The Effect of N-Terminal Cyclization on the Function of the HIV Entry Inhibitor 5P12-RANTES

Abstract

Despite effective treatment for those living with Human Immunodeficiency Virus (HIV), there are still two million new infections each year. Protein-based HIV entry inhibitors, being highly effective and specific, could be used to protect people from initial infection. One of the most promising of these for clinical use is 5P12-RANTES, a variant of the chemokine RANTES/CCL5. The N-terminal amino acid of 5P12-RANTES is glutamine (Gln; called Q0), a residue that is prone to spontaneous cyclization when at the N-terminus of a protein. It is not known how this cyclization affects the potency of the inhibitor or whether cyclization is necessary for the function of the protein, although the N-terminal region of RANTES has been shown to be critical for receptor interactions, with even small changes having a large effect. We have studied the kinetics of cyclization of 5P12-RANTES as well as N-terminal variations of the protein that either produce an identical cyclized terminus (Glu0) or that cannot similarly cyclize (Asn0, Phe0, Ile0, and Leu0). We find that the half life for N-terminal cyclization of Gln is roughly 20 h at pH 7.3 at 37 °C. However, our results show that cyclization is not necessary for the potency of this protein and that several replacement terminal amino acids produce nearly-equally potent HIV inhibitors while remaining CC chemokine receptor 5 (CCR5) antagonists. This work has ramifications for the production of active 5P12-RANTES for use in the clinic, while also opening the possibility of developing other inhibitors by varying the N-terminus of the protein.

Efficient production of fluorophore‐labeled CC chemokines for biophysical studies using recombinant enterokinase and recombinant sortase

“…Generally, for CC chemokines, it is most efficient to unfold the protein in denaturant such as guanidinium chloride and then refold it using any of a variety of conditions. [13,18,25,26] But there are still several impediments to the efficient production of fluorophore-labeled chemokine. One impediment is the cost of specific proteases to cleave the fusion tag, which can be hundreds of dollars per aliquot from commercial sources, but which is necessary because the activity of a CC chemokine is sensitive to the exact sequence at its N-terminus.…”

Regulation of Chemokine–Receptor Interactions and Functions

“…In their review article, Thompson et al [ 6 ] discuss this modification and its consequences for recognition of both chemokine receptors and GAGs. An additional modification, investigated by Nguyen et al [ 10 ], is the cyclization of an N-terminal glutamine residue to yield pyroglutamate. Considering the importance of the chemokine N-terminus for function, this modification has the potential to influence receptor interactions.…”