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.

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.

scholarly journals Targeted delivery of proteins across the blood–brain barrier

2007 ◽  
Vol 104 (18) ◽  
pp. 7594-7599 ◽  
Author(s):  
Brian J. Spencer ◽  
Inder M. Verma
Keyword(s):  
Blood Brain Barrier ◽  
Targeted Delivery ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Brain Barrier ◽  
Vector System ◽  
Blood Brain ◽  
Degenerative Disorders ◽  
Density Lipoprotein Receptor ◽  
General Method

Treatment of many neuronal degenerative disorders will require delivery of a therapeutic protein to neurons or glial cells across the whole CNS. The presence of the blood–brain barrier hampers the delivery of these proteins from the blood, thus necessitating a new method for delivery. Receptors on the blood–brain barrier bind ligands to facilitate their transport to the CNS; therefore, we hypothesized that by targeting these receptors, we may be able to deliver proteins to the CNS for therapy. Here, we report the use of the lentivirus vector system to deliver the lysosomal enzyme glucocerebrosidase and a secreted form of GFP to the neurons and astrocytes in the CNS. We fused the low-density lipoprotein receptor-binding domain of the apolipoprotein B to the targeted protein. This approach proved to be feasible for delivery of the protein and could possibly be used as a general method for delivery of therapeutic proteins to the CNS.

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.

scholarly journals Sclerostin inhibits Wnt signaling through tandem interaction with two LRP6 ectodomains

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jinuk Kim ◽  
Wonhee Han ◽  
Taeyong Park ◽  
Eun Jin Kim ◽  
Injin Bang ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Low Density Lipoprotein ◽  
Wnt Signaling Pathway ◽  
Density Lipoprotein ◽  
Interaction Sites ◽  
Wnt Proteins ◽  
C Terminus ◽  
Density Lipoprotein Receptor

Abstract Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the β-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2–SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling.

Sign in / Sign up

Export Citation Format

Share Document