Structural requirements for furin-induced cleavage and activation of Shiga toxin.
Shiga toxin has a protease-sensitive site in the disulfide loop region of the A-chain. Cleavage of this site by furin is essential for rapid intoxication of cells by Shiga toxin. We have here investigated whether in addition to the Arg-X-X-Arg sequence, there are other structural requirements in the disulfide loop region for furin cleavage. A toxin mutant (Shiga-2D toxin) still containing the consensus motif for cleavage by furin, but lacking ten amino acids in the disulfide loop, was generated. Trypsin was able to cleave Shiga-2D toxin in vitro, demonstrating that the protease-sensitive region is intact. However, Shiga-2D toxin was not efficiently cleaved by furin either in vitro or in vivo. Furthermore, unless it was precleaved with trypsin, Shiga-2D toxin was much less toxic than wild type Shiga toxin in LoVo cells expressing functional furin. In contrast, LoVo/neo cells lacking functional furin were unable to activate both wild type Shiga toxin and Shiga-2D toxin. In conclusion, an extended loop structure is required for furin-induced cleavage of Shiga toxin.
Kurmanova
Alma
A
W.A. Engelhardt Institute for Molecular Biology, The Russian Academy of Sciences, Moscow, Russia.
Llorente
Alicia
A
Polesskaya
Anna
A
Garred
Oystein
O
Olsnes
Sjur
S
Kozlov
Juri
J
Sandvig
Kirsten
K
D000595:Amino Acid Sequence; D045683:Furin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D022621:Shiga Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D022621:Shiga Toxin; D045683:Furin