A novel method of ligand peptidomics to identify peptide ligands binding to AQP2-expressing plasma membranes and intracellular vesicles of rat kidney

2008 ◽  
Vol 295 (1) ◽  
pp. F300-F309 ◽  
Author(s):  
Yu-Jung Lee ◽  
Hyo-Jung Choi ◽  
Jung-Suk Lim ◽  
Ji-Hyun Earm ◽  
Byung-Heon Lee ◽  
...  
Keyword(s):  
Phage Display ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Peptide Library ◽  
Peptide Ligands ◽  
High Affinity ◽  
Phage Display Technique ◽  
Affinity Peptide ◽  
Display Technique

Aquaporin-2 (AQP2), the vasopressin-regulated water channel in collecting duct principal cells, plays a key role in the regulation of body water balance. We aimed to isolate high-affinity peptide ligands that bind to immunoisolated AQP2-expressing plasma membrane (PM) or intracellular vesicle (ICV) preparations from rat kidney by the in vitro phage display technique. Immunoblotting revealed that AQP2 was exclusively expressed in the immunoisolated AQP2 membrane fractions (PM and ICV), compared with the nonimmunoisolated or preimmune IgG pulldown rat kidney samples. Moreover, AQP1 or H+-ATPase (B1 subunit) expression was minimal in the immunoisolated AQP2 membrane fractions, indicating the specificity of AQP2 membrane isolation. A phage peptide library based on T7 415-1b phage vector displaying CX7C was constructed. After three rounds of biopanning, seven phage clones of high frequency were selected, which showed high affinity to the AQP2-containing PM or ICV fractions compared with a nonrecombinant T7 insertless phage clone. In contrast, these phage clones showed lower affinity to H+-ATPase-containing fractions. Fluorescein-conjugated peptide labeling was associated with intracellular compartment and PM of primary cultured inner medullary collecting duct cells, relative to absent or very weak labeling with fluorescein-conjugated control peptide. Library analyses demonstrated proteins that had motifs homologous to the peptide ligands, albeit with a high probability of a random match due to short peptide sequences. In summary, we applied the in vitro phage display technique to identify high-affinity peptide ligands to AQP2-expressing membranes. Library analyses identified proteins having homologous motifs, which need to be examined for involvement in AQP2 trafficking and regulation.

Design of new high-affinity peptide ligands for human leukocyte antigen-DQ2 using a positional scanning peptide library

2010 ◽  
Vol 71 (5) ◽  
pp. 475-481 ◽  
Author(s):  
Ulrike Jüse ◽  
Yvonne van de Wal ◽  
Frits Koning ◽  
Ludvig M. Sollid ◽  
Burkhard Fleckenstein
Keyword(s):  
Human Leukocyte Antigen ◽  
Human Leukocyte ◽  
Peptide Library ◽  
Peptide Ligands ◽  
Leukocyte Antigen ◽  
High Affinity ◽  
Positional Scanning ◽  
Affinity Peptide

Identification of a porcine TLR2-targeting peptide ligands using a cell-based phage display combined with high-throughput sequencing

2020 ◽  
Author(s):  
Su-Bin An ◽  
Seo-Ho Oh ◽  
Jun-Yeong Lee ◽  
Kwang-Hwan Choi ◽  
Chang-Kyu Lee ◽  
...  
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Oral Route ◽  
Peptide Ligands ◽  
Mucosal Vaccine ◽  
Cell Targeting ◽  
M Cell ◽  
Phage Display Technique

Abstract M cell targeting is one of the critical issues to develop efficient mucosal vaccine design. In this study, peptide ligands with high affinity to porcine TLR2, which is highly expressed in M cells and play an important role in mucosal immune responses in pigs, were identified through the cell-based phage display technique combined with high-throughput sequencing. A random phage-peptide library was applied to the porcine TLR2 overexpressing cell line and total 85, 557 unique peptide sequences were identified from approximately 9.0 × 107 reads after three rounds of both subtractive and non-subtractive biopanning via high-throughput sequencing. Among the unique sequences, three candidate peptide sequences, NAGHLSQ, VPSKPGL, and RANLDGQ, were selected based on their abundance in the third round of biopanning. Consequently, NAGHLSQ showed the highest affinity exclusively to porcine TLR2 compared with other candidates and its binding mechanism was inferred to be directly associated with ligand binding site of the TLR2 through the in vitro competitive analysis. The peptide identified in this research could be used in development of effective porcine mucosal vaccine as an M cell targeting moiety to enhance the transport of antigens into the Peyer's patch via oral route.

Screening a specific Zn(ii)-binding peptide for improving the cognitive decline of Alzheimer's disease in APP/PS1 transgenic mice by inhibiting Zn2+-mediated amyloid protein aggregation and neurotoxicity

2019 ◽  
Vol 7 (12) ◽  
pp. 5197-5210 ◽  
Author(s):  
Xiaoyu Zhang ◽  
Manli Zhong ◽  
Pu Zhao ◽  
Xiancheng Zhang ◽  
You Li ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Phage Display ◽  
Cognitive Decline ◽  
Amyloid Protein ◽  
Binding Peptide ◽  
Phage Display Technique ◽  
Aβ Aggregation ◽  
Display Technique

PZn screen from phage display technique and PZn loaded nanoparticles inhibiting Aβ aggregation and neurotoxicity in vitro and in vivo.

scholarly journals Phage-Display Based Discovery and Characterization of Peptide Ligands against WDR5

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1225
Author(s):  
Jiawen Cao ◽  
Tiantian Fan ◽  
Yanlian Li ◽  
Zhiyan Du ◽  
Lin Chen ◽  
...  
Keyword(s):  
Phage Display ◽  
Cyclic Peptide ◽  
Protein Complexes ◽  
Peptide Ligands ◽  
Phage Library ◽  
Cancer Drug ◽  
Protein Protein Interaction ◽  
Phage Display Technique ◽  
Peptide Mimic ◽  
Human Proteins

WD40 is a ubiquitous domain presented in at least 361 human proteins and acts as scaffold to form protein complexes. Among them, WDR5 protein is an important mediator in several protein complexes to exert its functions in histone modification and chromatin remodeling. Therefore, it was considered as a promising epigenetic target involving in anti-cancer drug development. In view of the protein–protein interaction nature of WDR5, we initialized a campaign to discover new peptide-mimic inhibitors of WDR5. In current study, we utilized the phage display technique and screened with a disulfide-based cyclic peptide phage library. Five rounds of biopanning were performed and isolated clones were sequenced. By analyzing the sequences, total five peptides were synthesized for binding assay. The four peptides are shown to have the moderate binding affinity. Finally, the detailed binding interactions were revealed by solving a WDR5-peptide cocrystal structure.

Afferent arteriolar adenosine A2a receptors are coupled to KATP in in vitro perfused hydronephrotic rat kidney

1999 ◽  
Vol 277 (6) ◽  
pp. F926-F933 ◽  
Author(s):  
Lilong Tang ◽  
Michael Parker ◽  
Qing Fei ◽  
Rodger Loutzenhiser
Keyword(s):  
Rat Kidney ◽  
Afferent Arteriole ◽  
Adenosine A2a Receptors ◽  
A2a Receptors ◽  
High Affinity ◽  
Vasodilatory Response ◽  
Renal Microvasculature ◽  
Adenosine A2a ◽  
Arteriolar Vasodilation

Adenosine is known to exert dual actions on the afferent arteriole, eliciting vasoconstriction, by activating A1 receptors, and vasodilation at higher concentrations, by activating lower-affinity A2 receptors. We could demonstrate both of these known adenosine responses in the in vitro perfused hydronephrotic rat kidney. Thus, 1.0 μM adenosine elicited a transient vasoconstriction blocked by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and 10–30 μM adenosine reversed KCl-induced vasoconstriction. However, when we examined the effects of adenosine on pressure-induced afferent arteriolar vasoconstriction, we observed a third action. In this setting, a high-affinity adenosine vasodilatory response was observed at concentrations of 10–300 nM. This response was blocked by both 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM-241385) and glibenclamide and was mimicked by 2-phenylaminoadenosine (CV-1808) (IC50 of 100 nM), implicating adenosine A2a receptors coupled to ATP-sensitive K channels (KATP). Like adenosine, 5′- N-ethylcarboxamidoadenosine (NECA) elicited both glibenclamide-sensitive and glibenclamide-insensitive vasodilatory responses. The order of potency for the glibenclamide-sensitive component was NECA > adenosine = CV-1808. Our findings suggest that, in addition to the previously described adenosine A1 and low-affinity A2b receptors, the renal microvasculature is also capable of expressing high-affinity adenosine A2areceptors. This renal adenosine receptor elicits afferent arteriolar vasodilation at submicromolar adenosine levels by activating KATP.

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