Glycobiology - Advance Access

Identification of human hyaluronidase-4 as a novel chondroitin sulfate hydrolase that preferentially cleaves the galactosaminidic linkage in the trisulfated tetrasaccharide sequence

Kaneiwa, T., Mizumoto, S., Sugahara, K., Yamada, S. — Wed, 04 Nov 2009 08:05:15 PST

Human hyaluronidases have been considered to be the enzymes acting at the initial step in the catabolism of chondroitin sulfate (CS) in vivo. However, human hyaluronidase-1 digests CS more slowly than hyaluronan (HA), and its preferred substrate is HA rather than CS. We have identified a chondroitin hydrolase in Caenorhabditis elegans, which effectively degrades chondroitin but depolymerizes HA to a much lesser extent (Kaneiwa T, Yamada S, Mizumoto S, Montaño AM, Mitani S, Sugahara K. 2008. J Biol Chem. 283:14971–14979), suggesting the existence of CS-specific endoglycosidases in mammalian systems. In this study, human hyaluronidase-4 was demonstrated to be a CS-specific endo-β-N-acetylgalactosaminidase. This is the first demonstration of a CS hydrolase in higher organisms. The specificity of a purified recombinant form of the enzyme was investigated in detail through the characterization of degradation products. The best substrate of the CS hydrolase was the galactosaminidic linkage in the sequence of a trisulfated tetrasaccharide GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)-GlcUA-GalNAc(4-O- or 6-O-sulfate), where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. The disaccharide unit on the nonreducing side, GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate) (D unit), is rich in shark fin cartilage CS-D among various CS isoforms. CS hydrolase will be a useful tool for investigating CS-specific functions in tissues and cells. In addition, it may well be applicable to the treatment of acute spinal cord injuries as in the case of, or instead of, the bacterial CS lyase which has been used for recent clinical trials.

Fucosylated glycan inhibition of human hepatocellular carcinoma cell migration through binding to chemokine receptors

Wu, L. H., Shi, B. Z., Zhao, L. Q., Wu, X. Z. — Sun, 01 Nov 2009 22:38:37 PST

SMMC-7721 hepatocellular carcinoma cells (HCC) were incubated with fucosylated glycoproteins that had been isolated from retinoic acid-treated cells by affinity chromatography. HCC migration was significantly inhibited by AAL- and LCA-glycoproteins. Glycopeptides, obtained by digestion of the glycoproteins with trypsin and papain, were found to have a similar inhibitory effect on HCC migration as the corresponding glycoproteins. The inhibitory actions of the glycoproteins were almost abolished after digestion with -L-1,3/4- or -L-1,2-fucosidase. Induction of HCC migration with chemokines including interleukin-8 (IL-8), lymphotactin, monocyte chemoattractant protein-1, and stroma cell-derived factor-1 was examined and IL-8 was found to be the most potent. Interestingly, the isolated glycoproteins significantly inhibited HCC migration and F-actin aggregation induced by IL-8, whereas the glycans themselves did not induce F-actin assembly. From receptor binding analysis AAL-glycan was found to bind IL-8 receptors especially CXCR2 directly and such binding could be blocked by 3'- or 2'-fucosyllactose. After CXCR2 silence by target RNAi, the cells almost lost the response to AAL-glycan inhibition. Our findings suggest that fucosylation plays an important role in the interaction between IL-8 and its receptors inducing HCC migration.

Trans-sialidase activity of Photobacterium damsela {alpha}2,6-sialyltransferase and its application in the synthesis of sialosides

Cheng, J., Huang, S., Yu, H., Li, Y., Lau, K., Chen, X. — Fri, 30 Oct 2009 02:17:50 PDT

Trans-sialidases catalyze the transfer of a sialic acid from one sialoside to an acceptor to form a new sialoside. 2,3-Trans-sialidase activity was initially discovered in the parasitic protozoan Trypanosoma cruzi, and more recently was found in a multifunctional Pasteurella multocida sialyltransferase PmST1. 2,8-Trans-sialidase activity was also described for a multifunctional Campylobacter jejuni sialyltransferase CstII. We report here the discovery of the 2,6-trans-sialidase activity of a previously reported recombinant truncated bacterial 2,6-sialyltransferase from Photobacterium damsela (15Pd2,6ST). This is the first time that the 2,6-trans-sialidase activity has ever been identified. Kinetic studies indicate that 15Pd2,6ST-catalyzed trans-sialidase reaction follows a ping-pong bi-bi reaction mechanism. Cytidine 5’-monophosphate, the product of sialyltransferase reactions, is not required by the trans-sialidase activity of the enzyme but enhances the trans-sialidase activity modestly as a non-essential activator. Using chemically synthesized Neu5AcpNP and LacβMU, 2,6-linked sialoside Neu5Ac2,6LacβMU has been obtained in one-step in high yield using the trans-sialidase activity of 15Pd2,6ST. In addition to the 2,6-trans-sialidase activity, 15Pd2,6ST also has 2,6-sialidase activity. The multifunctionality is thus a common feature of many bacterial sialyltransferases.

Structures of two novel, serologically non-related core oligosaccharides of Yokenella regensburgei lipopolysaccharides differing only by a single hexose substitution.

Fri, 30 Oct 2009 08:41:52 PDT

Immunochemical analysis of the Yokenella regensburgei lipopolysaccharides (LPS) indicated the presence of the core oligosaccharide-related immunotypes among the investigated strains. The structure of the core oligosaccharide segment of the Y. regensburgei LPS has been investigated using chemical methods, mass spectrometry and ¹H, ¹³C NMR spectroscopy. It was concluded that the core oligosaccharides of the strains PCM 2476 and PCM 2477 are composed of an undecasaccharide [structures: see text]. The combined data revealed two immunotypes of the core oligosaccharide recognised by antibodies against the whole bacterial cells. The structural differences between the core oligosaccharides are limited to the outermost terminal hexopyranose residue. In the core oligosaccharide of the strain PCM 2476 it was identified as -d-Glcp and in that of the strain PCM 2477 as -d-Galp. This subtle difference between the glycoforms of the LPS core appeared to be essential for formation of the epitopes recognised by the specific antibodies directed against the Yokenella regensburgei whole bacterial cells. The oligosaccharides are not substituted by phosphate groups. Instead, the carboxyl groups of Kdo and galacturonic acid residues present in the core provide the negative charges. The undecasaccharides represent a novel core type of bacterial LPS, that is characteristic for Y. regensburgei.

Site specific analysis of N-linked oligosaccharides of recombinant lysosomal arylsulfatase A produced in different cell lines

Wed, 28 Oct 2009 10:04:16 PDT

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a deficiency of the lysosomal enzyme arylsulfatase A (ASA). Enzyme replacement therapy (ERT) is a therapeutic option for MLD and other lysosomal disorders. This therapy depends on N-linked oligosaccharide mediated delivery of intravenously injected recombinant enzyme to the lysosomes of patient cells. Because of the importance of N-linked oligosaccharide side chains in ERT we examined the composition of the three N-linked glycans of four different recombinant ASAs in a site specific manner. Depending on the culture conditions and the cell line expressing the enzyme, we detected a high variability of the high mannose type N-glycans which prevail at all glycosylation sites. Our data show that the composition of the glycans is largely determined by substantial trimming in the medium. The susceptibility for trimming is different for the glycans at the three N-glycosylation sites. Interestingly, which of the glycans is most susceptible to trimming also depends on production conditions. CHO cells cultured under bioreactor conditions yielded recombinant ASA with the most preserved N-glycan structures, the highest mannose-6-phosphate content and the highest similarity to non-recombinant enzyme. Notably, roughly one third of the N-glycans released from the three glycosylation sites were fucosylated. In the last years numerous recombinant lysosomal enzymes were used for preclinical ERT trials. Our data show that the oligosaccharide structures were very different in these trials making it difficult to draw common conclusions from the various investigations.

HABA- based ionic liquid matrices for UV-MALDI-MS analysis of heparin and heparan sulfate oligosaccharides

Mon, 26 Oct 2009 04:13:31 PDT

Polysulfated carbohydrates such as heparin (HP) and heparan sulfate (HS) are not easily amenable to usual ultraviolet matrix-assisted laser desorption/ionization-mass spectrometry (UV-MALDI)-MS analysis due to the thermal lability of their O- and N-SO₃ moieties, and their poor ionization efficiency with common crystalline matrices. Recently, ionic liquid matrices showed considerable advantages over conventional matrices for MALDI-MS of acidic compounds. Two new ionic liquid matrices (ILMs) based on the combination of 2-(4-hydroxyphenylazo)benzoic acid (HABA) with 1,1,3,3-tetramethylguanidine and spermine were evaluated in the study herein. Both ILMs were successfully applied to the analysis of synthetic heparin oligosaccharides of well-characterized structures as well as to heparan sulfate derived oligosaccharides from enzymatic depolymerization. HABA-based ILMs showed improved signal-to-noise ratio as well as a decrease of fragmentation/desulfation processes and cation exchange. Sulfated oligosaccharides were detected with higher sensitivity than with usual crystalline matrices, and their intact fully O- and N-sulfated species [M-Na]^– were easily observed on mass spectra. MALDI-MS characterization of analytes as much as challenging than heparin octasaccharide carrying 8-O and 4 N-sulfo groups, and heparin octadecasulfated dodecasaccharide were successfully achieved.

Comparison of the substrate specificities and catalytic properties of the sister N-acetylglucosaminyltransferases, GnT-V and GnT-Vb (IX)

Alvarez-Manilla, G., Troupe, K., Fleming, M., Martinez-Uribe, E., Pierce, M. — Wed, 21 Oct 2009 07:11:02 PDT

N-acetylglucosaminlyltransferase-V (GnT-V) synthesizes GlcNAc β1,6Man branched N-glycans both in vitro and in vivo. A paralog, GnT-Vb (or GnT-IX), has also been shown to synthesize both GlcNAcβ1,6Man branched N- and O-glycans. GnT-V is expressed in most human and rodent tissues while GnT-Vb expression is limited mainly to neural tissue and testes. It is of interest, therefore, to compare the catalytic properties and reaction kinetics of these sister enzymes. The results demonstrate that while GnT-V was fully active without exogenous cation and in the presence of EDTA, the activity of GnT-Vb was stimulated over 4-fold in the presence of 10 mM Mn⁺⁺. The pH optimum for GnT-V was in the range of 6.5–7.0, while that of GnT-Vb was 8.0. common for glycosyltransferases active in brain. Both enzymes transferred GlcNAcβ1,6 to the Man residue of the GlcNAcβ1,2Man moiety of glycan substrates, and both enzymes acted effectively on a synthetic GlcNAcβ1,2Man1,2Glc-O-octyl trisaccharide acceptor. Moreover, although both enzymes utilized an N-linked asialo-agalacto-biantennary glycan as acceptor, GnT-Vb displayed an almost 2.5-fold higher apparent K_m value compared to GnT-V. Conversely, GnT-Vb very efficiently glycosylated a synthetic glycopeptide, Ac-H₂N-Val-Glu-Pro-(GlcNAcβ1,2-Man-O-)Thr-Ala-Val-CO-Ac, while GnT-V showed relatively poor activity toward this O-Man-linked glycopeptide acceptor, with a K_m value of 20-fold higher than that of GnT-Vb. When the N-linked asialo-agalacto-biantennary glycan acceptor was utilized with GnT-Vb, the expected triantennary β1,6 branched product was observed up to 8 hr. incubation. An additional product with two β1,6-linked GlcNAc resides, however, was observed after prolonged (>8hr) incubation, consistent with an earlier report. This unusual tetraantennary product was observed with GnT-Vb only after substantial accumulation of the first triantennary product and not during the early stages of incubation.

Sialyltransferases of marine bacteria efficiently utilize glycosphingolipid substrates

Fri, 16 Oct 2009 09:28:17 PDT

Bacterial sialyltransferases (STs) from marine sources were characterized using glycosphingolipids (GSLs). Bacterial STs were found to be β-galacotoside STs. There were two types of STs: (1) ST obtained from strains such as ishi-224, 05JTC1 (#1), ishi-467, 05JTD2 (#2), and faj-16, 05JTE1 (#3), which form 2–3 sialic acid (Sia) linkages, named 2–3ST, (2) ST obtained from strains such as ISH-224, N1C0 (#4), pda-rec, 05JTB2 (#5), and pda-0160, 05JTA2 (#6), which form 2–6 Sia linkages, named 2–6ST. All STs showed affinity to neolacto- and lacto-series GSLs, particularly in neolactotetraosyl ceramide (nLc₄Cer). No large differences were observed in the pH and temperature profiles of enzyme activities. Kinetic parameters obtained by Lineweaver-Burk plot analysis showed that #3 and #4 STs had the practical synthetic activity and thus it became easily possible to achieve large-scale ganglioside synthesis (100~300µM) using these recombinant enzymes. Gangliosides synthesized from nLc₄Cer by 2–3 and 2–6STs were structurally characterized by several analytical and immunological methods, and they were identified as IV³NeuAc-nLc₄Cer(S2–3PG) and IV⁶NeuAc-nLc₄Cer (S2–6PG), respectively.

Further characterization of these STs using lactotetraosylceramide (Lc₄Cer), neolactohexaosylceramide (i antigen) and IV⁶kladoLc₈Cer (I antigen) showed the synthesis of corresponding gangliosides as well. Synthesized gangliosides showed binding activity to the influenza A virus {A/panama/2007/99 (H3N2)} at a similar level to purified S2–3PG and S2–6PG from mammalian sources.

The above evidence suggests that these STs have unique features, including substrate specificities restricted to lacto- and neolactoseries GSLs, as well as catalytic potentials for ganglioside synthesis. This demonstrates that efficient in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing Sias modifications, thereby permitting the exploration of unknown functions.

Direct Detection of HSulf-1 and HSulf-2 Activities on Extracellular Heparan Sulfate and their Inhibition by PI-88

Mon, 12 Oct 2009 09:59:40 PDT

Heparan sulfates (HS) bind a diversity of protein ligands on the cell surface and in the extracellular matrix and thus can modulate cell signaling. The state of sulfation in glucosamines and uronic acids within the chains strongly influences their binding. We have previously cloned and characterized two human extracellular endoglucosamine 6-sulfatases, HSulf-1 and HSulf-2, which selectively liberate the 6-O sulfate groups on glucosamines present in N, 6-O and 2-O trisulfated disaccharides of intact HS and heparins. These enzymes serve important roles in development and are upregulated in a number of cancers. To determine whether the Sulfs act on the trisulfated disaccharides that exist on the cell surface, we expressed HSulfs in cultured cells and performed a flow cytometric analysis with the RB4CD12, an anti-HS antibody that recognizes N- and O- sulfated HS saccharides. The endogenously expressed level of the cell surface RB4CD12 epitope was greatly diminished in CHO, HEK293 and HeLa cells transfected with HSulf-1 or HSulf-2 cDNA. In correspondence with the RB4CD12 finding, the N, 6-O and 2-O trisulfated disaccharides of the HS isolated from the cell surface/extracellular matrix were dramatically reduced in the Sulf-expressed HEK 293 cells. We then developed an ELISA and confirmed that the RB4CD12 epitope in immobilized heparin was degraded by purified recombinant HSulf-1 and HSulf-2, and conditioned medium (CM) of MCF-7 breast carcinoma cells, which contain a native form of HSulf-2. Furthermore, HSulf-1 and HSulf-2 exerted activity against the epitope expressed on microvessels of mouse brains. Both HSulf activities were potently inhibited by PI-88, a sulfated heparin mimetic with anti-cancer activities. These findings provide new strategies for monitoring the extracellular remodeling of HS by Sulfs during normal and pathophysiological processes.

An Echinococcus multilocularis Coproantigen Is a Surface Glycoprotein With Unique O- Gycosylation

Hulsmeier, A. J., Deplazes, P., Naem, S., Nonaka, N., Hennet, T., Kohler, P. — Mon, 12 Oct 2009 09:59:39 PDT

A major surface constituent of Echinococcus multilocularis adult worms, referred to as EmA9 antigen, was immunoaffinity purified and identified as a high molecular weight glycoconjugate. Labelling studies using the monoclonal antibody MAbEmA9 indicated that this antigen undergoes a regulated expression during the development from the larval to the adult parasite. Chemical modification of carbohydrate by periodate oxidation resulted in a reduced reactivity with antigen specific antibodies. Non-reductive β-elimination of the purified molecule indicated the presence of O-linked glycans attached to threonine residues. Carbohydrate compositional analyses indicated the presence of N- and O-glycans with the ratio of carbohydrate to protein being 1.5:1 (w/w). N- and O-linked glycans were released by hydrazinolysis and analysed as 2-aminobenzamide derivatised glycans by mass spectrometry together with HPLC and enzymatic sequencing. Novel linear O-linked saccharides with multiple β-HexNAc extensions of reducing end Gal were identified. N-linked glycans were also detected with oligomannose and mono-, bi-, tri- and tetra-antennary type structures, most of which were found to be core-fucosylated. Taken together, the results indicate that the EmA9 antigen is a glycoprotein located at the outer surface of the adult E. multilocularis. The observation that the EmA9 antigen expression is developmentally regulated suggests an involvement of this glycoprotein in the establishment of the parasite in its canine host.

MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas

Thu, 08 Oct 2009 13:19:41 PDT

Changes in mucin protein expression and in glycosylation are common features in pre-neoplastic lesions and cancer and are therefore used as cancer-associated markers. De novo expression of intestinal mucin MUC2 and cancer-associated sialyl-Tn antigen are frequently observed in intestinal metaplasia (IM) and gastric cancer. However, despite that these antigens often co-localize, MUC2 has not been demonstrated to be a carrier of sialyl-Tn. By using the in situ proximity ligation assay (in situ PLA) we herein could show that MUC2 is a major carrier of the sialyl-Tn antigen in all IM cases and in most gastric carcinoma cases. The requirement by in situ PLA for the presence of both antigens in close proximity increases the selectivity compared to measurement of co-localization, as determined by immunohistochemistry. To identify the mucin which is the carrier of a carbohydrate structure offers unique advantages for future development of more accurate diagnostic and prognostic markers.

An {alpha}2,6-sialyltransferase cloned from Photobacterium leiognathi strain JT-SHIZ-119 shows both sialyltransferase and neuraminidase activity

Wed, 30 Sep 2009 23:54:03 PDT

We cloned, expressed, and characterized a novel β-galactoside 2,6-sialyltransferase from Photobacterium leiognathi strain JT-SHIZ-119. The protein showed 56% to 96% identity to the marine bacterial 2,6-sialyltransferases classified into glycosyltransferase family 80. The sialyltransferase activity of the N-terminal truncated form of the recombinant enzyme was 1477 U/L of Escherichia coli culture. The truncated recombinant enzyme was purified as a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis through 3 column chromatography steps. The enzyme had distinct activity compared with known marine bacterial 2,6-sialyltransferases. Although 2,6-sialyltransferases cloned from marine bacteria, such as Photobacterium damselae strain JT0160, P. leiognathi strain JT-SHIZ-145, and Photobacterium sp. strain JT-ISH-224, show only 2,6-sialyltransferase activity, the recombinant enzyme cloned from P. leiognathi strain JT-SHIZ-119 showed both 2,6-sialyltransferase and 2,6-linkage-specific neuraminidase activity. Our results provide important information toward a comprehensive understanding of the bacterial sialyltransferases belonging to the group 80 glycosyltransferase family in the CAZy database.

Human sialidase NEU4 long and short are extrinsic proteins bound to outer mitochondrial membrane and the endoplasmic reticulum, respectively

Wed, 30 Sep 2009 23:54:02 PDT

Sialidases are widely distributed glycohydrolytic enzymes removing sialic acid residues from glycoconjugates. In mammals, several sialidases with different subcellular localizations and biochemical features have been described. NEU4, the most recently identified member of the human sialidase family, is found in two forms, NEU4 long and NEU4 short, differing in the presence of a 12 aminoacid sequence at the N-terminus. Contradictory data are present in the literature about the subcellular distribution of these enzymes, their membrane anchoring mechanism being still unclear.

In this work we investigate human NEU4 long and NEU4 short membrane anchoring mechanism and their subcellular localization. Protein extraction with Triton X-114 and sodium carbonate and cross-linking experiments demonstrate that both forms of NEU4 are extrinsic membrane proteins, anchored via protein-protein interactions. Moreover, through confocal microscopy and subcellular fractionation, we show that the long form localizes in mitochondria, while the short form is also associated with the endoplasmic reticulum. Finally, mitochondria subfractionation experiments suggest that NEU4 long is bound to the outer mitochondrial membrane.

Separation and identification of GM1b pathway Neu5Ac- and Neu5Gc gangliosides by on-line nanoHPLC-QToF MS and tandem MS: toward glycolipidomics screening of animal cell lines

Zarei, M., Muthing, J., Peter-Katalinic, J., Bindila, L. — Wed, 30 Sep 2009 23:54:01 PDT

Monosialoganglioside fraction of YAC-1 lymphoma cells was comprehensively analyzed and structurally defined by nano high performance liquid chromatography (nanoHPLC) in on-line conjunction with electrospray ionization time-of-flight mass spectrometry (ESI-QTOF MS). An efficient separation and sensitive detection of Neu5Gc – containing gangliosides from Neu5Ac – containing analogous was for the first time accomplished in single nanoHPLC/ESI-QTOF MS run, as demonstrated for mouse hybridoma cell GM3 fraction containing GM3(Neu5Ac) and GM3(Neu5Gc) species and further applied for the analysis of YAC-1 lymphoma cell monosialoganglioside fraction. New insights into YAC-1 monosialoganglioside mixture heterogeneity were obtained: 31 distinct species, comprising 18 Neu5Gc-containing gangliosides and 13 Neu5Ac – containing species of GM1b and GalNAc-GM1b type were found to be expressed by YAC-1 cell line. On-line structural elucidation of individually separated Neu5Ac- and Neu5Gc – containing gangliosides provided strong evidence on the "GM1b-pathway" sourcing for monosialoganglioside synthesis. Such an analytical method is documented as superior to the classical approaches by increased speed of analysis, sensitivity and level of information, being thus a viable glycolipidomic tool.

Lessons from GNE-deficient embryonic stem cells: Sialic acid biosynthesis is involved in proliferation and gene expression

Weidemann, W., Klukas, C., Klein, A., Simm, A., Schreiber, F., Horstkorte, R. — Wed, 30 Sep 2009 23:54:00 PDT

Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. They are involved in a variety of cellular functions, such as cell adhesion or signal recognition. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. Inactivation of GNE causes early embryonic lethality. In this study we analyzed wild type and GNE-deficient embryonic stem cells from mice. We found for the first time that proliferation is directly correlated with GNE-expression and the cellular sialic acid concentration. Furthermore we identified growth-related genes that are differentially expressed in GNE-deficient embryonic stem cells compared to wild type embryonic stem cells.

Protective effect of N-glycan bisecting GlcNAc residues on {beta}-amyloid production in Alzheimer's disease

Wed, 23 Sep 2009 00:35:14 PDT

Alteration of glycoprotein glycans often changes various properties of the target glycoprotein and contributes to a wide variety of diseases. Here we focused on the N-glycans of amyloid precursor protein whose cleaved fragment, β-amyloid, is thought to cause much of the pathology of Alzheimer's disease (AD). We previously determined the N-glycan structures of normal and mutant amyloid precursor proteins (the Swedish type and the London type). In comparison with normal amyloid precursor protein, mutant amyloid precursor proteins had higher contents of bisecting GlcNAc residues. Because N-acetylglucosaminyltransferase III (GnT-III) is the glycosyltransferase responsible synthesizing bisecting GlcNAc, the current report measured GnT-III mRNA expression levels in the brains of AD patients. Interestingly, GnT-III mRNA expression was increased in AD brains. Furthermore, β-amyloid treatment increased GnT-III mRNA expression in Neuro2a mouse neuroblastoma cells. We then examined the influence of bisecting GlcNAc on the production of β-amyloid. Both β-amyloid 40 and β-amyloid 42 were significantly decreased in GnT-III transfected cells. When secretase activities were analyzed in GnT-III transfectant cells, -secretase activity was increased. Taken together, these results suggest that up-regulation of GnT-III in AD brains may represent an adaptive response to protect them from additional β-amyloid production.

Characterization of GD3 Ganglioside as a Novel Biomarker of Mouse Neural Stem Cells

Nakatani, Y., Yanagisawa, M., Suzuki, Y., Yu, R. K. — Wed, 23 Sep 2009 00:35:14 PDT

Neural stem cells (NSCs) are undifferentiated neural cells characterized by their high proliferative potential and the capacity for self-renewal with retention of multipotency. Over the past two decades, there has been a huge effort to identify NSCs morphologically, genetically, and molecular biologically. It is still controversial, however, what bona fide NSCs are. To define and characterize NSCs more systematically, it is crucial to explore novel cell-surface marker molecules of NSCs. In this study, we focused on GD3, a b-series ganglioside that is enriched in the immature brain and the subventricular zone (SVZ) of the postnatal and adult brain, and evaluated the usefulness of GD3 as a cell-surface biomarker for identifying NSCs. We demonstrated that GD3 was expressed in more than 80% of NSCs prepared from embryonic, postnatal, and adult mouse brain tissue by the neurosphere culture method. The percentage of GD3-expressing NSCs in neurospheres was nearly the same as it was in neurospheres derived from embryonic, postnatal, and adult brains but decreased drastically to about 40% after differentiation. GD3⁺ cells isolated from embryonic mouse striata, postnatal and adult mouse SVZs by fluorescence-activated cell sorting with R24 anti-GD3 monoclonal antibody efficiently generated neurospheres compared with GD3^– cells. These cells possessed multipotency to differentiate into neurons, astrocytes, and oligodendrocytes. These data indicate that GD3 is a unique and powerful cell-surface biomarker to identify and isolate NSCs.

Structural basis of the affinity for oligomannosides and analogs displayed by BC2L-A, a Burkholderia cenocepacia soluble lectin

Sun, 20 Sep 2009 22:59:11 PDT

The opportunistic pathogen Burkholderia cenocepacia contains three soluble carbohydrate-binding proteins, related to the fucose-binding lectin PA-IIL from Pseudomonas aeruginosa. All contain a PA-IIL-like domain and two of them have an additional N-terminal domain that displays no sequence similarities with known proteins. Printed arrays screening performed on the shortest one, B. cenocepacia lectin A (BC2L-A), demonstrated the strict specificity for oligomannose-type N-glycan structures (Lameignere, et al. 2008). The disaccharides Man1–2Man, Man1–3Man and Man1–6Man and the trisaccharide, Man1–3(Man1–6)Man were tested by titration microcalorimetry in order to evaluate their affinity for BC2L-A in solution and to characterize the thermodynamics of the binding. Oligomannose analogs presenting two mannoside residues separated by either flexible or rigid spacer where also tested. Only the rigid one yields to high affinity binding with a fast kinetics of clustering, while the flexible analog and the trimannoside display moderate affinities and no clustering effect on short time scale. The crystal structures of BC2L-A has been obtained in complex with Man1–3Man disaccharide and Man1(Man1–6)-3Man trisaccharide. The lengthy time required for the co-crystallization with the trisaccharide allowed for the formation of cluster since in the BC2L-A-trimannose complex solved at 1.1 Å resolution, the sugar creates a bridge between two adjacent dimers, yielding to molecular strings. AFM experiments were performed in order to visualize the filaments formed in solution by this type of interaction.

The role of CDR H3 in Antibody Recognition of a Synthetic Analogue of a Lipopolysaccharide Antigen

Fri, 18 Sep 2009 01:44:58 PDT

In order to explore the structural basis for adaptability in near germline monoclonal antibodies (mAb), we have examined the specificity of the promiscuous mAb S67-27 to both naturally derived carbohydrate antigens and a variety of synthetic non-natural antigens based on the bacterial lipopolysaccharide component 3-deoxy--d-manno-oct-2-ulosonic acid (Kdo). One such analogue, a 7-O-methyl (7-O-Me) Kdo disaccharide, was found to bind to the antibody with at least 30 fold higher affinity than any other antigen tested. The structure of S67-27 in complex with this analogue and three other naturally occurring Kdo antigens revealed that the enhanced affinity of the mAb for the synthetic analogue was accomplished by the strategic positioning of CDR H3 away from a conserved Kdo binding pocket that allowed the formation of new antibody-antigen contacts. Furthermore, comparison of this structure with the structures of related mAbs revealed how the position and structure of CDR H3 influence the specificity or promiscuity of near-germline carbohydrate-recognizing antibodies by altering the architecture of the combining site.

GM3 synthase overexpression results in reduced cell motility and in caveolin-1 up-regulation in human ovarian carcinoma cells.

Wed, 16 Sep 2009 18:00:11 PDT

In this paper, we describe the effects of the expression of GM3 synthase at high levels in human ovarian carcinoma cells. Overexpression of GM3 synthase in A2780 cells consistently resulted in elevated ganglioside (GM3, GM2 and GD1a) levels. GM3 synthase overexpressing cells had a growth rate similar to wild type cells, but showed a strongly reduced in vitro cell motility accompanied by reduced levels of the epithelial-mesenchymal transition marker smooth muscle actin. A similar reduction of cell motility was observed upon treatment with exogenous GM3, GM2 and GM1, but not with GD1a. A photolabeling experiment using radioactive and photoactivable GM3 highlighted several proteins directly interacting with GM3. Among those, caveolin-1 was identified as a GM3-interacting protein in GM3 synthase overexpressing cells. Remarkably, caveolin-1 was markedly upregulated in GM3 synthase overexpressing cells. In addition, the motility of low GM3 synthase expressing cells was also reduced in the presence of a Src kinase inhibitor; on the other hand, higher levels of the inactive form of c-Src were detected in GM3 synthase overexpressing cells, associated with a ganglioside- and caveolin-rich detergent insoluble fraction.

Endomannosidase undergoes phosphorylation in the Golgi apparatus

Torossi, T., Guhl, B., Roth, J., Ziak, M. — Wed, 16 Sep 2009 10:01:11 PDT

Glucose residues from N-linked oligosaccharides are removed by glucosidase I and II in the endoplasmic reticulum (ER) or by the alternate endomannosidase pathway in the Golgi apparatus. Our morphological analysis demonstrates that recombinant rat endomannosidase exhibited a cis and medial Golgi localization alike the endogenous enzyme and its ER to Golgi transport is COP II mediated. Recombinant endomannosidase undergoes a posttranslational modification, which is not related to N-or O-glycosylation. A shift in molecular mass of recombinant endomannosidase was observed upon phosphatase digestion but not for ER-retained CHO cell endomannosidase. Furthermore, immunoprecipitation of ³⁵S- and ³³P-labeled endomannosidase expressed in CHO-K1 cells suggests that recombinant endomannosidase undergoes phosphorylation. Substitution of the single cytoplasmic threonine residue of rat endomannosidase by either an alanine or valine residue resulted in the same posttranslational modification alike the wild type enzyme. The subcellular localization and the in vivo activity of the mutant endomannosidase were not affected. Thus, endomannosidase phosphorylation is occurring in luminal sequences. Modification was prevented when endomannosidase was synthesized using reticulocyte lysates in the presence of canine microsomes. Treatment of cells with brefeldin A blocked the posttranslational modification of endomannosidase, suggesting that phosphorylation is occurring in the Golgi apparatus, the residence of endomannosidase.

Glycan Gimmickry by Parasitic Helminths: A Strategy for Modulating the Host Immune Response?

van Die, I., Cummings, R. D. — Wed, 16 Sep 2009 12:10:11 PDT

Parasitic helminths (worms) co-evolved with vertebrate immune systems to enable long-term survival of worms in infected hosts. Among their survival strategies, worms use their glycans within glycoproteins and glycolipids, which are abundant on helminth surfaces and in their excretory/secretory products, to regulate and suppress host immune responses. Many helminths express unusual and antigenic (nonhost-like) glycans, including those containing polyfucose, tyvelose, terminal GalNAc, phosphorylcholine, methyl groups, and sugars in unusual linkages. In addition, some glycan antigens are expressed that share structural features with those in their intermediate and vertebrate hosts (host-like glycans), including Le^X (Galβ1–4[Fuc1–3]GlcNAc-), LDNF (GalNAcβ1–4[Fuc1–3]GlcNAc-), LDN (GalNAcβ1–4GlcNAc-), and Tn (GalNAcá1-O-Thr/Ser) antigens. The expression of host-like glycan determinants is remarkable and suggests that helminths may gain advantages by synthesizing such glycans. The expression of host-like glycans by parasites previously led to the concept of "molecular mimicry", in which molecules are either derived from the pathogen or acquired from the host to evade recognition by the host immune system. However, recent discoveries into the potential of host glycan-binding proteins (GBPs), such as C-type lectin receptors and galectins, to functionally interact with various host-like helminth glycans provide new insights. Host GBPs through their interactions with worm-derived glycans participate in shaping innate and adaptive immune responses upon infection. We thus propose an alternative concept termed "glycan gimmickry", which is defined as an active strategy of parasites to use their glycans to target GBPs within the host to promote their survival.

Mammalian cell ganglioside-binding specificities of E. coli enterotoxins LT-IIb and variant LT-IIb(T13I)

Sat, 12 Sep 2009 05:22:39 PDT

LT-IIb, a type II heat-labile enterotoxin of E. coli, is a potent immunologic adjuvant with high affinity binding for ganglioside GD1a. Earlier study suggested that LT-IIb bound preferentially to the terminal sugar sequence NeuAc2–3Galβ1–3GalNAc. However, studies in our laboratory suggested a less restrictive binding epitope. LT-IIb(T13I), an LT-IIb variant, engineered by a single isoleucine-threonine substitution, retains biological activity, but with less robust inflammatory effects. We theorized that LT-IIb has a less restrictive binding epitope than previously proposed and that immunologic differences between LT-IIb and LT-IIb (T13I) correlate with subtle ganglioside binding differences. Ganglioside binding epitopes, determined by affinity overlay immunoblotting and enzymatic degradation of ganglioside components of RAW264.7 macrophages, indicated that LT-IIb bound to a broader array of gangliosides than previously recognized. Each possessed NeuAc2–3Galβ1–3GalNAc, although not necessarily as a terminal sequence. Rather, each had a requisite terminal or penultimate single sialic acid and binding was independent of ceramide composition. RAW264.7 enterotoxin-binding and non-binding ganglioside epitopes were definitively identified as GD1a and GM1a respectively, by enzymatic degradation and mass spectroscopy. Affinity overlay immunoblots, constructed to the diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIb bound NeuAc- and NeuGc-gangliosides with nearly equal affinity. However, LT-IIb(T13I) exhibited enhanced affinity for NeuGc-gangliosides and more restrictive binding. These studies further elucidate the binding epitope for LT-IIb and suggest that the diminished inflammatory activity of LT-IIb(T13I) is mediated by a subtle shift in ganglioside binding. These studies underscore the high degree of specificity required for ganglioside-protein interactions.

Hypoxic Regulation of Secreted Proteoglycans in Macrophages

Fri, 11 Sep 2009 04:07:00 PDT

Macrophages are prominent in hypoxic areas of atherosclerotic lesions and their secreted proteoglycans (PG), such as versican, can modulate the retention of lipoproteins and the activity of enzymes, cytokines, and growth factors involved in atherogenesis. In this study, we report the effects of hypoxia on PG secreted by human monocyte-derived macrophages (HMDM) and the potential regulation by the transcription factor hypoxia inducible factor (HIF-1 and HIF-2). We found that versican co-localized with HIF-1 in macrophage-rich areas in human advanced atherosclerotic lesions. Versican and perlecan mRNA expression increased after exposure to 0.5% O₂ (hypoxia) compared with 21% O₂ (control cells). Using precursors to GAG biosynthesis combined with immunoabsorption with a versican antibody an increased versican synthesis was detected at hypoxia. Furthermore, siRNA knockdown of HIF-1 and HIF-2 in THP-1 cells showed that the hypoxic induction of versican and perlecan mRNA expression involved HIF signaling. Versican expression was co-regulated by HIF-1 and HIF-2 but expression of perlecan was influenced only by HIF-1 and not by HIF-2 knockdown. The results show that oxygen concentration is an important modulator of PG expression in macrophages. This may be a novel component of the complex role of macrophages in atherosclerosis.

Characterization of Gene-activated Human Acid-{beta}-Glucosidase: Crystal Structure, Glycan Composition and Internalization into Macrophages

Wed, 09 Sep 2009 08:42:04 PDT

Gaucher disease, the most common lysosomal storage disease, can be treated with enzyme replacement therapy (ERT), in which defective acid-β-glucosidase (GlcCerase) is supplemented by recombinant, active enzyme. The X-ray structures of recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme®) and in transgenic carrot cells (prGCD) have been previously solved. We now describe the structure and characteristics of a novel form of GlcCerase under investigation for the treatment of Gaucher disease, Gene-Activated^TM human GlcCerase (velaglucerase alfa). In contrast to imiglucerase and prGCD, velaglucerase alfa contains the native human enzyme sequence. All three GlcCerases consist of three domains, with the active site located in domain III. The distances between the carboxylic oxygens of the catalytic residues, E340 and E235, are consistent with distances proposed for acid-base hydrolysis. Kinetic parameters (K_m and V_max) of velaglucerase alfa and imiglucerase, as well as their specific activities, are similar. However, analysis of glycosylation patterns shows that velaglucerase alfa displays distinctly different structures from imiglucerase and prGCD. The predominant glycan on velaglucerase alfa is a high-mannose type, with nine mannose units, while imiglucerase contains a chitobiose tri-mannosyl core glycan with fucosylation. These differences in glycosylation affect cellular internalization; the rate of velaglucerase alfa internalization into human macrophages is at least 2-fold greater than that of imiglucerase.

Analysis of Differential Expression of Glycosyltransferases in Healing Corneas by Glycogene Microarrays

Saravanan, C., Cao, Z., Head, S. R., Panjwani, N. — Mon, 07 Sep 2009 01:41:54 PDT

It is generally accepted that the glycans on the cell surface and extracellular matrix proteins play a pivotal role in the events that mediate re-epithelialization of wounds. Yet, the global alteration in the structure and composition of glycans, specifically occuring during corneal wound closure remains unknown. In this study, GLYCOv2 glycogene microarray technology was used for the first time to identify the differentially expressed glycosylation-related genes in healing mouse corneas. Of ~ 2000 glycogenes on the array, the expression of 11 glycosytransferase and glycosidase enzymes was upregulated and that of 19 was downregulated more than 1.5-fold in healing corneas compared with the normal, uninjured corneas. Among them, notably, glycosyltransferases, β3GalT5, T-synthase, and GnTIVb, were all found to be induced in the corneas in response to injury, whereas, GnTIII and many sialyltransferases were downregulated. Interestingly, it appears that the glycan structures on glycoproteins and glycolipids, expressed in healing corneas as a result of differential regulation of these glycosyltransferases, may serve as specific counterreceptors for galectin-3, a carbohydrate-binding protein, known to play a key role in re-epithelialization of corneal wounds. Additionally, many glycogenes including a proteoglycan, glypican-3, and cell adhesion proteins dectin-1 and -2, and mincle, and mucin 1 were identified for the first time to be differentially regulated during corneal wound healing. Results of glycogene microarray data were confirmed by qRT-PCR and lectin blot analyses. The differentially expressed glycogenes identified in the present study have not previously been investigated in the context of wound healing and represent novel factors for investigating the role of carbohydrate-mediated recognition in corneal wound healing.