Source file for Fig 7C and 7D.

Some types of collagens, including transmembrane MACIT collagens and C. elegans cuticle collagens, are N-terminally cleaved at a dibasic site that resembles the consensus for furin or other proprotein convertases of the subtilisin/kexin (PCSK) family. Such cleavage may release transmembrane collagens from the plasma membrane and affect extracellular matrix assembly or structure. However, the functional consequences of such cleavage are unclear and evidence for the role of specific PCSKs is lacking. Here, we used endogenous collagen fusions to fluorescent proteins to visualize the secretion and assembly of the first collagen-based cuticle in C. elegans and then tested the role of the PCSK BLI-4 in these processes. Unexpectedly, we found that cuticle collagens SQT-3 and DPY-17 are secreted into the extraembryonic space several hours before cuticle matrix assembly. Furthermore, this early secretion depends on BLI-4/PCSK; in bli-4 and cleavage-site mutants, SQT-3 and DPY-17 are not efficiently secreted and instead form large intracellular puncta. Their later assembly into cuticle matrix is reduced but not entirely blocked. These data reveal a role for collagen N-terminal processing in intracellular trafficking and the control of matrix assembly in vivo. Our observations also prompt a revision of the classic model for C. elegans cuticle matrix assembly and the pre-cuticle-to-cuticle transition, suggesting that cuticle layer assembly proceeds via a series of regulated steps and not simply by sequential secretion and deposition.

多种胶原蛋白（collagens），涵盖跨膜MACIT胶原蛋白（transmembrane MACIT collagens）以及秀丽隐杆线虫（C. elegans）角质层胶原蛋白，可在双碱基位点（dibasic site）发生N端剪切；该位点的识别序列与弗林蛋白酶（furin）及枯草溶菌素/克赛菌素（PCSK）家族的前蛋白转化酶（proprotein convertases）共识序列高度相似。此类剪切可将跨膜胶原蛋白从质膜（plasma membrane）上释放，并影响细胞外基质（extracellular matrix）的组装与结构。然而，此类剪切的功能后果尚未明确，且缺乏特定PCSK家族成员参与该过程的实验证据。

本研究利用内源性胶原蛋白与荧光蛋白（fluorescent proteins）的融合蛋白，可视化了秀丽隐杆线虫中首个胶原蛋白基角质层的分泌与组装过程，并探究了PCSK家族成员BLI-4在该过程中的作用。令人意外的是，我们发现角质层胶原蛋白SQT-3与DPY-17在角质层基质组装前数小时便已被分泌至胚外空间（extraembryonic space）。此外，这一早期分泌过程依赖于BLI-4/PCSK；在bli-4基因缺失或剪切位点突变的线虫中，SQT-3与DPY-17无法被有效分泌，反而形成大量细胞内点状聚集（intracellular puncta）。它们后续组装为角质层基质的过程虽受到抑制，但并未完全阻断。

本研究数据揭示了胶原蛋白N端加工在细胞内运输及体内基质组装调控中的重要作用。我们的研究结果也促使我们重新审视秀丽隐杆线虫角质层基质组装及前角质层向角质层转变的经典模型，提示角质层组装是一系列受严格调控的步骤共同完成的，而非仅通过简单的顺序分泌与沉积实现。