scholarly journals Structural and Functional Analysis of theXestia c-nigrum Granulovirus Matrix Metalloproteinase

2000 ◽  
Vol 74 (23) ◽  
pp. 11240-11246 ◽  
Author(s):  
Rinkei Ko ◽  
Kazuhiro Okano ◽  
Susumu Maeda
Keyword(s):  
Amino Acid ◽  
Matrix Metalloproteinase ◽  
Sea Urchin ◽  
Catalytic Domain ◽  
Fat Body ◽  
Distinct Pattern ◽  
Binding Motif ◽  
Reading Frame ◽  
Matrix Metalloproteinase 3 ◽  
Inactive Form

ABSTRACT Sequence analysis of the Xestia c-nigrum granulovirus (XcGV) genome identified an open reading frame encoding a 469-amino-acid (54-kDa) protein with over 30% amino acid sequence identity to a region of about 150 amino acids that includes the catalytic domains of human stromelysin 1 (Str1)/matrix metalloproteinase 3 (MMP-3) (EC 3.4.24.17 ) and sea urchin hatching enzyme (HE). Stromelysin homologs have not been reported from baculoviruses or other viruses. Unlike human Str1 and sea urchin HE, the putative XcGV-MMP does not have a signal peptide and lacks the peptide motif involved in the cysteine switch that maintains other MMPs in an inactive form. The putative XcGV-MMP, however, possesses a conserved zinc-binding motif in its putative catalytic domain. The XcGV-MMP homolog was cloned, and a recombinant Bombyx morinucleopolyhedrovirus (BmNPV) that expresses XcGV-MMP under the polyhedrin promoter was constructed. A distinct pattern of melanization was observed in B. mori larvae infected with MMP-expressing BmNPV. Fat body extracts from larvae overexpressing the 54-kDa recombinant MMP digested dye-impregnated collagen (Azocoll). The enzymatic activity was inhibited by two metalloproteinase inhibitors, EDTA and 1,10-phenanthroline. These results suggest that the XcGV MMP-3 gene homolog encodes a functional metalloproteinase.

scholarly journals Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp. Strain XY-234

2002 ◽  
Vol 184 (9) ◽  
pp. 2399-2403 ◽  
Author(s):  
Fumiyoshi Okazaki ◽  
Yutaka Tamaru ◽  
Shinnosuke Hashikawa ◽  
Yu-Teh Li ◽  
Toshiyoshi Araki
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Catalytic Domain ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Amino Acid Residues ◽  
Glycosyl Hydrolases ◽  
Binding Studies ◽  
Calculated Molecular Mass ◽  
Reading Frame

ABSTRACT A β-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the β-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed β-1,3-xylan but not other polysaccharides such as β-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or β-1,4-mannan. TxyA was capable of binding specifically to β-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the Kd and the maximum amount of protein bound to β-1,3-xylan were 4.2 μM and 18.2 μmol/g of β-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of β-1,3-xylan.

Analysis of the Binding of Hydroxamic Acid and Carboxylic Acid Inhibitors to the Stromelysin-1 (Matrix Metalloproteinase-3) Catalytic Domain by Isothermal Titration Calorimetry

Biochemistry ◽  
1999 ◽  
Vol 38 (41) ◽  
pp. 13592-13601 ◽  
Author(s):  
Matthew H. Parker ◽  
Elizabeth A. Lunney ◽  
Daniel F. Ortwine ◽  
Alexander G. Pavlovsky ◽  
Christine Humblet ◽  
...  
Keyword(s):  
Carboxylic Acid ◽  
Matrix Metalloproteinase ◽  
Isothermal Titration Calorimetry ◽  
Hydroxamic Acid ◽  
Catalytic Domain ◽  
Titration Calorimetry ◽  
Matrix Metalloproteinase 3

scholarly journals Dipeptidyl peptidase III is a zinc metallo-exopeptidase: Molecular cloning and expression

1998 ◽  
Vol 329 (2) ◽  
pp. 275-282 ◽  
Author(s):  
Katsuhiko FUKASAWA ◽  
M. Kayoko FUKASAWA ◽  
Makoto KANAI ◽  
Shingo FUJII ◽  
Junzo HIROSE ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dipeptidyl Peptidase ◽  
Cloning And Expression ◽  
Zinc Binding ◽  
Binding Motif ◽  
Ph Optimum ◽  
Reading Frame ◽  
Theoretical Molecular Mass ◽  
Cloned Cdna ◽  
Zinc Binding Domain

We have purified dipeptidyl peptidase III (EC 3.4.14.4) from human placenta. It had a pH optimum of 8.8 and readily hydrolysed Arg-Arg-β-naphthylamide. Monoamino acid-, Gly-Phe-, Gly-Pro- and Bz-Arg-β-naphthylamides were not hydrolysed at all. The enzyme was inhibited by p-chloromercuriphenylsulphonic acid, metal chelators and 3,4-dichloroisocoumarin and contained 1 mol of zinc per mol of enzyme. The zinc dissociation constant was 250 fM at pH 7.4 as determined by the zinc binding study. We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNA species, a cDNA clone with 2633 bp encoding the rat enzyme. The longest open reading frame encodes a 827-residue protein with a theoretical molecular mass of 92790 Da. Escherichia coli SOLR cells were infected with the pBluescript phagemid containing the cloned cDNA and established the overexpression of a protein that hydrolysed Arg-Arg-β-naphthylamide. The recombinant protein was purified and the amino acid sequence of the protein was confirmed. We presumed that the putative zinc-binding domain involved in catalysis was present in the recombinant enzyme. It was a novel zinc-binding motif in that one amino acid residue was inserted into the conserved HEXXH motif characteristic of the metalloproteinases.

Cloning of genetic loci involved in endoprotease activity in Streptomyces lividans 66: a novel neutral protease gene with an adjacent divergent putative regulatory gene

1992 ◽  
Vol 38 (9) ◽  
pp. 912-920 ◽  
Author(s):  
M. J. Butler ◽  
C. C. Davey ◽  
P. Krygsman ◽  
E. Walczyk ◽  
L. T. Malek
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Structural Gene ◽  
Protease Activity ◽  
Transcriptional Regulator ◽  
Streptomyces Lividans ◽  
Open Reading Frame ◽  
Binding Motif ◽  
Multicopy Plasmid ◽  
Reading Frame

A skimmed-milk clearing assay was used to identify, in a multicopy Streptomyces lividans 66 genomic library, DNA fragments that lead to increased expression of protease activity in S. lividans 66. Three independent loci were identified. The majority class (slpA, which represented 68 of 71 clones) produced large zones of clearing. Two other classes (designated slpB and slpC) showed smaller zones than slpA. Subcloning and deletion analysis of the slpA locus delineated the relevant DNA to within a 2.5 kilobase pair fragment. DNA sequence analysis revealed a structural gene associated with the appearance of an extracellular protein in the culture medium. The derived amino acid sequence indicated the presence of a zinc-binding motif, which was previously noted to be characteristic of metalloprotease enzymes. However, the relatively small size of the protein (apparent molecular weight 20 000 – 24 000) suggests that it represents a novel class of neutral proteases distinct from the thermolysin-type enzymes. An adjacent divergent open reading frame was identified and shown to cause a significant increase in protease activity when present together with the protease structural gene on a multicopy plasmid in S. lividans 66. The derived amino acid sequence of this open reading frame showed homology with previously characterized regulatory proteins of the LysR family of transcriptional regulator proteins. Key words: Streptomyces, extracellular proteases, putative transcriptional regulator.

scholarly journals Primary structure of the Saccharomyces cerevisiae GAL4 gene.

1984 ◽  
Vol 4 (2) ◽  
pp. 260-267 ◽  
Author(s):  
A Laughon ◽  
R F Gesteland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Dna Sequence ◽  
Primary Structure ◽  
Open Reading Frame ◽  
Binding Motif ◽  
Reading Frame ◽  
Activator Proteins ◽  
Inducible Genes

The GAL4 gene encodes a positive regulator of the galactose-inducible genes in Saccharomyces cerevisiae. Recently, GAL4 has been cloned and its 2.8-kilobase mRNA has been identified. We report here the DNA sequence of GAL4 and the mapping of the 5' and 3' ends of its transcripts. The region sequenced contains a single open reading frame, 881 codons long, which could encode a 99,350-dalton protein. The 5' ends of the GAL4 transcripts fall into two clusters. Transcripts which begin at the upstream cluster would encode the 99,350-dalton protein, whereas those starting at the downstream cluster may result in the synthesis of a shorter, 91,600-dalton protein. The putative GAL4 proteins contain an amino acid sequence near their amino termini which resembles a DNA-binding motif found in bacterial and phage repressors and gene activator proteins.

scholarly journals Cloning, expression and gene organization of a human Neu5Acα2–3Galβ1–3GalNAc α2,6-sialyltransferase: hST6GalNAc IV

2000 ◽  
Vol 352 (1) ◽  
pp. 37-48 ◽  
Author(s):  
Anne HARDUIN-LEPERS ◽  
David C. STOKES ◽  
Wim F.A. STEELANT ◽  
Bénédicte SAMYN-PETIT ◽  
Marie-Ange KRZEWINSKI-RECCHI ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Expressed Sequence Tag ◽  
Human Cancer ◽  
Cell Types ◽  
Gene Organization ◽  
Reading Frame ◽  
Human Cancer Cells

On the basis of the detection of expressed sequence tag (‘EST’) similar to the rat N-acetylgalactosamine α2,6-sialyltransferase (ST6GalNAc) III cDNA, we have identified a novel member of the human ST6GalNAc family. We have isolated a cDNA clone containing an open reading frame that codes for a type II membrane protein of 302 amino acids with a seven-amino-acid cytoplasmic domain, an 18-amino-acid transmembrane domain and the smallest described catalytic domain of 277 amino acids. This predicted sialyltransferase sequence is similar to the rat ST6GalNAc III (46.6%), but was found to be even more similar to the recently reported mouse ST6GalNAc IV (88.1%) on the basis of amino acid sequence identity. Northern-blot analysis showed that the newly identified gene is expressed constitutively in various adult human tissues as a 2.2kb transcript, but was also found to be expressed at lower levels in brain, heart and skeletal muscle as a 2.5kb transcript. Expression of the hST6GalNAc IV gene was investigated by reverse transcription PCR in various human cancer cells, and was found to be present in the majority of cell types with the exception of the carcinoma cell line T47D and pro-monocyte THP cells. The transient expression in COS-7 cells of the full-length cDNA led to the production of an active enzyme sharing the acceptor specificity of the ST6GalNAc family towards Neu5Acα2–3Galβ1–3GalNAcα-O-R (where ‘R’denotes H, benzyl, or a peptidic chain). Detailed analysisin vitro of substrate specificity revealed that the enzyme required the trisaccharide Neu5Acα2–3Galβ1–3GalNAc found on O-glycans and arylglycosides. In addition, we have clarified the genomic organization of ST6GalNAc IV gene.

A sea urchin homologue of the chordate Brachyury (T) gene is expressed in the secondary mesenchyme founder cells

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2747-2754 ◽  
Author(s):  
Y. Harada ◽  
H. Yasuo ◽  
N. Satoh
Keyword(s):  
Amino Acid ◽  
Sea Urchin ◽  
Larval Stage ◽  
Single Copy ◽  
Amino Acid Identity ◽  
Molecular Probe ◽  
Reading Frame ◽  
Acid Identity ◽  
Origin And Evolution ◽  
Founder Cells

Chordates are thought to have emerged from some common ancestor of deuterostomes by organizing shared anatomical and embryological features including a notochord, a dorsal nerve cord and pharyngeal gill slits. Because the notochord is the most prominent feature of chordates and because the Brachyury (T) gene is essential for notochord formation, the T gene is a key molecular probe with which to explore the origin and evolution of chordates. We investigated whether the sea urchin (echinoderm) conserves the T gene and, if so, where the sea urchin T gene is expressed. A cDNA clone for the sea urchin T (HpTa) gene contained a long open reading frame that encodes a polypeptide of 434 amino acids. Although the overall degree of amino acid identity was not very high (52%, sea urchin/mouse), in the T domain of the N terminus the amino acid identity was 73% (sea urchin/mouse). The HpTa gene is present as a single copy per haploid genome. As with the chordate T gene, the expression of HpTa is transient, being first detected in the swimming blastula, maximally transcribed in the gastrula, decreasing at the prism larval stage and barely detectable at the pluteus larval stage. HpTa transcripts were found in the secondary mesenchyme founder cells, vegetal plate of the mesenchyme blastula, extending tip of the invaginating archenteron and, finally, the secondary mesenchyme cells at the late-gastrula stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure of the Saccharomyces cerevisiae GAL4 gene

1984 ◽  
Vol 4 (2) ◽  
pp. 260-267
Author(s):  
A Laughon ◽  
R F Gesteland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Dna Sequence ◽  
Primary Structure ◽  
Open Reading Frame ◽  
Binding Motif ◽  
Reading Frame ◽  
Activator Proteins ◽  
Inducible Genes

The GAL4 gene encodes a positive regulator of the galactose-inducible genes in Saccharomyces cerevisiae. Recently, GAL4 has been cloned and its 2.8-kilobase mRNA has been identified. We report here the DNA sequence of GAL4 and the mapping of the 5' and 3' ends of its transcripts. The region sequenced contains a single open reading frame, 881 codons long, which could encode a 99,350-dalton protein. The 5' ends of the GAL4 transcripts fall into two clusters. Transcripts which begin at the upstream cluster would encode the 99,350-dalton protein, whereas those starting at the downstream cluster may result in the synthesis of a shorter, 91,600-dalton protein. The putative GAL4 proteins contain an amino acid sequence near their amino termini which resembles a DNA-binding motif found in bacterial and phage repressors and gene activator proteins.

scholarly journals Analysis of the primary sequence and microtubule-binding region of the Drosophila 205K MAP.

1990 ◽  
Vol 111 (6) ◽  
pp. 2563-2572 ◽  
Author(s):  
I Irminger-Finger ◽  
R A Laymon ◽  
L S Goldstein
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Consensus Sequence ◽  
Specific Protein ◽  
Peptide Sequence ◽  
Binding Motif ◽  
Cdna Clones ◽  
Microtubule Binding ◽  
Reading Frame ◽  
Amino Acid Region

We have sequenced cDNA clones encoding the Drosophila 205K microtubule-associated protein (MAP), a protein that may be the species specific homologue of mammalian MAP4. The peptide sequence deduced from the longest open-reading frame reveals a hydrophilic protein, which has basic and acidic regions that are similar in organization to mammalian MAP2. Using truncated forms of the 205K MAP, a 232-amino acid region could be defined that is necessary for microtubule binding. The amino acid sequence of this region shares no similarity with the binding motif of MAP2 or tau. We also analyzed several embryonic cDNA clones, which show the existence of differentially spliced mRNAs. Finally, we identified several potential protein kinase target sequences. One of these is distal to the microtubule-binding site and fits the phosphorylation consensus sequence of proteins phosphorylated by the mitosis specific protein kinase cdc2. Our data suggest that the 205K MAP uses a microtubule-binding motif unlike that found in other MAPs, and also raise the possibility that the activities of the 205K MAP may be regulated by alternative splicing and phosphorylation.

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