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PubMed2001

Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its characterization.

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-beta-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two L-glutamine residues. A substance P derivative with an N-acetyl-D-glucosamine residue attached to the fifth or sixth L-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-D-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the L-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the L-glutamine residue of the peptide was not liberated by peptide-N(4)-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F.

Amidohydrolases↗

PubMed1997

Localization of germination-specific spore-lytic enzymes in Clostridium perfringens S40 spores detected by immunoelectron microscopy.

The localization of germination-specific spore-lytic enzymes, an amidase and a muramidase, in Clostridium perfringens S40 spores was examined by immunoelectron microscopy with respective antisera raised against the enzymes and a colloidal gold-immunoglobulin G complex. For both antisera, immunogold particles were visualized on the outside of the cortex of dormant spores, and they were not detected in germinated spores and decoated spores.

Antibodies, Fungal↗

PubMed1998

Pleiotropic mutations alter the kinetics of calcium transport, competence regulation, autolysis and experimental virulence in Streptococcus pneumoniae.

Streptococcus pneumoniae is a pathogen in which the extracellular calcium concentration plays a major physiological role, in growth as well as in the induction of competence for genetic transformation and activation of autolysis. Both responses are under the control of a protein activator exported in the medium. We have checked the impact of mutations which alter the regulation of competence and autolysis on experimental virulence. Isogenic encapsulated derivatives carrying the relevant mutations were serotype 3 smooth clones, obtained by transformation of the relevant rough strains with DNA from a serotype 3 smooth isolate. Survival kinetics and bacterial clearance from the blood were followed after intraperitoneal infection of Swiss mice with the different bacterial cultures. In this model, mutants showing an attenuation of virulence relative to the wild type fell into two classes. In the first, represented by the lytA::ery mutant V1095 defective for calcium-induced autolysis, attenuated virulence could be correlated with rapid bacterial clearance from the blood. In the second, represented by the dmb mutants V2200 and V3300, attenuation was associated with delayed bacterial clearance from the blood, and correlated with altered kinetics of calcium transport and of regulation of competence and autolysis. It appeared unlikely that attenuation of virulence for strains V2200 and V3300 was a direct consequence of their competence phenotype, since the com::ery mutants V1008 and V1019, defective for the production of the competence activator, were as virulent as the wild-type strain. Autolysis involving an N-acetyl-muramyl-alanine amidase encoded by lytA was also regulated by calcium. The inserted allele lytA0::ery further reduced virulence in the dmb1 background (V2200). This additive effect of lytA- to dmb1 points to different routes of virulence regulation by LYT and DMB1 and suggests that the kinetics of calcium traffic controls several pathways involved in the virulence of pneumococcus.

Animals↗

PubMed1994

The three-dimensional structure of PNGase F, a glycosylasparaginase from Flavobacterium meningosepticum.

BACKGROUND: Peptide:N-glycosidase F (PNGase F) is an enzyme that catalyzes the complete removal of N-linked oligosaccharide chains from glycoproteins. Often called an endoglycosidase, it is more correctly termed an amidase or glycosylasparaginase as cleavage is at the asparagine-sugar amide linkage. The enzyme is widely used in structure-function studies of glycoproteins. RESULTS: We have determined the crystal structure of PNGase F at 1.8 A resolution. The protein is folded into two domains, each with an eight-stranded antiparallel beta jelly roll configuration similar to many viral capsid proteins and also found, in expanded form, in lectins and several glucanases. Two potential active site regions have been identified, both in the interdomain region and shaped by prominent loops from one domain. Exposed aromatic residues are a feature of one site. CONCLUSIONS: The finding that PNGase F is based on two jelly roll domains suggests parallels with lectins and other carbohydrate-binding proteins. These proteins either bind sugars on the concave face of the beta-sandwich structure (aided by loops) or amongst the loops themselves. Further analysis of the function and identification of the catalytic site should lead to an understanding of both the specificity of PNGase F and possibly also the recognition processes that identify glycosylation sites on proteins.

Amidohydrolases↗

PubMed1994

Glycosylation of bombesin receptors: characterization, effect on binding, and G-protein coupling.

In the present study, we investigated the nature and the importance of glycosylation of two mammalian bombesin receptors, the neuromedin B receptor (NMB-R) and the gastrin-releasing peptide receptor (GRP-R), using chemical cross-linking and enzymatic deglycosylation. [125I]-(D-Tyr0)NMB cross-linked to native NMB-R on rat C-6 glioblastoma cells or rat NMB-R transfected into BALB 3T3 cells revealed a single broad band, M(r) = 63,000, on both cell types that was not altered by DTT. NMB inhibited cross-linking specifically and saturably with an IC50 of 4.8 and 6.1 nM for C-6 and NMB-R transfected cells, respectively, and there was a close correlation between its ability to inhibit binding and its ability to inhibit cross-linking. A single broad band of M(r) = 82,000 was cross-linked with [125I]GRP on mouse GRP-R transfected BALB 3T3 cells. Peptide-N4-(N-acetyl-beta- glucosaminyl)asparagine amidase F (PNGase F) digestion increased the mobility of the original band in C-6, NMB-R, and GRP-R transfected cell membranes. Endoglycosidase H (Endo-H) and endoglycosidase F2 (Endo-F2) digestion had no effect on both transfected cells. Neuraminidase digestion slightly increased the mobility of the original band in NMB-R transfected cell membranes; however, it had no effect on GRP-R transfected cell membranes. Endo-alpha-N-acetylglucosaminidase (O-glycanase) digestion subsequent to neuraminidase treatment showed no additional effect on either receptor. Serial partial deglycosylation of cross-linked NMB-Rs with PNGase F treatment for different incubation periods revealed one band of partially glycosylated receptor (53 kDa) besides the fully glycosylated and fully deglycosylated ones, showing that NMB-R has two oligosaccharide chains. Similarly, three partially deglycosylated species (72, 62, and 52 kDa) are seen with the GRP-R, indicating that the GRP-R has four oligosaccharide chains. Treatment of unlabeled membranes with PNGase F followed by affinity labeling resulted in fully deglycosylated NMB-R or 75% deglycosylated GRP-R. Deglycosylation of the NMB-R did not alter its affinity for NMB or alter G-protein coupling; however, 75% deglycosylation of the GRP-R both decreased its affinity for GRP and altered its ability to couple to G-proteins. The present results demonstrate that NMB-R on native and transfected cells is an N-linked sialoglycoprotein with two triantenary and/or tetraantenary complex oligosaccharide chains. The apparent M(r) of this sialoglycoprotein is 63,000, and this protein does not contain disulfide-linked subunits or O-linked carbohydrates.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗

PubMed2005

Solution structure of the peptidoglycan binding domain of Bacillus subtilis cell wall lytic enzyme CwlC: characterization of the sporulation-related repeats by NMR.

Bacillus subtilis CwlC is a cell wall lytic N-acetylmuramoyl-l-alanine amidase that plays an important role in mother-cell lysis during sporulation. The enzyme consists of an N-terminal catalytic domain with C-terminal tandem repeats. The repeats [repeat 1 (residues 184-219) and repeat 2 (residues 220-255)] are termed CwlCr. We report on the solution structure of CwlCr as determined by multidimensional NMR, including the use of 36 (h3)J(NC)'-derived hydrogen bond restraints and 64 residual (1)D(NH) dipolar couplings. Two tandem repeats fold into a pseudo-2-fold symmetric single-domain structure consisting of a betaalphabetabetaalphabeta-fold containing numerous contacts between the repeats. Hydrophobic residues important for structural integrity are conserved between the repeats, and are located symmetrically. We also present NMR analysis of the circularly permuted repeat mutant of CwlCr. Secondary structure content from the chemical shifts and hydrogen bonds derived from (h3)J(NC)' show that the mutant folds into a structure similar to that of the wild type, suggesting that the repeats are exchangeable. This implies that conserved hydrophobic residues are crucial for maintaining the folding of the repeats. While monitoring the chemical shift perturbations following the addition of digested soluble peptidoglycan fragments, we identified two peptidoglycan interaction sites of CwlCr at the edges of the protein symmetrically, and they are located approximately 28 A from each other.

Amino Acid Sequence↗

PubMed1996

The Beta-subunit of the rabbit H,K-ATPase:a glycoprotein with all terminal lactosamine units capped with alpha-linked galactose residues.

The beta-subunit of the gastric H,K-ATPase is the most abundant glycoprotein in the tubulovesicular compartment of the acid-secreting parietal cells. The oligosaccharides of the beta-subunit have been shown to contain fucose, N-acetylglucosamine, mannose, galactose, and N-acetylgalactosamine. Previous studies have shown that the rabbit beta-subunit is devoid of N-acetylneuraminic acid. Here we report the structural features of the N-linked oligosaccharides of the beta-subunit from rabbit H,K-ATPase. We used glycosidase digestions and analysis by high-pH anion-exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption/ionization mass spectrometry to analyze the peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase (PNGase F)- and endo-beta-N-acetylglucosaminidase H (Endo H)-released oligosaccharides. The studies showed that the oligosaccharides of the beta-subunit are a mixture of both oligomannosidic and lactosamine-type structures. The high-mannose structures were identified as Man5Man8GlcNAc2 species. A striking finding was that all the branches of the lactosamine-type structures were terminated with Galalpha-->Galbeta-->GlcNAc extensions. All of the lactosamine-type structures were found to be core fucosylated and some of them contained one to three lactosamine repeats. We propose that a part of the adaptation of the gastric beta-subunit to the acidic environment of the stomach is through providing acid-stable terminal residues on the oligosaccharides.

Amidohydrolases↗

PubMed2004

Evaluation of magnetic polymer micro-beads as carriers of immobilised biocatalysts for selective and stereoselective transformations.

The kinetic, selective and stereoselective properties of enzyme immobilised on magnetic polymer beads with diameters in the range 1 microm was studied with penicillin amidase from E. coli. The enzyme was immobilised on epoxy and glutaraldehyde-activated poly(vinyl alcohol), poly(methylmetacrylate) and poly(vinyl acetate-divinylbenzene) magnetic beads. The amount of covalently bound active protein was dependent on the chemical modification of the matrix and increased at higher ionic strength of the immobilisation buffer. The small size of the magnetic beads, that reduces mass transfer limitations, and the decreased charge density in the electric double layer resulted in lower apparent Km values and higher efficiency for benzylpenicillin hydrolysis, higher stereoselectivity in condensation of R-phenylglycine amide with S- and R-Phe and in hydrolysis of racemic phenylacetyl-Phe and higher selectivity in kinetically controlled synthesis of cephalexin compared to the enzyme immobilised on larger and porous carriers.

Binding Sites↗

PubMed2005

Accumulation of partly folded states in the equilibrium unfolding of the pneumococcal choline-binding module C-LytA.

Choline-binding modules are present in some virulence factors and many other proteins of Streptococcus pneumoniae (Pneumococcus). The most extensively studied choline-binding module is C-LytA, the C-terminal moiety of the pneumococcal cell-wall amidase LytA. The three-dimensional structure of C-LytA is built up from six loop-hairpin structures forming a left-handed beta-solenoid with four choline-binding sites. The affinity of C-LytA for choline and other structural analogues allows its use as an efficient fusion tag for single-step purification of hybrid proteins. In the present study, we characterize the folding and stability of C-LytA by chemical and thermal equilibrium denaturation experiments. Unfolding experiments using guanidinium chloride at pH 7.0 and 20 degrees C suggest the existence of two partly folded states (I1 and I2) in the following model: N (native)-->I1<=>I2. The N-->I1 transition is non-co-operative and irreversible, and is significant even in the absence of a denaturant. In contrast, the I1<=>I2 transition is co-operative and reversible, with an associated freeenergy change (DeltaG(0)) of 30.9+/-0.8 kJ x mol(-1). The residual structure in the I2 state is unusually stable even in 7.4 M guanidinium chloride. Binding of choline stabilizes the structure of the native state, induces its dimerization and prevents the accumulation of the I1 species ([N]2<=>[I2]2, DeltaG(0)=50.1+/-0.8 kJ x mol(-1)). Fluorescence and CD measurements, gel-filtration chromatography and limited proteolysis suggest that I1 differs from N in the local unfolding of the N-terminal beta-hairpins, and that I2 has a residual structure in the C-terminal region. Thermal denaturation of C-LytA suggests the accumulation of at least the I1 species. These results might pave the way for an effective improvement of its biotechnological applications by protein engineering.

Choline↗

PubMed1997

The lytic enzyme of the pneumococcal phage Dp-1: a chimeric lysin of intergeneric origin.

We have localized, cloned and characterized the genes coding for the lytic system of the pneumococcal phage Dp-1. The lytic enzyme of this phage (Pal), previously identified as an N-acetyl-muramoyl-L-alanine amidase, shows a modular organization similar to that described for the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. The construction of chimeric enzymes between pneumococcus and bacteria (or phages) that belong to different Gram-positive families has shown that the interchange of functional domains switches enzyme specificity. Interestingly, Pal appears to be a natural chimeric enzyme of intergeneric origin, that is the N-terminal domain was highly similar to that of the murein hydrolase coded by a gene found in the phage BK5-T that infects Lactococcus lactis, whereas the C-terminal domain was homologous to those found in the lytic enzymes of the pneumococcal system that is responsible for the binding to the choline residues present in the cell wall substrate. Biochemical analysis of Pal revealed that this enzyme shares important properties with those of the major LytA101 autolysin found in an atypical, clinical pneumococcal isolate. These peculiar characteristics have been ascribed to a modified C-terminal domain. The natural chimeric enzyme described here provides further support for the theory of modular evolution of proteins and its characteristics also furnish interesting clues on the molecular mechanisms involved in the more invasive types of atypical pneumococci.

Amino Acid Sequence↗

PubMed2002

The role of hydrophobic active-site residues in substrate specificity and acyl transfer activity of penicillin acylase.

Penicillin acylase of Escherichia coli catalyses the hydrolysis and synthesis of beta-lactam antibiotics. To study the role of hydrophobic residues in these reactions, we have mutated three active-site phenylalanines. Mutation of alphaF146, betaF24 and betaF57 to Tyr, Trp, Ala or Leu yielded mutants that were still capable of hydrolysing the chromogenic substrate 2-nitro-5-[(phenylacetyl)amino]-benzoic acid. Mutations on positions alphaF146 and betaF24 influenced both the hydrolytic and acyl transfer activity. This caused changes in the transferase/hydrolase ratios, ranging from a 40-fold decrease for alphaF146Y and alphaF146W to a threefold increase for alphaF146L and betaF24A, using 6-aminopenicillanic acid as the nucleophile. Further analysis of the betaF24A mutant showed that it had specificity constants (kcat/Km) for p-hydroxyphenylglycine methyl ester and phenylglycine methyl ester that were similar to the wild-type values, whereas the specificity constants for p-hydroxyphenylglycine amide and phenylglycine amide had decreased 10-fold, due to a decreased kcat value. A low amidase activity was also observed for the semisynthetic penicillins amoxicillin and ampicillin and the cephalosporins cefadroxil and cephalexin, for which the kcat values were fivefold to 10-fold lower than the wild-type values. The reduced specificity for the product and the high initial transferase/hydrolase ratio of betaF24A resulted in high yields in acyl transfer reactions.

Aminobenzoates↗

PubMed2005

Competence-programmed predation of noncompetent cells in the human pathogen Streptococcus pneumoniae: genetic requirements.

Natural competence for genetic transformation is the best-characterized feature of the major human pathogen Streptococcus pneumoniae. Recent studies have shown the virulence of competence-deficient mutants to be attenuated, but the nature of the connection between competence and virulence remained unknown. Here we document the release, triggered by competent cells, of virulence factors (e.g., the cytolytic toxin pneumolysin) from noncompetent cells. This phenomenon, which we name allolysis, involves a previously undescribed bacteriocin system consisting of a two-peptide bacteriocin, CibAB, and its immunity factor, CibC; the major autolysin, LytA, and lysozyme, LytC; and a proposed new amidase, CbpD. We show that CibAB are absolutely required for allolysis, whereas LytA and LytC can be supplied either by the competent cells or by the targeted cells. We propose that allolysis constitutes a competence-programmed mechanism of predation of noncompetent cells, which benefits to the competent cells and contributes to virulence by coordinating the release of virulence factors.

Amidohydrolases↗

PubMed1996

Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance.

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 muramoyl-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.

Bacillus subtilis↗

PubMed1997

Detailed studies on substrate structure requirements of glycoamidases A and F.

Glycoamidases (peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, EC 3.5.1.52; also known as peptide: N-glycanases (PNGases) release N-linked oligosaccharides from glycopeptides and/or glycoproteins by hydrolyzing the glycosylated beta-amide bond of the asparagine side chain. The most widely used glycoamidases are those from Flavobacterium meningosepticum (glycoamidase F or PNGase F) and almond emulsin (glycoamidase A or PNGase A). To study the substrate structure requirement of these enzymes systematically, we synthesized >30 glycopeptides containing cellobiose, lactose, GlcNAc, and di-N,N'-acetylchitobiose (CTB). The length of the peptide was varied from one to five amino acids, and glycosylamines were linked to either Asn or Gln located at different positions in the peptide, including NH2 and COOH termini. Neither enzyme could cleave cellobiose and lactose glycopeptides, indicating that the 2-acetamido group on the Asn-linked GlcNAc is important in the recognition by both glycoamidases A and F. GlcNAc peptides could be cleaved by both enzymes, albeit not as effectively as CTB glycopeptides. Neither enzyme requires the Asn-Xaa-(Ser/Thr) sequence (required for N-glycosylation) for activity. Glycoamidase A could even hydrolyze a Gln-bound CTB glycopeptide, whereas the action of glycoamidase F on this substrate is minimal. While glycoamidase A could act on CTB dipeptides, glycoamidase F preferred a tripeptide or longer. The Km and Vmax values of glycoamidase A for t-butoxycarbonyl-(CTB)-Asn-Ala-Ser-OMe were 2.1 mM and 0.66 micromol/min/mg, respectively. A natural glycodipeptide, Man9-GlcNAc2-Asn-Phe, was also completely hydrolyzed by glycoamidase A.

Amidohydrolases↗

PubMed1999

Digestion of Streptococcus pneumoniae cell walls with its major peptidoglycan hydrolase releases branched stem peptides carrying proinflammatory activity.

The peptidoglycan of Gram-positive bacteria is known to trigger cytokine release from peripheral blood mononuclear cells (PBMCs). However, it requires 100-1000 times more Gram-positive peptidoglycan than Gram-negative lipopolysaccharide to release the same amounts of cytokines from target cells. Thus, either peptidoglycan is poorly active or only part of it is required for PBMC activation. To test this hypothesis, purified Streptococcus pneumoniae walls were digested with their major autolysin N-acetylmuramoyl-L-alanine amidase, and/or muramidase. Solubilized walls were separated by reverse phase high pressure chromatography. Individual fractions were tested for their PBMC-stimulating activity, and their composition was determined. Soluble components had a Mr between 600 and 1500. These primarily comprised stem peptides cross-linked to various extents. Simple stem peptides (Mr <750) were 10-fold less active than undigested peptidoglycan. In contrast, tripeptides (Mr >1000) were >/=100-fold more potent than the native material. One dipeptide (inactive) and two tripeptides (active) were confirmed by post-source decay analysis. Complex branched peptides represented </=2% of the total material, but their activity (w/w) was almost equal to that of LPS. This is the first observation suggesting that peptidoglycan stem peptides carry high tumor necrosis factor-stimulating activity. These types of structures are conserved among Gram-positive bacteria and will provide new material to help elucidate the mechanism of peptidoglycan-induced inflammation.

Cell Wall↗

PubMed2003

Capsular expression in Streptococcus pneumoniae negatively affects spontaneous and antibiotic-induced lysis and contributes to antibiotic tolerance.

Penicillin and vancomycin induce a lytic response in Streptococcus pneumoniae that requires the N-acetylmuramyl-l-alanine amidase LytA. We show that clinical isolates of pneumococci of capsular serotypes 1, 4, 6B, and 23F were generally less lytic to penicillin than pneumococci of serotypes 14 and 3. In addition, most 9V isolates were less lytic to vancomycin, compared with isolates of other serotypes. Parent-mutant pairs expressing and not expressing capsular serotypes 2, 4, and 9V were compared for antibiotic-induced lysis. The nonencapsulated variants were considerably more lytic after beta-lactam and/or vancomycin treatment, and antibiotic tolerance was seen only in the context of capsule expression. Conversion from a nonlytic to a lytic phenotype, after loss of capsule expression, required an intact lytA autolysin gene. Exogenous addition of purified LytA gave a lower lytic response in capsulated strains, compared with that in nonencapsulated mutants. Spontaneous autolysis in stationary phase also was negatively affected by capsule expression in an autolysin-dependent manner. Long-term starvation in the stationary phase of the vancomycin- and penicillin-tolerant isolate I95 yielded nonencapsulated mutants that had lost antibiotic tolerance and were lytic to penicillin and vancomycin. The 9V capsular locus of I95 and one of these stationary phase-selected mutants were completely sequenced. The only difference found was a 1-bp frameshift deletion in the cps9vE gene of the lytic mutant, encoding a uridine diphosphate-glucosyl-1-phosphate transferase. Two additional independently isolated lytic mutants of I95 from the stationary phase also contained mutations in the same region of cps9vE, which identified it as a mutational hot spot. This report demonstrates that capsular polysaccharides negatively influence the lytic process and contribute to antibiotic tolerance in clinical isolates of pneumococci.

Anti-Bacterial Agents↗

PubMed1992

Carbohydrate structure of Marburg virus glycoprotein.

Marburg virus was propagated in E6 cells, a cloned cell line of Vero cells, in the presence of [6-3H]glucosamine. Radiolabelled viral glycoprotein was digested with trypsin, and oligosaccharides were liberated by sequential treatment with endo-beta-N-acetylglucosaminidase H, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and O-glycosidase, by beta-elimination, and by alkaline hydrolysis. After fractionation by HPLC and gel filtration, glycans were characterized chromatographically, by digestion with exoglycosidases and, in part, by methylation analysis and liquid secondary ion mass spectrometry. The oligosaccharide structures thus established include oligomannosidic and hybrid-type N-glycans, as well as neutral fucosylated bi-, tri- and tetraantennary species, most of which carry an additional bisecting N-acetylglucosamine. In addition, high amounts of neutral mucin-type O-glycans with type-1 and type-2 core structures were detected. None of the glycans present in this viral glycoprotein carried sialic acid residues.

Amidohydrolases↗

PubMed1981

Deficiency of autolytic activity in Bacillus subtilis and Streptococcus pneumoniae is associated with a decreased permeability of the wall.

Autolytic-deficient mutants of Bacillus subtilis which grow as chains of non-separated bacilli have been isolated by a procedure involving filtration of mutagenized cultures through glass-sinter filters. The mutants obtained were some 80-90% deficient in both autolysins, N-acetylmuramoyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase. Treatment of one of these mutants with 5 M-LiCl extracted only about 20% of the protein obtained from an equivalent amount of the autolysin-containing bacilli. Similarly, a reduction of 40-60% in LiCl-extractable protein was obtained with autolytic-deficient Streptococcus pneumoniae whether these organisms lacked the autolytic enzyme or were phenotypically deficient by growth in ethanolamine-containing medium. Chromatography on Sephadex G-100 of the protein extracted from B. subtilis and S. pneumoniae revealed that the major difference between autolytic-deficient and parent organisms was a decrease in proteins of high molecular weight. Smaller differences were observed in a second fraction which contained low molecular weight material and proteins such as the autolysins, whose elution from the column was retarded by interaction with the Sephadex. Further examination of the fractionated protein from B subtilis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis confirmed that the major difference between the extracts was in the amount of protein present and did not result from marked changes in the size of the extracted proteins. These observations suggest that autolysin deficiency in B. subtilis and S. pneumoniae results in a change in the porosity (permeability) of the bacterial wall.

Acetylglucosaminidase↗