Abstract

Two inherent challenges in the mechanistic interpretation of protease-deficient phenotypes are defining the specific substrate cleavages whose reduction generates the phenotypes and determining whether the phenotypes result from loss of substrate function, substrate accumulation, or loss of a function(s) embodied in the substrate fragments. Hence, recapitulation of a protease-deficient phenotype by a cleavage-resistant substrate would stringently validate the importance of a proteolytic event and clarify the underlying mechanisms. Versican is a large proteoglycan required for development of the circulatory system and proper limb development, and is cleaved by ADAMTS proteases at the Glu441-Ala442 peptide bond located in its alternatively spliced GAGβ domain. Specific ADAMTS protease mutants have impaired interdigit web regression leading to soft tissue syndactyly that is associated with reduced versican proteolysis. Versikine, the N-terminal proteolytic fragment generated by this cleavage, restores interdigit apoptosis in ADAMTS mutant webs. Here, we report a new mouse transgene, VcanAA, with validated mutations in the GAGβ domain that specifically abolish this proteolytic event. VcanAA/AA mice have partially penetrant hindlimb soft tissue syndactyly. However, Adamts20 inactivation in VcanAA/AA mice leads to fully penetrant, more severe syndactyly affecting all limbs, suggesting that ADAMTS20 cleavage of versican at other sites or of other substrates is an additional requirement for web regression. Indeed, immunostaining with a neoepitope antibody against a cleavage site in the versican GAGα domain demonstrated reduced staining in the absence of ADAMTS20. Significantly, mice with deletion of Vcan exon 8, encoding the GAGβ domain, consistently developed soft tissue syndactyly, whereas mice unable to include exon 7, encoding the GAGα domain in Vcan transcripts, consistently had fully separated digits. These findings suggest that versican is cleaved within each GAG-bearing domain during web regression, and affirms that proteolysis in the GAGβ domain, via generation of versikine, has an essential role in interdigital web regression.

Highlights

  • Extracellular matrix (ECM) proteolysis is necessary for tissue remodeling during mammalian development

  • The findings suggest that cooperative versican processing by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteases generates a critical level of versikine to promote apoptosis of interdigital mesenchymal cells; when versican proteolysis is reduced below this hypothetical critical threshold, interdigit cells may be resistant to bone morphogenetic protein (BMP)-induced apoptosis [4]

  • In a prior in vitro analysis of versican cleavage by ADAMTS5, we demonstrated that mutation of the P1 and P3 glutamic acid residues

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Summary

Introduction

Extracellular matrix (ECM) proteolysis is necessary for tissue remodeling during mammalian development. With this preliminary evidence of specific point mutations that could abrogate versican processing, we used homologous recombination in mouse embryonic stem cells to replace E438 and E441 with A and generate transgenic mice with cleavage-resistant versican, an allele designated as VcanAA We used this new allele to rigorously test the biological relevance of versican proteolysis at these two sites in the context of web regression and along with analysis of web regression in Vcan exon 7- or exon 8specific mutants, we provide new insights on the role of ADAMTS proteases and versican in this process

Results
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