• BMB Reports
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• v.42 no.6
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• pp.380-386
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• 2009

In neurodegenerative diseases, such as Alzheimer' and Parkinson', microglial cell activation is thought to contribute to CNS injury by producing neurotoxic compounds. Prothrombin and kringle-2 increase levels of NO and the mRNA expression of iNOS, IL-1$\beta$, and TNF-$\alpha$ in microglial cells. In contrast, the human prothrombin kringle-2 derived peptide NSA9 inhibits NO release and the production of pro-inflammatory cytokines such as IL-1$\beta$, TNF-$\alpha$, and IL-6 in LPS-activated EOC2 microglia. In this study, we investigated the anti-inflammatory effects of NSA9 in human prothrombin- and kringle-2-stimulated EOC2 microglia. Treatment with 20-100 ${\mu}M$ of NSA9 attenuated both prothrombin- and kringle-2-induced microglial activation. NO production induced by MAPKs and NF-$\kappa$B was similarly reduced by inhibitors of ERK (PD98059), p38 (SB203580), NF-$\kappa$B (N-acetylcysteine), and NSA9. These results suggest that NSA9 acts independently as an inhibitor of microglial activation and that its effects in EOC2 microglia are not influenced by the presence of kringle-2.

• Biomolecules & Therapeutics
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• v.11 no.4
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• pp.205-213
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• 2003

Various angiogenesis inhibitors target vascular endothelial cells and block tumor angiogenesis. Angiostatin is a specific endogenous angiogenesis inhibitor in clinical trials, which contains only the first four triple loop structures, known as kringle domains. Its generated by proteolytic cleavage of its parent molecule plasminogen, which itself does not exhibit antiangiogenic activity. Kringle domains from prothrombin, apolipoprotein, hepatocyte growth factor, urokinase and tissue-type plasminogen activator also elicit anti-angiogenic or antitumor activities in several model systems, albeit low amino acid sequence identity between angiostatin and each individual kringle. However, the differential effects of each kringle domain on endothelial cell proliferation, and migration observed in these kringle domains, suggest that the amino acid sequence of the primary structure is still important although kringle architecture is essential for anti-mlgiogenic activity. If it is further studied as to how amino acid sequence and kringle architecture contributes in anti-angiogenic activity, with studies on underlying mechanisms of anti-angiogenesis by kringle-based angiogenesis inhibitors, it will provide basis for the development of new potent anti-angiogenesis inhibitors and improvement of the efficacy of angiogenesis inhibitors.

• BMB Reports
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• v.32 no.2
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• pp.147-153
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• 1999

Previously, we reported that the prothrombin kringle 2 (fragment 2), induced by LPS administration into rabbit, inhibited bFGF-stimulated BCE cell growth (Lee et al., 1998). In this study, we cloned and overexpressed the kringle 2 domain of rabbit and human prothrombin as a fusion protein with the pelB leader sequence in E. coli using the T7 promoter. The fusion protein was cleaved during translocation into the peri plasmic space, and cleaved recombinant protein was readily isolated from whole cell lysate by DEAE-Sepharose and Sephacryl S-200 gel filtration chromatography. Both the recombinant rabbit and human prothrombin kringle 2 showed very similar biochemical and functional characteristics to the rabbit prothrombin kringle 2 purified from rabbit serum, in terms of abnormal electrophoretic migration and endothelial cell growth inhibitory activity.

• Journal of Life Science
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• v.16 no.7 s.80
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• pp.1199-1206
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• 2006

Plasminogen kringle 5 is a potent inhibitor of endothelial tell proliferation like an endogenous angiogenesis inhibitor, angiostatin consisting of plasminogen kringles 1-4. In this study, we produced the recombinant protein of plasminogen kringle 5 (PK5) employing an Pichia expression system and examined its. effect on~endothelial cell migration and its possible inhibitory mechanism. PK5 was expressed in Pichia pastoris GS115 by fusion of the cDNA spanning from Thr456 to Phe546 to the secretion signal sequence of a-factor prepro-peptide. After methanol induction, the secreted PK5 was purified by using S-spin column. SDS-PACE analysis of the purified protein showed one major band of approximately 10kDa. In in vitro migration assays, the purified protein inhibited dose-dependently the migration of human umbilical endothelial cells (HUVECs) induced by basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) with an $IC_{50}$ of approximately 500nM. Accordingly, it inhibited bfGF-stimulated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in HUVECs at 500nM. In addition, it also potently inhibited bFGF-induced cytoskeletal rearrangement of HUVECs. Thus, these results suggest that Pichia-produced PK5 effectively inhibits endothelial cell migration, in part by suppression of ERK1/2 activation and blocking cytoskeleton rearrangement.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.591-597
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• 2003

Human urokinase kringle domain, sharing homology with angiostatin kringles, has been shown to be an inhibitor of angiogenesis, which can be used for the treatment of cancer, rheumatoid arthritis, psoriasis, and retinopathy. Here, the expression of the kringle domain of urokinase (UK1) as a secreted protein in high levels is reported. UK1 was expressed in the methylotrophic yeast Pichia pastoris GS115 by fusion of the cDNA spanning from Ser47 to Lys135 to the secretion signal sequence of ${\alpha}-factor$ prepro-peptide. In a flask culture, the secreted UK1 reached about 1 g/l level after 120h of methanol induction and was purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the purified UK1 revealed that it was cleaved at the Ste13 signal cleavage site. The molecular mass of UK1 was determined to be 10,297.01 Da. It was also confirmed that the purified UK1 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, or epidermal growth factor, in a dose-dependent manner. These results suggest that a P. pastoris sytem can be employed to obtain large amounts of soluble and active UK1.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.308-311
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• 2006

In an attempt to increase production of LK8, an 86-amino-acid kringle fragment of human apolipoprotein(a) with three disulfide linkages, protein disulfide isomerase (PDI) was coexpressed in recombinant Saccharomyces cerevisiae harboring the LK8 gene in the chromosome. Whereas overexpression of the LK8 gene without coexpressing PDI was detrimental to both host cell growth and LK8 production, coexpression of PDI increased the LK8 production level by 2.5-fold in batch cultivation and 5.0-fold in fed-batch cultivation compared with the control strain carrying only the genomic PDI gene.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.6
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• pp.523-527
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• 2004

Recombinant Saccharomyces cerevisiae expression systems were developed to pro­duce a novel human anti-angiogenic protein called LK8, an 86 amino-acid kringle fragment pro­tein with three disulfide linkages. Galactose-inducible LK8 expression plasmid was constructed, and LK8 production levels by four S. cerevisiae strains were compared in order to select an op­timal host strain. S. cerevisiae 2805 was the most efficient among the strains tested. Elevating the LK8 gene copy number through multiple integration using 8-sequences as target sites re­sulted in more than a two-fold increase in the LK8 production level compared with the plasmid­based expression system. The maximum LK8 protein concentration of 25 mg/L was obtained from batch cultivation of the yeast transformant that harbors 16 copies of the LK8 gene. In con­clusion, the strain integrated with the multiple LK8 gene secreted the protein with relatively high yield, although, the increased LK8 gene dosage over 11 copies did not lead to further en­hancement in batch cultivations.

• Bulletin of the Korean Chemical Society
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• v.25 no.12
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• pp.1863-1868
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• 2004

Kringle 5 (K5), located outside of angiostain (K1-4) in human plasminogen, displays more potent antiangiogenic activity on endothelial cell proliferation than angiostatin itself. Using a yeast two-hybrid system in vivo, we have recently identified Rgl2 (guanine nucleotide dissociation stimulator (RalGDS)-like factor 2) as a binding protein of human K5. In order to confirm in vitro protein interaction between K5 and Rgl2, we developed bacterial recombinant expression systems for them. K5 and Rgl2 proteins were expressed in high yields and purified into pure forms with His tags and GST fusion, respectively. GST-pull down experiments clearly demonstrated that K5 interacts specifically with Rgl2 in vitro. These results indicate that Rgl2 functions as a receptor protein for K5 in vitro as well as in vivo, leading to anti-angiogenesis through regulating Ras signaling pathways.

• Proceedings of the PSK Conference
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• 2001.10a
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• pp.94-95
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• 2001

• Proceedings of the Microbiological Society of Korea Conference
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• 2006.05a
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• pp.111-112
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• 2006