A new mouse mutant with cleavage-resistant versican and isoform-specific versican mutants demonstrate that proteolysis at the Glu(441)-Ala(442) peptide bond in the V1 isoform is essential for interdigital web regression.
A new mouse mutant with cleavage-resistant versican and isoform-specific versican mutants demonstrate that proteolysis at the Glu(441)-Ala(442) peptide bond in the V1 isoform is essential for interdigital web regression.
复制标题
一个新的小鼠突变体，具有抗切割的verscan和异构体特异的verscan突变体，证明了V1亚型中Glu(441)-Ala(442)肽键的蛋白质分解对于叉指网络回归是必不可少的。
DOI：
10.1016/j.mbplus.2021.100064
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发表时间：
2021-06
影响因子：
--
通讯作者：
Apte SS
中科院分区：
文献类型：
--
作者：
Nandadasa S;Burin des Roziers C;Koch C;Tran-Lundmark K;Dours-Zimmermann MT;Zimmermann DR;Valleix S;Apte SS
·新的Vcan小鼠等位基因VcanAA具有ADAMTS蛋白酶抗性多功能蛋白聚糖。· VcanAA/AA小鼠是存活的并且发展出软组织并指（STS）·缺乏多功能蛋白聚糖GAGβ结构域但不缺乏GAGα结构域的小鼠也具有STS。·多功能蛋白聚糖GAGβ蛋白水解片段versikine是web回归所必需的。在蛋白酶缺陷表型的机制解释中的两个固有挑战是定义其还原产生表型的特定底物裂解，以及确定表型是否由底物功能丧失、底物积累或底物片段中体现的功能丧失引起。因此，通过抗切割底物再现蛋白酶缺陷表型将严格验证蛋白水解事件的重要性并阐明潜在机制。Versican是循环系统发育和正常肢体发育所需的一种大蛋白聚糖，并在位于其可变剪接的GAGβ结构域中的Glu 441-Ala 442肽键处被ADAMTS蛋白酶切割。特定的ADAMTS蛋白酶突变体具有受损的趾间蹼退化，导致与减少的多功能蛋白聚糖蛋白水解相关的软组织并指畸形。Versikine，由这种切割产生的N-末端蛋白水解片段，恢复ADAMTS突变网中的指间凋亡。在这里，我们报告了一种新的小鼠转基因，VcanAA，在GAGβ结构域中具有经验证的突变，可以特异性地消除这种蛋白水解事件。VcanAA/AA小鼠具有部分伸出的后肢软组织并指畸形。然而，在VcanAA/AA小鼠中，Adamts 20失活导致完全渗透，更严重的并指影响所有肢体，这表明ADAMTS 20在其他位点或其他底物的多功能蛋白聚糖裂解是蹼消退的额外要求。事实上，用针对多功能蛋白聚糖GAGα结构域中切割位点的新表位抗体进行免疫染色，证明在不存在ADAMTS 20的情况下染色减少。值得注意的是，Vcan外显子8（编码GAGβ结构域）缺失的小鼠始终发生软组织并指，而Vcan转录物中不能包含外显子7（编码GAGα结构域）的小鼠始终具有完全分离的手指。这些发现表明，在蹼退化期间，多功能蛋白聚糖在每个GAG承载结构域内被切割，并确认GAGβ结构域中的蛋白水解，通过生成versikine，在趾间蹼退化中具有重要作用。
• A novel Vcan mouse allele, VcanAA, has ADAMTS protease-resistant versican. • VcanAA/AA mice are viable and develop soft tissue-syndactyly (STS) • VcanAA/AA STS is rendered more severe in combination with Adamts20Bt/Bt. • Mice lacking the versican GAGβ domain, but not the GAGα domain, also have STS. • The versican GAGβ proteolytic fragment versikine is necessary for web regression. 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.