CST-II’s recognition domain for acceptor substrates in α-(2→8)-sialylations

CST-II is a bacterial sialyltransferase known for its ability to perform α-(2→8)-sialylations using GM₃ related trisaccharide substrates. Previously, we probed the enzyme’s substrate specificity and developed an efficient synthesis for α-(2→8)-oligosialosides, and we suggested that CST-II could have a very small substrate recognition domain. Here we report our full studies on CST-II’s recognition feature for acceptor substrates. The current study further demonstrates the versatility of CST-II in preparing complex oligosaccharides that contain α-(2→8)-oligosialyl moieties.     

Residues Met~(76) and Gln~(79) in HLA-G α1 domain involved in KIR2DL4 recognition

INTRODUCTIONThe non-classical HLA class I antigen HLA-G is mainlyexpressed on extravillous cytotrophoblasts that invade de-ciduae in uterine pregnancy. Furthermore, HLA-G canmodulate the function of most immune component cellssuch as NK cells, T cells and…     

Residues Met^76 and Gin^79 in HLA-G α1 domain involved in KIR2DIA recognition

Human leukocyte antigen-G (HLA-G) has long been speculated as a beneficial factor for a successful pregnancy for its restricted expression on fetal-matemal extravillous cytotrophoblasts and its capability of modulating uterine natural killer cell (uNK) function such as cytotoxicity and cytokine production through NK cell receptors. HLA class I α1 domain is an important killer cell Ig-like receptor (KIR) recognition site and the Met^76 and Gln^79 are unique to HLA-Gin this region. NK cell receptor KIR2DL4 is a specific receptor for HLA-G, yet the recognition site on HLA-G remains unknown. In this study, retroviral transduction was applied to express the wild type HLA-G (HLA-wtG), mutant HLA-G(HLA-mG) on the chronic myelogenous leukemia cell line K562 cells and KIR2DL4 molecule on NK-92 cells,respectively. KIR2DL4-IgG Fc fusion protein was generated to determine the binding specificity between KIR2DL4 and HLA-G. Our results showed that residue Met76, Gln79 mutated to Ala^76.79 in the α1 domain of HLA-G protein could affect the binding affinity between KIR2DL4 and HLA-G, meanwhile, the KIR2DL4 transfected NK-92 cells (NK-92-2DL4) showed a considerably different cytolysis ability against the HLA-wtG and HLA-mG transfected K562 targets.Taken together, our data indicated that residue Met^76 and Gin^79 in HLA-G α1 domain plays a critical role in the recognition of KIR2DL4, which could be an explanation for the isoforms of HLA-G, all containing the α1 domain, with the potential to regulate NK functions.     

MMC and LD simulations of α-D-Glcp-(1→2)-α-D-Glcp-(1→3)-α-D-Glcp-OMe. A model for the terminal trisaccharide in glycoprotein precursors

The conformational flexibility and the dynamics of -D-Glcp-(12)--D-Glcp-(13)--D-Glcp-OMe (I) has been investigated by Metropolis-Monte Carlo with the HSEA (Hard Sphere Exo-Anomeric) force field and Langevin dynamics simulations employing two different CHARMm (Chemistry at HARvard Molecular Mechanics) force fields, CHEAT95 and PARM22. The conformational space spanned by the molecule is similar for the two former force fields but differ significantly for the latter. Hydrogen bonding between O2 and O4 of the title compound is analysed in comparison to NMR and preliminary results from X-ray powder diffraction studies. © 1998 Rapid Science Ltd     

Conformational analysis of a Chlamydia-specific disaccharide α-Kdo-(2→8)-α-Kdo-(2→O)-allyl in aqueous solution and bound to a monoclonal antibody: Observation of intermolecular transfer NOEs

The disaccharide -Kdo-(28)--Kdo (Kdo: 3-deoxy-d-manno-oct-2-ulosonic acid) represents a genus-specific epitope of the lipopolysaccharide of the obligate intracellular human pathogen Chlamydia. The conformation of the synthetically derived disaccharide -Kdo-(28)--Kdo-(2O)-allyl was studied in aqueous solution, and complexed to a monoclonal antibody S25-2. Various NMR experiments based on the detection of NOEs (or transfer NOEs) and ROEs (or transfer ROEs) were performed. A major problem was the extensive overlap of almost all ¹H NMR signals of -Kdo-(28)--Kdo-(2O)-allyl. To overcome this difficulty, HMQC-NOESY and HMQC-trNOESY experiments were employed. Spin diffusion effects were identified using trROESY experiments, QUIET-trNOESY experiments and MINSY experiments. It was found that protein protons contribute to the observed spin diffusion effects. At 800 MHz, intermolecular trNOEs were observed between ligand protons and aromatic protons in the antibody binding site. From NMR experiments and Metropolis Monte Carlo simulations, it was concluded that -Kdo-(28)--Kdo-(2O)-allyl in aqueous solution exists as a complex conformational mixture. Upon binding to the monoclonal antibody S25-2, only a limited range of conformations is available to -Kdo-(28)--Kdo-(2O)-allyl. These possible bound conformations were derived from a distance geometry analysis using transfer NOEs as experimental constraints. It is clear that a conformation is selected which lies within a part of the conformational space that is highly populated in solution. This conformational space also includes the conformation found in the crystal structure. Our results provide a basis for modeling studies of the antibody–disaccharide complex.     

Synthesis of the acceptor analog αFuc(1→2)αGal-O(CH2)7 CH3: A probe for the kinetic mechanism of recombinant human blood group B glycosyltransferase

We report the chemical synthesis of Fuc(12)Gal-O(CH₂)₇CH₃ (1) an analog of the natural blood group (O)H disaccharide Fuc(12)Gal-OR. Compound 1 was a good substrate for recombinant blood group B glycosyltransferase (GTB) and was used as a precursor for the enzymatic synthesis of the blood group B analog Gal(3)[Fuc(12)]Gal-O(CH₂)₇CH₃ (2). To probe the mechanism of the GTB reaction, kinetic evaluations were carried out employing compound 1 or the natural acceptor disaccharide Fuc(12)Gal-O(CH₂)₇CH₃ (3) with UDP-Gal and UDP-GalNAc donors. Comparisons of the kinetic constants for alternative donor and acceptor pairs suggest that the GTB mechanism is Theorell-Chance where donor binding precedes acceptor binding. GTB operates with retention of configuration at the anomeric center of the donor. Retaining reactions are thought to occur via a double-displacement mechanism with formation of a glycosyl-enzyme intermediate consistent with the proposed Theorell-Chance mechanism.     

Synthese von O-(3-desoxy-α-d-manno-2-octulopyranosylonsäure)-(2→4)-O-(3-desoxy-α-d-manno-2-octulopyranosylonsäure)- (2→6)-O-(2-amino-2-desoxy-β-d-glucopyranosyl)-(1→6)-2-amino-2-desoxy-d-glucopyranose

Benzyl-3-O-benzyl-2-benzyloxycarbonylamino-6-O-[2-benzyloxycarbonyl-amino-2-deoxy-3,4-O-(tetraisopropyldisiloxane-1,3-diyl)- β-d-glucopyranosyl]-2-deoxy-α-d-glucopyranoside was coupled with methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-α-d-manno-2-octulopyranosyl bromide)onate (13) to yield the α-glycosidically linked trisaccharide. After deacetylation and selective introduction of a second 7′,8′-O-tetraisopropyldisiloxane group, a further glycosidation reaction with 13 led regioselectively to the tetrasaccharide benzyl O-[methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-α-d-manno-2-octulopyranosyl)onate]-(2→4)-O-{methyl [3-deoxy-7,8-O-(tetraisopropyldisiloxane-1,3-diyl)-α-d-manno-2-octulopyranosyl]-onate}-(2→6)-O- [2-benzyloxycarbonylamino-2-deoxy-3,4-O-(tetraisopropyldisiloxane-1,3-diyl)-β-d-glucopyranosyl]- (1→6)-3-O-benzyl-2-benzyloxycarbonyl-amino-2-deoxy-α-d-glucopyranoside. A series of deblocking steps gave O-(3-deoxy-α-d-manno-2-octulopyranosylonic acid)-(2→4)-O-(3-deoxy-α-d-manno-2-octulopyranosylonic acid)- (2→6)-O-(2-amino-2-deoxy-β-d-glucopyranosyl)-(1→6)-2-amino-2-deoxy-d-glucopyranose which was identical with a tetrasaccharide that had been isolated by hydrazinolysis of the lipopolysaccharide from Salmonella minnesota R 595. Hence, synthetic proof is provided for the linkages in this part of the inner core region of lipopolysaccharides.     

Synthesis of Some New α- and β-(l→2) Linked Disaccharides Containing L-Quinovose (6-Deoxy L-Glucose)

Hepta-O-acetyl-2-0-β-l-quinovopyranosyl-α-d-glucose (VI) and hepta-O-acetyl-2-O-α-l-quinovopyranosyl-β-d-gIucose (VIII) were prepared by the coupling of 2,3,4-tri-O-acetyl-α-l-quinovopyranosyl bromide (IV) with l,3,4,6-tetra-O-acetyl-α-D-glucose (V) in the presence of mercuric cyanide and mercuric bromide in absolute acetonitrile.

Similarly, hepta-O-acetyW-O-α-l-quinovopyranosyl-α-d-galactose (X) and hepta-O-acetyl-2-O-β-L-quinovopyranosyl-α-d-galactose (XI) were prepared by the reaction of IV with 1,3,4,6-tetra-O-acetyl-α-d-galactose (IX).

Removal of the protecting groups of VI, VIII, X and XI afforded the corresponding disaccharides. On treatment with hydrogen bromide, VI, VIII, X and XI gave the corresponding acetobromo derivatives.     

Identification,characterization, and developmental expression of a novel α2 → 8-KDN-transferase which terminates elongation of α2 → 8-linked oligo-polysialic acid chain synthesis in trout egg polysialoglycoproteins

A novel glycosyltransferase which catalyses transfer of deaminated neuraminic acid, KDN (2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) from CMP-KDN to the non-reducing termini of oligo-polysialyl chains of polysialoglycoprotein (PSGP), was discovered in the ovary of rainbow trout (Oncorhynchus mykiss). The KDN-transferase activity was optimal at neutral pH, and stimulated 2 to 2.5-fold by 2–5mm Mg²⁺ or Mn²⁺. Expression of KDN-transferase was developmentally regulated in parallel with expression of the 2 8-polysialytransferase, which catalyses synthesis of the oligo-polysialyl chains in PSGP. Incorporation of the KDN residues into the oligo-polysialyl chains prevented their further elongation, resulting in capping of the oligo-polysialyl chains. This is the first example of a glycosyltransferase that catalyses termination of 2 8-polysialylation in glycoproteins.Abbreviations KDN 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid or naturally occurring deaminated neuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ge N-glycolylneuraminic acid - CMP-KDN cytidine 5-(3-deoxy-d-glycero-d-galacto-2-nonulosonic phosphate) or cytidine 5-KDN phosphate - CMP-NeuAc cytidine 5-Neu5Ac phosphate; oligo-polySia, oligo- and/or polysialic acid - PSGP rainbow trout egg polysialoglycoprotein comprising 2 8-linked oligo- polyNeu5Gc - PSGP (low Sia) a precursor of PSGP present at early stages of oogenesis which contains mostly the disialyl group, Sia2 8Sia2 6- - *K-PSGP [¹⁴C]KDN-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]KDN with the immature cortical vesicle fraction P1 containing KDN-transferase - *A-PSGP [¹⁴C]Neu5Ac-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]Neu5Ac with the P1 fraction - A-*K-PSGP andK-*K-PSGP the products obtained after incubating *K-PSGP with P1 fraction and unlabelled CMP-Neu5Ac or CMP-KDN, respectively - *K-PSGP _cho ,A-*K-PSGP _cho , andK-*K-PSGP _cho mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of *K-PSGP,A-*K-PSGP, and K-*K-PSGP, respectively - *A-PSGP _cho a mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of [¹⁴C]Neu5Ac-labelled PSGP - Endo-N endo-N-acylneuraminidase - DP degree of polymerization - GLC gas-liquid chromatography - HPLC high performance liquid chromatography - TLC thin layer chromatography     

Remarkable enantioselective α-amination in 1-phenyl-2-(N-alkylamino)-1-propanol

(1R,2R)-1-Phenyl-1-alkyl/arylamino-2-(N-alkylamino)propane hydrochloride salts have been synthesized with high degree of enantiomeric purity from (1S,2R)-(+)-1-phenyl-2-(N-alkylamino)-1-propanol through the corresponding chloro derivatives. This reaction sequence involves three inversions with overall inversion of configuration at C-1.     

A recombinant α-(2→3)-sialyltransferase with an extremely broad acceptor substrate specificity from Photobacterium sp. JT-ISH-224 can transfer N-acetylneuraminic acid to inositols

We confirmed that a recombinant α-(2→3)-sialyltransferase cloned from Photobacterium sp. JT-ISH-224 recognizes inositols having a structure corresponding to the C-3 and C-4 of a galactopyranoside moiety, such as epi-, 1d-chiro, myo-, and muco-inositol, as acceptor substrates, and that the enzyme can transfer N-acetylneuraminic acid (Neu5Ac) from cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) to them. After purifying the reaction products, the structures were confirmed by use of NMR spectroscopy and mass spectrometry. From these results, it was clearly shown that the α-(2→3)-sialyltransferase from Photobacterium sp. JT-ISH-224 recognizes acceptor substrates through the cis-diol structure corresponding to the 3- and 4-position of the galactopyranoside moiety.     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

A role for Leu247 residue within transmembrane domain 2 in ginsenoside-mediated α7 nicotinic acetylcholine receptor regulation

Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of Panax ginseng, inhibit subsets of nAChR channel currents, but not α7, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of α7 nAChR induces alterations in channel gating properties and converts α7 nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside Rg₃ (Rg₃) activity against the α7 nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to Rg₃. We further characterized Rg₃ regulation of L247T receptors. We found that Rg₃ inhibition of mutant α7 nAChR channel currents was reversible and concentration-dependent. Rg₃ inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between Rg₃ and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in Rg₃ interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that Rg₃ forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas Rg₃ localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for Rg₃ at the channel pore.     

The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca(2+)-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein-protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit.     

Protein recognition of hetero-/homoleptic ruthenium(II) tris(bipyridine)s for α-chymotrypsin and cytochrome c

We examined the relationship between the structures of hetero-/homoleptic ruthenium(II) tris(bipyridine) metal complexes (Ru(II)(bpy)(3)) and their binding properties for α-chymotrypsin (ChT) and cytochrome c (cyt c). Heteroleptic compound 1a binds to both ChT and cyt c in 1:1 ratio, whereas homoleptic 2 forms 1:2 protein complex with ChT but 1:1 complex with cyt c. These results suggest that the structure of the recognition cavity in Ru(II)(bpy)(3) can be designed for shape complementarity to the targeted proteins. In addition, Ru(II)(bpy)(3) complexes were found to be potent inhibitors of cyt c reduction and to permeate A549 cells.     

Lipoarabinomannan biosynthesis in Corynebacterineae: the interplay of two α(1→2)-mannopyranosyltransferases MptC and MptD in mannan branching

Lipomannan (LM) and lipoarabinomannan (LAM) are key Corynebacterineae glycoconjugates that are integral components of the mycobacterial cell wall, and are potent immunomodulators during infection. LAM is a complex heteropolysaccharide synthesized by an array of essential glycosyltransferase family C (GT-C) members, which represent potential drug targets. Herein, we have identified and characterized two open reading frames from Corynebacterium glutamicum that encode for putative GT-Cs. Deletion of NCgl2100 and NCgl2097 in C. glutamicum demonstrated their role in the biosynthesis of the branching α(1→2)-Manp residues found in LM and LAM. In addition, utilizing a chemically defined nonasaccharide acceptor, azidoethyl 6-O-benzyl-α-D-mannopyranosyl-(1→6)-[α-D-mannopyranosyl-(1→6)](7) -D-mannopyranoside, and the glycosyl donor C(50) -polyprenol-phosphate-[(14) C]-mannose with membranes prepared from different C. glutamicum mutant strains, we have shown that both NCgl2100 and NCgl2097 encode for novel α(1→2)-mannopyranosyltransferases, which we have termed MptC and MptD respectively. Complementation studies and in vitro assays also identified Rv2181 as a homologue of Cg-MptC in Mycobacterium tuberculosis. Finally, we investigated the ability of LM and LAM from C. glutamicum, and C. glutamicumΔmptC and C. glutamicumΔmptD mutants, to activate Toll-like receptor 2. Overall, our study enhances our understanding of complex lipoglycan biosynthesis in Corynebacterineae and sheds further light on the structural and functional relationship of these classes of polysaccharides.     

Potential implications of the glycosylation patterns in collagen α1(I) and α2(I) chains for fibril assembly and growth

O-Glycosylation of hydroxylysine (Hyl) in collagen occurs at an early stage of biosynthesis before the triple-helix has formed. This simple post-translational modification (PTM) of lysine by either a galactosyl or glucosylgalactosyl moiety is highly conserved in collagens and depends on the species, type of tissue and the collagen amino acid sequence. The structural/functional reason why only specific lysines are modified is poorly understood, and has led to increased efforts to map the sites of PTMs on collagen sequences from different species and to ascertain their potential role in vivo. To investigate this, we purified collagen type I (Col1) from the skins of four animals, then used mass spectrometry and proteomic techniques to identify lysines that were oxidised, galactosylated, glucosylgalactosylated, or glycated in its mature sequence. We found 18 out of the 38 lysines in collagen type Iα1, (Col1A1) and 7 of the 30 lysines in collagen type Iα2 (Col1A2) were glycosylated. Six of these modifications had not been reported before, and included a lysine involved in crosslinking collagen molecules. A Fourier transform analysis of the positions of the glycosylated hydroxylysines showed they display a regular axial distribution with the same d-period observed in collagen fibrils. The significance of this finding in terms of the assembly of collagen molecules into fibrils and of potential restrictions on the growth of the collagen fibrils is discussed.     

Molecular characterization and identification of surrogate substrates for diacylglycerol lipase α

Diacylglycerol lipase α is the key enzyme in the formation of the most prevalent endocannabinoid, 2-arachidonoylglycerol in the brain. In this study we identified the catalytic triad of diacylglycerol lipase α, consisting of serine 472, aspartate 524 and histidine 650. A truncated version of diacylglycerol lipase α, spanning residues 1-687 retains complete catalytic activity suggesting that the C-terminal domain is not required for catalysis. We also report the discovery and the characterization of fluorogenic and chromogenic substrates for diacylglycerol lipase α. Assays performed with these substrates demonstrate equipotent inhibition of diacylglycerol lipase α by tetrahydrolipastatin and RHC-20867 as compared to reactions performed with the native diacylglycerol substrate. Thus, confirming the utility of assays using these substrates for identification and kinetic characterization of inhibitors from pharmaceutical collections.