution of small proteins.     

Dendritic cell maturation results in pronounced changes in glycan expression affecting recognition by siglecs and galectins

Dendritic cells (DC) are the most potent APC in the organism. Immature dendritic cells (iDC) reside in the tissue where they capture pathogens whereas mature dendritic cells (mDC) are able to activate T cells in the lymph node. This dramatic functional change is mediated by an important genetic reprogramming. Glycosylation is the most common form of posttranslational modification of proteins and has been implicated in multiple aspects of the immune response. To investigate the involvement of glycosylation in the changes that occur during DC maturation, we have studied the differences in the glycan profile of iDC and mDC as well as their glycosylation machinery. For information relating to glycan biosynthesis, gene expression profiles of human monocyte-derived iDC and mDC were compared using a gene microarray and quantitative real-time PCR. This gene expression profiling showed a profound maturation-induced up-regulation of the glycosyltransferases involved in the expression of LacNAc, core 1 and sialylated structures and a down-regulation of genes involved in the synthesis of core 2 O-glycans. Glycosylation changes during DC maturation were corroborated by mass spectrometric analysis of N- and O-glycans and by flow cytometry using plant lectins and glycan-specific Abs. Interestingly, the binding of the LacNAc-specific lectins galectin-3 and -8 increased during maturation and up-regulation of sialic acid expression by mDC correlated with an increased binding of siglec-1, -2, and -7.