scholarly journals Plasmin-dependent elimination of the growth-factor-like domain in urokinase causes its rapid cellular uptake and degradation

2001 ◽  
Vol 355 (3) ◽  
pp. 639-645 ◽  
Author(s):  
Alexei POLIAKOV ◽  
Vsevolod TKACHUK ◽  
Tatyana OVCHINNIKOVA ◽  
Natalia POTAPENKO ◽  
Svyatoslav BAGRYANTSEV ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Single Chain ◽  
Surface Binding ◽  
Receptor Associated Protein ◽  
Type Plasminogen Activator ◽  
Subsequent Degradation ◽  
Density Lipoprotein Receptor

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) act in concert to mediate pericellular proteolysis and to stimulate intracellular signalling responsible for cell migration and proliferation. uPA is composed of three domains, a proteolytic domain (PD), a kringle domain (KD) and a growth-factor-like domain (GFD), the last of which mediates the interaction with uPAR. We demonstrate that uPA, associated with the surface of U937 cells, undergoes plasmin-mediated cleavage of the Lys46-Ser47 bond with elimination of the GFD. Using recombinant forms of uPA, we show that a uPA variant lacking the GFD (r-uPA∆GFD) and unable to associate with uPAR is rapidly cleared from the cell surface. Binding and internalization of r-uPA∆GFD are markedly decreased in the presence of 39kDa receptor-associated protein (RAP), the antagonist of several endocytic receptors of the low-density lipoprotein receptor family, suggesting that this protein clearance pathway is used for r-uPA∆GFD. In contrast with rapidly internalized r-uPA∆GFD, the intact recombinant single-chain urokinase with wild-type structure (r-uPAwt) bound to uPAR is retained on the cell surface. Soluble uPAR protects uPA from cleavage by plasmin that results in the elimination of GFD, suggesting that uPAR might protect cell-bound urokinase from plasmin-mediated cleavage between the GFD and KD and subsequent degradation.

The Role of the Low-Density Lipoprotein Receptor-Related Protein (LRP) in the Plasma Clearance and Liver Uptake of Recombinant Single-chain Urokinase-type Plasminogen Activator in Rats

1997 ◽  
Vol 77 (04) ◽  
pp. 710-717 ◽  
Author(s):  
Marieke E van der Kaaden ◽  
Dingeman C Rijken ◽  
J Kar Kruijt ◽  
Theo J C van Berkel ◽  
Johan Kuiper
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Single Chain ◽  
Liver Uptake ◽  
Type Plasminogen Activator ◽  
Parenchymal Liver ◽  
Urokinase Type Plasminogen Activator ◽  
Density Lipoprotein Receptor

SummaryUrokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91 % and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= deltal25- rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-deltal25-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

Native and Non-glycosylated Recombinant Single-chain Urokinase-type Plasminogen Activator Are Recognized by Different Receptor Systems on Rat Parenchymal Liver Cells

1995 ◽  
Vol 74 (02) ◽  
pp. 722-729 ◽  
Author(s):  
Marieke E van der Kaaden ◽  
Dingeman C Rijken ◽  
Eleonore Groeneveld ◽  
Theo J C van Berkel ◽  
Johan Kuiper
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Liver Cells ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Parenchymal Liver ◽  
Urokinase Type Plasminogen Activator ◽  
Density Lipoprotein Receptor ◽  
Parenchymal Cells ◽  
Lipoprotein Receptor Related Protein

SummaryThe recognition systems mediating the clearance of glycosylated high molecular weight single-chain urokinase-type plasminogen activator (HMW-scu-PA, produced in human embryonic kidney cells) and recombinant non-glycosylated scu-PA (rscu-PA, produced in E. coli) were analyzed by studying their binding charactaristics to freshly isolated rat parenchymal liver cells.The binding of 125I-HMW-scu-PA at 4° C was calcium-dependent and of high affinity (Kd = 37.6 nM) and could be inhibited by low molecular weight two-chain u-PA (LMW-tcu-PA) and lactose, but not by the low density lipoprotein receptor-related protein (LRP)-associated 39-kDa protein (RAP), rscu-PA or a mutant form lacking amino acids 11-135 (Delta 125-rscu-PA). Removal of the carbohydrate side chain of HMW-scu-PA by treatment with N-glycosidase F, completely reduced the specific binding to the parenchymal cells and strongly reduced its competition with 125I-HMW-scu-PA in cell binding.Recombinant scu-PA also bound with high affinity (Kd= 38.7 nM) to the parenchymal liver cells. The binding of 125I-rscu-PA could be competed for by unlabeled rscu-PA while Delta 125-rscu-PA, LMW-tcu-PA or lactose were ineffective. In contrast to HMW-scu-PA, binding of 125I-rscu-PA could be effectively inhibited by RAP (Ki = 1.1 nM), while also its association and degradation, as determined at 37° C, were inhibited by RAP. Pretreatment of the parenchymal cells with proteinase K supplied further evidence for the involvement of two different receptor systems. The binding of rscu-PA was decreased for 91%, while that of HMW-scu-PA showed a decrease of 51%.It is suggested that native HMW-scu-PA is bound and degraded by the rat parenchymal liver cells via a lectin-like recognition site, while non-glycosylated recombinant scu-PA is bound and degraded by rat parenchymal liver cells via the low density lipoprotein receptor-related protein (LRP). The differences in recognition system for native and recombinant proteins by liver cells suggest that the glycosylation of recombinant proteins, as obtained in mammalian expression systems, can be important for their physiological fate and their pharmacological application.

scholarly journals Improved scFv Anti-LOX-1 Binding Activity by Fusion with LOX-1-Binding Peptides

2017 ◽  
Vol 2017 ◽  
pp. 1-9
Author(s):  
Wei Hu ◽  
Qiuhong Xie ◽  
Hongyu Xiang
Keyword(s):  
Targeted Delivery ◽  
Structural Characteristics ◽  
Low Density Lipoprotein ◽  
Three Dimensional ◽  
Density Lipoprotein ◽  
Binding Activity ◽  
Single Chain ◽  
C Terminus ◽  
Density Lipoprotein Receptor ◽  
Therapeutic Molecules

The oxidized low-density lipoprotein receptor-1 (LOX-1) targeted single-chain variable fragment (scFvs) is a promising molecule for the targeted delivery of imaging and therapeutic molecules of atherosclerotic diseases; however, its applications are limited by the inherent low antigen affinity. In this study, the three-dimensional (3D) model of the anti-LOX-1 scFv was constructed and its docking with the LOX-1 protein was developed. To improve the LOX-1-binding activity, the anti-LOX-1 scFv was designed to fuse with one of three LOX-1-binding heptapeptides, LTPATAI, FQTPPQL, and LSIPPKA, at its N-terminus and C-terminus and in the linker region, which have different LOX-1-binding interfaces with the anti-LOX-1 scFv analyzed by an array of computational approaches. These scFv/peptide fusions were constructed, successfully expressed in Brevibacillus choshinensis hosts, and purified by a two-step column purification process. The antigen binding activity, structural characteristics, thermal stability, and stability in serum of these fusion proteins were examined. Results showed that the scFv with N-terminal fusing peptides proteins demonstrated increased LOX-1-binding activity without decrease in stability. These findings will help increase the application efficacy of LOX-1 targeting scFv in LOX-1-based therapy.

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