Scleral matrix metalloproteinases, serine proteinase activity and hydrational capacity are increased in myopia induced by retinal image degradation

In the avian model of myopia, retinal image degradation quickly leads to ocular enlargement. We now give evidence that regionally specific changes in ocular size are correlated with both biomechanical indices of scleral remodeling, e.g. hydration capacity and with biochemical changes in proteinase activities. The latter include a 72 kDa matrix metalloproteinase (putatively MMP-2), other gelatin-binding MMPs, an acid pH MMP and a serine protease. Specifically, we have found that increases in scleral hydrational capacity parallel increases in collagen degrading activities. Gelatin zymography reveals that eyes with 7 days of retinal image degradation have elevated levels (1.4-fold) of gelatinolytic activities at 72 and 67 kDa M(r) in equatorial and posterior pole regions of the sclera while, after 14 days of treatment, increases are no longer apparent. Lower M(r) zymographic activities at 50, 46 and 37 kDa M(r) are collectively increased in eyes treated for both 7 and 14 days (1.4- and 2.4-fold respectively) in the equator and posterior pole areas of enlarging eyes. Western blot analyses of scleral extracts with an antibody to human MMP-2 reveals immunoreactive bands at 65, 30 and 25 kDa. Zymograms incubated under slightly acidic conditions reveal that, in enlarging eyes, MMP activities at 25 and 28 kDa M(r) are increased in scleral equator and posterior pole (1.6- and 4.5-fold respectively). A TIMP-like protein is also identified in sclera and cornea by Western blot analysis. Finally, retinal-image degradation also increases (~2.6-fold) the activity of a 23.5 kDa serine proteinase in limbus, equator and posterior pole sclera that is inhibited by aprotinin and soybean trypsin inhibitor. Taken together, these results indicate that eye growth induced by retinal-image degradation involves increases in the activities of multiple scleral proteinases that could modify the biomechanical properties of scleral structural components and contribute to tissue remodeling and growth.

A serine proteinase inhibitor from frog eggs with bacteriostatic activity

By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K-i) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs. (C) 2007 Elsevier Inc. All rights reserved.

Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain

Serine proteinase inhibitors (SPIs) play important roles in host physiological and immunological processes in all multicellular organisms. A novel Kazal-type SPI gene was cloned from the Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) encoding a polypeptide of 509 amino acids with a putative signal peptide of 22 amino acids. The deduced amino acid sequence of CfKZSPI contained 12 tandem Kazal domains with high similarity to other Kazal-type SPIs. The temporal expression of CfKZSPI in hemocytes after Vibrio anguillorum challenge was recorded by quantitative real-time RT-PCR. The relative mRNA expression level of CfKZSPI was up-regulated and reached 43.6-fold at 3 h post-challenge. After a decrease at 6 h, the expression Level increased again and reached 207.8-fold at 12 h post-challenge. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCf KZSPI-1 2) showed significant inhibitory activity against trypsin but no activity against thrombin. When the molar ratio of inhibitor to trypsin reached 1:1, almost 90% of the enzyme activity could be inhibited, which suggested that one molecule of rCfKZSPI-12 was able to inhibit one molecule of trypsin. Kinetics analysis with Dixon plot showed that the inhibition constant (K-i) of rCfKZSPI-12 to trypsin was 173 nmol L-1. These results indicated that CfKZSPI was a novel Kazal-type SPI with significant inhibitory activity against trypsin, and was suspected to be involved in scallop immune response. (c) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a serine proteinase homolog prophenoloxidase-activating factor in the swimming crab Portunus trituberculatus

Serine proteinase homologues (SPHs), as one of prophenoloxiase-activating factors (PPAFs), play critical roles in innate immunity of crabs. Based on an EST from the eyestalk full length cDNA library, the complete cDNA (designated as PtSPH) and genomic DNA of SPH from the swimming crab Portunus trituberculatus were cloned in this study. The estimated molecular weight of mature PtSPH (354 amino acids) was 38.7 kDa and its isoelectric point was 5.08. Multiple sequence alignment revealed that PtSPH lacked a catalytic residue with a substitution of Ser in the active site triad to Gly. Phylogenetic analysis indicated PtSPH together with PPAFs of Callinectes sapidus (AAS60227), Eriocheir sinensis (ACU65942), Penaeus monodon (ABE03741, ACP19563) and Pacifastacus leniusculus (ACB41380), formed a distinct cluster which only included clip-SPHs. As the first analyzed genomic structure of PPAFs in crustaceans, two introns were found in the open reading frame region of this gene. The mRNA transcripts of PtSPH could be detected in all the examined tissues, and were higher expressed in the eyestalk than that in gill, hepatopancreas, haemocytes and muscle. Accompanied with the change in phenoloxidase (PO) activity and total haemocyte counts, the temporal expression of PtSPH gene in haemocytes after Vibrio alginolyticus challenge demonstrated a clear time-dependent expression pattern with two peaks within the experimental period of 32 h. These findings suggest that PtSPH is involved in the antibacterial defense mechanism of Portunus tritubercualtus crab. (C) 2010 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge. (c) 2005 Elsevier Ltd. All rights reserved.

Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets

SILVA, Fatima C. B. L. et al. Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets. Insect Biochemistry and Molecular Biology, v. 36, p. 561-569, 2006.ISSN: 0965-1748.DOI: 10.1016/j.ibmb.2006.04.004.

Suppression of breast cancer growth and metastasis by a serpin myoepithelium-derived serine proteinase inhibitor expressed in the mammary myoepithelial cells

A serpin was identified in normal mammary gland by differential cDNA sequencing. In situ hybridization has detected this serpin exclusively in the myoepithelial cells on the normal and noninvasive mammary epithelial side of the basement membrane and thus was named myoepithelium-derived serine proteinase inhibitor (MEPI). No MEPI expression was detected in the malignant breast carcinomas. MEPI encodes a 405-aa precursor, including an 18-residue secretion signal with a calculated molecular mass of 46 kDa. The predicted sequence of the new protein shares 33% sequence identity and 58% sequence similarity to plasminogen activator inhibitor (PAI)-1 and PAI-2. To determine whether MEPI can modulate the in vivo growth and progression of human breast cancers, we transfected a full-length MEPI cDNA into human breast cancer cells and studied the orthotopic growth of MEPI-transfected vs. control clones in the mammary fat pad of athymic nude mice. Overexpression of MEPI inhibited the invasion of the cells in the in vitro invasion assay. When injected orthotopically into nude mice, the primary tumor volumes, axillary lymph node metastasis, and lung metastasis were significantly inhibited in MEPI-transfected clones as compared with controls. The expression of MEPI in myoepithelial cells may prevent breast cancer malignant progression leading to metastasis.

The hepatitis C virus NS3 serine proteinase and NS4A cofactor: establishment of a cell-free trans-processing assay.

The hepatitis C virus RNA genome encodes a long polyprotein that is proteolytically processed into at least 10 products. The order of these cleavage products in the polyprotein is NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. A serine proteinase domain located in the N-terminal one-third of nonstructural protein NS3 mediates cleavage at four downstream sites (the 3/4A, 4A/4B, 4B/5A, and 5A/5B sites). In addition to the proteinase catalytic domain, the NS4A protein is required for processing at the 4B/5A site but not at the 5A/5B site. These cleavage events are likely to be essential for virus replication, making the serine proteinase an attractive antiviral target. Here we describe an in vitro assay where the NS3-4A polyprotein, NS3, the serine proteinase domain (the N-terminal 181 residues of NS3), and the NS4A cofactor were produced by cell-free translation and tested for trans-processing of radiolabeled substrates. Polyprotein substrates, NS4A-4B or truncated NS5A-5B, were cleaved in trans by all forms of the proteinase, whereas NS4A was also required for NS4B-5A processing. Proteolysis was abolished by substitution mutations previously shown to inactivate the proteinase or block cleavage at specific sites in vivo. Furthermore, N-terminal sequence analysis established that cleavage in vitro occurred at the authentic 4A/4B site. Translation in the presence of microsomal membranes enhanced processing for some, but not all, proteinase-substrate combinations. Trans-processing was both time and temperature dependent and was eliminated by treatment with a variety of detergents above their critical micelle concentrations. Among many common proteinase inhibitors tested, only high (millimolar) concentrations of serine proteinase inhibitors tosyllysyl chloromethyl ketone and 4-(2-aminoethyl)benzenesulfonyl fluoride inactivated the NS3 proteinase. This in vitro assay should facilitate purification and further characterization of the viral serine proteinase and identification of molecules which selectively inhibit its activity.

A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene.

The squamous cell carcinoma antigen (SCCA) is a member of the ovalbumin family of serine proteinase inhibitors (serpins). A neutral form of the protein is found in normal and some malignant squamous cells, whereas an acidic form is detected exclusively in tumor cells and in the circulation of patients with squamous cell tumors. In this report, we describe the cloning of the SCCA gene from normal genomic DNA. Surprisingly, two genes were found. They were tandemly arrayed and flanked by two other closely related serpins, plasminogen activator inhibitor type 2 (PAI2) and maspin at 18q21.3. The genomic structure of the two genes, SCCA1 and SCCA2, was highly conserved. The predicted amino acid sequences were 92% identical and suggested that the neutral form of the protein was encoded by SCCA1 and the acidic form was encoded by SCCA2. Further characterization of the region should determine whether the differential expression of the SCCA genes plays a causal role in development of more aggressive squamous cell carcinomas.

Negative regulation of prophenoloxidase (proPO) activation by a clip-domain serine proteinase homolog (SPH) from endoparasitoid venom

Most parasitic wasps inject maternal factors into the host hemocoel to suppress the host immune system and ensure successful development of their progeny. Melanization is one of the insect defence mechanisms against intruding pathogens or parasites. We previously isolated from the venom of Cotesia rubecula a 50 kDa protein that blocked melanization in the hemolymph of its host, Pieris rapae [Insect Biochem. Mol. Biol. 33 (2003) 1017]. This protein, designated Vn50, is a serine proteinase homolog (SPH) containing an amino-terminal clip domain. In this work, we demonstrated that recombinant Vn50 bound P. rapae hemolymph components that were recognized by antisera to Tenebrio molitor prophenoloxidase (proPO) and Manduca sexta proPO-activating proteinase (PAP). Vn50 is stable in the host hemolymph-it remained intact for at least 72 It after parasitization. Using M. sexta as a model system, we found that Vn50 efficiently down-regulated proPO activation mediated by M. sexta PAP-1, SPH-1, and SPH-2. Vn50 did not inhibit active phenoloxidase (PO) or PAP-1, but it significantly reduced the proteolysis of proPO. If recombinant Vn50 binds P. rapae proPO and PAP (as suggested by the antibody reactions), it is likely that the molecular interactions among M. sexta proPO, PAP-1, and SPHs were impaired by this venom protein. A similar strategy might be employed by C rubecula to negatively impact the proPO activation reaction in its natural host. (C) 2004 Elsevier Ltd. All rights reserved.

Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets

SILVA, Fatima C. B. L. et al. Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets. Insect Biochemistry and Molecular Biology, v. 36, p. 561-569, 2006.ISSN: 0965-1748.DOI: 10.1016/j.ibmb.2006.04.004.

Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets

SILVA, Fatima C. B. L. et al. Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets. Insect Biochemistry and Molecular Biology, v. 36, p. 561-569, 2006.ISSN: 0965-1748.DOI: 10.1016/j.ibmb.2006.04.004.

Effects of inhibitors of plasminogen activator, serine proteinases, and collagenase IV on the invasion of basement membranes by metastatic cells

Using both human and murine cell lines, we show that malignant cells are able to invade through basement membrane and also secrete elevated amounts of collagenase IV, an enzyme implicated in the degradation of basement membranes. Using serine proteinase inhibitors and antibodies to plasminogen activators as well as a newly described collagenase inhibitor we demonstrate that a protease cascade leads to the activation of an enzyme(s) that cleaves collagen IV. Inhibition at each step reduces the invasion of the tumor cells through reconstituted basement membrane in vitro. Treatment with a collagenase inhibitor reduced the incidence of lung lesions in mice given i.v. injections of malignant melanoma cells.

Molecular and functional characterizations of a Kunitz-type serine protease inhibitor FcKuSPI of the shrimp Fenneropenaeus chinensis

Serine proteinase inhibitors play important and diverse roles in biological processes such as coagulation, defense mechanisms, and immune responses. Here, we identified and characterized a Kunitz-type proteinase inhibitor, designated FcKuSPI, of the BPTI/Kunitz family of serine proteinase inhibitors from the hemocyte cDNA library of the shrimp Fenneropenaeus chinensis. The deduced amino acid sequence of FcKuSPI comprises 80 residues with a putative signal peptide of 15 amino acids. The predicted molecular weight of the mature peptide is 7.66 kDa and its predicted isoelectric point is 8.84. FcKuSPI includes a Kunitz domain containing six conserved cysteine residues that are predicted to form three disulfide bonds. FcKuSPI shares 44e53% homology with BPTI/Kunitz family members from other species. FcKuSPI mRNAwas expressed highly in the hemocytes and moderately in muscle in healthy shrimp. Recombinant FcKuSPI protein demonstrated anti-protease activity against trypsin and anticoagulant activity against citrated human plasma in a dose-dependent manner in in vitro assays.