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E A Auerswald

Publications and source records attributed to E A Auerswald.

At least 37 records · Page 2Linked to original sources
PubMed1997

Quail cystatin: isolation and characterisation of a new member of the cystatin family and its hypothetical interaction with cathepsin B.

Quail cystatin, a new cysteine proteinase inhibitor protein of the cystatin superfamily, was purified from egg albumen of Japanese quail Coturnix coturnix japonica. Amino acid sequencing and mass spectrometry revealed the complete 116 amino acid residue primary structure of a phosphorylated form (13,173 Da). The inhibitor has a 90% sequence identity with chicken cystatin. Its interaction with papain is rapid and tight (Ki = 4.4 pM; k(on) = 1.8x10(7) M(-1) s(-1); k(off) = 0.8x10(-4) s(-1)) and very similar to that of chicken cystatin. Surprisingly, however, cathepsin B was inhibited 15-fold more strongly by quail cystatin (Ki = 47 pM; k(on) = 19x10(7) M(-1) s(-1); k(off) = 9x10(-4) s(-1)) than by chicken cystatin (Ki = 784 pM; k(on) = 2.9x10(7) M(-1) s(-1); k(off) = 24x10(-4) s(-1)). Intuitive comparative conformational inspection of related inhibitors and of cognate enzymes suggest that: (i) the 3D structure of quail cystatin is nearly identical to that of chicken cystatin, (ii) quail cystatin can interact with cathepsin B analogous to the stefin B-papain interaction, if the 'occluding loop' of cathepsin B possesses an 'open' conformation, (iii) the greater inhibition of cathepsin B by quail cystatin compared to chicken cystatins probably arises from two additional ionic interactions between residues Arg15 and Lys112 of the inhibitor and Glu194 and Asp124 of the enzyme, respectively. The two potential salt bridges are located outside of the known contact regions between cystatins and peptidases of the papain family.

Amino Acid Sequence↗
PubMed1997

A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase.

CFTR-NBF-2 expressed and purified in fusion with the maltose-binding protein was shown to catalyse the reaction ATP-->ADP+Pi by three different assays, monitoring ATP turnover, formation of ADP and release of Pi (Km 86 microM, rate constant 0.37 min(-1)). The reaction product ADP inhibits this ATPase activity. In a similar manner the hydrolysis of GTP to GDP and Pi was demonstrated (Km 40 microM, rate constant 0.29 min(-1)). In the presence of AMP the ATPase reaction was superseded by the formation of two ADP from ATP and AMP. As typical for adenylate kinases a distinct AMP-binding site could be verified for CFTR-NBF-2 by the inability of TNP-ATP and AMP to compete for binding. All three enzymatic activities were inhibited by the symmetric double-substrate-mimicking inhibitor Ap5A. As NBF-2 plays a central role in CFTR channel opening and closing the results reported here are fundamental in understanding mechanisms of CFTR channel activity regulation.

Adenosine Diphosphate↗
PubMed1997

Proteolytic enzymes in yolk-sac membrane of quail egg. Purification and enzymatic characterisation.

Degradation of yolk protein is essential for the early development of the avian embryo. In Japanese quail (Coturnix coturnix japonica), proteolysis in the surrounding tissue of the yolk, the yolk-sac membrane, can be inhibited by class-specific inhibitors of cysteine proteinases as well as of aspartic proteinases. Purification of the enzymes leads to one cysteine proteinase and one aspartic proteinase with an apparent molecular mass of 29 kD and 44 kD, respectively. Both enzymes were purified in a two-chain form, although a single-chain form is also present in the homogenate of yolk-sac membrane. The cysteine proteinase was identified by NH2-terminal sequence analysis as well as by kinetic studies as a new cathepsin B from quail. Like mammalian cathepsin B, this avian cathepsin B exhibits two different kinds of proteolytic activity, an endopeptidase activity and a dipeptidyl carboxypeptidase activity. Chicken egg white cystatin, a protein-aceous cysteine proteinase inhibitor, inhibits quail cathepsin B with an equilibrium dissociation constant (Ki) of 3.3 nM. Likewise the aspartic proteinase was identified as a new cathepsin D from quail. This avian cathepsin D has a different processing site to all known mammalian cathepsins D. In quail cathepsin D one NH2-termini is homologous to amino acids 211-230 in mammalian cathepsin D. This is more than 100 amino acids downstream of the mammalian processing site. Comparison of the enzymatic properties of quail and bovine cathepsin D indicate that the different processing site has no influence on the enzymatic properties.

Amino Acid Sequence↗
PubMed1996

A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP-binding protein.

Association reactions of a recombinant CFTR-NBF-2 polypeptide fused to glutathione S-transferase with guanine nucleotides were monitored quantitatively by recording the fluorescence enhancement of excited trinitrophenol (TNP)-labelled GTP after binding to NBF-2. Binding of TNP-GTP to the recombinant NBF-2 polypeptide was characterized by a Kd value of 3.9 microM. The corrected Kd values for unlabelled guanine nucleotides were determined to be 33 microM for GTP, 92 microM for GDP and 217 microM for GMP. TNP-ATP bound to NBF-2 was competitively displaced by GTP indicating a common binding site for both nucleotides. The recombinant NBF-2 did not show an intrinsic GTPase activity above a detection limit of 0.007 min(-1). Our findings provide the first experimental evidence that NBF-2 can act as a GTP-binding subunit that would favor the release of GDP after GTP hydrolysis.

Adenosine Triphosphate↗
PubMed1996

Hybrids of chicken cystatin with human kininogen domain 2 sequences exhibit novel inhibition of calpain, improved inhibition of actinidin and impaired inhibition of papain, cathepsin L and cathepsin B.

Chicken cystatin and human kininogen domain 2 are members of the cystatin superfamily of protein-type cysteine proteinase inhibitors. They show structural and functional similarities, but only human kininogen domain 2 inhibits calpain. Using recombinant chicken cystatin as a scaffold for hybrid cassette analysis, the known reactive-site regions (N-terminus, first hairpin loop and second hairpin loop) were substituted by the corresponding sequences of human kininogen domain 2 in a single and combined manner. Seven hybrids were expressed, purified to homogeneity, characterized protein-chemically, and their inhibition of papain, actinidin, human cathepsin B, human cathepsin L and calpain (80-kDa subunit of rabbit skeletal muscle calpain II and porcine erthrocyte calpain 1) was determined. Strong but temporary inhibition of calpain by chicken cystatin hybrids carrying the N-terminus alone (variant sc1-KD2) or the N-terminus together with the first hairpin loop (variant sc1/2-KD2) was observed; hybrids of the second hairpin loop (sc3-KD2, sc1/3-KD2, sc2/3-KD2, sc1/2/3-KD2) were less strong calpain inhibitors. These data indicate that the inhibiton of calpain by human kininogen domain 2 requires the correct conformation and combination of several contact sites, and suggest that the N-terminus and the first hairpin loop play a major role in this ensemble. Remarkably, hybrid sc2-KD2 exhibited 5 or 150 times stronger inhibition of actinidin compared to native chicken cystatin or to proteolytically isolated human kininogen domain 2, respectively. This indicates an important role of the first hairpin loop of cystatins in the interaction with actinidin. Along with the impaired inhibition of cathepsin L, papain, actinidin, cathepsin B and calpain by the hybrids sc1/3-KD2, sc2/3-KD2 and sc1/2/3-KD2, these results support our hypothesis that all three predicted contact regions of kininogen domain 2 contribute to binding in the active-site clefts of papain-like enzymes in a finely balanced manner.

Amino Acid Sequence↗
PubMed1996

Sequence of a new Bowman-Birk inhibitor from Torresea acreana seeds and comparison with Torresea cearensis trypsin inhibitor (TcTI2).

TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds of Torresea acreana, one of the two known species of Torresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears as M(r) 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). The putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. The antibodies raised against TcTI2, Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. The inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. The latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.

Amino Acid Sequence↗
PubMed1996

Amino acid sequence of a lectin-like protein from Lachesis muta stenophyrs venom.

The primary structure of the lectin-like protein from Lachesis muta stenophyrs venom was deduced from analysis of the N-terminus and the sequence of peptides obtained after digestion with trypsin, Arg-C enzyme, Staphylococcus aureus V8 protease and endoproteinase Asp-N. Peptides generated by cleavage of the lectin with cyanogen bromide and o-iodosobenzoic acid were also sequenced. Comparison of the complete 135 amino acid residues sequence with those of the lectin from the venom of Crotalus atrox, with platelet coagglutinin from Bothrops jararaca beta-fragment and with the anticoagulant B protein chain from Trimeresurus flavoviridis venom, revealed 92, 46 and 29% identity, respectively. Significant homology was also found with C-type carbohydrate-recognition domain-like structures from invertebrate and vertebrate lectins. To our knowledge, this is the second known primary structure of a lectin-like protein from snake venom.

Amino Acid Sequence↗
PubMed1996

Plant serine proteinase inhibitors. Structure and biochemical applications on plasma kallikrein and related enzymes.

The action of two Bowman-Birk and several plant Kunitz-type inhibitors were studied on trypsin, chymotrypsin, plasma kallikrein and factor XII. The primary structure of some of them was completely defined. The results showed that the Bowman-Birk type inhibitors, although potent inhibitors for trypsin (Ki in the range of 1-2 nM), are not able to inhibit plasma kallikrein. Factor XII (Ki = 1.4 microM) and chymotrypsin (Ki = 5.0 nM) are inhibited by Torresea cearensis trypsin inhibitor (TcTI) but not by Dioclea glabra trypsin inhibitor (DgTI). Both inhibitors reactive site regions are highly homologous, and the amino acid residues in P1 position are the same, Lys and His; major differences are in the charge of the C-terminal portion of the molecules. The studied Kunitz-type inhibitors were all able to inhibit plasma kallikrein (Ki between 4 and 80 nM), with the exception of Schizolobium parahyba chymotrypsin inhibitor (SpCI), that is specific for chymotrypsin. All Kunitz-type inhibitors inactivate chymotrypsin, but with a dissociation constant in the range of 0.1 to 0.6 microM. Factor XIIf is inhibited with Ki in the range of 0.1 microM. Bauhinia bauhinioides trypsin inhibitor (BbTI) did not promote factor XIIf inhibition. The Kunitz-type inhibitors are a highly homologous, sharing 60% identity in the N-terminal portion of the loop containing the reactive site, and 28.6% identity in the C-terminal portion of the same loop.

Amino Acid Sequence↗
PubMed1995

Functional display and expression of chicken cystatin using a phagemid system.

A recombinant phage antibody system has been used to display functionally active chicken cystatin, a small protein-type inhibitor of papain-like cysteine proteinases. A synthetic gene, AEF-[S1M, M29I, M89L] chicken cystatin, was ligated into phagemid pCANTAB 5E, cloned, sequenced and displayed on the tips of filamentous phage as a fusion protein with protein III (gpIII). The gene was expressed also as soluble inhibitor in a non-suppressor E.coli strain. Recombinant phages were selected by binding to carboxymethylated-papain. The positive phages showed binding to papain in dose dependent manner. The chicken cystatin-gpIII fusion protein was detected with anti-chicken cystatin antibody and with anti-protein III antibody by western blot analysis. Unfused soluble inhibitor was identified in the periplasma and showed strong papain inhibitory activity. The results demonstrate that functionally active cystatin can be displayed on the phage surface. This technology can be employed further to select inhibitor variants which differ in their binding affinity to papain and papain-like enzymes.

Animals↗
PubMed1995

Expression and functional properties of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator fused to glutathione-S-transferase.

CFTR-NBF-2 was expressed in Escherichia coli in fusion with glutathione-S-transferase, the soluble portion was purified and identified as a structured protein by its CD spectrum. Association reactions of the recombinant NBF-2 with adenine nucleotides were monitored qualitatively by demonstrating its ability to bind specifically to ATP-, ADP- and AMP-affinity agarose and quantitatively by recording the fluorescence enhancement of excited trinitrophenol (TNP)-labelled adenine nucleotides occurring as a result of binding to NBF-2. Best-fit monophasic binding curves to the fluorescence data indicated Kd values of 22 microM for TNP-ATP, 39 microM for TNP- ADP and 2.1 microM for TNP-AMP. The corrected Kd values for unlabelled adenine nucleotides competing with the fluorophores were determined to be 37 microM for ATP, 92 microM for ADP and 12 microM for AMP. The recombinant NBF-2 did not show any hydrolytic activity on ATP (detection limit 0.001 s-1). Our findings support the concept of a central role of NBF-2 in CFTR activity regulation acting as an allosteric switch between channel opening and closing and give the first experimental evidence that the channel inhibitor AMP could act via NBF-2.

Adenosine Diphosphate↗
PubMed1995

Temporary inhibition of papain by hairpin loop mutants of chicken cystatin. Distorted binding of the loops results in cleavage of the Gly(9)-Ala10 bond.

Temporary inhibition of the cysteine proteinases papain and cathepsin L was observed with several hairpin loop mutants of recombinant chicken cystatin at enzyme concentrations above nanomolar. Kinetic modelling of inhibition data, gel electrophoresis and amino acid sequencing revealed that reappearance of papain activity is due to selective cleavage of the Gly(9)-Ala10 bond in the N-terminal binding area of the chicken cystatin variants, resulting in truncated inhibitors of lower affinity. Cleavage of the same bond by contaminating papaya proteinase IV was ruled out by previous purification of papain and suitable control experiments. According to the proposed kinetic model, cleavage occurs within the enzyme-inhibitor complex with first order rate constants ktemp of 2.3 x 10(-3) up to 5 x 10(-1) s-1. A similar ktemp/Km ratio was found for all mutants (0.7 x 10(6)-2.1 x 10(6) s-1.M-1); it is almost identical with the kcat/Km ratio of the peptide substrate Z-Phe-Arg-NHMec. These results suggest that distorted contacts of one of the hairpin loops affect binding of the N-terminal contact area in a way that covalent interaction of the Gly(9)-Ala10 bond with the active-site Cys residue of papain can occur and the bond is cleaved in a substrate-like manner.

Alanine↗
PubMed1995

Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain.

Five recombinant hairpin loop variants of chicken cystatin (delta V55, delta V55-S56, delta P103-L105, delta I102-Q107, loop2-KD2) were constructed by cassette mutagenesis, expressed in E. coli, purified to homogeneity, characterized by protein-chemical means and by their inhibitory properties. The variant forms, modified in two of the three postulated cysteine proteinase binding regions, were inhibitorily active. However, the equilibrium dissociation constants of the complexes between papain as well as human cathepsin B or L and the cystatin variants show a weaker affinity for all three enzymes compared with recombinant chicken cystatin. These results prove the contribution of both hairpin loops to complex formation with the three enzymes. Furthermore, the kinetic constants indicate discrete differences in the molecular mechanism of interaction between chicken cystatin and papain, cathepsin B, and cathepsin L. Inhibition of cathepsin L was much less affected than inhibition of papain or cathepsin B by the modifications achieved in the five variants. Remarkably, at high enzyme concentration (above 0.5 nM) inhibition of papain by these variants was 'temporary', that means, active papain was released from the enzyme-inhibitor complex within minutes to hours (compare [1]).

Amino Acid Sequence↗
PubMed1994

Structure of leech derived tryptase inhibitor (LDTI-C) in solution.

The three-dimensional solution structure of the leech derived tryptase inhibitor form C (LDTI-C), an inhibitor of 46 amino acids which contains 3 disulfide bridges, has been determined using 2D NMR spectroscopy. The 3D structure was determined on the basis of 262 interresidue interproton distance constraints derived from nuclear Overhauser enhancement measurements and 25 phi angles, supplemented by 3 psi and 15 chi 1 angles. The core of LDTI-C is very well defined and consists of a short 3(10)-helix-loop and a short two-stranded antiparallel beta-sheet between residues 13-14 and 20-21. The N-terminus is fixed to the core by two disulfide bridges, while the C-terminus is connected to the beta-sheet via the third disulfide bridge. The binding loop in LDTI exhibits lowest energy conformations belonging to the canonical conformation of serine proteinase inhibitors.

Amino Acid Sequence↗
PubMed1994

Production, inhibitory activity, folding and conformational analysis of an N-terminal and an internal deletion variant of chicken cystatin.

Two deletion variants of chicken cystatin were produced after cassette mutagenesis of the recombinant Arg-Glu-Phe-[Met1, Ile29, Leu89]-chicken egg white cystatin gene in Escherichia coli. The variant des-Ser1-Pro11-[Ala12, Glu13, Phe14, Met15, Ile29, Leu89]-chicken cystatin (N-del 2) and the variant Arg-Glu-Phe-[Met1, Ile29]-des-Cys71-Met89-chicken cystatin (del-helix II) were purified and characterized by inhibition kinetics, far-ultraviolet-CD and fluorescence spectroscopy, and their folding in guanidine hydrochloride (Gdn/HCl) was studied. The del-helix II variant, shortened by 19 amino acids, is a basic, stefin-like mini-cystatin with one disulfide bridge. Its inhibitory properties are identical to chicken cystatin and its stability against Gdn/HCl is similar. The folding of the del-helix II variant corresponds best to a single step process. In contrast to this, the reversible folding of natural and recombinant chicken cystatin is more complex when recorded by either tryptophan fluorescence or far-ultraviolet-CD. With increasing Gdn/HCl concentration, a stabilization of secondary-structural elements is initially observed, followed by unfolding with minor but distinct intermediate states. The N-del 2 variant has a neutral pI and shows folding behaviour very similar to natural and recombinant chicken cystatin. However its inhibition constants with papain, actinidin and cathepsin B and L are 1000-100,000-fold higher than those obtained with natural and recombinant chicken cystatin.

Amino Acid Sequence↗
PubMed1994

Structure/function implications for the aminopeptidase specificity of aleurain.

The cysteine protease aleurain, a member of the papain superfamily, was characterized by its specificity constants, kcat/Km, for the hydrolysis of different substrates of the type H-P1-NH-Mec (NH-Mec, 4-methylcoumaryl-7-amide). The determined constants for the different substrates decrease in the order citrulline > Arg = Phe >> Ala. A 75-fold loss of specificity was observed when the substrate Bz-Arg-NH-Mec (Bz, benzoyl), with a blocked N-terminus, was used instead of H-Arg-NH-Mec. The pH dependence of kcat/Km for H-Arg-NH-Mec was bell-shaped with pKa1 and pKa2 values of 5.81 and 7.27, respectively, at 25 degrees C. The residue corresponding to a pKa1 value of 5.81 could be identified by its ionisation enthalpy, delta Hion, of 15 kJ/mol as a carboxylate group of the enzyme interacting electrostatically with the residue with pKa2 7.27, attributed to the alpha-amino group of the substrate by its delta Hion value of 48 kJ/mol. Aleurain can be titrated at the active site with L-trans-epoxy-succinylleucylamido(4-guanidino)butane, and the reaction was characterized by its association rate constant of 19,000 M-1.s-1. Native chicken cystatin inhibited aleurain competitively with Ki 133 nM. Recombinant chicken cystatin variants Ala-Glu-Phe-[Met1, Ile29, Leu89] chicken egg-white cystatin, (variant 1) and the N-terminally truncated form des-(S1-P11)-[Ala12, Glu12, Phe14, Met15, Leu89]-chicken egg-white cystatin, (variant 2), inhibited aleurain competitively with Ki values of 125 nM and 5 microM, respectively. Implications for the aminopeptidase activity of aleurain are discussed using cathepsin H for comparison.

Aminopeptidases↗
PubMed1994

A Kazal-type inhibitor of human mast cell tryptase: isolation from the medical leech Hirudo medicinalis, characterization, and sequence analysis.

Human tryptase, a tetrameric proteinase expressed by mast cells, is virtually unique among the serine proteinases as it is not inhibited by any proteinaceous inhibitor tested so far. We have now isolated, sequenced, and characterized an inhibitor of human tryptase from the medical leech Hirudo medicinalis. LDTI (Leech-Derived Tryptase Inhibitor) was purified to apparent homogeneity by cation exchange and affinity chromatography. Amino acid sequencing of the protein consisting of 46 residues (M(r) 4738) revealed a high degree of similarity to the non-classical Kazal-type inhibitors bdellin B-3 and rhodniin, inhibitors isolated from the medical leech and the insect Rhodnius prolixus, respectively. LDTI is a tight-binding and relatively specific inhibitor of human tryptase; it inhibits only trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) with similar affinities. Inhibition studies using small chromogenic substrates revealed that LDTI inhibits the amidolytic activity of tryptase by approximately 50%, suggesting that most likely due to steric hindrance LDTI binds to and inhibits only 2 of 4 active sites of tryptase. LDTI appears useful as a prototype of inhibitors of human tryptase and as a pharmacological tool for the investigation of the role of tryptase in health and disease.

Amino Acid Sequence↗