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A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers.

ABSTRACT: The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ? 400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE(373) neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.

SUBMITTER: Kosasih HJ

PROVIDER: S-EPMC4751368 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers.

Kosasih Hansen J HJ Last Karena K Rogerson Fraser M FM Golub Suzanne B SB Gauci Stephanie J SJ Russo Vincenzo C VC Stanton Heather H Wilson Richard R Lamande Shireen R SR Holden Paul P Fosang Amanda J AJ

The Journal of biological chemistry 20151214 7

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains ...[more]

PMID: 26668318

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Project description:ObjectiveA disintegrin and metallopeptidase with thrombospondin motifs type 8 (ADAMTS8) is crucial for diverse physiological processes, such as inflammation, tissue morphogenesis, and tumorigenesis. The chicken ADAMTS8 (chADAMTS8) gene was differentially expressed in the kidney following exposure to different calcium concentrations, suggesting a pathological role of this protein in metabolic diseases. We aimed to examine the molecular characteristics of chADAMTS8 and analyze the gene-expression differences in response to toll-like receptor 3 (TLR3) stimulation.MethodsThe ADAMTS8 mRNA and amino acid sequences of various species (chicken, duck, cow, mouse, rat, human, chimpanzee, pig, and horse) were retrieved from the Ensembl database and subjected to bioinformatics analyses. Reverse-transcription polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) experiments were performed with various chicken tissues and the chicken fibroblast DF-1 cell line, which was stimulated with polyinosinic-polycytidylic acid (poly[I:C]; a TLR3 ligand).ResultsThe chADAMTS8 gene was predicted to contain three thrombospondin type 1 (TSP1) domains, whose amino acid sequences shared homology among the different species, whereas sequences outside the TSP1 domains (especially the amino-terminal region) were very difffferent. Phylogenetic analysis revealed that chADAMTS8 is evolutionarily clustered in the same clade with that of the duck. chADAMTS8 mRNA was broadly expressed in chicken tissues, and the expression was significantly up-regulated in the DF-1 cells in response to poly(I:C) stimulation (p<0.05). These results showed that chADAMTS8 may be a target gene for TLR3 signaling.ConclusionIn this report, the genetic information of chADAMTS8 gene, its expression in chicken tissues, and chicken DF-1 cells under the stimulation of TLR3 were shown. The result suggests that chADAMTS8 expression may be induced by viral infection and correlated with TLR3-mediated signaling pathway. Further study of the function of chADAMTS8 during TLR3-dependent inflammation (which represents RNA viral infection) is needed and it will also be important to examine the molecular mechanisms during different regulation, depending on innate immune receptor activation.

Project description:ImportanceA Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 is hypothesized to play a role in the pathogenesis of invasive infection, but studies in sepsis are lacking.ObjectivesTo study A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 protein level in pediatric sepsis and to study the association with outcome.DesignData from two prospective cohort studies.Setting and participantsCohort 1 is from a single-center study involving children admitted to PICU with meningococcal sepsis (samples obtained at three time points). Cohort 2 includes patients from a multicenter study involving children admitted to the hospital with invasive bacterial infections of differing etiologies (samples obtained within 48 hr after hospital admission).Main outcomes and measuresPrimary outcome measure was mortality. Secondary outcome measures were PICU-free days at day 28 and hospital length of stay.ResultsIn cohort 1 (n = 59), nonsurvivors more frequently had A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 levels above the detection limit than survivors at admission to PICU (8/11 [73%] and 6/23 [26%], respectively; p = 0.02) and at t = 24 hours (2/3 [67%] and 3/37 [8%], respectively; p = 0.04). In cohort 2 (n = 240), A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 levels in patients within 48 hours after hospital admission were more frequently above the detection limit than in healthy controls (110/240 [46%] and 14/64 [22%], respectively; p = 0.001). Nonsurvivors more often had detectable A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 levels than survivors (16/21 [76%] and 94/219 [43%], respectively; p = 0.003), which was mostly attributable to patients with Neisseria meningitidis.Conclusions and relevanceIn children with bacterial infection, detection of A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 within 48 hours after hospital admission is associated with death, particularly in meningococcal sepsis. Future studies should confirm the prognostic value of A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 and should study pathophysiologic mechanisms.

Dataset Information

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers.

Publications

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers.

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