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C A Sprecher

Publications and source records attributed to C A Sprecher.

At least 19 recordsLinked to original sources
PubMed1999

Caspase-1 (interleukin-1beta-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9.

The regulation of caspases, cysteine proteinases that cleave their substrates after aspartic residues, is poorly understood, even though they are involved in tightly regulated cellular processes. The recently discovered serpin analogue proteinase inhibitor 9 (PI9) is unique among human serpin analogues in that it has an acidic residue in the putative specificity-determining position of the reactive-site loop. We measured the ability of PI9 to inhibit the amidolytic activity of several caspases. The hydrolysis of peptide substrates by caspase-1 (interleukin-1beta-converting enzyme), caspase-4 and caspase-8 is inhibited by PI9 in a time-dependent manner. The rate of reaction of caspase-1 with PI9, as well as the rate of substrate hydrolysis of the initial caspase-PI9 complex, shows a hyperbolic dependence on the concentration of PI9, indicative of a two-step kinetic mechanism for inhibition with an apparent second-order rate constant of 7x10(2) M(-1).s(-1). The hydrolysis of a tetrapeptide substrate by caspase-3 is not inhibited by PI9. The complexes of caspase-1 and caspase-4 with PI9 can be immunoprecipitated but no complex with caspase-3 can be detected. No complex can be immunoprecipitated if the active site of the caspase is blocked with a covalent inhibitor. These results show that PI9 is an inhibitor of caspase-1 and to a smaller extent caspase-4 and caspase-8, but not of the more distantly related caspase-3. PI9 is the first example of a human serpin analogue that inhibits members of this class of cysteine proteinases.

Caspase 3↗
PubMed1999

Inhibitory properties of human recombinant Arg24-->Gln type-2 tissue factor pathway inhibitor (R24Q TFPI-2).

Human type-2 tissue factor pathway inhibitor (TFPI-2), also known as placental protein 5, is a 32-kDa serine proteinase inhibitor consisting of three tandemly arranged Kunitz-type domains homologous to tissue factor pathway inhibitor. TFPI-2 inhibits a variety of serine proteinases involved in coagulation and fibrinolysis through an arginine residue (R24) in its first Kunitz-type domain, which constitutes a putative P1 residue for the substrate recognition sites of these proteinases. As recent studies have shown that this P1 residue to be a glutamine in murine TFPI-2, we constructed, expressed, and purified a human TFPI-2 mutant with glutamine substituted for arginine at position 24 (R24Q TFPI-2). R24Q TFPI-2 lost approximately 90% of its inhibitory activity towards bovine trypsin and virtually all inhibitory activity towards human plasmin and the factor VIIa-tissue factor complex, emphasizing the importance of the P1 Arg24 residue in the inhibition of these serine proteinases. However, whereas wild-type TFPI-2 is a relatively weak inhibitor of human factor Xa amidolytic activity (IC50 approximately 1 microM), R24Q TFPI-2 exhibited enhanced inhibitory activity towards the amidolytic and coagulant activities of this proteinase with a Ki of 18 nM. While the molecular basis for the enhanced inhibition of human factor Xa by R24Q TFPI-2 is unknown, these data provide suggestive evidence that murine TFPI-2 may function as a serine proteinase inhibitor in spite of the absence of a P1 Arg or Lys residue.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of a novel class I cytokine receptor.

The human gp130 cDNA sequence was used as a query to search an expressed sequence tag database (dbEST) to identify cDNA sequences with similarity to the cytokine class I receptor family. A novel class I cytokine receptor was identified in a human infant brain cDNA library and was named WSX-1. Full-length cDNA sequences for human and murine WSX-1 were isolated and characterized. The WSX-1 cDNA encodes a 636 amino acid transmembrane protein with an extracellular domain of 482 amino acids and a cytoplasmic domain of 96 amino acids. The structure of the WSX-1 protein most closely resembles that of gp130. Northern blot analysis indicates high levels of expression in thymus, spleen, lymph node, and peripheral blood leukocytes, suggesting a role for WSX-1 in modulation of the immune response.

Amino Acid Sequence↗
PubMed1996

Inhibitory properties of a novel human Kunitz-type protease inhibitor homologous to tissue factor pathway inhibitor.

In a previous report, we described the molecular cloning, expression, and partial characterization of a second human tissue factor pathway inhibitor (TFPI), which we designated as TFPI-2 [Sprecher, C. A., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3353-3357]. Recombinant TFPI-2 inhibited the amidolytic activity of trypsin as well as that of factor VIIa in complex with tissue factor. TFPI-2 recently has been shown to be identical to placental protein 5 (PP5), a glycoprotein originally isolated from placenta that exhibits serine protease inhibitory activity. In the present study, we have examined TFPI-2/PP5 for its ability to inhibit a number of serine proteases involved in blood coagulation and fibrinolysis, inasmuch as TFPI-2/PP5 prolonged the coagulation time of human plasma induced by either tissue factor or contact activation in a dose-dependent manner. In addition to its ability to inhibit the amidolytic and proteolytic activities of the factor VIIa-tissue factor complex, TFPI-2/PP5 strongly inhibited the amidolytic activities of human factor XIa, human plasma kallikrein, and human plasmin with Ki values of 15, 25, and 3 nM, respectively. TFPI-2/PP5 was also a weak inhibitor of the activation of factor X by a complex of human factor IXa and poly(lysine) with an apparent Ki of 410 nM. Heparin markedly enhanced the ability of TFPI-2/PP5 to inhibit factor VIIa-tissue factor both in the solution phase and on cell surfaces. In addition, heparin augmented the inhibition of human factor Xa amidolytic activity at relatively high levels (10-100 nM) of TFPI-2/PP5. No significant inhibition of glandular kallikrein, urinary plasminogen activator, tissue plasminogen activator, human activated protein C, human factor Xa, human thrombin, or leukocyte elastase was observed when these proteases were incubated with TFPI-2 in the absence of heparin.

Amino Acid Sequence↗
PubMed1995

Molecular cloning, expression, and partial characterization of two novel members of the ovalbumin family of serine proteinase inhibitors.

A human placental lambda gt11 cDNA library was screened for sequences encoding proteins related to human proteinase inhibitor 6 (PI6), and two plaques were identified that displayed weak hybridization at high stringency. Isolation and characterization of the DNA inserts revealed two novel sequences encoding proteins composed of 376 and 374 amino acids with predicted molecular masses of approximately 42 kDa. The novel proteins displayed all of the structural features unique to the ovalbumin family of intracellular serpins including the apparent absence of a cleavable N-terminal signal sequence. The degree of amino acid sequence identity between the novel serpins and PI6 (63-68%) significantly exceeds that of any other combination of known intracellular serpins. The two novel serpins encoded by the two novel cDNA sequences have been designated as proteinase inhibitor 8 (PI8) and proteinase inhibitor 9 (PI9). The putative reactive center P1-P1' residues for PI8 and PI9 were identified as Arg339-Cys340 and Glu340-Cys341, respectively. PI9 appears to be unique in that it is the first human serpin identified with an acidic residue in the reactive center P1 position. In addition, the reactive center loop of PI9 exhibits 54% identity with residues found in the reactive center loop of the cowpox virus CrmA serpin. Two PI8 transcripts of 1.4 kilobases (kb) and 3.8 kb were detected by Northern analysis in equal and greatest abundance in liver and lung, while the 1.4-kb mRNA was in excess over the 3.8-kb mRNA in skeletal muscle and heart. Two PI9 transcripts of 3.4 and 4.4 kb were detected in equal and greatest abundance in lung and placenta and were weakly detected in all other tissues. PI8 and PI9 were expressed in baby hamster kidney and yeast cells, respectively. Immunoblot analyses using rabbit anti-PI6 IgG indicated the presence of PI8 in the cytosolic fraction of stably transfected cells that formed an SDS-stable 67-kDa complex with human thrombin. PI9 was purified to homogeneity from the yeast cell lysate by a combination of heparin-agarose chromatography and Mono Q fast protein liquid chromatography and migrated as a single band in SDS-polyacrylamide gel electrophoresis with an apparent molecular mass of 42 kDa. Purified recombinant PI9 failed to inhibit the amidolytic activities of trypsin, papain, thrombin, or Staphylococcus aureus endoproteinase Glu-C and did not form an SDS-stable complex when incubated with thrombin. The cognate intracellular proteinases that interact with PI8 and PI9 are unknown.

Amino Acid Sequence↗
PubMed1994

Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization.

Thrombopoietin (TPO), a lineage-specific cytokine affecting the proliferation and maturation of megakaryocytes from committed progenitor cells, is believed to be the major physiological regulator of circulating platelet levels. Recently we have isolated a cDNA encoding a ligand for the murine c-mpl protooncogene and shown it to be TPO. By employing a murine cDNA probe, we have isolated a gene encoding human TPO from a human genomic library. The TPO locus spans over 6 kb and has a structure similar to that of the erythropoietin gene (EPO). Southern blot analysis of human genomic DNA reveals a hybridization pattern consistent with a single gene locus. The locus was mapped by in situ hybridization of metaphase chromosome preparations to chromosome 3q26-27, a site where a number of chromosomal abnormalities associated with thrombocythemia in cases of acute myeloid leukemia have been mapped. A human TPO cDNA was isolated by PCR from kidney mRNA. The cDNA encodes a protein with 80% identity to previously described murine TPO and is capable of initiating a proliferative signal to murine interleukin 3-dependent BaF3 cells expressing the murine or human TPO receptor.

Amino Acid Sequence↗
PubMed1994

The use of conserved cellulase family-specific sequences to clone cellulase homologue cDNAs from Fusarium oxysporum.

Five cDNAs from the cellulolytic fungi Fusarium oxysporum that code for five distinct cellulase homologues have been cloned and sequenced. The cloning strategy exploited the hydrophobic cluster analysis-based cellulase family classification of Henrissat and Bairoch [Biochem. J. 293 (1993) 781-788] to design degenerate oligodeoxyribonucleotides (oligos) that encoded amino-acid sequences conserved in an intra-family, but not inter-family, manner among cellulases from different species. Polymerase chain reaction (PCR) experiments using F. oxysporum genomic DNA primed with these 'family-specific' oligos were used to rapidly generate PCR fragments which were in turn used to probe cDNA libraries. Two distinct cDNAs coding for cellulase C-family homologues and one cDNA each coding for homologues to the B, F and K families, were isolated in this manner. This approach is an example of the power of multiple sequence analysis to generate cross-species, homology-based probes to rapidly clone homologues in a species of interest.

Amino Acid Sequence↗
PubMed1994

Molecular cloning, expression, and partial characterization of a second human tissue-factor-pathway inhibitor.

Previous studies have shown that tissue-factor-pathway inhibitor (TFPI) is an important regulator of the extrinsic pathway of blood coagulation through its ability to inhibit factor Xa and factor VIIa-tissue factor activity. We describe the molecular cloning and expression of a full-length cDNA that encodes a molecule, designated TFPI-2, that has a similar overall domain organization and considerable primary amino acid sequence homology to TFPI. After a 22-residue signal peptide, the mature protein contains 213 amino acids with 18 cysteines and two canonical N-linked glycosylation sites. The deduced sequence of mature TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxyl-terminal tail highly enriched in basic amino acids. Northern analysis indicates that TFPI-2 is transcribed in umbilical vein endothelial cells, liver, and placenta. TFPI-2 was expressed in baby hamster kidney cells and purified from the serum-free conditioned medium by a combination of heparin-agarose chromatography, Mono Q FPLC, Mono S FPLC, and Superose 12 FPLC. Purified TFPI-2 migrated as a single band in SDS/PAGE and exhibited a molecular mass of 32 kDa in the presence and absence of reducing agent. The amino-terminal sequence of recombinant TFPI-2 was identical to that predicted from the cDNA. Despite its structural similarity to TFPI, the purified recombinant TFPI-2 failed to react with polyclonal anti-TFPI IgG. Preliminary studies indicated that purified recombinant TFPI-2 strongly inhibited the amidolytic activities of trypsin and the factor VIIa-tissue factor complex. In addition, the inhibition of factor VIIa-tissue factor amidolytic activity by recombinant TFPI-2 was markedly enhanced in the presence of heparin. TFPI-2 at high concentrations weakly inhibited the amidolytic activity of human factor Xa, but had no measurable effect on the amidolytic activity of human thrombin.

Amino Acid Sequence↗
PubMed1994

The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization.

Characterization of the human glucagon-receptor-encoding gene (GGR) should provide a greater understanding of blood glucose regulation and may reveal a genetic basis for the pathogenesis of diabetes. A cDNA encoding a complete functional human glucagon receptor (GGR) was isolated from a liver cDNA library by a combination of polymerase chain reaction and colony hybridization. The cDNA encodes a receptor protein with 80% identity to rat GGR that binds [125I]glucagon and transduces a signal leading to increases in the concentration of intracellular cyclic adenosine 3',5'-monophosphate. Southern blot analysis of human DNA reveals a hybridization pattern consistent with a single GGR locus. In situ hybridization to metaphase chromosome preparations maps the GGR locus to chromosome 17q25. Analysis of the genomic sequence shows that the coding region spans over 5.5 kb and is interrupted by 12 introns.

Amino Acid Sequence↗
PubMed1993

Molecular cloning of the cDNA for a human amyloid precursor protein homolog: evidence for a multigene family.

Alzheimer's disease is a degenerative neurological disorder characterized by neural loss and brain lesions associated with plaques containing large amounts of the beta/A4 amyloid peptide. Molecular cloning of the cDNA for this peptide from human brain has shown it to be derived by proteolysis from a much larger precursor called the amyloid precursor protein (APP). The biological role of the precursor is unknown, but it has been shown to be transcribed in many human tissues in addition to brain. In the present report, we describe the molecular cloning from a human placental library of a full-length cDNA for a molecule closely related to APP. This novel molecule, which we have called amyloid precursor protein homolog (APPH), shares overall domain organization with APP. It is 763 amino acids in length and appears to encode a signal peptide, a large apparent extracellular domain including a Kunitz inhibitor domain, a transmembrane region, and a short cytoplasmic domain. Northern analysis indicates that it occurs in at least two molecular forms and is transcribed in human brain, heart, lung, liver, and kidney, in addition to placenta. On the basis of its extensive sequence similarity and conservation of domain structure, APPH is the nearest relative of APP yet identified in an emerging multigene family.

Amino Acid Sequence↗
PubMed1993

Expression cloning and signaling properties of the rat glucagon receptor.

Glucagon and the glucagon receptor are a primary source of control over blood glucose concentrations and are especially important to studies of diabetes in which the loss of control over blood glucose concentrations clinically defines the disease. A complementary DNA clone for the glucagon receptor was isolated by an expression cloning strategy, and the receptor protein was expressed in several kidney cell lines. The cloned receptor bound glucagon and caused an increase in the intracellular concentration of adenosine 3', 5'-monophosphate (cAMP). The cloned glucagon receptor also transduced a signal that led to an increased concentration of intracellular calcium. The glucagon receptor is similar to the calcitonin and parathyroid hormone receptors. It can transduce signals leading to the accumulation of two different second messengers, cAMP and calcium.

Amino Acid Sequence↗
PubMed1991

Endoproteolytic processing of the human protein C precursor by the yeast Kex2 endopeptidase coexpressed in mammalian cells.

The human protein C precursor undergoes extensive co- and posttranslational modification during its biosynthesis in the liver. These modifications include glycosylation, gamma-carboxylation, and beta-hydroxylation of specific amino acids and endoproteolytic processing to remove the pre- and propeptides as well as the pair of basic amino acids which connect the light and heavy chains in the precursor. Previous studies with a recombinant mammalian expression system have indicated that the endopeptidase in several mammalian cell types which recognizes and cleaves this dibasic site has a substrate specificity for sites which also include a basic amino acid in the -4 position (Foster et al., 1990). Since the human protein C precursor has His154 in the -4 position, it is poorly and incompletely cleaved in BHK and several other mammalian cell lines and also apparently secreted from the liver as a mixed population of mature two-chain and precursor one-chain molecules. In the present study, a mammalian expression system has been used to study the effect of coexpressing the protein C precursor together with the yeast Kex2 endopeptidase which is known to recognize and process dibasic pairs within peptide precursors in yeast. Coexpression of the KEX2 gene resulted in complete conversion of the protein C precursor to the mature two-chain form. Amino-terminal sequencing of the cleavage products has indicated that the cleavage occurs in the correct location and that this site is preferentially recognized by the yeast endopeptidase within the context of the mammalian cell secretory pathway.

Amino Acid Sequence↗
PubMed1990

Endoproteolytic processing of the dibasic cleavage site in the human protein C precursor in transfected mammalian cells: effects of sequence alterations on efficiency of cleavage.

The human protein C precursor undergoes extensive co- and posttranslational modification during its biosynthesis in the liver. These modifications include glycosylation, gamma-carboxylation and beta-hydroxylation of specific amino acids, and endoproteolytic processing to remove the pre- and propeptides and also to remove the pair of basic amino acids that connect the light and heavy chains in the precursor. Specific molecular signals have been elucidated which direct several of these modifications; however, the mechanism for cleavage and removal of the basic amino acid pair has not been established. In the present study, a recombinant mammalian expression system has been used to study the molecular signals that direct removal of this basic amino acid pair. Mutations were introduced by site-directed mutagenesis either to insert additional basic amino acids or to alter the sequence adjacent to the basic pair by point mutations. The mutant protein precursors were expressed and analyzed for the degree of processing to 2-chain form and also for the location of the cleavage site (by N-terminal sequencing) and subsequent removal of the basic amino acids from the newly formed C terminus of the light chain. These experiments have shown that human protein C can be readily synthesized and secreted in several mammalian cell lines. However, cell lines vary considerably in their capacity to remove the dibasic pair in the protein C precursor and, like the liver, secrete a mixed population of 1-chain and 2-chain forms of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Propeptide of human protein C is necessary for gamma-carboxylation.

Protein C is one of a family of vitamin K dependent proteins, including blood coagulation factors and bone proteins, that contains gamma-carboxyglutamic acid. Sequence analysis of the cDNAs for these proteins has revealed the presence of a prepro leader sequence that contains a pre sequence or hydrophobic signal sequence and a propeptide containing a number of highly conserved amino acids. The pre region is removed from the growing polypeptide chain by signal peptidase, while the pro region is subsequently removed from the protein prior to secretion. In the present study, deletion mutants have been constructed in the propeptide region of the cDNA for human protein C, and the cDNAs were then expressed in mammalian cell culture. These deletions included the removal of 4, 9, 12, 15, 16, or 17 amino acids comprising the carboxyl end of the leader sequence of 42 amino acids. The mutant proteins were then examined by Western blotting, barium citrate adsorption and precipitation, amino acid sequence analysis, and biological activity and compared with the native protein present in normal plasma. These experiments have shown that protein C is readily synthesized in mammalian cell cultures, processed, and secreted as a two-chain molecule with biological activity. Furthermore, the pre portion or signal sequence in human protein C is 18 amino acids in length, and the pro portion of the leader sequence is 24 amino acids in length. Also, during biosynthesis and secretion, the amino-terminal region of the propeptide (residues from about -12 through -17) is important for gamma-carboxylation of protein C, while the present data and those of others indicate that the carboxyl-terminal portion of the propeptide (residues -1 through -4) is important for the removal of the pro leader sequence by proteolytic processing.

Amino Acid Sequence↗