Expression of the non-glycosylated kringle domain of tissue type plasminogen activator in Pichia and its anti-endothelial cell activity

Lee, Sang Bae; Oh, Hyeyoung; Kim, Hyun-Kyung; Joe, Yeonsoo

doi:10.1016/j.pep.2006.06.002

Cited by 3 publications

(4 citation statements)

References 29 publications

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“…The insert DNA was cut with SacI and introduced into Pichia pastoris GS115 (pPICZα‐C/PK5) or X‐33 (pPICZα‐C/endostatin) by electroporation. After the selection of PK5 or endostatin‐expressing clones, expression in a large‐scale culture and purification were performed by a similar method to that reported previously for the recombinant kringles 1–2 of tissue‐type plasminogen activator (TK1–2) protein [14].…”

Section: Methodsmentioning

confidence: 99%

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Outgrowing endothelial progenitor‐derived cells display high sensitivity to angiogenesis modulators and delayed senescence

Kim

et al. 2007

FEBS Letters

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Outgrowing endothelial progenitor-derived cells (EPDCs) originate from a novel hierarchy of endothelial progenitor cells. In this study, EPDCs isolated from human cord blood were examined for phenotype and functional features upon aging. Young or aged EPDCs were similar to human umbilical vein endothelial cells (HUVECs), in exhibiting typical endothelial phenotypes. However, EPDCs were more sensitive to angiogenesis inducers or inhibitors in proliferation and migration. In addition, EPDCs underwent senescence markedly slowly with sustained endothelial NO synthase expression and activation, and their ability to undergo capillary morphogenesis was retained throughout longterm culture. Thus, these results suggest that a homogenous population of EPDCs derived from clonogenic expansion may provide an effective vasculogenesis tool.

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Section: Methodsmentioning

confidence: 99%

“…The migration of EPDCs and HUVECs was evaluated in a modified Boyden chamber assay as described previously [14].…”

Section: Methodsmentioning

confidence: 99%

Outgrowing endothelial progenitor‐derived cells display high sensitivity to angiogenesis modulators and delayed senescence

Kim

et al. 2007

FEBS Letters

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“…Work by Collen et al [31] have shown the increased catalytic efficiency for plasminogen activation by site directed mutagenesis of tPA in the kringle domains and recently a paper by Lee et al [32] showed antiendothelial property of just the kringle 1 and 2 domains in Pichia pastoris with no glycosylation are research work carried out on similar lines. This fibrin affinity is believed to be due to interactions of the finger and kringle 2 domain with fibrin.…”

Section: Discussionmentioning

confidence: 99%

and : Critical Residues for In Vitro Biological Activity of Reteplase

Mandi

Sundaram

Tandra

et al. 2010

Advances in Hematology

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Reteplase (rPA) is a thrombolytic agent used for the treatment of acute myocardial infarction. We studied the expression of rPA and its selected asparagine mutants after integration into the Pichia genome. Though methanol induction of the native and the rPA mutants showed similar expression levels (~200–250 mg/L), the mutants displayed significant loss of protease activity. Strikingly, the clot lysis activities of these mutants were considerably different. While mutation of Asn12 (N12P) of the Kringle 2 domain showed delayed clot lysis activity (t 1/2 = 38 min) compared to the native rPA (t 1/2 = 33 min), a faster rate of clot lysis (t 1/2 = 27 min) was observed when the Asn278 (N278S) of the serine protease domain was mutated. Interestingly, the slowest clot lysis activity (t 1/2 = 49 min) demonstrated by the double mutant (N12P, N278S) suggests the dominant role of Asn12 in regulating the fibrinolytic activity of rPA. The results presented in this paper indicate that the fibrinolytic and the proteolytic activities of rPA are independent of each other.

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“…Annexin A2/S100A10 also binds plasminogen at the site of the cytoplasmic membrane and is a source of plasminogen activation on the cell surface [117]. Binding to endothelial cells by t-PA’s kringle domains has been shown to inhibit neoangiogenesis [[118], [119], [120]]. The binding of t-PA to the epidermal growth factor receptor protects neurons in ischemic conditions [60].…”

Section: Tissue Plasminogen Activatormentioning

confidence: 99%

Structural Biology and Protein Engineering of Thrombolytics

Mičan

Toul

Bednář

et al. 2019

Computational and Structural Biotechnology Journal

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Myocardial infarction and ischemic stroke are the most frequent causes of death or disability worldwide. Due to their ability to dissolve blood clots, the thrombolytics are frequently used for their treatment. Improving the effectiveness of thrombolytics for clinical uses is of great interest. The knowledge of the multiple roles of the endogenous thrombolytics and the fibrinolytic system grows continuously. The effects of thrombolytics on the alteration of the nervous system and the regulation of the cell migration offer promising novel uses for treating neurodegenerative disorders or targeting cancer metastasis. However, secondary activities of thrombolytics may lead to life-threatening side-effects such as intracranial bleeding and neurotoxicity. Here we provide a structural biology perspective on various thrombolytic enzymes and their key properties: (i) effectiveness of clot lysis, (ii) affinity and specificity towards fibrin, (iii) biological half-life, (iv) mechanisms of activation/inhibition, and (v) risks of side effects. This information needs to be carefully considered while establishing protein engineering strategies aiming at the development of novel thrombolytics. Current trends and perspectives are discussed, including the screening for novel enzymes and small molecules, the enhancement of fibrin specificity by protein engineering, the suppression of interactions with native receptors, liposomal encapsulation and targeted release, the application of adjuvants, and the development of improved production systems.

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Expression of the non-glycosylated kringle domain of tissue type plasminogen activator in Pichia and its anti-endothelial cell activity

Cited by 3 publications

References 29 publications

Outgrowing endothelial progenitor‐derived cells display high sensitivity to angiogenesis modulators and delayed senescence

Outgrowing endothelial progenitor‐derived cells display high sensitivity to angiogenesis modulators and delayed senescence

and : Critical Residues for In Vitro Biological Activity of Reteplase

Structural Biology and Protein Engineering of Thrombolytics

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