ABSTRACT
The potent angiogenesis inhibitor known as human plasminogen Kringle 5 has shown promise in the treatment of vascular disorders and malignancies. The study aimed to investigate the recognition and interaction between Kringle 5 and the A2M domain of human complement component C5 using bio-specific methodologies and molecular dynamics (MD) simulation. Initially, the specific interaction between Kringle 5 and A2M was confirmed and characterized through Ligand Blot and ELISA, yielding the dissociation constant (Kd) of 1.70 × 10-7 mol/L. Then, Kringle 5 showcased a dose-dependent inhibition of the production of C5a in lung cancer A549 cells, consequently impeding their proliferation and migration. Following the utilization of frontal affinity chromatography (FAC), it was revealed that there exists a singular binding site with the binding constant (Ka) of 3.79 × 105 L/mol. Following the implementation of homology modeling and MD optimization, the detailed results indicate that only a specific segment of the N-terminal structure of the A2M molecule engages in interaction with Kringle 5 throughout the binding process and the principal driving forces encompass electrostatic force, hydrogen bonding, and van der Waals force. In conclusion, the A2M domain of human complement C5 emerges as a plausible binding target for Kringle 5 in vivo.
Subject(s)
Molecular Dynamics Simulation , Plasminogen , Protein Binding , Humans , Plasminogen/chemistry , Plasminogen/metabolism , Binding Sites , Complement C5a/chemistry , Complement C5a/metabolism , A549 Cells , Protein Domains , Cell Proliferation/drug effects , Cell Movement/drug effects , Peptide FragmentsABSTRACT
The binding and molecular recognition between α-chain of human complement C3b (α-chain of C3b) and human plasminogen Kringle 5 (Kringle 5) were studied and explored by frontal chromatography and dynamics simulation in the combination of bio-specific technologies. The specific interaction between the α-chain of C3b and Kringle 5 was initially confirmed by ligand blot and ELISA (Kd = 4.243×10-6 L/mol). Furthermore, the binding determination conducted via frontal chromatography showed that the presence of a single binding site between them, with the binding constant of 2.98 × 105 L/mol. Then the molecular recognition by dynamics simulation and molecular docking showed that there were 9 and 13 amino acid residues respective in the Kringle 5 and α-chain of C3b directly implicated in the binding and the main stabilizing forces were electrostatic force (-55.99 ± 11.82 kcal/mol) and Van der Waals forces (-42.70 ± 3.45 kcal/mol). Additionally, a loop structure (65-71) in Kringle 5 underwent a conformational change from a random structure to an α-helix and a loop structure (417-425) in α-chain of C3b was closer to the molecular center, both of them were more conducive to the binding between them. Meanwhile, the involvement of the lysine binding site of Kringle 5 played an important role in the binding process. In addition, the erythrocyte-antibody complement rosette assay substantiated that the presence of Kringle 5 hindered the transportation of α-chain of C3b to antigen-antibody complex in a dose-dependent manner. These findings collectively indicated that the α-chain of C3b is very likely a receptor protein for Kringle 5, which provides a methodology for other similar investigations and valuable insights into expansion of the pharmacological effects and potential application of Kringle 5 in immune-related diseases.
Subject(s)
Chromatography , Peptide Fragments , Plasminogen , Humans , Protein Binding , Amino Acid Sequence , Molecular Docking Simulation , Binding Sites , Peptide Fragments/metabolism , Protein ConformationABSTRACT
Objective:To investigate the effects and mechanism of exosome (EXO)-loaded kringle V11 (KV11) delivery on corneal neovascularization (CNV).Methods:KV11 was bound to the surface of endothelial cell-derived exosomes by using CP05, an EXO-targeting anchoring peptide, to produce EXO-KV11.The binding efficiency and optimal concentration ratio were determined using the Apogee flow system.A total of 100 8-week-old healthy male SPF grade SD rats were selected, 10 of which were randomly selected as a normal control group without any treatment.The CNV model was established by alkali burn in the other 90 rats, which were randomly divided into three groups, EXO-KV11 group, KV11 group, and normal saline group by the random number table method, with 30 rats in each group.Each group was injected subconjunctivally with 100 μl of EXO-KV11 (25 μg), KV11 (25 μg), or normal saline every other day from the first day after the alkali burn, respectively.The CNV of rats was observed on days 1, 4, 7, and 14 after alkali burn.The CNV area was calculated by ventricular perfusion with fluorescein isothiocyanate-dextran (FITC-dextran) and corneal angiography.The amount of CNV lumen was observed by hematoxylin and eosin staining.The distribution of CD31 in rat corneas was determined by immunohistochemical method.The expression levels of voltage-dependent anion channel 1(VDAC1), endoplasmic reticulum stress, autophagy and apoptosis-associated proteins were detected by Western blot.This study was approved by the Animal Ethics Committee of Peking University People's Hospital (No.20210019). All animal procedures complied with the regulations of the Vision and Ophthalmology Association and the Animal Protection and Use Committee of Peking University.Results:The optimal concentration ratio of KV11 to EXO was 4∶1 and the binding affinity reached up to 87.5% by Apogee flow cytometers.On days 7 and 14 after alkali burn, there were significant differences in CNV area among the four groups ( F=4.613, 15.590; both at P<0.05). On day 7 after alkali burn, the CNV area was smaller in EXO-KV11 group than in KV11 and normal saline groups, with statistically significant differences (both at P<0.05). On day 14 after alkali burn, the CNV area was smaller in EXO-KV11 and KV11 groups than in normal saline group, and smaller in EXO-KV11 group than in KV11 group, showing statistically significant differences (all at P<0.05). The results of quantitative analysis of corneal fluorescence mounts showed that the relative CNV fluorescence area of the normal saline group, KV11 group and EXO-KV11 group were (8.3±1.7)%, (5.2±1.6)%and (3.4±0.7)%, respectively, showing a statistically significant overall comparison difference ( F=11.735, P<0.01). The relative CNV fluorescence area was larger in KV11 and normal saline groups than in EXO-KV11 group, and larger in normal saline group than in KV11 group, showing statistically significant differences (all at P<0.05). On day 14 after alkali burn, massive neovascular lumens were observed in the matrix of the normal saline group.The number of neovascular lumens in KV11 group was smaller than that in normal saline group.The corneal structure appeared normal in EXO-KV11 group, and neovascular lumens were rare.Numerous CD31-positive cells were observed in the corneal stroma of the normal saline group, which formed into lumen structures.The number of lumens surrounded by CD31-positive cells in the corneal stroma was smaller in KV11 group than in normal saline group, and smaller in EXO-KV11 group than in KV11 group.There were significant differences in the relative expression levels of VDAC1, protein kinase R-like endoplasmic reticulum kinase (PERK), p62, cleaved caspase 3 among the four groups ( F=35.960, 8.947, 17.791, 101.168; all at P<0.01). The relative expression levels of VDAC1, PERK, p62, cleaved caspase 3 were higher in EXO-KV11 group than in KV11 and normal saline groups, showing statistically significant differences (all at P<0.001). There was no significant difference in the relative expression of microtubule-associated proteins 1A/1B light chain 3B (LC3B)Ⅱ/LC3BⅠ protein among all four groups ( F=0.445, P=0.727). Conclusions:EXO-KV11 can inhibit CNV more remarkably than KV11.EXO-KV11 inhibits CNV by promoting the expression of VDAC1 and PERK and suppressing the autophagic flux.
ABSTRACT
BACKGROUND: Cells can die through a process called apoptosis in both pathological and healthy conditions. Cancer development and progression may result from abnormal apoptosis. The 78-kDa glucose-regulated protein (GRP78) is increased on the surface of cancer cells. Kringle 5, a cell apoptosis agent, is bound to GRP78 to induce cancer cell apoptosis. Kringle 5 was docked to GRP78 using ClusPro 2.0. The interaction between Kringle 5 and GRP78 was investigated. RESULTS: The interacting amino acids were found to be localized in three areas of Kringle 5. The proposed peptide is made up of secondary structure amino acids that contain Kringle 5 interaction residues. The 3D structure of the peptide model amino acids was created using the PEP-FOLD3 web tool. CONCLUSIONS: The proposed peptide completely binds to the GRP78 binding site on the Kringle 5, signaling that it might be effective in the apoptosis of cancer cells.
Subject(s)
Endoplasmic Reticulum Chaperone BiP , Neoplasms , Heat-Shock Proteins/metabolism , Kringles , Peptides/pharmacology , Apoptosis , Amino AcidsABSTRACT
Plasminogen Kringle 5 is one of the most potent cytokines identified to inhibit the proliferation and migration of vascular endothelial cells. Herein, six aptamer candidates that specifically bind to Kringle 5 were generated by the systematic evolution of ligands by exponential enrichment (SELEX). After 10 rounds of screening against Kringle 5, a highly enriched ssDNA pool was sequenced and the representative aptamers were subjected to binding assays to evaluate their affinity and specificity. The preferred aptamer KG-4, which demonstrated a low dissociation constant (Kd) of â¼ 432 nM and excellent selectivity for Kringle 5. A conserved "motif" of eight bases located at the stem-loop intersection, common to the aptamer, was further confirmed as the recognition element for binding with Kringle 5. The bulge formed by the motif and depression on the lysine binding site of Kringle 5 were both located at the binding interface, and the "induced fit" between their structures played a central role in the recognition process. Kringle 5 interacts KG-4 primarily through enthalpy-driven van der Waals forces and hydrogen bond. The key nucleotides A34 and C35 at motif on KG-4 and the positively charged amino acids in the loop 1 and loop 4 regions on Kringle 5 play a major role in the interaction. Furthermore, KG-4 dose-dependently reduced the proliferation inhibition of vascular endothelial cells by Kringle 5 and had a blocking effect on the function of Kringle 5 in inhibiting migration and promoting apoptosis of vascular endothelial cells in vitro. This study put a new light on protein-aptamer binding mechanism and may provide insight into the treatment of ischemic diseases by target depletion of Kringle 5.
Subject(s)
Aptamers, Nucleotide , Endothelial Cells , Humans , Endothelial Cells/metabolism , Plasminogen/chemistry , Plasminogen/metabolism , Binding Sites , Aptamers, Nucleotide/chemistryABSTRACT
Given the significant clinical potential of human plasminogen Kringle 5 on tumours, it is crucial to seek its receptors for a thorough comprehension of its physiological functions and mechanism. Eleven candidates have been screened out in our previous works. In the present work, we further inquired whether the candidate, von Willebrand factor type A domain 1 in coagulation factor C homology protein (abbr. vWA1), was a potential receptor of Kringle 5, and investigated their binding mechanism by bio-specific experiments, frontal affinity analysis (FA), and molecular dynamic simulation (MDS). After the potential was validated by bio-specific experiments, the FA results stated that vWA1 exhibited a strong interaction towards Kringle 5 in the proportion of 1:1 with the binding constant of 4.18 × 104 L/mol. The MDS results showed that the binding was mainly driven by electrostatic and Van der Waals forces and occurred spontaneously, during which vWA1 and Kringle 5 mutually fit each other by conformational changing into more flexible and suitable structures including fluctuations for five loops and partial transformation into a random coil for α6-helix in vWA1. Moreover, lysine binding site Leu71-Tyr74 was speculated responsible for Kringle 5 in binding and Tyr72 to be the key amino acid residue. In short, this work not only confirmed vWA1 as a potential Kringle 5 receptor but also provided valuable information on the detailed binding, facilitating the application development of Kringle 5 in regulating immune or inhibiting tumour migration through vWA1.
Subject(s)
Extracellular Matrix Proteins , Molecular Dynamics Simulation , Amino Acid Sequence , Binding Sites , Humans , Ligands , Magnetic Resonance Spectroscopy , Peptide Fragments , Plasminogen , Protein Binding , Protein ConformationABSTRACT
Voltage-dependent anion channel-l (VDAC-1) can bind with plasminogen Kringle 5 as the cell surface receptor and induce cell apoptosis, but the detailed information of binding is not clear yet. Thus, the mutual recognition and binding were investigated here utilizing frontal affinity chromatography, surface plasma resonance, mutation analysis combining molecular dynamics simulation. The results showed that Kringle 5 binds with VDAC-1 in equimolar driven mainly by electrostatic force, with 15 amino acid residues participating in Kringle 5 and 21 in VDAC-1. The observed conformational changes indicated the automatic structure regulation providing these two proteins suitable conformations and spatial surroundings for the tighter and stabler binding. Moreover, Glu29 in Kringle 5 was speculated as the key residue maintaining the largest energy contribution. Therefore, this work provided precise information for the recognition and binding of Kringle 5 with VDAC-1 that is valuable for the corresponding treatment of tumours or other angiogenic diseases.
Subject(s)
Molecular Dynamics Simulation , Voltage-Dependent Anion Channel 1 , Voltage-Dependent Anion Channels , Binding Sites , Humans , Peptide Fragments , Plasminogen , Protein Binding , Voltage-Dependent Anion Channel 1/metabolism , Voltage-Dependent Anion Channels/metabolismABSTRACT
Human plasminogen Kringle 5 is known to pose a more potent anti-angiogenesis effect by inducing endothelial cell apoptosis. Our previous studies have identified the peptide IGNSNTL as a binding sequence of Kringle 5 using Ph.D.-7 phage display peptide library and enzyme-linked immunosorbent assay. Here, eleven proteins were screened and summarized by BLAST, laminin α3 chain G1 domain (LG1) was considered as the most potential receptor based on E value and domain function. The specific interaction of them was directly revealed through ligand blot and a strong concentration-dependent manner occurred between them (Ka 4.30 × 105 L mol-1) in frontal chromatography observation. Moreover, R10A/P83R substitution Kringle 5 decreased the affinity capacity to LG1. Furthermore, a remarkable conformational change from random coil3 to α helix and α1 helix to random coil were observed to the structural compactness and stability for LG1. Surface loops and coils also showed fluctuations up to some extent, giving the binding surface greater flexibility and correspondingly allowing for induced-fit binding, which was -23.87 kcal mol-1 of the free energy with electrostatic force as a main driver. Taken together, not only effective theoretical prediction and experiment validated that LG1 is receptor of Kringle 5, but also give an new perspective of the binding mechanism of Kringle 5 and its specific receptor and could facilitate the development of novel agent targeted toward pathologic angiogenesis.
Subject(s)
Endothelial Cells/metabolism , Laminin/chemistry , Molecular Dynamics Simulation , Peptide Fragments/metabolism , Plasminogen/metabolism , Amino Acid Sequence , Binding Sites , Humans , Kinetics , Ligands , Mutant Proteins/chemistry , Peptide Library , Protein Binding , Protein Domains , ThermodynamicsABSTRACT
The previous pharmacokinetic methods can be only limited to drug analysis in vitro, which provide less information on the distribution and metabolismof drugs, and limit the interpretation and assessment of pharmacokinetics, the determination of metabolic principles, and evaluation of treatment effect. The objective of the study was to investigate the pharmacokinetic characteristics of gene recombination angiogenesis inhibitor Kringle 5 in vivo. The SPECT/CT and specific 131I-Kringle 5 marked by Iodogen method were both applied to explore the pharmacokinetic characteristics of 131I-Kringle 5 in vivo, and to investigate the dynamic distributions of 131I-Kringle 5 in target organs. Labeling recombinant angiogenesis inhibitor Kringle 5 using 131I with longer half-life and imaging in vivo using SPECT instead of PET, could overcome the limitations of previous methods. When the doses of 131I-Kringle 5 were 10.0, 7.5 and 5.0 g/kg, respectively, the two-compartment open models can be determined within all the metabolic process in vivo. There were no significant differences in t1/2α, t1/2ß, apparent volume of distribution and CL between those three levels. The ratio of AUC(0~∞) among three different groups of 10.0, 7.5 and 5.0 g/kg was 2.56:1.44:1.0, which was close to the ratio (2:1.5:1.0). It could be clear that in the range of 5.0-10.0 g/kg, Kringle 5 was characterized by the first-order pharmacokinetics. Approximately 30 min after 131I-Kringle 5 was injected, 131I-Kringle 5 could be observed to concentrate in the heart, kidneys, liver and other organs by means of planar imaging and tomography. After 1 h of being injected, more radionuclide retained in the bladder, but not in intestinal. It could be concluded that 131I-Kringle 5 is mainly excreted through the kidneys. About 2 h after the injection of 131I-Kringle 5, the radionuclide in the heart, kidneys, liver and other organs was gradually reduced, while more radionuclide was concentrated in the bladder. The radionuclide was completely metabolized within 24 h, and the distribution of radioactivity in rats was similar to normal levels. In our study, the specific marker 131I-Kringle 5 and SPECT/CT were successfully used to explore pharmacokinetic characteristics of Kringle 5 in rats. The study could provide a new evaluation platform of the specific, in vivo and real-time functional imaging and pharmacokinetics for the clinical application of 131I-Kringle 5.
ABSTRACT
The interactions between angiogenesis inhibitor Kringle 5 and its five specific ligands were investigated by frontal affinity chromatography in combination with fluorescence spectra and site-directed molecular docking. The binding constants of trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), epsilon-aminocaproic acid (EACA), benzylamine, 7-aminoheptanoic acid (7-AHA) and L-lysine to Kringle 5 were 19.0×10(3), 7.97×10(3), 6.45×10(3), 6.07×10(3) and 4.04×10(3) L/mol, respectively. The five ligands bound to Kringle 5 on the lysine binding site in equimolar amounts, which was pushed mainly by hydrogen bond and Van der Waals force. This binding affinity was believed to be dependent on the functional group and flexible feature in ligands. This study will provide an important insight into the binding mechanism of angiogenesis inhibitor Kringle 5 to its specific ligands.
Subject(s)
Chromatography, Affinity , Kringles/physiology , Angiogenesis Inhibitors/metabolism , Binding Sites , Hydrogen Bonding , Ligands , Lysine/chemistry , Protein BindingABSTRACT
The previous pharmacokinetic methods can be only limited to drug analysis in vitro, which provide less information on the distribution and metabolismof drugs, and limit the interpretation and assessment of pharmacokinetics, the determination of metabolic principles, and evaluation of treatment effect. The objective of the study was to investigate the pharmacokinetic characteristics of gene recombination angiogenesis inhibitor Kringle 5 in vivo. The SPECT/CT and specific 131I-Kringle 5 marked by Iodogen method were both applied to explore the pharmacokinetic characteristics of 131I-Kringle 5 in vivo, and to investigate the dynamic distributions of 131I-Kringle 5 in target organs. Labeling recombinant angio-genesis inhibitor Kringle 5 using 131I with longer half-life and imaging in vivo using SPECT instead of PET, could overcome the limitations of previous methods. When the doses of 131I-Kringle 5 were 10.0, 7.5 and 5.0 g/kg, respectively, the two-compartment open models can be determined within all the metabolic process in vivo. There were no significant differences in t1/2α, t1/2β, apparent volume of distribution and CL between those three levels. The ratio of AUC(0 ? 1) among three different groups of 10.0, 7.5 and 5.0 g/kg was 2.56:1.44:1.0, which was close to the ratio (2:1.5:1.0). It could be clear that in the range of 5.0–10.0 g/kg, Kringle 5 was characterized by the first-order pharmacokinetics. Approximately 30 min after 131I-Kringle 5 was injected, 131I-Kringle 5 could be observed to concentrate in the heart, kidneys, liver and other organs by means of planar imaging and tomography. After 1 h of being injected, more radionuclide retained in the bladder, but not in intestinal. It could be concluded that 131I-Kringle 5 is mainly excreted through the kidneys. About 2 h after the injection of 131I-Kringle 5, the radionuclide in the heart, kidneys, liver and other organs was gradually reduced, while more radionuclide was concentrated in the bladder. The radionuclide was completely metabolized within 24 h, and the distribution of radioactivity in rats was similar to normal levels. In our study, the specific marker 131I-Kringle 5 and SPECT/CT were suc-cessfully used to explore pharmacokinetic characteristics of Kringle 5 in rats. The study could provide a new evaluation platform of the specific, in vivo and real-time functional imaging and pharmacokinetics for the clinical application of 131I-Kringle 5.
ABSTRACT
In this work, a novel method was established to isolate and purify Human plasminogen Kringle 5 (HPK5) as a histidine-tagged fusion protein expressed in Escherichia coli BL21 (DE3). This method consisted of sample extraction using a Ni-chelated Sepharose Fast-Flow affinity column, ammonium sulfate salting-out and Sephadex G-75 size-exclusion column in turn. The purity analysis by SDS-PAGE, high-performance size-exclusion and reversed-phase chromatographies showed that the obtained recombinant fusion HPK5 was homogeneous and its purity was higher than 96%; the activity analysis by chorioallantoic membrane model of chicken embryos revealed that the purified recombinant HPK5 exhibited an obvious anti-angiogenic activity under the effective range of 5.0-25.0 µg/mL. Through this procedure, about 19 mg purified recombinant fusion HPK5 can be obtained from 1 L of original fermentation solution. Approximate 32% of the total recombinant fusion HPK5 can be captured and the total yield was approximately 11%.
Subject(s)
Ammonium Sulfate/chemistry , Chromatography, Gel/methods , Peptide Fragments/isolation & purification , Plasminogen/isolation & purification , Recombinant Proteins/isolation & purification , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/isolation & purification , Angiogenesis Inhibitors/pharmacology , Animals , Chemical Precipitation , Chick Embryo , Electrophoresis, Polyacrylamide Gel , Escherichia coli/isolation & purification , Humans , Neovascularization, Physiologic/drug effects , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Plasminogen/chemistry , Plasminogen/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacologyABSTRACT
Objective To construct baculovirus expression system for the human plasminogen kringle 5(hK5) gene,and detect its expression in Spodoptera frugiperda 21(sf21). Methods Baculovirus Bacmid-rhK5 was prepared with bac-to-bac baculovirus expression system,and sf21 cells were infected.The expression of rhK5 in sf21 cells was determined by Western blot.The activity of purified protein was detected by vascular endothelial cell inhibition test. Results The pFastBac HTB-rhK5 was successfully constructed,and sf21 cells were transfected with the constructed vector.The output of rhK5 obtained was 90 ug/L,and the protein possessed the inhibitory activity of endothelial cell proliferation.The median effective dose(ED50) was 4 ug/mL. Conclusion The rhK5 baculovirus expression vector is highly expressed in sf21 cells,which lays a foundation for the the expression of rhK5 protein with biologic activities.