Matrix Biology Program, Benaroya Research Institute, Seattle, WA, USA.
Department of Anatomy and Medical Imaging, University of Auckland, Auckland, New Zealand.
Matrix Biol. 2020 Jul;89:27-42. doi: 10.1016/j.matbio.2020.01.002. Epub 2020 Jan 27.
Versican is a large extracellular matrix (ECM) chondroitin sulfate (CS) proteoglycan found in most soft tissues, which is encoded by the VCAN gene. At least four major isoforms (V0, V1, V2, and V3) are generated via alternative splicing. The isoforms of versican are expressed and accumulate in various tissues during development and disease, where they contribute to ECM structure, cell growth and migration, and immune regulation, among their many functions. While several studies have identified the mRNA transcript for the V3 isoform in a number of tissues, little is known about the synthesis, secretion, and targeting of the V3 protein. In this study, we used lentiviral generation of doxycycline-inducible rat V3 with a C-terminal tag in stable NIH 3T3 cell lines and demonstrated that V3 is processed through the classical secretory pathway. We further show that N-linked glycosylation is required for efficient secretion and solubility of the protein. By site-directed mutagenesis, we identified amino acids 57 and 330 as the active N-linked glycosylation sites on V3 when expressed in this cell type. Furthermore, exon deletion constructs of V3 revealed that exons 11-13, which code for portions of the carboxy region of the protein (G3 domain), are essential for V3 processing and secretion. Once secreted, the V3 protein associates with hyaluronan along the cell surface and within the surrounding ECM. These results establish critical parameters for the processing, solubility, and targeting of the V3 isoform by mammalian cells and establishes a role for V3 in the organization of hyaluronan.
蛋白聚糖聚糖（CS）是一种大型细胞外基质（ECM），在大多数软组织中都有发现，由 VCAN 基因编码。至少有四种主要的同种型（V0、V1、V2 和 V3）通过选择性剪接产生。同种型的蛋白聚糖在发育和疾病过程中在各种组织中表达和积累，在这些组织中，它们有助于 ECM 结构、细胞生长和迁移以及免疫调节等多种功能。虽然有几项研究已经在许多组织中鉴定出 V3 同种型的 mRNA 转录本，但对于 V3 蛋白的合成、分泌和靶向知之甚少。在这项研究中，我们使用慢病毒生成了带有 C 末端标签的可诱导表达的大鼠 V3，并在稳定的 NIH 3T3 细胞系中进行了研究，证明了 V3 是通过经典的分泌途径进行加工的。我们进一步表明，N-连接糖基化是蛋白质有效分泌和可溶性所必需的。通过定点突变，我们确定了在该细胞类型中表达时 V3 的 57 位和 330 位氨基酸为活性 N-连接糖基化位点。此外，V3 的外显子缺失构建体表明，编码蛋白羧基区域部分（G3 结构域）的外显子 11-13 对于 V3 的加工和分泌是必不可少的。一旦分泌，V3 蛋白就会与透明质酸一起沿着细胞表面和周围的 ECM 聚集。这些结果确定了哺乳动物细胞对 V3 同种型进行加工、可溶性和靶向的关键参数，并确立了 V3 在透明质酸组织中的作用。
