A new mouse mutant with cleavage-resistant versican and isoform-specific versican mutants demonstrate that proteolysis at the Glu441-Ala442 peptide bond in the V1 isoform is essential for interdigital web regression

Nandadasa, Sumeda; Koch, Christopher D.; Tran‐Lundmark, Karin; Dours-Zimmermann, Marı́a T.; Zimmermann, Dieter R.; Valleix, Sophie; Apte, Suneel S.

doi:10.1016/j.mbplus.2021.100064

Cited by 17 publications

(14 citation statements)

References 54 publications

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“…Because of the diversity of molecules and networks that comprise the ECM and existence of numerous secreted proteases, we hypothesized that selective proteolysis by specific proteases having preferred ECM substrates, may have a crucial role in ECM proteostasis in specific contexts of embryonic development. One example of a specific ECM proteostatic phenomenon mediated by ADAMTS proteases that influences embryonic cell behavior and is strongly supported by mouse genetic models is their role in versican turnover during interdigital web regression ( Dubail et al, 2014 ; McCulloch et al, 2009 ; Nandadasa et al, 2021 ). Other than this example, little is known about the fate of macromolecular ECM complexes, especially those formed by homologous proteins with different stoichiometry in the embryo versus adult, and their proteostatic mechanisms.…”

Section: Discussionmentioning

confidence: 99%

Proteolysis of fibrillin-2 microfibrils is essential for normal skeletal development

Mead

Martin

Wang

et al. 2022

eLife

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The embryonic extracellular matrix (ECM) undergoes transition to mature ECM as development progresses, yet few mechanisms ensuring ECM proteostasis during this period are known. Fibrillin microfibrils are macromolecular ECM complexes serving structural and regulatory roles. In mice, Fbn1 and Fbn2, encoding the major microfibrillar components, are strongly expressed during embryogenesis, but fibrillin-1 is the major component observed in adult tissue microfibrils. Here, analysis of Adamts6 and Adamts10 mutant mouse embryos, lacking these homologous secreted metalloproteases individually and in combination, along with in vitro analysis of microfibrils, measurement of ADAMTS6-fibrillin affinities and N-terminomics discovery of ADAMTS6-cleaved sites, identifies a proteostatic mechanism contributing to postnatal fibrillin-2 reduction and fibrillin-1 dominance. The lack of ADAMTS6, alone and in combination with ADAMTS10 led to excess fibrillin-2 in perichondrium, with impaired skeletal development defined by a drastic reduction of aggrecan and cartilage link protein, impaired BMP signaling in cartilage, and increased GDF5 sequestration in fibrillin-2-rich tissue. Although ADAMTS6 cleaves fibrillin-1 and fibrillin-2 as well as fibronectin, which provides the initial scaffold for microfibril assembly, primacy of the protease-substrate relationship between ADAMTS6 and fibrillin-2 was unequivocally established by reversal of the defects in Adamts6-/- embryos by genetic reduction of Fbn2, but not Fbn1.

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Section: Discussionmentioning

confidence: 99%

Proteolysis of fibrillin-2 microfibrils is essential for normal skeletal development

Mead

Martin

Wang

et al. 2022

eLife

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“…Mice with limb-specific conditional deletion of ADAMTS9 develop soft-tissue syndactyly (16). This defect is also seen in mice with elimination of a canonical ADAMTS cleavage site in the versican core protein, validating versican as an ADAMTS9 and ADAMTS20 substrate in digit separation (30). However, mice with cleavage-resistant versican lack other defects seen in Adamts9 and Adamts20 mutants, some of which may reflect defective cilium-mediated signaling, or reduced cleavage of other ECM substrates that are yet to be defined.…”

Section: Introductionmentioning

confidence: 67%

“…However, mice with cleavage-resistant versican lack other defects seen in Adamts9 and Adamts20 mutants, some of which may reflect defective cilium-mediated signaling, or reduced cleavage of other ECM substrates that are yet to be defined. Other confirmed ADAMTS9 substrates are aggrecan and fibronectin, both components of the ECM (30). A prior degradomic analysis of skin from a hypomorphic mouse Adamts9 mutant identified several putative ECM proteins as ADAMTS9 substrates, but these remain unvalidated and their cleavage by ADAMTS20 was not previously tested (31).…”

Section: Introductionmentioning

confidence: 90%

Degradomic identification of membrane type 1-matrix metalloproteinase (MT1-MMP/MMP14) as an ADAMTS9 and ADAMTS20 substrate

Nandadasa¹,

Martin²,

Deshpande³

et al. 2022

Preprint

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The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix (ECM) proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative N-terminomics method, terminal amine isotopic labeling of substrates (TAILS) was used to compare parental and gene-edited cells and their medium to identify ADAMTS9 substrates. Among differentially abundant N-terminally labeled internal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease MT1-MMP, whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge i.e., between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by re-expression of ADAMTS9 and ADAMTS20, and was dependent on hinge O-glycosylation. Since MT1-MMP is a type I transmembrane protein, identification of an N-terminally labeled peptide in the medium suggested additional downstream cleavage sites in its ectodomain. Indeed, a C-terminally semi-tryptic MT1-MMP peptide with greater abundance in wild-type RPE-1 medium identified by a targeted search indicated a cleavage site in the hemopexin domain. Consistent with retention of MT1-MMP catalytic domain on the surface of gene-edited cells, pro-MMP2 activation, which requires cell-surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell-surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell-surface activity of MT1-MMP and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.

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“…Cleavage of the aggregating proteoglycans VCAN and Aggrecan (ACAN) also generates bioactive fragments referred to as Versikine [ 8 ] and Aggrekine [ 9 , 10 ], respectively. Use of mouse models deficient in ECM proteoglycan proteases (i.e., proteoglycanases) together with neo-epitope specific antibodies that identify the corresponding cleaved ECM substrates [ 11 , 12 , 13 ] has uncovered critical in vivo ECM proteolytic events emphasizing the importance of the degradome [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ]. Specifically with respect to cardiac valve development, mice deficient in A Disintegrin and Metalloproteinase with Thrombospondin motifs-5 (ADAMTS5) or ADAMTS9 proteoglycanases exhibit enlarged malformed cardiac valves due to excess of their proteoglycan substrates VCAN and ACAN [ 14 , 23 ]; the VCAN and ACAN proteolytic profiles, as identified by neo-epitope specific antibodies, are also disrupted [ 8 , 9 , 10 ].…”

Section: Introductionmentioning

confidence: 99%

Increased Proteoglycanases in Pulmonary Valves after Birth Correlate with Extracellular Matrix Maturation and Valve Sculpting

Dupuis

Evins

Abell

et al. 2023

JCDD

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Increased mechanical forces on developing cardiac valves drive formation of the highly organized extracellular matrix (ECM) providing tissue integrity and promoting cell behavior and signaling. However, the ability to investigate the response of cardiac valve cells to increased mechanical forces is challenging and remains poorly understood. The developmental window from birth (P0) to postnatal day 7 (P7) when biomechanical forces on the pulmonary valve (PV) are altered due to the initiation of blood flow to the lungs was evaluated in this study. Grossly enlarged PV, in mice deficient in the proteoglycan protease ADAMTS5, exhibited a transient phenotypic rescue from postnatal day 0 (P0) to P7; the Adamts5−/− aortic valves (AV) did not exhibit a phenotypic correction. We hypothesized that blood flow, initiated to the lungs at birth, alters mechanical load on the PV and promotes ECM maturation. In the Adamts5−/− PV, there was an increase in localization of the proteoglycan proteases ADAMTS1, MMP2, and MMP9 that correlated with reduced Versican (VCAN). At birth, Decorin (DCN), a Collagen I binding, small leucine-rich proteoglycan, exhibited complementary stratified localization to VCAN in the wild type at P0 but colocalized with VCAN in Adamts5−/− PV; concomitant with the phenotypic rescue at P7, the PVs in Adamts5−/− mice exhibited stratification of VCAN and DCN similar to wild type. This study indicates that increased mechanical forces on the PV at birth may activate ECM proteases to organize specialized ECM layers during cardiac valve maturation.

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A new mouse mutant with cleavage-resistant versican and isoform-specific versican mutants demonstrate that proteolysis at the Glu441-Ala442 peptide bond in the V1 isoform is essential for interdigital web regression

Abstract: Highlights • A novel Vcan mouse allele, Vcan AA , has ADAMTS protease-resistant versican. • Vcan AA/AA mice are viable and develop soft tissue-syndactyly (STS) • Vcan AA/AA STS is rendered more severe in combination with Adamts20 … Show more

Cited by 17 publications

References 54 publications

Proteolysis of fibrillin-2 microfibrils is essential for normal skeletal development

Proteolysis of fibrillin-2 microfibrils is essential for normal skeletal development

Degradomic identification of membrane type 1-matrix metalloproteinase (MT1-MMP/MMP14) as an ADAMTS9 and ADAMTS20 substrate

Increased Proteoglycanases in Pulmonary Valves after Birth Correlate with Extracellular Matrix Maturation and Valve Sculpting

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