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W D Schleuning

Publications and source records attributed to W D Schleuning.

At least 37 records · Page 2Linked to original sources
PubMed1995

Structural features mediating fibrin selectivity of vampire bat plasminogen activators.

The distinguishing characteristic of vampire bat (Desmodus rotundus) salivary plasminogen activators (DSPAs) is their strict requirement for fibrin as a cofactor. DSPAs consist of structural modules known from urokinase (u-PA) and tissue-type plasminogen activator (t-PA) such as finger (F), epidermal growth factor (E), kringle (K), and protease (P), combining to four genetically and biochemically distinct isoenzymes, exhibiting the formulas FEKP (DSPA alpha 1 and alpha 2) and EKP and KP (DSPA beta and DSPA gamma). Only DSPA alpha 1 and alpha 2 bind to fibrin. All DSPAs are single-chain molecules, displaying substantial amidolytic activity. In a plasminogen activation assay, all four DSPAs are almost inactive in the absence of fibrin but strongly stimulated by fibrin addition. The catalytic efficiency (kcat/Km) of DSPA alpha 1 increases 10(5)-fold, whereas the corresponding value of t-PA is only 550. The ratio of the bimolecular rate constants of plasminogen activation in the presence of fibrin versus fibrinogen (fibrin selectivity) of DSPA alpha 1, alpha 2, beta, gamma, and t-PA was found to be 13,000, 6500, 250, 90, and 72, respectively. Whereas all DSPAs are therefore more fibrin dependent and fibrin selective than t-PA, the extent depends on the respective presence of the various domains. The introduction of a plasmin-sensitive cleavage site in a position akin to the one in t-PA partially obliterates fibrin cofactor requirement. Fibrin dependence and fibrin selectivity of DSPAs are accordingly mediated by fibrin binding, which involves the F domain, as yet undefined determinants within the K and P domains, and by the absence of a plasmin-sensitive activation site. These findings transcend the current understanding of fibrin-mediated stimulation of plasminogen activation: in addition to fibrin binding, specific protein-protein interactions come into play, which stabilize the enzyme in its active conformation.

Aminocaproic Acid↗
PubMed1995

Low-M(r) urokinase-type plasminogen activator as a reporter protein.

A reporter plasmid coding for the low-molecular-weight (low-M(r)) form of single-chain urokinase-type plasminogen activator (low-M(r) u-PA; Leu144-Leu411) has been constructed and used to analyze promoter activity. Vectors containing the low-M(r) u-PA cDNA coupled to hormone-responsive promoters were introduced to mammalian cells. Following hormone treatment, the activity of the secreted reporter protein was determined in aliquots of cell culture supernatants. The assay is based on plasminogen activation by low-M(r) u-PA and subsequent cleavage of the plasmin-specific tripeptide substrate, S-2251. The resulting chromophore, p-nitroanilide, was quantified colorimetrically at 405 nm. Transient and stable expression of the low-M(r) u-PA reporter gene in different eukaryotic cells demonstrates the suitability of the system for the quantification of the activity of eukaryotic promoter elements in a rapid and highly sensitive manner, while maintaining cell integrity.

Animals↗
PubMed1995

Isolation and characterization of the androgen-dependent mouse cysteine-rich secretory protein-3 (CRISP-3) gene.

The mRNA for cysteine-rich secretory protein-3 (CRISP-3) was originally identified in the mouse salivary gland as an androgen-dependent transcript, and is closely related to CRISP-1 and CRISP-2 which are abundantly expressed in the epididymis and testis respectively. Overlapping phage clones encompassing the entire length of the CRISP-3 gene were isolated from a lambda EMBL3 genomic library and analysed. DNA sequencing revealed that the gene consisted of eight exons ranging between 55 and 740 bp in size, and seven introns. All exon-intron junctions conformed to the GT/AG rule established for eukaryotic genes. The length of the introns was determined by PCR and was found to vary between 1.0 and 3.7 kb, indicating that the gene spans over 20 kb of the mouse genome. Primer extension allowed the mapping of the major transcription initiation site to an adenine located at the appropriate position downstream of a bona fide TATA box, in a region corresponding well to the eukaryotic consensus sequence. Over 800 bp of CRISP-3 promoter region were determined and two regions almost exactly matching the androgen-responsive element consensus RGWACANNNTGTWCY detected. In addition, sequences described in the Drosophila melanogaster Sgs-3 gene as being involved in its salivary gland-specific expression as well as two putative OTF- and GATA-binding elements were also found.

Androgens↗
PubMed1995

Expression of active recombinant pallidipin, a novel platelet aggregation inhibitor, in the periplasm of Escherichia coli.

The platelet aggregation inhibitor pallidipin is a protein present in the saliva of the blood-sucking triatomine bug Triatoma pallidipennis. Expression of recombinant pallidipin in the periplasm of Escherichia coli was achieved by placing its coding sequence downstream of the alkaline phosphatase (APase) or trc promoter in frame with bacterial leader peptide DNA sequences derived from APase or from the periplasmic form of cyclophilin (Cph). In each case the DNA sequence of mature pallidipin was merged to the leader peptide coding part, either directly, or while introducing additional amino acids, in order to assess their influence on the activity of the leader peptidase and on the biological activity of the recombinant protein. All tested constructs gave rise to abundant periplasmic expression of pallidipin, which was then purified by a combination of cation- and anion-exchange chromatography followed by size-exclusion gel chromatography. Recombinant pallidipin had the expected molecular mass (approximately 19 kDa) and was correctly processed, as demonstrated by SDS/PAGE and N-terminal amino acid sequencing. The highest expression levels were obtained with the three APase-derived expression plasmids. Platelet aggregation tests revealed that E. coli-derived pallidipin was fully active, with an IC50 of 33-89 nM, comparable with that of the native protein, except when an additional N-terminal lysyl-isoleucyl dipeptide was present, which resulted in an IC50 more than ten times higher.

Amino Acid Sequence↗
PubMed1995

Production of vampire bat plasminogen activator DSPA alpha 1 in CHO and insect cells.

Salivary plasminogen activator from the vampire bat Desmodus rotundus (DSPA alpha 1) is a promising new thrombolytic agent. Continuous growth of a stably transfected, methotrexate amplified, dhfr- CHO cell line yields up to 60 mg l-1 of DSPA alpha 1. Utilizing an engineered baculovirus 10 mg l-1 were produced in batches of Sf9 insect cells. Recombinant DSPA alpha 1 is purified from both sources using a one-step purification protocol. Although differences in glycosylation were detected, enzymatic activity and fibrin cofactor dependency are unaffected when DSPA alpha 1 derived from the two expression systems is compared.

Animals↗
PubMed1995

Splice variants of the human EP3 receptor for prostaglandin E2.

The EP3 receptor for prostaglandin E2 (PGE2) mediates various biological activities such as uterine contraction, inhibition of gastric acid secretion, presynaptic inhibition of neurotransmitter release and potentiation of platelet aggregation. In an attempt to understand the molecular basis of this diversity of biological function, we cloned full-length cDNAs encoding EP3 receptors for PGE2 from human uterus cDNA libraries. Seven cDNA variants were identified which code for six distinct EP3-receptor isoforms. Sequencing revealed that the receptor isoforms differ in their intracellular C-terminal domains. Southern blot experiments indicate that the isoforms are generated by alternative splicing. The EP3-receptor gene is expressed in various tissues with high expression in kidney and pancreas, as demonstrated by Northern blot analysis. All receptors, stably expressed in baby hamster kidney (BHK) cells, bind PGE2 specifically with similar Kd of 2.2-5.8 nM. The binding of [3H]PGE2 is competed with by unlabelled prostaglandins in the order sulprostone (a PGE2-like agonist) approximately PGE2 >> PGF2 alpha > Iloprost (a prostacyclin analogue) > PGD2, which is specific for EP3 receptors. Analysis of the signal-transduction pathways demonstrated that all receptors respond with inhibition of forskolin-induced cAMP accumulation with an IC50 of 0.1-3 nM PGE2. In addition, some isoforms induce an increase in intracellular free calcium ([Ca2+]i) at PGE2 concentrations greater than or equal to 10 nM. These results may offer an explanation for the different physiological responses observed in various tissues following activation of EP3 receptors.

Amino Acid Sequence↗
PubMed1995

Mouse androgen-dependent epididymal glycoprotein CRISP-1 (DE/AEG): isolation, biochemical characterization, and expression in recombinant form.

In the rat, the secretory glycoprotein DE/AEG is one of the main constituents of the epididymal fluid. We have recently reported the cloning of the cDNA for the related cysteine-rich secretory protein-1 (CRISP-1) from murine epididymis (Haendler et al., 1993; Endocrinology 133:192-198). The protein has now been isolated from the same organ and its N-terminal amino acid sequence has been determined. CRISP-1 exhibited an isoelectric point of approximately 6.8. High levels of CRISP-1 antigen were detected in the corpus and cauda of the epididymis, vas deferens, seminal vesicle, prostate, and in the salivary gland by immunohistochemistry. A quantitative analysis of the cauda epididymal fluid by sandwich ELISA revealed that CRISP-1 represented approximately 15% of the total protein. For heterologous expression, the CRISP-1 coding sequence was introduced into the pMPSV/CMV vector before transfection of baby hamster kidney (BHK) cells and selection with puromycin and neomycin. Expression in insect cells was achieved by co-transfection of Sf9 cells with a transfer vector and baculovirus DNA. Recombinant CRISP-1 was isolated in quantities sufficient for structural analysis. Ethyl maleimide treatment showed that all 16 cysteines were engaged in disulfide bonds. Proteolytic digestion demonstrated that the six cysteines localized in the N-terminal moiety formed three bonds with each other, suggesting the existence of two discrete domains in the protein.

Amino Acid Sequence↗
PubMed1995

A general route to fingerprint analyses of peptide-antibody interactions using a clustered amino acid peptide library: comparison with a phage display library.

We provide a general route to fingerprint analyses of peptide-antibody interactions using a novel chemically synthesized peptide library. A combinatorial clustered amino acid peptide library XO1O2O3O4X (O = one of six amino acid clusters [APG], [DE], [HKR], [NQST], [FYW] and [ILVM]; X = randomized position) bound to a continuous cellulose membrane support was designed to overcome the problem of combinatorial explosion in the synthesis of peptide libraries. This library served as the starting point for the identification and detailed characterization of a TGF alpha peptide epitope recognized by the antibody Tab2. By analysing 1728 hexapeptide mixtures and 1600 single hexapeptides we identified a large number of structurally different high affinity Tab2 binding molecules. Our data provide a detailed picture of the structural basis of this antibody-peptide interaction. In addition to the detection of key amino acids involved in Tab2 binding we observed a high variability of Tab2 binding sequences supporting an induced fit mechanism in antibody-peptide recognition. In contrast, a phage display hexapeptide library led to the detection of only one dominant Tab2 binding peptide. The data obtained also demonstrate the influence of phage proteins on the interaction between the antibody and the displayed peptide. Comparing both approaches with regard to ease of handling and identified sequences, the chemical libraries are clearly favored to study antibody-peptide interactions.

Amino Acid Sequence↗
PubMed1995

Primary structure and properties of helothermine, a peptide toxin that blocks ryanodine receptors.

Helothermine, a protein from the venom of the Mexican beaded lizard (Heloderma horridum horridum), was found to inhibit [3H]ryanodine binding to cardiac and skeletal sarcoplasmic reticulum, to block cardiac and skeletal ryanodine receptor channels incorporated into planar bilayers, and to block Ca(2+)-induced Ca2+ release triggered by photolysis of nitr-5 in saponin-permeabilized trabeculae from rat ventricle. Cloning of the helothermine cDNA revealed that the protein is composed of 223 amino acids with a molecular mass of 25,376 daltons, and apparently is stabilized by eight disulfide bridges. The peptide sequence showed significant homology with a family of cysteine-rich secretory proteins found in the male genital tract and in salivary glands. The interaction of helothermine and ryanodine receptors should serve to define functional domains within the channel structure involved in the control of Ca2+ release from sarcoplasmic reticulum.

Amino Acid Sequence↗
PubMed1994

Mutational analysis of human endothelin receptors ETA and ETB identification of regions involved in the selectivity for endothelin 3 or cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu).

Two endothelin(ET)-receptor subtypes have been identified in mammals. They differ in their affinity towards the ET isopeptides with ETA displaying an ET-1-selective profile and ETB a non-selective one. To identify the regions responsible for the differential selectivity, chimeric forms were engineered by sequentially exchanging extracellular regions together with their flanking transmembrane domains. Two sets of reciprocal receptor mutants were thereby generated and analysed by expression in COS-7 cells. The recombinant receptor chimeras were characterised by direct and competitive radioligand-binding analysis. COS-7 cells transfected with vectors for the mutant receptors exhibited specific saturable [3-125I]iodotyrosyl ET-1 (125I-ET-1) binding, with affinities comparable to those of the wild-type receptors (apparent Ki approximately 1-6 x 10(-9) M). An average of 10(5)-10(6) binding sites/cell was calculated for the wild-type and mutant forms. In competition experiments using 125I-ET-1 and unlabeled ET-3, an ETB-selective agonist, we detected a clear switch from an ET-1-selective profile to a non-isopeptide-selective profile in ETA chimeras where the second extracellular loop and the flanking transmembrane domains IV and V, or the third extracellular loop and the flanking transmembrane domains VI and VII, had been exchanged for the corresponding parts of ETB. The opposite effect, namely a switch from a non-isopeptide-selective to an ET-1-selective binding, was observed for the mirror ETB chimeras where the symmetrical exchange had been operated. Using 125I-ET-1 and the ETA-specific antagonist cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu) (BQ123), we were able to map the main determinants responsible for this selectivity to the N-terminal moiety of this receptor. Therefore, the ability for the interaction with ET-3 or BQ123 is governed by two different regions of the ET receptors.

Amino Acid Sequence↗
PubMed1994

Induction of c-fos gene expression by urokinase-type plasminogen activator in human ovarian cancer cells.

Binding of urokinase-type plasminogen activator (u-PA) to u-PA receptor (u-PAR) induces the rapid and transient expression of c-fos in OC-7 ovarian carcinoma cells. The pretreatment of the cells with protein tyrosine kinase (PTK) inhibitors, but not the inactivation of the u-PA active site by DFP (diisopropyl fluorophosphate), abrogates this effect. A soluble u-PAR fragment, expressed in baculovirus-infected Sf9 cells and purified by affinity chromatography, competes for binding of u-PA to u-PAR and inhibits c-fos induction. We conclude that activation of u-PAR after interaction with u-PA at the cell surface initiates a transmembrane signal, most likely in conjunction with other still unknown protein(s). This signal generates PTK activity feeding into a signal transduction pathway which activates nuclear transcription factors.

Binding Sites↗
PubMed1994

An inhibitor of collagen-induced platelet aggregation from the saliva of Triatoma pallidipennis.

The saliva of Triatoma pallidipennis, a blood-sucking triatomine bug (Hemiptera, family Reduviidae, subfamily Triatominae) was found to contain a factor that specifically inhibits collagen-induced platelet aggregation. The 19-kDa protein was purified to homogeneity and named pallidipin. Collagen-mediated aggregation of platelets in plasma and of washed platelets was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effectors (ADP, thrombin, thromboxane A2 mimetic U46619, phorbol ester) was detected. Pallidipin had no effect on platelet adhesion to collagen but inhibited ATP release from platelets. It interacted reversibly with platelets and may share with collagen a common target on them. The protein exhibits a unique primary structure (predicted from cDNA clones) with no significant similarity to other previously described sequences. The protein produced in recombinant baby hamster kidney cells had antiaggregatory effects similar to those of native pallidipin. Availability of recombinant pallidipin will allow further investigation of the precise mechanism of action.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

Purification of human big endothelin 1 derived through cleavage with collagenase and dipeptidylpeptidase IV from a fusion protein expressed in Escherichia coli.

The cDNA coding for human big endothelin 1 (bigET-1), preceded by an optimized collagenase recognition sequence and followed by a stop codon, was fused in frame to the C-terminal region of alkaline phosphatase (AP). The fusion protein (AP-bigET), expressed in Escherichia coli K12 upon the lowering of organic phosphate concentrations, consisted of alkaline phosphatase (1-447), the collagenase cleavage site (Gly-Pro-Ala)4, and glycylprolyl-bigET-1. AP-bigET accumulated intracellularly in the form of inclusion bodies that were extensively washed and finally extracted by 8 M urea to yield highly enriched AP-bigET. Upon digestion of the fusion protein with collagenase, two disulfide conformeres of glycylprolyl-bigET-1 (bigET-1A and bigET-1B) could be purified by reverse-phase FPLC. Upon treatment with dipeptidylpeptidase IV to remove the N-terminal glycylprolyl-dipeptide, the later-eluting form of bigET-1 (bigET-1B) coeluted with authentic human bigET-1 on reverse-phase HPLC. BigET-1A and bigET-1B were formed at a ratio of 1:3. After reduction and S-pyridylethylation, both conformers coeluted with authentic but reduced bigET-1. Their amino acid sequences were identical. Both forms were converted by digestion with pepsin to the respective ET-1 conformeres (ET-1A and ET-1B) that were purified. In vasoconstriction assays, ET-1B but not ET-1A, at 10(-8) M, evoked a maximal response indistinguishable from that of authentic ET-1.

Alkaline Phosphatase↗
PubMed1994

Rational design of hirulog-type inhibitors of thrombin.

The two crystal structures of thrombin complexed with its most potent natural inhibitor hirudin and with the active-site inhibitor D-Phe-Pro-Arg-CH2Cl [Rydel, T.J. et al., J. Mol. Biol., 221 (1991) 583; Bode, W. et al., EMBO J., 8 (1989) 3467] were used as a basis to design a new inhibitor, combining the high specificity of the polypeptide hirudin with the simpler chemistry of an organic compound. In the new inhibitor, the C-terminal amino acid residues 53-65 of hirudin are linked by a spacer peptide of four glycines to the active-site inhibitor NAPAP (N alpha-(2-naphthyl-sulfonyl-glycyl)-DL-p-amidinophenylalanyl-piperi dine). Energy minimization techniques served as a tool to determine the preferred configuration at the amidinophenylalanine and the modified piperidine moiety of the inhibitor. The predictions are supported by the interaction energies determined for D- and L-NAPAP in complex with thrombin, which are in good agreement with experimentally determined dissociation constants. The conformational flexibility of the linker peptide in the new inhibitors was investigated with molecular dynamics techniques. A correlation between the Pl' position and the interactions of the linker peptide with the protein is suggested. Modifications of the linker peptide are proposed based on the distribution of its main-chain torsion angles in order to enhance its binding to thrombin.

Amino Acid Sequence↗
PubMed1994

Inhibition of acrosin by protein C inhibitor and localization of protein C inhibitor to spermatozoa.

Protein C inhibitor (PCI) is synthesized by cells throughout the male reproductive tract and is present in high concentrations (220 micrograms/ml) in seminal plasma. Seminal plasma as well as the acrosome of spermatozoa are rich in possible target proteases for PCI. We analyzed the interaction of PCI with acrosin, a serine protease stored in its zymogen form in the acrosome of spermatozoa. Purified human PCI inhibited the amidolytic activity of purified boar acrosin with an apparent second-order rate constant of 3.7 x 10(4) M-1.s-1. Inhibition was paralleled by the degradation of PCI from its 57- to its 54-kDa form. Human PCI also inhibited the amidolytic activity of activated human sperm extracts and formed complexes with acrosin as determined by an enzyme-linked immunosorbent assay. Immunocytochemistry revealed that morphologically abnormal spermatozoa stained for PCI antigen, whereas morphologically normal spermatozoa were negative. In immunoelectron microscopy, PCI was exclusively localized in the immediate vicinity of disrupted acrosomal membranes of sperm heads. These data suggest that PCI might function as a scavenger of prematurely activated acrosin, thereby protecting intact surrounding cells and seminal plasma proteins from possible proteolytic damage.

Acrosin↗
PubMed1994

Coronary thrombolysis with Desmodus salivary plasminogen activator in dogs. Fast and persistent recanalization by intravenous bolus administration.

BACKGROUND: DSPA (Desmodus salivary plasminogen activator) is a new thrombolytic agent corresponding to a natural plasminogen activator discovered in the saliva of the vampire bat Desmodus rotundus. Compared with tissue plasminogen activator (TPA), DSPA, produced in a recombinant cell line, is more fibrin cofactor dependent than TPA. METHODS AND RESULTS: The thrombolytic properties of DSPA and TPA were compared in a canine model of copper coil-induced coronary thrombosis. All dogs received heparin 200 IU/kg IV and SC. Whereas controls did not reperfuse within 180 minutes (none of six), intravenous bolus administration of DSPA at 25, 50, and 100 micrograms/kg resulted in a 100% incidence (6 of 6) of recanalization within 37, 23, and 18 minutes, respectively. TPA at 63 and 125 micrograms/kg reopened the coronaries in 33% (two of six) and 50% (three of six) of cases within 40 minutes. Eighty-three percent (5 of 6) of the arteries were still patent 3 hours after 50 and 100 micrograms/kg DSPA, whereas only 20% (one of five) of all coronaries originally recanalized with both doses of TPA were still open at 3 hours. Plasma levels of alpha 2-antiplasmin decreased significantly only with 125 micrograms/kg TPA. The clearance of DSPA (2.3 to 3.5 mL.min-1.kg-1) was lower compared with TPA (11.4 to 20 mL.min-1.kg-1) due to a prolonged terminal half-life. CONCLUSIONS: In a canine coronary thrombosis model, DSPA exhibited higher potency and recanalized coronary arteries faster and with a lower incidence of reocclusion than TPA. Its properties may translate into a higher efficacy in patients compared with available thrombolytic agents. The long half-life of DSPA may allow for single bolus administration in the treatment of acute myocardial infarction.

Animals↗
PubMed1993

Stable expression of human endothelin receptors ETA and ETB by transfected baby hamster kidney cells.

The cDNAs for human endothelin receptors ETA and ETB were subcloned into the eukaryotic expression vector pMPSV/CMV and transfected, in parallel with plasmids carrying resistances for hygromycin B and puromycin, into baby hamster kidney (BHK) cells. Cell clones constitutively expressing high levels of either receptor were obtained through the combined selective pressure of both antibiotics. This was further confirmed by Northern blot analysis using ETA- or ETB-specific oligonucleotide probes. The calculated KD for endothelin (ET)-1 binding to ETA and ETB were 2.2 x 10(-10) M and 5.3 x 10(-10) M, respectively. Competitive binding experiments using the different ET forms showed the expected isopeptide-selective and non-isopeptide-selective profiles for ETA and ETB, respectively. BQ 123, a specific ETA antagonist, competed with ET-1 for binding to ETA but not to ETB.

Animals↗