Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing.
ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu(373)-Ala(374) bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu(1480)-Gly(1481) bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the C-terminal spacer domain deletion mutant more effectively hydrolyzed both the Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein. Finding of ADAMTS-4 in the interleukin-1alpha-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered.
Kashiwagi
Masahide
M
Kennedy Institute of Rheumatology Division, Imperial College London, United Kingdom.
Enghild
Jan J
JJ
Gendron
Christi
C
Hughes
Clare
C
Caterson
Bruce
B
Itoh
Yoshifumi
Y
Nagase
Hideaki
H
D051722:ADAM Proteins; D000071121:ADAMTS4 Protein; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry; D002358:Cartilage, Articular / Q000378:metabolism; D020134:Catalytic Domain; D002417:Cattle; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000737:chemistry; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D058575:Decorin; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D016326:Extracellular Matrix Proteins; D000071235:Fibromodulin; D017353:Gene Deletion; D005822:Genetic Vectors; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007375:Interleukin-1 / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011348:Procollagen N-Endopeptidase; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D014168:Transferrin / Q000737:chemistry
D002352:Carrier Proteins; C546878:DCN protein, human; D018076:DNA, Complementary; D058575:Decorin; D000939:Epitopes; D016326:Extracellular Matrix Proteins; C000604330:FMOD protein, human; D007375:Interleukin-1; D011509:Proteoglycans; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D014168:Transferrin; D000071235:Fibromodulin; D018698:Glutamic Acid; D002809:Chondroitin Sulfates; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human; D000409:Alanine