Engineered surfaces for binding carbamylated proteins

  • Author / Creator
    Ma, Yuhao
  • For patients with decreased kidney function, waste materials accumulate within the blood compartment. Although not all of these chemical species have been directly linked to a specific toxicity, they are generally referred to as uremic toxins. Of particular interest are protein bound uremic toxins for they are extremely difficult to remove using membrane technology. One type of protein bound uremic toxin involves protein carbamylation. Carbamylation of albumin (cHSA) has shown that both extent of carbamylation and concentration of carbamylated albumin are associated with significantly elevated risk of all-cause mortality. Currently there has been no effective method for specifically targeting and removing cHSA from the blood. We believe small peptides, stabilized through immobilization on surfaces, can be employed as high affinity, cheap ligands to target and remove cHSA from blood.
    For the first phase we studied the effect of carbamylation on three proteins, cHSA, carbamylated fibrinogen (cFgn) and carbamylated α-lactalbumin (cLA), in terms of physiological aspects. While the molecular integrity and secondary structure were preserved after carbamylation, it was found carbamylation increases the surface charge negativity of protein and significantly altered its tertiary structure. For HSA and LA, carbamylation increased the accessibility of fluorescence quencher. For Fgn, carbamylation increased the fraction of accessible tryptophan. Also, all studied carbamylated proteins were found to have reduced interaction with surrounding water. Protein adsorption studies revealed that carbamylated proteins generally had a lower amount of adsorption, and the type of substrates influenced the overall amount of adsorption.
    After this study, phage display was performed against carbamylated HSA to determine the potential ligand for carbamylated protein. A total of seven candidate phage clones were identified from phage display. ELISA results showed that these phage clones had specificity towards cHSA compared to native HSA. Isothermal titration calorimetry was used to determine that the peptide GSAARTISPSLL (cH2-p1) had a dissociation constant (Kd) of 1.0 × 10-4 M when binding to cHSA, and was almost one magnitude lower than its Kd to native HSA. The binding affinity for cH2-p1 towards cHSA was found to be little influenced by the extent of carbamylation. Moreover, cH2-p1 was found to preferentially bind cFgn, with a similar Kd (8.4×10–5 M) as cHSA while there seems to be no interaction between cH2-p1 and native Fgn. All above results suggest that the candidate peptide cH2-p1 can specifically bind to carbamylated protein and recognize only homocitrulline residues as the main target.
    The peptide, cH2p1, was immobilized so as to investigate the possibility of using this ligand for selective binding and removal of cHSA. A p(HEMA) coating was formed, and the peptide cH2p1 was tethered to the polymer film through either C terminal (NH-cH2p1) or N terminal (CO-cH2p1) binding. To investigate the surface binding carbamylated and native HSA or carbamylated and native Fgn were labeled with radioactive 125I. Both cH2-p1 functionalized surfaces show selective binding features towards cHSA and cFgn, compared to their native protein form, with NH-cH2p1 of superior selectivity than CO-cH2p1. Both surfaces also show a high carbamylated protein binding ability in diluted plasma, with an ultralow Fgn adsorption observed for NH-cH2p1.
    The results of the study suggest that the candidate peptide cH2-p1 immobilized p(HEMA) surface had specific affinity to carbamylated cHSA. The potential of the proof-of-concept surface platform shown in this work makes it possible to build adsorbent materials based on surface immobilized peptide ligands for removing carbamylated proteins from CKD patients and benefit hemodialysis treatment.

  • Subjects / Keywords
  • Graduation date
    Spring 2021
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.