Characterization of a peptide containing the major heparin binding domain of human hepatic lipase

Abstract

Human hepatic lipase (hHL) is a cell surface associated enzyme that hydrolyzes triacylglycerols and phospholipids within circulating lipoproteins. We hypothesized that an amino acid sequence mimicking the major heparin binding domain (HBD) of hHL will displace hHL from cell surfaces. To test this hypothesis, we generated a recombinant protein of thioredoxin linked with a cleavable, tagged sequence containing amino acids 442 to 476 of the mature hHL sequence, which contains the major HBD of hHL. The recombinant protein associated with heparin‐sepharose, and its peak elution from heparin‐sepharose occurred in the presence of 0.5 M NaCl. We cleaved and purified the tagged sequence containing the HBD from the recombinant protein and tested the ability of the peptide to displace full‐length hHL from HEK‐293 cells. The peptide indeed displaced hHL from cell surfaces, while no significant displacement was observed in the presence of a peptide with a scrambled sequence. Finally, we obtained structural information for the peptide containing the HBD. 1H‐ and 15N‐NMR spectra of the peptide indicate the peptide is largely unstructured, although not completely random coil. The addition of heparin to the peptide induced some changes in chemical shift, suggesting changes in peptide structure and/or specific interactions with heparin. Molecular simulations confirm the largely unstructured nature of the isolated peptide, but they also indicate weak tendencies for both α‐ and β‐structure formation in different parts of the chain. Overall, these data provide a proof‐of‐principle for the use of mimetic peptides for the displacement of cell surface associated lipases.

The binding of heparin to spike glycoprotein inhibits SARS-CoV-2 infection by three mechanisms

“…In these experimental conditions, heparin prevents spike cleavage by furin, indicating that it physically associates with the immobilized spike fragment. On the other hand, a 2M NaCl wash, which is known to disrupt heparin/protein interactions ( 23 , 24 ), completely detached heparin from the spike fragment, restoring cleavage by furin ( Fig. 3 D).…”

Structural fluctuations and mechanical stabilities of the metamorphic protein RfaH

“…To this end, we use Monte Carlo simulations in combination with a computationally efficient all‐atom protein model 36 . This model has previously been applied to a range of protein processes, including folding, 37 conformational fluctuations of disordered proteins, 38,39 and protein‐peptide binding 40 . To probe local mechanical stability properties we apply the technique of Das et al, 41 which was developed to mechanically characterize proteins that are prone to misfolding 42 .…”