Identification of an ADAMTS-4 cleavage motif using phage display leads to the development of fluorogenic peptide substrates and reveals matrilin-3 as a novel substrate.
ADAMTS-4 and ADAMTS-5 are aggrecanases responsible for the breakdown of cartilage aggrecan in osteoarthritis. Multiple ADAMTS-4 cleavage sites have been described in several matrix proteins including aggrecan, versican, and brevican, but no concise predictive cleavage motif has been identified for this protease. By screening a 13-mer peptide library with a diversity of 10(8), we have identified the ADAMTS-4 cleavage motif E-(AFVLMY)-X(0,1)-(RK)-X(2,3)-(ST)-(VYIFWMLA), with Glu representing P1. Several 13-mer peptides containing this motif, including DVQEFRGVTAVIR and HNEFRQRETYMVF, were shown to be substrates for ADAMTS-4. These peptides were found to be specific substrates for ADAMTS-4 as they were not cleaved by ADAMTS-5. Modification of these peptides with donor (6-FAM) and acceptor (QSY-9) molecules resulted in the development of fluorescence-based substrates with a Km of approximately 35 microM. Furthermore, the role of Glu at P1 and Phe at P1' in binding and catalysis was studied by exploring substitution of these amino acids with the D-isomeric forms. Substitution of P1 with dGlu was tolerable for binding, but not catalysis, whereas substitution of P1' with dPhe precluded both binding and catalysis. Similarly, replacement of Glu with Asp at P1 abolished recognition and cleavage of the peptide. Finally, BLAST results of the ADAMTS-4 cleavage motif identified matrilin-3 as a new substrate for ADAMTS-4. When tested, recombinant ADAMTS-4 effectively cleaved intact matrilin-3 at the predicted motif at Glu435/Ala436 generating two species of 45 and 5 kDa.
Hills
Robert
R
Pfizer Global Research and Development, St. Louis, Missouri 63017, USA. Robert.L.Hills@Pfizer.com
Mazzarella
Richard
R
Fok
Kam
K
Liu
Min
M
Nemirovskiy
Olga
O
Leone
Joseph
J
Zack
Marc D
MD
Arner
Elizabeth C
EC
Viswanathan
Malini
M
Abujoub
Aida
A
Muruganandam
Arumugam
A
Sexton
Daniel J
DJ
Bassill
Gary J
GJ
Sato
Aaron K
AK
Malfait
Anne-Marie
AM
Tortorella
Micky D
MD
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000071121:ADAMTS4 Protein; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D064235:Matrilin Proteins; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011348:Procollagen N-Endopeptidase / Q000235:genetics / Q000378:metabolism*; D040081:Protein Array Analysis; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D016326:Extracellular Matrix Proteins; D064235:Matrilin Proteins; D019151:Peptide Library; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D018698:Glutamic Acid; D051722:ADAM Proteins; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein