Establishment of an abalone digestive gland cell line secreting various glycosidases in protein-free culture

A cell line designated as ADG was established from an abalone digestive gland using ERDF medium supplemented with 8% fetal bovine serum (FBS), 8% abalone hemolymph, and high concentrations of NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂. ADG cells proliferated better in protein-free medium than in FBS-supplemented medium. Among 9 kinds of media examined, ERDF medium was shown to be optimal for cell growth. ADG cells secreted 13 different kinds of glycosidases in protein-free medium: α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, N-acetyl-α-D-galactosaminidase, N-acetyl-β-D-galactosaminidase, α-D-glucosidase, β-D-glucosidase, N-acetyl-α-D-glucosaminidase, N-acetyl-β-D-glucosaminidase, α-D-mannosidase, β-D-mannosidase, β-D-xylosidase, and 1-3 xylanase. When ADG cells were cultured in Grace’s insect cell medium, the activity of some secreted glycosidases increased 25-fold to 65-fold per cell as compared with control cells cultured in ERDF medium. ADG - abalone digestive gland; ERDF - enriched RDF; FBS - fetal bovine serum; L-15 - Leibovitz’s L-15 media; DME - Dulbecco’s modified Eagle medium; F-12 - nutrient mixture (Ham); LDF - L-15; DME: F-12 = 10 : 7 : 3; MEM - minimum essential medium; RPMI - RPMI medium 1640; 199 - media 199; GIC - Grace’s insect cell medium; pNP -p -nitrophenol. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enzyme production by lactobacilli and the potential link with infective endocarditis

H.J. OAKEY, D.W.S. HARTY AND K.W. KNOX. 1995. Fifty-six strains of lactobacilli were examined for the production of glycosidases and proteases (arylamidases) that could be associated with the ability to grow in vivo and/or be a factor in the pathogenesis of endocarditis. The strains were from seven species, with an emphasis on Lactobacillus rhamnosus and Lact. paracasei subsp. paracasei , both of which have been associated with endocarditis and provided 12 of the 13 strains isolated from cases of the disease. Other species were Lact. acidophilus, Lact. plantarum, Lact. salivarius, Lact. fermentum and Lact. oris.
Commonly expressed glycosidase activities were α-D-galactosidase and β- N -acetyl-D-glucosaminidase followed by β-D-glucosidase and α-L-fucosidase. The combined production of β- N -acetyl-D-glucosaminidase and α-D-galactosidase was a feature of the endocarditis isolates. In contrast, β-D-galactosidase was produced by very few of the strains within species implicated in endocarditis but most of the strains of Lact. salivarius, Lact. fermentum and Lact. oris.
The most commonly produced arylamidases active against substrates employed for testing human blood clotting cascade were activated protein C(Ca)-like, activated factor X(Xa)-like and Hageman factor-like followed by kallikrein-like and chymotrypsin-like enzymes. Kallikrein-like enzyme activity was shown more frequently by strains from species commonly isolated from cases of endocarditis ( Lact. rhamnosus and Lact. paracasei subsp. paracasei ) than from other oral species ( Lact. plantarum, Lact. salivarius, Lact. fermentum and Lact. oris ).
The data indicate that some lactobacilli can produce enzymes that would enable the breakdown of human glycoproteins and the synthesis and lysis of human fibrin clots, characteristics which aid the colonization and survival of bacteria infecting an endocarditis vegetation.     

O-glycosylhydrolases of embryos of the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> and effect of some natural substances on their biosynthesis

Embryos of the sea urchin Strongylocentrotus intermedius have been found to contain o-glycosyl hydrolases: highly active 1,3-β-D-glucanase and β-D-mannosidase as well as a lower activity of β-D-glucosidase and β-D-galactosidase. Dynamics of changes of the enzyme activities has been studied at various stages of the sea urchin development. There has also been studied effects of some substances (natural fucoidans, β-1.3; 1.6-glucans formed by enzymatic synthesis as well as protein inhibitor of marine mollusc endo-1,3-β-D-glucanases) on development of the embryos and biosynthesis of 1,3-β-D-glucanase and α-D-mannosidase.     

Isolation of a bacterium that causes anaaki disease of the red algae Porphyra yezoensis

Anaaki disease causes severe damage to the red algae Porphyra yezoensis from which the Japanese traditional food 'nori'is produced. The causative agent of anaaki disease was isolated by several repeats of single-colony isolation and infection experiments, and was identified as Flavobacterium sp. LAD-1. The bacterium showed hydrolytic activity toward porphyran but not toward other polysaccharides composing the thallus of Porphyra , such as β-1,3-xylan or β-1,4-mannan. The bacterium also showed β-D-galactosidase activity.     

GLYCOSIDASES IN NORMAL AND SCRAPIE MOUSE BRAIN

Abstract— The pH optima of ten glycosidases have been determined in normal and scrapie-affected mouse brain. The enzymes α-mannosidase, α-glucosidase and β-glucosidase displayed two peaks of enzyme activity over the pH range examined.
There is a significant increase in the activity of the enzymes α-mannosidase, β-glucuronidase, N -acetyl β-D-glucosaminidase, N -acetyl β-galactosaminidase, β-glucosidase (pH 4.1), α-fucosidase and β-xylosidase in the brains of mice clinically affected with scrapie, whilst only α-mannosidase (pH 4.1), β-glucuronidase, N -acetyl-β-D-glucosinidase and N- acetyl-β-D-gaiactosaminidase are elevated before mice exhibit signs of the disease.     

Coinduction of α-L-arabinofuranosidase and α-D-galactosidase formation in Trichoderma reesei RUT C-30

Abstract Formation of α-L-arabinosidase can be induced in Trichoderma reesei by growing the fungus on L-arabinose or dulcitol, and by adding L-arabinose, L-arabitol, D-galactose, or dulcitol ot non-growing mycelia. The same conditions also stimulated the formation of α-D-galactosidase, but not that of various other enzymes involved in hemicellulose degradation. The optimal inducer concentration with all compounds was 4 mM for both enzymes. Using L-arabinose and D-galactose, the induction efficiency was highest at pH 6.5, whereas induction by arabitol and dulcitol was more efficient at low pH (2.5). The addition of 50 mM glucose did not repress α-L-arabinosidase or α-D-galactosidase formation. These findings suggest coregulation of two hemicellulose side-chain cleaving enzymes in T. reesei .     

Possible interference of lactose-fermenting marine vibrios in coliform -D-galactosidase assays

C.M. DAVIES, S.C. APTE AND S.M. PETERSON. 1995. An investigation into possible interferences in β-D-galactosidase-based assays for coliform bacteria in marine waters was carried out. A rapid instrumental fluorescence assay for β-D-galactosidase activity, using 4-methylumbelliferyl-β-D-galactosidase as a substrate, was used to investigate activities of this enzyme in non-coliform bacterial isolates from coastal waters. Only 2% of isolates showed slight enzyme activity after a 1-h incubation period at 44.5βC. At a lower incubation temperature of 20βC, 51% and 94% of the isolates showed some enzyme activity within 6 h and 48 h, respectively. Fifty-nine out of 67 of these isolates were identified as Vibrio species. A lac⁺ strain of Vibrio vulnificus was found to produce β-D-galactosidase which caused significant false-positive reactions in the Colilert-Marine Water assay when present at concentrations of 10 cfu ml⁻¹ or greater. This interference could be overcome by addition of the vibriostatic agent O/129. The high fluorescence of this reagent, however, precluded the simultaneous determination of Escherichia coli in the Colilert test and also its use in instrumental fluorescence assays. It was concluded that in assays employing high temperatures and short incubation times, Vibrio species are unlikely to cause significant interferences.     

Structure of the mannose-rich oligosaccharide chains of concanavalin A-binding glycopeptides derived from beef brain glycoproteins

Abstract: A neutral, mannose-rich, concanavalin A (Con A)-binding glycopeptide fraction was obtained by proteolytic digestion of defatted beef brain tissue. Hydrazinolysis followed by gel filtration of the reaction products provided three oligosaccharides. A portion of each oligosaccharide was treated by exhaustive digestion with α-mannosidase. Another portion was subjected to selective acetolysis of Manαl-6Man linkages, providing two fragments that were recovered by gel filtration. The structure of the intact oligosaccharides, as well as the fragments obtained by selective acetolysis and enzymatic treatment, were resolved by gas-liquid chromatographic-mass spectrometric analysis. The structures of the three oligosaccharides were: (a) Manαl-2Manαl-6(Manαl -3)Manαl-6(Manαl-2Manαl-2Manαl 3)Manβ1-4- N -acetylglucosamine (GlcNAc)β - 4N- acetylglucosaminitol (GlcOLNAc); (b) Manαl -2Manαl -6(Manαl -3)Manαl-6(Manαl-2Manαl-3)-Manβ1-4GlcNAcβl -4GlcOLNAc; and (c) Manαl -6(Manαl-3) Manαl - 6(Manαl - 3)Manβl -4GlcNAc-βl - 4GlcOLNAc. These structures account for 15–20% of the glycoprotein-carbohydrate of whole beef brain and most of the oligosaccharides that demonstrate a high affinity for Con A. In view of the large number of Con A-binding glycoproteins in brain tissue, it appears that many of these different glycoproteins must contain structurally identical oligosaccharides.     

SIALOGLYCOPROTEINS AND SEVERAL GLYCOSIDASES IN DEVELOPING RAT BRAIN

Abstract— The amount of sialoglycoproteins expressed as μmol of sialic acid per g of lipid-free residue remained fairly constant in developing rat brain. However, the activity of various enzymes which may be involved in glycoprotein metabolism varied in an inconstant fashion during the period of development. The specific activity of a neuraminidase increased, N -acetyl-β-glucosaminidase remained relatively constant, while the specific activities of α-mannosidase and α-fucosidase decreased.     

Isoform-Specific Up-Regulation by Ouabain of Na+,K+-ATPase in Cultured Rat Astrocytes

Abstract: There are two α-subunit isoforms (α1 and α2) and two β-subunit isoforms (β1 and β2) of Na⁺,K⁺-ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5–1.0 m M ) increased the levels of α1 and β1 mRNAs, whereas it decreased those of α2 and β2 mRNAs in cultured rat astrocytes. The increases in α1 and β1 mRNAs were observed at 6–48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased α1 and β1, but not α2 and β2, proteins, and that the isoforms in control and ouabain-treated cultures were of glial origin. Low extracellular K⁺ and monensin (20 µ M ) mimicked the effect of ouabain on α1 mRNA. The ouabain-induced increase in α1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 µ M ), the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N',N' -tetraacetic acid tetraacetoxymethyl ester (30 µ M ), and the calcineurin inhibitor FK506 (1 n M ). These findings indicate that chronic inhibition of Na⁺,K⁺-ATPase up-regulates the α1 and β1, but not α2 and β2, isoforms in astrocytes, suggesting a functional coupling of α1β1 complex. They also suggest that intracellular Na⁺, Ca²⁺, and calcineurin may be involved in ouabain-induced up-regulation of the enzyme in astrocytes.     

Purification and Chemical Characterization of β-Trace Protein from Human Cerebrospinal Fluid: Its Identification as Prostaglandin D Synthase

Abstract: β-Trace protein from pooled human CSF was purified to homogeneity. An apparent molecular mass of 23–29 kDa was determined for the polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequencing of the polypeptide yielded the unique amino acid sequence APEAQVSVQPNFQQDKFLGRWFSA²⁴. Alignment of amino acid sequences obtained from tryptic peptides with the sequence previously deduced from a cDNA clone isolated by other investigators allowed the identification of β-trace protein as prostaglandin D synthase [prostaglandin-H₂ D-isomerase; (5 Z , 13 E )-(15 S )-9α, 11 a-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase; EC 5.3.99.2]. A conservative amino acid exchange (The instead of Ser) was detected at amino acid position 154 of the β-trace polypeptide chain in the corresponding tryptic peptide. The two N -glycosylation sites of the polypeptide were shown to be almost quantitatively occupied by carbohydrate. Carbohydrate compositional as well as methylation analysis indicated that Asn²⁹and Asn⁵⁶ bear exclusively complex-type oligosaccharide structures (partially sialylated with α2–3- and/or α2–6-linked N -acetylneuraminic acid) that are almost quantitatively α1-6 fucosylated at the proximal N -acetylglucosamine; ∼70% of these molecules contain a bisecting N -acetylglucosamine. Agalacto structures as well as those with a peripheral fucose are also present.     

Role of Group III Metabotropic Glutamate Receptors in Excitotoxin-Induced Cerebellar Granule Cell Death

Abstract: The neuronal effects of the metabotropic glutamate receptor agonist (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid have been studied in cultured rat cerebellar granule cells, and compared with those of the endogenous excitotoxin glutamate, and the dietary excitotoxin β- N -methylamino- l -alanine. Glutamate, β- N -methylamino- l -alanine, and (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid all caused concentration-dependent cerebellar granule cell death over a 24-h exposure period. The metabotropic antagonist ( RS )-α-methyl-4-carboxyphenylglycine reduced glutamate-, β- N -methylamino- l -alanine-, and (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid-induced death by 50, 37, and 90%, respectively. (1 S ,3 R )-Aminocyclopentane-1,3-dicarboxylic acid-induced death was unaffected by the group I antagonist ( RS )-1-aminoindan-1,5-dicarboxylic acid, increased by the group II antagonist ethylglutamic acid, and markedly decreased by the group III antagonist ( RS )-α-methylserine- O -phosphate. Neither (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid nor the group I agonist ( RS )-3,5-dihydroxyphenylglycine caused an increase in intracellular free calcium levels. The group III agonist l -(+)-2-amino-4-phosphonobutyric acid also induced concentration-dependent cerebellar granule cell death, and so it was suggested that the group III metabotropic glutamate receptors were responsible for (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid-induced death. Blocking these receptors with ( RS )-α-methylserine- O -phosphate also prevented a proportion of glutamate- and β- N -methylamino- l -alanine-induced death.     

From alpha to beta: identification of amino acids required for the N-acetyllactosamine-specific lectin-like activity of bundlin

Bundle-forming pili (BFP) promote the adherence of typical enteropathogenic Escherichia coli (EPEC) to human intestinal epithelial cells. BFP are polymers of bundlin and nine bundlin alleles have been identified in EPEC isolated from diverse sources. These alleles are divided into two main groups, α and β, based on their amino acid sequences. Alpha bundlins are also N -acetyllactosamine- (LacNAc) specific lectins and bind to HEp-2 cells, whereas β bundlins do not display these characteristics. The four surface-exposed regions of amino acid sequence heterogeneity between α and β bundlin were therefore investigated as potential LacNAc-specific carbohydrate-binding domains in a bundlin. Mutation of one of these domains, 137-GENNI-141, in α₁ bundlin to that of β bundlin (136-SPDST-140) resulted in BFP that no longer bound to LacNAc or HEp-2 cells. Conversely, mutating the β₃ bundlin gene to encode the α bundlin sequence at this domain resulted in the gain of HEp-2 cell adherence. The importance of this domain in carbohydrate binding is supported by the finding that introducing the mutation GENNI→GENNT altered the α₁ bundlin carbohydrate-binding specificity from LacNAc to the Lewis X glycan sequence.     

ON THE STRUCTURE OF TWO NEW GANGLIOSIDES FROM BEEF BRAIN

Abstract— Two new gangliosides were isolated in pure form from beef brain. They were provisionally named ganglioside G5a and G5b. Ganglioside GSa is a monosialoganglioside containing fucose. Its basic neutral glycosphingolipid core is the gangliotetraose ceramide: Gal (1 —> 3) GalNac (1—> 4) Gal (1 —> 4) Glc (1—>) ceramide, most likely with β-linkages. Fucose is linked to the 2-position of external galactose, N -acetylneuraminic acid to the 3- position of internal galactose. Ganglioside G5b is a mixture of at least two isomeric disialogangliosides containing N -acetylneuraminic acid and N -glycolylneura-minic acid. The major isomer has the following structure: NeuNac (α,2—>3) Gal (β,1—>3) GalNac (β, 1 —> 4) (NeuNglα, 2 —> 3) gal (β,1—>4) Glc (β,1 —>)-ceramide. The minor isomer contains N -acetylneur-aminic acid and N -glycolylneuraminic acid in an inverted linkage position.     

Sequences in the Amino Termini of GABA ρ and GABAA Subunits Specify Their Selective Interaction In Vitro

Abstract: Molecular cloning has revealed that there are six classes of subunits capable of forming GABA-gated chloride channel receptors. GABA_A receptors are composed of α, β, γ, δ, and ε/χ subunits, whereas GABA_C receptors appear to contain ρ subunits. However, retinal cells exhibiting GABA_C responses express α, β, and ρ subunits, raising the possibility that GABA_C receptors may be a mixture of subunit classes. Using in vitro translated protein, we determined that human GABA_A receptor subunits α1, α5, and β1 did not coimmunoprecipitate with full-length ρ1, ρ2, or the N-terminal domain of ρ1 that contains signals for ρ-subunit interaction. To explore the molecular mechanism underlying these apparently exclusive combinations, chimeric subunits were created and tested for interaction with the wild-type subunits. Transfer of the N terminus of β1 to ρ1 created a β1ρ1 chimera that coimmunoprecipitated with the α1 subunit but not with the ρ2 subunit. Furthermore, exchanging the N terminus of the ρ1 subunit with the corresponding region of β1 produced a ρ1β1 chimera that interfered with ρ1 receptor expression in Xenopus oocytes, whereas the full-length β1 subunit had no effect. Together, these results indicate that sequences in the N termini direct assembly of ρ subunits and GABA_A subunits into GABA_C and GABA_A receptors, respectively.     

Scanning mutagenesis of regions in the Gα protein Gpa1 that are predicted to interact with yeast mating pheromone receptors

The mechanism by which receptors activate heterotrimeric G proteins was examined by scanning mutagenesis of the Saccharomyces cerevisiae pheromone-responsive Gα protein (Gpa1). The juxtaposition of high-resolution structures for rhodopsin and its cognate G protein transducin predicted that at least six regions of Gα are in close proximity to the receptor. Mutagenesis was targeted to residues in these domains in Gpa1, which included four loop regions (β2–β3, α2–β4, α3–β5, and α4–β6) as well as the N and C termini. The mutants displayed a range of phenotypes from nonsignaling to constitutive activation of the pheromone pathway. The constitutive activity of some mutants could be explained by decreased production of Gpa1, which permits unregulated signaling by Gβγ. However, the constitutive activity caused by the F344C and E335C mutations in the α2–β4 loop and F378C in the α3–β5 loop was not due to decreased protein levels, and was apparently due to defects in sequestering Gβγ. The strongest loss of the function mutant, which was not detectably induced by a pheromone, was caused by a K314C substitution in the β2–β3 loop. Several other mutations caused weak signaling phenotypes. Altogether, these results suggest that residues in different interface regions of Gα contribute to activation of signaling.     

Human Brain Cerebroside β-Galactosidase: Deficiency of Transgalactosidic Activity in Krabbe's Disease

Abstract: Under experimental conditions optimal for the assay of D-galactosyl- N -acylsphingosine galactohydrolase (EC 3.2.1.46) activity, homog-enates of neurologically normal human brain tissue could transfer galactose from galactosyl ceramide (gal-cer), lactosyl ceramide (lac-cer), 4-methylumbelliferyl- β-galactoside (4-MU-gal), or p -nitrophenyl- β-galactoside (PNP-gal) to [1-¹⁴C]oleoyl sphingosine, but homogenates of brain tissue from patients with Krabbe's disease lacked this ability. The rate of hydrolysis of ganglioside G_M1 and to a lesser extent, of PNP-gal by homogenates of Krabbe's brain tissue was also decreased. Activity of PNP- β-galactosidase in normal brain tissue, like that of cerebroside β-galactosidase from the same source, was considerably more heat-stable than the activity of either 4-MU- β-galactosidase or the predominant G_M1β-D-galactosidase (EC 3.2.1.23). Lac-cer and G_M1, as well as 4-MU-gal and PNP-gal, were competitive inhibitors of human-brain cerebroside β-galactosidase. These findings confirm the ability of mammalian cerebroside β-galactosidase to catalyze a transgalactosylation reaction and provide additional information on the substrate specificity of human brain cerebroside β-galactosidase.     

Enzyme Activity Profiles in Mouse Teratocarcinomas

Abstract. Nine tumour lines with different developmental capacities were derived from spontaneous as well as from one induced teratocarcinoma: three teratocarcinoma-derived rhabdomyosarcomas TDR 602, TDR 694, and TDR 114; two teratocarcinoma-derived neuroblastomas TDN 2151 and TDN 2283; two teratocarcinoma-derived endodermal tumours TDE 274 and TDE 113; one multipotential teratocarcinoma OTT 2289, and one undifferentiated teratocarcinoma OTT 2158. Quantitative analyses of ten catabolic enzymes, i.e. alkaline and acid phosphatase, α- and β-galactosidase, α- and β-glucosidase, α-mannosidase, α-fucosidase, β-glucuronidase, and hexosaminidase were carried out at the 20-cell level, and specific enzyme activity profiles were established for each of the tumour lines studied. These profiles may be used for the biochemical identification of a tumour type at the single cell level in addition to morphological and biological criteria.     

Structural Analysis of the Sixth Immunoglobulin-Like Domain of Mouse Neural Cell Adhesion Molecule L1 and Its Interactions with αvβ3, αIIbβ3, and α5β1 Integrins

Abstract: Previous experiments suggested that the human cell adhesion molecule L1 interacts with different integrins via its sixth immunoglobulin-like domain in an RGD-dependent manner. Here we have described the expression of this domain from early postnatal mouse brain, analyzed the structure of the recombinant protein by circular dichroism and fluorescence spectroscopy, and performed solid-phase binding studies to αvβ3, αIIbβ3, and α5β1 integrins. The domain was found to have the expected β-sheet organization, which was lost in the presence of guanidine hydrochloride. The midpoint of the single-step transition occurred at 1.5 M guanidine hydrochloride. The sixth immunoglobulin-like domain of mouse brain L1 contains two RGD motifs and was found to bind in a concentration-dependent and saturable way to αvβ3, αIIbβ3, and α5β1 integrins, suggesting specific interactions with these ligands. However, only the interaction to αvβ3 could be inhibited in a concentration-dependent manner by an RGD-containing peptide, and the IC₅₀ was determined to be ∼20 n M . Mutants of the domain, which lack either one or both of the RGD sites, demonstrated that the RGD site comprising residues 562–564 is involved in the interaction to αvβ3. Our findings indicate an RGD-independent mechanism for the interactions to αIIbβ3 and α5β1, as no involvement of any RGD motif could be demonstrated.