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The current research investigated mindfulness, savoring and temporal balance and how they are related to temporal perspective (TP) in materialistic individuals in a sample of 404 University students. Recent research on materialism and TP has found that highly materialistic individuals are more likely to have a past-negative or present fatalistic perspective rather than a balanced temporal perspective which is considered the most adaptive. Given these findings, it was hypothesized that materialism is negatively related to mindfulness and savoring, which involve having a clear focus on the present moment. It was also hypothesized that materialism is positively associated with the unbalanced types of TP, the "reminiscer", "futurist" and "time restrictive" and that materialism is associated with deviation from the balanced time perspective (DBTP). In addition, it was hypothesized that mindfulness and savoring mediate the relationship between DBTP and materialism. The results are consistent with these hypotheses as mindfulness was negatively related to materialism and savoring the moment was related to lower levels of materialism. Materialism was also associated with higher DBTP scores and the "reminiscer" and "futurist" unbalanced temporal styles. Mindfulness, savoring the moment and anticipation-savoring were found to be mediators in the DBTP and materialism relationship. [ABSTRACT FROM AUTHOR]

Psychological flexibility, or the ability to flexibly respond to psychological events, may be associated with peoples' preferences for material versus experiential purchases, their attachment to objects and experiences, and their motivation for non-material purchases. The present research tested predictions that greater psychological flexibility and lesser psychological inflexibility are associated with lower materialism and non-attachment (Study 1) and with a greater preference for, and internal motivation toward, experiential versus material purchases (Study 2). Study 1 (N = 298 undergraduates) revealed relationships between psychological inflexibility, high materialism, and attachment, and between psychological flexibility, low materialism, and non-attachment. Non-attachment was a meditator in the relationships between both flexibility and inflexibility and materialism. Study 2 (N = 299 undergraduates) revealed that psychological flexibility was correlated with both experiential buying and autonomous reasons for experiential buying, whereas inflexibility was related to controlled and amotivated reasons for experiential buying. We discuss the research and practice implications of the current findings. • Psychological flexibility/inflexibility & non-attachment correlate with materialism. • Non-attachment mediates the relationship between flexibility and materialism. • Psychological flexibility is related to experiential over material purchases. • Flexibility is related to autonomous motivation for experiential purchases. • Inflexibility is related to heteronomous motivation for experiential purchases. [ABSTRACT FROM AUTHOR]

Legionaminic acids (Leg) are bacterial analogs of neuraminic acid, with the same stereochemistry but different substituents at C5, C7 and C9. Hence they may be incorporated into useful analogs of sialoglycoconjugates, and we previously reported two sialyltransferases that could utilize cytidine monophosphate (CMP)-Leg5Ac7Ac for preparation of Leg glycoconjugates, which were resistant to sialidases [Watson DC, Leclerc S, Wakarchuk WW, Young NM. 2011. Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid. Glycobiology. 21:99-108.]. These were the porcine ST3Gal1 and Pasteurella multocida sialyltransferases. We now report two additional sialyltransferases with superior Leg-transferase properties to the previous two. These are (i) a truncated form of a Photobacterium a2,6-sialyltransferase with an Ala-Met mutation in its active site, and (ii) an a2,3-sialyltransferase from Neisseria meningitidis MC58 with a higher transferase activity than the P. multocida enzyme, with either CMP-Neu5Ac or CMP-Leg5Ac7Ac as the donor. These enzymes will enable the production of useful Leg5Ac7Ac glycoconjugate derivatives with either a2,6 or a2,3 linkages and unique biological properties. [ABSTRACT FROM AUTHOR]

New functions can be incorporated into anti-hapten or anti-protein antibodies by mutating selected residues in the binding-site region either to Cys, to allow alkylation with reagents bearing the desired functional groups, or to His, to create metal-binding sites or to make antigen binding pH-sensitive. However, choosing suitable sites for these mutations has been hampered by the lack of antibodies with these features, to serve as models. Remarkably, the anti-carbohydrate antibody Se155-4, specific for the Salmonella group B lipopolysaccharide, already has a Cys and two pairs of His residues close to the antigen-binding pocket in its structure, and shows pH-dependent antigen binding. We therefore investigated modification of its Cys94L in an scFv version of the antibody with the aims of creating a ‘reagentless’ fluorescent sensor and attaching a metal-binding group that might confer lyase activity. These groups were successfully introduced, as judged by mass spectrometry, and had only slightly reduced antigen binding in enzyme-linked immunosorbent assay. The fluorescent product was sensitive to addition of antigen in a solution format, unlike a modification of a more distant Cys introduced into the VH CDR4 loop. Two other routes to modulate antigen binding were also explored, metal binding by the His pair alongside the antigen-binding pocket and insertions into CDR4 to extend the antigen-contact area. His residues adjacent to the antigen-binding pocket bound copper, causing a 5-fold decrease in antigen binding. In CDR4 of the VH domain, the preferred insert length was four residues, which gave stable antigen-binding products but did not improve overall antigen affinity. [ABSTRACT FROM AUTHOR]

Highlights: [•] Agreeableness, neuroticism and materialism were investigated. [•] High levels of materialism were found with high neuroticism and low agreeableness. [•] High neurotic and agreeable and low neurotic and agreeable were medium materialist. [•] Low levels of materialism were found with low neuroticism and high agreeableness. [•] Agreeableness did not moderate relationship between neuroticism and materialism. [Copyright &y& Elsevier]

The relationship between self-reported gossip, evaluative conversation about other people, and the self was examined with 243 university students completing questionnaires measuring gossip from 2 theoretical viewpoints: as an individual-difference characteristic, or as having social functions. The self was examined using several perspectives: self-concept clarity, self-efficacy, locus of control, and self-monitoring. Using structural equation modeling, gossip was related to external locus of control, high-self-monitoring, low self-concept clarity, and low self-efficacy. The final model is that high self-monitoring and locus of control mediate the relationship between self-clarity/efficacy and gossip. The study demonstrates the important role of self-monitoring and locus of control in gossip and that negative gossip may be associated with a more externalized, unclear sense of self. [ABSTRACT FROM AUTHOR]

In addition to sialic acid, bacteria produce several other nonulosonic acids, including legionaminic acid (Leg). This has exactly the same stereochemistry as sialic acid, with the added features of 9-deoxy and 7-amino groups. In order to explore the biological effects of replacing sialic acid residues (Neu5Ac) in glycoconjugates with Leg in its diacetylated form, diacetyllegionaminic acid (Leg5Ac7Ac), we tested CMP-Leg5Ac7Ac as a donor substrate with a selection of bacterial and mammalian sialyltransferases. The CMP-Leg5Ac7Ac was synthesized in vitro by means of cloned enzymes from the bacillosamine portion of the Campylobacter jejuni N-glycan pathway and from the Leg pathway of Legionella pneumophila. Using fluorescent derivatives of lactose, Galβ1,4GlcNAcβ and T-antigen (Galβ1,3GalNAcα) as acceptors, we tested eight different sialyltransferases and found that the Pasteurella multocida PM0188h and porcine ST3Gal1 sialyltransferases were significantly active with CMP-Leg5Ac7Ac, showing ∼60% activity when compared with CMP-Neu5Ac. The Photobacterium α2,6 sialyltransferase was weakly active, with ∼6% relative activity. The Leg5Ac7Ac-α-2,3-lactose product was then tested as a substrate with six sialidases of viral, bacterial and mammalian origin. All showed much lower activities than with the corresponding sialic acid substrate, with the influenza virus N1 being the most active and human NEU2 being the least active. These results show the feasibility of producing glycoconjugates with Leg5Ac7Ac residues as the terminal sugars, which should display novel biological properties. [ABSTRACT FROM AUTHOR]

The question of whether emotion control and coping are separate independent concepts or overlapping constructs was explored with a sample of 422 university students using the Emotional Control Questionnaire and the Coping Inventory for Stressful Situations. In addition, the relationship between emotion control, coping, and psychological symptoms was explored using the Brief Symptom Inventory. Evidence was found for both independence and overlap between emotion control and coping. Emotion focused coping was a strong predictor of psychopathology. Rehearsal and benign control were associated with the set of psychological symptoms. Aggression control was related to hostility and interpersonal sensitivity. Coping-emotion control combinations and gender differences were also examined in terms of the psychological symptoms. [ABSTRACT FROM AUTHOR]

Legionaminic acid is a nine-carbon α-keto acid that is similar in structure to other members of the sialic acid family that includes neuraminic acid and pseudaminic acid. It is found as a component of the lipopolysaccharide in several bacterial species and is perhaps best known for its presence in the O-antigen of the causative agent of Legionnaires' disease, Legionella pneumophila. In this work, the enzymes responsible for the biosynthesis and activation of N,N′-diacetyllegionaminic acid are identified for the first time. A cluster of three L. pneumophila genes bearing homology to known sialic acid biosynthetic genes (neuA,B,C) were cloned and overexpressed in Escherichia coli. The NeuC homologue was found to be a hydrolyzing UDP-N,N′-diacetylbacillosamine 2-epimerase that converts UDP-N,N′- diacetylbacillosamine into 2,4-diacetamido-2,4,6-trideoxymannose and UDP. Stereochemical and isotopic labeling studies showed that the enzyme utilizes a mechanism involving an initial anti elimination of UDP to form a glycal intermediate and a subsequent syn addition of water to generate product. This is similar to the hydrolyzing UDP-N-acetylglucosamine 2-epimerase (NeuC) of sialic acid biosynthesis, but the L. pneumophila enzyme would not accept UDP-GIcNAc as an alternate substrate. The NeuB homologue was found to be a N,N′-diacetyllegionaminic acid synthase that condenses 2,4-diacetamido-2,4,6- trideoxymannose with phosphoenolpyruvate (PEP), although the in vitro activity of the recombinant enzyme. (isolated as a MalE fusion protein) was very low. The synthase activity was dependent on the presence of a divalent metal ion, and the reaction proceeded via a C-O bond cleavage process, similar to the reactions catalyzed by the sialic acid and pseudaminic acid synthases. Finally, the NeuA homologue was shown to possess the CMP-N,N′-diacetyllegionaminic acid synthetase activity that generates the activated form of legionaminic acid used in lipopolysaccharide biosynthesis. Together, the three enzymes constitute a pathway that converts a UDP-linked bacillosamine derivative into a CMP-linked legionaminic acid derivative. [ABSTRACT FROM AUTHOR]

This cross-cultural research study aimed to explore the relationship of stress and coping with psychological illness or symptoms among university students in Canada and India. The predictor variables were stress (hassles and life experience), 8 ways of coping (Folkman & Lazarus, 1988), and selected personal-social variables, namely, locus of control, self-esteem, and social support. The criterion variables were 9 psychological symptoms of the Brief Symptom Inventory (BSI; Derogatis & Spencer, 1982). The results revealed that the Indian students reported more psychological symptoms compared to the Canadian students. Stepwise multiple-regression analyses also revealed considerable differences between the 2 samples with respect to the contribution of predictor variables in accounting for variance in the BSI scales. [ABSTRACT FROM AUTHOR]

Coolidge (2001) has developed a short-form, the short Coolidge axis-two inventory (SCATI), a 70-item version of the original 225-item Coolidge axis-II inventory (CATI). In a normative study of this instrument, the test was found to have many of the psychometric properties of the original. Multivariate analysis revealed that the internal structure of the SCATI is similar to the original. [ABSTRACT FROM AUTHOR]

Abstract: Conceptualizations of procrastination as self-regulation failure have received considerable empirical support. However, procrastination has only occasionally been examined in relation to variables emphasized in models of self-regulated learning. We examined relations between procrastination, achievement goal orientations, and learning strategies. One hundred and seventy undergraduates completed measures of procrastination, goal orientation, and learning strategies usage. Bivariate correlations revealed that procrastination related negatively to a mastery-approach goal orientation and positively to a mastery-avoidance goal orientation. Procrastination was also related to greater disorganization and less use of cognitive and meta-cognitive strategies. Multiple regression analyses revealed that disorganization and cognitive strategies usage were most predictive of procrastination. Results are interpreted within a self-regulation perspective. [Copyright &y& Elsevier]

Abstract: Using a series of computer-based assignments, we examined whether students’ submission patterns revealed a hyperbolic pattern of temporal discounting, such that few assignments are submitted far ahead of the deadline and submission of assignments accelerates at an increasing rate as the deadline becomes imminent. We further examined whether variables related to self-regulation – namely, self-reported procrastination, implementation intentions, say-do correspondence, and perceived academic control – correlated with behavioural postponement. Results revealed strong behavioural evidence of temporal discounting, especially among those who identified themselves as procrastinators. Among the self-regulation measures, only say-do correspondence consistently correlated with procrastination. [Copyright &y& Elsevier]

Recent research has established a pattern of impairment and distress associated with the symptoms of DSM-IV Axis-II disorders such that personality disorders occurring more commonly among males are associated with more social and occupational impairment, but less personal distress, than personality disorders occurring more commonly among females. The current study examined whether a similar pattern exists for DSM-IV Axis-I disorders. Lay judges (N = 206) rated the social impairment, occupational impairment, and personal distress associated with the symptoms of six male-typed and six female-typed Axis-I disorders. Impairment and distress were associated with male-typed and female-typed Axis-I disorders in the same manner as personality disorders. Reasons for the emphasis of social and occupational impairment among male-typed disorders and distress among female-typed disorders are discussed. © 2004 Wiley Periodicals, Inc. J Clin Psychol. [ABSTRACT FROM AUTHOR]

Dysfunction in personality disorder symptoms was assessed using multivariate techniques to analyse lay judges' (N = 216) ratings of occupational impairment, social impairment, and personal distress. Factor analysis revealed that ratings of occupational impairment and social impairment loaded onto distinct factors. Personal distress ratings loaded onto two separate factors: high distress and low distress. Multidimensional scaling revealed two dimensions for overall dysfunction among personality disorders: severity of dysfunction and internalization-externalization. The dimensions were independence-dependence and severity of dysfunction for occupational impairment, interpersonal involvement and dominance-submission for social impairment, and internalization-externalization and severity for personal distress. [ABSTRACT FROM AUTHOR]

This research examined the relative contribution of 20 defence mechanisms assessed by the 40-item Defence Style Questionnaire (DSQ-40) as discriminating variables in predicting membership in personality disorder (PD) clusters. The odd, dramatic and anxious clusters of DSM-III--R and the detached, dependent, independent and ambivalent groups of Millon were used as PD types. PDs were assessed by the Coolidge Axis II Inventory and the Millon Clinical Multiaxial Inventory--II in a non-clinical sample. Results of discriminant function analysis provide empirical support for the usefulness of both DSM and Millon classification schemes. The DSM's odd cluster and Millon's ambivalent types are classified most accurately with the CATI. The MCMI-II classifies most accurately the DSM's odd and dramatic, and Millon's independent and ambivalent, personality types. [ABSTRACT FROM AUTHOR]

Examines the complex correlation of personality disorders with self-esteem, locus of control, interpersonal and five-factor models of personality. Discussion of psychology terms; Use of Coolidge Axis-II Inventory, Millon Clinical Multiaxial Inventory-II and other rating scales; Classification of personality disorders.

Presents a study in which a number of university students who were administered the Coolidge Axis II Inventory (CATI), were examined for comorbidity personality disorders. Comparison of comorbidity patterns with clinical data reported by other researchers; What the results confirm; How the DSM-IV personality disorders were conceived; Results of the study.

The complete amino acid sequence of a basic non-histone protein, H6, isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells, has been determined. Protein H6, first described by D. T. Wigle and G. H. Dixon [J. Biol. Chem. 246, 5636-5644 (1971)] was extracted with 5% trichloracetic acid and purified by ion-exchange chromatography on carboxymethylcellulose (CM-52). Sequence analysis was performed by automatic Edman degradation of the amino terminus of the intact protein and a series of large fragments derived by cleavage with chymotrypsin, staphylococcal protease and with mild acid to cleave at aspartic acid residues. Protein H6 possesses 69 residues and shows considerable similarities to the 89-residue calf thymus HMG-17 protein previously sequenced [Walker, J. M., Hastings, J. R. B. & Johns, E. W. (1977) Eur. J. Biochem. 76, 461-468]. B. Levy W. and G. H. Dixon [Proc. Natl Acad. Sci. U.S.A. 74, 2810-2814 (1977)] have shown that H6 is selectively solubilized when trout testis nuclei (or chromatin) are digested with DNase I under conditions which preferentially hydrolyze that portion of DNA enriched in transcribed sequences [Levy, W. B. & Dixon, G. H. (1977) Nucleic Acids Res. 4, 883-898]. Recently H6 has been located as a stoichiometric component of a distinct subset of trout testis nucleosomes that are complexed with a core nucleosome comprising 140 base pairs of DNA and the inner histones H2A, H2B, H3 and H4 [Levy, W. B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem., in the press]. [ABSTRACT FROM AUTHOR]

A specific non-histone chromatin-associated protein, having a high content of both acidic and basic amino acids has been isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells. The protein has been prepared by the extraction of chromatin with 0.35 M sodium chloride and purified by chromatography on carboxymethyl-cellulose with a gradient of lithium chloride at pH 9.0. The amino acid sequence of the first 29 residues of the amino-terminal region has been determined using an automatic protein sequencer. The primary structure of this protein differs from that of any of the histones yet sequenced and therefore, cannol be a degradation product of any of them. Moreover, the N-terminal amino acid sequence shows considerable similarity to the HMG-1 and HMG-2 chromosomal proteins described by Goodwin et al. [Eur. J. Biochem. 38. t4-19 (1973)] whose N-terminal sequences were also determined in this laboratory. [ABSTRACT FROM AUTHOR]

Two tryptic phosphopeptides containing the sites on the α and β subunits of phosphorylase kinase which are phosphorylated by protein kinase, dependent on adenosine 3': 5'-monophosphate (cyclic AMP), have been isolated and their amino acid sequences have been determined. 32p-labelled phosphorylase kinase, containing 1.9 mol phosphate per mol enzyme, was digested with an equimolar quantity of trypsin for 2.5 min at pH 7.0, 20 °C. This treatment released nearly all the 32P radioactivity associated with the β subunit as trichloroacetic-acid-soluble material. Only a small proportion of the 32P radioactivity associated with the α subunit was solubilised, the remainder being removed in the trichloroacetic acid pellet. The β-subunit tryptic phosphopeptide was completely resolved from traces of the α-subunit phosphopeptide by gel filtration on Sephadex G-25. Further purification by peptide mapping separated the phosphopeptide into four components, each derived from the same nine-amino-acid segment of the βchain, which was found to possess the sequence:Gln-Ser-Gly-Ser(P)-ValIle-TyrPro-Leu-Lys. The four components were produced by the partial cyclisation of the N-terminal glutaminyl residue, and by the presence of two alleles for the β subunit in the rabbit population, which led to a valine-isoleucine ambiguity. The α-subunit phosphopeptide was liberated from the trichloroacetic acid pellet by redigestion with trypsin. It was the largest component in the digest which remained soluble in 5 % trichloreacetic acid, and obtained in a highly purified form by a single filtration on Sephadex G-50. The peptide comprised 39 amino acids of which nine were serine and three were threonine residues. Only one residue, the serine at position three from the amino terminus, was phosphorylated. The amino-terminal sequence of the peptide was shown to be: Arg-Leu-Ser(P)-Ile-Ser-Thr-GluSer-Glx-Pro-Asx-Gly. The sequences confirm the stoichiometry of the reaction and the absolute specificity of cyclicAMP-dependent protein kinase for just two of the 200 serine residues in the enzyme. These results and an inspection of the rate of phosphorylation of a number of skeletal muscle proteins, including each enzyme of the glycolytic pathway, lead to the conclusion that cyclic-AMP-dependent · protein kinase is an extremely specific enzyme. The molecular basis of this specificity is discussed. [ABSTRACT FROM AUTHOR]

Presents information on a study of gender, age, and cultural differences using the Defense Style Questionnaire-40. Definition of Defense Style Questionnaire-40; Classes of persons from whom cross-cultural comparison was made; Reason differences were noted; Findings about internal structure of defense styles; Indepth look at study.

The Coolidge Axis-II Inventory (CATI) is a 200-item self-report inventory that is designed to measure DSM-IV personality disorders (PD's). Norms with a sample of 1,790 Canadian college students are compared with a US sample of 573 college students and 36 elderly subjects. Comparisons for the 11 DSM-IV personality disorder scales are made with several age groups and with gender. Some interesting similarities and differences were observed. With the Canadian sample, a gender difference was found on the antisocial scale. Age differences were found for on several PD scales in that younger respondents (17–24 years) scored higher than the older ones (25–57 years). This information is likely to be helpful to clinicians in monitoring age related behavioral changes in personality disorder. [ABSTRACT FROM AUTHOR]

Oncomodulin is an acidic calcium-binding protein of 108 amino acids which has been detected only in tumors. The complete amino acid sequence of rat oncomodulin has been established, and the N-terminus shown to be N-acetylserine. Homology of the oncomodulin sequence with members of the troponin C superfamily of calcium-binding proteins was demonstrated, and putative calcium-binding domains were located. A further degree of homology was established between oncomodulin and the parvalbumins, including rat parvalbumin. Oncomodulin was therefore considered to be a parvalbumin-like calcium-binding protein. [ABSTRACT FROM AUTHOR]

ABSTRACT: PEB3 is a glycoprotein adhesin from Campylobacterjejuni whose structure suggested a role in transport. We have investigated potential ligands for PEB3 and characterized their binding properties using biophysical methods in solution and by X-ray crystallography. A thermal aggregation assay of PEB3 with a library of physiological compounds identified three possible ligands [3-phosphoglycerate (3-PG), phosphoenolpyruvate (PEP), and aconitate], which stabilized wild-type PEB3 but did not stabilize either a PEB3 form containing two mutations at the ligand-binding site, T138A/S139A, or a second PEB3 mutant, K I 35E, at a site ∼14 eeÅ away. Fluorescence titration experiments and cocrystal structures with various ligands were used to characterize the binding of 3-PG, PEP, and phosphate to PEB3. Further, a C. jejuni growth experiment in minimal medium supplemented with 3-PG showed that this molecule enhances the growth of wild-type C. jejuni, but not of the PEB3 mutants. Crystallographic analysis of PEB3 complexes revealed that the Serl7l-G1n180 region in the presence of 3-PG or other phosphates is helical and similar to those of other transport proteins, but it is nonhelical when citrate is bound. The K135E mutation resulted in expression of a more highly glycosylated form of PEB3 in vivo, and its crystal structure showed the conformation of the first two residues of the glycan. On the basis of our findings, we suggest that PEB3 is a transport protein that may function in utilization of 3-PG or other phosphate-containing molecules from the host. [ABSTRACT FROM AUTHOR]

The structure and specificity of the recombinant α-2,3-sialyltransferase from Neisseria meningitidis are reported. This enzyme showed an unusual acceptor specificity in that it could use a-terminal and β-terminal Ga] residues as acceptors, in addition (β1→4]-linked and (β1→3)-linked terminal Gal served as acceptors. These properties distinguish the bacterial enzyme from the more widely investigated mammalian equivalents. The protein was expressed as a membrane-associated protein in Escherichia coli at a level of 750 U/I(≈250 mg/l). The protein could be extracted with buffers containing 0.2% Triton X-100 and purified to homogeneity using immobilized-metal-affinity chromatography. Electrospray-ionization mass spectrometry of peptides obtained by cleavage with cyanogen bromide and trypsin confirmed over 95 % of the deduced amino acid sequence. When used for enzymatic synthesis in coupled reactions with recombinant CMP-Neu5Ac synthetase, the α-2,3-sialyltransferase could sialylate fluorescent derivatives of N-acetyllactosamine with N-acetylneuraminic acid. N-propionylneuraminic acid and N-glycoloylneuraminic acid. [ABSTRACT FROM AUTHOR]

The amino acid sequence of peanut (Arachis hypogaea) agglutinin was determined from three major fragments obtained by mild acid cleavage at Asp-Pro peptide bonds. The sequence of 236 amino acids has residues identical to those that form the metal-binding site and the hydrophobic pocket in concanavalin A and other lectins, although the overall similarity is only 42%. In the segments of peanut agglutinin that correspond to the four loops that form the carbohydrate-binding site in concanavalin A and ravin, several central residues are homologous, while others show changes to smaller side chains, such as Tyr → Gly. The carbohydrate-binding site of peanut agglutinin may therefore have a similar peptide-backbone architecture, but form a considerably more open cleft. [ABSTRACT FROM AUTHOR]

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH 2 and 59°C. This extremophile is remarkable by the absence of a cell wall or an S-layer. Treating the cells with Triton X-100 at pH 3 allowed the extraction of all of the cell surface glycoproteins while keeping cells intact. The extracted glycoproteins were partially purified by cation-exchange chromatography, and we identified five glycoproteins by N-terminal sequencing and mass spectrometry of in-gel tryptic digests. These glycoproteins are positive for periodic acid-Schiff staining, have a high content of Asn including a large number in the Asn-X-Ser/Thr sequon and have apparent masses that are 34–48% larger than the masses deduced from their amino acid sequences. The pooled glycoproteins were digested with proteinase K and the purified glycopeptides were analyzed by NMR. Structural determination showed that the carbohydrate part was represented by two structures in nearly equal amounts, differing by the presence of one terminal mannose residue. The larger glycan chain consists of eight residues: six hexoses, one heptose and one sugar with an unusual residue mass of 226 Da which was identified as 6-deoxy-6-C-sulfo-d-galactose (6-C-sulfo-d-fucose). Mass spectrometry analyses of the peptides obtained by trypsin and chymotrypsin digestion confirmed the principal structures to be those determined by NMR and identified 14 glycopeptides derived from the main glycoprotein, Ta0280, all containing the Asn-X-Ser/Thr sequons. Thermoplasma acidophilum appears to have a “general” protein N-glycosylation system that targets a number of cell surface proteins. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is highly unusual among bacteria in forming N-linked glycoproteins. The heptasaccharide produced by its pgl system is attached to protein Asn through its terminal 2,4-diacetamido- 2,4,6-trideoxy-D-Glc (QuiNAc4NAc or N,N'-diacetylbacillosamine) moiety. The crucial, last part of this sugar's synthesis is the acetylation of UDP-2-acetamido-4-amino-2,4,6-trideoxy-D-Glc by the enzyme PglD, with acetyl-CoA as a cosubstrate. We have determined the crystal structures of PglD in CoA-bound and unbound forms, refined to 1.8 and 1.75 Å resolution, respectively. PglD is a trimer of subunits each comprised of two domains, an N-terminal α/β-domain and a C-terminal left-handed β-helix. Few structural differences accompany CoA binding, except in the C-terminal region following the β-helix (residues 189-195), which adopts an extended structure in the unbound form and folds to extend the β-helix upon binding CoA. Computational molecular docking suggests a different mode of nucleotide–sugar binding with respect to the acetyl-CoA donor, with the molecules arranged in an "L-shape", compared with the "in-line" orientation in related enzymes. Modeling indicates that the oxyanion intermediate would be stabilized by the NH group of Gly143', with His125' the most likely residue to function as a general base, removing H+ from the amino group prior to nucleophilic attack at the carbonyl carbon of acetyl-CoA. Site-specific mutations of active site residues confirmed the importance of His125', Glu124', and Asn118. We conclude that Asn118 exerts its function by stabilizing the intricate hydrogen bonding network within the active site and that Glu124' may function to increase the pKa of the putative general base, His 125'. [ABSTRACT FROM AUTHOR]

The Campylobacter jejuni pgl locus encodes an N-linked protein glycosylation machinery that can be functionally transferred into Escherichia coli. In this system, we analyzed the elements in the C. jejuni N-glycoprotein AcrA required for accepting an N-glycan. We found that the eukaryotic primary consensus sequence for N-glycosylation is N terminally extended to D/E-Y-N-X-S/T (Y, X≠P) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were N-glycosylated when they were either artificially introduced or when they were present in non-C. jejuni proteins. We were able to produce recombinant glycoproteins with engineered N-glycosylation sites and confirmed the requirement for a negatively charged side chain at position −2 in C. jejuni N-glycoproteins. N-glycosylation of AcrA by the eukaryotic OST in Saccharomyces cerevisiae occurred independent of the acidic residue at the −2 position. Thus, bacterial N-glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence. [ABSTRACT FROM AUTHOR]