Dual Roles of the Cardin-Weintraub Motif in Multimeric Sonic Hedgehog
Dual Roles of the Cardin-Weintraub Motif in Multimeric Sonic Hedgehog
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DOI：
10.1074/jbc.m110.206474
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发表时间：
2011-07-01
影响因子：
4.8
通讯作者：
Grobe, Kay
中科院分区：
文献类型：
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作者：
Farshi, Pershang;Ohlig, Stefanie;Grobe, Kay
果蝇形态发生素刺猬蛋白（Hh）及其哺乳动物同源物音猬蛋白、印度刺猬蛋白和沙漠刺猬蛋白是分泌型信号分子，它们在胚胎发生过程中调节组织模式形成，并在成年个体的组织内稳态和再生中发挥作用。所有Hh家族成员的功能在产生细胞表面的形态发生素多聚化、多聚体释放、多聚体向靶细胞扩散以及信号接收等层面受到调控。已知这些机制都依赖于带正电荷的Hh氨基酸（卡丹 - 温特劳布（CW）基序）与带负电荷的硫酸乙酰肝素（HS）糖胺聚糖链的相互作用。然而，对于这些相互作用的精确机制理解仍然缺乏。在这项工作中，我们对多聚体音猬蛋白（称为ShhNp）以及缺失一个或多个CW残基的突变形式的离子型HS相互作用进行了表征。我们发现，所有五个CW残基的缺失以及CW残基Lys(33)、Arg(35)和Lys(39)（小鼠命名法）的定点突变消除了HS结合。相比之下，CW残基Arg(34)和Lys(38)对HS结合没有贡献。对Shh晶格接触的分析和验证为这一发现提供了解释。我们证明CW残基Arg(34)和Lys(38)与多聚体中相邻分子上的酸性凹槽接触，这表明这些残基在ShhNp多聚化而非HS结合中具有新的功能。因此，重组单体形态发生素（称为ShhN）在依赖CW的HS结合和生物活性方面与生理相关的ShhNp多聚体不同，这为ShhN和ShhNp之间观察到的功能差异提供了新的解释。
The fly morphogen Hedgehog (Hh) and its mammalian orthologs, Sonic, Indian, and Desert hedgehog, are secreted signaling molecules that mediate tissue patterning during embryogenesis and function in tissue homeostasis and regeneration in the adult. The function of all Hh family members is regulated at the levels of morphogen multimerization on the surface of producing cells, multimer release, multimer diffusion to target cells, and signal reception. These mechanisms are all known to depend on interactions of positively charged Hh amino acids (the Cardin-Weintraub (CW) motif) with negatively charged heparan sulfate (HS) glycosaminoglycan chains. However, a precise mechanistic understanding of these interactions is still lacking. In this work, we characterized ionic HS interactions of multimeric Sonic hedgehog (called ShhNp) as well as mutant forms lacking one or more CW residues. We found that deletion of all five CW residues as well as site-directed mutagenesis of CW residues Lys(33), Arg(35), and Lys(39) (mouse nomenclature) abolished HS binding. In contrast, CW residues Arg(34) and Lys(38) did not contribute to HS binding. Analysis and validation of Shh crystal lattice contacts provided an explanation for this finding. We demonstrate that CW residues Arg(34) and Lys(38) make contact with an acidic groove on the adjacent molecule in the multimer, suggesting a new function of these residues in ShhNp multimerization rather than HS binding. Therefore, the recombinant monomeric morphogen (called ShhN) differs in CW-dependent HS binding and biological activity from physiologically relevant ShhNp multimers, providing new explanations for functional differences observed between ShhN and ShhNp.