Synthetic peptide inhibitors of complement serine proteases—I. Identification of functionally equivalent protease inhibitor sequences in serpins and inhibition of C1s and D

Gi, Glover; Cs, Schasteen; Liu, WS; Rp, Levine

doi:10.1016/0161-5890(88)90040-5

Cited by 20 publications

(7 citation statements)

References 30 publications

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“…The decapeptide representing the cleavage site of the serpin failed to block sperm-egg binding. This finding was consistent with previous reports that peptides based on the variable active cleavage centre may not act as protease inhibitors because this region does not contribute significantly to binding to the enzyme (Glover et al, 1988) or they are not presented in the appropriate conformation.…”

Section: G I S N V F T S H a D L S R I S N H S N~p V S E M V H K A V supporting

confidence: 93%

Human sperm–egg binding is inhibited by peptides corresponding to core region of an acrosomal serine protease inhibitor

Moore

Penfold

Johnson

et al. 1993

Molecular Reproduction Devel

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An antiserum, designated R4 and raised against denatured hamster acrosomes, was shown to localize specifically to the acrosomal region of hamster, rat, mouse, and human spermatozoa, and to inhibit both hamster and human sperm-oocyte binding in vitro. Following screening of a human testis lambda gt11 cDNA expression library with the antiserum R4, a series of cDNA clones were isolated. One (cDNA 134) was selected based on the ability of the beta-galactosidase fusion protein to inhibit human and hamster sperm-zona binding in vitro. The fusion protein was also shown to inhibit the penetration of zona-free hamster oocytes by human spermatozoa. Sequence analysis revealed that cDNA 134 coded for a portion of a serine protease inhibitor (serpin) closely related to plasma Protein C inhibitor. Sequencing of an additional cDNA clone (261) and Northern blot analysis confirmed that a Protein C inhibitor-like mRNA is synthesised in the human testis. Affinity-purified anti-134 antibody specifically localized to the acrosomal region of both hamster and human sperm. Synthetic peptides corresponding to the conserved core region responsible for the interaction of the serpin with its cognate protease also blocked human sperm-zona binding in vitro. The results suggest that this acrosomally located inhibitor plays an important role in the series of binding events that results in human fertilization.

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Section: G I S N V F T S H a D L S R I S N H S N~p V S E M V H K A V supporting

confidence: 93%

Human sperm–egg binding is inhibited by peptides corresponding to core region of an acrosomal serine protease inhibitor

Moore

Penfold

Johnson

et al. 1993

Molecular Reproduction Devel

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“…Crystal structure analysis has predicted that residues 359-365 of the post-complex form of a1-AT are particularly well exposed at the surface of the molecule (10). These residues are adjacent to a region which is highly conserved among the serpin family (12). We synthesized several peptides overlapping the sequence of this region ( Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Peptides were synthesized by the solid-phase method (11,12), purified, and subjected to amino acid composition and sequence analysis. Peptides were dissolved in water and then added to cell culture fluid.…”

Section: Methodsmentioning

confidence: 99%

Identification of a serpin-enzyme complex receptor on human hepatoma cells and human monocytes.

Perlmutter

Glover

Rivetna

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

176

107

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Formation of the covalently stabilized complex of a1-antitrypsin (a,-AT) with neutrophil elastase, the archetype of serine proteinase inhibitor serpin-enzyme complexes, is associated with structural rearrangement of the a,-AT molecule and hydrolysis of a reactive-site peptide bond.An =4-kDa carboxyl-terminal cleavage fragment is generated. a1-AT-elastase complexes are biologically active, possessing chemotactic activity and mediating increases in expression of the a,-AT gene in human monocytes and macrophages. This suggested that structural rearrangement of the a,-AT molecule, during formation of a complex with elastase, exposes a domain that is recognized by a specific cell surface receptor or receptors. To test this hypothesis, the known three-dimensional structure of al-AT and comparisons of the primary structures of the serpins were used to select a potentially exteriorly exposed and highly conserved region in the complexed form of Several recent studies suggest that a1-AT-elastase complexes have intrinsic functional activities. These complexes stimulate neutrophil chemotaxis (5) and mediate increases in expression of the a1-AT gene in human monocytes and macrophages (6, 7). a1-AT-elastase complexes are more rapidly cleared from the circulation than the corresponding native proteins. In fact, in vivo clearance of a1-AT-elastase complexes is blocked by other serpin-enzyme complexes (8, 9), suggesting the presence of a common recognition system. Taken together, these observations suggest that structural rearrangement of the a1-AT molecule, during formation of a complex with elastase, exposes a domain that is recognized by a specific cell surface receptor, or receptors. In the following study we used synthetic peptides based on the sequence of the carboxyl-terminal fragment of a1-AT as candidate mediators for regulation of a1-AT synthesis and as candidate ligands for cell surface binding. This region of the a1-AT molecule was selected because it had been previously implicated in the chemotactic activity of a1-AT-elastase complexes (5) and because crystal structure analysis (10) predicted that a domain within this region was exteriorly exposed after formation of a complex. EXPERIMENTAL PROCEDURESMaterials. Peptides were synthesized by the solid-phase method (11, 12), purified, and subjected to amino acid composition and sequence analysis. Peptides were dissolved in water and then added to cell culture fluid. Preparation of purified human plasma a1-AT, leukocyte elastase, and leukocyte cathepsin G has been previously described (6). Purified human plasma AT III and a,-ACT were purchased from Athens Research and Technology (Athens, GA). Purified human thrombin was purchased from Boehringer-MannAbbreviations: a1-AT, a1-antitrypsin; AT III, antithrombin III; a,-ACT, a1-antichymotrypsin; serpin, serine proteinase inhibitor; SEC, serpin-enzyme complex.tTo whom reprint requests should be addressed at:

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“…These peptides were based on the C3 convertase cleavage site in C3 (47), the factor D cleavage site in factor B (48), sites of length polymorphism in C3 or C5 (49), or the C terminus of the serine protease inhibitor (48). None of these approaches has yielded a potent peptide that can inhibit complement activation.…”

Section: Biotransformation Of Compstatinmentioning

confidence: 99%

Binding Kinetics, Structure-Activity Relationship, and Biotransformation of the Complement Inhibitor Compstatin

Sahu

Soulika

Morikis

et al. 2000

The Journal of Immunology

103

180

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We have previously identified a 13-residue cyclic peptide, Compstatin, that binds to complement component C3 and inhibits complement activation. Herein, we describe the binding kinetics, structure-activity relationship, and biotransformation of Compstatin. Biomolecular interaction analysis using surface-plasmon resonance showed that Compstatin bound to native C3 and its fragments C3b and C3c, but not C3d. While binding of Compstatin to native C3 was biphasic, binding to C3b and C3c followed the 1:1 Langmuir binding model; the affinities of Compstatin for C3b and C3c were 22- and 74-fold lower, respectively, than that of native C3. Analysis of Compstatin analogs synthesized for structure-function studies indicated that 1) the 11-membered ring between disulfide-linked Cys2-Cys12 constitutes a minimal structure required for optimal activity; 2) retro-inverso isomerization results in loss of inhibitory activity; and 3) some residues of the type I β-turn segment also interact with C3. In vitro studies of Compstatin in human blood indicated that a major pathway of biotransformation was the removal of Ile1, which could be blocked by N-acetylation of the peptide. These findings indicate that acetylated Compstatin is stable against enzymatic degradation and that the type I β-turn segment is not only critical for preservation of the conformational stability, but also involved in intermolecular recognition.

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Synthetic peptide inhibitors of complement serine proteases—I. Identification of functionally equivalent protease inhibitor sequences in serpins and inhibition of C1s and D

Cited by 20 publications

References 30 publications

Human sperm–egg binding is inhibited by peptides corresponding to core region of an acrosomal serine protease inhibitor

Human sperm–egg binding is inhibited by peptides corresponding to core region of an acrosomal serine protease inhibitor

Identification of a serpin-enzyme complex receptor on human hepatoma cells and human monocytes.

Binding Kinetics, Structure-Activity Relationship, and Biotransformation of the Complement Inhibitor Compstatin

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