Heparin enhances the furin cleavage of HIV-1 gp160 peptides

Abstract

Infectious HIV-1 requires gp160 cleavage by furin at the REKR511 downward arrow motif (site1) into the gp120/gp41 complex, whereas the KAKR503 (site2) sequence remains uncleaved. We synthesized 41mer and 51mer peptides, comprising site1 and site2, to study their conformation and in vitro furin processing. We found that, while the previously reported 19mer and 13mer analogues represent excellent in vitro furin substrates, the present extended sequences require heparin for optimal processing. Our data support the hypothesis of a direct binding of heparin with site1 and site2, allowing selective exposure/accessibility of the REKR sequence, which is only then optimally cleaved by furin.

Figure 1

Table 1 Peptide sequences

Figure 2

CD spectra of: (A) 19mer, (B) 41mer, and (C) 51mer in (——) water, (‐ ‐ ‐ ‐ ‐) Phosphate buffer pH 7, (‐·‐·‐·‐) 14 mM SDS, and (– – –) 98%TFE/H₂O; (D) water (black), 20 μM heparin (dark gray) in H₂O and phosphate buffer pH 7.2 (light gray).

Figure 3

CD spectra of: (A) 19mer, (B) 41mer, and (C) 51mer in (black) 0.15 M NaCl and 25 mM Tris–HCl buffer pH 7, and (gray, arrow) 20 μM heparin in 0.15 M NaCl and 25 mM Tris–HCl buffer pH 7.

Figure 4

RP‐HPLC profiles of: (A) 13mer digestion and (B) 19mer digestion obtained on a Varian C₁₈ column with UV detector (214 nm). Arrows indicate the fragments.

Figure 5

RP‐HPLC profiles of the digestion of the (A) 41mer and (B) 51mer obtained on a Varian C₁₈ column with UV detector (214 nm). 20 μL of the enzymatic assay solution was taken (top) immediately after the addition of the substrate and upon overnight incubation (bottom). Arrows indicate the fragments, (∗) being LGVAPTKAKRRVVQREKR for the 41mer and (∗∗) being AVGIGALFLGFLGAAGSTMGAAS.

Figure 6

Initial rate versus concentration of the 18mer to assess its effect on the furin cleavage of: (A) Pyr‐RTKR‐MCA, and (B) 19mer.

Figure 7

Effect of heparin on the processing of the 41mer and 51mer. Upper panel controls: (left) RFU released versus time upon Pyr‐RTKR‐MCA cleavage by furin in (black) the absence, or presence of 20 μM (dark gray) or 100 μM (light gray) heparin; (upper central panel): incubation of the 41mer peptide with recombinant vaccinia WT‐infected culture supernatant (control); (upper right panel) effect of decanoyl‐RVKR‐cmk on the processing of the 41mer by furin. processing. Lower panels: 20 μL of the enzymatic assay solution containing 2.5 μM heparin was taken (top) immediately after the addition of the substrate and (bottom) after overnight incubation. Arrows indicate the fragments, (∗) being LGVAPTKAKRRVVQREKR for the 41mer peptide or YKYKVVKIEPLGVAPTKAKRRVVQREKR for the 51mer peptide and (∗∗) being AVGIGALFLGFLGAAGSTMGAAS.