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

Sumeda Nandadasa; Cyril Burin des Roziers; Christopher Koch; Karin Tran-Lundmark; María T Dours-Zimmermann; Dieter R Zimmermann; Sophie Valleix; Suneel S Apte

doi:10.1016/j.mbplus.2021.100064

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

Matrix Biol Plus. 2021 May 14:10:100064. doi: 10.1016/j.mbplus.2021.100064. eCollection 2021 Jun.

Authors

Sumeda Nandadasa¹, Cyril Burin des Roziers², Christopher Koch¹, Karin Tran-Lundmark³, María T Dours-Zimmermann⁴, Dieter R Zimmermann⁴, Sophie Valleix², Suneel S Apte¹

Affiliations

¹ Department of Biomedical Engineering-ND20, Cleveland Clinic Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195, United States.
² Institut Cochin, Inserm U1016 - CNRS UMR8104 - Paris Descartes University Medical School, 24, Rue du faubourg Saint Jacques, 75014 Paris, France.
³ Department of Experimental Medical Science and Wallenberg Center for Molecular Medicine, Lund University, Lund, Sweden.
⁴ Department of Pathology and Molecular Pathology, University Hospital Zurich, Zurich, Switzerland.

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, Vcan ^AA, with validated mutations in the GAGβ domain that specifically abolish this proteolytic event. Vcan ^AA/AA mice have partially penetrant hindlimb soft tissue syndactyly. However, Adamts20 inactivation in Vcan ^AA/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.

Keywords: ADAMTS; Extracellular matrix; Limb development; Metalloprotease; Proteoglycan; Syndactyly.