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. 2021 Jan 25;6(4):3079-3089.
doi: 10.1021/acsomega.0c05481. eCollection 2021 Feb 2.

Phage-Display-Derived Peptide Specific to Carbamylated Protein

Yuhao Ma  1 , Meng Wu  2 , Shuhui Li  2 , Marcello Tonelli  3 , Larry D Unsworth  1   2
Affiliations

Phage-Display-Derived Peptide Specific to Carbamylated Protein

Yuhao Ma et al. ACS Omega. .

Abstract

Protein carbamylation has been linked with diseases commonly associated with patients with reduced kidney function. Carbamylated human serum albumin (cHSA), which has been proven to be nephrotoxic and associated with heart failure for chronic kidney disease (CKD) patients, was chosen for our study. Through phage display against cHSA, one specific peptide sequence (cH2-p1) was identified with higher selectivity toward cHSA over native HSA. The cH2-p1 peptide was synthesized, and its target binding was analyzed through isothermal titration calorimetry (ITC). The result showed that cH2-p1 was able to bind cHSA of different levels of carbamylation with a similar dissociation constant of ∼1.0 × 10-4 M. This peptide also showed a binding specificity to carbamylated fibrinogen (cFgn), while not binding to native Fgn at all. For better understanding of the binding mechanism of cH2-p1, competitive binding of cH2-p1 and anti-homocitrulline to cHSA was performed, and the result revealed that cH2-p1 may bind to homocitrulline residues in a similar manner to the antibody. A molecular docking study was further performed to investigate the favored binding conformation of homocitrulline residue to cH2-p1. This work demonstrates the potential of peptides as a specific binding element to carbamylated proteins.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Affinity of candidate phages obtained through the phage display biopanning targeting cHSA to native HSA and carbamylated HSA (cHSA-1) (data represent mean ± 1 standard deviation (SD), n = 3; *, p < 0.01; **, p < 0.001).
Figure 2
Figure 2
Affinity of candidate phages obtained through the phage display biopanning targeting cHSA to native HSA, carbamylated HSA (cHSA-2), Fgn, and carbamylated Fgn (cFgn) (data represent mean ± 1 SD, n = 3; *, p < 0.01; **, p < 0.001).
Figure 3
Figure 3
ITC raw titration data and fitting of integrated heat plots of peptide cH2-p1 titration into (A) native HSA and (B) cHSA-2.
Figure 4
Figure 4
ITC raw titration data and fitting of integrated heat plots of peptide cH2-p4 titration into (A) native HSA and (B) cHSA-2.
Figure 5
Figure 5
ITC raw titration data and fitting of integrated heat plots of peptide cH2-p6 titration into (A) native HSA and (B) cHSA-2.
Figure 6
Figure 6
(a) ζ-Potential and (b) isoelectric point of native HSA and carbamylated HSA.
Figure 7
Figure 7
ITC raw titration data and fitting of integrated heat plots of peptide cH2-p1 titration into (A) cHSA-1, (B) cHSA-2, and (C) cHSA-3.
Figure 8
Figure 8
ITC raw titration data and fitting of integrated heat plots of peptide cH2-p1 titration into (A) native Fgn and (B) cFgn.
Figure 9
Figure 9
ITC raw titration data and fitting of integrated heat plots of peptide cH2-p1 titration into cHSA with cHSA-Abs and without cHSA been incubated with anti-Hcit.
Figure 10
Figure 10
Two ways, (a) pose a and (b) pose b, of docking the designed model Hcit-res (Pink carbon chain) into the candidate pocket of cH2-p1 (green carbon chain) by AutoDock Vina. The surface of cH2-p1 is shown with an electrostatic potential map.
Figure 11
Figure 11
(a) Modeling conformation of cH2-p1. (b) Top-ranked potential binding site, cH2-p1-C1, of cH2-p1.
Figure 12
Figure 12
Model molecule Hcit-res to mimic homocitrulline residue.
See this image and copyright information in PMC

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