Identification and in silico structural and functional analysis of a trypsin-like protease from shrimp Macrobrachium carcinus

Macrobrachium carcinus (Linnaeus, 1758) is a species of freshwater shrimp widely distributed from Florida southwards to southern Brazil, including southeast of Mexico. In the present work, we identified a putative trypsin-like protease cDNA fragment of 736 nucleotides from M. carcinus hepatopancreas tissue by the 3′RACE technique and compared the deduced amino acid sequence to other trypsin-related proteases to describe its structure and function relationship. The bioinformatics analyses showed that the deduced amino acid sequence likely corresponds to a trypsin-like protease closely related to brachyurins, which comprise a subset of serine proteases with collagenolytic activity found in crabs and other crustacea. The M. carcinus trypsin-like protease sequence showed a global sequence identity of 94% with an unpublished trypsin from Macrobrachium rosenbergii (GenBank accession no. AMQ98968), and only 57% with Penaeus vannamei trypsin (GenBank accession no. CAA60129). A detailed analysis of the amino acid sequence revealed specific differences with crustacean trypsins, such as the sequence motif at the beginning of the mature protein, activation mechanism of the corresponding zymogen, amino acid residues of the catalytic triad and residues responsible for substrate specificity.

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The complete amino acid sequence of rat submaxillary gland tonin does contain the aspartic acid at the active site: confirmation by protein sequence analyses

Biochemistry and Cell Biology ◽

10.1139/o87-042 ◽

1987 ◽

Vol 65 (4) ◽

pp. 321-337 ◽

Cited By ~ 11

Author(s):

C. Lazure ◽

R. Leduc ◽

N. G. Seidah ◽

G. Thibault ◽

J. Genest ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Active Site ◽

Serine Proteases ◽

Submaxillary Gland ◽

Catalytic Triad ◽

Complete Amino Acid Sequence ◽

Molecular Weights ◽

Amino Acid Peptide ◽

Close Relationship

The revised amino acid sequence of rat submaxillary gland tonin, a serine protease, does contain the active site Asp residue. The active site of this kallikrein-related enzyme is thus made up of the same catalytic triad (Asp, Ser, and His) found in all known serine proteases. The important Asp residue has now been localized in a 16 amino acid peptide previously reported as missing in the tonin sequence. The complete amino acid sequence thus contains 235 residues corresponding to a molecular weight of 25 658, more in agreement with previously reported molecular weights. Moreover, the revised structure led (a) to the assignment of Arg, Asn, and Val residues instead of His, Asp, and Gly at positions 63, 165, and 169, respectively; (b) to the assignment of residues occupying an overlapping sequence at positions 165–171, and finally (c) to the localization of two N-glycosylation sites at positions 82 and 165. These results further document the close relationship of tonin to the ever expanding kallikrein family.

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Determination of amino acid sequence and site of mRNA expression of four vitellins in the giant freshwater prawn,Macrobrachium rosenbergii

Journal of Experimental Zoology ◽

10.1002/1097-010x(20001101)287:6<413::aid-jez2>3.0.co;2-v ◽

2000 ◽

Vol 287 (6) ◽

pp. 413-422 ◽

Cited By ~ 69

Author(s):

Wei-Jun Yang ◽

Tsuyoshi Ohira ◽

Naoaki Tsutsui ◽

Thanumalayaperumal Subramoniam ◽

Do Thi Thanh Huong ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Mrna Expression ◽

Macrobrachium Rosenbergii ◽

Freshwater Prawn ◽

Giant Freshwater Prawn

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Analysis of Htra Gene from Zebrafish (Danio Rerio)

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.7554 ◽

2015 ◽

Vol 10 (2) ◽

Author(s):

M. Murwantoko ◽

Chio Oka ◽

Masashi Kawaichi

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Danio Rerio ◽

Serine Proteases ◽

Catalytic Domain ◽

Fruit Fly ◽

Wide Range ◽

Igf Binding ◽

Bacterial Homolog

HtrA which is characterized by the combination of a trypsin-like catalytic domain with at least one C-terminalPDZ domain is a highly conserved family of serine proteases found in a wide range of organisms. However theidentified HtrA family numbers varies among spesies, for example the number of mammalian, Eschericia coli,fruit fly-HtrA family are 4, 3 and 1 gene respectively. One gene is predicted exist in zebrafish. Since no completeinformation available on zebrafish HtrA, in this paper zebrafish HtrA (zHtrA) gene was analyzed. The zHtrA isbelonged to HtrA1 member and predicted encodes 478 amino acids with a signal peptide, a IGF binding domain,a Kazal-type inhibitor domain in the up stream of HtrA-bacterial homolog. At the amino acid sequence the zHtrA1showed the 69%, 69%, 68%, 54% and 54% with the rat HtrA1, mouse HtrA1, human HtrA1, human HtrA3 andmouse HtrA4 respectively. The zHtrA1 is firstly expressed at 60 hpf and mainly in the vertebral rudiments in thetail region.

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Identification and expression of Pen c 2, a novel allergen from Penicillium citrinum

Biochemical Journal ◽

10.1042/bj3410051 ◽

1999 ◽

Vol 341 (1) ◽

pp. 51-59 ◽

Cited By ~ 8

Author(s):

Lu-Ping CHOW ◽

Ning-Yuan SU ◽

Chia-Jung YU ◽

Bor-Luen CHIANG ◽

Horng-Der SHEN

Keyword(s):

Amino Acid ◽

Serine Proteases ◽

Catalytic Triad ◽

Penicillium Citrinum ◽

E Coli ◽

Acid Protein ◽

Amino Acid Signal Peptide ◽

Cloned Cdna ◽

Allergic Disorders ◽

Amino Acid Protein

The mould genus, Penicillium, is known to be a significant source of environmental aero-allergens. One important allergen from Penicillium citrinum, Pen c 2, has been identified by means of two-dimensional immunoblotting using IgE-containing patients' sera. This novel allergen was cloned, sequenced and expressed in Escherichia coli. The cloned cDNA encodes a large 457-amino acid protein precursor containing a 16-amino acid signal peptide, a 120-amino acid propeptide and the 321-amino acid mature protein. Comparison of the Pen c 2 sequence with known protein sequences revealed shared high sequence similarities with two vacuolar serine proteases from Aspergillus niger and Saccharomyces cerevisiae. Asp-46, His-78 and Ser-244 were found to constitute the catalytic triad of the 39-kDa Pen c 2. The DNA coding for Pen c 2 was cloned into vector PQE-30 and expressed in E. coli as a His-tag fusion protein that bound serum IgE from Penicillium-allergic patients on immunoblots. Recombinant Pen c 2 could therefore be used effectively for diagnosis and also potentially for the treatment of mould-derived allergic disorders.

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Genetic Organization and Molecular Analysis of the EcoVIII Restriction-Modification System of Escherichia coli E1585-68 and Its Comparison with Isospecific Homologs

Applied and Environmental Microbiology ◽

10.1128/aem.69.5.2638-2650.2003 ◽

2003 ◽

Vol 69 (5) ◽

pp. 2638-2650 ◽

Cited By ~ 10

Author(s):

Iwona Mruk ◽

Tadeusz Kaczorowski

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Start Codon ◽

Sequence Motif ◽

Specific Sequence ◽

Modification System ◽

Acid Protein ◽

Restriction Modification ◽

Amino Acid Protein

ABSTRACT The EcoVIII restriction-modification (R-M) system is carried by the Escherichia coli E1585-68 natural plasmid pEC156 (4,312 bp). The two genes were cloned and characterized. The G+C content of the EcoVIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or E. coli genomic DNA (50.8%). The difference suggests that there is a possibility that the EcoVIII R-M system was recently acquired by the genome. The 921-bp EcoVIII endonuclease (R · EcoVIII) gene (ecoVIIIR) encodes a 307-amino-acid protein with an M r of 35,554. The convergently oriented EcoVIII methyltransferase (M · EcoVIII) gene (ecoVIIIM) consists of 912 bp that code for a 304-amino-acid protein with an M r of 33,930. The exact positions of the start codon AUG were determined by protein microsequencing. Both enzymes recognize the specific palindromic sequence 5′-AAGCTT-3′. Preparations of EcoVIII R-M enzymes purified to homogeneity were characterized. R · EcoVIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5′ protruding ends. M · EcoVIII functions as a monomer and modifies the first adenine residue at the 5′ end of the specific sequence to N 6-methyladenine. These enzymes are thus functionally identical to the corresponding enzymes of the HindIII (Haemophilus influenzae Rd) and LlaCI (Lactococcus lactis subsp. cremoris W15) R-M systems. This finding is reflected by the levels of homology of M · EcoVIII with M · HindIII and M · LlaCI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m6 N-adenine β-class methyltransferases. The deduced amino acid sequence of R · EcoVIII shows weak homology with its two isoschizomers, R · HindIII (26%) and R · LlaCI (17%). A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R · EcoVIII (D108X12DXK123), as well as in the primary structures of R · LlaCI and R · HindIII. Polyclonal antibodies raised against R · EcoVIII did not react with R · HindIII, while anti-M · EcoVIII antibodies cross-reacted with M · LlaCI but not with M · HindIII. R · EcoVIII requires Mg(II) ions for phosphodiester bond cleavage. We found that the same ions are strong inhibitors of the M · EcoVIII enzyme. The biological implications of this finding are discussed.

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Determination of Factor Xa Activity by Means of Chromogenic Substrates Based on the Primary Structure of Prothrombin

10.1055/s-0039-1689426 ◽

1975 ◽

Cited By ~ 1

Author(s):

L. Aurell ◽

A. Olausson ◽

G. Claeson

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Primary Structure ◽

Serine Proteases ◽

Factor Xa ◽

Amino Acid Sequences ◽

Peptide Substrate ◽

Chromogenic Substrates ◽

Special Regard

Through the work of Magnusson and co-workers leading to the elucidation of the primary structure of prothrombin including the amino acid sequences around the two bonds split by factor Xa it has been possible to design a synthetic chromogenic peptide substrate. Bz-Ile-Glu-Gly-Arg-pNA, specifically intended for the determination of factor Xa. Furthermore, additional substrates have been synthezised with various alterations in the amino acid sequence. The activity of factor Xa and other serine proteases within the coagulation and fibrinolytic systems towards these substrates will be discussed with special regard to their possible use in coagulation studies.

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Human diadenosine 5′,5′′′-P1,P4-tetraphosphate pyrophosphohydrolase is a member of the MutT family of nucleotide pyrophosphatases

Biochemical Journal ◽

10.1042/bj3110717 ◽

1995 ◽

Vol 311 (3) ◽

pp. 717-721 ◽

Cited By ~ 42

Author(s):

N M H Thorne ◽

S Hankin ◽

M C Wilkinson ◽

C Nuñez ◽

R Barraclough ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Expressed Sequence Tag ◽

Sequence Motif ◽

Diadenosine Tetraphosphate ◽

Expressed Sequence

The cDNA and derived amino acid sequence of human diadenosine 5′,5‴-P1,P4-tetraphosphate pyrophosphohydrolase have been determined with the aid of the GenBank Expressed Sequence Tag database. This enzyme possesses a modification of the MutT sequence motif found in certain nucleotide pyrophosphatases. It is unrelated to the enzymes of diadenosine tetraphosphate catabolism found in prokaryotes and fungi.

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Extracellular-matrix degradation at acid pH. Avian osteoclast acid collagenase isolation and characterization

Biochemical Journal ◽

10.1042/bj2900873 ◽

1993 ◽

Vol 290 (3) ◽

pp. 873-884 ◽

Cited By ~ 32

Author(s):

H C Blair ◽

S L Teitelbaum ◽

L E Grosso ◽

D L Lacey ◽

H L Tan ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Substrate Specificity ◽

Cathepsin B ◽

Type I Collagen ◽

Sequence Similarity ◽

Bone Matrix ◽

Collagenolytic Activity ◽

Type I ◽

Isolation And Characterization

Osteoclasts degrade bone matrix, which is mainly type I collagen and hydroxyapatite, in an acidic extracellular compartment. Thus we reasoned that osteoclasts must produce an acid collagenase. We purified this enzyme, a 31 kDa protein, from avian osteoclast lysates (in 100 mM acetate/1 mM CHAPS/1 mM dithiothreitol, pH 4.4), fractionated by (NH2)2SO4 precipitation, gelatin-affinity, cation exchange, and gel filtration. Fraction activity was measured using diazotized collagen or 3H-labelled cross-linked collagen (decalcified and trypsin-treated metabolically L-[4,5-3H]proline-labelled bone) as substrates. Iodoacetate, leupeptin, antipain, pepstatin and mercurials inhibited collagenolysis by the isolated proteinase; mercurial derivatives could not be re-activated by dithiothreitol. Collagen degradation was maximal at pH 4.4; purified proteinase reproduced the collagenolytic activity of cell lysates. The N-terminal amino acid sequence from the isolated protein and its CNBr degradation fragments showed sequence similarity to mammalian cathepsin Bs, and near-identity with avian liver cathepsin B. Peptide substrate specificity of the osteoclastic enzyme resembled those of mammalian cathepsin B and its avian liver counterpart, but degradation of low-molecular-mass substrates by the osteoclastic enzyme was slower, reflecting generally lower kcat. values. Further, kcat/Km varied less between arginine-containing substrates than for previously reported cathepsin Bs, indicating different substrate specificity of the osteoclast enzyme. Polyclonal antibody raised to a 25 kDa fragment of the enzyme recognized a single 31 kDa band in SDS/PAGE of osteoclast lysates blotted to poly(vinylidene difluoride), adsorbed collagenolytic activity of osteoclast lysates, and stained avian osteoclasts in tissue sections. Degenerate sense- and antisense-oligonucleotide primers, predicted from segments of primary amino acid sequence, amplified a 486 bp DNA fragment; this was cloned and sequenced. Of 162 amino acids encoded, 77% are identical with those of human cathepsin B; hybridization identified a 2.4 kb RNA in osteoclast lysates. We conclude that the major avian osteoclast collagenolytic enzyme is a cathepsin B, whose activity varies from other enzymes of its class.

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