Angiostatin formation involves disulfide bond reduction and proteolysis in kringle 5 of plasmin.
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Plasmin is processed in the conditioned medium of HT1080 fibrosarcoma cells producing fragments with the domain structures of the angiogenesis inhibitor, angiostatin, and microplasmin. Angiostatin consists of kringle domains 1-4 and part of kringle 5, while microplasmin consists of the remainder of kringle 5 and the serine proteinase domain. Our findings indicate that formation of angiostatin/microplasmin involves reduction of plasmin by a plasmin reductase followed by proteolysis of the reduced enzyme. We present evidence that the Cys461-Cys540 and Cys511-Cys535 disulfide bonds in kringle 5 of plasmin were reduced by plasmin reductase. Plasmin reductase activity was secreted by HT1080 and Chinese hamster ovary cells and the human mammary carcinoma cell lines MCF-7, MDA231, and BT20 but not by the monocyte/macrophage cell line THP-1. Neither primary foreskin fibroblasts, blood monocyte/macrophages, nor macrovascular or microvascular endothelial cells secreted detectable plasmin reductase. In contrast, cultured bovine and rat vascular smooth muscle cells secreted small but reproducible levels of plasmin reductase. Reduction of the kringle 5 disulfide bonds triggered cleavage at either Arg529-Lys530 or two other positions C-terminal of Cys461 in kringle 5 by a serine proteinase. Plasmin autoproteolysis could account for the cleavage, although another proteinase was mostly responsible in HT1080 conditioned medium. Three serine proteinases with apparent Mr of 70, 50, and 39 were purified from HT1080 conditioned medium, one or more of which could contribute to proteolysis of reduced plasmin. (+info)
Macrophage metalloelastase, MMP-12, cleaves human apolipoprotein(a) in the linker region between kringles IV-4 and IV-5. Potential relevance to lipoprotein(a) biology.
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In this study we found that macrophage metalloelastase, MMP-12 cleaves, in vitro, apolipoprotein(a) (apo(a)) in the Asn3518-Val3519 bond located in the linker region between kringles IV-4 and IV-5, a bond immediately upstream of the Ile3520-Leu3521 bond, shown previously to be the site of action by neutrophil elastase (NE). We have also shown that human apo(a) injected into the tail vein of control mice undergoes degradation as reflected by the appearance of immunoreactive fragments in the plasma and in the urine of these animals. To define whether either or both of these enzymes may be responsible for the in vivo apo(a) cleavage, we injected intravenously MMP-12(-/-), NE -/- mice and litter mates, all of the same strain, with either lipoprotein(a) (Lp(a)), full-length free apo(a), or its N-terminal fragment, F1, obtained by the in vitro cleavage of apo(a) by NE. In the plasma of Lp(a)/apo(a)-injected mice, F1 was detected in control and NE -/- mice but was virtually absent in the MMP-12(-/-) mice. Moreover, fragments of the F1 type were present in the urine of the animals except for the MMP-12(-/-) mice. These fragments were significantly smaller in size than those observed in the plasma. All of the animals injected with F1 exhibited small sized fragments in their urine. These observations provide evidence that, in the mouse strain used, MMP-12 plays an important role in the generation of F1 from injected human Lp(a)/apo(a) and that this fragment undergoes further cleavage during renal transit via a mechanism that is neither NE- nor MMP-12-dependent. Thus, factors influencing the expression of MMP-12 may have a modulating action on the biology of Lp(a). (+info)
Optimization of apolipoprotein(a) genotyping with pulsed field gel electrophoresis.
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BACKGROUND: Increased lipoprotein(a) is a risk factor for atherosclerosis, and its concentration in serum is inversely correlated with the size of the apoliprotein(a) [apo(a)] component. The size of the apo(a) gene is determined mainly by the Kringle IV size polymorphism. We have optimized and characterized pulsed field gel electrophoresis (PFGE) for apo(a) genotyping. METHODS: Established PFGE protocols were adjusted. The changes included the following: (a) increased DNA yields by the use of all leukocytes for isolation from either 3 mL of fresh EDTA whole blood or 250 microL of frozen buffy coats; (b) increased efficiency of Kpn1 digestion by the inclusion of a digestion buffer wash; (c) reduction of assay time by the use of capillary blotting; (d) increased sensitivity by the use of four digoxigenin-labeled apo(a) probes; and (e) identification using a single film by the inclusion of a digoxigenin-labeled lambda marker probe in addition to apo(a) probes in the hybridization mix. RESULTS: In older Caucasians, 93% (buffy coats, n=468) were heterozygous for apo(a) gene size. An inverse correlation between serum lipoprotein(a) and the sum of Kringle IV alleles was found (y = -23x + 1553; r = -0.442; n = 468). Gel-to-gel variation was minimal (3%). Imprecision (SD) was one Kringle IV repeat (control sample containing eight fragments of 72-233 kb; n=34 electrophoretic runs). CONCLUSIONS: The practicality and sensitivity of the apo(a) genotyping technique by PFGE were improved, and accuracy and reproducibility were preserved. The optimized procedure is promising for apo(a) genotyping on frozen buffy coats from large epidemiological studies. (+info)
Enhancement through mutagenesis of the binding of the isolated kringle 2 domain of human plasminogen to omega-amino acid ligands and to an internal sequence of a Streptococcal surface protein.
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In the background of the recombinant K2 module of human plasminogen (K2(Pg)), a triple mutant, K2(Pg)[C4G/E56D/L72Y], was generated and expressed in Pichia pastoris cells in yields exceeding 100 mg/liter. The binding affinities of a series of lysine analogs, viz. 4-aminobutyric acid, 5-aminopentanoic acid, epsilon-aminocaproic acid, 7-aminoheptanoic acid, and t-4-aminomethylcyclohexane-1-carboxylic acid, to this mutant were measured and showed up to a 15-fold tighter interaction, as compared with wild-type K2(Pg) (K2(Pg)[C4G]). The variant, K2(Pg)[C4G/E56D], afforded up to a 4-fold increase in the binding affinity to these same ligands, whereas the K2(Pg)[C4G/L72Y] mutant decreased the same affinities up to 5-fold, as compared with K2(Pg)[C4G]. The thermal stability of K2(Pg)[C4G/E56D/L72Y] was increased by approximately 13 degrees C, as compared with K2(Pg)[C4G]. The functional consequence of up-regulating the lysine binding property of K2(Pg) was explored, as reflected by its ability to interact with an internal sequence of a plasminogen-binding protein (PAM) on the surface of group A streptococci. A 30-mer peptide of PAM, containing its K2(Pg)-specific binding region, was synthesized, and its binding to each mutant of K2(Pg) was assessed. Only a slight enhancement in peptide binding was observed for K2(Pg)[C4G/E56D], compared with K2(Pg)[C4G] (K(d) = 460 nM). A 5-fold decrease in binding affinity was observed for K2(Pg)[C4G/L72Y] (K(d) = 2200 nM). However, a 12-fold enhancement in binding to this peptide was observed for K2(Pg)[C4G/E56D/L72Y] (K(d) = 37 nM). Results of these PAM peptide binding studies parallel results of omega-amino acid binding to these K2(Pg) mutants, indicating that the high affinity PAM binding by plasminogen, mediated exclusively through K2(Pg), occurs through its lysine-binding site. This conclusion is supported by the 100-fold decrease in PAM peptide binding to K2(Pg)[C4G/E56D/L72Y] in the presence of 50 mM 6-aminohexanoic acid. Finally, a thermodynamic analysis of PAM peptide binding to each of these mutants reveals that the positions Asp(56) and Tyr(72) in the K2(Pg)[C4G/E56D/L72Y] mutant are synergistically coupled in terms of their contribution to the enhancement of PAM peptide binding. (+info)
Angiostatin binds to smooth muscle cells in the coronary artery and inhibits smooth muscle cell proliferation and migration In vitro.
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Angiostatin is an inhibitor of angiogenesis that is known to reduce endothelial cell proliferation and consequently prevent the progression of tumor metastases. However, the modest effect of angiostatin on endothelial cell proliferation raises the possibility that angiostatin might exert its effects on other cells. To determine the cellular distribution of angiostatin binding in tissues with neovasculature (atherosclerotic coronary arteries), we developed a fusion protein consisting of placental alkaline phosphatase and the first 3 kringles of plasminogen. Angiostatin binding colocalized with smooth muscle cells and could be inhibited by a 50-fold molar excess of plasminogen and 10 mmol/L epsilon-amino-n-caproic acid. The fusion protein also bound to smooth muscle cells in culture. Angiostatin inhibited hepatocyte growth factor-induced proliferation and migration of smooth muscle cells, suggesting that they are a target for the antiangiogenic effect of angiostatin. (+info)
Molecular basis of congenital lp(a) deficiency: a frequent apo(a) 'null' mutation in caucasians.
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High plasma concentrations of lipoprotein(a) [Lp(a)], a covalent low-density lipoprotein-apolipoprotein(a) [apo(a)] complex, are associated with coronary heart disease and stroke. Heritability of Lp(a) levels is high and the major locus determining Lp(a) concentrations is the apo(a) gene. We here demonstrate that a G-->A substitution at the +1 donor splice site of the apo(a) kringle (K) IV type 8 intron occurs with a high frequency ( approximately 6%) in Caucasians but not in Africans and is associated with congenital deficiency of Lp(a) in plasma. This mutation alone accounts for a quarter of all 'null' apo(a) alleles in Caucasians. RT-PCR analysis based on apo(a) illegitimate transcription in lympho- blastoid cells demonstrated that the donor splice site mutation results in an alternative splicing of the K IV type 8 intron and encodes a truncated form of apo(a). Expression of the alternatively spliced cDNA analogue in HepG2 cells showed that the truncated apo(a) form is secreted but is unable to form the covalent Lp(a) complex. Immunoprecipitated plasma apo(a) from homozygotes for the mutation was almost completely fragmented. Taken together, our data indicate that a failure in complex formation followed by fast degradation in plasma of the truncated free apo(a) is one mechanism which underlies the null Lp(a) type associated with the donor splice site mutation. (+info)
Effect of plasminogen activators on human recombinant apolipoprotein(a) having the plasminogen activation cleavage site.
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The serine-proteinase domain in human apolipoprotein(a) [apo(a)] and plasminogen exhibit 89% sequence identity including the catalytic triad. Cleavage of the Arg(561)-Val(562) activation site in plasminogen by either tissue- or urokinase-type plasminogen activator results in formation of the fibrinolytic enzyme plasmin. Apo(a) does not contain measurable amidolytic activity nor can it be activated by plasminogen activators. It has been suggested that the latter finding might be explained by the substitution of the plasminogen Arg-Val activation site by Ser-Ile in apo(a). To investigate if introduction of the Arg-Val activation site in apo(a) might result in sensitivity towards plasminogen activators, we expressed wild-type and Arg-Val mutant recombinant apo(a) [r-apo(a)] in human embryonic kidney and hepatocyte cell lines. Free r-apo(a) and lipoprotein-like particles [r-Lp(a)] were obtained in the culture supernatants of transfected 293 and HepG2 cells, respectively. Incubation of mutant r-apo(a)/r-Lp(a) with plasminogen activators produced neither plasmin-like activity nor cleavage at the Arg-Val activation site, even in the presence of various stimulators of plasminogen activation. Our data suggest that the high selectivity of activators for plasminogen activation requires interactions with regions in plasminogen distant from the activation disulfide loop which are not present in apo(a). (+info)
Inhibition of tumor growth correlates with the expression level of a human angiostatin transgene in transfected B16F10 melanoma cells.
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Although the therapeutic value of angiostatin, a proteolytic fragment of plasminogen, has been recognized for the treatment of cancer, the production of bioactive angiostatin remains a difficult task. Here we report that expression of a cDNA encoding a secreted, four-kringle human angiostatin inhibited tumor growth of B16F10 melanoma cells in mice but did not suppress tumor cell growth in culture. After transfection and selection, stable expression of the angiostatin cDNA was demonstrated in several B16F10 clones by quantitative mRNA analysis using the Taqman method. Cells that expressed angiostatin at either a low, medium, or high level were injected into C57BL/6 mice. s.c. Growth of B16F10 tumors was diminished by the angiostatin transgene, and the inhibition was directly proportional to the expression level of angiostatin in the transfected cells. However, suppression of s.c. tumor growth was transient, and eventually, tumors emerged with a strongly decreased expression of the transgene. Angiostatin expression also reduced lung metastasis from i.v.-injected B16F10 cells. Our data indicate that a cDNA encoding bioactive human angiostatin is potentially useful for gene therapy of human cancers, but the delivery of the transgene may require repeated dosing to achieve sustained dormancy of primary tumors and cancer metastases. (+info)