Galp Residues Research Articles

Frost Grape Polysaccharide (FGP), an Emulsion-Forming Arabinogalactan Gum from the Stems of Native North American Grape Species Vitis riparia Michx.

A new arabinogalactan is described that is produced in large quantity from the cut stems of the North American grape species Vitis riparia (Frost grape). The sugar composition consists of l-arabinofuranose (l-Araf, 55.2%) and d-galactopyranose (d-Galp 30.1%), with smaller components of d-xylose (11.2%), d-mannose (3.5%), and glucuronic acid (GlcA, ∼2%), the latter linked via a galactosyl residue. Permethylation identified 3-linked Galp residues, some substituted at the 2-position with Galp or Manp, terminal Araf and Xylp, and an internal 3-substituted Araf. NMR (HSQC, TOCSY, HMBC, DOSY) identified βGalp and three αAraf spin systems, in an Araf-α1,3-Araf-α1,2-Araf-α1,2-Galp structural motif. Diffusion-ordered NMR showed that the FGP has a molecular weight of 1-10 MDa. Unlike gum arabic, the FGP does not contain a hydroxyproline-rich protein (HPRP). FGP forms stable gels at >15% w/v and at 1-12% solutions are viscous and are excellent emulsifiers of flavoring oils (grapefruit, clove, and lemongrass), giving stable emulsions for ≥72 h. Lower concentrations (0.1% w/v) were less viscous, yet still gave stable grapefruit oil/water emulsions. Hence, FGP is a β1,3-linked arabinogalactan with potential as a gum arabic replacement in the food and beverage industries.

Chemical structure of the arabinogalactan protein from gum ghatti and its interaction with bovine serum albumin.

Exudate gums, because of their beneficial properties, have been significant items of international trade in various industries for centuries. This manuscript sets out to gain insight into the fine structural details of an arabinogalactan protein (AGP) of gum ghatti (Anogeissus latifolia gum). The presence of a highly branched 554 kDa AGP having 1,6-linked Galp, 1,2-linked Manp, 1,3-linked Araf and 1,4-linked GlcpA main chain, substituted at O-4,6 of 1,2-linked Manp, and O-3/O-3,4 of 1,6-linked Galp residues by Araf, Arap and Galp units was revealed by chemical, chromatographic, ESMS, and NMR analyses. In particular, ESMS analysis of per acetylated oligomeric fragments derived from AGP by Smith degradation followed by acetylation was described as a commanding tool for providing critical structural information on a spectrum of glycerol tagged oligosaccharides. In addition, formation of an electrostatically driven complex between the isolated AGP and bovine serum albumin resulting in changes in the microenvironment around the tryptophan residues of BSA was established. A moderate radical scavenging activity comparable with those of standard antioxidants was observed from the AGP fraction (∼94% at 1 mg/mL) that could be valuable in foods or pharmaceutical products as alternatives to synthetic antioxidants.

Structural studies of arabinan-rich pectic polysaccharides from Abies sibirica L. Biological activity of pectins of A. sibirica

Highly branched arabinan-rich pectic polysaccharides, containing 84% of arabinose, was extracted from wood greenery of Abies sibirica L. The structure of arabinan was studied by the 1D and 2D NMR spectroscopy. The macromolecule backbone was represented mainly by RG-I (molar ratio GalA:Rha∼1.3:1) patterns with high degree of rhamnose branching. Side chains were comprised of 1,5-linked α-l-Araf residues (the major part of polymer mass), 1,3,5-di-O- and 1,2,3,5-tri-O-linked α-l-Araf residues, confirming the presence of highly branched 1,5-α-l-arabinan. Although most l-Araf were in α-anomeric form, minor terminal β-l-Araf-(1→… was detected. 1,4-β-d-linked Galp residues found in the side chains account for minor AG-I or 1,4-galactan, as compared to arabinan. A tentative structure was proposed.Polysaccharides obtained from Siberian fir greenery were screened for biological activity. Galacturonan had a strongest stimulating effect on germination and growth rate of seeds, germs and roots of Triticum aestivum, Avena sativa, and Secale cereale.

An arabinogalactan from flowers of Chrysanthemum morifolium: structural and bioactivity studies

A water-soluble polysaccharide (P2) with a molecular weight of 1.7×105Da was isolated from the hot aqueous extract of flowers of the Chrysanthemum morifolium. It was homogeneous for there was only a symmetrical peak on the spectrum of high-performance gel permeation chromatography. Monosaccharide analysis revealed that P2 is an arabinogalactan containing arabinose (38.4% w/w), galactose (58.8% w/w), and glucose (2.8% w/w) in a ratio of 1:1.53:0.07. The GC–MS results of the derived alditol acetates from the permethylated P2 showed some separate peaks corresponding to (1→6)-linked Gal, (1→3,6)-linked Gal, terminal Ara, (1→5)-linked Ara, and (1→3,5)-linked Ara units in a molar ratio of nearly 1.12:1.90:1.84:0.35:0.12. Terminal Glc unit also existed in a trace amount. 1H, 13C, DEPT-135 and 2D NMR (including HMQC and HMBC), as well as graded acid hydrolysis experiment, indicated that the core structure features include a backbone chain consisting of (1→6)-linked and (1→3,6)-linked Galp residues. Side chains which were composed of terminal Glcp, terminal, (1→5)-linked and (1→3,5)-linked Araf residues were located at position 3. Additionally, P2 showed good immunological activity as it significantly enhanced splenocyte proliferation in relatively low 25 and 50μg/mL doses. However, P2 showed little effects of thymocyte proliferation, indicating that it may have two modes of action.

Structural Studies of the O‐Acetyl‐Containing O‐Antigen from a Shigella flexneri Serotype 6 Strain and Synthesis of Oligo­saccharide Fragments Thereof

AbstractExtensive analysis by NMR spectroscopy of the delipidated lipopolysaccharide of Shigella flexneri serotype 6 strain MDC 2924‐71 confirmed the most recently reported structure of the O‐antigen repeating unit as {→4)‐β‐D‐GalpA‐(1→3)‐β‐D‐GalpNAc‐(1→2)‐α‐L‐Rhap3Ac/4Ac‐(1→2)‐α‐L‐Rhap‐(1→}, and revealed the non‐stoichiometric acetylation at O‐3C/4C. Input from the CASPER program helped to ascertain the fine distribution of the three possible patterns of O‐acetylation. The non‐O‐acetylated repeating unit (ABCD) corresponded to about 2/3 of the population, while 1/4 was acetylated at O‐3C (3AcCDAB), and 1/10 at O‐4C (4AcCDAB). Di‐ to tetrasaccharides with a GalpA residue (A) at their reducing end were synthesized as their propyl glycosides following a multistep linear strategy relying on late‐stage acetylation at O‐3C. Thus, the 3C‐O‐acetylated and non‐O‐acetylated targets were synthesized from common protected intermediates. Rhamnosylation was most efficiently achieved by using imidate donors, including at O‐4 of a benzyl galacturonate acceptor. In contrast, a thiophenyl 2‐deoxy‐2‐trichloroacetamido‐D‐galactopyranoside precursor was preferred for chain elongation involving residue B. Final Pd/C‐mediated deprotection ensured O‐acetyl stability. All of the target molecules represent parts of the O‐antigen of S. flexneri 6, a prevalent serotype. Non‐O‐acetylated oligosaccharides are also fragments of the Escherichia coli O147 O‐antigen.

Structural elucidation of an immunoenhancing heteroglycan isolated from Russula albonigra (Krombh.) Fr.

A water soluble heteroglycan (PS-II) of average molecular weight ∼1.45×105Da was isolated from the aqueous extract of an ectomycorrhizal edible mushroom, Russula albonigra (Krombh.) Fr. Structural characterization of PS-II was carried out using acid hydrolysis, methylation analysis, periodate oxidation, and 1D/2D NMR studies. Structural analysis revealed that PS-II was composed of terminal 2-O-methyl-Fucp, terminal Manp, (1→2)-Fucp, (1→3)-Glcp, (1→3,4)-Glcp, (1→6)-Galp, and (1→2,6)-Galp residues in a relative proportion of approximately 1:1:1:1:1:1:1. The proposed repeating unit of the PS-II had a backbone consisting of two (1→3)-β-d-glucopyranosyl, two (1→6)-α-d-galactopyranosyl, and one (1→2)-α-l-fucopyranosyl residues, out of which one (1→3)-β-d-glucopyranosyl residue was branched at O-4 position with terminal 2-O-methyl-α-l-fucopyranosyl and one (1→6)-α-d-galactopyranosyl residue was branched at O-2 position with terminal α-d-mannopyranosyl residue. This PS-II showed in vitro macrophage activation by NO production as well as splenocytes and thymocytes proliferation.

Addition of α-O-GlcNAc to threonine residues define the post-translational modification of mucin-like molecules in Trypanosoma cruzi.

Trypanosoma cruzi, an intracellular protozoan etiologic agent of Chagas disease is covered by a dense coat of mucin-type glycoproteins, which is important to promote the parasite entry and persistence in the mammalian host cells. The O-glycosylation of T. cruzi mucins (Tc-mucins) is initiated by enzymatic addition of α-O-N-acetylglucosamine (GlcNAc) to threonine (Thr) by the UDP-GlcNAc:polypeptide α-N-acetylglucosaminyltransferase (pp-α-GlcNAcT) in the Golgi. The Tc-mucin is characterized by the presence of a high structural diversity of O-linked oligosaccharides found among different parasite strains, comprising two O-glycan Cores. In the Core 1, from strains principally associated with the domestic transmission cycle of Chagas disease, the GlcNAc O-4 is substituted with a β-galactopyranose (βGalp) unit, and in the most complex oligosaccharides the GlcNAc O-6 is further processed by the addition of β1 → 2-linked Galp residues creating a short linear Galp-containing chain. In the Core 2 structures, expressed by strains isolated from T. cruzi sylvatic hosts, the GlcNAc O-4 carries a β-galactofuranose (βGalf) unit and the GlcNAc O-6 can carry a branched Galpβ1 → 3[Galpβ1 → 2]Galpβ1 → 6 motif. The O-glycans carrying nonreducing terminal βGalp are available for sialylation by a surface T. cruzi trans-sialidase activity. Based on structural results, this review summarizes available data on the highly conserved process, which adds the GlcNAc unit in α-linkage to Thr residues the basis of the post-translational modification system in T. cruzi mucins. In addition, a mechanism unique employed by the parasite to transfer exogenous sialic acid residues to Tc-mucins is presented.

Structural analysis of a pectic polysaccharide from boat-fruited sterculia seeds

The chemical structure of a water-soluble pectic polysaccharide (sterculia APS) isolated from boat-fruited sterculia seeds (Semen Sterculiae Lychnophorae) was elucidated using partial acid hydrolysis, methylation analysis and 1D/2D nuclear magnetic resonance (NMR) spectroscopy. The results of partial acid hydrolysis revealed that sterculia APS contained galacturonic acids and rhamnose units in the backbone, and arabinose, galactose and xylose residues in the branched chains. Combined the methylation analysis results with NMR spectroscopy, a possible structure of APS was proposed as follow: the backbone consisted of the homogalacturonan (HG) named “smooth” regions by linear linkage of 1,4-α-d-GalpA, and the rhamnogalacturonan-I (RG-I) called “hairy” ones by the linkage of →4)-α-d-GalpA-(1→2)-α-l-Rhap-(1→partially substituted at O-4 of rhamnose units. The GalpA residues were partially methyl esterified and O-acetylated on C-2 and/or C-3. The side chains were mainly composed of araban and arabinogalactan by the linkages of T-, 1,3-, 1,5-l-Araf and T-, 1,4-, 1,6-, 1,3,6-, 1,3,4-d-Galp, attached to O-4 of the backbone rhamnose units.

Structural characterization of a homogalacturonan from Capparis spinosa L. fruits and anti-complement activity of its sulfated derivative

A water-soluble polysaccharide CSPS-2B-2 with a molecular mass of 8.8 kDa, was obtained from the fruits of Capparis spinosa L. Chemical and NMR spectral analysis verified CSPS-2B-2 was a linear poly-(1-4)-α-D-galactopyranosyluronic acid in which 12.9±0.4% of carboxyl groups existed as methyl ester and 2.6±0.1% of D-GalpA residues were acetylated. A sulfated derivative Sul-2B-2 with a sulfation degree of 0.88±0.02 was prepared via the substitution of C-2 and/or C-3 of GalpA residues in CSPS-2B-2. Bioassay on the complement and coagulation system demonstrated that Sul-2B-2 (CH(50): 3.5±0.2 μg/mL) had a stronger inhibitory effect on the activation of complement system through the classic pathway than that of heparin (CH(50): 8.9±0.3 μg/mL). Interestingly, Sul-2B-2 at low dose even middle dose (for example 52 μg/mL) had no effect on coagulation system, which was totally different from heparin. Thus, our observation indicated that Sul-2B-2 was more efficient than heparin in inhibiting the activation of the complement system through classical pathway and exhibiting a relatively less anti-coagulant activity. These results suggested that the sulfated derivative Sul-2B-2 prepared from the homogalacturonan in the fruits of Capparis spinosa L, might be a promising drug candidate in case of necessary therapeutic complement inhibition.

Structural Features Affecting Trafficking, Processing, and Secretion of Trypanosoma cruzi Mucins

Trypanosoma cruzi is wrapped by a dense coat of mucin-type molecules encoded by complex gene families termed TcSMUG and TcMUC, which are expressed in the insect- and mammal-dwelling forms of the parasite, respectively. Here, we dissect the contribution of distinct post-translational modifications on the trafficking of these glycoconjugates. In vivo tracing and characterization of tagged-variants expressed by transfected epimastigotes indicate that although the N-terminal signal peptide is responsible for targeting TcSMUG products to the endoplasmic reticulum (ER), the glycosyl phosphatidylinositol (GPI)-anchor likely functions as a forward transport signal for their timely progression along the secretory pathway. GPI-minus variants accumulate in the ER, with only a minor fraction being ultimately released to the medium as anchorless products. Secreted products, but not ER-accumulated ones, display several diagnostic features of mature mucin-type molecules including extensive O-type glycosylation, Galf-based epitopes recognized by monoclonal antibodies, and terminal Galp residues that become readily sialylated upon addition of parasite trans-sialidases. Processing of N-glycosylation site(s) is dispensable for the overall TcSMUG mucin-type maturation and secretion. Despite undergoing different O-glycosylation elaboration, TcMUC reporters yielded quite similar results, thus indicating that (i) molecular trafficking signals are structurally and functionally conserved between mucin families, and (ii) TcMUC and TcSMUG products are recognized and processed by a distinct repertoire of stage-specific glycosyltransferases. Thus, using the fidelity of a homologous expression system, we have defined some biosynthetic aspects of T. cruzi mucins, key molecules involved in parasite protection and virulence.

Demonstration of the presence of acetylation and arabinose branching as structural features of locust bean gum galactomannans

Galactomannans from locust bean gum (LBG) are polysaccharides containing single Galp residues as side chains in a β-(1→4)-Manp backbone. Nevertheless, sugars other than mannose and galactose are always detected, namely, arabinose and glucose, although in small amounts. In order to demonstrate that LBG galactomannans contain other structural features than those usually reported in literature, the galactomannans from LBG obtained from two different commercial sources were submitted to a selective degradation by Aspergillus niger endo-β-d-mannanase and the oligosaccharides (OS) obtained were fractioned by size exclusion chromatography using a Biogel-P2 (100–1800Da). A. niger endo-β-d-mannanase is able to hydrolyse the β-(1→4)-linked mannan backbone when the mannose residues are not hindered by the presence of substituents. This enables collection of fragments of the substituted mannan polysaccharide in the different chromatographic fractions that can be analysed by Electrospray Tandem Mass Spectrometry (ESI-MSn). Both samples showed the presence of hexose OS (Hex3–6), acetylated hexoses (AcHex3–6), hexoses substituted with a pentose unit (PentHex3–6), and OS composed by acetylated hexoses substituted with a pentose unit (PentAcHex3–6). These structures were confirmed by GC–qMS of reduced and permethylated oligosaccharides. Sugar and glycosidic-linkage analysis allowed to assign the pentose unit to a terminally-linked arabinofuranose residue at the O-6 of a (1→4)-Manp residue. The presence of arabinose substituted OS and acetylated mannoses have never been reported to occur in LBG.

Characterization of an arabinogalactan-protein from suspension culture of Echinacea purpurea

Suspension cultures of Echinacea purpurea have been established in MS medium supplemented with 2,4-D and an arabinogalactan-protein (AGP) was purified from the secreted soluble polymers by precipitation with ethanol, followed by precipitation with β-glucosyl Yariv reagent. It revealed typical features of AGPs: a high amount of polysaccharide (90% w/w) with the dominating monosaccharides galactose and arabinose and some glucuronic acid, and a small protein moiety (10% w/w) with the main amino acids Ala, Hyp, Glx, Ser, Asx and Thr. Linkage- and NMR-analyses showed the polysaccharide part to be composed of a branched core-polysaccharide of 3-, 6- and 3,6-linked Galp residues with terminal Araf, Arap, Galp and GlcAp residues. Compared to an AGP from pressed juice of the aerial parts of Echinacea purpurea, differences particularly in terminal arabinose mono- and oligosaccharides in arabinogalactan (AG) side branches could be detected. Testing of different AGP-antibodies with both AGPs confirmed the results of the analytical investigations. Binding of AGPs from plant and cell cultures to LM2, a monoclonal AGP-antibody reacting with a GlcA containing epitope, was comparable. The reactivity of a monoclonal antibody raised against the AGP from the plant recognizing a galactan epitope was also nearly similar with both AGPs. In contrast, polyclonal antibodies raised against the AGP from the plant and directed against an Araf-containing epitope of the AG side branches showed nearly no cross reactivity with the AGP from cell culture.

Structural features of an immunostimulating and antioxidant acidic polysaccharide from the seeds of Cuscuta chinensis.

Three crude polysaccharide fractions, named CS-A, CS-B and CS-C, were prepared from the seeds of Cuscuta chinensis by hot water extraction and diluted alkali extraction subsequently, then EtOH precipitation, and tested for lymphocyte proliferation activity. CS-A showed a stimulating effect on concanavalin A or lipopolysaccharide induced mitogenic activity of lymphocytes. An acidic polysaccharide (CS-A-3beta) was purified from CS-A by anion exchange and gel filtration chromatography. The polysaccharide showed potent stimulating effects on lymphocyte proliferation and antibody production, but did not significantly influence the serum IgG level. Its structural features were elucidated by methylation analysis, partial acid hydrolysis, 1D and 2D NMR and ESI-mass spectroscopy. The data obtained indicated that CS-A-3beta had a backbone consisting of alpha-D-1,4-linked GalpA residues and beta-L-1,2-linked Rhap residues with branches at C-4 of Rhap residues and C-3 of GalpA residues, composed of arabinogalactan and glucobiose. This structure is typical for a pectic polysaccharide of the rhamnogalacturonan type. In addition, the effect of CS-A, CS-B, CS-C and CS-A-3beta on hydrogen peroxide induced cell lesion in rat pheochromocytoma line PC 12 was investigated. The results indicated that, besides its immunostimulating activity, CS-A-3beta had a protective effect against free radical-induced cell toxicity.

An arabinogalactan-protein from cell culture of Malva sylvestris.

An arabinogalactan-protein (AGP) from suspension culture medium of Malva sylvestris was isolated by precipitation with beta-glucosyl Yariv reagent, followed by gel-permeation chromatography. It revealed characteristic features of AGPs: a high amount of polysaccharide with a ratio of galactose to arabinose of 1.9 : 1, some uronic acids, and a small protein moiety with the main amino acids serine, alanine and hydroxyproline. The molecular weight was estimated to be 1.3 x 10(6) Da. Linkage analyses showed that the AGP is composed of a highly branched core polysaccharide of 3-, 6-, and 3,6-linked Galp residues with terminal Araf, GlcAp and Galp. Partial acid hydrolysis resulted in loss of Araf residues at the periphery of the molecule and heavily reduced its reactivity with beta-glucosyl Yariv antigen.

CHEMISTRY, PROPERTIES, AND PHYLOGENETIC IMPLICATIONS OF THE METHYLATED CARRAGEENANS FROM RED ALGAE OF THE GENUS ARESCHOUGIA (ARESCHOUGIACEAE, GIGARTINALES, RHODOPHYTA)

The three Australian‐endemic species comprising the genus Areschougia have been examined to determine the structure of their nonfibrillar wall components. The polysaccharide extracted from the most widely distributed species, A. congesta (Turner) J. Agardh, was shown by compositional analyses, Fourier transform infrared (FTIR) spectroscopy, linkage analysis, and 13C‐NMR spectroscopy to be a carrageenan composed predominantly of the repeating disaccharides 6′‐O‐meth‐ ylcarrabiose 2,4′‐disulfate, carrabiose 2,4′‐disulfate (the repeating unit of ι‐carrageenan), 4′,6′‐O‐(1‐carboxyethylidene)carrabiose 2‐sulfate, and 6′‐O‐methylcarrabiose 2‐sulfate. The carrageenan also contained small amounts of 4‐linked Galp residues, some bearing methyl ether substitution at O‐3 and some possibly bearing sulfate ester and/or glycosyl substitutions at O‐3. The A. congesta carrageenan had unique rheological properties, its gels having some similarities to those of commercial ι‐carrageenan but with the viscosity of commercial λ‐carrageenan. Polysaccharides from A. ligulata Harvey ex J. Agardh and A. stuartii Harvey were shown by constituent sugar and FTIR analyses to be sulfated galactans rich in mono‐O‐methylgalactose. The carrageenan structures of Areschougia spp. were consistent with those of the genera Rhabdonia, Erythroclonium, and Austroclonium, the other genera constituting the family Areschougiaceae.

Complete NMR Structural Elucidation of the Capsular Polysaccharide Adjuvant from Klebsiella I‐714

The capsular polysaccharide (CPS) produced by the non-pathogenic Klebsiella strain I-714, selected for its immunomodulating activity, has been purified and almost completely detoxified in a previous study [Adam, O., Vercellone, A., Paul, F., Monsan, P. F. & Puzo, G. (1995) Anal. Biochem. 225, 321-327]. The present report concerns the structural elucidation of this CPS by several one-dimensional and two-dimensional 1H-NMR and 13C-NMR experiments performed on the native molecule. It was found to be a high molecular-mass branched polymer constituted by a hexasaccharide repeating unit of following structure: 4)-alpha-L-Rhap-(1-->3)-beta-D-Galp-(1-->2)-alpha-L-Rhap-(1- ->4)-beta-D- GlcpA-(1-->3)-¿alpha-L-Rhap-(1-->2)-¿-alpha-D-Galp-(1. The presence of two glycosidic substitutions on the Galp residue resulted in a strong overlapping of its proton signals, preventing direct assignment of both proton and carbon resonances. However, assignment could be achieved using the two-bond and three-bond 1H-13C heteronuclear coupling observed in the 1H-13C heteronuclear multiple-bond correlation (HMBC) spectrum.

Biosynthesis of lipophosphoglycan from Leishmania major: solubilization and characterization of a (beta 1-3)-galactosyltransferase.

Lipophosphoglycan (LPG), is the major cell surface molecule of promastigotes of all Leishmania species. It is comprised of three domains: a conserved glycosylphosphatidylinositol anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6Gal beta 1-4Man alpha 1-) backbone and capped with a neutral oligosaccharide. In Leishmania major the backbone is substituted at the C(O)3 of the Galp residue with side chains containing Galp, Glcp and Arap residues whereas in Leishmania donovani the backbone is unsubstituted. We report the solubilization of a (beta 1-3) galactosyltransferase [(beta 1-3)GalT] from a L. major microsomal preparation using Triton X-100. Solubilization occurs with a 10-fold stimulation of enzyme activity. This (beta 1-3)GalT specifically transfers Gal residues from UDP-Gal to exogenously added L. donovani LPG acceptor. Depolymerization of the [14C]Gal-labelled LPG product with mild acid and analysis by high-performance anion-exchange chromatography detected only the phosphotrisaccharide. (P3; PO4-6([14C]Gal beta 1-3-4Man alpha 1-) found in L. major LPG. This contrasts with the activity of the membrane-bound enzyme which also synthesizes the larger phosphosaccharide units[Ng, Handman and Bacic (1994) Glycobiology 4, 845-853]. This suggests that more than one (beta 1-3)GalT is involved in the addition of these Gal units and that the solubilized activity is the (beta 1-3)GalT that adds the first beta Gal residue to the acceptor. The (beta 1-3)GalT was partially purified by lectin-affinity chromatography and used to establish the K(m) values for UDP-Gal (445 microM) and L. donovani acceptor (280 microM as P2 molar equivalent) in kinetic assays. Inhibition studies with various glycosides and mono- and di-saccharides established the P2 repeating unit as the minimum acceptor structure recognized by (beta 1-3)GalT. The detergent-solubilized (beta 1-3)GalT was reversibly inactivated by millimolar concentrations of univalent anionic salts. The (beta 1-3)GalT had an absolute requirement for Mn2+ and also required Mg2+ for optimum activity; Mg2+ cannot substitute for Mn2+, which is loosely bound to beta (1-3)GalT and is probably involved in the correct folding of the enzyme. The (beta 1-3)GalT was unaffected by Ca2+ ions, but were irreversibly inactivated by micromolar levels of transition metal ions (Cu2+ > Zn2+ > Ni2 > Co2+). The (beta 1-3)GalT activity was also inhibited by diethyl pyrocarbonate, but not by N-ethylmaleimide or iodoacetamide, suggesting that active-site histidine residues, rather than cysteine residue(s), are important for enzyme activity.

Structures of the O-antigens of Klebsiella serotypes 02 (2a,2e), 02 (2a,2e,2h), and 02 (2a,2f,2g), members of a family of related D-galactan O-antigens in Klebsiella spp

The structures of the lipopolysaccharide O-antigens from Klebsiella serotypes 02(2a,2f,2g), O2(2a,2e) and O2(2a,2e,2h) have been determined. These O-polysaccharides are part of a family of related structures, which share a D-galactan I backbone. D-galactan I has the repeating unit structure: [→3)-β-D-Gal f-(1→3)-α-D-Gal p-(1→]. The O-polysaccharide of serotype O2(2a,2f,2g) differs from other known O-polysaccharides in Klebsiella spp. Each of the main-chain Gal p residues in the 02(2a,2f,2g) O-polysaccharide is substituted with an α-(1→4)-linked D-Gal p residue, to form a trisaccharide repeating unit. The LPS O-polysaccharides of serotypes O2(2a,2e) and O2(2a,2e,2h) both contain α-(1→2)-linked D-Galp substituents on the main-chain Galp residues, and resemble serotype O9. The only difference between the 09, O2(2a,2e), and O2(2a,2e,2h) carbohydrate structures involves the stoichiometry of addition of side chain α-D-Gal p residues. However, the polymers from serotypes O2(2a,2e), O2(2a,2e,2h) and 09 are all modified by O-acetylation and these modifications may contribute to altered antigenic factors. The structures reported here resolve ambiguities between previous chemical and serological analyses of 02 antigens. Genetic analyses showed that enzymes involved in the addition of α-D-Gal p residues are encoded by genes outside the rfbO9 (O-antigen biosynthesis) region and this provides an explanation for occasional non-stoichiometric addition of side chain α-D-Galp residues.