Inhibition of HIV-1 gp160-dependent membrane fusion by a furin-directed alpha 1-antitrypsin variant.
Furin is a membrane-associated calcium-dependent serine endoprotease that cleaves proproteins on the carboxyl side of the consensus sequence -Arg-X-Lys/Arg-Arg-. Using site-directed mutagenesis, a variant alpha 1-antitrypsin (alpha 1-AT) was constructed which contains in its reactive site -Arg-X-X-Arg-, the minimal sequence required for efficient processing by furin (Molloy, S. S., Bresnahan, P. A., Leppla, S. H., Klimpel, K. R., and Thomas, G. (1992) J. Biol. Chem. 267, 16396-16402). This alpha 1-AT variant, [Arg355 Arg358]alpha 1-AT (alpha 1-PDX), is greater than 3,000-fold more effective than [Arg358]alpha 1-AT (alpha 1-AT Pittsburgh, alpha 1-PIT) at inhibiting furin in vitro (K0.5 = 0.03 microgram/ml). Furthermore, the P4 Arg in alpha 1-PDX greatly attenuates the thrombin inhibitory properties of this serpin (> 300-fold) compared with alpha 1-PIT (which contains a P4 Ala), thus increasing the selectivity of alpha 1-PDX for furin. Expression studies show that alpha 1-PDX, and not alpha 1-PIT, blocks the processing of two furin substrates, pro-beta-nerve growth factor and human immunodeficiency virus (HIV)-1 gp160 in transfected cells. In addition, a syncytium assay shows that alpha 1-PDX blocks the membrane fusogenic properties of HIV-1 gp160. The potential use of alpha 1-PDX in manipulating the activation of proproteins in a tissue- and time-specific manner is discussed.
Anderson
E D
ED
Vollum Institute, Oregon Health Sciences University, Portland 97201.
Thomas
L
L
Hayflick
J S
JS
Thomas
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D045683:Furin; D015686:Gene Products, env / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019302:HIV Envelope Protein gp160; D015497:HIV-1 / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion / Q000187:drug effects*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013381:Subtilisins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000494:pharmacology*
D015686:Gene Products, env; D019302:HIV Envelope Protein gp160; D011498:Protein Precursors; D000515:alpha 1-Antitrypsin; D013381:Subtilisins; D045683:Furin