The Role of Exopolysaccharides in Oral Biofilms.

The oral cavity contains a rich consortium of exopolysaccharide-producing microbes. These extracellular polysaccharides comprise a major component of the oral biofilm. Together with extracellular proteins, DNA, and lipids, they form the biofilm matrix, which contributes to bacterial colonization, biofilm formation and maintenance, and pathogenesis. While a number of oral microbes have been studied in detail with regard to biofilm formation and pathogenesis, the exopolysaccharides have been well characterized for only select organisms, namely Streptococcus mutans and Aggregatibacter actinomycetemcomitans. Studies on the exopolysaccharides of other oral organisms, however, are in their infancy. In this review, we present the current research on exopolysaccharides of oral microbes regarding their biosynthesis, regulation, contributions to biofilm formation and stability of the matrix, and immune evasion. In addition, insight into the role of exopolysaccharides in biofilms is highlighted through the evaluation of emerging techniques such as pH probing of biofilm colonies, solid-state nuclear magnetic resonance for macromolecular interactions within biofilms, and super-resolution microscopy analysis of biofilm development. Finally, exopolysaccharide as a potential nutrient source for species within a biofilm is discussed.

Role of de-N-acetylase PgaB from Aggregatibacter actinomycetemcomitans in exopolysaccharide export in biofilm mode of growth.

Aggregatibacter actinomycetemcomitans, a Gram-negative bacterium, is the causative agent of localized aggressive periodontitis. Attachment to a biotic surface is a critical first step in the A. actinomycetemcomitans infection process for which exopolysaccharides have been shown to be essential. In addition, the pga operon, containing genes encoding for biosynthetic proteins for poly-N-acetyl glucosamine (PNAG), plays a key role in A. actinomycetemcomitans virulence, as a mutant strain lacking the pga operon induces significantly less bone resorption. Among the genes in the pga operon, pgaB codes for a de-N-acetylase that is responsible for the deacetylation of the PNAG exopolysaccharide. Here we report the role of PgaB in regulation of virulence genes using a markerless, scarless deletion mutant targeting the coding region of the N-terminal catalytic domain of PgaB. The results demonstrate that the N-terminal, catalytic domain of PgaB is crucial for exopolysaccharide export.

Structural and functional analysis of de-N-acetylase PgaB from periodontopathogen Aggregatibacter actinomycetemcomitans.

The oral pathogen Aggregatibacter actinomycetemcomitans uses pga gene locus for the production of an exopolysaccharide made up of a linear homopolymer of ß-1,6-N-acetyl-d-glucosamine (PGA). An enzyme encoded by the pgaB of the pga operon in A. actinomycetemcomitans is a de-N-acetylase, which is used to alter the PGA. The full length enzyme (AaPgaB) and the N-terminal catalytic domain (residues 25-290, AaPgaBN) from A. actinomycetemcomitans were cloned, expressed and purified. The enzymatic activities of the AaPgaB enzymes were determined using 7-acetoxycoumarin-3-carboxylic acid as the substrate. The AaPgaB enzymes displayed significantly lower de-N-acetylase activity compared with the activity of the deacetylase PdaA from Bacillus subtilis, a member of the CE4 family of enzymes. To delineate the differences in the activity and the active site architecture, the structure of AaPgaBN was determined. The AaPgaBN structure has two metal ions in the active site instead of one found in other CE4 enzymes. Based on the crystal structure comparisons among the various CE4 enzymes, two residues, Q51 and R271, were identified in AaPgaB, which could potentially affect the enzyme activity. Of the two mutants generated, Q51E and R271K, the variant Q51E showed enhanced activity compared with AaPgaB, validating the requirement that an activating aspartate residue in the active site is essential for higher activity. In summary, our study provides the first structural evidence for a di-nuclear metal site at the active site of a member of the CE4 family of enzymes, evidence that AaPgaBN is catalytically active and that mutant Q51E exhibits higher de-N-acetylase activity.

Surface display of Aggregatibacter actinomycetemcomitans autotransporter Aae and dispersin B hybrid act as antibiofilm agents.

Among the various proteins expressed by the periodontopathogen Aggregatibacter actinomycetemcomitans, two proteins play important roles for survival in the oral cavity. The autotransporter Aae facilitates the attachment of the pathogen to oral epithelial cells, which act as a reservoir, while the biofilm-degrading glycoside hydrolase dispersin B facilitates the movement of daughter cells from the mature biofilm to a new site. The objective of this study was to use the potential of these two proteins to control biofilms. To this end, we generated a hybrid construct between the Aae C-terminal translocating domain and dispersin B, and mobilized it into Escherichia coli Rosetta (DE3) pLysS cells. Immunofluorescence analysis of the modified E. coli cells confirmed the presence of dispersin B on the surface. Further, the membrane localization of the displayed dispersin B was confirmed with Western blot analysis. The integrity of the E. coli cells displaying the dispersin B was confirmed through FACS analysis. The hydrolytic activity of the surface-displayed dispersin B was confirmed by using 4-methylumbelliferyl-ß-d-glucopyranoside as the substrate. The detachment ability of the dispersin B surface-displaying E. coli cells was shown using Staphylococcus epidermidis and Actinobacillus pleuropneumoniae biofilms in a microtiter assay. We concluded that the Aae ß-domain is sufficient to translocate foreign enzymes in the native folded form and that the method of Aae-mediated translocation of surface displayed enzymes might be useful for control of biofilms.

BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis.

Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca(2+)-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca(2+) overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC ß-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavß subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain-binding pocket in Cavß and interferes with the association between Cavß and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavß via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca(2+) channel activity at the plasma membrane, resulting in the inhibition of Ca(2+)-evoked exocytosis. Thus, BARP can modulate the localization of Cavß and its association with the Cavα1 subunit to negatively regulate VGCC activity.

Modeling and biochemical analysis of the activity of antibiofilm agent Dispersin B.

Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix (PGA), which is a linear polymer of beta(1,6)-linked N-acetylglucosamine (GlcNAc) residues. Dispersin B (DspB), a soluble glycoside hydrolase produced by the periodontal pathogen Actinobacillus actinomycetemcomitans degrades PGA. The enzyme DspB is an alpha/beta TIM-barrel protein and belongs to family 20 glycosyl hydrolases members. The enzyme activity of DspB with regard to its substrate specificity towards beta(1,6)-linked GlcNAc polymers and its endo/exo character was investigated through ligand docking and the hydrolysis of synthetic oligosaccharides. Ligand docking analysis suggested that beta(1,6)-linked GlcNAc oligosaccharide bound to the active site better that beta(1,4)-linked GlcNAc oligosaccharide. Our combined results indicate that DspB is an exo-acting enzyme that hydrolyzes beta(1,6)-linked N-acetylglucosamine oligomers.

Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport.

The members of the RGK small GTP-binding protein family, Kir/Gem, Rad, Rem and Rem2, are multifunctional proteins that regulate voltage-gated calcium channel activity and cell shape remodeling. Calmodulin (CaM) or CaM 14-3-3 are regulators of RGK functions and their association defines the subcellular localization of RGK proteins. Abolition of CaM association results in the accumulation of RGK proteins in the nucleus, whereas 14-3-3 binding maintains them in the cytoplasm. Kir/Gem possesses nuclear localization signals (NLS) that mediate nuclear accumulation through an importin alpha5-dependent pathway (see Mahalakshmi RN, Nagashima K, Ng MY, Inagaki N, Hunziker W, Béguin P. Nuclear transport of Kir/Gem requires specific signals and importin alpha5 and is regulated by Calmodulin and predicted service phosphorylations. Traffic 2007; doi: 10.1111/j.1600-0854.2007.00598.x). Because the extent of nuclear localization depends on the RGK protein and the cell type, the mechanism and regulation of nuclear transport may differ. Here, we extend our analysis to the other RGK members and show that Rem also binds importin alpha5, whereas Rad associates with importins alpha3, alpha5 and beta through three conserved NLS. Predicted phosphorylation of a serine residue within the bipartite NLS affects, as observed for Kir/Gem, nuclear accumulation of Rem, but not that of Rad or Rem2. We also identify an additional regulatory phosphorylation for all RGK proteins that prevents binding of 14-3-3 and thereby interferes with their cytosolic relocalization by 14-3-3. Functionally, nuclear localization of RGK proteins contributes to the suppression of RGK-mediated cell shape remodeling. Importantly, we show that endogenous RGK proteins are localized predominantly in the nucleus of individual cells of the brain cortex 'in situ' as well as in primary hippocampal cells, indicating that transport between the nucleus and their site of action in the cytoplasm (i.e., cytoskeleton, endoplasmic reticulum or plasma membrane) is of physiological relevance for the regulation of RGK protein function.

Nuclear transport of Kir/Gem requires specific signals and importin alpha5 and is regulated by calmodulin and predicted serine phosphorylations.

Kir/Gem, together with Rad, Rem and Rem2, is a member of the RGK small GTP-binding protein family. These multifunctional proteins regulate voltage-gated calcium channel (VGCC) activity and cell-shape remodeling. Calmodulin and 14-3-3 binding modulate the functions of RGK proteins. Intriguingly, abolishing the binding of calmodulin or calmodulin and 14-3-3 results in nuclear accumulation of RGK proteins. Under certain conditions, the Ca(v)beta3-subunit of VGCCs can be translocated into the nucleus along with the RGK proteins, resulting in channel inactivation. The mechanism by which nuclear localization of RGK proteins is accomplished and regulated, however, is unknown. Here, we identify specific nuclear localization signals (NLS) in Kir/Gem that are both required and sufficient for nuclear transport. Importin alpha5 binds to Kir/Gem, and its depletion using RNA interference impairs nuclear translocation of this RGK protein. Calmodulin and predicted phosphorylations on serine residues within or in the vicinity of a C-terminal bipartite NLS regulate nuclear translocation by interfering with the association between importinalpha5 and Kir/Gem. These predicted phosphorylations, however, do not affect Kir/Gem-mediated calcium channel downregulation but rather, as shown in the accompanying paper (Mahalakshmi RN, Ng MY, Guo K, Qi Z, Hunziker W, Béguin P. Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport. Traffic 2007; doi:10.1111/j.1600-0854.2007.00599.x), interfere with cell-shape remodeling.

Detachment and killing of Aggregatibacter actinomycetemcomitans biofilms by dispersin B and SDS.

The periodontopathogen Aggregatibacter actinomycetemcomitans forms tenacious biofilms on abiotic surfaces in vitro. The objective of the present study was to measure the susceptibility of A. actinomycetemcomitans biofilms to detachment and killing by the anionic surfactant sodium dodecyl sulfate (SDS). We found that biofilms formed by a wild-type strain were resistant to detachment by SDS. In contrast, biofilms formed by an isogenic mutant strain that was deficient in the production of PGA (poly-N-acetyl-glucosamine), a biofilm matrix polysaccharide, were sensitive to detachment by SDS. Pre-treatment of wild-type biofilms with dispersin B, a PGA-degrading enzyme, rendered them sensitive to detachment by SDS and resulted in a > 99% increase in SDS-mediated cell killing. We concluded that PGA protects A. actinomycetemcomitans cells from detachment and killing by SDS. Dispersin B and SDS may be useful agents for treating chronic infections caused by A. actinomycetemcomitans and other PGA-producing bacteria.

Tyrosine sulfation of statherin.

Tyrosylprotein sulfotransferase (TPST), responsible for the sulfation of a variety of secretory and membrane proteins, has been identified and characterized in submandibular salivary glands (William et al. Arch Biochem Biophys 1997; 338: 90-96). In the present study we demonstrate the sulfation of a salivary secretory protein, statherin, by the tyrosylprotein sulfotransferase present in human saliva. Optimum statherin sulfation was observed at pH 6.5 and at 20 mm MnCl(2). Increase in the level of total sulfation was observed with increasing statherin concentration. The K(m)value of tyrosylprotein sulfotransferase for statherin was 40 microM. Analysis of the sulfated statherin product on SDS-polyacrylamide gel electrophoresis followed by autoradiography revealed (35)S-labelling of a 5 kDa statherin. Further analysis of the sulfated statherin revealed the sulfation on tyrosyl residue. This study is the first report demonstrating tyrosine sulfation of a salivary secretory protein. The implications of this sulfation of statherin in hydroxyapatite binding and Actinomyces viscosus interactions are discussed.

RGK small GTP-binding proteins interact with the nucleotide kinase domain of Ca2+-channel beta-subunits via an uncommon effector binding domain.

RGK proteins (Kir/Gem, Rad, Rem, and Rem2) form a small subfamily of the Ras superfamily. Despite a conserved GTP binding core domain, several differences suggest that structure, mechanism of action, and functional regulation differ from Ras. RGK proteins down-regulate voltage-gated calcium channel activity by binding in a GTP-dependent fashion to the Cavbeta subunits. Mutational analysis combined with homology modeling reveal a novel effector binding mechanism distinct from that of other Ras GTPases. In this model the Switch 1 region acts as an allosteric activator that facilitates electrostatic interactions between Arg-196 in Kir/Gem and Asp-194, -270, and -272 in the nucleotide-kinase (NK) domain of Cavbeta3 and wedging Val-223 and His-225 of Kir/Gem into a hydrophobic pocket in the NK domain. Kir/Gem interacts with a surface on the NK domain that is distinct from the groove where the voltage-gated calcium channel Cavalpha1 subunit binds. A complex composed of the RGK protein and the Cavbeta3 and Cav1.2 subunits could be revealed in vivo using coimmunoprecipitation experiments. Intriguingly, docking of the RGK protein to the NK domain of the Cavbeta subunit is reminiscent of the binding of GMP to guanylate kinase.

Susceptibility of staphylococcal biofilms to enzymatic treatments depends on their chemical composition.

Bacterial infections are serious complications after orthopaedic implant surgery. Staphylococci, with Staphylococcus epidermidis as a leading species, are the prevalent and most important species involved in orthopaedic implant-related infections. The biofilm mode of growth of these bacteria on an implant surface protects the organisms from the host's immune system and from antibiotic therapy. Therapeutic agents that disintegrate the biofilm matrix would release planktonic cells into the environment and therefore allow antibiotics to eliminate the bacteria. An addition of a biofilm-degrading agent to a solution used for washing-draining procedures of infected orthopaedic implants would greatly improve the efficiency of the procedure and thus help to avoid the removal of the implant. We have previously shown that the extracellular staphylococcal matrix consists of a poly-N-acetylglucosamine (PNAG), extracellular teichoic acids (TAs) and protein components. In this study, we accessed the sensitivity of pre-formed biofilms of five clinical staphylococcal strains associated with orthopaedic prosthesis infections and with known compositions of the biofilm matrix to periodate, Pectinex Ultra SP, proteinase K, trypsin, pancreatin and dispersin B, an enzyme with a PNAG-hydrolysing activity. We also tested the effect of these agents on the purified carbohydrate components of staphylococcal biofilms, PNAG and TA. We found that the enzymatic detachment of staphylococcal biofilms depends on the nature of their constituents and varies between the clinical isolates. We suggest that a treatment with dispersin B followed by a protease (proteinase K or trypsin) could be capable to eradicate biofilms of a variety of staphylococcal strains on inert surfaces.

Structural analysis of dispersin B, a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans.

Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix that holds the bacteria together in a mass and firmly attaches the bacterial mass to the underlying surface. A major component of the extracellular polysaccharide matrix in several phylogenetically diverse bacteria is PGA, a linear polymer of N-acetylglucosamine residues in beta(1,6)-linkage. PGA is produced by the Gram-negative periodontopathogen Actinobacillus actinomycetemcomitans as well as by the Gram-positive device-associated pathogen Staphylococcus epidermidis. We recently reported that A.actinomycetemcomitans produces a soluble glycoside hydrolase named dispersin B, which degrades PGA. Here, we present the crystal structure of dispersin B at 2.0A in complex with a glycerol and an acetate ion at the active site. The enzyme crystallizes in the orthorhombic space group C222(1) with cell dimensions a=41.02A, b=86.13A, c=185.77A. The core of the enzyme consists a (beta/alpha)(8) barrel topology similar to other beta-hexosaminidases but significant differences exist in the arrangement of loops hovering in the vicinity of the active site. The location and interactions of the glycerol and acetate moieties in conjunction with the sequence analysis suggest that dispersin B cleaves beta(1,6)-linked N-acetylglucosamine polymer using a catalytic machinery similar to other family 20 hexosaminidases which cleave beta(1,4)-linked N-acetylglucosamine residues.

14-3-3 and calmodulin control subcellular distribution of Kir/Gem and its regulation of cell shape and calcium channel activity.

Individual members of the RGK family of Ras-related GTPases, which comprise Rad, Gem/Kir, Rem and Rem2, have been implicated in important functions such as the regulation of voltage-gated calcium channel activity and remodeling of cell shape. The GTPase Kir/Gem inhibits the activity of calcium channels by interacting with the beta-subunit and also regulates cytoskeleton dynamics by inhibiting the Rho-Rho kinase pathway. In addition, Kir/Gem interacts with 14-3-3 and calmodulin, but the significance of this interaction on Kir/Gem function is poorly understood. Here, we present a comprehensive analysis of the binding of 14-3-3 and calmodulin to Kir/Gem. We show that 14-3-3, in conjunction with calmodulin, regulates the subcellular distribution of Kir/Gem between the cytoplasm and the nucleus. In addition, 14-3-3 and calmodulin binding modulate Kir/Gem-mediated cell shape remodeling and downregulation of calcium channel activity. Competition experiments show that binding of 14-3-3, calmodulin and calcium channel beta-subunits to Kir/Gem is mutually exclusive, providing a rationale for the observed regulatory effects of 14-3-3 and calmodulin on Kir/Gem localization and function.

The roles of histidine residues at the starch-binding site in streptococcal-binding activities of human salivary amylase.

Human salivary alpha-amylase participates in the initial digestion of starch and may be involved in the colonization of viridans streptococci in the mouth. To elucidate the role of histidine residues located near the starch-binding site on the streptococcal-binding activity, the wild type and three histidine mutants, H52A, H299A and H305A were constructed and expressed in a baculovirus system. While His52 is located near the non-reducing end of the starch-binding pocket (subsite S3/S4), the residues His299 and His305 are located near the subsites S1/S1'. For the wild type, the cDNA encoding the leader and secreted sequences of human salivary amylase was amplified by polymerase chain reaction from a human submandibular salivary-gland cDNA library, and subcloned into the baculovirus shuttle vector pVL1392 downstream of the polyhedrin promoter. Oligonucleotide-based, site-directed mutagenesis was used to generate the mutants expressed in the baculovirus system. Replacing His52 or His299 or His305 to Ala residue did not alter the bacterial-binding activity significantly, but these mutants did show differences in their catalytic activities. The mutant H52A showed negligible reduction in enzymatic activity compared to that of wild type for the hydrolysis of starch and oligosaccharides. In contrast, the H299A and H305A mutants showed a 12 to 13-fold reduction (90-92%) in starch-hydrolysing activity. In addition, the k(cat) for the hydrolysis of oligosaccharides by H299A decreased by as much as 11-fold for maltoheptaoside. This reduction was even higher (40-fold) for the hydrolysis of p-nitrophenyl maltoside, with a significant change in K(M). The mutant H305A, however, exhibited a reduction in k(cat) only, with no changes in the K(M) for the hydrolysis of oligosaccharides. The reduction in the k(cat) for the H305A mutant was almost 93% for maltoheptaoside hydrolysis. The pH activity profile for the H305A mutant was also significantly different from that of the wild type and the other two mutants. These results suggest that, although histidines at the starch-binding site of salivary amylase are involved in starch binding and catalysis, they may not participate in Streptococcus gordonii G9B binding.

Candidacidal activity prompted by N-terminus histatin-like domain of human salivary mucin (MUC7)1.

Histidine-rich peptides (histatins, Hsn) in saliva are thought to provide a non-immune defense against Candida albicans. Sequence homology search of the human salivary mucin, MUC7, against histatins revealed a domain at the N-terminus (R3-Q17) having 53% identity to Hsn-5. To determine its candidacidal activity, this 15 residue basic histidine-rich domain of MUC7 (I) was prepared by solid-phase Fmoc chemistry. Various N- and C-terminal protected derivatives of I were also synthesized to correlate the effect of peptide overall charge in exhibiting cidal potency. Candidacidal activity measurement of I and its variants showed considerable ED50 values (effective dosage required to kill 50% of candida cells), albeit greater than Hsn-5 (ED50 approximately 4-6 microM). Of the various analogs tested, N-terminal free acid (I, ED50 approximately 40 microM) and amide (V, ED50 approximately 16 microM) exhibited appreciable candidacidal activities suggesting the possible role of peptide net charge in cidal action. Blocking of N-terminus with a bulky octanoyl group showed only marginal effect on the cidal activity of I or V, indicating that hydrophobicity of these synthetic constructs may not be important for exerting such activities. Membrane-induced conformational transition from random coil to helical structures of all the test peptides implied their tendency to adapt order structures at the lipid-membrane interface similar to that of Hsn-5. However, comparison of propensity for helical structure formation vs. ED50 indicated that cidal potency of MUC7 Hsn-like peptides depends largely on electrostatic interactions irrespective of secondary structural elements. Delineation of solution structure of the most active peptide (V) by 2D-NMR revealed essentially a non-structured conformation in aqueous medium, which further supported the fact that the peptide helical structure may not be a prerequisite for posing candidacidal activity. The formation of smaller truncated peptides and/or Hsn-like fragments on proteolytic degradation of intact MUC7 in the presence of oral flora provided indirect evidence that mucin could serve as a backup candidacidal agent to salivary Hsn.

A concise methodology for the stereoselective synthesis of O-glycosylated amino acid building blocks: complete 1H NMR assignments and their application in solid-phase glycopeptide synthesis.

A facile strategy for the stereoselective synthesis of suitably protected O-glycosylated amino acid building blocks, namely, Nalpha-Fmoc-Ser-[Ac4-beta-D-Gal-(1-3)-Ac2-alpha or beta-D-GalN3]-OPfp and Nalpha-Fmoc-Thr-[Ac4-beta-D-Gal-(1-3)-Ac2-alpha or beta-D-GalN3]-OPfp is described. What is new and novel in this report is that Koenigs-Knorr type glycosylation of an aglycon serine/threonine derivative (i.e. Nalpha-Fmoc-Ser-OPfp or Nalpha-Fmoc-Thr-OPfp) with protected beta-D-Gal(1-3)-D-GalN3 synthon mediated by silver salts resulted in only alpha- and/or beta-isomers in excellent yields under two different reaction conditions. The subtle differences in stereoselectivity were demonstrated clearly when glycosylation was carried out using only AgClO4 at -40 degrees C which afforded a-isomer in a quantitative yield (alpha:beta = 5:1). On the other hand, the beta-isomer was formed exclusively when the reaction was performed in the presence of Ag2CO3/AgClO4 at room temperature. A complete assignment of 1H resonances to individual sugar ring protons and the characteristic anomeric alpha-1 H and beta-1 H in Ac4Galbeta(1-3)Ac2GalN3 alpha and/or beta linked to Ser/Thr building blocks was accomplished unequivocally by two-dimensional double-quantum filtered correlated spectroscopy and nuclear Overhauser enhancement and exchange spectroscopy NMR experiments. An unambiguous structural characterization and documentation of chemical shifts, including the coupling constants for all the protons of the aforementioned alpha- and beta-isomers of the O-glycosylated amino acid building blocks carrying protected beta-D-Gal(1-3)-D-GalN3, could serve as a template in elucidating the three-dimensional structure of glycoproteins. The synthetic utility of the building blocks and versatility of the strategy was exemplified in the construction of human salivary mucin (MUC7)-derived, O-linked glycopeptides with varied degrees of glycosylation by solid-phase Fmoc chemistry. Fmoc/tert-butyl-based protecting groups were used for the peptidic moieties in conjunction with acetyl sugar protection. The transformation of the 2-azido group into the acetamido derivative was carried out with thioacetic acid on the polymer-bound glycopeptides before the cleavage step. After cleaving the glycopeptide from the resin, the acetyl groups used for sugar OH-protection were removed with sodium methoxide in methanol. Finally, the glycopeptides were purified by reversed-phase high-performance liquid chromatography and their integrity was confirmed by proton NMR as well as by mass spectral analysis. Secondary structure analysis by circular dichroism of both the glycosylated and nonglycosylated peptides revealed that carbohydrates did not exert any profound structural effect on the peptide backbone conformation.

Structural features of the human salivary mucin, MUC7.

Human salivary mucin (MUC7) is characterized by a single polypeptide chain of 357 aa. Detailed analysis of the derived MUC7 peptide sequence reveals five distinct regions or domains: (1) an N-terminal basic, histatin-like domain which has a leucine-zipper segment, (2) a moderately glycosylated domain, (3) six heavily glycosylated tandem repeats each consisting of 23 aa, (4) another heavily glycosylated MUC1- and MUC2-like domain, and (5) a C-terminal leucine-zipper segment. Chemical analysis and semi-empirical prediction algorithms for O-glycosylation suggested that 86/105 (83%) Ser/Thr residues were O-glycosylated with the majority located in the tandem repeats. The high (approximately 25%) proline content of MUC7 including 19 diproline segments suggested the presence of polyproline type structures. CD studies of natural and synthetic diproline-rich peptides and glycopeptides indicated that polyproline type structures do play a significant role in the conformational dynamics of MUC7. In addition, crystal structure analysis of a synthetic diproline segment (Boc-Ala-Pro-OBzl) revealed a polyproline type II extended structure. Collectively, the data indicate that the polyproline type II structure, dispersed throughout the tandem repeats, may impart a stiffening of the backbone and could act in consort with the glycosylated segments to keep MUC7 in a semi-rigid, rod shaped conformation resembling a 'bottle-brush' model.

Crystal structures of the copper and nickel complexes of RNase A: metal-induced interprotein interactions and identification of a novel copper binding motif.

We report the crystal structures of the copper and nickel complexes of RNase A. The overall topology of these two complexes is similar to that of other RNase A structures. However, there are significant differences in the mode of binding of copper and nickel. There are two copper ions per molecule of the protein, but there is only one nickel ion per molecule of the protein. Significant changes occur in the interprotein interactions as a result of differences in the coordinating groups at the common binding site around His-105. Consequently, the copper- and nickel-ion-bound dimers of RNase A act as nucleation sites for generating different crystal lattices for the two complexes. A second copper ion is present at an active site residue His-119 for which all the ligands are from one molecule of the protein. At this second site, His-119 adopts an inactive conformation (B) induced by the copper. We have identified a novel copper binding motif involving the alpha-amino group and the N-terminal residues.

Crystal structure of a dipeptide Boc-Aib-Phe-OMe.

In order to understand the effect of the restrictions posed by the Aib residue on peptide conformation we studied the crystal structure of a dipeptide tBoc-Aib-Phe-OMe. Crystals of this compound are triclinic, space group P1 with a = 9.600(1) A, b = 10.262(1) A, c = 10.799(1) A, alpha = 98.43 degrees (1), beta = 99.18 degrees (1), gamma = 98.87 degrees (1), V = 1021.69(18) A3 and Z = 2. The structure was solved by direct methods and refined to an R-factor of 4.98%. The backbone conformational angles for the Aib residue in molecule A are in the left-handed helical region, while in molecule B they are in the right-handed helical region. The Phe residue in molecule A is in the right-handed helical conformation, while in molecule B it is in the beta-region. The peptide units are trans and show significant deviation from planarity [(omega 1 = 166.67(5) degrees and omega 2 = -177.9(5)].