Two Clip Domain Family of Serine Proteases and a Serine Protease Homolog from the Fall Webworm, Hyphantria cunea; cDNA Cloning and Characterization

Two types of serine proteases and a serine protease homologue cDNAs were isolated from Hyphantria cunea larvae induced immune response due to an injection of a microorganism through RT‐PCR and cDNA library screening, and their characteristics were examined. The isolated cDNAs are composed 2.1 kb, 2.2 kb, and 2.5 kb nucleotide each, which encoded 388, 390, 580 amino acid residues, and were designated as HcPE‐1, HcPE‐2 and HcPE‐3, respectively. They were revealed as serine proteases or a serine protease homologue with the clip domain through a database search. The deduced amino acid sequence comparison showed high homology of 72‐78% among them. Six Cys residues of the N‐terminal clip domain forming the disulfide bond, Cys residues of the catalytic domain, and Cys residues forming inter‐bridge between clip domain and catalytic domain were also well preserved. Three amino acid residues, His, Asp, and Ser, within the active site were perfectly conserved in HcPE‐2 and HcPE‐3, however, His was replaced with Gln¹⁷⁸ in HcPE‐1. The Arg residues (HcPE‐1, Arg¹³²; HcPE‐2, Arg¹³⁴; HcPE‐3, Arg³²⁵) known as the activation sites by proteolytic cleavage were preserved well in all three types of protein. In case of HcPE‐3, three continuous clip‐like domains existed in the N terminal. As the result of phylogenetic analysis, three clip domain family of protein from H. cunea make groups with arthropod proclotting enzyme precursor. Northern blot analysis showed all three genes were induced through an injection of Escherichia coli, but expression patterns were varied.     

丝氨酸蛋白酶抑制剂的研究及应用

丝氨酸蛋白酶抑制剂（serpin）是一类结构、序列同源的蛋白酶抑制剂,它是体内许多蛋白水解级联反应的调节因子,其遗传性结构或分泌异常将导致许多疾病．因此对于其结构及作用机理的研究将为临床应用提供依据．     

Nonconventional Protease Catalysis in Frozen Aqueous Solutions

During the past decade proteases have been widely used as catalysts in peptide synthesis. Unfortunately, they are not ideal ligases. Enzymatic peptide synthesis in frozen aqueous systems has been developed as an approach towards the suppression of competitive reactions. This paper summarizes reports concerning the behaviour of non-enzymatic as well as of enzyme-catalysed reactions when the reaction mixture is frozen. The advantages of freezing the reaction mixture in serine and cysteine protease-catalysed peptide synthesis, the influence of modified reaction conditions and the possible reasons for the yield-increasing effect of freezing are discussed.     

Isolation and characterization of two cDNAs from atlantic cod encoding two distinct psychrophilic elastases

The cDNAs encoding two different Atlantic cod elastases have been isolated and sequenced. The predicted amino acid sequences revealed two preproelastases, consisting of a signal peptide, an activation peptide and a mature enzyme of 242 and 239 amino acids. Amino acid sequence identity between the two cod elastases was 60.1% and identity with mammalian elastases ranged from 50–64%. The two cod elastases contain all the major structural features common to serine proteases, such as the catalytic triad His57, Asp102 and Ser195. Both cod elastases have a high content of methionine, consistent with previous findings in psychrophilic fish enzymes.     

A Protease Inhibitor of the Serpin Family Is a Major Protein in Carp Perimeningeal Fluid: I. Protein Purification and Characterization

Abstract: Two isoforms of a protease inhibitor of the serpin family (p62) have been purified from bighead carp perimeningeal fluid. Both isoforms migrate with an apparent molecular mass of 62 kDa on reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gels. Both proteins inhibited the activities of bovine trypsin, bovine chymotrypsin, and porcine pancreatic elastase. They also formed complexes with these proteases that were resistant to sodium dodecyl sulfate treatment. p62 exists in the extracts of all tissues examined, including brain, head kidney, kidney, liver, muscle, ovary, pituitary, and spleen. It is also present in serum, ovarian fluid, and milt as well as perimeningeal fluid. The protease inhibitor is a glycoprotein, and its carbohydrate moiety could be removed by endoglycosidase F. Because p62 resembles mammalian α₁-antitrypsin in many aspects, it is likely a fish equivalent of α₁-antitrypsin.     

Purification and characterization of a 22-kDa protein in chloroplasts from green spores of the fern Osmunda japonica

Changes in the polypeptide composition of chloroplasts were investigated during germination of green spores of the fern Osmunda japonica . The polypeptide composition of chloroplasts was appreciably changed during a germination time course of 48 h. Levels of five polypeptides with apparent molecular masses of 47, 44, 42, 22 and 18.5 kDa in the soluble fraction of chloroplasts and three polypeptides with molecular masses of 24, 22 and 15 kDa in the thylakoid membranes decreased during germination. In contrast, no decrease of chloroplast polypeptides was observed in the spores incubated with cycloheximide for 48 h. A new 22-kDa protein was isolated from thylakoid membranes of spores and the amino-terminal sequence of the purified protein was determined. High levels of alanine and glycine were found in the basic protein (pl > 10.3). This protein, with a native molecular mass of 80 kDa, was characterized by a subunit band observed at a molecular mass of 22 kDa on SDS-PAGE and by the disappearance of the band during spore germination. Protease activity against the 22-kDa protein was observed in an extract prepared from chloroplasts of quiescent spores. A hypothetical cytosolic proteinaceous factor is implicated in the regulation of protein degradation in chloroplasts.     

Characterization of a cDNA encoding the 55 kDa B regulatory subunit of Arabidopsis protein phosphatase 2A

Type 2A serine/threonine protein phosphatases (PP2A) are key components in the regulation of signal transduction and control of cell metabolism. The activity of these protein phosphatases is modulated by regulatory subunits. While PP2A activity has been characterized in plants, little is known about its regulation. We used the polymerase chain reaction to amplify a segment of a cDNA encoding the B regulatory subunit of PP2A from Arabidopsis. The amplified DNA fragment of 372 nucleotides was used as a probe to screen an Arabidopsis cDNA library and a full-length clone (AtB) of 2.1 kbp was isolated. The predicted protein encoded by AtB is 43 to 46% identical and 53 to 56% similar to its yeast and mammalian counterparts, and contains three unique regions of amino acid insertions not present in the animal B regulatory subunit. Genomic Southern blots indicate the Arabidopsis genome contains at least two genes encoding the B regulatory subunit. In addition, other plant species also contain DNA sequences homologous to the B regulatory subunit, indicating that regulation of PP2A activity by the 55 kDa B regulatory subunit is probably ubiquitous in plants. Northern blots indicate the AtB mRNA accumulates in all Arabidopsis tissues examined, suggesting the protein product of the AtB gene performs a basic housekeeping function in plant cells.     

Genetic control of PI and GC variants in the American Mink

Genetic polymorphism of the serum α-protease inhibitor (PI) and group-specific component (GC) in minks was revealed using one-dimensional polyacrylamide gel electrophoresis and immunoblotting. Two codominant alleles were identified at each of the two loci. The data ruled out the possibility of any linkage between the PI, GC and the coat colour gene Crystal ( Cr ).     

N-terminal sequence analysis of atrial granule serine proteinase purified by affinity chromatography

Atrial granule serine proteinase is considered the leading candidate endoproteolytic processing enzyme of pro-atrial natriuretic factor. Its cleavage specificity is directed toward a monobasic amino acid processing site, and as such, the atrial enzyme is distinguished from the family of prohormone convertases which act at dibasic amino acid processing sites. To delineate the molecular mechanisms which distinguish monobasic from dibasic amino acid-directed processing enzymes, pure atrial enzyme is needed for sequence determination leading to molecular cloning, and for preparation of antisera. An affinity chromatography purification scheme seemed a logical modification of our established procedures to yield suitable amounts of enzyme for further studies. Surprisingly, pseudo-peptide bond inhibitors of the atrial enzyme [Damodaran and Harris (1995),J. Protein Chem., this issue] formed ineffective affinity ligands, even though these compounds contain essential residues on either side of what would be the scissile bond in a peptide substrate. On the other hand, tripeptide aldehydes (based on the substrate recognition sequence of the atrial enzyme) linked to Sepharose formed effective affinity matrices, permitting purification of the enzyme in a single step from a subcellular fraction enriched for atrial granules and lysosomes. Hence, the enzyme was purified 2000-fold in 90% overall yield, and subjected to N-terminal sequence analysis through 26 residues. The sequence determined, XXPEAAGLPG[R, L]GNPVP[F, G]R[Q, I]XY[G, E]XR(N, A]V, indicates that the atrial enzyme is unique, showing little sequence homology to other proteins in the database.Abbreviations AGSP atrial granule serine proteinase - ANF atrial natriuretic factor - BSA bovine serum albumin - Bz benzoyl - EACA 6()-aminocaproic acid - HEPES N-2-hydroxyethylpiperazine-N'-propanesulfonic acid - HPLC high-performance liquid chromatography - PEG polyethylene glycol-3350 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Single-letter abbreviations are used to denote amino acids     

Preparations of Ψ-peptide bond and peptide-aldehyde inhibitors of atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor

Pseudo-peptide bond inhibitors (-bond inhibitors) and peptide-aldehyde inhibitors of atrial granule serine proteinase, the candidate processing enzyme of pro-atrial natrieuretic factor, are prepared in high yield and purity by novel synthetic routes. The -bond compounds retain essential residues for enzyme binding, but place the enzyme inhibition site in the midst of the peptide sequence. Thus, Bz-APR--LR and Bz-APR--SLRR can be considered readthrough inhibitors of atrial granule serine proteinase. The most potent -peptide, Bz-APR--SLRR (IC₅₀=250 M), is about fivefold less potent than the best peptide-aldehyde inhibitor (EACA-APR-CHO), and both the -bond and peptide-aldehyde compounds are competitive, reversible inhibitors of the enzyme. The -bond peptides containing two C-terminal Arg residues are three-to tenfold more potent than the analogous compounds containing only one C-terminal Arg residue, confirming the importance of both Arg residues in the enzyme processing recognition site. As expected, because of their moderate potencies, the -peptides are not useful affinity ligands for purification of atrial granule serine proteinase, but both peptide aldehydes are effective affinity ligands [Damodaran and Harris (1995),J. Protein Chem., this issue].Abbreviations AGSP atrial granule serine proteinase - ANF atrial natriuretic factor - Bz benzoyl - DIEA diisopropylethylamine - DIPCDI diisopropylcarbodiimide - DMF dimethylformamide - DMSO dimethylsulfoxide - EACA 6(e)-aminocaproic acid - EtOAc ethyl acetate - HEPES N-2-hydroxyethylpiperazine-N-propanesulfonic acid - HOBt N-hydroxybenzotriazole - HPLC high-performance liquid chrornatography - NMR nuclear magnetic resonance - PEG polyethylene glycol-3350 - PyBOP benzotriazole-1-yl-oxy-trispyrrolidino-phosphonium-hexafluorophospate - TEA triethylamine - TFA trifluoroacetic acid - THF tetrahydrofuran - TLC thin-layer chromatography - UV ultraviolet - pseudo-peptide bond -CH₂-NH-. Single-letter abbreviations are used to denote amino acids