Phospho-beta-glucosidase from Fusobacterium mortiferum: purification, cloning, and inactivation by 6-phosphoglucono-delta-lactone.

6-Phosphoryl-beta-D-glucopyranosyl:6-phosphoglucohydrolase (P-beta-glucosidase, EC 3.2.1.86) has been purified from Fusobacterium mortiferum. Assays for enzyme activity and results from Western immunoblots showed that P-beta-glucosidase (Mr, 53,000; pI, 4.5) was induced by growth of F. mortiferum on beta-glucosides. The novel chromogenic and fluorogenic substrates, p-nitrophenyl-beta-D-glucopyranoside-6-phosphate (pNPbetaGlc6P) and 4-methylumbelliferyl-beta-D-glucopyranoside-6-phosphate (4MUbetaGlc6P), respectively, were used for the assay of P-beta-glucosidase activity. The enzyme hydrolyzed several P-beta-glucosides, including the isomeric disaccharide phosphates cellobiose-6-phosphate, gentiobiose-6-phosphate, sophorose-6-phosphate, and laminaribiose-6-phosphate, to yield glucose-6-phosphate and appropriate aglycons. The kinetic parameters for each substrate are reported. P-beta-glucosidase from F. mortiferum was inactivated by 6-phosphoglucono-delta-lactone (P-glucono-delta-lactone) derived via oxidation of glucose 6-phosphate. The pbgA gene that encodes P-beta-glucosidase from F. mortiferum has been cloned and sequenced. The first 42 residues deduced from the nucleotide sequence matched those determined for the N terminus by automated Edman degradation of the purified enzyme. From the predicted sequence of 466 amino acids, two catalytically important glutamyl residues have been identified. Comparative alignment of the amino acid sequences of P-beta-glucosidase from Escherichia coli and F. mortiferum indicates potential binding sites for the inhibitory P-glucono-delta-lactone to the enzyme from F. mortiferum.     

Cellobiose-6-phosphate hydrolase (CelF) of Escherichia coli: characterization and assignment to the unusual family 4 of glycosylhydrolases

The gene celF of the cryptic cel operon of Escherichia coli has been cloned, and the encoded 6-phospho-beta-glucosidase (cellobiose-6-phosphate [6P] hydrolase; CelF [EC 3.2.1.86]) has been expressed and purified in a catalytically active state. Among phospho-beta-glycosidases, CelF exhibits unique requirements for a divalent metal ion and NAD(+) for activity and, by sequence alignment, is assigned to family 4 of the glycosylhydrolase superfamily. CelF hydrolyzed a variety of P-beta-glucosides, including cellobiose-6P, salicin-6P, arbutin-6P, gentiobiose-6P, methyl-beta-glucoside-6P, and the chromogenic analog, p-nitrophenyl-beta-D-glucopyranoside-6P. In the absence of a metal ion and NAD(+), purified CelF was rapidly and irreversibly inactivated. The functional roles of the cofactors have not been established, but NAD(+) appears not to be a reactant and there is no evidence for reduction of the nucleotide during substrate cleavage. In solution, native CelF exists as a homotetramer (M(w), approximately 200,000) composed of noncovalently linked subunits, and this oligomeric structure is maintained independently of the presence or absence of a metal ion. The molecular weight of the CelF monomer (M(r), approximately 50,000), estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is in agreement with that calculated from the amino acid sequence of the polypeptide (450 residues; M(r) = 50,512). Comparative sequence alignments provide tentative identification of the NAD(+)-binding domain (residues 7 to 40) and catalytically important glutamyl residues (Glu(112) and Glu(356)) of CelF.     

Purification from Fusobacterium mortiferum ATCC 25557 of a 6-phosphoryl-O-alpha-D-glucopyranosyl:6-phosphoglucohydrolase that hydrolyzes maltose 6-phosphate and related phospho-alpha-D-glucosides.

6-Phosphoryl-O-alpha-D-glucopyranosyl:6-phosphoglucohydrolase (6-phospho-alpha-glucosidase) has been purified from Fusobacterium mortiferum ATCC 25557. p-Nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alpha Glc6P) served as the chromogenic substrate for detection and assay of enzyme activity. The O2-sensitive, metal-dependent phospho-alpha-glucosidase was stabilized during purification by inclusion of dithiothreitol and Mn2+ ion in chromatography buffers. Various 6-phosphoryl-O-alpha-linked glucosides, including maltose 6-phosphate, pNP alpha Glc6P, trehalose 6-phosphate, and sucrose 6-phosphate, were hydrolyzed by the enzyme to yield D-glucose 6-phosphate and aglycone moieties in a 1:1 molar ratio. 6-Phospho-alpha-glucosidase (M(r) of approximately 49,000; pI of approximately 4.9) is activated by Fe2+, Mn2+, Co2+, and Ni2+, and the maximum rate of pNP alpha Glc6P hydrolysis occurs at 40 degrees C within the pH range 7.0 to 7.5. The sequence of the first 32 amino acids of 6-phospho-alpha-glucosidase exhibits 67% identity (90% similarity) to that deduced for the N terminus of a putative phospho-beta-glucosidase (designated ORF f212) encoded by glvG in Escherichia coli. Western blots involving highly specific polyclonal antibody against 6-phospho-alpha-glucosidase and spectrophotometric analyses with pNP alpha Glc6P revealed only low levels of the enzyme in glucose-, mannose-, or fructose-grown cells of F. mortiferum. Synthesis of 6-phospho-alpha-glucosidase increased dramatically during growth of the organism on alpha-glucosides, such as maltose, alpha-methylglucoside, trehalose, turanose, and palatinose.     

Sucrose fermentation by Fusobacterium mortiferum ATCC 25557: transport, catabolism, and products.

Studies of sucrose utilization by Fusobacterium mortiferum ATCC 25557 have provided the first definitive evidence for phosphoenolpyruvate-dependent sugar:phosphotransferase activity in the family Bacteroidaceae. The phosphoenolpyruvate-dependent sucrose:phosphotransferase system and the two enzymes required for the dissimilation of sucrose 6-phosphate are induced specifically by growth of F. mortiferum on the disaccharide. Monomeric sucrose 6-phosphate hydrolase (M(r), 52,000) and a dimeric ATP-dependent fructokinase (subunit M(r), 32,000) have been purified to electrophoretic homogeneity. The physicochemical and catalytic properties of these enzymes have been examined, and the N-terminal amino acid sequences for both proteins are reported. The characteristics of sucrose 6-phosphate hydrolase and fructokinase from F. mortiferum are compared with the same enzymes from both gram-positive and gram-negative species. Butyric, acetic, and D-lactic acids are the end products of sucrose fermentation by F. mortiferum. A pathway is proposed for the translocation, phosphorylation, and metabolism of sucrose by this anaerobic pathogen.     

Characteristics of Fusobacterium ulcerans,a New and Unusual Species Compared with Fusobacterium varium and Fusobacterium mortiferum

Fusobacterium ulcerans is a newly described obligately anaerobic Gram-negative, non-spore-forming rod [1] that has been isolated from tropical ulcers. Two morphotypes were described: one resemblingFusobacterium varium and the other Fusobacterium mortiferum[1]. Because of the weak or negative fermentation reactions of most fusobacteria, the standard carbohydrate tests used for identification of anaerobe organisms are of little use for identification, and other rapid and simple methods are needed. We characterized eight F. ulcerans strains using conventional biochemical testing. We further analysed these strains by PCR employing a single non-specific primer AP3 and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell proteins. PCR using a self-designed pair of primers for the amplification of the spacer (intergenic) region between the 16S and 23S rRNA genes, led to the development of genetic markers for species identification. All F. ulcerans clinical isolates appeared very similar to each other in all the test parameters, but were distinctly different from the type strains of the two phenotypically similar species, F. mortiferum and F. varium. High similarity in PCR- and protein-profiles also raise the possibility that all these F. ulcerans strains came from one clone. We noted significant differences among the strains of F. mortiferum and F. varium.     

Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment.

A 2112-bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3-kb mRNA from human liver and HL-60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12-bp 5' non-coding region, an open reading frame of 1269 bp and an 831-bp 3' non-coding region with a putative polyadenylation signal 25 bp upstream of a 3' poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N-terminal amino acid residues of human prostatic acid phosphatase and the N-terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.     

Phosphoenolpyruvate-dependent maltose:phosphotransferase activity in Fusobacterium mortiferum ATCC 25557: specificity, inducibility, and product analysis.

Phosphoenolypyruvate-dependent maltose:phosphotransferase activity was induced in cells of Fusobacterium mortiferum ATCC 25557 during growth on maltose. The disaccharide was rapidly metabolized by washed cells maintained under anaerobic conditions, but fermentation ceased immediately upon exposure of the cell suspension to air. Coincidentally, high levels of a phosphorylated derivative accumulated within the cells. Chemical and enzymatic analyses, in conjunction with data from 1H, 13C, and 31P nuclear magnetic resonance spectroscopy, established the structure of the purified compound as 6-O-phosphoryl-alpha-D-glucopyranosyl-(1-4)-D-glucose (maltose 6-phosphate). A method for the preparation of substrate amounts of this commercially unavailable disaccharide phosphate is described. Permeabilized cells of F. mortiferum catalyzed the phosphoenolpyruvate-dependent phosphorylation of maltose under aerobic conditions. However, the hydrolysis of maltose 6-phosphate (to glucose 6-phosphate and glucose) by permeabilized cells or cell-free preparations required either an anaerobic environment or addition of dithiothreitol to aerobic reaction mixtures. The first step in dissimilation of the phosphorylated disaccharide appears to be catalyzed by an oxygen-sensitive maltose 6-phosphate hydrolase. Cells of F. mortiferum, grown previously on maltose, fermented a variety of alpha-linked glucosides, including maltose, turanose, palatinose, maltitol, alpha-methylglucoside, trehalose, and isomaltose. Conversely, cells grown on the separate alpha-glucosides also metabolized maltose. For this anaerobic pathogen, we suggest that the maltose:phosphotransferase and maltose 6-phosphate hydrolase catalyze the phosphorylative translocation and cleavage not only of maltose but also of structurally analogous alpha-linked glucosides.     

Molecular cloning, expression and characterization of the Canis familiaris interleukin-4.

Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. The rapid amplification of cDNA ends (RACE) was used to clone the canine IL-4 gene. It was expressed in mammalian cells and Escherichia coli. Monoclonal antibodies were raised to rcIL-4 for use in an enzyme-linked immunosorbent assay (ELISA). This will facilitate studies on the role of cIL-4 in inflammatory diseases, particularly rheumatoid arthritis.     

The cloning,expression, and comparative analysis of peroxiredoxin 6 from various sources

Human, rat, Xenopus, and Drosophila (DPx2540 and DPx6005) peroxiredoxin cDNAs were cloned and expressed in Escherichia coli. The recombinant enzymes were compared with respect to enzymatic activity toward various substrates and protection of plasmid DNA from the Fenton reaction products. The activity toward H₂O₂ decreased in the following order: DPx2540 > human Prx6 > Xenopus Prx6 > rat Prx6 > DPx6005. The activity toward tret-butyl hydroperoxide decreased in the following order: DPx2540 = DPx6005 > rat Prx6 > Xenopus Prx6 > human Prx6. The efficiency of plasmid DNA protection from oxidative damage mediated by the Fenton reaction decreased in the order of DPx2540 > DPx6005 = rat Prx6 = human Prx6 > Xenopus Prx6. The optimal temperature for activity of all enzymes was 37°C. Peroxiredoxins from rat, Xenopus, and Drosophila (DPx6005) retained no less than 50% of their activity in a wider temperature range (10–50°C) as compared with the human and Drosophila (DPx2540) enzymes (25–45°C). The thermostability of the enzymes decreased in the following order: DPx6005 = rat > human > Xenopus > DPx2540. The results confirmed a negative correlation between the activity and stability of peroxiredoxin 6, especially in the case of the Xenopus and Drosophila enzymes.     

46 kd mannose 6-phosphate receptor: cloning, expression, and homology to the 215 kd mannose 6-phosphate receptor

We have isolated cDNA clones encoding the entire sequence of the bovine 46 kd cation-dependent mannose 6-phosphate (CD Man-6-P) receptor. Translation of CD Man-6-P receptor mRNA in Xenopus laevis oocytes results in a protein that binds specifically to phosphomannan-Sepharose, thus demonstrating that our cDNA clones encode a functional receptor. The deduced 279 amino acid sequence reveals a single polypeptide chain that contains a putative signal sequence and a transmembrane domain. Trypsin digestion of microsomal membranes containing the receptor and the location of the five potential N-linked glycosylation sites indicate that the receptor is a transmembrane protein with an extracytoplasmic amino terminus. This extracytoplasmic domain is homologous to the approximately 145 amino acid long repeating domains present in the 215 kd cation-independent Man-6-P receptor.     

Human M2-type pyruvate kinase: cDNA cloning, chromosomal assignment and expression in hepatoma

Two overlapping clones, covering the entire coding sequence of human M2-type pyruvate kinase (PK) cDNA, were isolated and sequenced. Nucleotide sequencing results showed that they contained the 109-bp 5'-untranslated region, the 1593-bp coding region and the 585-bp 3'-untranslated region. Nucleotide sequence homology was 90% and 69% with rat M2-type and L-type PK cDNA, respectively. In situ hybridization using the human M2-type PK cDNA probe disclosed that the gene for M2-type PK is located at band q22 on chromosome 15. Northern blot analysis with RNA from human hepatoma demonstrated that M2-type PK was predominantly expressed in hepatoma cells, whereas L-type PK was preferentially expressed in the non-tumor portion of the liver.     

LTCI, a novel chymotrypsin inhibitor of the potato I family from the earthworm Lumbricus terrestris. Purification, cDNA cloning, and expression

A novel chymotrypsin inhibitor of the potato I protease inhibitor family from the earthworm Lumbricus terrestris was purified. The inhibitor, named LTCI, was isolated by methanol extraction, affinity chromatography on immobilized methylchymotrypsin, and ion exchange chromatography followed by RP–HPLC. The 7076 Da inhibitor consists of a single polypeptide chain of 64-amino-acid residues without disulfide bridges. LTCI is the first of the potato I protease inhibitors with Tyr in position P1 of the reactive site. cDNA analysis revealed that LTCI is produced as a 86-amino-acid precursor with a 22-amino-acid secretory signal peptide. RT–PCR analysis demonstrates that LTCI mRNA is expressed in body wall, intestine, and coelomocytes. The recombinant LTCI was produced in Escherichia coli as a fusion protein with intein and chitin binding domain using IMPACT™–CN system.     

Characterization, chromosomal assignment, and tissue expression of a novel human gene belonging to the ARF GAP family

We have identified and characterized a novel human ADP-ribosylation factor GTPase-activating protein (ARFGAP1) gene that is related to other members of the ARF GAP family. The full-length cDNA for human ARFGAP1 was cloned following the identification of an EST obtained by large-scale cDNA library sequencing through a Blast search of public databases. Structurally, ARFGAP1 encodes a polypeptide of 516 amino acids, which contained a typical GATA-1-type zinc finger motif (CXXCX(16)CXXC) with the four cysteine residues that are highly conserved among other members of the ARF GAP family. The conserved ARF GAP domain may emphasize the biological importance of this gene. The ARFGAP1 gene, which contained 16 exons ranging from 0.5 to 9.3 kb, was mapped to human chromosome 22q13.2-q13.3 using radiation hybridization and in silico analyses. ARFGAP1 is strongly expressed in endocrine glands and testis. Interestingly, the expression of ARFGAP1 in testis is about sixfold higher than that in ovary, indicating a possible role of ARFGAP1 in the physiological function of sperm. Expression of ARFGAP1 in four human fetal tissues and seven cancer cell lines was also detected.     

Human connexin 30 (GJB6), a candidate gene for nonsyndromic hearing loss: molecular cloning, tissue-specific expression, and assignment to chromosome 13q12

Mutations in connexin 26 are responsible for approximately 20% of genetic hearing loss and 10% of all childhood hearing loss. However, only about 75% of the mutations predicted to be in Cx26 are actually observed. While this may be due to mutations in noncoding regulatory regions, an alternative hypothesis is that some cases may be due to mutations in another gene immediately adjacent to Cx26. Another gap junction gene, connexin 30 (HGMW-approved symbol GJB6), is found to lie on the same PAC clone that hybridizes to chromosome 13q12. Human connexin 26 and connexin 30 are expressed in the same cells of the cochlea. Cx26 and Cx30 share 77% identity in amino acid sequence but Cx30 has an additional 37 amino acids at its C-terminus. These considerations led us to hypothesize that mutations in Cx30 might also be responsible for hearing loss. Eight-eight recessive nonsyndromic hearing loss families from both American and Japanese populations were screened for mutations. In addition, 23 dominant hearing loss families and 6 singleton families presumed to be recessive were tested. No significant mutation has been found in the dominant or recessive families.