4510
Biochemistry 1998, 37, 4510-4517
Retroviral Envelope Glycoprotein Processing: Structural Investigation of the Cleavage Site Maxime Moulard,*,‡ Laurent Challoin,§ Ste´phane Canarelli,| Kamel Mabrouk,| and Herve´ Darbon§ C.I.M.L., Marseille, France, Laboratoire AFMB, IBSM, Marseille, France, and Laboratoire de Biochimie IFR Jean Roche, CNRS UMR6560, Marseille, France ReceiVed October 27, 1997; ReVised Manuscript ReceiVed January 22, 1998
ABSTRACT: Proteolytic activation of retroviral envelope glycoprotein precursors occurs at the carboxyl side of a consensus motif consisting of the amino acid sequence (Arg/Lys)-Xaa-(Arg/Lys)-Arg. Synthetic peptides spanning the processing sites of HIV-1/2 and SIV glycoprotein precursors were examined for their ability to be cleaved by the subtilisin-like endoproteases kexin and furin. To determine the potential role of secondary structure on proteolytic activation, we examined the secondary structure of synthetic peptides by circular dichroism and NMR spectroscopy. The results indicate that (i) the peptides were correctly cleaved by kexin and furin and therefore could be used as specific substrates for the purification and characterization of the lymphocyte endoprotease(s) responsible for proteolytic processing of precursors; (ii) the regions surrounding the cleavage sites could be characterized by their flexibility in aqueous solutions. However, a loop has been shown to be a determinant for the specificity of the interaction between the enzyme and its substrate as determined by molecular modeling. Furthermore, we determine and propose a possible structure of the cleavage site which fits to the active site of the modeled furin.
Viral envelope glycoproteins are synthesized as precursors which are cleaved by a cellular endoprotease in a late Golgi compartment (1, 2), probably by a kexin-like protease (35). The mature envelope glycoproteins are generated from an inactive precursor by selective and limited endoproteolysis which occurs at the carboxyl side of a consensus motif consisting of (Arg/Lys)-Xaa-(Arg/Lys)-Arg (6). Except for the presence of this sequence, proteolytic processing sites in precursors of viral glycoproteins exhibit only minor similarities at the amino acid sequence, suggesting that other parameters are likely to be involved in the viral glycoprotein precursor processing. The HIV-1 glycoprotein precursor (gp160) undergoes a proteolytic cleavage at Arg508-Glu-Lys-Arg511 (site 1) (7). The cleavage generates the external glycoprotein gp120 associated noncovalently to the transmembrane glycoprotein gp41. The gp120 is responsible for the interaction of the viral particle with CD4 receptor-coreceptor complexes, and gp41 is required for the fusion of viral and cellular lipid membranes. Site-directed mutagenesis suggested that basic residues within the cleavage recognition sequence are crucial in determining the efficiency of the cleavage (8, 9). For example, replacement of Arg511 with Ser completely abolishes cleavage leading to the production of noninfectious viral particles (10). In addition to Arg511, Arg508, and Lys510 of site 1, there is another area of potential enzyme * Correspondence to: Centre d’Immunologie CNRS-INSERM de Marseille-Luminy, Parc Scientifique de Luminy, Case 906, F-13288 Marseille Cedex 9, France. Phone: (33) 491 26 94 94. Fax: (33) 491 26 94 30. E-mail:
[email protected]. ‡ C.I.M.L. § Laboratoire AFMB. | Laboratoire de Biochimie.
sensitivity corresponding to Lys500-Ala-Lys-Arg-Arg504 (site 2). Basic amino acids of site 2 are also shown to be important for proteolytic processing at site 1 (10). Replacement of each of the four basic residues of site 2 by neutral amino acid abolished the cleavage, suggesting that, in addition to the sequence Arg508-Glu-Lys-Arg511, a contribution of surrounding amino acids is required. The newly discovered kexin-like enzyme family is considered to be a candidate for the intracellular processing of the HIV envelope glycoprotein (11, 12). Among the members of this family, furin and PC7 have been shown to cleave correctly the HIV-1 gp160 (13) as well as the HIV-2 gp140 (11) in vitro using the recombinant vaccinia viruses for expression of each of the proteins. In the case of HIV-1 gp160, of the seven dibasic sites which constitute potential substrates for proprotein convertases, cleavage occurs selectively at site 1, generating biologically active glycoproteins. Taken together, these data strongly suggest that the amino acid sequence of site 1 alone is not sufficient to allow for correct processing of gp160. To investigate the importance of secondary structure in determining the processing of such precursors, we have addressed several questions concerning the role of these structures in the catalytic process. Indeed, we have made use of a series of synthetic peptides mimicking precursor processing sites of gp160 HIV-1, gp140 HIV-2, and gp140 SIV to measure their reactivity for purified kexin from Saccharomyces cereVisiae and human recombinant soluble furin. The cleavability and the cleavage selectivity for each peptide is presented. Secondary structures of the synthetic peptides have also been investigated by CD and NMR. Experimental data from NMR demonstrates the peptide p511 (gp140 SIV) is likely to adopt turn structures. Therefore,
S0006-2960(97)02662-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 03/10/1998
Furin and HIV Envelope Glycoprotein Precursor Interaction Table 1: Amino Acid Sequence of Synthetic Peptides Used in This Study Peptide p510 (HIV-1) Val510-Val-Gln-Arg-Glu-Lys-Arg-Ala-Val-Gly-Ile-Gly521 Peptides Gp4 and Gp1 (HIV-2) (Gp4) Val487-Glu-Ile-Thr-Pro-Ile-Gly-Glu-Ala-Pro-Thr-Lys-GluLys-Arg-Tyr-Ser-Ser-Ala-His-Gly507 (Gp1) Tyr502-Ser-Ser-Ala-His-Gly507 Peptide p511 (SIV) Pro511-Thr-Asp-Val-Lys-Arg-Tyr-Thr-Thr-Gly-Gly-Thr-SerArg-Asn-Lys-Arg-Gly-Val-Phe530
we attempted to demonstrate that the obtained NMR structure of p511 is relevant to a potential structure in gp160 since p511 fits into the molecular model of the active site of furin. MATERIALS AND METHODS Enzymes. Recombinant soluble kexin was purified from media of kexin secreting cells by concentration and elution on Q-Sepharose followed by Mono-Q chromatography (14). The enzyme was 90 µg/mL and 1000 units/µL corresponding to 1.1 × 107 units/mg (1 unit can cleave 1 pmole boc-GlnArg-Arg per minute under our standart conditions) and was from R. Fuller (University of Michigan Medical Center, Ann Arbor, MI). Recombinant soluble kexin was purified as published previously (15) and was from G. Thomas (Vollum Institute, Portland, OR). The concentration of the enzyme was 160 ng/µL. Peptide Substrates. Synthetic peptides which mimic the processing cleavage sites of the envelope glycoproteins used in this study are listed in Table 1. The substrate peptide Gp4 (HIV-2 ROD) and its N-terminal peptide Gp1 were synthesized by solid-phase procedures following the general method of Merrifield (16). Stepwise elongation of these peptides was carried out on an automated peptide synthesizer (Applied Biosystems Inc., model 430 A) utilizing NMPHOBt (N-methylpyrrolidone-2 hydroxybenzotriazole) as adapted by Applied Biosystems. After anhydrous hydrogen fluoride cleavage, the crude peptides were highly purified (>99%) by C18 reversed-phase medium-pressure liquid chromatography. Homogeneous fractions were pooled, lyophilized, and characterized by analytical HPLC and by amino acid analysis after acid hydrolysis for 24 h. The substrate peptides p510 (HIV-1) and p511 (SIV mac251) where obtained from Neosystem (Strasbourg, France). HPLC and Amino Acid Analysis. Peptides were fractionated by HPLC using a 5-mm RP C18 endcapped column (5 × 250 mm) as described elsewhere (17). Amino acid analysis was performed with a Beckman model 6300 apparatus. Protein Substrates. Recombinant gp160 (rgp160)1 was derived from the enV gene of HIV-1 IIIB expressed in baculovirus-infected insect cells. rgp160 including the two potential cleavage sites (L494GVAPTKAKRRVVQREKRAVGIGALF) was supplied in PBS (AGMED, Bedford, MA). Before enzymatic proteolysis, rgp160 was extensively dia1 Abbreviations: DQF-COSY, double quantum filtered correlation spectroscopy; HIV, human immunodeficiency virus; NOESY, nuclear Overhauser effect spectroscopy; rgp, recombinant glycoprotein; SIV, simian immunodeficiency virus; TFE, 2,2,2-trifluoroethanol; TOCSY, total correlation spectroscopy.
Biochemistry, Vol. 37, No. 13, 1998 4511 lyzed against a buffer composed of 20 mM Tris-HCl, pH 7.4, and 2 mM CaCl2. Enzyme Assay. Synthetic peptides (1 mg/mL) were dissolved in H2O. Ten nanomoles of each peptide underwent proteolysis in a 0.1 mL reaction volume containing 2 µL of kexin or 5 µL of the purified furin (0.08 µg) in 20 mM TrisHCl buffer, pH 7.4, containing 2 mM CaCl2 and 0.5% (v/v) Triton X-100. The reaction mixture was incubated at room temperature for 0-24 h. Products were identified by HPLC purification followed by amino acid analysis. Western Blotting. Transfer of the proteins from the SDS polyacrylamide gel (8%) to nitrocellulose membranes was conducted for 3 h at 200 mA. Immunodetection of cleaved fragments was performed with a 10 µg/mL dilution of a monoclonal antibody directed against gp120 (mAb 5.5) or gp41 (mAb 41.A) obtained from F. Traincard (Hybridolab, Paris, France). Membranes were then incubated with peroxidase-conjugated goat anti-mouse IgG and developed with chemiluminescence reagents from Amersham following suppliers instructions. CD Measurements. CD spectra were acquired on a JobinYvon Mark IV spectrophotometer (Longjumeau, France). The spectra were recorded at 25 °C by using 0.5 mm path length cell, with a 2 s time constant and a scan rate of 0.5 nm/s. The instrument was calibrated with (+)-10-camphosulfonic acid. The peptide concentration in the solutions, based on the absorption spectra, was from 0.5 to 1 mg/mL. The spectra were cumulated 5-fold in water or in water-TFE (2,2,2-trifluoroethanol) and automatically averaged. NMR. Proton NMR spectra were routinely recorded at 25 °C on a Bruker AMX400 spectrometer in the phase sensitive mode by time-proportional phase incrementation of the first pulse [TPPI] (18). A double-quantum-filtered 2-D correlation spectrum (DQF-COSY) (19), a total correlation spectrum (TOCSY) was acquired with a mixing time of 100 ms using TPPI, and a phase-sensitive 2-D nuclear Overhauser effect (NOE) spectrum (NOESY) (20) with mixing times of 300 and 400 ms were acquired. The solvent OH resonance was suppressed by low-power irradiation during the relaxation delay and, for NOESY, during the mixing time. After the two-dimensional data matrixes were zero-filled to 2K in both dimensions (600, 400, and 800 experiments with 1K data points were recorded for, respectively, COSY, TOCSY, and NOESY spectra) and multiplied by a shifted sine-bell window in both dimensions, twodimensional spectra were Fourier transformed and baseline corrected with FTTOOL software (Eccles, personal communication) running on an IRIS 4D-380 Silicon Graphics computer. Identification of the amino acid spin systems and sequential assignment were achieved using the well-known general strategy described by Wu¨thrich (21) with the help of EASY software (22) and gave rise to a nearly complete proton assignment. Identification of spin systems was obtained by analysis and comparison of 2D-DQF-COSY and TOCSY recorded in water. NOE intensities, used as input for the structure calculations were obtained from a NOESY spectrum recorded with 100 ms mixing time on the fully protonated sample, and the peaks integrated by the peakintegration routine of the EASY software (22), running on an IPC SUN station. The intra-residual and sequential NOE’s were partitioned into three categories (
