Rearranging exosites in noncatalytic domains can redirect the substrate specificity of ADAMTS proteases.
ADAMTS proteases typically employ some combination of ancillary C-terminal disintegrin-like, thrombospondin-1, cysteine-rich, and spacer domains to bind substrates and facilitate proteolysis by an N-terminal metalloprotease domain. We constructed chimeric proteases and substrates to examine the role of C-terminal domains of ADAMTS13 and ADAMTS5 in the recognition of their physiological cleavage sites in von Willebrand factor (VWF) and aggrecan, respectively. ADAMTS5 cleaves Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds in bovine aggrecan but does not cleave VWF. Conversely, ADAMTS13 cleaves the Tyr(1605)-Met(1606) bond of VWF, which is exposed by fluid shear stress but cannot cleave aggrecan. Replacing the thrombospondin-1/cysteine-rich/spacer domains of ADAMTS5 with those of ADAMTS13 conferred the ability to cleave the Glu(1615)-Ile(1616) bond of VWF domain A2 in peptide substrates or VWF multimers that had been sheared; native (unsheared) VWF multimers were resistant. Thus, by recombining exosites, we engineered ADAMTS5 to cleave a new bond in VWF, preserving physiological regulation by fluid shear stress. The results demonstrate that noncatalytic thrombospondin-1/cysteine-rich/spacer domains are principal modifiers of substrate recognition and cleavage by both ADAMTS5 and ADAMTS13. Noncatalytic domains may perform similar functions in other ADAMTS family members.
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Weiqiang
W
Gao
Jian
J
Zhu
Lisa A
LA
Westfield
Elodee A
EA
Tuley
Patricia J
PJ
Anderson
J Evan
JE
Sadler
D051722:ADAM Proteins / Q000737:chemistry / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D000071116:ADAMTS5 Protein; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D059748:Proteolysis; D013379:Substrate Specificity; D014841:von Willebrand Factor / Q000378:metabolism
D017931:DNA Primers; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human