Project description:Background: Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. Two similar matrix-degrading metalloproteases, ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each have mild cardiac anomalies, but their combined genetic inactivation is precluded by tight linkage. We coupled Adamts1 inactivation with pharmacologic ADAMTS5 blockade to uncover stage-specific cooperative roles and mechanisms in mouse cardiac development. Methods: ADAMTS5 blockade was achieved in Adamts1 null mouse embryos using an activity-blocking monoclonal antibody during distinct developmental windows covering myocardial compaction or cardiac septation and outflow tract rotation. Synchrotron imaging, RNA in situ hybridization, immunofluorescence microscopy and electron microscopy were used to determine the impact on cardiac development and compared to Gpc6 and ADAMTS-cleavage resistant mouse mutants. Mass spectrometry-based N-terminomics was used to identify relevant substrates. Results: Combined inactivation of ADAMTS1 and ADAMTS5 prior to 12.5 days of gestation led to dramatic accumulation of versican-rich cardiac jelly and inhibited formation of compact and trabecular myocardium, which we also observed in mice with ADAMTS cleavage-resistant versican. Subsequently, combined knockout impaired outflow tract development and ventricular septal closure, generating a tetralogy of Fallot-like defect independently of versican proteolysis. N-terminomics of combined ADAMTS knockout and wild-type hearts identified a cleaved glypican-6 peptide only in the wild-type and showed that ADAMTS1 and ADAMTS5 each cleaved glypican-6. Paradoxically, ADAMTS1 and ADAMTS5 inactivated hearts lacked glypican-6 despite unaltered Gpc6 transcription. Gpc6-/- mice demonstrated similar rotational defects as the combined ADAMTS knockout and both had reduced Hedgehog signaling. Conclusions: ADAMTS1 and ADAMTS5 ensure proper cardiac development via cleavage of distinct proteoglycans, each with independent roles in cardiac development. Whereas versican clearance in cardiac jelly is required for proper ventricular cardiomyogenesis, glypican-6 cleavage may activate/stabilize this cell-surface proteoglycan which is required for Hedgehog signaling during outflow tract development.

Project description:In mammals, the A Disintegrin-like And Metalloproteinase domain with Thrombospondin type 1 motifs (ADAMTS) family comprises 19 secreted proteases, including 6 members with the ability to cleave large aggregating proteoglycans: ADAMTS1, 4, 5, 9, 15, and 20. ADAMTS9 has an essential, non-redundant role during embryogenesis, as Adamts9 null embryos die prior to completing gastrulation and Adamts9 haploinsufficiency results in cardiovascular and ocular anomalies. ADAMTS9 proteolytic activity is required for proteostasis of versican, a widely distributed large aggregating proteoglycan/hyalectan in the provisional extracellular matrix present during early development. Despite its importance, ADAMTS9 proteoglycanase activity has not been directly compared to that of ADAMTS1, 4, and 5, due to difficulties in expressing and purifying the >200 kDa full-length form of ADAMTS9. Like ADAMTS1, 4, and 5, ADAMTS9 cleaves versican V1 isoform at the Glu441-Ala442 site, but unlike them, cleavages at other sites are unknown. Here, we expressed a truncated ADAMTS9 variant (ADAMTS9 MDTCS) consisting of all ADAMTS ‘core domains’ that render it more comparable in size and domain structure to ADAMTS1, 4, and 5 and compared its activity against versican, aggrecan and the small leucine-rich proteoglycan biglycan. A catalytically inactive ADAMTS9 MDTCS variant (ADAMTS9 EQ) was used as the control in all experiments. We identified cleavage sites in versican (V1 and V2 isoforms) and biglycan by all 4 ADAMTS family members using a quantitative label-free proteomics strategy. Moreover, using a quantitative substrate cleavage assay, we established that ADAMTS9 MDTCS versicanase activity at the Glu441-Ala442 site is 175-fold lower than ADAMTS5, 9-fold lower than ADAMTS4, and 5.5-fold higher lower than ADAMTS1. These provide systematic analysis of the proteoglycanase activity in the ADAMTS family and highlight differences and similarities in cleavage site specificities that could potentially be leveraged to design selective small molecule inhibitors for therapeutic applications.

Dataset Information

Effect of genetic modification of extracellular matrix-related genes on B16F1 melanoma progression

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets