A quantitative assay for aggrecanase activity.
Aggrecanase activities of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteinases were measured with a recombinant aggrecan fragment and two monoclonal antibodies. Recombinant human aggrecan interglobular domain was first incubated in the presence of ADAMTS enzymes. The aggrecan peptide with the N-terminal sequence ARGSVIL released upon hydrolysis was then quantified in an enzyme-linked immunosorbent assay (ELISA) with an anti-neoepitope antibody specific for the N-terminal ARGSVIL sequence and a second anti-aggrecan peptide antibody. For higher sensitivity of the assay, P1-P5 residues of the aggrecanase site within the aggrecan substrate were changed by in vitro mutagenesis. Specific activities of recombinant truncated ADAMTS1 and ADAMTS4 estimated with authentic aggrecan interglobular domain amounted to 2.4 +/- 0.4 and 21.7 +/- 9.5 nmoles hydrolyzed substrate/min.mg, respectively. The values were 10.3 +/- 5.1 and 151.5 +/- 93.5 nmoles/min.mg for hydrolysis of the modified substrate. The aggrecanase activity assay can be used for (1) kinetic characterization of aggrecanase activities of human and animal ADAMTS, (2) screening of inhibitors for aggrecan hydrolyzing ADAMTS, and (3) estimation of aggrecanase activities in biological samples.
Will
Horst
H
Invitek GmbH, Berlin, Germany. proteinexpression@invitek.de
Dettloff
Matthias
M
Bendzkô
Peter
P
Sveshnikov
Peter
P
D051722:ADAM Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000071097:ADAMTS1 Protein; D000595:Amino Acid Sequence; D001681:Biological Assay / Q000379:methods; D015153:Blotting, Western; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000235:genetics; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013582:Synovial Fluid / Q000201:enzymology
D018076:DNA, Complementary; D000939:Epitopes; D010450:Endopeptidases; D051722:ADAM Proteins; D000071097:ADAMTS1 Protein; C103859:ADAMTS1 protein, human; C083641:aggrecanase