Many extracellular matrix (ECM) associated proteins that influence ECM properties have Thrombospondin type 1 repeats (TSRs) which are modified with O-linked fucose. The O-fucose is added in the endoplasmic reticulum to folded TSRs by the enzyme Protein O-fucosyltransferase-2 (POFUT2) and is proposed to promote efficient trafficking of substrates. The importance of this modification for function of TSR-proteins is underscored by the early embryonic lethality of mouse embryos lacking Pofut2. To overcome early lethality and investigate the impact of the Pofut2 knockout on the secretion of POFUT2 substrates and on extracellular matrix properties in vivo, we deleted Pofut2 in the developing limb mesenchyme using Prrx1-Cre recombinase. Loss of Pofut2 in the limb mesenchyme caused significant shortening of the limbs, long bones and tendons and stiff joint resembling the musculoskeletal dysplasias in human and in mice with mutations in ADAMTS or ADAMTSL proteins. Limb shortening was evident at embryonic day 14.5 where loss of O-fucosylation led to an accumulation of fibrillin 2 (FBN2), decreased BMP and IHH signaling, and increased TGF-β signaling. Consistent with these changes we saw a decrease in the size of the hypertrophic zone with lower levels of Collagen-X. Unexpectedly, we observed minimal effects of the Pofut2 knockout on secretion of two POFUT2 substrates, CCN2 or ADAMTS17, in the developing bone. In contrast, CCN2 and two other POFUT2 substrates important for bone development, ADAMTS6 and 10, showed a decrease in secretion from POFUT2-null HEK293T cells in vitro. These combined results suggest that the impact of the Pofut2 mutation is cell-type specific. In addition, these observations raise the possibility that the O-fucose modification on TSRs extends beyond promoting efficient trafficking of POFUT2 substrates and has the potential to influence their function in the extracellular environment.

许多影响 ECM 特性的细胞外基质 (ECM) 相关蛋白具有用O连接的岩藻糖修饰的 1 型血小板反应蛋白重复序列​​ (TSR)。O-岩藻糖通过酶蛋白O-岩藻糖基转移酶 2 (POFUT2)在内质网中添加到折叠的 TSR 中，并被提议用于促进底物的有效运输。这种修饰对 TSR 蛋白功能的重要性通过缺乏Pofut2 的小鼠胚胎的早期胚胎致死率得到了强调。为了克服早期致死性并研究Pofut2敲除对 POFUT2 底物分泌和体内细胞外基质特性的影响，我们删除了 Pofut2使用Prrx1-Cre重组酶在发育中的肢体间充质中。肢体间充质中Pofut2的缺失导致四肢显着缩短，长骨和肌腱和关节僵硬，类似于人类和 ADAMTS 或 ADAMTSL 蛋白突变的小鼠的肌肉骨骼发育不良。在胚胎第 14.5 天，肢体缩短很明显，此时O-岩藻糖基化的丧失导致原纤维蛋白 2 (FBN2) 的积累，BMP 和 IHH 信号传导减少，以及 TGF-β 信号传导增加。与这些变化一致，我们看到肥大区的大小随着胶原蛋白-X 水平的降低而减小。出乎意料的是，我们观察到Pofut2的影响很小在发育中的骨骼中敲除两种 POFUT2 底物 CCN2 或 ADAMTS17 的分泌。相比之下，CCN2 和其他两种对骨发育很重要的 POFUT2 底物 ADAMTS6 和 10在体外显示出POFUT2无效 HEK293T 细胞的分泌减少。这些综合结果表明，Pofut2突变的影响是细胞类型特异性的。此外，这些观察结果提出了这样的可能性，即TSR 上的岩藻糖修饰超出了促进 POFUT2 底物的有效运输，并有可能影响它们在细胞外环境中的功能。