A biochemical chimera suggesting the existence of at least two dolichol-P-glucose:dolichol-P-P-oligosaccharide glucosyltransferases

As previously reported, incubation of liver dolichol-P, UDP-[14C]Gal, and a particulate preparation of Acetobacter xylinum leads to the synthesis of dolichol-P-[14C]Gal (P. Romero, R. Garcia, and M. Dankert (1977) Mol. Cell. Biochem. 16, 205-212). It is now reported that upon incubation of the latter with rat liver microsomes, [14C-galactose]-Gal1Man9GlcNAc2-P-P-dolichol and [14C-galactose]Gal1Glc1Man9GlcNAc2-P-P-dolichol are formed. The galactosyl residues appeared to be (1,3)-linked in the same positions as the glucose units in the respective physiological compounds. No lipid-linked Gal1Glc2Man9GlcNAc2 was formed, thus strongly suggesting the presence of at least two dolichol-P-Glc:dolichol-P-P-oligosaccharide glucosyltransferases, only one of which is able to use dolichol-P-Gal as substrate. Incubation of the galactosylated dolichol-P-P derivatives with rat liver microsomes led to the transfer of the oligosaccharides to microsomal proteins. No endogenous, membrane-bound glycosidases were able to remove the galactose residues but mannose units were excised by endogenous neutral mannosidases.     

Structural characterization and in vitro antitumor activity of a novel polysaccharide isolated from the fruiting bodies of Pleurotus ostreatus

A novel water-soluble polysaccharide (POPS-1) was obtained from the fruiting bodies of Pleurotus ostreatus by hot water extraction, ethanol precipitation, and fractionated by DEAE-cellulose ion exchange chromatography and sepharose CL-6B gel filtration chromatography using an ATKA explore 100 purifier. The structure characterization and antitumor activity of the POPS-1 were evaluated in this paper. According to GC analysis, HPGPC, FT-IR, partial acid hydrolysis, periodate oxidation and Smith degradation, methylation and GC-MS analysis, the results indicate POPS-1 (M(w)=31 kDa) was composed of Man; Gal; Glc with a molar ratio of 1:2.1:7.9, it had a backbone of beta-(1-->3)-linked glucose residues, which occasionally branches at O-6. The branches were composed of (1-->3)-linked Glc, (1-->4)-linked Gal, (1-->4)-linked Man, and terminated with Glc and Gal residues. Cytotoxicity assay showed POPS-1 presented significantly higher antitumor activity against Hela tumor cell in vitro, in a dose-dependent manner, and exhibited significantly lower cytotoxicity to human embryo kidney 293T cells than Hela tumor cells compared with 5-Fu. The results suggest POPS-1 may be considered as a potential candidate for developing a novel low toxicity antitumor agent.     

Active-site mapping of a Populus xyloglucan endo-transglycosylase with a library of xylogluco-oligosaccharides

Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and +1). Donor binding is dominated by the higher affinity of the XXXG moiety (G=Glcbeta(1-->4) and X=Xylalpha(1-->6)Glcbeta(1-->4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K(m) values for the donor substrates showed that a GG-unit on negative and -XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal=Galbeta(1-->4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.     

Isolation and characterization of an endo-beta-galactosidase from Bacteroides fragilis.

Six strains of Bacteroides fragilis were examined and all found to produce endo-beta-galactosidase, an enzyme that hydrolyses internal beta-galactosidic linkages of oligosaccharides belonging to the poly-N-acetyl-lactosamine series, with the common structure GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc/Glc. The enzyme was produced without the addition of an inducer such as keratan sulphate. It was purified 7000-fold from the culture supernatant and obtained with a yield 4-10-fold greater than from sources described previously. The specificity of the enzyme towards bovine corneal keratan sulphate, milk oligosaccharides and the glycolipids lacto-N-neotetraosylceramide and lacto-N-tetraosylceramide closely resembled that of the endo-beta-galactosidase isolated from Escherichia freundii. A novel observation was that both enzymes hydrolysed the type 2 sequence, Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc, at about twice the rate of the type 1 isomer, Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc. Because of the ease of purification of the enzyme and high yield in the absence of contaminating glycosidases and proteinases, Bacteroides fragilis is a valuable source of endo-beta-galactosidase for the structural analysis of carbohydrate chains.     

A small region of the natural killer cell receptor, Siglec-7, is responsible for its preferred binding to alpha 2,8-disialyl and branched alpha 2,6-sialyl residues. A comparison with Siglec-9.

Siglec-7 is a sialic acid-binding lectin recently identified as an inhibitory receptor on natural killer cells. Here we characterize the sugar-binding specificity of Siglec-7 expressed on Chinese hamster ovary cells using polyvalent streptavidin-based glyco-probes. Glyco-probes carrying unique oligosaccharide structures such as GD3 (NeuAc alpha 2,8NeuAc alpha 2,3Gal beta 1,4Glc) and LSTb (Gal beta 1,3[NeuAc alpha 2,6]GlcNAc beta 1,3Gal beta 1,4Glc) oligosaccharides bound to Siglec-7 better than those carrying LSTc (NeuAc alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal beta 1,4Glc) or GD1a (NeuAc alpha 2,3Gal beta 1,3GalNAc beta 1,4[NeuAc alpha 2,3]Gal beta 1,4Glc) oligosaccharides. In contrast, Siglec-9, which is 84% identical to Siglec-7, did not bind to the GD3 and LSTb probes but did bind to the LSTc and GD1a probes. To identify a region(s) responsible for their difference in binding specificity, we prepared a series of V-set domain chimeras between Siglecs-7 and -9. Substitution of a small region, Asn(70)-Lys(75), of Siglec-7 with the equivalent region of Siglec-9 resulted in loss of Siglec-7-like binding specificity and acquisition of Siglec-9-like binding properties. In comparison, a Siglec-9-based chimera, which contains Asn(70)-Lys(75) with additional amino acids derived from Siglec-7, exhibited Siglec-7-like specificity. These results, combined with molecular modeling, suggest that the C-C' loop in the sugar-binding domain plays a major role in determining the binding specificities of Siglecs-7 and -9.     

Studies of the binding activity of phage G13 to synthetic trisaccharides analogous to binding structures in Salmonella typhimurium and Escherichia coli C core saccharide. Correlation between conformation and binding activity.

Phage G13 binds to the carbohydrate part of lipopolysaccharides from rough mutants of Salmonella and Escherichia coli as the first event of infection. Equilibrium dialysis inhibition studies with native and synthetic trisaccharides as inhibitors suggested that phage G13 recognizes branched oligosaccharides having 6-O-alpha- or 7-O-alpha-glycosyl groups with alpha-Man(1----3) [alpha-Man(1----6)]Man (Man[Man]Man) and alpha-Glc(1----3)-[alpha-Hep(1----7)] alpha-Hep(1----3) alpha-Hep(1----5)Kdo as the smallest saccharides with inhibitory activity (Wollin et al., 1989). Of four synthetic analogues to Man[Man]Man only Man(1----3)[alpha-Gal(1----6)]alpha-Man-OMe (Man[Gal]-Man) and alpha-Glc(1----3)[alpha-Hep(1----7)]alpha-Hep-OMe (Glc[Hep]Hep) inhibited the binding of labelled E. coli C core nonasaccharide ligand to G13 with activities which were 10- and 15-fold lower than Man[Man]Man. The trisaccharides alpha-Man(1----3)[alpha-Glc(1----6)[alpha-Man-OMe (Man[Glc]Mann) and alpha-Man(1---3)[alpha-Tal(1----6)]alpha-Man-OMe (Man[Tal]Man) showed no inhibition at concentrations 75-fold higher than Man[Man]Man. Minimum energy conformation calculations of the saccharides using the GESA method showed that the 6-O-alpha-Man group in Man[Man]Man and the 7-O-alpha-Hep group SL805 pentasaccharide expose their OH-2 and OH-3 groups in a similar way and these are postulated to be key structural features for binding activity. The importance of hydroxy groups at certain positions is implied from the fact that both manno- and galacto-isomers are active. We also conclude that the O6-C6-C5-O5-C1 region of the 3-O-alpha-glycosyl group in the Man[Man]Man trisaccharide, or part of it, is important for the G13 binding activity.     

Chemical characterisation of the oligosaccharides in a sample of milk of a white-nosed coati, Nasua narica (Procyonidae: Carnivora).

Carbohydrates were extracted from a sample of coati milk and the component oligosaccharides were separated and partially purified by gel filtration and preparative thin layer chromatography. Their structures were determined by 1H-NMR. Fuc alpha 1-->2Gal beta 1-->4Glc Gal alpha 1-->3Gal beta 1-->4Glc Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc Fuc alpha 1-->2Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc Gal alpha 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc The two pentasaccharides are novel sugars. In addition, higher oligosaccharides, whose core units were lacto-N-neohexaose, were found in coati milk. Free lactose constituted only about one-third of the total free milk saccharides. The results are discussed in terms of comparisons with the milk sugars of bears and other species.     

Evidence of Regional Differences in the Lectin Histochemistry Along the Ductus Epididymis of the Lizard, Podarcis Sicula Raf.

The regional difference in the carbohydrate components of the ductus epididymis epithelium of a lizard was delineated by means of 13 lectins. Basal cells expressed only N-acetylglucosamine (GlcNAc). Throughout the ductus, the secretory cells showed oligosaccharides with terminal N-acetylneuraminic acid (Neu5Ac)α(2,6)galactose (Gal)/N-acetylgalactosamine (GalNAc) and internal mannose (Man) and/or glucose (Glc) in the whole cytoplasm, oligosaccharides terminating in Neu5Acα(2,6)Galβ(1,3)GalNAc, Neu5Acα(2,6)Galβ (1,4)GlcNAc, GalNAc, GlcNAc, and fucose (Fuc) in the supra-nuclear zone, and also glycans terminating in Neu5Acα(2,3)Galβ (1,4)GlcNAc, Neu5Acα(2,6)Galβ(1,3)GalNAc, Galβ (1,4)GlcNAc on the luminal surface. In the caput and corpus regions, the supra-nuclear cytoplasm was characterized by terminal Galβ(1,4)GlcNAc and αGalNAc, the luminal surface by αGalNAc and Gal. The Golgi zone, showing oligosaccharides with terminal Neu5Acα(2,3)Galβ (1,4)GlcNAc, Neu5Acα(2,6)Galβ (1,3)GalNAc, Neu5Acα(2,6)Galβ (1,4)GlcNAc, and internal GlcNAc, expressed terminal Galβ (1,4)GlcNAc and αGalNAc in the caput, and terminal β GalNAc in the corpus. The granules showed all the investigated carbohydrates in their peripheral zone except terminal βGalNAc and Fuc, whereas internal Man/Glc and terminal Gal were expressed in the central core, and Fuc throughout the ductus, terminal GlcNAc in the caput and corpus, and terminal αGalNAc only in the corpus.     

Probing the substrate specificity of four different sialyltransferases using synthetic beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) (CH(2))7CH3 analogues general activating effect of replacing N-acetylglucosamine by N-propionylglucosamine

The acceptor specificities of ST3Gal III, ST3Gal IV, ST6Gal I and ST6Gal II were investigated using a panel of beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH(2))(7)CH(3) analogues. Modifications introduced at either C2, C3, C4, C5, or C6 of terminal D-Gal, as well as N-propionylation instead of N-acetylation of subterminal D-GlcN were tested for their influence on the alpha-2,3- and alpha-2,6-sialyltransferase acceptor activities. Both ST3Gal enzymes displayed the same narrow acceptor specificity, and only accept reduction of the Gal C2 hydroxyl function. The ST6Gal enzymes, however, do not have the same acceptor specificity. ST6Gal II seems less tolerant towards modifications at Gal C3 and C4 than ST6Gal I, and prefers beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc (LacdiNAc) as an acceptor substrate, as shown by replacing the Gal C2 hydroxyl group with an N-acetyl function. Finally, a particularly striking feature of all tested sialyltransferases is the activating effect of replacing the N-acetyl function of subterminal GlcNAc by an N-propionyl function.     

Galactomannans with novel structures from the lichen Roccella decipiens Darb

Two homogeneous galactomannan fractions were isolated from the lichen, Roccella decipiens, one (FP) containing Man and Gal in an 81:19 molar ratio and the other (RFS), having Man, Gal, and Glc in a 43:56:1 molar ratio. FP consisted of a main chain with (1-->4)-linked alpha-D-Manp units, most of which were substituted at O-2 with side chains consisting of nonreducing end-, 2-O- and 6-O-substituted alpha-Manp units. The latter appeared to be substituted by single-unit beta-D-Galf nonreducing ends. RFS contained a similar alpha-D-Manp core structure, but with side chains containing nonreducing end, 5-O-, 6-O-, and 5,6-di-O-substituted beta-D-Galf units. Such polysaccharide structures have not been previously reported.     

Substrate conformation modulates aggrecanase (ADAMTS-4) affinity and sequence specificity. Suggestion of a common topological specificity for functionally diverse proteases

Protease-substrate interactions are governed by a variety of structural features. Although the substrate sequence specificities of numerous proteases have been established, "topological specificities," whereby proteases may be classified based on recognition of distinct three-dimensional structural motifs, have not. The aggrecanase members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family cleave a variety of proteins but do not seem to possess distinct sequence specificities. In the present study, the topological substrate specificity of ADAMTS-4 (aggrecanase-1) was examined using triple-helical or single-stranded poly(Pro) II helical peptides. Substrate topology modulated the affinity and sequence specificity of ADAMTS-4 with K(m) values indicating a preference for triple-helical structure. In turn, non-catalytic ADAMTS-4 domains were critical for hydrolysis of triple-helical and poly(Pro) II helical substrates. Comparison of ADAMTS-4 with MMP-1 (collagenase 1), MMP-13 (collagenase 3), trypsin, and thermolysin using triple-helical peptide (THP) and single-stranded peptide (SSP) substrates demonstrated that all five proteases possessed efficient "triple-helical peptidase" activity and fell into one of two categories: (k(cat)/K(m))(SSP) > (k(cat)/K(m))(THP) (thermolysin, trypsin, and MMP-13) or (k(cat)/K(m))(THP) > or = (k(cat)/K(m))(SSP) and (K(m))(SSP) > (K(m))(THP) (MMP-1 and ADAMTS-4). Overall these results suggest that topological specificity may be a guiding principle for protease behavior and can be utilized to design specific substrates and inhibitors. The triple-helical and single-stranded poly(Pro) II helical peptides represent the first synthetic substrates successfully designed for aggrecanases.     

Further characterization of the binding properties of two monoclonal antibodies recognizing human Tn red blood cells

The terminal alpha anomeric Ga1NAc residue is an essential sugar for the Tn glycotope, human blood group A determinant, and Forssman antigen. In a previous study [King M.J., Parson S.F., Wu A,M., Jones N., Transfusion 31: 142-149, 1991] we defined two monoclonal antibodies (MoAbs, BRIC66 and BRIC111) reacting with human Tn red blood cells. However, more advanced studies of these two MoAbs were hampered by the lack of availability of Gal/GalNAc related glycotopes. In order to use these antibodies as powerful probes to elucidate structural changes during life processes, we have characterized in detail the combining sites of these two MoAbs using enzyme-linked immunosorbent (ELISA) and inhibition assays with an extended glycan/ligand collection. From the results, it has been established that BRIC66 demonstrated multiple specificities and its reactivity towards glycotopes was defined as: Ga1NAc alpha1-->Ser/Thr (Tn) > or = Ga1NAc alpha1-->3(LFuc alpha1-->2)Gal (Ah) > Ga1NAcalpha1-->3Galbeta1-->4Glc (AL) > Ga1NAalpha1-->3Gal (A) GalNAc alpha1-->3GalNAc > Gal or Glc. Another MoAb, BRIC111, mainly bound Tn-glycophorin. The best ligand for this MoAb was Tn-containing glycopeptides (M.W. < 3.0 x 10(3) Da) from asialo ovine salivary mucin (OSM), which was approximately 70 and 58 times more active than Ga1NAc and monomeric Ga1NAc alpha1-->Ser/Thr (Tn), respectively, suggesting that the active glycotopes present in glycophorin for BRIC111 binding also exist in OSM. The N-acetyl group at carbon-2 and configuration at carbon-2 and carbon-4 of the alpha anomeric Ga1NAc are required for the binding of either MoAb. Identification of these binding properties should aid in the selection of these MoAbs and the conditions required for biological studies and clinical applications.     

Glycosphingolipids in insects. The amphoteric moiety, N-acetylglucosamine-linked phosphoethanolamine, distinguishes a group of ceramide oligosaccharides from the pupae of Calliphora vicina (Insecta: Diptera)

A group of Calliphora vicina pupal glycolipids could be segregated from the neutral glycosphingolipids, according to their two-dimensional TLC migration properties and positive reactions toward ninhydrin and fluorescamine spray reagents. These classified zwitterionic glycolipids were isolated by silica-gel column chromatography and characterized by the presence of a N-acetyl-glucosamine-bound phosphoethanolamine residue. The structural elucidation of the oligosaccharide moieties was performed by the determination of constituent carbohydrates as alditol acetates, linkage analysis by permethylation, exoglycosidase cleavage, fast-atom-bombardment mass spectrometry and NMR spectroscopy. The dominant fatty acid and sphingoid base species of the ceramide moieties were C20:0 (arachidic acid) and C14:1 (tetradecasphing-4-enine), respectively. The chemical structures of the zwitterionic, biogenetic glycosphingolipid series were determined as: (PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1- 4)Glc beta Cer; Gal(beta 1-3)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1- 3)Man(beta 1-4)Glc beta Cer; GlcNAc(beta 1-3)Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn- 6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer.     

Mutational analysis provides molecular insight into the carbohydrate-binding region of calreticulin: pivotal roles of tyrosine-109 and aspartate-135 in carbohydrate recognition

Calreticulin (CRT) is a lectin chaperone present in the lumen of the endoplasmic reticulum. It interacts with various glycoproteins by binding via their attached Glc(1)Man(9)GlcNAc(2) moiety. To provide further insight into these lectin-glycan interactions, we are investigating the interaction of CRT with various sugars. We have earlier modeled the complex between CRT and the Glc(1)Man(3) tetrasaccharide, a derivative of the native Glc(1)Man(9)GlcNAc(2) sugar moiety. Here, we have systematically mutated the residues implicated by the model in the interaction of CRT to its sugar substrates and categorized the role played by each of the subsites of calreticulin toward the glycan binding. The CRT mutants Y109F and D135L did not show any binding to the sugar substrates interacting with the wild-type protein, demonstrating the great importance of these residues in the carbohydrate-binding site of CRT. Also, D317L and M131A showed weak affinity toward the trisaccharide. The mutation of residues from the primary binding site of CRT, i.e., those interacting with glucose, appears to be far less tolerated as compared to mutations in residues that interact with the mannose residues of the glycan. Also, methyl-2-deoxy-glucopyranosyl-alpha(1-->3)-mannopyranoside failed to bind, asserting to the significance of the interactions between the primary binding site of CRT and the 2'-OH of the glucose residue of the oligosaccharide substrate in generating specificity for this recognition. These studies provide detailed molecular insight into the sugar binding specificity of CRT.     

Glycosphingolipids in insects. Chemical structures of ceramide tetra-, penta-, hexa-, and heptasaccharides from Calliphora vicina pupae (Insecta: Diptera)

Four neutal fraction glycosphingolipids, designated components 4-7, were purified from the pupae of Calliphora vicina and isolated by the use of high performance liquid chromatography. Their chemical structures were determined to be: GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer and Gal(alpha 1-3)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; and GlcNAC(beta 1-3)Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer. By the use of specific exoglycosidases, it was possible to assign anomeric configurations to all the sugar residues present. Analysis of the ceramide moiety by electron-impact mass spectrometry revealed the dominant fatty acid and sphingoid to be arachidic acid (C20:0) and tetradecasphing-4-enine, respectively.     

Cleavage of the O antigen 4, 5, 12 of Salmonella typhimurium by hydrofluoric acid

The effect of hydrofluoric acid (aqueous 48% HF) upon different lipopolysaccharides (LPS) was studied, employing conditions (48 h at + 4°C) that are commonly used to dephosphorylate LPS. From the LPS of Salmonella typhimurium having the O antigen 4,5,12 almost all of the O-antigenic sugars (Abe, Gal, Glc, Man, Rha) were liberated in dialysable form, whereas the saccharide chains of Salmonella LPS with O antigen 6,7 (Man, Glc, GlcNAc) were resistant to HF. The lability towards HF was shown to be due to the presence of the deoxysugar L-rhamnose in the saccharide backbone of the O antigen 4,5,12, since only Rha was found as the terminal sugar in the corresponding dialysable material. Hydrofluoric acid can thus be used to specifically cleave Rha-containing polysaccharides.     

Mutation of arginine 228 to lysine enhances the glucosyltransferase activity of bovine beta-1,4-galactosyltransferase I

Beta-1,4-galactosyltransferase I (beta4Gal-T1) normally transfers Gal from UDP-Gal to GlcNAc in the presence of Mn(2+) ion (Gal-T activity) and also transfers Glc from UDP-Glc to GlcNAc (Glc-T activity), albeit at only 0.3% efficiency. In addition, alpha-lactalbumin (LA) enhances this Glc-T activity more than 25 times. Comparison of the crystal structures of UDP-Gal- and UDP-Glc-bound beta4Gal-T1 reveals that the O4 hydroxyl group in both Gal and Glc moieties forms a hydrogen bond with the side chain carboxylate group of Glu317. The orientation of the O4 hydroxyl of glucose causes a steric hindrance to the side chain carboxylate group of Glu317, accounting for the enzyme's low Glc-T activity. In this study, we show that mutation of Arg228, a residue in the vicinity of Glu317, to lysine (R228K-Gal-T1) results in a 15-fold higher Glc-T activity, which is further enhanced by LA to nearly 25% of the Gal-T activity of the wild type. The kinetic parameters indicate that the main effect of the mutation of Arg228 to lysine is on the k(cat) of Glc-T, which increases 3-4-fold, both in the absence and in the presence of LA; simultaneously, the k(cat) for the Gal-T reaction is reduced 30-fold. The crystal structure of R228K-Gal-T1 complexed with LA, UDP-Gal, and Mn(2+) determined at 1.9 A resolution shows that the Asp318 side chain exhibits a minor alternate conformation, compared to that in the wild type. This alternate conformation now causes a steric hindrance to the O4 hydroxyl group of the Gal moiety of UDP-Gal, probably causing the dissociation of UDP-Gal and the reduced k(cat) of the Gal-T reaction.     

Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site

Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins.The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework.     

Cleavage of the O antigen 4,5,12 of Salmonella typhimurium by hydrofluoric acid

The effect of hydrofluoric acid (aqueous 48% HF) upon different lipopolysaccharides (LPS) was studied, employing conditions (48 h at + 4°C) that are commonly used to dephosphorylate LPS. From the LPS of Salmonella typhimurium having the O antigen 4,5,12 almost all of the O-antigenic sugars (Abe, Gal, Glc, Man, Rha) were liberated in dialysable form, whereas the saccharide chains of Salmonella LPS with O antigen 6,7 (Man, Glc, GlcNAc) were resistant to HF. The lability towards HF was shown to be due to the presence of the deoxysugar L-rhamnose in the saccharide backbone of the O antigen 4,5,12, since only Rha was found as the terminal sugar in the corresponding dialysable material. Hydrofluoric acid can thus be used to specifically cleave Rha-containing polysaccharides.     

Defining the carbohydrate specificities of aplysia gonad lectin exhibiting a peculiar D-galacturonic acid affinity

Aplysia gonad lectin (AGL), which has been shown to stimulate mitogenesis in human peripheral lymphocytes, to suppress tumor cells, and to induce neurite outgrowth and improve cell viability in cultured Aplysia neurons, exhibits a peculiar galacturonic acid/galactose specificity. The carbohydrate binding site of this lectin was characterized by enzyme-linked lectino-sorbent assay and by inhibition of AGL-glycan interactions. Examination of the lectin binding with 34 glycans revealed that it reacted strongly with the following glycoforms: most human blood group precursor (equivalent) glycoproteins (gps), two Galalpha1-->4Gal-containing gps, and two d-galacturonic acid (GalUA)-containing polysaccharides (pectins from apple and citrus fruits), but poorly with most human blood group A and H active and sialylated gps. Among the GalUA and mammalian saccharides tested for inhibition of AGL-glycan binding, GalUA mono- to trisaccharides were the most potent ones. They were 8.5 x 10(4) times more active than Gal and about 1.5 x 10(3) more active than the human blood group P(k) active disaccharide (E, Galalpha1-->4Gal). This disaccharide was 6, 28, and 120 times more efficient than Galbeta1-->3GlcNAc(I), Galbeta1-->3GalNAc(T), and Galbeta1--> 4GlcNAc (II), respectively, and 35 and 80 times more active than melibiose (Galalpha1-->6Glc) and human blood group B active disaccharide (Galalpha1-->3Gal), respectively, showing that the decreasing order of the lectin affinity toward alpha-anomers of Gal is alpha1-->4 > alpha1-->6 > alpha1-->3. From the data provided, the carbohydrate specificity of AGL can be defined as GalUAalpha1-->4 trisaccharides to mono GalUA > branched or cluster forms of E, I, and II monomeric E, I, and II, whereas GalNAc is inactive.