Molecular cloning, sequencing, and expression of cDNA encoding serine protease with fibrinolytic activity from earthworm

An earthworm, Lumbricus rubellus, produces alkaline trypsin-like proteases that are greater than trypsins in their stability and strong tolerance to organic solvents. cDNAs encoding strong fibrinolytic proteases (F-III-2 and F-III-1) in the six isozymes were cloned and sequenced to study their stability-structure relationship. The cDNAs of F-III-2 and F-III-1 comprised 1011 and 973 bp and included open reading frames that encode polypeptides of 245 and 246 amino acid residues, respectively. The deduced amino acid sequences of F-III-2 and F-III-1 have 7 and 8 activation peptides in the N-termini respectively, indicating that they are translated as proenzymes and modified to active forms by posttranslational processing. They showed similarity to mammalian serine proteases and conserved the catalytic amino acid residues, however, neither arginine nor lysine residues were present in the autolysis region. The gene encoding the native form of F-III-2 was expressed in Pichia pastoris to produce and secrete the earthworm protease in the culture medium, which dissolves an artificial fibrin plate.     

Molecular biology of 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase enzymes

There are two steroid 11β-hydroxylase isozymes encoded by the CYP11B1 and CYP11B2 genes on human chromosome 8q. The first is expressed at high levels in the normal adrenal gland, has 11β-hydroxylase activity and is regulated by ACTH. Mutations in the corresponding gene cause congenital adrenal hyperplasia due to 11β-hydroxylase deficiency; thus, this isozyme is required for cortisol biosynthesis. The second isozyme is expressed at low levels in the normal adrenal gland but at higher levels in aldosterone-secreting tumors, and has 11β-hydroxylase, 18-hydroxylase and 18-oxidase activities. The corresponding gene is regulated by angiotensin II, and mutations in this gene are found in persons who are unable to synthesize aldosterone due to corticosterone methyloxidase II deficiency. Thus, this isozyme is required for aldosterone biosynthesis.

Cortisol and aldosterone are both effective ligands of the “mineralocorticoid” receptor in vitro, but only aldosterone is a potent mineralocorticoid in vivo. This apparent specificity occurs because 11β-hydroxysteroid dehydrogenase in the kidney converts cortisol to cortisone, which is not a ligand for the receptor. This enzyme is a “short-chain” dehydrogenase which is encoded by a single gene on human chromosome 1. It is possible that mutations in this gene cause a form of childhood hypertension called apparent mineralocorticoid excess, in which the mineralocorticoid receptor is not protected from high concentrations of cortisol.     

Thiophilic adsorption chromatography: purification of Equ c2 and Equ c3, two horse allergens from horse sweat

Purification of two allergens from horse (Equus caballus) sweat, Equ c2 and Equ c3, by means of salt-promoted chromatography on a “thiophilic” (T-gel) adsorbent is described. Immobilization of these proteins was found to be dependent on the presence of water-structure-forming salts where the ammonium sulphate concentration in the equilibration buffer was 2 M. Equ c2 showed higher affinity towards the thiophilic matrix than Equ c3. Their molecular mass (M_r) values established by SDS–polyacrylamide gel electrophoresis were for Equ c2 ≈17 000 and for Equ c3 ≈16 000, and both proteins showed a low isoelectric point of ≈3.8. Their allergenic properties were also investigated using sera from horse-sensitized patients, where it was demonstrated that these proteins exhibited an IgE antibody binding capacity. In this report we show the broad potential applications of thiophilic adsorption chromatography for the efficient purification of allergens.     

Molecular cloning and characterization of cDNA encoding fibrinolytic enzyme-3 from earthworm Eisenia foetida

Earthworm fibrinolytic enzyme (EFE), a multi-com-ponent protease purified from some earthworm breeds,belongs to serine protease family with fibrinolytic activity[1]. It has been used in prevention and treatment of cardiacand cerebrovascular diseases in Ch…     

An isozyme of earthworm serine proteases acts on hydrolysis of triacylglycerol

An enzyme catalyzing the hydrolysis of triacylglycerol was purified from an earthworm. The N-terminal amino acid sequence and the catalytic function of the purified enzyme were identical to those of Isozyme C, an isozyme of the earthworm-serine proteases. No other lipase proteins were found in the earthworm cells. The isozyme might act on the hydrolysis of triacylglycerol as well as the protein decomposition.     

蚯蚓纤溶酶分子生物学进展

蚯蚓纤溶酶是近年发现的一种新型的溶解血栓物质,属丝氨酸蛋白酶,不同种属的蚯蚓中均可分离到,具纤溶活性和溶栓活性。有较好的热稳定性,多为单体酶,多数兼有纤溶活性和纤溶酶原激活活性。不同种属的蚯蚓分离的纤溶酶性质上有一定差别。已获得多种纤溶酶的N端序列及部分核酸序列,相互之间及与某些蛋白酶之间有一定的同源性。纤溶酶通过降解目的蛋白的特定位点而起作用 。     

Carnitine palmitoyltransferases 1 and 2: biochemical, molecular and medical aspects

Carnitine palmitoyltransferase (CPT) deficiencies are common disorders of mitochondrial fatty acid oxidation. The CPT system is made up of two separate proteins located in the outer (CPT1) and inner (CPT2) mitochondrial membranes. While CPT2 is an ubiquitous protein, three tissue-specific CPT1 isoforms––the so-called “liver” (CPT1-A), “muscle” (CPT1B) and «brain» (CPT1-C) CPT1s––have been shown to exist. Amino acid and cDNA nucleotide sequences have been identified for all of these proteins. CPT1-A deficiency presents as recurrent attacks of fasting hypoketotic hypoglycemia. Twenty four CPT1A mutations have been reported to date. CPT1-B and -C deficiencies have not been hitherto identified. CPT2 deficiency has several clinical presentations. The “benign” adult form (more than 200 families reported) is characterized by episodes of rhabdomyolysis triggered by prolonged exercise. The prevalent S113L mutation is found in about 50% of mutant alleles. The infantile-type CPT2 presents as severe attacks of hypoketotic hypoglycemia, occasionally associated with cardiac damage commonly responsible for sudden death before 1 year of age. In addition to these symptoms, features of brain and kidney dysorganogenesis are frequently seen in the neonatal-onset CPT2 deficiency, almost always lethal during the first month of life. Around 40 CPT2 mutations (private missense or truncating mutations) have hitherto been detected. Treatment is based upon avoidance of fasting and/or exercise, a low fat diet enriched with medium chain triglycerides and carnitine. Prenatal diagnosis may be offered for pregnancies at a 1/4 risk of infantile/severe-type CPT2 deficiency.     

Characterization of Potent Fibrinolytic Enzymes in Earthworm,Lumbricus rubellus

Stable and potent fibrinolytic enzymes (six homogeneous proteins) were purified to homogeneity from extracts of the lyophilized powder of an earthworm, Lumbricus rubellus. The molecular weight of each enzyme estimated by SDS–polyacrylamide gel electrophoresis was different from those by gel filtration chromatography in the six purified proteins. The exact molecular weight of each enzyme (F-III-2, F-III-1, F-II, F-I-2, F-I-l, and F-I-0) measured by ionspray MS analysis was 29, 662, 29, 667, 24, 664, 24, 220, 24, 196, and 23,013, respectively. The isoelectric point (pI) of each enzyme was 3.40, 3.60, 4.20, 4.00, 4.30, and 4.85, respectively. The enzymes were single polypeptide chains. They had a very strong fibrinolytic activity and the maximum reactivity for chromogenic substrates from pH 9-11. The enzymes, acidic proteins that had abundant asparagine and aspartic acid, and low lysine in their amino acid composition, did not contain component sugars. The enzymes were stable at from pH 1-11 and up to 60°C. Studies on substrate specificity and inhibition indicated that these enzymes were alkaline trypsin-like serine proteases. N-Terminal amino acid sequences of the enzymes had local similarities to those of trypsin-like enzymes such as elastase and coagulation factor IX. From the results of amino acid sequence, amino acid composition analyses and immunological analyses, it was suggested that these six enzyme proteins were derived as isozyme(s) from at least four different genes.     

In situ localization and substrate specificity of earthworm protease-II and protease-III-1 from Eisenia fetida

Recently, the function in fibrinolysis of earthworm proteases has been studied. In our experiments, earthworm protease-II (EfP-II) and earthworm protease-III-1 (EfP-III-1) were isolated and purified from Eisenia fetida. As shown by the assay of sections of the earthworm on fibrin plates, the enzymic activity was mainly detected around the clitellum. In the presence of anti-EfP-II or anti-EfP-III-1 serum, the immunological signals of the two isozymes were clearly found in the anterior alimentary mucosa, suggesting that EfP-II and -III-1 are localized and expressed in intestinal epithelial cells. The Michaelis-Menten constant (K(m)) for EfP-III-1 reacting with BAEE is smaller (1.7x10(-5)M) in comparison with the K(m) values of other substrates such as Chromozym-Try and -TH (3.3-6.0x10(-5)M). This indicates that EfP-III-1 is a trypsin-like protein. EfP-II shows a strong trypsin-like, moderate elastase-like and weak chymotrypsin-like serine function. The relative broad substrate specificity of EfP-II and EfP-III-1 is consistent with their localization in the anterior alimentary canal where different micro-organisms and ingested proteins require to be digested.     

Polysaccharides in desert reclamation: Compositions of exocellular proteoglycan complexes produced by filamentous blue-green and unicellular green edaphic algae

The filamentous blue-green alga Nostoc calcicola Geitler, strain 79WA01, showed a peak production of 70% of its biomass as a mixture of exocellular proteoglycan complexes, containing 3–30% of a polypeptide moiety and>70% of a complex glycuronoglycan. The former contained high proportions of Asp, Glu, Arg, and amido-NH₃, in addition to 35% of “hydrophobic” amino-acids. The latter varied in composition in different fractions: GalA (2.5–10.3%), GlcA (4.7–11.5%), Glc (11.7–39.0%), Xyl (5.7–17.9%), Man (2.7–9.5%), Gal (5.7–9.5%), Fuc (1.5–11.1%), Ara (1.9–4.3%), and Rha (1.4–4.4%). None of the fractions showed a stoichiometric ratio of sugar residues.

Palmelloid cells of three unicellular green soil-algae of the genus Chlamydomonas yielded 70% of their dry weight as capsular mucilage. About 50% of the sodium salt of this material was soluble in water, and contained 3–12% of polypeptide and 88–97% of glycuronoglycan (GlcA:Glc:Xyl = 1:1:3 for C. humicola Lucksch, and GlcA:Gal = 1:2 for C. peterfii Gerloff and C. sajao Lewin). These categorical differences in sugar composition, together with narrow composition distributions, suggested regular structures for the glycuronoglycans. The remainder of the mucilages contained essentially the same glycuronoglycan chains, but a higher proportion of polypeptide. These materials did not form true solutions in water, but dispersed as microgel particles.     

Molecular characterization of Lma-p54, a new epicuticular surface protein in the cockroach Leucophaea maderae (Dictyoptera,oxyhaloinae)

The epicuticular surface protein Lma-p54 is imbedded in the “cuticular waxes” which cover the abdominal surface of the adult Leucophaea maderae. Natural Lma-p54 was purified and the complete cDNA sequence was determined by RT–PCR using primers based on Edman degradation fragments. Northern blot and in situ hybridization analyses showed that Lma-p54 was expressed in the adult abdominal epidermis and in the chemical sense organs of both sexes. Sequence alignment indicates that Lma-p54 is closely related to aspartic proteases (EC 3.4.23). However, there are critical amino acid substitutions at the level of the active site and, since no proteolytic activity was detected in the abdominal secretion, the protein is likely inactive. As an inactive aspartic protease, Lma-p54 is related to pregnancy-associated glycoproteins (PAGs) which still present a peptide-binding ability. According to recent experiments using whole tergal protein secretions, a role in intraspecific contact recognition was proposed for this surface protein.     

Human interferon-γ lacking 23 COOH-terminal amino acids is biologically active

We constructed five mutated cDNAs encoding human interferon-γ (IFN-γ) derivatives lacking 19–23 COOH-terminal residues and expressed them in Escherichia coli. All the derivatives were purified to homogeneity. They showed substantially the same order of antiviral activity in vitro as the intact molecule, and behaved as a dimer. The far- and near-UV circular dichroism spectra of the derivatives were quite similar to those of the intact one. These results indicate that the 23 COOH-terminal amino acids at least are not essential for achieving the full antiviral activity and constructing the higher structure of human IFN-γ.     

Eisenstasin,new antistasin family inhibitor from the earthworm

A couple of new antistasin family serine protease inhibitors have been isolated from the non-hematophagous earthworm, Eisenia andrei. These novel inhibitors have been designated as eisenstasin I and II. Similar to other antistasin family inhibitors, eisenstasin I and II feature 3 and 4 internal repeats, respectively, of a 24–29 amino acid sequence, both of which exhibit a conserved pattern of 6-cysteine/2-glycine at an identical position between the third and fourth cysteine residues. This suggests that the eisenstasins isolated from the earthworm are members of the antistasin family. The eisenstasins are 82% similar with regard to amino acid sequences and exhibit over 70% similarity with the antistasins from the earthworm Lumbricus rubellus, while also displaying less than 40% sequence similarity with the leech antistasins. Earthworm eisenstasins are basic proteins, primarily due to the frequent occurrence of arginine residues in their structure, especially at the C-terminal region. As arginine is a key residue for the substrate specificity of some serine proteases including FXa, it is thought that these multiple arginine residues may play a role in the inhibitory characteristics of the eisenstasins. Considering the structure and number of the internal repeats derived from a variety of animal species, the deletion as well as the duplication of all or part of an internal repeat may be implicated in the evolution of the structure and function of the antistasin family inhibitors.     

Malat-dehydrogenase isoenzymbanden als potentielles chemotaxonomisches kriterium für cyanophyceen-species

Malate dehydrogenase isoenzymes from eight cyanophycean species were investigated with polyacrylamide disc gel electrophoresis. Using 7% acrylamide the pherograms from each species showed 5–8 zones with malate dehydrogenase activity. It was demonstrated that in Anabaena flos-aquae there are 8 isoenzyme bands which include 3 forms of equal molecular weight, two of which consist of several isomers differing in their net charge. The MDH zymograms of the blue-green algae investigated can be used as “fingerprints”. The isoenzyme pattern of the MDHs of Anacystis nidulans makes its position in the order Chroococcales uncertain.

Résumé

Wäßrige Extrakte aus acht Cyanophyceenarten wurden einer Polyacrylamid-Discelektrophorese unterzogen und die erhaltenen Elektropherogramme auf Malat-Dehydrogenase (MDH)-Aktivität geprüft. Dabei ergab sich, daß unter unseren Versuchsbedingungen die MDH der getesteten Cyanophyceen in 5–8 Isoenzymbanden aufspaltet. Für Anabaena flos-aquae konnte gezeigt warden, daß sich die 8 Isoenzymbanden auf wenigstens 3 Molekulargewichts-Isomere zurückführen lassen, von denen zwei noch mehrere Ladungsisomere bilden. Die erhaltenen Zymogramme zeigen “fingerprint”-Charakter. was ihre mögliche Verwendbarkeit für die Chemotaxonomie der Cyanophyta nahelegt. Die Stellung von Anacystis nidulans innerhalb der CyanophyceenOrdnungen wird diskutiert.     

Cloning,Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

A lumbrokinase gene encoding a blood-clot dissolving protein was cloned from earthworm (Eisenia fetida) by RT-PCR amplification. The gene designated as CST1 (GenBank No. {"type":"entrez-nucleotide","attrs":{"text":"AY840996","term_id":"56718408","term_text":"AY840996"}}AY840996) was sequence analyzed. The cDNA consists of 888 bp with an open reading frame of 729 bp, which encodes 242 amino acid residues. Multiple sequence alignments revealed that CST1 shares similarities and conserved amino acids with other reported lumbrokinases. The amino acid sequence of CST1 exhibits structural features similar to those found in other serine proteases, including human tissue-type (tPA), urokinase (uPA), and vampire bat (DSPAα1) plasminogen activators. CST1 has a conserved catalytic triad, found in the active sites of protease enzymes, which are important residues involved in polypeptide catalysis. CST1 was expressed as inclusion bodies in Escherichia coli BL21(DE3). The molecular mass of recombinant CST1 (rCST) was 25 kDa as estimated by SDS–PAGE, and further confirmed by Western Blot analysis. His-tagged rCST1 was purified and renatured using nickel-chelating resin with a recovery rate of 50% and a purity of 95%. The purified, renatured rCST1 showed fibrinolytic activity evaluated by both a fibrin plate and a blood clot lysis assay. rCST1 degraded fibrin on the fibrin plate. A significant percentage (65.7%) of blood clot lysis was observed when blood clot was treated with 80 mg/mL of rCST1 in vitro. The antithrombotic activity of rCST1 was 912 units/mg calculated by comparison with the activity of a lumbrokinase standard. These findings indicate that rCST1 has potential as a potent blood-clot treatment. Therefore, the expression and purification of a single lumbrokinase represents an important improvement in the use of lumbrokinases.     

Presence of intestinal, liver and heart/adipocyte fatty-acid-binding protein types in the liver of a chimaera fish

Five fatty-acid-binding proteins from the liver of the elephant fish (Callorhynchus callorhynchus), a chimaera fish that belongs—together with the elasmobranchs—to the ancient chondrichthyes class were isolated and characterized. The purification procedures for these proteins involved gel filtration, anion-exchange chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a last step. They were submitted to “in gel” tryptic or cyanogen bromide digestion and the resulting peptides were separated by high performance liquid chromatography and then sequenced by Edman degradation. According to their partial amino acid sequences, one of them presents the highest identity with fatty-acid-binding proteins from human and catfish liver, another three with those from mammalian heart or adipose tissue and the fifth with the mammalian intestinal fatty-acid-binding protein. The presence of various members of this protein family, as now found in elephant fish and previously in catfish (Rhamdia sapo) liver, does not occur in mammalian liver which expresses only one a characteristic fatty-acid-binding protein.     

Biochemical properties of purified cathepsin B from Schistosoma mansoni

A previously described “major acidic proteinase” of adult Schistosoma mansoni, believed to play a key role in the parasite's metabolism, has been identified as a cathepsin B (Sm31). Purified Sm cathepsin B was not recognized by anti-Sm32 or anticathepsin L antibodies. The enzyme hydrolyzes the synthetic protease substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC as well as protein substrates. Its pH optimum is 3.0 with serum albumin, 4.0–5.0 with globin and 5.5–6.0 with the synthetic substrates. The enzyme was inactivated by cysteine proteinase inhibitors. Its activity against protein substrates would support the hypothesis that it plays a role in schistosome nutrition.     

Characterization of two extracellular proteinases from Aspergillus terreus and their role in the formation of low molecular weight endoglucanases

Two extracellular proteolytic activities from the wood degrading fungus Aspergillus terreus have been characterized. Proteinase I (serine thiol-dependent enzyme) was active over a broad pH range (7·0–10·0) and at 55°C. The second proteinase (metalloproteinase) showed optimal activity at pH 6·0–7·0 and at 65–70°C. Both proteins had isoelectric points at acid pH and contained carbohydrate moieties. The metalloproteinase possessed a uniquely high content of serine and threonine and an extremely low percentage of glutamate and aspartate. The metalloproteinase was involved in the formation of the low molecular mass endoglucanases of A. terreus.