Sumio Tanase

Activation of Human Matrix Metalloproteinases by Various Bacterial Proteinases

Matrix metalloproteinases (MMPs) are zinc-containing proteinases that participate in tissue remodeling under physiological and pathological conditions. To test the involvement of bacterial proteinases in tissue injury during bacterial infections, we investigated the activation potential of various bacterial proteinases against precursors of MMPs (proMMPs) purified from human neutrophils (proMMP-8 and −9) and from human fibrosarcoma cells (proMMP-1). Each proMMP was subjected to treatment with a series of bacterial proteinases at molar ratios of 0.01-0.1 (bacterial proteinase to proMMP), and activities of MMPs generated were determined. Among six different bacterial proteinases, thermolysin family enzymes (family M4) such as Pseudomonas aeruginosa elastase, Vibrio cholerae proteinase, and thermolysin strongly activated all three proMMPs via limited proteolysis to generate active forms of the MMPs. N-terminal sequence analysis of the active MMPs revealed that cleavage occurred at the Val82-Leu83 and Thr90-Phe91 bonds of proMMP-1 and proMMP-9, respectively, which are located near the N terminus of the catalytic domain of MMPs. In contrast, Serratia 56-kDa proteinase and Pseudomonas alkaline proteinase, both of which are classified as members of the serralysin subfamily of zinc metalloproteinases (family M10), and Serratia 73-kDa thiol proteinase did not evidence proteolytic processing or activation of proMMP-1, −8, and −9 under these experimental conditions. These results indicate that bacterial proteinases may play an important role in tissue destruction and disintegration of extracellular matrix at the site of infections.

Ethylene Production by Micro-organisms

Publisher Summary Ethylene is the simplest of unsaturated hydrocarbons and the most important starting material in petroleum chemistry. It is also a plant hormone involved in a number of physiological processes, such as fruit ripening and plant senescence. The chapter summarizes the biosynthetic pathways to ethylene found in micro-organisms and compares them with results from higher plants. The chapter discusses the characteristics of the reaction catalyzed by the ethylene-forming enzyme. Micro-organisms and higher plants synthesize ethylene by different biochemical pathways. In micro-organisms, there are two biosynthetic pathways for ethylene formation—namely, the KMBA and the 2-oxoglutarate pathways. Using the purified enzyme, the ethylene-forming reactions were surveyed and it was found that the purified enzyme simultaneously catalyzes two reactions— namely, formation of ethylene and of succinate from 2-oxoglutarate. In a comparison with the ethylene-forming enzymes of higher plants, the homology score for the entire amino acid-residue sequence of the enzyme from Ps. syringae compared with the enzymes from plants, or with 2-oxoglutarate-dependent dioxygenases, was low. However, functionally significant regions appear to be conserved. The chapter also discusses the genes for the ethylene-forming enzyme of Ps. syringae and higher plants from the evolutionary point of view. It is expected that the gene in Ps. syringae will be very useful for production of ethylene, and following further research, practicable new processes for production of ethylene will be established and will herald a new age free from the threat of shortages of petroleum resources.

Induction of vascular leakage through release of bradykinin and a novel kinin by cysteine proteinases from Staphylococcus aureus

Staphylococcus aureus is a major pathogen of gram-positive septic shock and frequently is associated with consumption of plasma kininogen. We examined the vascular leakage (VL) activity of two cysteine proteinases that are secreted by S. aureus. Proteolytically active staphopain A (ScpA) induced VL in a bradykinin (BK) B2-receptor–dependent manner in guinea pig skin. This effect was augmented by staphopain B (SspB), which, by itself, had no VL activity. ScpA also produced VL activity from human plasma, apparently by acting directly on kininogens to release BK, which again was augmented significantly by SspB. Intravenous injection of ScpA into a guinea pig caused BK B2-receptor–dependent hypotension. ScpA and SspB together induced the release of leucyl-methionyl-lysyl-BK, a novel kinin with VL and blood pressure–lowering activities that are equivalent to BK. Collectively, these data suggest that production of BK and leucyl-methionyl-lysyl-BK by staphopains is a new mechanism of S. aureus virulence and bacterial shock. Therefore, staphopain-specific inhibitors and kinin-receptor antagonists could be used to treat this disease.

Monocyte Chemotactic Factor in Rheumatoid Arthritis Synovial Tissue PROBABLY A CROSS-LINKED DERIVATIVE OF S19 RIBOSOMAL PROTEIN

The extracts of rheumatoid arthritis-synovial lesions from seven patients possessed a strong chemotactic activity for monocytes and a negligible one for polymorphonuclear leukocytes. These results are consistent with a prominent histological feature of the synovial lesion, the mononuclear cell predominant infiltration. The major monocyte chemotactic factor in the synovial tissue extracts was purified to a single protein peak in reverse phase high performance liquid chromatography with a C4 column. NH-terminal amino acid analysis of the initial 20 residues yielded a single sequence. Surprisingly, this sequence was completely identical to that of S19 ribosomal protein. The purified sample demonstrated two protein bands in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 34 and 68 kDa. These sizes were 2 and 4 times that of S19 ribosomal protein, suggesting that the chemotactic factor would be a dimer or tetramer of S19 ribosomal protein cross-linked by factor XIIIa. A recombinant human S19 ribosomal protein was prepared as a fusion protein with a maltose binding protein in Escherichia coli. After treatment with factor XIIIa, cross-linked recombinant S19 ribosomal protein exhibited the monocyte chemotactic activity, although the untreated recombinant protein did not.

Two reactions are simultaneously catalyzed by a single enzyme: The arginine-dependent simultaneous formation of two products, ethylene and succinate, from 2-oxoglutarate by an enzyme from Pseudomonas syringae

A single enzyme isolated from Pseudomonas syringae pv. phaseolicola PK2 simultaneously catalyzed two reactions, namely, the formation of ethylene and succinate from 2-oxoglutarate, at a molar ratio of 2:1. In the main reaction, 2-oxoglutarate was dioxygenated to produce one molecule of ethylene and three molecules of carbon dioxide. In the sub-reaction, both 2-oxoglutarate and L-arginine were mono-oxygenated to yield succinate plus carbon dioxide and L-hydroxyarginine, respectively, the latter being further transformed to guanidine and L-delta 1-pyrroline-5-carboxylate. We propose a dual-circuit mechanism for the entire reaction, in which the binding of L-arginine and 2-oxoglutarate in a Schiff-base structure generates a common intermediate for two reactions.

Conformational stability and warfarin-binding properties of human serum albumin studied by recombinant mutants

Correctly folded recombinant wild-type human serum albumin and the single-residue mutants K199A, W214A, R218H and H242Q were produced with the use of a yeast expression system. The changes in R218H resulted in a pronounced decrease in intrinsic fluorescence. Thermodynamic parameters for thermal denaturation of the present mutants and of five additional mutants have been determined, showing small increases in stability for two mutants (R218H and H242Q) and a larger decrease in stability for one (W214A). In the last of these, denaturation was a heterogeneous process starting at physiological temperature. The high-affinity binding constant for warfarin at pH 7.4 was determined by fluorescence spectroscopy: there was a significant increase in affinity for binding of warfarin to H242Q and K199A and a smaller decrease in affinity for W214A and R218H. The findings show that Trp-214 is not as essential for the high-affinity binding of warfarin as has previously been thought.

In vitro and in vivo properties of recombinant human serum albumin from pichia pastoris purified by a method of short processing time

AbstractPurpose. Recombinant human serum albumin (rHSA), secreted by a Pichia pastorisexpression system, was purified by a fast and efficient method, the essential feature of which is strong but reversible binding of the protein to Blue Sepharose. The structural characteristics, stability, and ligand-binding properties of the resulting protein were examined, and pre-clinical studies were performed. Methods. Protein structure was investigated by amino acid sequencing, sodium polyacrylamide gel electrophoresis, CD spectroscopy and chromatography. Stability was examined by denaturation by guanidine hydrochloride and by calorimetry, and ligand binding was studied by ultrafiltration. Rat experiments were performed with 125I-labeled albumin. Results. Far-ultraviolet and near-ultraviolet CD spectra of rHSA were identical to those of human serum albumin isolated from serum (HSA). Mercaptalbumin and non-mercaptalbumin were separated by high-performance liquid chromatography using an N-methylpyridinium polymer-based column. 60% of rHSA existed as mercaptalbumin, a content that is higher than that of a commercial preparation of HSA. Fatty acids, N-acetyl-L-tryptophan and pasteurization had similar effects on the conformational stability of rHSA and HSA. Stereoselective ligand-binding properties (warfarin, phenprocoumon, pranoprofen and ibuprofen) of rHSA were the same as those of HSA. The effect of the neutral to base transition on warfarin (site I-ligand) and dansylsarcosine (site II-ligand) binding to rHSA was also similar to HSA. In vivo studies showed comparable half-lives, excretion and tissue distributions of the two albumin preparations. Conclusion. The present yeast expression system and purification procedure result in rHSA with structural and functional properties very similar to those of HSA.

Molecular cloning in Escherichia coli, expression, and nucleotide sequence of the gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2

The gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2 was found to be encoded by an indigenous plasmid, designated pPSP1. The gene for the ethylene-forming enzyme was cloned and expressed in Escherichia coli JM109. Nucleotide sequence analysis of the clone revealed an open reading frame that encodes 350 amino acids (mol. wt. 39,444). In a comparison with other proteins, the homology score for the entire amino-acid sequence of the ethylene-forming enzyme of Pseudomonas syringae versus ethylene-forming enzymes from plants and 2-oxoglutarate-dependent dioxygenases was low. However, functionally significant regions are conserved.

Functional Analysis of Recombinant Human Serum Albumin Domains for Pharmaceutical Applications

AbstractPurpose. Functional analysis of the three recombinant human serum albumin (rHSA) domains and their potential as stand-alone proteins for use as drug delivery protein carriers. Methods. Protein structure was examined by fluorescence and CD spectroscopy. Ligand binding was estimated by ultrafiltration. Antioxidant activity was estimated by measuring the quenching of dihydrorhodamine 123. Esterase-like activity and enolase-like activity were estimated from the rate of hydrolysis of p-nitrophenyl acetate and conversion of dihydrotestosterone from the 3-keto to 3-enol form, respectively. The domains of human serum albumin (HSA) were radiolabeled with 111In to evaluate their pharmacokinetics. Results. The ligand binding ability of subsites Ia and Ib could not be detected in domain II. However, the binding of ligands to subsite Ic and site II were preserved in domain II and domain III, respectively. Domain III retained about 45% of its esterase-like activity, and weaker esterase-like activity was also observed in domain I. All domains showed low enolase-like activity in a pH 7.4 phosphate buffer, but domain II had higher activity in a pH 9.2 carbonate buffer. Domain I exhibited antioxidant activity comparable to that of rHSA. All three of the 111In-radiolabeled domains were rapidly eliminated from HSA, with high accumulation in the kidneys. Conclusion. Domain I of HSA has great potential for further development as a drug delivery protein carrier, due to its favorable properties and the presence of a free cysteine residue.

Organization of the Chemokine Gene Cluster on Human Chromosome 17q11.2 Containing the Genes for CC Chemokine MPIF-1, HCC-2, HCC-1, LEC, and RANTES

To understand the organization of the human CC chemokine gene cluster on chromosome 17q11.2, we determined the nucleotide sequence of a region 181 kb long containing five CC chemokine genes, MPIF-1 (SCYA23), HCC-2 (SCYA15), HCC-1 (SCYA14), LEC (SCYA16), and RANTES (SCYA5), by the random shot-gun method. The four CC chemokine genes, MPIF-1, HCC-2, HCC-1, and LEC, are clustered within a region 40 kb long, whereas the RANTES gene is located approximately 10 kb apart from the four chemokine gene minicluster. These chemokine genes are arranged in the same orientation, and their sizes are relatively long, 3.1 (HCC-1)-8.8 kb (RANTES) when compared with other CC chemokine genes, such as MIP-1alpha/LD78alpha (SCYA3) (1.9 kb) and MCP-1 (SCYA2) (1.5 kb). In contrast to most other human CC chemokine genes that consist of three exons, the MPIF-1 and HCC-2 genes, separated by 12 kb, have four exons. When the nucleotide sequences of the MPIF-1 and HCC-2 genes are compared, they are well conserved, including introns and flanking sequences, except for the middle region of the long first intron, indicating that they have been generated recently in evolutionary terms by duplication. In addition to the CC chemokine genes, more than 30 exons are identified in the sequenced region by similarity search against expressed sequence tags (ESTs) and also by the gene prediction program GenScan. This indicates that the chemokine cluster sequenced in this study is a gene-rich region in the human genome.