O-fucosylation of thrombospondin type I repeats is dispensable for trafficking thrombospondin 1 to platelet secretory granules.

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 ß3glucosyltransferase (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.

O-fucosylation of thrombospondin type 1 repeats is essential for ECM remodeling and signaling during bone development.

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-ß 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.

ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.

Peters plus syndrome (MIM #261540 PTRPLS), characterized by defects in eye development, prominent forehead, hypertelorism, short stature and brachydactyly, is caused by mutations in the ß3-glucosyltransferase (B3GLCT) gene. Protein O-fucosyltransferase 2 (POFUT2) and B3GLCT work sequentially to add an O-linked glucose ß1-3fucose disaccharide to properly folded thrombospondin type 1 repeats (TSRs). Forty-nine proteins are predicted to be modified by POFUT2, and nearly half are members of the ADAMTS superfamily. Previous studies suggested that O-linked fucose is essential for folding and secretion of POFUT2-modified proteins and that B3GLCT-mediated extension to the disaccharide is essential for only a subset of targets. To test this hypothesis and gain insight into the origin of PTRPLS developmental defects, we developed and characterized two mouse B3glct knockout alleles. Using these models, we tested the role of B3GLCT in enabling function of ADAMTS9 and ADAMTS20, two highly conserved targets whose functions are well characterized in mouse development. The mouse B3glct mutants developed craniofacial and skeletal abnormalities comparable to PTRPLS. In addition, we observed highly penetrant hydrocephalus, white spotting and soft tissue syndactyly. We provide strong genetic and biochemical evidence that hydrocephalus and white spotting in B3glct mutants resulted from loss of ADAMTS20, eye abnormalities from partial reduction of ADAMTS9 and cleft palate from loss of ADAMTS20 and partially reduced ADAMTS9 function. Combined, these results provide compelling evidence that ADAMTS9 and ADAMTS20 were differentially sensitive to B3GLCT inactivation and suggest that the developmental defects in PTRPLS result from disruption of a subset of highly sensitive POFUT2/B3GLCT targets such as ADAMTS20.

Genetic and biochemical evidence that gastrulation defects in Pofut2 mutants result from defects in ADAMTS9 secretion.

Protein O-fucosyltransferase 2 (POFUT2) adds O-linked fucose to Thrombospondin Type 1 Repeats (TSR) in 49 potential target proteins. Nearly half the POFUT2 targets belong to the A Disintegrin and Metalloprotease with ThromboSpondin type-1 motifs (ADAMTS) or ADAMTS-like family of proteins. Both the mouse Pofut2 RST434 gene trap allele and the Adamts9 knockout were reported to result in early embryonic lethality, suggesting that defects in Pofut2 mutant embryos could result from loss of O-fucosylation on ADAMTS9. To address this question, we compared the Pofut2 and Adamts9 knockout phenotypes and used Cre-mediated deletion of Pofut2 and Adamts9 to dissect the tissue-specific role of O-fucosylated ADAMTS9 during gastrulation. Disruption of Pofut2 using the knockout (LoxP) or gene trap (RST434) allele, as well as deletion of Adamts9, resulted in disorganized epithelia (epiblast, extraembryonic ectoderm, and visceral endoderm) and blocked mesoderm formation during gastrulation. The similarity between Pofut2 and Adamts9 mutants suggested that disruption of ADAMTS9 function could be responsible for the gastrulation defects observed in Pofut2 mutants. Consistent with this prediction, CRISPR/Cas9 knockout of POFUT2 in HEK293T cells blocked secretion of ADAMTS9. We determined that Adamts9 was dynamically expressed during mouse gastrulation by trophoblast giant cells, parietal endoderm, the most proximal visceral endoderm adjacent to the ectoplacental cone, extraembryonic mesoderm, and anterior primitive streak. Conditional deletion of either Pofut2 or Adamts9 in the epiblast rescues the gastrulation defects, and identified a new role for O-fucosylated ADAMTS9 during morphogenesis of the amnion and axial mesendoderm. Combined, these results suggested that loss of ADAMTS9 function in the extra embryonic tissue is responsible for gastrulation defects in the Pofut2 knockout. We hypothesize that loss of ADAMTS9 function in the most proximal visceral endoderm leads to slippage of the visceral endoderm and altered characteristics of the extraembryonic ectoderm. Consequently, loss of input from the extraembryonic ectoderm and/or compression of the epiblast by Reichert's membrane blocks gastrulation. In the future, the Pofut2 and Adamts9 knockouts will be valuable tools for understanding how local changes in the properties of the extracellular matrix influence the organization of tissues during mammalian development.

Development of a conditional Mesd (mesoderm development) allele for functional analysis of the low-density lipoprotein receptor-related family in defined tissues.

The Low-density lipoprotein receptor-Related Protein (LRP) family members are essential for diverse processes ranging from the regulation of gastrulation to the modulation of lipid homeostasis. Receptors in this family bind and internalize a diverse array of ligands in the extracellular matrix (ECM). As a consequence, LRPs regulate a wide variety of cellular functions including, but not limited to lipid metabolism, membrane composition, cell motility, and cell signaling. Not surprisingly, mutations in single human LRPs are associated with defects in cholesterol metabolism and development of atherosclerosis, abnormalities in bone density, or aberrant eye vasculature, and may be a contributing factor in development of Alzheimer's disease. Often, members of this diverse family of receptors perform overlapping roles in the same tissues, complicating the analysis of their function through conventional targeted mutagenesis. Here, we describe development of a mouse Mesd (Mesoderm Development) conditional knockout allele, and demonstrate that ubiquitous deletion of Mesd using Cre-recombinase blocks gastrulation, as observed in the traditional knockout and albino-deletion phenotypes. This conditional allele will serve as an excellent tool for future characterization of the cumulative contribution of LRP members in defined tissues.