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

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