Enzymatic action of human glandular kallikrein 2 (hK2). Substrate specificity and regulation by Zn2+ and extracellular protease inhibitors.
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Human glandular kallikrein 2 (hK2) is a serine protease expressed by the prostate gland with 80% identity in primary structure to prostate-specific antigen (PSA). Recently, hK2 was shown to activate the zymogen form of PSA (proPSA) in vitro and is likely to be the physiological activator of PSA in the prostate. hK2 is also able to activate urokinase and effectively cleave fibronectin. We studied the substrate specificity of hK2 and regulation of its activity by zinc and extracellular protease inhibitors present in the prostate and seminal plasma. The enzymatic activity and substrate specificity was studied by determining hK2 cleavage sites in the major gel proteins in semen, semenogelin I and II, and by measuring hydrolysis of various tripeptide aminomethylcoumarin substrates. HK2 cleaves substrates C-terminal of single or double arginines. Basic amino acids were also occasionally found at several other positions N-terminal of the cleavage site. Therefore, the substrate specificity of hK2 fits in well with that of a processor of protein precursors. Possible regulation mechanisms were studied by testing the ability of Zn2+ and different protease inhibitors to inhibit hK2 by kinetic measurements. Inhibitory constants were determined for the most effective inhibitors PCI and Zn2+. The high affinity of PCI for hK2 (kass = 2.0 x 10(5) M-1 x s-1) and the high concentrations of PCI (4 microM) and hK2 (0.2 microM) in seminal plasma make hK2 a very likely physiological target protease for PCI. hK2 is inhibited by Zn2+ at micromolar concentrations well below the 9 mM zinc concentration found in the prostate. The enzymatic activity of hK2 is likely to be reversibly regulated by Zn2+ in prostatic fluid. This regulation may be impaired in CAP and advanced metastatic cancer resulting in lack of control of the hK2 activity and a need for other means of control. (+info)
Structural characterization of the oligosaccharide chains of native and crystallized boar seminal plasma spermadhesin PSP-I and PSP-II glycoforms.
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The PSP-I/PSP-II heterodimer is the major protein of boar seminal plasma. Both subunits are glycoproteins of the spermadhesin family and each contains a single N-glycosylation site. After enzymatic release of the oligosaccharides from isolated PSP-I and PSP-II, mainly neutral and monosialylated oligosaccharides, and small amounts of disialylated oligosaccharides, were recovered from both proteins. Twenty-two neutral oligosaccharides, 11 monosialylated glycans and three disialylated carbohydrate chains were characterized using mass spectrometric and NMR techniques. PSP-I and PSP-II share the same glycans but differ in their relative molar ratios. Most glycan structures are proximally alpha1-6-fucosylated, diantennary complex-type bearing nonsialylated or alpha2-6-sialylated N-acetyllactosamine or di-N-acetyllactosamine antennae. The majority of nonsialylated N-acetyllactosamine antennae bear terminal alpha1-3-linked Gal residues. In addition, the N-acetylglucosamine residue of nonsialylated N-acetyl and di-N-acetyllactosamine antennae can be modified by an alpha1-3-linked fucose residue. Structures of higher antennarity, as well as structures 3,6-branched at galactose residues, were found in smaller amounts. In one oligosaccharide, N-acetylneuraminic acid is substituted by N-glycolylneuraminic acid. Mass spectrometric analysis of PSP-I and PSP-II glycoforms isolated from crystallized PSP-I/PSP-II heterodimer showed the coexistence of major PSP-I and PSP-II glycoforms in the hexagonal crystals. Oligosaccharides with the NeuNAcalpha2-6GalNAcbeta1-4GlcNAc-R motif block adhesive and activation-related events mediated by CD22, suggesting a possible immunoregulatory activity for PSP-I/PSP-II. (+info)
Interaction of antimicrobial peptides with biological and model membranes: structural and charge requirements for activity.
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Species right across the evolutionary scale from insects to mammals use peptides as part of their host-defense system to counter microbial infection. The primary structures of a large number of these host-defense peptides have been determined. While there is no primary structure homology, the peptides are characterized by a preponderance of cationic and hydrophobic amino acids. The secondary structures of many of the host-defense peptides have been determined by a variety of techniques. The acyclic peptides tend to adopt helical conformation, especially in media of low dielectric constant, whereas peptides with more than one disulfide bridge adopt beta-structures. Detailed investigations have indicated that a majority of these host-defense peptides exert their action by permeabilizing microbial membranes. In this review, we discuss structural and charge requirements for the interaction of endogenous antimicrobial peptides and short peptides that have been derived from them, with membranes. (+info)
Aprotinin binding to amyloid fibrils.
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Different low molecular mass ligands have been used to identify amyloid deposits. Among these markers, the dyes Thioflavin T and Congo Red interact specifically with the beta-sheet structure arranged in a cross-beta conformation, which is characteristic of amyloid. However, the molecular details of this interaction remain unknown. When labelled with technetium-99m, the proteinase inhibitor aprotinin has been shown to represent a very important radiopharmaceutical agent for in vivo imaging of extra-abdominal deposition of amyloid in amyloidosis of the immunoglobulin type. However, no information is available as to whether aprotinin binds other types of amyloid fibrils and on the nature and characteristics of the interaction. The present work shows aprotinin binding to insulin, transthyretin, beta-amyloid peptide and immunoglobulin synthetic amyloid fibrils by a specific dot-blot ligand-binding assay. Aprotinin did not bind amorphous precipitates and/or the soluble fibril precursors. A Ka of 2.9 microM-1 for the binding of aprotinin to insulin amyloid fibrils was determined by Scatchard analysis. In competition experiments, analogues such as an aprotinin variant, a spermadhesin and the soybean trypsin inhibitor were tested and results suggest that both aprotinin and the spermadhesin interact with amyloid fibrils through pairing of beta-sheets of the ligands with exposed structures of the same type at the surface of amyloid deposits. An electrostatic component may also be involved in the binding of aprotinin to amyloid fibrils because important differences in binding constants are observed when substitutions V15L17E52 are introduced in aprotinin; on the other hand beta-sheet containing acidic proteins, such as the soybean trypsin inhibitor, are unable to bind amyloid fibrils. (+info)
A novel heat-labile phospholipid-binding protein, SVS VII, in mouse seminal vesicle as a sperm motility enhancer.
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SVS VII, one of seven major proteins in mouse seminal vesicle secretion, was purified to homogeneity. Neither glycoconjugate nor free thiol group was detected in the protein. The primary structure deduced from the corresponding cDNA was confirmed using amino acid sequence determination, which supported the finding that SVS VII consists of 76 amino acid residues with five disulfide bridges. Accordingly, it has a theoretical molecular mass of 8538, which was proven using the mass spectrum of SVS VII. The CD spectrum of SVS VII in 50 mm phosphate buffer at pH 7.4 appeared as one negative band arising from the beta form at 217 nm and several fine structures due to nonpeptide chromophores including a prominent band for the disulfide bond at 250 nm. This, together with the predicted secondary structures, indicated no helices but a mixture of beta form, beta turn, and unordered form in SVS VII. A cytochemical study illustrated the presence of the SVS VII-binding region on the entire surface of mouse sperm. The SVS VII-sperm binding was inhibited by the dispersed sperm lipids. The results of TLC overlay assay for the binding of (125)I-SVS VII to phospholipids and the interaction between SVS VII and phospholipid liposomes demonstrated a specific binding of this protein to both phosphatidylethanolamine and phosphatidylserine. The SVS VII-sperm binding greatly enhanced sperm motility but did not induce sperm capacitation. Heating the protein solution for 10 min at 90 degrees C unfolded the protein molecule, and the unfolded SVS VII immobilized the sperm. (+info)
Semenogelins are ectopically expressed in small cell lung carcinoma.
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Two proteins recovered from cell surface adhesion complexes in a small cell lung carcinoma (SCLC) cell line were identified as fragments of the seminal plasma proteins semenogelin I and semenogelin II. Association of both proteins with the adhesion complexes was induced by epidermal growth factor. Expression of semenogelins was previously thought to be highly specific to seminal vesicles, but Western blot analysis demonstrated that semenogelin II is widely expressed in SCLC cell lines and occasionally in other malignant cell lines. Although semenogelin expression is normally restricted to males, two SCLC cell lines from female patients were also positive for semenogelin II expression. Immunohistochemical analysis demonstrated diffuse expression of semenogelins in 12 of 13 SCLC tumors and focal expression in a minority of lung squamous and adenocarcinomas. Semenogelins were secreted into the medium by cultured SCLC cells, which suggested that these proteins may be useful markers for detecting residual tumor burden or recurrence of SCLC after treatment. (+info)
Synthesis of novel anti-inflammatory peptides derived from the amino-acid sequence of the bioactive protein SV-IV.
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SV-IV is a basic, thermostable, secretory protein of low Mr (9758) that is synthesized by rat seminal vesicle (SV) epithelium under strict androgen transcriptional control. This protein is of obvious pharmacological interest because it has potent nonspecies-specific immunomodulatory, anti-inflammatory, and pro-coagulant activities. In evaluating the clinical relevance and the possible use in medicine of SV-IV, we became interested in the study of its structure-function relationships and aimed to identify in its polypeptide chain specific peptide fragments possessing the marked anti-inflammatory properties of the protein not associated with other biological activities (pro-coagulation and immunomodulation) typical of this molecule. By using two different experimental approaches (the fragmentation of the protein into peptide derivatives by chemical methods and the organic synthesis on solid phase of selected peptide fragments), data were obtained showing that in this protein: (a) the immunomodulatory activity is related to the structural integrity of the whole molecule; (b) the anti-inflammatory activity is located in the N-terminal region of the molecule, the 8-16 peptide fragment being the most active; (c) the identified anti-inflammatory peptide derivatives do not seem to possess pro-coagulant activity, even though this particular function has been located in the 1-70 segment of the molecule. (+info)
Semenogelin II gene is replaced by a truncated line 1 repeat in the cotton-top tamarin.
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The human seminal vesicles secrete two proteins, semenogelin I and semenogelin II, at very high concentrations. It has previously been shown that the cotton-top tamarin (Sanguinus oedipus), a New World monkey, is lacking the semenogelin II gene. We have now determined the nucleotide sequence of DNA located 5--13 kilobases (kb) downstream of the tamarin semenogelin I gene---a region that in man is occupied by the semenogelin II gene. Two regions with homology to the human semenogelin II gene were identified in the tamarin DNA. The first region, of 3.5 kb, is homologous to DNA upstream of the human gene, and the second region, of 0.6 kb, is mainly derived from the second intron. Between these regions, equivalent to 594 base pairs (bp) upstream of the transcription initiation site to 12 bp downstream of the stop codon in the human semenogelin II gene, the cotton-top tamarin DNA carries a truncated LINE1 repeat. In another set of experiments, the tamarin DNA hybridizing to the mouse semenoclotin gene was investigated. It was concluded that hybridization is with the second intron of the semenoclotin gene, but very likely, the material does not represent a cotton-top tamarin semenoclotin gene. Thus, a mammalian ancestor probably carried a single gene that in the rodent lineage developed into the semenoclotin gene and in the primate lineage into a progenitor of the semenogelin genes. (+info)