Polymorphism and evolution of collagenolytic serine protease genes in crustaceans.

Two genomic DNA fragments encoding crustacean collagenolytic serine protease genes show coding fragments that span 1522-1526 base pairs and contain seven exons encoding the complete amino acid sequence of two enzymes, CHYA and CHYB. As in serine protease genes from other organisms, the region coding for the residues around the active site is split by two introns. Although the introns differ from those of other organisms in size and nucleotide sequence, their number and location are more or less the same as found in mammalian chymotrypsin or elastase genes that evolved lately, but different for trypsin genes. Meanwhile, the junction that occurs between the propeptide and the maturation site is only found in the shrimp genes. This is also the case for the junction located 13 amino acids after the active site aspartic acid in these genes. Between 40 and 50 copies of the genes are reported by Southern analysis. Seven different genes within ChyA Pv family present 0-6% base changes, whereas five different genes belonging to ChyB Pv family show changes of up to 27% in the short studied portion of exon 4. This last family presents a mosaic organization of the coding parts, which are also expressed in the hepatopancreas of the shrimp as the variant PVC5 cDNA.     

Molecular structure of Rarobacter faecitabidus protease I. A yeast-lytic serine protease having mannose-binding activity.

Rarobacter faecitabidus protease I (RPI) is a serine protease exhibiting lytic activity toward living yeast cells. RPI is similar to elastase in its substrate specificity and has a lectin-like affinity for mannose. The gene encoding RPI was cloned to elucidate its structure and function. And its nucleotide sequence revealed that it contains an open reading frame encoding a 525-amino acid protein. Homology comparison indicated that pre-pro-RPI consists of three domains: (1) an NH2-terminal prepro domain not found in the mature form of RPI, (2) a protease domain homologous to the trypsin family of serine proteases, and (3) a COOH-terminal domain homologous to the COOH-terminal part of Oerskovia xanthineolytica beta-1,3-glucanase and the NH2-terminal part of the ricin B chain, a lectin isolated from the part of the ricin B chain, a lectin isolated from the castor bean. The RPI gene and its mutant were subsequently expressed in Escherichia coli under its beta-galactosidase promoter to investigate the function of the COOH-terminal domain. The mutant RPI, whose COOH-terminal domain was truncated by site-directed mutagenesis, lost both its mannose-binding and yeast-lytic activity, although the protease activity was not affected. These findings suggest that the COOH-terminal domain actually participates in the mannose-binding activity and is required for yeast-lytic activity.     

Molecular evolution of the wound-induced serine protease inhibitor wip1 in Zea and related genera

Plant defense mechanisms have been the subject of intensive investigation. However, little is known about their long-term evolutionary dynamics. We investigated the molecular diversity of a wound-induced serine protease inhibitor, wip1, in the genus Zea, as well as the divergence of wip1 among four genera, Zea, Tripsacum, Sorghum, and Oryza, in order to gain insight into the long-term evolution of plant defense. The specific objectives of this study were to determine (1) whether wip1 has a history of positive or balancing selection, as has been shown for genes involved in plant defense against pathogens, and (2) if the evolutionary histories of wip1 inhibitory loops, which come into closest contact with proteases, differ from the evolutionary history of other parts of this gene. The Zea polymorphism data are consistent with a neutral evolutionary history. In contrast, relative-rate tests suggest a nonneutral evolutionary history. This inconsistency may indicate that selection acting on wip1 is episodic or that wip1 evolves in response to selection favoring novel alleles. We also detected significant heterogeneity in the evolutionary rates of the two inhibitory loops of wip1-one inhibitory loop is highly conserved, whereas the second has diverged rapidly. Because these two inhibitory loops are predicted to have very similar biochemical functions, the significantly different evolutionary histories suggest that these loops have different ecological functions.     

New insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina

Background  

Subtilisin-like serine proteases play an important role in pathogenic fungi during the penetration and colonization of their hosts. In this study, we perform an evolutionary analysis of the subtilisin-like serine protease genes of subphylum Pezizomycotina to find if there are similar pathogenic mechanisms among the pathogenic fungi with different life styles, which utilize subtilisin-like serine proteases as virulence factors. Within Pezizomycotina, nematode-trapping fungi are unique because they capture soil nematodes using specialized trapping devices. Increasing evidence suggests subtilisin-like serine proteases from nematode-trapping fungi are involved in the penetration and digestion of nematode cuticles. Here we also conduct positive selection analysis on the subtilisin-like serine protease genes from nematode-trapping fungi.     

Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity.

A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease.     

Molecular evolution of type 1 serine/threonine protein phosphatases

Type 1 serine/threonine protein phosphatases (PP1s) play key roles in many cellular processes. To understand the evolutionary relationships among PP1s from various kingdoms and to provide a valid basis to evaluate the structure-function relationships of these phosphatases, 44 PP1 sequences were aligned, revealing a high sequence similarity among PP1 homologs. About one-third of the total amino acids are conserved in all the sequences studied. Most of these conserved amino acids are located within a 270-amino-acid core region. They include most sites critical to the activity and regulation of PP1s based on three-dimensional structural studies of mammalian PP1s. Positional variation analysis using a sliding window approach revealed two variable blocks in the 270-amino-acid core region. The major variable block corresponds to a subdomain composed of three alpha-helices (alphaG, alphaH, and alphaI) and three beta-sheets (beta7, beta8, and beta9). Phylogenetic analyses suggested that plant and animal PP1s form distinct monophyletic groups. The plant PP1 family contains several subgroups that may be older than the monocot-dicot divergence. In the animal PP1 family, different vertebrate isoforms appear to form distinct subgroups. Relative substitution rate studies indicated that plant PP1s are more diverse than animal PP1s, with an average substitution rate 1.5 times as large as that of animal PP1s. The possible involvement of PP1s in the establishment of multicellularity is discussed.     

Molecular recognition of chymotrypsin by the serine protease inhibitor ecotin from Yersinia pestis

Resistance to antibiotics is a problem not only in terms of healthcare but also biodefense. Engineering of resistance into a human pathogen could create an untreatable biothreat pathogen. One such pathogen is Yersinia pestis, the causative agent of plague. Previously, we have used a bioinformatic approach to identify proteins that may be suitable targets for antimicrobial therapy and in particular for the treatment of plague. The serine protease inhibitor ecotin was identified as one such target. We have carried out mutational analyses in the closely related Yersinia pseudotuberculosis, validating that the ecotin gene is a virulence-associated gene in this bacterium. Y. pestis ecotin inhibits chymotrypsin. Here, we present the structure of ecotin in complex with chymotrypsin to 2.74 Å resolution. The structure features a biologically relevant tetramer whereby an ecotin dimer binds to two chymotrypsin molecules, similar to what was observed in related serine protease inhibitor structures. However, the vast majority of the interactions in the present structure are distinctive, indicating that the broad specificity of the inhibitor for these proteases is based largely on its capacity to recognize features unique to each of them. These findings will have implications for the development of small ecotin inhibitors for therapeutic use.     

Molecular architecture of the ATP-dependent CodWX protease having an N-terminal serine active site

CodWX in Bacillus subtilis is an ATP-dependent, N-terminal serine protease, consisting of CodW peptidase and CodX ATPase. Here we show that CodWX is an alkaline protease and has a distinct molecular architecture. ATP hydrolysis is required for the formation of the CodWX complex and thus for its proteolytic function. Remarkably, CodX has a 'spool-like' structure that is formed by interaction of the intermediate domains of two hexameric or heptameric rings. In the CodWX complex, CodW consisting of two stacked hexameric rings (WW) binds to either or both ends of a CodX double ring (XX), forming asymmetric (WWXX) or symmetric cylindrical particles (WWXXWW). CodWX can also form an elongated particle, in which an additional CodX double ring is bound to the symmetric particle (WWXXWWXX). In addition, CodWX is capable of degrading EzrA, an inhibitor of FtsZ ring formation, implicating it in the regulation of cell division. Thus, CodWX appears to constitute a new type of protease that is distinct from other ATP-dependent proteases in its structure and proteolytic mechanism.     

Effect of a serine protease on isolated myofibrils.

Morphological changes occurred in myofibrils prepared from the glycerinated psoas muscle of rabbit during incubation with a serine protease crystallized from rat skeletal muscle. Two notable phenomena were observed: (1) loss of the Z band in the early stage of incubation and (2) complete disappearance of the A band after swelling of the myofibrils. The results indicate that the serine protease has an action on myofibrils different from that of Ca2+-dependent neutral protease.     

Neutral inhibitors of the serine protease factor Xa.

A neutral inhibitor of the serine protease factor Xa was identified via a high-throughput screen of a commercial library. The initial lead 1 demonstrated reversible and competitive inhibition kinetics for factor Xa and possessed a high degree of selectivity versus other related serine proteases. Initial modeling efforts and the generation of a series of analogues of 1 are described.     

Molecular characterization of the catalytic domains of human complement serine protease C1r

Limited cleavages of human C1r by extrinsic proteases of various specificity (plasmin, elastase, chymotrypsin, thermolysin) yield dimeric associations of two globular domains, each comprised of the intact B chain disulfide linked to gamma, the C-terminal fragment of the A chain. These (gamma-B)2 domains, which are homologous to those obtained from C1r by autolytic cleavage [Villiers, C. L., Arlaud, G. J., & Colomb, M. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4477-4481], represent the core of the C1r molecule and are associated with the catalytic properties of the serine active site. V8 protease also yields (gamma-B)2 associations, although additional cleavages occur in the B chain. Sequence analysis shows that all cleavages generating the gamma fragments occur within a 13-residue sequence extending from positions 274 to 286 of the C1r A chain. Chemical cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide of the (gamma-B)2 catalytic domains obtained from C1r autolytic cleavage indicates that each gamma-B domain interacts with its neighbor in a "head to tail" configuration, the gamma region of one domain interacting with the B chain of the other domain, and conversely. No evidence is found of gamma-gamma or B-B interactions. Such a head to tail configuration, placed in the context of the model proposed for the C1s-C1r-C1r-C1s catalytic subunit of C1 [Colomb, M. G., Arlaud, G. J., & Villiers, C. L. (1984) Philos. Trans. R. Soc. London, B 306, 283-292], is compatible with autolytic activation of C1r through an intramolecular cross-mechanism and with subsequent activation of C1s by activated C1r.     

Complement factor D, a novel serine protease.

Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of factor D activity is instead attained by a novel mechanism that depends on reversible conformational changes for expression and control of catalytic activity. These conformational changes are believed to be induced by the single natural substrate, C3bB, and to result in realignment of the catalytic triad, the specificity pocket, and the nonspecific substrate binding site, all of which have atypical conformations. Mutational studies have defined structural determinants responsible for these unique structural features of factor D and for the resultant low reactivity with synthetic esters.     

Neuroserpin, an axonally secreted serine protease inhibitor.

We have identified and chromatographically purified an axonally secreted glycoprotein of CNS and PNS neurons. Several peptides derived from it were microsequenced. Based on these sequences, a fragment of the corresponding cDNA was amplified and used as a probe to isolate a full length cDNA from a chicken brain cDNA library. Because the deduced amino acid sequence qualified the protein as a novel member of the serpin family of serine protease inhibitors, we called it neuroserpin. Analysis of the primary structural features further characterized neuroserpin as a heparin-independent, functional inhibitor of a trypsin-like serine protease. In situ hybridization revealed a predominantly neuronal expression during the late stages of neurogenesis and in the adult brain in regions which exhibit synaptic plasticity. Thus, neuroserpin might function as an axonally secreted regulator of the local extracellular proteolysis involved in the reorganization of the synaptic connectivity during development and synapse plasticity in the adult.     

Molecular characterization of zyme/protease M/neurosin (PRSS9), a hormonally regulated kallikrein-like serine protease

The cDNA for the zyme/protease M/neurosin gene (HGMW-approved symbol PRSS9) has recently been identified. Zyme appears to play a role in Alzheimer disease as well as in breast cancer. In this paper, we describe the complete genomic organization of the zyme gene. Zyme spans 10.5 kb of genomic sequence on chromosome 19q13.3-q13.4. The gene consists of seven exons, the first two of which are untranslated. All splice junctions follow the GT/AG rule, and the intron phases are identical to those of many other genes belonging to the same family, i.e., the kallikreins, NES1, and neuropsin. Fine-mapping of the genomic locus indicates that zyme lies upstream of the NES1 gene and downstream from the PSA and KLK2 genes. Tissue expression studies indicate that zyme is expressed mainly in brain tissue, including spinal cord and cerebellum, in mammary gland, and in kidney and uterus. Zyme is regulated by steroid hormones in the breast carcinoma cell line BT-474. Estrogens and progestins, and to a lesser extent androgens, up-regulate the zyme gene in a dose-dependent manner.     

Electronic aspects of serine protease catalysis

A new electrostatic approach is applied to serine protease catalysis. It is is based upon the demonstration that the polarities, or partial charges, of the atomic components of the molecules involved in the reaction alternate in sign. When the atomic components of opposite polarities of the enzyme and substrate approach close to each other during the catalysis, the electrostatic interactions between them increase in intensity. These increasing interactions are related to the decrease in the energy barrier. When the serine protease--catalyzed reaction is followed from this perspective, it is shown to result in a marked simplification of the catalytic mechanism. A number of concerted proton transfers and electron density displacements around the active site are indicated. This approach is not inconsistent with other electrostatic methods, and is supported by independent partial charge calculations.     

Specificity of the serine protease inhibitor, phenylmethylsulfonyl fluoride.

Clarified cell-free extracts were prepared from rapidly dividing $\text{Bacillus subtilis}$ cells and from rabbit liver cells. These extracts were treated with [³H]-phenylmethylsulfonyl fluoride (PMSF) and analyzed by electrophoresis in isoelectric focusing polyacrylamide gels or detergent gels. Not less than 14 proteins in the $\text{B. subtilis}$ extracts and not less than 15 proteins in rabbit liver extracts reacted covalently with PMSF. These results suggest that PMSF is not as specific for serine proteases as sometimes supposed, and its effects in physiological experiments should be interpreted with caution.     

Characterization of a membrane-associated serine protease in Escherichia coli.

Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively (M. Pacaud, J. Bacteriol. 149:6-14, 1982). The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. We termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [3H]diisopropylfluorophosphate as an active-site labeling reagent, we determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, and endogenous, periplasmic inhibitor of trypsin.