Retroviral envelope glycoprotein processing: structural investigation of the cleavage site.
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.
Moulard
M
M
C.I.M.L., Marseille, France, Laboratoire AFMB, IBSM, Marseille, France. moulard@ciml.univ-mrs.fr
Chaloin
L
L
Canarelli
S
S
Mabrouk
K
K
Darbon
H
H
Challoin
L
L
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D045683:Furin; D015686:Gene Products, env / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D019302:HIV Envelope Protein gp160 / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D043484:Proprotein Convertases; D011487:Protein Conformation; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015302:Simian Immunodeficiency Virus / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism; D054299:env Gene Products, Human Immunodeficiency Virus
D015686:Gene Products, env; D019302:HIV Envelope Protein gp160; D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D054299:env Gene Products, Human Immunodeficiency Virus; C066315:gp140 envelope protein, Human immunodeficiency virus 2; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae; D045683:Furin