Earticle

The attachment of O-linked β-N-acetylglucosamine (O-GlcNAc) to proteins is an abundant and reversible modification that involves many cellular processes including transcription, translation, cell proliferation, apoptosis, and signal transduction. Here, we found that the O-GlcNAc modification pattern was altered during all-trans retinoic acid (tRA)-induced neurite outgrowth in the MN9D neuronal cell line. We identified several O-GlcNAcylated proteins using mass spectrometric analysis, including α- and β-tubulin. Further analysis of α- and β-tubulin revealed that O-GlcNAcylated peptides mapped between residues 173 and 185 of α-tubulin and between residues 216 and 238 of β-tubulin, respectively. We found that an increase in α-tubulin O-GlcNAcylation reduced heterodimerization and that O-GlcNAcylated tubulin did not polymerize into microtubules. Consequently, when O-GlcNAcase inhibitors were co-incubated with tRA, the extent of neurite outgrowth was decreased by 20% compared to control. Thus, our data indicate that the O-GlcNAcylation of tubulin negatively regulates microtubule formation.

Modification of nuclear and cytosolic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is ubiquitous in cells. The in vivo function of the protein O-GlcNAcylation, however, is not well understood. Here, we genetically manipulated the cellular O-GlcNAcylation level in Drosophila and found that it promotes developmental growth by enhancing insulin signaling. This alteration of growth is due to cell growth and apoptosis and not to cell proliferation, and is mediated, at least in part, through O-GlcNAcylation of Akt. These results indicate that O-GlcNAcylation is one of the crucial mechanisms involved in control of insulin signaling in normal Drosophila development.

Protein O-phosphorylation can occur reciprocal with O-GlcNAc modification and represents a regulatory principle for proteins. O-phosphorylation of serine by GSK-3β on Snail1, a transcriptional repressor of E-cadherin and a key regulator of the epithelial-mesenchymal transition (EMT) program, results in its proteasomal degradation. We show that Snail1 carries O-GlcNAc at serine112 that stabilizes it by suppressing O-phosphorylation-mediated degradation. Stabilization by O-GlcNAc of Snail1 results in attenuation of E-cadherin mRNA expression. Enhanced O-GlcNAc modification occurred under hyperglycemic conditions and initiated EMT by transcriptional suppression of E-cadherin through Snail1. Thus, a molecular link exists between cellular glucose metabolism and the control of EMT by dynamic reciprocal O-phosphorylation and O-GlcNAc modification of Snail1.

Carbohydrate-protein recognition events play key roles in physiological and pathological processes. The understanding of these biomolecular interactions provides deep insight into glycan-mediated biological processes and enables the development of more effective therapeutic agents and diagnostic tools. Carbohydrate microarrays, which are composed of diverse glycans orderly and densely attached to a single chip substrate, have been extensively developed as reliable and efficient tools for the rapid analysis of carbohydrate-based biomolecular interactions. The most general method for construction of these microarrays involves site-specific and covalent immobilization of chemically modified glycans to properly derivatized surfaces. This immobilization strategy requires the use of properly functionalized sugars, which are typically prepared by multi-step synthetic sequences. In order to avoid the need for functionalized glycans, immobilization strategies that employ unmodified sugars have been developed. In recent investigations directed at this goal, we have developed a novel, direct, site-specific technique for immobilization of unmodified carbohydrates, including simple carbohydrates, oligosaccharides and polysaccharides, on hydrazide-derivatized surfaces. This method eliminates the need for laborious and tedious glycan derivatization. To demonstrate the scope and applicability of this approach, carbohydrate microarrays containing a variety of glycans (fifty eight glycans) have been constructed by using one-step, direct attachment of free carbohydrates to a hydrazide-coated surface. These microarrays have been employed for analysis of sugar binding specificities of lectins, antibodies and bacterial cells. In addition, this microarray format has been applied to the determination of binding affinities between proteins and glycans.

Microarray technologies have received considerable attention owing to the fact that they serve as effective tools for high-throughput analysis of boimolecular interactions and the identification of bioactive substances that bind to biomolecules. Most of the current methods used to construct microarrays rely on the immobilization of substances on properly derivatized surfaces. Among various functional groups used for this purpose, the N-hydroxysuccinimide (NHS) ester group has been largely employed since it can be readily reacted with amine or hydrazide functionalities in substances of interest. However, the NHS ester group is usually introduced onto the surface of a glass slide by employing inconvenient and time consuming multi-step processes. In recent studies, it was developed an efficient, single step method for derivatization of glass surfaces with NHS ester groups that takes advantage of an acid-mediated reaction of NHS ester functionalized dimethallysilanes with silanols on the glass surface. Conditions for the surface modification procedure that utilize TfOH rather than Sc(OTf)3 were found to be superior. Protein and RNA-binding experiments show that glass surfaces modified by employing this method are suitable for efficient immobilization of various substances that are appended by amine, hydrazide and alcohol functionalities. The newly developed acid-mediated, glass surface modification method should be generally applicable to the preparation of various functional group-modified surfaces.

Cell-surface glycans are involved in both physiological and pathological processes through their interactions with proteins. Carbohydrate microarrays have become powerful tools for studies of these biomolecular interactions in a high-throughput manner. A major driving force for theexpanded use of glycan microarrays is the development of new experimental protocols in which they can be applied. A potentially important application of these microarrays is to assay glycosyltransferase activities. To date, only a few examples of enzymatic reactions taking place on the carbohydrate microarrays have been reported. Herein, we present the results of our recent studies which show that carbohydrate microarrays can be used for assaying galactosyltransferase activity. The approach requires only atiny amount of immobilized acceptor substrates (picomoles), and enzymatic catalytic activities are readily assessed by measuring the amount of a product formed in a time-dependent manner. The level of time-dependent product conversion is determined by using fluorescence detection of lectin recognition of carbohydrate products.

Cancer that is one of serious diseases for human causes angiogenesis, metastasis, and abnormal cell divisions including genetic change. Thus, lots of researchers have been investigated to inhibit cancer. Human MAC-2 binding protein (or human 90K, h90K) has very crucial role for inhibition of cancer metastasis. h90K is a glycoprotein having 7 N-glycosylation sites. In this work, we performed expression of recombinant h90K glycoprotein in Drosophila S2 cell system. We constructed stably-transfected S2-h90K cells by antibiotic selection and confirmed h90K expression using Western Blot analyses with hexa histidine tag and bioactivity check. And we compared with Chinese Hamster Ovary (CHO) cell-deived human MAC-2 binding protein for expression and activity check. We also identified glycosylation patterns of recombinant h90K glycoprotein using HPLC and MALDI-MS. This results and application technology will contribute for scientific and medical engineering.

Lipid rafts are membrane microdomains enriched in saturated phospholipid, sphingolipid, and cholesterol. Pattern Recognition Receptors (PRRs) such as Toll-like Receptors (TLRs), dectin-1 or DC-SIGN are expressed on lipid rafts. SIGN-R1, a PRR, is a homologue of human DC-SIGN and expressed at high levels on macrophages within the marginal zone of the spleen and lymph node medulla, mediating the uptake of pneumococcal capsular polysaccharides (CPSs). Here we showed that SIGN-R1 was expressed in lipid rafts of SIGN-R1 transfectant cell lines in vitro as well as in lipid rafts of spleen or lymphnodes in vivo. It was identified that SIGN-R1 recruited its ligands such as dextran and pneumococcal capsular polysaccharides in vitro. Additionally, it was found that SIGN-R1 was multimerized in lipid rafts with the intravenous administration of S. pneumoniae type 14 or its capsular polysaccharide (CPS14) in vivo. However, the multimerization of SIGN-R1 was not occurred against CPS-depleted S. pneumoniae type 14 or another gram-positive bacteria, Streptococcus aureus. All of these results suggested that the localization of SIGN-R1 in lipid rafts might be important for protecting host against S. pneumoniae, activation its defense mechanism including the multimerization of SIGN-R1. Currently, further studies are in investigation as follows. 1) Whether SIGN-R1 could mediate complement activation against its ligands in lipid rafts? 2) Whether SIGN-R1 mediates a specific signaling from lipid rafts against its ligand? 3) Whether SIGN-R1 mediates the generation of a specific cytokine from lipid rafts against its ligands?

Fucoidans, a group of marine sulfated polysaccharides of the cell-wall matrix of brown algae, exhibit a wide range of biological activities, such as anti-inflammatory, antiviral, antioxidant, anticoagulant, antitumor, and anti-angiogenesis activities. In spite of their interest as biologically active compounds, no convenient commercial sources with fucoidanolytic activities are available yet. we have searched for a bacterial strain capable of degrading a fucoidan which was purified in our lab from the sporopyll of Korean Undaria pinnatifida. Among isolates, a strain, tentatively named as Sphingomonas paucimobilis PF-1 (KCTC 11130BP) fucoidanase activity producing low-molecular weight fuco-oligosaccharides (LMFOs) ranging from 1,389 to 3,749 Da was partially purified from Sphingomonas paucimobilis PF-1. Resulting LMFOs were investigated for immunomodulating activity on murine macrophages and splenocytes. Treatment of fucoidan and LMFOs resulted in inhibition of the growth of murine macrophage RAW 264.7 cells, but its cytotoxicity was not observed in normal murine splenocytes. Treatment of fucoidan and LMFOs induced TNF-a in a dose-dependent manner from two types of macrophages, RAW 264.7 cells and murine peritoneal macrophages. The TNF-a-inducing activity of LMFOs was higher than that of fucoidan. LMFOs also activated murine splenocytes to produce cytokine (IL-6) and chemokines (RANTES and MIP-1a). These results indicate that fucoidan and LMFOs stimulate early innate immune system with no cytotoxicity.

Sialic acids are typically found as terminal monosaccharides attached to the cell surface glycoconjugates and play important roles in many physiological and pathological processes, including microbe binding that leads to infections, regulation of the immune response, the progression and spread of human malignancies, differentiation, development, canceration, and degenerative diseases (1-3). UDP-GlcNAc is a metabolic precursor of sialic acids. Thus, glucose metabolism and regulation of metabolic flux of UDT-GlcNAc critically affect the expression of cell surface sialic acid and nervous system function (4,5). Here, we investigated the expression of sialic acid in human neuroblastoma cell, SK-N-SH, in response to glucose deprivation. When subjected to a glucose-free environment for 24 h, SK-N-SH cells showed severe defects in neurite development and eventually died. During glucose limitation, the expression level of sialic acid of total cellular proteins was examined by lectin blotting using Maackia amunrensis(MAA) lectin and Sambucus nigra(SNA) lectin, which are specific for α-2,3 sialic acid and α-2,6 sialic acid, respectively. Glucose deprivation caused increase in MAA binding to several major proteins (especially, 45 and 75 kDa), suggesting an increased α-2,3 sialylation on these proteins. Lectin histochemistry using MAA lectin also showed a significant increase in surface α,2-3 sialylation upon glucose deprivation. In addition, glucose deprivation resulted in a dose-dependant increase in the mRNA level of ST3GAL4 sialyltransferase by 2.3 folds comparing to that cultured in 2 mg/ml glucose medium, whereas expressions of other sialyltransferases (ST3GAL3, ST3GAL6, and ST6GAL1) were not significantly influenced. These results suggest that glucose deprivation upregulates the α,2-3 sialylation on several cell surface glycoproteins and this phenomenon may be involved in neural cell death by glucose deprivation.

A novel polysialic acid-specific endo-sialidase referred to Endo-PS was purified from the culture filtrate of Pseudomonas fluorescens JK-0412 to apparent homogeneity and has a subunit molecular mass of 20 kDa. The purified enzyme Endo-PS has a molecular weight of 110 kDa by native-PAGE, suggesting that the active enzyme is a hexamer. Although 12 residues of N-terminal amino acid sequence of Endo-PS showed 75% identity with 21-kDa chitin binding protein CBP21 of Serratia marcescens 2170, no significant homology with other known proteins was observed. The specific activity of Endo-PS toward an artificial substrate 4-methylumbelliferyl-sialic acid (4-MU-Neu5A) was determined to be 16 nmole/mg/min with Km value of 0.08 mM. The enzyme was maximally active at an optimal temperature of 37℃ and pH 8. It possesses the ability to only hydrolyze natural substrate α2-8 linked homo-polysialic acid (or colominic acid), but not α2-3 or α2-6 sialyllactoses. Under optimal condition, sialyloligosaccharides ranging from dimer up to 30 residues long liberated from the cleavage of polysialic acid in the function of time were identified by high performance anion-exchange chromatography (HPAEC). Taken together these results suggest that the biochemical properties of Endo-PS purified from the culture filtrate of P. fluorescens JK-0412 are specificfor polysialic acid, referred as a novel endo-polysialidase, reporting first about the properties of non-pathogenic bacterial-derived endo-sialidase.

A chitinolytic enzyme from the culture filtrates of Pseudomonas fluorescens was purified to apparent homogeneity and its kinetic properties were characterized. The molecular weight of the purified enzyme (NagA) was estimated by SDS–PAGE to be approximately 50 kDa. The optimum pH, temperature, and pH stability of NagA on synthetic substrate pNP-GlcNAc were determined to be pH 6.5, 37oC and pH 6–9, respectively. Its kinetic parameters on pNP-GlcNAc and pNP-(GlcNAc)2 were Km = 0.13 mM and 0.82 mM, and Vmax = 245 and 40 pmol/㎍/min, respectively. Chitinolytic activity of the purified NagA toward natural substrates (GlcNAc)n, n=2-5 was also determined by high performance anion-exchange chromatography with pulsed electrochemical detection (HPAEC-PED). The results suggest that NagA is an exo-type β-N-acetylglucosaminidase yielding GlcNAc as the final product from the chitooligosaccharides. Taken together, this is the first report of an exo-type chitinolytic enzyme based on the comparison of its peptide sequence from other chitinolytic enzyme produced by other microorganisms.