On the biosynthesis of alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid catalyzed by the sialyltransferase of Escherichia coli Bos-12.

Escherichia coli Bos-12 synthesizes a heteropolymer of sialic acids with alternating alpha-2,9/alpha-2,8 glycosidic linkages (1). In this study, we have shown that the polysialyltransferase of the E. coli Bos-12 recognizes an alpha-2,8 glycosidic linkage of sialic acid at the nonreducing end of an exogenous acceptor of either the alpha-2,8 homopolymer of sialic acid or the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid and catalyzes the transfer of Neu5Ac from CMP-Neu5Ac to this residue. When the exogenous acceptor is an alpha-2,8-linked oligomer of sialic acid, the main product synthesized is derived from the addition of a single residue of [14C]Neu5Ac to form either an alpha-2,8 glycosidic linkage or an alpha-2,9 glycosidic linkage at the nonreducing end, at an alpha-2, 8/alpha-2,9 ratio of approximately 2:1. When the acceptor is the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid, chain elongation takes place four to five times more efficiently than the alpha-2,8-linked homopolymer of sialic acid as an acceptor. It was found that the alpha-2,9-linked homopolymer of sialic acid and the alpha-2,8/alpha-2,9-linked hetero-oligomer of sialic acid with alpha-2,9 at the nonreducing end not only failed to serve as an acceptor for the E. coli Bos-12 polysialyltransferase for the transfer of [14C]Neu5Ac, but they inhibited the de novo synthesis of polysialic acid catalyzed by this enzyme. The results obtained in this study favor the proposal that the biosynthesis of the alpha-2, 9/alpha-2,8 heteropolymer of sialic acid catalyzed by the E. coli Bos-12 polysialyltransferase involves a successive transfer of a preformed alpha-2,8-linked dimer of sialic acid at the nonreducing terminus of the acceptor to form an alpha-2,9 glycosidic linkage between the incoming dimer and the acceptor. The glycosidic linkage at the nonreducing end of the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid produced by E. coli Bos-12 should be an alpha-2,8 glycosidic bond and not an alpha-2,9 glycosidic linkage.     

DFT studies of the disaccharide, alpha-maltose: relaxed isopotential maps

The disaccharide, alpha-maltose, forms the molecular basis for the analysis of the structure of starch, and determining the conformational energy landscape as the molecule oscillates around the glycosidic bonds is of importance. Thus, it is of interest to determine, using density functionals and a medium size basis set, a relaxed isopotential contour map plotted as a function of the phi(H) and psi(H) dihedral angles. The technical aspects include the method of choosing the starting conformations, the choice of scanning step size, the method of constraining the specific dihedral angles, and the fitting of data to obtain well defined contour maps. Maps were calculated at the B3LYP/6-31+G( *) level of theory in 5 degrees intervals around the (phi(H),psi(H))=(0 degrees ,0 degrees ) position, out to approximately +/-30 degrees or greater, for gg-gg'-c, gg-gg'-r, gt-gt'-c, gt-gt'-r, tg-tg'-c, and tg-tg'-r conformers, as well as one-split gg(c)-gg'(r) conformer. The results show that the preferred conformation of alpha-maltose in vacuo depends strongly upon the hydroxyl group orientations ('c'/'r'), but the energy landscape moving away from the minimum-energy position is generally shallow and transitions between conformational positions can occur without the addition of significant energy. Mapped deviations of selected parameters such as the dipole moment; the C1-O1-C4', H1-C1-O1, and H4'-C4'-O1 bond angles; and deviations in hydroxymethyl rotamers, O5-C5-C6-O6, O5'-C5'-C6'-O6', C5-C6-O6-H, and C5'-C6'-O6'-H', are presented. These allow visualization of the structural and energetic changes that occur upon rotation about the glycosidic bonds. Interactions across the bridge are visualized by deviations in H(O2)...O3', H(O3')...O2, and H1...H4' distances and the H(O2)-O2-C2-C1 and H'(O3')-O3'-C3'-C4' hydroxyl dihedral angles.     

Structural determination of the sialic acid polysaccharide antigens of Neisseria meningitidis serogroups B and C with carbon 13 nuclear magnetic resonance.

The application of 13-C nuclear magnetic resonance to the analysis of some sialic acid-containing meningococcal polysaccharide antigens is described. Complete assignments of the spectra of both the native serogroup B and the de-O-acetylated serogroup C polysaccharides have been made. These assignments were based on the corresponding data for some related monomers (sialic acid and its alpha-and beta-methylglycosides) and on supportive chemical evidence. The data indicate that the serogroup B polysaccharide is a 2 yields 8-alpha-linked homopolymer of sialic acid, identical in structure with colominic acid from Escherichia coli, whereas the de-O-acetylated serogroup C polysaccharide is a 2 yield 9-alpha-linked homopolymer. The native serogroup C polysaccharide is O-acetylated (1.16 mol of O-acetyl per sialic acid residue), all the O-acetyl substituents being located only at C-7 and C-8 of the sialic acid residues, and in addition contains unacetylated residues (24%). The polysaccharide contains di-O-acetylated residues (O-acetyl on C-7 and C-8), and at least one of the possible monoacetylated residues at C-7 or C-8.     

CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the biosynthesis of polysialosyl units in neural cell adhesion molecules

Prokaryotic derived probes that specifically recognize alpha-2,8-ketosidically linked polysialosyl units were developed to identify and study the temporal expression of these unique carbohydrate moieties in developing neural tissue (Vimr, E. R., McCoy, R. D., Vollger, H. F., Wilkison, N. C., and Troy, F. A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1971-1975). These polysialosyl units cap N-linked oligosaccharides of the complex-type on neural cell adhesion molecules (N-CAM). A Golgi-enriched fraction from 20-day-old fetal rat brain contains a membrane-associated sialyltransferase that catalyzes the incorporation of [14C]N-acetylneuraminic acid [( 14C]NeuNAc) from CMP-[14C] NeuNAc into polymeric products. At pH 6.0, 84 pmol of NeuNAc mg of protein-1 h-1 were incorporated. In sodium dodecyl sulfate-polyacrylamide gels, the major radiolabeled species migrated with a mobility expected for N-CAM. A bacteriophage-derived endoneuraminidase specific for polysialic acid was used to demonstrate that at least 20-30% of the [14C]NeuNAc was incorporated into alpha-2,8-linked polysialosyl units. This was confirmed by structural studies which showed that the endoneuraminidase-sensitive brain material consisted of multimers of sialic acid. The addition of a partially purified preparation of chick N-CAM to the membranous sialyltransferase stimulated sialic acid incorporation 3-fold. The product of this reaction was also sensitive to endoneuraminidase and contained alpha-2,8-linked polysialosyl chains, thus showing that N-CAM can serve as an exogenous acceptor for sialylation in vitro. Sialic acid incorporated into adult rat brain membranes was resistant to endoneuraminidase, indicating that the poly-alpha-2,8-sialosyl sialyltransferase activity is restricted to an early developmental epoch. It is recommended that the enzyme described here be designated CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the trivial name poly-alpha-2,8-sialosyl sialyltransferase be adopted.     

Conformational preferences of cyclopropyl peptides. Crystal structure of (E)-DL-1-benzamido-1-methoxycarbonyl-2-chlorcyclopropane (BCP)

Crystal structure analysis of (E)-DL-1-benzamido-1-methoxycarbonyl-2-chlorocyclopropane (C12H12NO3Cl) is reported. The phi' (about N1-C1 bond) and psi' (about C1-C11 bond) torsional angles for this compound are -62.5 degrees and -33.0 degrees, respectively, and are close to the phi, psi values of the 3(10) helix and the alpha-helix. Semi-empirical potential energy calculations are performed on a cyclopropyl dipeptide which is a special case of alpha,alpha-disubstituted dipeptide where the alpha-carbon and the two substituent carbon atoms form a 3-membered ring. Our calculations show that different types of helics: alpha-, gamma-, pi-, omega-, 3(10-) and delta-helices, are energetically favorable. Another interesting possibility is the formation of a cyclic pentapeptide with five-fold symmetry. The effect of substitutions on C beta atom are also studied with the help of potential energy maps. Selective substitutions on C beta atom may be used effectively to restrict either phi or psi values into a very narrow range.     

Biophysical studies on molecular mechanism of abortifacient action of prostaglandins. VI. Conformation energy calculation on PGE2, PGF2 alpha and 15-(s)-methyl PGF2 alpha

This paper reports the conformation energy (CE) calculations on PGE2, PGE2 alpha and 15-(s)-methyl PGE2 alpha on the basis of empirical potential energy functions for the simultaneous rotations around C7-C8 (psi), C12-C13 (theta) and C14-C15 (phi) bonds. The variation of the minimum conformation energy E for each isoenergy map in the psi theta plane with respect to phi gives two minima around 90 degrees and 240 degrees in PGE2, 60 degrees and 245 degrees in PGF2 alpha, and 60 degrees and 150 degrees in 15-(s)-methyl PGF2 alpha. The latter two forms also have a small dip at 270 degrees. The pattern of allowed low energy conformations of PGF2 alpha and 15-(s)-methyl PGF2 alpha is quite similar and is characterized by the existence of six low energy regions.     

Cell-free biosynthesis of the O-acetylated N-acetylneuraminic acid capsular polysaccharide of group C meningococci.

A cell-free system was established to study the biosynthesis of group C meningococcal capsular polysaccharide, an alpha-2 leads to 9-linked N-acetylneuraminic acid (NeuAc) homopolymer containing O-acetyl groups at either C7 or C8. Sialyltransferase activity, isolated from group C meningococcus strain C-11, catalyzed incorporation of [14C]NeuAc from CMP (CMP--[14C]NeuAc) into polymeric form. This sialyltransferase was stimulated by addition of meningococcus group C and Escherichia coli K92 capsular polysaccharides, the latter being an alpha-2 leads to 8- and alpha-2 leads to 9-linked NeuAc heteropolymer. Group C meningococcal sialyltransferase did not require divalent ions but was stimulated by Mn2+. Attempts to demonstrate a lipid-soluble intermediate in the biosynthesis of this NeuAc polymer were unsuccessful. Meningococcal group C sialyltransferase incorporated NeuAc into a membrane-associated product. The polysaccharide can be extracted from the membrane-bound fraction with Triton X-100. The newly synthesized polysaccharide coprecipitates with authentic group C antigen in meningococcal group C antiserum and is degraded by sodium metaperiodate, indicating that the NeuAc polymer synthesized by the cell-free system consists of alpha-2 leads to 9 linkage. Meningococcal group C spheroplast membranes contain an O-acetylase that can catalyze the transfer of acetyl groups from acetyl coenzyme A to the in vitro-synthesized polysaccharide.     

Use of a bacteriophage-derived endo-N-acetylneuraminidase and an equine antipolysialyl antibody to characterize the polysialyl residues in salmonid fish egg polysialoglycoproteins. Substrate and immunospecificity studies

Polysialoglycoproteins (PSGP), a class of glycoproteins containing oligo(poly)sialylglycan chains, are the major glycoprotein components in cortical alveoli of a number of Salmonidae fish eggs. Lake trout, Salvelinus namaycush, egg PSGP (PSGP(Sn)) differs from rainbow trout, Salmo gairdneri, egg PSGP (PSGP(Sg)) in its sialic acid composition; the former contains both N-acetyl- and N-glycolyl-D-neuraminic acid residues, designated Neu5Ac and Neu5Gc, while the latter contains only Neu5Gc residues. Fragmentation analysis of oligo(poly)sialyl chains in lake trout PSGP(Sn) has established that there are two distinct types of oligo(poly)sialyl structures in this PSGP molecule, namely alpha-2,8-linked oligo/poly(Neu5Ac) and alpha-2,8-linked oligo/poly(Neu5Gc). No hybrid structure having both Neu5Ac and Neu5Gc residues in the fragment oligosialic acids was detected. These two distinct PSGP preparations from eggs of lake trout and rainbow trout have been used to compare their immunoreactivity with anti-polysialyl antibodies (H.46) and sensitivity to a bacteriophage-derived (Escherichia coli K1F) endo-N-acetylneuraminidase (Endo-N). H.46 was found to cross-react only with lake trout PSGP(Sn) in immunodiffusion assays but not with rainbow trout PSGP(Sg), indicating that H.46 is a specific probe for alpha-2,8-linked poly(Neu5Ac) but not for poly(Neu5Gc). In contrast, Endo-N was found to catalyze the hydrolysis of both alpha-2,8-linked poly (Neu5Ac) and poly(Neu5Gc), so that this enzyme can be used as a diagnostic reagent for detecting both types of polysialic acids. H.46 was used in indirect immunofluorescence experiments to localize PSGP(Sn) in cortical alveoli isolated from lake trout eggs.     

Structural studies of the pectic polysaccharide from duckweed Lemna minor L

The pectic polysaccharide of duckweed Lemna minor L. termed lemnan (LM) was shown to contain the ramified, "hairy" region. Using partial acid hydrolysis and Smith degradation followed by NMR spectroscopy of the fragments obtained, some structural features of the hairy region of LM were elucidated. Partial acid hydrolysis of LM afforded the crude polysaccharide fraction LMH that was separated into two polysaccharide fractions: LMH-1 and LMH-2. In addition, the oligosaccharide fraction LMH-3 contained 97% D-apiose was obtained from the supernatant. A further more rigorous acidic hydrolysis of LMH led to the crude polysaccharide fraction LMHR which was separated in to two fractions: LMHR-1 and LMHR-2. Smith degradation of LMH afforded the polysaccharide fragment LMHS differed in low contents of apiose residues. Unfortunately, NMR-spectroscopy failed to provide significant evidence concerning the structure of LMH-1 due to the complexity of the macromolecule. The structure of the 1H/13C-NMR spectroscopy including the correlation 2D NMR spectroscopy. As a result, alpha-1,4-D-galactopyranosyluronan was confirmed to be the main constituent of the LM backbone. In addition, the ramified, "hairy" region of the macromolecule appeared to contain segments consisting of residues of terminal and beta-1,5-linked apiofuranose, terminal and alpha-1,5-linked arabinofuranose, terminal and beta-1,3- and beta-1,4- linked galactopyranose, the terminal and beta-1,4-linked xylopyranose, and beta-1,4-linked 2-mono-O-methyl xylopyranose. Analytical and NMR-spectral data of LMHS confirmed the presence of considerable amounts of the non-oxidized of 1,4-linked D-galactopyranosyl uronic acid residues. Thus, some side chains of the ramified region of lemnan appeared to attach to D-galactopyranosyl uronic acid residues of the backbone.     

Determinant specificities of the groups B and C polysaccharides of Neisseria meningitidis

A meningococcal group B-specific horse antiserum contains at least two distinct populations of antibodies with specificities for determinants on the group B capsular polysaccharide antigen. These two populations were differentiated on the basis of the ability of only one of them to be absorbed from the antiserum by the structurally related colominic acid. The nature of the colominic acid-specific determinant was elucidated by a radioimmunoassay inhibition technique with the use of a series of linear alpha-(2----8)-linked oligomers of sialic acid as inhibitors. Colominic acid was labeled by prior removal of its N-acetyl groups, followed by their replacement with the use of [3H]acetic anhydride. The conformational nature of the determinant was proposed because of the unusually large size (10 sialic acid residues) of the oligomer required to function as an efficient inhibitor. The structure of the determinant responsible for the second population of group B-specific antibodies has not been determined, but it is obviously based on an as yet undefined conformational or structural feature peculiar to the group B meningococcal polysaccharide. In contrast to the colominic acid-specific group B determinant, the determinant responsible for the group C polysaccharide-specific rabbit antibodies proved to be more conventional. Inhibitory properties of the alpha-(2----9)-linked oligomers maximized with those containing four or five sialic acid residues, which is consistent with the approximate estimated maximal size of an antibody site.     

Role of azaamino acid residue in beta-turn formation and stability in designed peptide.

The structural perturbation induced by C(alpha)-->N(alpha) exchange in azaamino acid-containing peptides was predicted by ab initio calculation of the 6-31G* and 3-21G* levels. The global energy-minimum conformations for model compounds, For-azaXaa-NH2 (Xaa=Gly, Ala, Leu) appeared to be the beta-turn motif with a dihedral angle of phi= +/- 90 degrees, psi=0 degrees. This suggests that incorporation of the azaXaa residue into the i+2 position of designed peptides could stabilize the beta-turn structure. The model azaLeu-containing peptide, Boc-Phe-azaLeu-Ala-OMe, which is predicted to adopt a beta-turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using 1H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (phi i+1, psi i+1) (phi i+2, psi i+2)] of the Phe-azaLeu fragment in the model peptide, Boc-Phe-azaLeu-Ala-OMe, as [(-59 degrees, 127 degrees) (107 degrees, -4 degrees)]. This solution conformation supports a betaII-turn structural preference in azaLeu-containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i+2 position in synthetic peptides is expected to provide a stable beta-turn formation, and this could be utilized in the design of new peptidomimetics adopting a beta-turn scaffold.     

Conformational studies of cyclic tetrapeptides. Evidence for a bis gamma-turn conformation for chlamydocin and Ala4-chlamydocin in nonpolar solvents

The conformations of chlamydocin and cyclo (Ala-Aib-Phe-D-Pro) (Ala4-chlamydocin) in chloroform have been investigated by nuclear magnetic resonance, infrared and circular dichroism spectroscopy. The data obtained from these experiments establish an all transoid, bis gamma-turn conformation for both compounds in chloroform with the following torsional angles (+/- 20 degrees): Ala4-chlamydocin: Aib, phi + 60 degrees, psi - 50 degrees; omega + 160 degrees; Phe phi - 120 degrees, psi + 120 degrees, omega - 160 degrees; D-Pro phi + 60 degrees, psi - 55 degrees, omega + 160 degrees; Ala phi - 110 degrees, psi + 110 degrees, omega - 160 degrees. Chlamydocin adopts a closely related conformation in neat chloroform. Nuclear Overhauser Effect (NOE) data are utilized to assign amide bond geometries in the cyclic tetrapeptide ring system.     

Significant conformational changes in an antigenic carbohydrate epitope upon binding to a monoclonal antibody.

Transferred nuclear Overhauser enhancement spectroscopy (TRNOE) was used to observe changes in a ligand's conformation upon binding to its specific antibody. The ligands studied were methyl O-beta-D-galactopyranosyl(1----6)-4-deoxy-4-fluoro-beta-D-galactopyra nos ide (me4FGal2) and its selectively deuteriated analogue, methyl O-beta-D-galactopyranosyl(1----6)-4-deoxy-2-deuterio-4-fluoro-beta -D- galactopyranoside (me4F2dGal2). The monoclonal antibody was mouse IgA X24. The solution conformation of the free ligand me4F2dGal2 was inferred from measurements of vicinal 1H-1H coupling constants, long-range 1H-13C coupling constants, and NOE cross-peak intensities. For free ligand, both galactosyl residues adopt a regular chair conformation, but the NMR spectra are incompatible with a single unique conformation of the glycosidic linkage. Analysis of 1H-1H and 1H-13C constants indicates that the major conformer has an extended conformation: phi = -120 degrees; psi = 180 degrees; and omega = 75 degrees. TRNOE measurements on me4FGal2 and me4F2dGal2 in the presence of the specific antibody indicate that the pyranose ring pucker of each galactose ring remains unchanged, but rotations about the glycosidic linkage occur upon binding to X24. Computer calculations indicate that there are two sets of torsion angles that satisfy the observed NMR constraints, namely, phi = -152 +/- 9 degrees; psi = -128 +/- 7 degrees; and omega = -158 +/- 6 degrees; and a conformer with phi = -53 +/- 6 degrees; psi = 154 +/- 10 degrees; and omega = -173 +/- 6 degrees. Neither conformation is similar to any of the observed conformations of the free disaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis, crystal structure, and molecular conformation of peptide N-Boc-L-Pro-dehydro-Phe-L-Gly-OH

The peptide N-Boc-L-Pro-dehydro-Phe-L-Gly-OH was synthesized by the usual workup procedure and finally coupling the N-Boc-L-Pro-dehydro-Phe to glycine. The peptide crystallizes in monoclinic space group P2(1) with a = 8.951(4) A, b = 5.677(6) A, c = 21.192(11) A, beta = 96.97(4) degrees, V = 1069(1) A3, Z = 2, dm = 1.295(5) Mgm-3, and dc = 1.297(4) Mgm-3. The structure was determined by direct methods using SHELXS86. The structure was refined by the block-diagonal least-squares procedure to an R value of 0.074 for 1002 observed reflections. The C alpha 2-C beta 2 distance of 1.33(2) A is an appropriate double bond length. The angle C alpha 2-C beta 2-C gamma 2 is 133(1) degrees. The peptide backbone torsion angles are theta 1 = -167(1) degrees, omega 0 = 179(1) degrees, phi 1 = -48(1) degrees, psi 1 = 137(1) degrees, omega 1 = 175(1) degrees, phi 2 = 65(2) degrees, psi 2 = 15(2) degrees, omega 2 = -179(1) degrees, and phi 3 = -166(1) degrees. These values show that the Boc group has a trans-trans conformation while the peptide backbone adopts a beta-turn II conformation, which is stabilized by an intramolecular hydrogen bond of length of 3.05(1) A. The structures of dehydro-Phe containing peptides suggest that the dehydro-Phe promotes the beta-turn II conformation. The five-membered pyrrolidine ring of the Pro residue adopts an ideal C gamma-exo conformation with torsion angles chi 1(1) = -24(1) degrees, chi 2(1) = 34(1) degrees, chi 3(1) = -30(1) degrees, chi 4(1) = 15(1) degrees, and theta 0(1) = 6(1) degrees. The side-chain torsion angles in dehydro-Phe are chi 1(2) = -1(2) degrees, chi 2,1(2) = -176(1) degrees, and chi 2,2(2) = 8(2) degrees. The plane of C alpha 2-C beta 2-C gamma 2 is rotated with respect to the plane of the phenyl ring at 7(1) degrees, which indicates that the atoms of the side chain of dehydro-Phe are essentially coplanar. The molecules form a 2(1) screw axis related hydrogen-bonded rows along the b axis.     

Structural analysis of a pectic polysaccharide from the leaves of Diospyros kaki

A pectic polysaccharide DL-2A with a molar mass of 8.5 x 10(5), was obtained from the boiling water extract of Diospyros kaki leaves. It had [alpha]20D -21.8 degrees (c 0.22, H2O) and consisted of rhamnose, arabinose, galactose, xylose and galacturonic acid units in the molar ratio of 0.4:3.4:2.4:1.0:0.8, along with traces of glucuronic acid. About 16.7% of galacturonic acid existed as the methyl ester. A combination of linkage analyses, periodate oxidation, partial acid hydrolysis, selective lithium-degraded reaction, ESIMS, 1H- and 13C- NMR spectral analyses revealed its structural features. It was found that DL-2A possessed an alpha-(1-->4)-galacturonan backbone with some insertions of alpha-1,2-Rhap residues. The side-chains of arabino-3,6-galactan were attached to the backbone via O-4 of Rhap residues and O-3 of GalAp residues, while 4-linked xylose residues (forming short linear chains) were directly linked to O-4 of rhamnose residues, not as part of the xylogalacturonan. These novel structural features enlarge the knowledge on the fine structure of pectic substances in the plant kingdom.     

The conformation of the T-antigen disaccharide bound to Maclura pomifera agglutinin in aqueous solution

The complex of Maclura pomifera agglutinin with the T-antigen disaccharide (beta-d-Gal-(1-->3)-alpha-d-GalNAc-(1-->O)-Me) was investigated by NMR spectroscopy in aqueous solution. Intramolecular transferred nuclear Overhauser enhancement (NOE) effects between the monosaccharide moieties were used to derive the ligand conformation in the lectin-bound state. Ligand protons in contact with the protein were identified by saturation transfer difference experiments and intermolecular transferred NOE effects. It is demonstrated that structural differences exist for the ligand-lectin complex in aqueous solution as compared with the previously published crystal structure (Lee, X., Thompson, A., Zhiming, Z., Ton-that, H., Biesterfeldt, J., Ogata, C., Xu, L., Johnston, R. A. Z. , and Young, N. M. (1998) J. Biol. Chem. 273, 6312-6318). In order to accommodate the O-methyl group of the disaccharide, the amino acid side chain of Tyr-122 has to rotate from its position in the crystal. The NMR data are in accord with two conformational families at the beta-(1-->3)glycosidic linkage in the solution complex with interglycosidic angles phi/psi = 45/-65 degrees and -65/-18 degrees. These differ from the bound conformation of the ligand in the crystal (phi/psi = 39/-8 degrees ) and are not highly populated by the ligand in the free state. The reason for the structural differences at the beta-(1-->3)glycosidic linkage are hydrogen bonds that stabilize the relative orientation of the monosaccharide units in the crystal. Our results demonstrate that the crystallization of a protein-carbohydrate complex can interfere with the delicate process of carbohydrate recognition in solution.     

Structural studies on the sialic acid polysaccharide antigen of Escherichia coli strain Bos-12.

A polysaccharide, antigenically related to group C meningococcus, has been isolated from Escherichia coli strain Bos-12 (016; K92; NM). Like groups B and C meningococcal polysaccharide, the Bos-12 antigen is a pure polymer of sialic acid. 13C NMR studies on the meningococcal group B and C polysaccharides have indicated that the former consists of sialic acid units linked 2 leads to 8- alpha, whereas the latter contains the sialic acid residues linked 2 leads to 9-alpha (Bhattacharjee, A.K., Jennings, H.J., Kenny, C.P., Martin, A., and Smith, I.C.P. (1975), J. Biol. Chem. 250, 1926). Comparison of natural abundance 13C NMR spectra of the Bos-12 polysaccharide with group B and C meningococcal polysaccharides established that Bos-12 was either (a) an equimolar mixture of 2 leads to 8-alpha linked sialic acid homopolymers or (b) a 2 leads to 8-alpha/2 leads to 9-alpha heteropolymer. These possibilities were distinguished in the following manner. The fact that Bos-12 polysaccharide precipitated with anti-group C serum but not with anti-group B serum would seem to exclude a. Further, chemical studies (periodate oxidation followed by tritiated NaBH4 reduction) gave saccharides with a radioactive-labeling pattern expected for alternating 2 leads to 8-alpha/2 leads to 9-alpha sialic acid linkages. Bos-12 is thus an 2 leads to 8/2 lead to 9-alpha heteropolymer.     

Role of sialic acid in bovine sperm-zona pellucida binding

Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.     

Peptide crystal growth via vapor diffusion. Crystal structure of glycyl-L-tyrosyl-L-alanine dihydrate.

The structure of a dihydrated form of glycyl-L-tyrosyl-L-alanine (GYA) has been determined as part of a series of peptide structural investigations and development of microscale vapor diffusion experiments for peptide crystal growth. Crystals were grown by the hanging-drop method against sodium acetate. The tripeptide is a zwitterion in the crystal, adopting an extended conformation through glycine, a nearly perpendicular bend at tyrosine and a reverse turn for the C-terminal carboxylate. Principal backbone torsion angles are psi 1 175(1) degrees, omega 2 173(1) degrees, phi 2 -119(1) degrees, psi 2 120(1) degrees, omega 3 172(1) degrees, phi 3 -73(1) degrees, psi 31 -9(1) degrees, psi 32 171(1) degrees. The tyrosyl side chain adopts an unusual orientation (chi 1/2 = -86(1) degrees). The relationship of the GYA.2H2O structure to GYA sequences in proteins is examined, particularly as regards its helix-forming potential. Crystal data: C14H19N3O4.2H2O, M(r) = 345.36, orthorhombic, P2(1)2(1)2(1), a = 4.810 (4), b = 11.400(7), c = 30.162(23)A, V = 1653.8(24)A-3, Z = 4, Dx = 1.387 Mgm-3, lambda(CuK- alpha) = 1.540 A, mu = 9.053 mm-1, F(000) = 736, T = 199 K, R = 0.041 for 1458 observations with I greater than or equal to 3 sigma(I).     

Crystal structure and conformation of a highly constrained linear tetrapeptide Boc-Leu-dehydro Phe-Ala-Leu-OCH3.

The conformation of a tetrapeptide containing a dehydro amino acid, delta ZPhe, in its sequence has been determined in the crystalline state using X-ray crystallographic techniques. The tetrapeptide, Boc-Leu-delta ZPhe-Ala-Leu-OCH3, crystallizes in the orthorhombic space group P2(1)2(1)2(1) with four molecules in a unit cell of dimensions a = 11.655(1) A, b = 15.698(6) A and c = 18.651(3) A V = 3414.9 A and Dcalc = 1.12 g/cm-3. The asymmetric unit contains one tetrapeptide molecule, C30H46N4O7, a total of 41 nonhydrogen atoms. The structure was determined using the direct methods program SHELXS86 and refined to an R-factor of 0.049 for 3347 reflections (I3.0(I). The linear tetrapeptide in the crystal exhibits a double bend of the Type III-I, with Leu1 (phi = -54.1 degrees, psi = -34.5 degrees) and delta ZPhe2 (phi = -59.9 degrees, psi = -17.1 degrees) as the corner residues of Type III turn and delta ZPhe2 (phi = -59.9 degrees, psi = -17.1 degrees) and Ala3 (phi = -80.4 degrees, psi = 0.5 degrees) residues occupying the corners of Type I turn, with delta ZPhe as the common residue in the double bend. The turn structures are further stabilized by two intramolecular 4----1 type hydrogen bonds.