Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Because the cytokine interleukin-1 beta (IL-1 beta) lacks a classical hydrophobic signal sequence, it has been unclear how it is released from cells, and whether release proceeds via a novel mechanism or through non-specific leakage. To address this issue, we have examined the secretion of the recombinant forms of human IL-1 beta from COS monkey kidney cells, which express low levels of endogenous IL-1 beta. Four proteins were expressed: precursor and mature IL-1 beta and precursor and mature IL-1 beta fused to an amino terminal hydrophobic signal sequence from human tissue plasminogen activator. By monitoring the appearance of a known cytosolic protein (ATP citrate lyase) in the medium, we find that the unmodified IL-1 beta s are non-specifically released in very small quantities from the cytosol. On the other hand, the signal sequence-modified IL-1 beta s are glycosylated and efficiently secreted by the ER/Golgi pathway. The secreted, modified-mature protein is also biologically active, suggesting that this pathway has been bypassed for reasons other than maintaining the structural integrity of IL-1 beta. More likely the alternative pathway is a critical aspect of IL-1 biology. The differences in kinetics and quantity of IL-1 beta release from monocytic and COS cells suggest that COS cells lack critical components for the rapid release seen in monocytes.
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PMID:Further aspects of IL-1 beta secretion revealed by transfected monkey kidney cells. 163 62

Complementary DNAs coding for four Desmodus rotundus salivary plasminogen activators (DSPAs) were isolated and characterized. The predicted amino acid sequences display structural features also found in tissue-type plasminogen activator. The largest forms (DSPA alpha 1 and -alpha 2) contain a signal peptide, a finger (F), an epidermal growth factor (EGF), a kringle, and a serine protease domain, whereas DSPA beta and -gamma lack the F and F-EGF domains, respectively. Additional differences between the four forms suggest that distinct genes code for the members of the DSPA family. Transfection of DSPA-encoding cDNAs, placed under the control of the simian virus 40 late promoter, into COS-1 cells resulted in the secretion of highly fibrin-dependent PAs.
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PMID:The plasminogen activator family from the salivary gland of the vampire bat Desmodus rotundus: cloning and expression. 193 19

Senile plaques, often surrounded by abnormally grown neurites, are characteristic of Alzheimer's diseased brain. The core of the plaque is mainly composed of amyloid beta protein (beta-AP), two of whose three precursors (APP) have serine proteinase inhibitor regions (APPI). APPI derivatives containing 60, 72 or 88 amino-acid fragments (APPI-60, APPI-72 and APPI-88, respectively) of the longest APP were produced in COS-1 cell culture medium, with the APPI cDNA ligated to the signal sequence of tissue plasminogen activator. The secreted APPIs were purified by sequential acetone precipitation followed by affinity chromatography using immobilized trypsin. These three APPIs and O-glycosylation-site-mutated APPI showed similar inhibitory activity against trypsin, chymotrypsin and plasmin. The purified APPI-72 was found to inhibit trypsin (Ki = 1.1 x 10(-10) M) and chymotrypsin (Ki = 5.8 x 10(-9) M) most strongly, and to inhibit leukocyte elastase (Ki = 7.9 x 10(-7) M) and several blood coagulation proteinases (Ki = 0.46-12 x 10(-7) M), but not urokinase or thrombin. The observed inhibition pattern was quite different from that of protease nexin I, one of serine proteinase inhibitors possessing neurite outgrowth activity. This suggests that the physiological roles of APPI are different from those of protease nexin I, and that APPI could not cause aberrant growth of neurite into the plaque. The presence of APPI having strong inhibitory activity in the brain might lead to the formation of amyloid deposits by preventing complete degradation of APPs.
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PMID:Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of Alzheimer's disease: different properties compared with protease nexin I. 218 Apr 85

An expression construct for human tissue-type plasminogen activator (t-pA) cDNA [containing a simian virus 40 (SV40) origin of replication] was introduced into CV1, COS-7 and COSts2 cells; in the latter cell line the amount of functionally active large T antigen of SV40 is regulated by the temperature. In a transient system, the expression in COSts2 cells at the permissive temperature for large T antigen was improved sixfold compared to COS-7 cells. By cotransfection with a plasmid conferring resistance to G418 into COSts2 cells, a cell line (COSts2Glob t-pA) could be isolated with barely detectable expression of t-pA at the semi-permissive and non-permissive temperature and inducible secretion of t-pA at the permissive temperature. The kinetics of induction, inducibility after continued propagation at the semi-permissive temperature and the influence of the temperature during previous propagation on inducibility were investigated. The biological activity of the secreted material was demonstrated by a functional assay. Inducibility of t-pA by temperature was accompanied by a dramatic increase of the copy number of episomal plasmids (up to 2000 copies per cell).
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PMID:Establishment of a temperature-inducible cell line for human plasminogen activator (tissue-type) by transfection of monkey cells with expression constructs. 312 86

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) mediates endocytosis of a number of structurally unrelated ligands, including complexes of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA), free t-PA, single-chain urokinase (pro-u-PA), alpha 2-macroglobulin-protease complexes, and lipoprotein lipase. So far, all ligands have in common the fact that they bind to the receptor in a Ca(2+)-dependent way and the fact that binding to the receptor can be inhibited by a 39-40-kDa protein, termed the receptor-associated protein. To obtain inhibitory antibodies for the analysis of the structure and function of the receptor we applied the combinatorial immunoglobulin repertoire cloning technique in order to specifically select monoclonal Fab fragments directed against Ca(2+)-dependent epitopes. In this report we describe the isolation of a Fab fragment (Fab A8) showing a high relative affinity for the receptor (0.5 nM). The binding of this Fab fragment to purified LRP is inhibited in the presence of 5 mM EDTA, receptor-associated protein, and lipoprotein lipase (IC50 values of 1.4 and 31 nM, respectively). By immunoblotting of CNBr-digested LRP it is shown that Fab A8 binds to a fragment that harbors the second cluster of cysteine-rich complement-type repeats flanked by epidermal growth factor repeats. Binding studies using 125I-labeled ligands and immobilized receptor show that Fab A8 partially inhibits the binding of [125I]u-PA.PAI-1 complexes (IC50 = 1.1 nM) and completely inhibits the binding of [125I]pro-u-PA to the receptor (IC50 = 2.2 nM). No inhibition was observed for the binding of 125I-labeled methylamine-activated alpha 2-macroglobulin or [125I]t-PA.PAI-1 to LRP. Degradation of [125I]u-PA.PAI-1 complexes by COS-1 cells was also partially (43%) inhibited by Fab A8. Our results provide evidence for the presence of an interaction site for pro-u-PA localized in the second cluster of cysteine-rich repeats that is unrelated to the t-PA.PAI-1 or methylamine-activated alpha 2-macroglobulin interaction sites.
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PMID:Analysis of the binding of pro-urokinase and urokinase-plasminogen activator inhibitor-1 complex to the low density lipoprotein receptor-related protein using a Fab fragment selected from a phage-displayed Fab library. 753 22

Three t-PA mutants, t-PA del K1 (with deletion of K1 domain), t-PA del (296-302) (with deletion of PAI-1 binding site) and their combination mutant t-PA del (K1, 296-302), were constructed by DNA recombination and site-directed mutagenesis techniques. Then the three t-PA mutants were transiently expressed in COS-7 cells, and in addition, the combination mutant t-PA del (K1, 296-302) was stably expressed in CHO cells. The biological analysis of the expression products demonstrated that t-PA del (296-302) and t-PA del (K1, 296-302) had obtained the resistance to inhibition by PAI-1. In addition, the half-life of t-PA del (K1, 296-302) in rat plasma was increased 6 fold while the mutant affinity for fibrin was only slightly affected. Therefore, it was reasonable to consider that the mutant t-PA del (K1, 296-302) may become a potent candidate of new thrombolytic agent.
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PMID:Construction, expression and the characterization of t-PA mutants with increased plasma half-life and resistance to inhibition by PAI-1. 754 67

A series of t-PA cDNA mutants containing different parts of 3'-UTR sequences have been constructed. In vitro translation of t-PA transcripts in rabbit reticulocyte lysates and its expression in COS-7 cells show that the 3'-UTR sequence has a very strong inhibitory effect on t-PA translation. The deletion of 3'-UTR results in 3-8-fold increase of t-PA expression. Further study shows that an AU-rich sequence of some 200 nt at 3' end of 3'-UTR is responsible for the translational inhibition. RNA stability experiment reveals that the AU-rich segment leads to a 3-fold decrease of t-PA mRNA stability. The insertion of this segment into the 3'-UTR of luciferase gene results in an obvious inhibition of Luc expression. A model is proposed for the regulation of t-PA expression.
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PMID:Inhibitory effect of 3'-untranslated region (3'-UTR) of human tissue-plasminogen activator (ht-PA) mRNA on its expression. 855 75

Three tissue-type plasminogen activator (t-PA) mutants were constructed by recombinant and site-directed mutagenesis techniques. They are del(296-302) with deletion of PAI-1 binding site, N117Q/N184Q with deglycosylation of K1 and K2 domains, and their combination mutant designated as GGI. Then these three mutants were successfully transiently expressed in COS-7 cells, and GGI was further stably expressed in CHO cells. The biological characterization of the expression products indicated that del(296-302) and GGI possessed the resistance to inhibition by PAI-1. In addition, the specific activity of GGI was increased by about 46%, the plasma half-life was prolonged by about one fold, while its affinity for fibrin was not affected.
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PMID:Construction, expression and characterization of tissue-type plasminogen activator mutants. 874 32

To tailor tissue-type plasminogen activator (tPA) to possess an affinity for the integrins, several mutants were constructed by introducing the Arg-Gly-Asp (RGD) sequene into the tPA molecule. These mutants were expressed in COS-1 cells and partially purified by lysine-Sepharose chromatography. The RGD-dependent binding of the mutants to platelet integrin, integrin alpha IIb beta 3, was evaluated by subtracting the nonspecific binding in the presence of 10 mM EDTA (or 1 mg/ml GRGDSP). The binding assay showed that two tPA mutants possess high affinity for the integrin in an RGD-dependent manner. One mutant is 148RGD-tPA with RGDS in place of DRDS (residues 148 to 151) in the loop region of the kringle 1 domain of tPA, and the other is 270RGD-tPA with RGDS in place of SQPQ (residues 270 to 273) in the linker region between the kringle 2 and protease domains. Using the chromogenic substrate Spectrozyme tPA, the 148RGD-tPA mutant was shown to possess amidolytic activity comparable with that of native tPA, while the 270RGD-tPA mutant exhibited several-fold lower activity. In addition, the 148RGD-tPA exhibited full tPA activity even when interacting with the integrin alpha IIb beta 3. These results suggest that the bifunctional 148RGD-tPA molecule might be useful as an improved thrombolytic agent specific for the platelet integrin, the integrin alpha IIb beta 3.
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PMID:Integrin-specific tissue-type plasminogen activator engineered by introduction of the Arg-Gly-Asp sequence. 892 Sep 10

The human endogenous retrovirus type K (HERV-K) family codes for the human teratocarcinoma-derived retrovirus (HTDV) particles. The existence of the envelope protein (ENV) of HERV-K encoded by the subgenomic env mRNA has not yet been demonstrated. To study the genetic requirements for successful expression of ENV, we have constructed a series of recombinant HERV-K env expression vectors for infection and transfection experiments in insect cells and mammalian cells, respectively. Six baculovirus constructs bearing full-length or truncated HERV-K env with or without homologous or heterologous signal peptides were used for infections of insect cells. All recombinant baculoviruses yielded ENV proteins with the expected molecular masses. The full-length 80- to 90-kDa HERV-K ENV protein including the cORF leader sequence was glycosylated in insect cells. In addition, the 14-kDa cORF protein was expressed due to splicing of the full-length env mRNA. The ENV precursor protein is not cleaved to the surface (SU) and transmembrane (TM) glycoproteins; it does not appear on the surface of infected insect cells and is not secreted into the medium. For ENV expression in COS cells, plasmid vectors harboring the cytomegalovirus immediate-early promoter/intron A element and the tissue plasminogen activator (t-PA) signal peptide or the homologous HERV-K signal peptide upstream of the env gene were employed. Glycosylated and uncleaved ENV was expressed as in GH teratocarcinoma cells but at higher levels. The heterologous t-PA signal sequence was instrumental for expression of HERV-K ENV on the cell surface. Hence, we have shown for the first time that the HERV-K env gene has the potential to be expressed as a full-length envelope protein with appropriate glycosylation. In addition, our data provide explanations for the lack of infectivity of HERV-K/HTDV particles.
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PMID:Expression of human endogenous retrovirus type K envelope glycoprotein in insect and mammalian cells. 906 Jun 28


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