Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using ¹³C₆-glucose and ¹³C₂-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS.Metabolic labeling of cultured cells with ¹³C₆-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a ¹³C₆-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells.Using ¹³C₂-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.Protein glycosylation, which is the most abundant post-translational modification, has important roles in many biological processes by modulating conformation and stability, whereas its dysregulation is associated with various diseases such as diabetes and cancer (1, 2). Glycosylation is regulated by various factors including glucose metabolism, the availability and localization of nucleotide sugars, and the expression and localization of glycosyltransferases (3, 4). Thus, ideally all of these components should be considered when detecting changes in a dynamic fashion; namely, it is necessary not only to take a snapshot but also to make movies of the dynamic changes in glycan metabolism.Glucose is used by living cells as an energy source via the glycolytic pathway as well as a carbon source for various metabolites including nucleotide sugars (e.g. UDP-GlcNAc and CMP-NeuAc). These nucleotide sugars are transported into the Golgi apparatus, and added to various glycans on proteins. UDP-GlcNAc is the donor substrate for N-acetylglucosaminyl (GlcNAc)¹ transferases; alternatively, it is used in the cytosol for O-GlcNAc modification (i.e. O-GlcNAcylation) of intracellular proteins (5). The UDP-GlcNAc synthetic pathway is complex as it is a converging point of glucose, nucleotide, fatty acid and amino acid metabolic pathways. Thus, the metabolic flow of glucose modulates the branching patterns of N-glycans via UDP-GlcNAc concentrations because many of the key GlcNAc transferases that determine the branching patterns have widely different K_m values for UDP-GlcNAc ranging from 0.04 mm to 11 mm (6, 7). Indeed, it was demonstrated that the branching formation of N-glycans in T cells is stimulated by the supply from the hexosamine pathway, whereby it regulates autoimmune reactions promoted by T cells (8).UDP-GlcNAc is also used for the synthesis of CMP-NeuAc, the donor substrate for sialyltransferases (9). The CMP-NeuAc concentration is controlled by the feedback inhibition of UDP-GlcNAc epimerase/ManNAc kinase by the final product CMP-NeuAc, and hence a high CMP-NeuAc level reduces metabolic flow in CMP-NeuAc de novo synthesis (10). However, there is still only limited information about how the levels of nucleotide sugars dynamically change in response to the environmental cues, and how such changes are reflected in the glycosylation of proteins.Stable isotope labeling is a promising approach to quantify metabolic changes in response to external cues (11, 12). For example, the use of nuclear magnetic resonance to obtain isotopomer signals of metabolically labeled molecules has been applied to trace the flux in glycolysis and fatty acid metabolism (13). An approach based on the mass isotopomers of labeled metabolites with ¹³C₆-glucose has been developed to monitor the UDP-GlcNAc synthetic pathway (13–15). The method based on the labeling ratio of each metabolite related to UDP-GlcNAc synthesis has clarified the contribution of each metabolic pathway (14). Moseley reported a novel deconvolution method for modeling UDP-GlcNAc mass isotopomers (15).Previous studies into the use of nucleotide sugars in glycosylation have relied on the specific detection of metabolically radiolabeled glycans (16). It is possible not only to deduce the glycan structures but also to trace their relative contributions to glycan synthesis without MS. On the other hand, mass isotopomer analysis of glycans labeled with stable isotope provides the ratios of labeled versus unlabeled molecules from MS spectra and structural details of the glycans. However, there are only a limited number of publications reporting the application of stable isotope labeling of glycans for monitoring the dynamics of glycans (17). To date, there have been no reports describing a systematic method for tracing cellular GlcNAc biosynthesis and use based on mass isotopomer analysis.The aim of this study was to extend our knowledge of the synthesis and metabolism of UDP-GlcNAc as well as its use in the synthesis of CMP-NeuAc, N- and O-glycans. We recently developed a conventional HPLC method for simultaneous determination of nucleotide sugars including unstable CMP-NeuAc (18). We first established an LC-MS method for isotopomer analysis of ¹³C₆-glucose labeled nucleotide sugars for tracing UDP-GlcNAc metabolism from synthesis to use, because previous methods were not suitable for estimating UDP-GlcNAc use in CMP-NeuAc de novo synthesis (15). We also established a method for isotopomer analysis of labeled N- and O-glycan to monitor the metabolic flow of hexosamine into glycans. Using these two methods, we demonstrated the differences in the use of hexosamines between hepatoma and pancreatic insulinoma cell lines. Our approach may be useful for identifying a metabolic “bottleneck” that governs the turnover speed and patterns of cellular glycosylation, which may be relevant for various applications including glycoprotein engineering and discovery of disease biomarkers.     

Polysialic acid and mucin type o-glycans on the neural cell adhesion molecule differentially regulate myoblast fusion

Polysialic acid attached to the neural cell adhesion molecule (NCAM) is thought to play a critical role in development. NCAM in muscle tissue contains a muscle-specific domain (MSD) to which mucin type O-glycans are attached. In the present study, using the C2C12 myoblast system, we show that NCAM containing MSD is increasingly expressed on the cell surface as myotubes form. Polysialic acid is primarily attached to N-glycans of NCAM, and polysialylated NCAM is expressed on the outer surface of myotube bundles. By transfecting cDNAs encoding wild type and mutant forms of NCAM, we found that NCAM containing MSD facilitates myoblast fusion, and this effect is diminished by mutating O-glycosylation sites at MSD. By contrast, forced expression of polysialic acid in early differentiation stages reduces myotube formation and delays the expression of NCAM containing the MSD domain. Strikingly, inhibition of polysialic acid synthesis by antisense DNA approach induced differentiation in both human rhabdomyosarcoma cells, which overexpress polysialic acid, and C2C12 cells. These results indicate that polysialic acid and mucin type O-glycans on NCAM differentially regulate myoblast fusion, playing critical roles in muscle development.     

Biosynthesis of HNK-1 glycans on O-linked oligosaccharides attached to the neural cell adhesion molecule (NCAM): the requirement for core 2 beta 1,6-N-acetylglucosaminyltransferase and the muscle-specific domain in NCAM

The HNK-1 glycan, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R, is highly expressed in neuronal cells and apparently plays critical roles in neuronal cell migration and axonal extension. The HNK-1 glycan synthesis is initiated by the addition of beta1,3-linked GlcA to N-acetyllactosamine followed by sulfation of the C-3 position of GlcA. The cDNAs encoding beta1,3-glucuronyltransferase (GlcAT-P) and HNK-1 sulfotransferase (HNK-1ST) have been recently cloned. Among various adhesion molecules, the neural cell adhesion molecule (NCAM) was shown to contain HNK-1 glycan on N-glycans. In the present study, we first demonstrated that NCAM also bears HNK-1 glycan attached to O-glycans when NCAM contains the O-glycan attachment scaffold, muscle-specific domain, and is synthesized in the presence of core 2 beta1,6-N-acetylglucosaminyltransferase, GlcAT-P, and HNK-1ST. Structural analysis of the HNK-1 glycan revealed that the HNK-1 glycan is attached on core 2 branched O-glycans, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAc. Using synthetic oligosaccharides as acceptors, we found that GlcAT-P and HNK-1ST almost equally act on oligosaccharides, mimicking N- and O-glycans. By contrast, HNK-1 glycan was much more efficiently added to N-glycans than O-glycans when NCAM was used as an acceptor. These results are consistent with our results showing that HNK-1 glycan is minimally attached to O-glycans of NCAM in fetal brain, heart, and the myoblast cell line, C2C12. These results combined together indicate that HNK-1 glycan can be synthesized on core 2 branched O-glycans but that the HNK-1 glycan is preferentially added on N-glycans over O-glycans of NCAM, probably because N-glycans are extended further than O-glycans attached to NCAM containing the muscle-specific domain.     

The mitochondrial DNA of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>: complete sequence,gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564?bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4?kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii.     

Synthesis of neoglycoconjugates containing deaminated neuraminic acid (KDN) using rat liver alpha2,6-sialyltransferase

2-Keto-3-deoxy-D- glycero -D- galacto -nononic acid (KDN) was introduced into asialotransferrin and N -acetyllactosamine (LacNAc) from CMP-KDN by using rat liver Galbeta1-->4GlcNAc alpha2, 6- sialyltransferase to form KDN-transferrin and KDN-LacNAc. These structures contain terminal KDNalpha2-->6Gal-residues, a glycotope that has not yet been described in natural glycoconjugates. KDN was transferred to all four Gal residues in asialotransferrin by this enzyme. The incorporation efficiency of KDN from CMP-KDN into asialotransferrin was about half that of Neu5Ac from CMP-Neu5Ac, based on the V max/ K m values for these donor substrates, 0.0527 min-1and 0.119 min-1, respectively. The KDNalpha2-->6Gal linkage was resistant to exosialidase treatment, in contrast to the sensitivity of the Neu5Acalpha2-->6Gal linkage. Interestingly, Sambucus sieboldiana agglutinin (SSA) was shown to prefer KDN-transferrin to the corresponding Neu5Ac-transferrin, as estimated by slot-blot analysis. The use of an alpha2,6-sialyltransferase to synthesize neoglycoproteins containing KDN has not been previously reported. Their facile synthesis using CMP-KDN and sialyltransferases with different specificities offers new possibilities to study the function of neo-KDN- glycoconjugates, and to explore their use in glycotechnology.      

Investigation by imaging mass spectrometry of biomarker candidates for aging in the hair cortex

Background

Human hair is one of the essential components that define appearance and is a useful source of samples for non-invasive biomonitoring. We describe a novel application of imaging mass spectrometry (IMS) of hair biomolecules for advanced molecular characterization and a better understanding of hair aging. As a cosmetic and biomedical application, molecules whose levels in hair altered with aging were comprehensively investigated.

Methods

Human hair was collected from 15 young (20±5 years old) and 15 older (50±5 years old) volunteers. Matrix-free laser desorption/ionization IMS was used to visualize molecular distribution in the hair sections. Hair-specific ions displaying a significant difference in the intensities between the 2 age groups were extracted as candidate markers for aging. Tissue localization of the molecules and alterations in their levels in the cortex and medulla in the young and old groups were determined.

Results

Among the 31 molecules detected specifically in hair sections, 2—one at m/z 153.00, tentatively assigned to be dihydrouracil, and the other at m/z 207.04, identified to be 3,4-dihydroxymandelic acid (DHMA)—exhibited a higher signal intensity in the young group than in the old, and 1 molecule at m/z 164.00, presumed to be O-phosphoethanolamine, displayed a higher intensity in the old group. Among the 3, putative O-phosphoethanolamine showed a cortex-specific distribution. The 3 molecules in cortex presented the same pattern of alteration in signal intensity with aging, whereas those in medulla did not exhibit significant alteration.

Conclusion

Three molecules whose levels in hair altered with age were extracted. While they are all possible markers for aging, putative dihydrouracil and DHMA, are also suspected to play a role in maintaining hair properties and could be targets for cosmetic supplementation. Mapping of ion localization in hair by IMS is a powerful method to extract biomolecules in specified regions and determine their tissue distribution.     

Polysialic acid facilitates tumor invasion by glioma cells

Polysialic acid (PSA) is thought to attenuate neural cell adhesion molecule (NCAM) adhesion, thereby facilitating neural cell migration and regeneration. Although the expression of PSA has been shown to correlate with the progression of certain tumors such as small cell lung carcinoma, there have been no studies to determine the roles of PSA in gliomas, the most common type of primary brain tumor in humans. In this study, we first revealed that among patients with glioma, PSA was detected more frequently in diffuse astrocytoma cells, which spread extensively. To determine directly the role of PSA in glioma cell invasion, we transfected C6 glioma cells with polysialyltransferases to express PSA. In those transfected cells, PSA is attached mainly to NCAM-140, whereas the mock-transfected C6 cells express equivalent amounts of PSA-free NCAM-140. Both PSA negative and positive C6 cell lines exhibited almost identical growth rates measured in vitro. However, PSA positive C6 cells exhibited increased invasion to the corpus callosum, where the mock-transfected C6 glioma cells rarely invaded when inoculated into the brain. By contrast, the invasion to the corpus callosum by both the mock-transfected and PSA positive C6 cells was observed in NCAM-deficient mice. These results combined indicate that PSA facilitates tumor invasion of glioma in the brain, and that NCAM-NCAM interaction is likely attenuated in the PSA-mediated tumor invasion.     

CMP substitutions preferentially inhibit polysialic acid synthesis

It is widely reported that derivatives of sugar moieties can be used to metabolically label cell surface carbohydrates or inhibit a particular glycosylation. However, few studies address the effect of substitution of the cytidylmonophosphate (CMP) portion on sialyltransferase activities. Here we first synthesized 2'-O-methyl CMP and 5-methyl CMP and then asked if these CMP derivatives are recognized by alpha2,3-sialyltransferases (ST3Gal-III and ST3Gal-IV), alpha2,6-sialyltransferase (ST6Gal-I), and alpha2,8-sialyltransferase (ST8Sia-II, ST8Sia-III, and ST8Sia-IV). We found that ST3Gal-III and ST3Gal-IV but not ST6Gal-I was inhibited by 2'-O-methyl CMP as potently as by CMP, while ST3Gal-III, ST3Gal-IV, and ST6Gal-I were moderately inhibited by 5-methyl CMP. Previously, it was reported that polysialyltransferase ST8Sia-II but not ST8Sia-IV was inhibited by CMP N-butylneuraminic acid. We found that ST8Sia-IV as well as ST8Sia-II and ST8Sia-III are inhibited by 2'-O-methyl CMP as robustly as by CMP and moderately by 5-methyl CMP. Moreover, the addition of CMP, 2'-O-methyl CMP, and 5-methyl CMP to the culture medium resulted in the decrease of polysialic acid expression on the cell surface and NCAM of Chinese hamster ovary cells. These results suggest that 2'-O-methyl CMP and 5-methyl CMP can be used to preferentially inhibit sialyltransferases, in particular, polysialyltransferases in vitro and in vivo. Such inhibition may be useful to determine the function of a carbohydrate synthesized by a specific sialyltransferase such as polysialyltransferase.     

High affinity sugar ligands of C-type lectin receptor langerin

Background

Langerin, a C-type lectin receptor (CLR) expressed in a subset of dendritic cells (DCs), binds to glycan ligands for pathogen capture and clearance. Previous studies revealed that langerin has an unusual binding affinity toward 6-sulfated galactose (Gal), a structure primarily found in keratan sulfate (KS). However, details and biological outcomes of this interaction have not been characterized. Based on a recent discovery that the disaccharide L4, a KS component that contains 6-sulfo-Gal, exhibits anti-inflammatory activity in mouse lung, we hypothesized that L4-related compounds are useful tools for characterizing the langerin-ligand interactions and their therapeutic application.