POFUT2基因敲除HEK293细胞

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.

Thrombospondin type-1 repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by β3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization are poorly understood. Here, we conducted molecular dynamics (MD) simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER.

Thrombospondin 1 (THBS1) is a secreted extracellular matrix glycoprotein that regulates a variety of cellular and physiological processes. THBS1's diverse functions are attributed to interactions between the modular domains of THBS1 with an array of proteins found in the extracellular matrix. THBS1's three Thrombospondin type 1 repeats (TSRs) are modified with O-linked glucose-fucose disaccharide and C-mannose. It is unknown whether these modifications impact trafficking and/or function of THBS1 in vivo. The O-fucose is added by Protein O-fucosyltransferase 2 (POFUT2) and is sequentially extended to the disaccharide by β3glucosyltransferase (B3GLCT). The C-mannose is added by one or more of four C-mannosyltransferases. O-fucosylation by POFUT2/B3GLCT in the endoplasmic reticulum has been proposed to play a role in quality control by locking TSR domains into their three-dimensional fold, allowing for proper secretion of many O-fucosylated substrates. Prior studies showed the siRNA knockdown of POFUT2 in HEK293T cells blocked secretion of TSRs 1-3 from THBS1. Here we demonstrated that secretion of THBS1 TSRs 1-3 was not reduced by CRISPR-Cas9-mediated knockout of POFUT2 in HEK293T cells and demonstrated that knockout of Pofut2 or B3glct in mice did not reduce the trafficking of endogenous THBS1 to secretory granules of platelets, a major source of THBS1. Additionally, we demonstrated that all three TSRs from platelet THBS1 were highly C-mannosylated, which has been shown to stabilize TSRs in vitro. Combined, these results suggested that POFUT2 substrates with TSRs that are also modified by C-mannose may be less susceptible to trafficking defects resulting from the loss of the glucose-fucose disaccharide.