Autocatalytic cleavage of ADAMTS-4 (Aggrecanase-1) reveals multiple glycosaminoglycan-binding sites.
ADAMTS-4, also referred to as aggrecanase-1, is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans (GAGs) attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In the present study, we demonstrate that full-length ADAMTS-4 (M(r) approximately 68,000) undergoes autocatalytic C-terminal truncation to generate two discrete isoforms (M(r) approximately 53,000 and M(r) approximately 40,000), which exhibit a marked reduction in affinity of binding to sulfated GAGs. C-terminal sequencing and mass analyses revealed that the GAG-binding thrombospondin type I motif was retained following autocatalysis, indicating that sites present in the C-terminal cysteine (cys)-rich and/or spacer domains also effect binding of full-length ADAMTS-4 to sulfated GAGs. Binding-competition experiments conducted using native and deglycosylated aggrecan provided direct evidence for interaction of the ADAMTS-4 cysteine-rich/spacer domains with aggrecan GAGs. Furthermore, synthetic peptides mimicking putative (consensus) GAG-binding sequences located within the ADAMTS-4 cysteine-rich and spacer domains competitively blocked binding of sulfated GAGs to full-length ADAMTS-4, thereby identifying multiple GAG-binding sites, which may contribute to the regulation of ADAMTS-4 function.
Wyeth Research, Cambridge, Massachusetts 02140, USA. cflannery@wyeth.com
Carl R
CR
Flannery
Weilan
W
Zeng
Chris
C
Corcoran
Lisa A
LA
Collins-Racie
Priya S
PS
Chockalingam
Tracy
T
Hebert
Stewart A
SA
Mackie
Thomas
T
McDonagh
Tara K
TK
Crawford
Kathy N
KN
Tomkinson
Edward R
ER
LaVallie
Elisabeth A
EA
Morris
D051722:ADAM Proteins; D000071121:ADAMTS4 Protein; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine; D006025:Glycosaminoglycans / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011348:Procollagen N-Endopeptidase; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D006025:Glycosaminoglycans; D007527:Isoenzymes; D011994:Recombinant Proteins; C020341:glucosaminoglycans; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human; D003545:Cysteine