Punctin/MADD-4, a member of the ADAMTSL extracellular matrix protein family, was identified as an anterograde synaptic organizer in the nematode Caenorhabditis elegans. At GABAergic neuromuscular junctions, the short isoform MADD-4B binds the ectodomain of neuroligin NLG-1, itself a postsynaptic organizer of inhibitory synapses. To identify the molecular bases of their partnership, we generated recombinant forms of the two proteins and carried out a comprehensive biochemical and biophysical study of their interaction, complemented by an in vivo localization study. We show that spontaneous proteolysis of MADD-4B first generates a shorter N-MADD-4B form, which comprises four thrombospondin (TSP) domains and one Ig-like domain and binds NLG-1. A second processing event eliminates the C-terminal Ig-like domain along with the ability of N-MADD-4B to bind NLG-1. These data identify the Ig-like domain as the primary determinant for N-MADD-4B interaction with NLG-1 in vitro. We further demonstrate in vivo that this Ig-like domain is essential, albeit not sufficient per se, for efficient recruitment of GABAA receptors at GABAergic synapses in C. elegans. The interaction of N-MADD-4B with NLG-1 is also disrupted by heparin, used as a surrogate for the extracellular matrix component, heparan sulfate. High-affinity binding of heparin/heparan sulfate to the Ig-like domain may proceed from surface charge complementarity, as suggested by homology three-dimensional modeling. These data point to N-MADD-4B processing and cell-surface proteoglycan binding as two possible mechanisms to regulate the interaction between MADD-4B and NLG-1 at GABAergic synapses.

中文翻译：

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Punctin/MADD-4 的 Ig 样结构域是与神经配体 NLG-1 胞外域相互作用的主要决定因素。

Punctin/MADD-4 是 ADAMTSL 细胞外基质蛋白家族的成员，被鉴定为线虫秀丽隐杆线虫中的顺行突触组织者。在 GABA 能神经肌肉接头处，短同种型 MADD-4B 结合神经配体 NLG-1 的胞外域，NLG-1 本身是抑制性突触的突触后组织者。为了确定它们伙伴关系的分子基础，我们生成了两种蛋白质的重组形式，并对它们的相互作用进行了全面的生化和生物物理研究，并辅以体内定位研究。我们表明 MADD-4B 的自发蛋白水解首先产生较短的 N-MADD-4B 形式，其包含四个血小板反应蛋白 (TSP) 结构域和一个 Ig 样结构域并结合 NLG-1。第二个处理事件消除了 C 端 Ig 样域以及 N-MADD-4B 结合 NLG-1 的能力。这些数据表明 Ig 样结构域是 N-MADD-4B 与 NLG-1 体外相互作用的主要决定因素。我们在体内进一步证明，这种 Ig 样结构域对于在秀丽隐杆线虫的 GABA 能突触处有效募集 GABAA 受体是必不可少的，尽管本身还不够。N-MADD-4B 与 NLG-1 的相互作用也被肝素破坏，肝素用作细胞外基质成分硫酸乙酰肝素的替代物。正如同源三维模型所表明的，肝素/硫酸乙酰肝素与 Ig 样结构域的高亲和力结合可能源于表面电荷互补性。