Comparing ATPase activity of ATP-binding cassette subfamily C member 4, lamprey CFTR, and human CFTR using an antimony-phosphomolybdate assay

Introduction: ATP-binding cassette (ABC) transporters use the hydrolysis of ATP to power the active transport of molecules, but paradoxically the cystic fibrosis transmembrane regulator (CFTR, ABCC7) forms an ion channel. We previously showed that ATP-binding cassette subfamily C member 4 (ABCC4) is the closest mammalian paralog to CFTR, compared to other ABC transporters. In addition, Lamprey CFTR (Lp-CFTR) is the oldest known CFTR ortholog and has unique structural and functional features compared to human CFTR (hCFTR). The availability of these evolutionarily distant orthologs gives us the opportunity to study the changes in ATPase activity that may be related to their disparate functions.Methods: We utilized the baculovirus expression system with Sf9 insect cells and made use of the highly sensitive antimony-phosphomolybdate assay for testing the ATPase activity of human ABCC4 (hABCC4), Lp-CFTR, and hCFTR under similar experimental conditions. This assay measures the production of inorganic phosphate (Pi) in the nanomolar range.Results: Crude plasma membranes were purified, and protein concentration, determined semi-quantitatively, of hABCC4, Lp-CFTR, and hCFTR ranged from 0.01 to 0.36 μg/μL. No significant difference in expression level was found although hABCC4 trended toward the highest level. hABCC4 was activated by ATP with the equilibrium constant (Kd) 0.55 ± 0.28 mM (n = 8). Estimated maximum ATPase rate (Vmax) for hABCC4 was about 0.2 nmol/μg/min when the protein was activated with 1 mM ATP at 37°C (n = 7). Estimated maximum ATPase rate for PKA-phosphorylated Lp-CFTR reached about half of hCFTR levels in the same conditions. Vmax for both Lp-CFTR and hCFTR were significantly increased in high PKA conditions compared to low PKA conditions. Maximum intrinsic ATPase rate of hABCC4 in the absence of substrate was twice that of hCFTR when activated in 1 mM ATP.Conclusion: The findings here suggest that while both ABCC4 and hCFTR bear one consensus and one degenerate ATPase site, the hCFTR exhibited a reduced intrinsic ATPase activity. In addition, ATPase activity in the CFTR lineage increased from Lp-CFTR to hCFTR. Finally, the studies pave the way to purify hABCC4, Lp-CFTR, and hCFTR from Sf9 cells for their structural investigation, including by cryo-EM, and for studies of evolution in the ABC transporter superfamily

Protein-Polymer Conjugates Synthesized Using Water-Soluble Azlactone-Functionalized Polymers Enable Receptor-Specific Cellular Uptake Toward Targeted Drug Delivery

Conjugation of proteins to drug-loaded polymeric structures is an attractive strategy for facilitating target-specific drug delivery for a variety of clinical needs. Polymers currently available for conjugation to proteins generally have limited chemical versatility for subsequent drug loading. Many polymers that do have chemical functionality useful for drug loading are often insoluble in water, making it difficult to synthesize functional protein–polymer conjugates for targeted drug delivery. In this work, we demonstrate that reactive, azlactone-functionalized polymers can be grafted to proteins, conjugated to a small-molecule fluorophore, and subsequently internalized into cells in a receptor-specific manner. Poly(2-vinyl-4,4-dimethylazlactone), synthesized using reversible addition–fragmentation chain transfer polymerization, was modified post-polymerization with substoichiometric equivalents of triethylene glycol monomethyl ether to yield reactive water-soluble, azlactone-functionalized copolymers. These reactive polymers were then conjugated to proteins holo-transferrin and ovotransferrin. Protein gel analysis verified successful conjugation of proteins to polymer, and protein–polymer conjugates were subsequently purified from unreacted proteins and polymers using size exclusion chromatography. Internalization experiments using a breast cancer cell line that overexpresses the transferrin receptor on its surface showed that the holo-transferrin–polymer conjugate was successfully internalized by cells in a mechanism consistent with receptor-mediated endocytosis. Internalization of protein–polymer conjugate demonstrated that the protein ligand maintained its overall structure and function following conjugation to polymer. Our approach to protein–polymer conjugate synthesis offers a simple, tailorable strategy for preparing bioconjugates of interest for a broad range of biomedical applications