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Bacterial endospores derive much of their longevity and resistance properties from the relative dehydration of their protoplasts. The spore cortex, a peptidoglycan structure surrounding the protoplasm, maintains, and is postulated to have a role in attaining, protoplast dehydration. A structural modification unique to the spore cortex is the removal of all or part of the peptide side chains from the majority of the muramic acid residues and the conversion of 50% of the muramic acid to muramic lactam. A mutation in the cwlD gene of Bacillus subtilis, predicted to encode a muramoyi-L-alanine amidase, results in the production of spores containing no muramic lactam. These spores have normally dehydrated protoplasts but are unable to complete the germination/outgrowth process to produce viable cells. Addition of germinants resulted in the triggering of germination with loss of spore refractility and the release of dipicolinic acid but no degradation of cortex peptidoglycan. Germination in the presence of lysozyme allowed the cwlD spores to produce viable cells and showed that they have normal heat resistance properties. These results (i) suggest that a mechanical activity of the cortex peptidoglycan is not required for the generation of protoplast dehydration but rather that it simply serves as a static structure to maintain dehydration, (ii) demonstrate that degradation of cortex peptidoglycan is not required for spore solute release or partial spore core rehydration during germination, (iii) indicate that muramic lactam is a major specificity determinant of germination lytic enzymes, and (iv) suggest the mechanism by which the spore cortex is degraded during germination while the germ cell wall is left intact.

Peptidoglycans was prepared from purified Bacillus subtilis spores of wild-type and several mutant strains. Reversed-phase high-pressure liquid chromatography, amino acid and amino sugar analysis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were utilized to study the peptidoglycan structure in wild-type and mutant spores. In wild-type spores, 50% of the muramic acid was converted to lactam. The remaining 25% had tetrapeptide or tripeptide side chains. Spores of a dacB mutant lacked the sporulation-specific penicillin-binding protein. Only 46% of the muramic acid in these spores was in lactam form.

Abstract Background Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses. Results The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Lex and H type 2 antennae in sialylated complex-type N-glycans. Conclusion The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.

Research has been conducted on Lactococcus lactis subsp. lactis nisin. The authors report that His-tagged nisin precursors from L. lactis mutant strains have been purified and analyzed, and demonstrate that NisB is required for dehydration reactions and that NisC is required for lanthionine formation in nisin biosynthesis.

We have recently identified two novel cysteine proteinase inhibitorsfrom the skin of Atlantic salmon (Salmo salar L.), namedsalmon kininogen and salarin. In preliminary experiments, the proteinswere found to be both N- as well as O-glycosylated. In the presentstudy we show that both proteins carry biantennary α2,3-sialylatedN-glycans. A very high amount of O-acetylated Neu5Ac units are present inthe N-glycans, comprising about 60% di-O-acetylated species.Non-O-acetylated Neu5Ac make up less than 5% of the sialicacids in the N-glycans. A small number of Neu5Acα2-8Neu5Acstructures were observed in the N-glycans as well. O-glycans fromboth proteins were recovered by reductive beta-elimination and wereidentified by mass spectrometric methods as mono- and disialylatedcore type 1 tri- and tetrasaccharides. The method used for O-glycan isolationprevented the identification of possible O-acetylation in the O-glycan-boundsialic acids, but O-acetylation was observed in one O-glycosylatedpeptide isolated from trypsin digest of salarin. The chemical natureof the sialic acid modifications was further studied by liquid chromatographytandem mass spectrometry of 1,2-diamino-4,5-methylenedioxybenzene–derivatizedsialic acids, revealing 7-, 8-, and 9- but no 4-O-acetylation. Toour knowledge, these are the first observations of sialic acid O-acetylation inN-glycans on fish species and represent clearly the most extensiveN-glycan O-acetylation described on any species. [ABSTRACT FROM PUBLISHER]

Human α3-fucosyltransferases(Fuc-Ts) are known to convert N-acetyllactosamine to Galβ1-4(Fucα1-3)GlcNAc (Lewis x antigen); someof them transfer fucose also to GalNAcβ1-4GlcNAc,generating GalNAcβ1-4(Fucα1-3)GlcNAc determinants.Here, we report that recombinant forms of Fuc-TV and Fuc-TVI aswell as Fuc-Ts of human milk converted chitin oligosaccharides of2–4 GlcNAc units efficiently to products containing a GlcNAcβ1-4(Fucα1-3)GlcNAcβ1-4R determinant at the nonreducingterminus. The product structures were identified by mass spectrometry andnuclear magnetic resonance experiments; rotating frame nuclear Overhauserspectroscopy data suggested that the fucose and the distal N-acetylglucosamineare stacked in the same way as the fucose and the distal galactose ofthe Lewis x determinant. The products closely resembled a nodulationfactor of Mesorhizobium loti but were distinct fromnodulation signals generated by NodZ-enzyme. [ABSTRACT FROM PUBLISHER]

Fucosylation of glycans on glycoproteins and -lipids requires the enzymatic activity of relevant fucosyltransferases and GDP-L-fucose as the donor. Due to the biological importance of fucosylated glycans, a readily accessible source of GDP-L-fucose would be required. Here we describe the construction of a stable recombinant S.cerevisiae strain expressing the E.coli genes gmd and wcaG encoding the two enzymes, GDP-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GMER(FX)) respectively, needed to convert GDP-mannose to GDP-fucose via the de novo pathway. Taking advantage of the rich inherent cytosolic GDP-mannose pool in S.cerevisiae cells we could easily produce 0.2 mg/l of GDP-L-fucose with this recombinant yeast strain without addition of any external GDP-mannose. The GDP-L-fucose product could be used as the fucose donor for α1,3fucosyltransferase to synthesize sialyl Lewis x (sLex), a glycan crucial for the selectin-dependent leukocyte traffic. [ABSTRACT FROM PUBLISHER]

Toxins from three Bacillus licheniformis strains connected to a fatal food poisoning were isolated and their structures elucidated. Toxins were purified from methanol extracts of the B. licheniformis biomass using boar sperm cells as the toxicity indicator. The HPLC purified toxins showed protonated masses m/z 1007, 1021 and 1035 in MALDI-TOF-MS. The toxins isolated from the strains of different origins contained the same three components of which and each had a same amino-acid residues l-Gln, l-Leu, d-Leu, l-Val, l-Asp, d-Leu and l-Ile in that order. Toxins were identified as lichenysin A, a cyclic lactonic heptalipopeptide in which the main 3-hydroxy fatty acids are 13–15 carbons in length. We showed that the toxins from food and food poisoning isolates of B. licheniformis were identical to lichenysin A both in the structure and in the toxic symptoms induced to boar spermatozoa. Confocal laser scanning microscopy showed that the acrosome and the plasma membrane of boar spermatozoa were the targets of lichenysin A toxicity. [ABSTRACT FROM AUTHOR]

We report that isomeric monofucosylhexasaccharides, Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAc, Galβ1–4GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4GlcNAc and Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc, and bifucosylhexasaccharides Galβ1–4GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAc, Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAc and Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4GlcNAc can be isolated in pure form from reaction mixtures of the linear hexasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc with GDP‐fucose and α1,3‐fucosyltransferases of human milk. The pure isomers were characterized in several ways; 1H‐NMR spectroscopy, for instance, revealed distinct resonances associated with the Lewis x group [Galβ1–4(Fucα1–3)GlcNAc] located at the proximal, middle, and distal positions of the polylactosamine chain. Chromatography on immobilized wheat germ agglutinin was crucial in the separation process used; the isomers carrying the fucose at the reducing end GlcNAc possessed particularly low affinities for the lectin. Isomeric monofucosyl derivatives of the pentasaccharides GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc and Galα1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc and the tetrasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc were also obtained in pure form, implying that the methods used are widely applicable. The isomeric Lewis x glycans proved to be recognized in highly variable binding modes by polylactosamine‐metabolizing enzymes, e.g., the midchain β1,6‐GlcNAc transferase (Leppänen et al., Biochemistry, 36, 13729–13735, 1997). [ABSTRACT FROM PUBLISHER]

Focuses on the enzymatic synthesis of alpha'3sialylated and multiply alpha3fucosylated biantennary polylactosamines. Construction of novel polylactosamine glycans; Effects of glycans on l-selectin-dependent, lymphocyte-to-endothelium adhesion processes; Presence of fucose units on the sialylated LN units of the inhibitor saccharide.

We have previously isolated and partially characterised the components of a highly purified interferon-α (IFN-α) preparation produced by Sendai-virus-induced human peripheral blood leukocytes. Nine IFN-α species were identified, and two of these were found to be glycosylated [Nyman, T. A., Tölö, H., Parkkinen, J. & Kalkkinen, N. (1998) Identification of nine interferon-α subtypes produced by Sendai-virus-induced human peripheral blood leukocytes, Biochem. J. 329, 295-302]. Here, we isolated the N-linked oligosaccharides of IFN-α14c and the O-linked chains of IFN-α2b, and the glycans were characterised by electrospray tandem mass spectrometry and by specific glycosidase digestions monitored by matrix-assisted laser desorption ionisation time of flight mass spectrometry. The IFN-α14c N-glycans were shown to exhibit core-fucosylated biantennary glycans, with about 10 % carrying an additional α1,3-linked fucose unit at the antennae. The IFN-α2b was shown to carry about 50 % core type-1 disialyltetrasaccharides, 30 % core type-1 monosialyltrisaccharides and 20 % core type-2 monosialylpentasaccharides. [ABSTRACT FROM AUTHOR]

Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLe(x)). It is thought that multivalent 6-sulfo SLe(x) expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLe(x) in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin-binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLe(x) epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLe(x). N-glycans containing potential 6-sulfo SLe(x) epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLe(x) epitopes on O-glycans that are important for its recognition by L-selectin.