Disaccharide Chain Research Articles

Membrane lipid composition of Carnobacterium maltaromaticum CNCM I-3298, a highly cryoresistant lactic bacterium

The growing consumption of fermented products has led to an increasing demand for lactic acid bacteria (LAB), especially for LAB tolerant to freezing/thawing conditions. Carnobacterium maltaromaticum is a psychrotrophic and freeze-thawing resistant lactic acid bacterium. The membrane is the primary site of damage during the cryo-preservation process and requires modulation to improve cryoresistance. However, knowledge about the membrane structure of this LAB genus is limited. We presented here the first study of the membrane lipid composition of C. maltaromaticum CNCM I-3298 including the polar heads and the fatty acid compositions of each lipid family (neutral lipids, glycolipids, phospholipids). The strain CNCM I-3298 is principally composed of glycolipids (32%) and phospholipids (55%). About 95% of glycolipids are dihexaosyldiglycerides while less than 5% are monohexaosyldiglycerides. The disaccharide chain of dihexaosyldiglycerides is composed of α-Gal(1−2)-α-Glc chain, evidenced for the first time in a LAB strain other than Lactobacillus strains. Phosphatidylglycerol is the main phospholipid (94%). All polar lipids are exceptionally rich in C18:1 (from 70% to 80%). Regarding the fatty acid composition, C. maltaromaticum CNCM I-3298 is an atypical bacterium within the genus Carnobacterium due to its high C18:1 proportion but resemble the other Carnobacterium strains as they mostly do not contain cyclic fatty acids.

Proteoglycan Sulphation in the Function of the Mature Central Nervous System.

Chondroitin sulphate and heparan sulphate proteoglycans (CSPGS and HSPGs) are found throughout the central nervous system (CNS). CSPGs are ubiquitous in the diffuse extracellular matrix (ECM) between cells and are a major component of perineuronal nets (PNNs), the condensed ECM present around some neurons. HSPGs are more associated with the surface of neurons and glia, with synapses and in the PNNs. Both CSPGs and HSPGs consist of a protein core to which are attached repeating disaccharide chains modified by sulphation at various positions. The sequence of sulphation gives the chains a unique structure and local charge density. These sulphation codes govern the binding properties and biological effects of the proteoglycans. CSPGs are sulphated along their length, the main forms being 6- and 4-sulphated. In general, the chondroitin 4-sulphates are inhibitory to cell attachment and migration, while chondroitin 6-sulphates are more permissive. HSPGs tend to be sulphated in isolated motifs with un-sulphated regions in between. The sulphation patterns of HS motifs and of CS glycan chains govern their binding to the PTPsigma receptor and binding of many effector molecules to the proteoglycans, such as growth factors, morphogens, and molecules involved in neurodegenerative disease. Sulphation patterns change as a result of injury, inflammation and ageing. For CSPGs, attention has focussed on PNNs and their role in the control of plasticity and memory, and on the soluble CSPGs upregulated in glial scar tissue that can inhibit axon regeneration. HSPGs have key roles in development, regulating cell migration and axon growth. In the adult CNS, they have been associated with tau aggregation and amyloid-beta processing, synaptogenesis, growth factor signalling and as a component of the stem cell niche. These functions of CSPGs and HSPGs are strongly influenced by the pattern of sulphation of the glycan chains, the sulphation code. This review focuses on these sulphation patterns and their effects on the function of the mature CNS.

A novel strategy for the discovery and validation of allergic component and its action mechanism in Red Ginseng

Red ginseng (a processed product of ginseng) is a famous herbal medicine or healthcare product widely used in Asia. However, there are many reports about anaphylactoid reaction of red ginseng. This study aims to establish a novel strategy for discovering and validating allergic component and its action mechanism in red ginseng. Firstly, RBL-2H3 cell degranulation experiment was performed by real-time cell analysis (RTCA) system and β-hexosaminase release test to illustrate the anaphylactoid reaction induced by red ginseng. Then, combined with ultra-performance liquid chromatography quadrupole/time of flight mass spectrometry (UPLC-Q/TOF-MS) analysis, the main components of red ginseng for inducing degranulation of RBL-2H3 cells was explored. Furthermore, the compounds with the parent nucleus structure of allergic component were studied to figure out whether they could induce RBL-2H3 cell degranulation, and the relationship between the structures of compounds and degranulation was further investigated. Finally, the underlying mechanism of allergic components was explored based on network toxicology and molecular docking method, which was further verified by western blotting analysis. Our findings demonstrated that red ginseng extract could induce degranulation of RBL-2H3 cells, and ginsenoside Ro (an oleanane-type triterpene saponin), was confirmed to be the main allergic component. Meanwhile, both ginsenoside Ro (Ro) and other oleanane-type triterpene saponins could induce degranulation of RBL-2H3 cells, and the compound with disaccharide chain on C3 position had the strongest activity of degranulation. And their mechanism of inducing degranulation of RBL-2H3 cells involved in PI3K-Akt pathway. The results would provide a reference for the quality control and evaluation of safety of herb medicines or healthcare products containing oleanane-type triterpenoid saponins. Our method offers a novel strategy for screening allergic components in red ginseng and promoting further study of safety evaluation of red ginseng.

Biosynthesis of a rosavin natural product in Escherichia coli by glycosyltransferase rational design and artificial pathway construction

Phytochemicals are rich resources for pharmaceutical and nutraceutical agents. A key challenge of accessing these precious compounds can present significant bottlenecks for development. The cinnamyl alcohol disaccharides also known as rosavins are the major bioactive ingredients of the notable medicinal plant Rhodiola rosea L. Cinnamyl-(6′-O-β-xylopyranosyl)-O-β-glucopyranoside (rosavin E) is a natural rosavin analogue with the arabinopyranose unit being replaced by its diastereomer xylose, which was only isolated in minute quantity from R. rosea. Herein, we described the de novo production of rosavin E in Escherichia coli. The 1,6-glucosyltransferase CaUGT3 was engineered into a xylosyltransferase converting cinnamyl alcohol monoglucoside (rosin) into rosavin E by replacing the residue T145 with valine. The enzyme activity was further elevated 2.9 times by adding the mutation N375Q. The synthesis of rosavin E from glucose was achieved with a titer of 92.9 mg/L by combining the variant CaUGT3T145V/N375Q, the UDP-xylose synthase from Sinorhizobium meliloti 1021 (SmUXS) and enzymes for rosin biosynthesis into a phenylalanine overproducing E. coli strain. The production of rosavin E was further elevated by co-overexpressing UDP-xylose synthase from Arabidopsis thaliana (AtUXS3) and SmUXS, and the titer in a 5 L bioreactor with fed-batch fermentation reached 782.0 mg/L. This work represents an excellent example of producing a natural product with a disaccharide chain by glycosyltransferase engineering and artificial pathway construction.

Coupling Liquid Chromatography and Tandem Mass Spectrometry to Electrophoresis for In-Depth Analysis of Glycosaminoglycan Drugs: Heparin and the Multicomponent Sulodexide

Glycosaminoglycans (GAGs) contribute to the treatment of many human diseases, especially in the field of thrombosis, because of their anticoagulant activity. GAGs interrupt the coagulation process by interacting with multiple coagulation factors through defined sequences within their linear and negatively charged chains, which are not fully elucidated. Numerous methods have been developed to characterize the structure of pharmaceutical GAGs, including intravenously or subcutaneously administered heparin and orally administered sulodexide. However, most currently available methods only focus on the oligosaccharide portion or analyze the whole mixture because longer-chain polysaccharides are extremely difficult to resolve by chromatographic separation. We have established two novel electrophoresis-mass spectrometry methods to provide a panoramic view of the structures of pharmaceutical GAGs. In the first method, an in-gel digestion procedure was developed to recover GAGs from the polyacrylamide gels, while in the second method, a strong anion exchange ultrafiltration procedure was developed to extract multiple GAG species from the agarose gels. Both procedures are compatible with liquid chromatography-tandem mass spectrometry, and structural information, such as disaccharide composition and chain length, can be revealed for each GAG fraction. The applications of these two methods on analysis of two different GAG drugs, heparin and sulodexide, were demonstrated. The current study offers the first robust tool to directly elucidate the structure of larger GAG chains with more biological importance rather than obtaining a vague picture of all chains as a mixture, which is fundamental for better understanding the structure-activity relationship and quality control of the GAG drugs.

Re-construction of Co-expression Network of Genes Involved in Bacterial Cell Wall Synthesis and Their Role in Penicillin Resistance

Background: Peptidoglycan (Murein), which consists of disaccharide and amino acid chain subunits, has a key role in bacterial survival and ranks first in the line defense system against drug therapy. In addition, the transpeptidase enzyme plays an important role in cross-linking in bacterial cell walls. In Escherichia coli bacteria, cross-linking happens by proteins that have a D-D transpeptidase role and bond two amino acids of D-alanine together. These proteins are characterized by their affinity for and binding of penicillin thus they are called penicillin-binding proteins (PBPs). It should be noted that this bonding formation is prevented by the beta-lactam family as they have a similar structure to the above-mentioned proteins. The product of the idtD gene by characteristics such as L-D transpeptidase can catalyze the peptidoglycan structure in the bacterial cell wall in the presence of beta-lactam antibiotics. Methods: In this study, around 426 interactions were identified between genes and approximately 20 genes with a key role in the process of bacterial cell wall synthesis by the reconstruction of 44 genes involved in bacterial cell wall synthesis. Results: The idtD gene locus at the reconstructed network clearly shows that its catalytic activity is the side activity, and there won’t be a lag or disturbance in the procedure cell wall synthesis by removing it from the cycle. However, this side process causes the strengthening of the bacterial cell wall synthesis process against disorders arising by the presence of beta-lactam antibiotics. Conclusions: These five genes in E. coli that furnish L-D transpeptidase properties include IdtA, IdtC, IdtD, IdtE, and mrdA out of which, IdtD is the most important gene in this process.

Deciphering the Biosynthesis of TDP-β-l-oleandrose in Avermectin.

Avermectin (AVM) refers to eight macrolides containing a common l-oleandrosyl disaccharide chain indispensable to their antiparasitic bioactivities. We delineated the biosynthetic pathway of TDP-β-l-oleandrose (1), the sugar donor of AVM, by characterizing AveBVIII, AveBV, and AveBVII as TDP-sugar 3-ketoreductase, 5-epimerase, and 3-O-methyltransferase, respectively. On the basis of this pathway, we successfully reconstituted the biosynthesis of 1 in Escherichia coli. Our work completes the biosynthetic pathway of AVM and lays a solid foundation for further studies.

Involvement of Heparan Sulfate and Heparanase in Neural Development and Pathogenesis of Brain Tumors.

Brain tumors are aggressive and devastating diseases. The most common type of brain tumor, glioblastoma (GBM), is incurable and has one of the worst five-year survival rates of all human cancers. GBMs are invasive and infiltrate healthy brain tissue, which is one main reason they remain fatal despite resection, since cells that have already migrated away lead to rapid regrowth of the tumor. Curative therapy for medulloblastoma (MB), the most common pediatric brain tumor, has improved, but the outcome is still poor for many patients, and treatment causes long-term complications. Recent advances in the classification of pediatric brain tumors reveal distinct subgroups, allowing more targeted therapy for the most aggressive forms, and sparing children with less malignant tumors the side-effects of massive treatment. Heparan sulfate proteoglycans (HSPGs), main components of the neurogenic niche, interact specifically with a large number of physiologically important molecules and vital roles for HS biosynthesis and degradation in neural stem cell differentiation have been presented. HSPGs are composed of a core protein with attached highly charged, sulfated disaccharide chains. The major enzyme that degrades HS is heparanase (HPSE), an important regulator of extracellular matrix (ECM) remodeling which has been suggested to promote the growth and invasion of other types of tumors. This is of clinical interest because GBM are highly invasive and children with metastatic MB at the time of diagnosis exhibit a worse outcome. Here we review the involvement of HS and HPSE in development of the nervous system and some of its most malignant brain tumors, glioblastoma and medulloblastoma.

Library of Recombinant Heparan Sulfates from Mammalian Cell Culture: Paving the Way to Bioengineered Heparin

Heparan sulfate is a polysulfated, unbranched polysaccharide that plays a role in numerous developmental, physiological and pathophysiological processes via its interaction with hundreds of heparan sulfate‐binding proteins. Heparin is a highly sulfated variant of heparan sulfate, used routinely in the clinic as an anticoagulant. Both molecules share a common biosynthetic pathway that includes numerous glycosyltransferases, sulfotransferases and an epimerase. Researchers in the field typically use heparan sulfate purified from porcine intestines or bovine kidney for experimentation. This material suffers from contamination, batch‐to‐batch variation and fails to reproduce the diversity of heparan sulfate structures found in nature. Heparin used in the clinic is also purified from pig intestines with variable compositions, risks of contamination and supply deficits. TEGA Therapeutics is developing a library of heparan sulfate products derived from cell culture. Genetic engineering gives rise to a diversity of heparan sulfate compositions. Each type of heparan sulfate is characterized with respect to disaccharide composition, chain length and protein binding affinity. This work paves the way for development of recombinant heparin bioengineered in cultured cells with a secure supply chain and the potential for enhanced properties and reduced side effects.Support or Funding InformationThis work is supported by NSF STTR Phase I grant 1622959 to C.G and S.S.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The eukaryotic translation initiation factor eIF4E harnesses hyaluronan production to drive its malignant activity.

The microenvironment provides a functional substratum supporting tumour growth. Hyaluronan (HA) is a major component of this structure. While the role of HA in malignancy is well-defined, the mechanisms driving its biosynthesis in cancer are poorly understood. We show that the eukaryotic translation initiation factor eIF4E, an oncoprotein, drives HA biosynthesis. eIF4E stimulates production of enzymes that synthesize the building blocks of HA, UDP-Glucuronic acid and UDP-N-Acetyl-Glucosamine, as well as hyaluronic acid synthase which forms the disaccharide chain. Strikingly, eIF4E inhibition alone repressed HA levels as effectively as directly targeting HA with hyaluronidase. Unusually, HA was retained on the surface of high-eIF4E cells, rather than being extruded into the extracellular space. Surface-associated HA was required for eIF4E's oncogenic activities suggesting that eIF4E potentiates an oncogenic HA program. These studies provide unique insights into the mechanisms driving HA production and demonstrate that an oncoprotein can co-opt HA biosynthesis to drive malignancy.

Humanized chondroitinase ABC sensitizes glioblastoma cells to temozolomide.

Malignant gliomas (glioblastomas; GBMs) are extremely aggressive and have a median survival of approximately 15months. Current treatment modalities, which include surgical resection, radiation and chemotherapy, have done little to prolong the lives of GBM patients. Chondroitin sulfate proteoglycans (CSPG) are critical for cell-cell and cell-extracellular matrix (ECM) interactions and are implicated in glioma growth and invasion. Chondroitinase (Chase) ABC is a bacterial enzyme that cleaves chondroitin sulfate disaccharide chains from CSPGs in the tumor ECM. Wild-type Chase ABC has limited stability and/or activity in mammalian cells; therefore, we created a mutant humanized version (Chase M) with enhanced function in mammalian cells. We hypothesized that disruption of cell-cell and cell-ECM interactions by ChaseM and temozolomide (TMZ) will enhance the chemotherapeutic availability and sensitivity of glioma cells. Utilizing primary patient-derived neurospheres, we found that ChaseM decreases glioma neurosphere aggregation in vitro. Furthermore, an oncolytic HSV-1 virus expressing secreted ChaseM (OV-ChaseM) enhanced viral spread and glioma cell killing compared to OV-Control, in vitro. OV-ChaseM plus TMZ combinatorial treatment resulted in a significant synergistic enhancement of glioma cell killing accompanied by an increase in apoptotic cell death. Intracellular flow cytometric analysis revealed a significant reduction in the phosphorylation of the pro-survival AKT protein following OV-ChaseM plus TMZ treatment. Lastly, in nude mice bearing intracranial GBM30 glioma xenografts, intratumoral OV-ChaseM plus TMZ (10mg/kg by oral gavage) combination therapy resulted in a significant (p<0.02) enhancement of survival compared to each individual treatment alone. These data reveal that OV-ChaseM enhances glioma cell viral susceptibility and sensitivity to TMZ.

Engineering precursor metabolite pools for increasing production of antitumor mithramycins in Streptomyces argillaceus

Mithramycin (MTM) is a polyketide antitumor compound produced by Streptomyces argillaceus constituted by a tricyclic aglycone with two aliphatic side chains, a trisaccharide and a disaccharide chain. The biosynthesis of the polyketide aglycone is initiated by the condensation of ten malonyl-CoA units to render a carbon chain that is modified to a tetracyclic intermediate and sequentially glycosylated by five deoxysugars originated from glucose-1-phosphate. Further oxidation and reduction render the final compound. We aimed to increase the precursor supply of malonyl-CoA and/or glucose-1-phosphate in S. argillaceus to enhance MTM production. We have shown that by overexpressing either the S. coelicolor phosphoglucomutase gene pgm or the acetyl-CoA carboxylase ovmGIH genes from the oviedomycin biosynthesis gene cluster in S. argillaceus, we were able to increase the intracellular pool of glucose-1-phosphate and malonyl-CoA, respectively. Moreover, we have cloned the S. argillaceus ADP-glucose pyrophosphorylase gene glgCa and the acyl-CoA:diacylglycerol acyltransferase gene aftAa, and we showed that by inactivating them, an increase of the intracellular concentration of glucose-1-phosphate/glucose-6-phosphate and malonyl-CoA/acetyl-CoA was observed, respectively. Each individual modification resulted in an enhancement of MTM production but the highest production level was obtained by combining all strategies together. In addition, some of these strategies were successfully applied to increase production of four MTM derivatives with improved pharmacological properties: demycarosyl-mithramycin, demycarosyl-3D-β-D-digitoxosyl-mithramycin, mithramycin SK and mithramycin SDK.

Two novel furostanol saponins from the tubers of Ophiopogon japonicus

Phytochemical investigation of the fresh tubers of Ophiopogon japonicus led to the isolation of two new furostanol saponins (1 and 2) together with two known steroidal saponins (3 and 4). Comprehensive spectroscopic analysis allowed the chemical structures of two new compounds to be elucidated as (25R)-26-O-[β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranosyl]-5-ene-furost-1β,3β,22α,26-tetraol-3-O-α-l-rhamnopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 4)]-β-d-glucopyranoside (1, ophiopogonin P) and (25R)-26-O-[β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl]-5-ene-furost-1β,3β,22α,26-tetraol-3-O-α-l-rhamnopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 4)]-β-d-glucopyranoside (2, ophiopogonin Q). Furostanol saponins with the disaccharide chain linked at C-26 hydroxy group of the aglycone have been rarely reported from natural sources.

Sulf1 modulates BMP signaling and is required for somite morphogenesis and development of the horizontal myoseptum

Heparan sulfate proteoglycans (HSPGs) are glycosylated extracellular or membrane-associated proteins. Their unbranched heparan sulfate (HS) disaccharide chains interact with many growth factors and receptors, modifying their activity or diffusion. The pattern of HS sulfation can be altered by the enzymes Sulf1 and Sulf2, secreted extracellular 6-O endosulfatases, which remove specific sulfate groups from HS. Modification by Sulf enzymes changes the binding affinity of HS for protein such as ligands and receptors, affecting growth factor gradients and activities. The precise expression of these sulfatases are thought to be necessary for normal development. We have examined the role of the sulf1 gene in trunk development of zebrafish embryos. sulf1 is expressed in the developing trunk musculature and as well as in midline structures such as the notochord, floorplate and hypochord. Knockdown of sulf1 with antisense morpholinos results in poor differentiation of the somitic trunk muscle, loss of the horizontal myoseptum, lack of pigmentation along the mediolateral stripe, and improper migration of the lateral line primordium. sulf1 knockdown results in a decrease in the number of Pax7-expressing dermomyotome cells, particularly along the midline where the horizontal myoseptum develops. It also leads to decreased sdf1/cxcl12 expression along the mediolateral trunk musculature. Both the Pax7 and cxcl12 expression can be restored by inhibition pharmacological inhibition of BMP signaling, which also restores formation of the myoseptum, fast muscle development, and pigmentation patterning. Lateral line migration and neuromast deposition depend on sdf1/cxcl12 and FGF signaling respectively, both of which are disrupted in sulf1 morphants. Pharmacological activation of FGF signaling can rescue the spacing of neuromast deposition in these fish. Together this data indicate that sulf1 plays a crucial role in modulating both BMP and FGF signaling along the developing myoseptum to coordinate the morphogenesis of trunk musculature, associated pigment cells, and lateral line neuromasts.

New steroidal saponins from the bulbs of Lilium brownii var. viridulum

Phytochemical investigation of the bulbs of Lilium brownii var. viridulum led to the isolation of seven new steroidal saponins (1–7), along with eight known analogues (8–15). The new steroidal saponins were identified as 27-O-[(3S)-3-O-β-d-glucopyranosyl 3-methylglutaroyl]isonarthogenin 3-O-[α-l-rhamnopyranosyl-(1→2)]-β-d-glucopyranoside (1), (24S,25S)-3β,17α,24-trihydroxy-5α-spirostan-6-one 3-O-[α-l-rhamnopyranosyl-(1→2)]-β-d-glucopyranoside (2), tenuifoliol 3-O-[β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside (3), 26-O-β-d-glucopyranosylnuatigenin (4), 26-O-β-d-glucopyranosylnuatigenin 3-O-β-d-glucopyranoside (5), 26-O-β-d-glucopyranosylnuatigenin 3-O-{α-l-rhamnopyranosyl-(1→2)-[β-d-glucopyranosyl-(1→6)]}-β-d-glucopyranoside (6), 26-O-[β-d-glucopyranosyl-(1→2)]-β-d-glucopyranosylnuatigenin 3-O-[α-l-rhamnopyranosyl-(1→2)]-β-d-glucopyranoside (7), using a combination of spectroscopic evidence and chemical methods. The carbohydrate chain of a sugar linked to C-3 of the HMG group (3-hydroxy-3-methylglutarate) in compound 1 is rarely found in nature. Compound 2 possesses a new (24S,25S)-3β,17α,24-trihydroxy-5α-spirostan-6-one aglycon moiety. The disaccharide chain linked to C-26 hydroxy group of the furospirostanol in compound 7 has not been observed from natural sources.

Design, Synthesis and Characterisation of Guanosine-Based Amphiphiles

A small library of sugar-modified guanosine derivatives has been prepared, starting from a common intermediate, fully protected on the nucleobase. Insertion of myristoyl chains and of diverse hydrophilic groups, such as an oligoethylene glycol, an amino acid or a disaccharide chain, connected through in vivo reversible ester linkages, or of a charged functional group provided different examples of amphiphilic guanosine analogues, named G1-G7 herein. All of the sugar-modified derivatives were positive in the potassium picrate test, showing an ability to form G-tetrads. CD spectra demonstrated that, as dilute solutions in CHCl(3), distinctive G-quadruplex systems may be formed, with spatial organisations dependent upon the structural modifications. Two compounds, G1 and G2, proved to be good low-molecular-weight organogelators in polar organic solvents, such as methanol, ethanol and acetonitrile. Ion transportation experiments through phospholipid bilayers were carried out to evaluate their ability to mediate H(+) transportation, with G5 showing the highest activity within the investigated series. Moreover, G3 and G5 exhibited a significant cytotoxic profile against human MCF-7 cancer cells in in vitro bioassays.

Crystal Structures of Two Vancomycin Complexes with Phosphate and N-Acetyl–d-Ala. Structural Comparison between Low-Affinity and High-Affinity Ligand Complexes of Vancomycin

Abstract Crystal structures of two vancomycin complexes with phosphate and N-acetyl–d-Ala (AcDA) were determined. Each complex involves two crystallographically independent vancomycin molecules (V1 and V2) in the asymmetric unit, which form a usually observed back-to-back arranged vancomycin dimer V1–V2 with two disaccharide chains packed in a head-to-head manner, but only one of the two ligand-binding sites is occupied. Comparison of the published crystal structures of low-affinity (small in molecular size) ligand complexes of vancomycin with high-affinity (large) ligand complexes reveals that when the high-affinity ligand binds, three structural factors (hydrogen-bonding interactions between the two peptide-backbones and hydrophobic intra-dimer sugar–ring and ring (face)–ring (edge) interactions) work to enhance the stabilization of the back-to-back dimer-interface, an important factor that is believed to promote antibacterial activity. It has also been revealed, by examining the high-affinity ligand complexes (including N-acetyl–d-Ala–d-Ala), that sugar–ligand interaction could cause different affinities of the two halves of the dimer; this is a factor responsible for the failure of the ligand binding to V1 in the AcDA complex. Possible scenarios for the formation of vancomycin complexes with low-affinity as well as high-affinity ligands are presented.

Epitope Specificities of the Group Y and W-135 Polysaccharides ofNeisseria meningitidis

Previous studies have identified the length dependency of several polysaccharide (PS) protective epitopes. We have investigated whether meningococcal polysaccharides Y and W-135 possess such epitopes. Oligosaccharides (OSs) consisting of one or more disaccharide repeating units (RU) were derived from the capsular PSs of group Y and W-135 meningococci (GYMP and GWMP, respectively) by mild acid hydrolysis. The relative affinities of anticapsular antibodies binding to derivative OSs of different chain lengths were measured in inhibition enzyme-linked immunosorbent assays. As OS size increased from two to three RU, there was a notable increase in binding inhibition of rabbit anti-group Y antiserum. This pattern of antibody binding inhibition was also observed for rabbit antiserum to group W-135, though the inhibition increase was much more pronounced. In the cases of both OS species, the concentration of inhibiting antigen required to achieve 50% inhibition of rabbit immunoglobulin binding increased progressively as the inhibiting disaccharide chain length increased from 1 RU through greater than 50 RU. These data suggest that antibodies directed against both of these meningococcal PSs recognize conformational epitopes only fully expressed in higher-molecular-weight forms of these antigens.

Deoxysugar Transfer during Chromomycin A 3 Biosynthesis in Streptomyces griseus subsp. griseus : New Derivatives with Antitumor Activity

Chromomycin A3 is an antitumor drug produced by Streptomyces griseus subsp. griseus. It consists of a tricyclic aglycone with two aliphatic side chains and two O-glycosidically linked saccharide chains, a disaccharide of 4-O-acetyl-D-oliose (sugar A) and 4-O-methyl-D-oliose (sugar B), and a trisaccharide of D-olivose (sugar C), D-olivose (sugar D), and 4-O-acetyl-L-chromose B (sugar E). The chromomycin gene cluster contains four glycosyltransferase genes (cmmGI, cmmGII, cmmGIII, and cmmGIV), which were independently inactivated through gene replacement, generating mutants C60GI, C10GII, C10GIII, and C10GIV. Mutants C10GIV and C10GIII produced the known compounds premithramycinone and premithramycin A1, respectively, indicating the involvement of CmmGIV and CmmGIII in the sequential transfer of sugars C and D and possibly also of sugar E of the trisaccharide chain, to the 12a position of the tetracyclic intermediate premithramycinone. Mutant C10GII produced two new tetracyclic compounds lacking the disaccharide chain at the 8 position, named prechromomycin A3 and prechromomycin A2. All three compounds accumulated by mutant C60GI were tricyclic and lacked sugar B of the disaccharide chain, and they were named prechromomycin A4, 4A-O-deacetyl-3A-O-acetyl-prechromomycin A4, and 3A-O-acetyl-prechromomycin A4. CmmGII and CmmGI are therefore responsible for the formation of the disaccharide chain by incorporating, in a sequential manner, two D-oliosyl residues to the 8 position of the biosynthetic intermediate prechromomycin A3. A biosynthetic pathway is proposed for the glycosylation events in chromomycin A3 biosynthesis.

Specific Structural Features of Heparan Sulfate Proteoglycans Potentiate Neuregulin-1 Signaling

Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. We examined the structural specificity needed for neuregulin-1-heparin interactions using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 required eight or more heparin disaccharides; however, as few as two disaccharides were still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduced erbB receptor activation by neuregulin-1 but had no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling.