A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Abstract

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulfate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

DPP4 Inhibitors and COVID-19–Holy Grail or Another Dead End?

“…Surprisingly, the marginal role, if any, of DPP4 in SARS-Cov-2 infection was concluded based on the results of experiments with hamster cells transiently transfected with human DPP4 (Hoffman et al 2020 ). Similar conclusion originates from another artificial system with human DPP4 expressed in insect cells (Xi et al 2020 ). However, both aforementioned experimental models have some limitations, which do not allow to exclude the role of DPP4 in SARS-CoV-2 invasion definitely.…”

Diabetes, obesity, metabolism, and SARS-CoV-2 infection: the end of the beginning

“…The role of DPP-4 as a MERS-CoV receptor spurred considerable speculation as to whether DPP-4i might interfere with SARS-CoV-2 infectivity ( Drucker, 2020 ). Nevertheless, experimental data do not support and actually refute the contention that human recombinant DPP-4 binds SARS-CoV-2 ( Xi et al., 2020 ). Serum circulating levels of sDPP-4 were reduced by ~50% in 7 individuals hospitalized with COVID-19 ( Schlicht et al., 2020 ); however, given the substantial intra- and inter-individual variability of sDPP-4 in humans, this preliminary observation must be confirmed in larger datasets ( Baggio et al., 2020 ).…”

Disentangling conflicting evidence on DPP-4 inhibitors and outcomes of COVID-19: narrative review and meta-analysis

“…Thus, DPP4 inhibition might block the entering of the virus in host cells. However, to date, no data firmly support that DPP4 is also a receptor for SARS-CoV-2 [ 10 , 11 ]. DPP4i has been identified as a potential candidate by structural studies [ 12 , 13 ] which so far await confirmation in human cells [ 10 ].…”

“…However, to date, no data firmly support that DPP4 is also a receptor for SARS-CoV-2 [ 10 , 11 ]. DPP4i has been identified as a potential candidate by structural studies [ 12 , 13 ] which so far await confirmation in human cells [ 10 ]. Furthermore, it has been hypothesized that DPP4 inhibition may antagonize SARS-CoV-2 virulence through a reduction of the cytokine storm [ 7 ] and lung inflammation [ 14 ].…”