O-glycosylation of Notch epidermal growth factor-like (EGF) repeats has long been studied in the context of protein folding, secretion from cells, and protein function. Originally, the protein O-glucosyltransferase, POGLUT1, was thought to be the only enzyme adding an O-glucose modification to EGFs, specifically to a serine between cysteine one and two of a six cysteine containing EGF repeat. The POGLUT1 O-glucose can be elongated on Notch EGFs with xyloses by GXYLT1/2 and XXYLT1, forming a trisaccharide. Mouse knockouts of Poglut1 are embryonic lethal with Notch1-related phenotypes. Recently, protein O-glucosyltransferases POGLUT2 and POGLUT3 were shown to add an O-glucose modification to a serine located between cysteine three and four, distinct from the POGLUT1 modification. This modification was first discovered on EGF11 of NOTCH1, then subsequently mapped on extracellular matrix proteins fibrillin-1 (FBN1), fibrillin-2 (FBN2), and latent transforming growth factor beta-binding protein 1 (LTBP1). Poglut2/3 double knockout mice exhibit neonatal lethality like Fbn1 or Ltbp1 knockouts. In addition, Poglut2/3 double knockout mice display syndactyly, similar to Fbn2 knockouts. These studies also showed decreased secretion of FBN1 and 2 from fibroblasts and incorporation into the extracellular matrix. Site mapping established a putative POGLUT2/3 consensus sequence: C3-x-N-T-x-G-S-F/Y-x-C4. Alanine variants at conserved residues redefined consensus to: C3-x-x-x-x-x-S-x-x-C4. Marfan syndrome (MFS) is caused by FBN1 variants, and MFS variants in the POGLUT2/3 consensus display aberrant O-glucosylation. With the neonatal lethality of mouse Poglut2/3 double knockouts and the changes in O-glucosylation caused by MFS variants, further examination of the effects of O-glucose on FBN1 function are needed.

BACKGROUND:Fibrillins provide a scaffold for elastic fiber formation, which enables lung recoil and aortic compliance. Abnormal fibrillin microfibrils, as in Marfan syndrome, lead to enlarged alveoli, vascular stiffening, and aneurysms. Our earlier studies suggested that fibrillin function depends on O-glucosylation of its epidermal growth factor (EGF)-like repeats by protein O-glucosyltransferase 2 (POGLUT2) and 3 (POGLUT3). Mice lacking both enzymes (Poglut2/3 DKO) die soon after birth. Rare survivors are runted, have digit 2-3 syndactyly, and variable eye defects. Here, we wanted to determine whether O-glucosylation was important for fibrillin organization/function during lung and aorta development. RESULTS:Fibrillin microfibrils and elastic fibrils were reduced and disorganized in the Poglut2/3 DKO lung and aorta. In the lung, loss of O-glucosylation impaired airway branching, delayed sacculation, and postnatally caused mildly enlarged alveolar airspaces and reduced lipofibroblasts and capillary endothelial cells. In the ascending aorta, the Poglut2/3 DKO led to disorganized smooth muscle layers with increased nuclear YAP, potentially indicating a change in matrix stiffness. CONCLUSIONS:The lung and aorta defects in Poglut2/3 DKO resembled mouse Fbn1 and Ltbp mutant models, suggesting a common microfibril-driven mechanism. Future studies should examine how loss of fibrillin O-glucosylation affects microfibril-dependent regulation of TGF-β and BMP signaling and matrix biomechanics.

The separation of individual digits is dependent on establishment of digit-interdigit periodicity, remodeling of the interdigital mesenchyme, and invagination of interdigital epithelial tongues. In Protein O-glucosyltransferase 2 and 3 double knockout (Poglut2/3 DKO) mice, digits 2 and 3 are fused, suggesting a defect in one or more processes. POGLUT2/3 add O-linked glucose to epidermal growth factor-like (EGF) repeats. Syndactyly is also observed when genes encoding the POGLUT2/3 substrates fibrillin 2 (FBN2) or both Nidogen 1 and 2 (NID1/2) are knocked out, suggesting that O-glucosylation is important for their function or localization. In this study, we evaluated the distribution of these substrates during digit separation and the effects of the Poglut2/3 DKO on their localization and cell behavior. During digit separation, the FBNs underwent a dramatic reorganization. Aberrant levels and distribution of the FBNs were observed in the Poglut2/3 DKO and microfibrils isolated from Poglut2/3 DKO skin showed altered periodicity in Fibrillin microfibrils. In contrast, the Poglut2/3 DKO had no effect on the levels or localization of NID1. In Poglut2/3 DKOs, bone morphogenetic protein (BMP) signaling was reduced during digit development, especially in the anterior autopod. Early anterior reduction of BMP signaling could potentially affect spacing of digits 2 & 3. While later reduction of BMP signaling in the Poglut2/3 DKO in the digit 2-3 region was likely responsible for defects in clearance of interdigital mesenchyme and interdigital tongue morphogenesis. These results highlight the importance of POGLUT2/3 mediated O-glucosylation for FBN microfibril organization and raise the possibility that O-glucose modulates the biological or physical properties of the FBN microfibril network.

Fibrillin microfibrils play a critical role in the formation of elastic fibers, tissue/organ development, and cardiopulmonary function. These microfibrils not only provide structural support and flexibility to tissues, but they also regulate growth factor signaling through a plethora of microfibril-binding proteins in the extracellular space. Mutations in fibrillins are associated with human diseases affecting cardiovascular, pulmonary, skeletal, and ocular systems. Fibrillins consist of up to 47 epidermal growth factor (EGF)-like repeats, of which more than half are modified by protein O-glucosyltransferase 2 (POGLUT2) and/or POGLUT3. Loss of these modifications reduces secretion of N-terminal fibrillin constructs overexpressed in vitro. Here, we investigated the role of POGLUT2 and POGLUT3 in vivo using a Poglut2/3 double knockout (DKO) mouse model. Blocking O-glucosylation caused neonatal death with skeletal, pulmonary, and eye defects reminiscent of fibrillin/elastin mutations. Proteomic analyses of DKO dermal fibroblast medium and extracellular matrix provided evidence that fibrillins were more sensitive to loss of O-glucose compared to other POGLUT2/3 substrates. This conclusion was supported by immunofluorescent analyses of late gestation DKO lungs where FBN levels were reduced and microfibrils appeared fragmented in the pulmonary arteries and veins, bronchioles, and developing saccules. Defects in fibrillin microfibrils likely contributed to impaired elastic fiber formation and histological changes observed in DKO lung blood vessels, bronchioles, and saccules. Collectively, these results highlight the importance of POGLUT2/3-mediated O-glucosylation in vivo and open the possibility that O-glucose modifications on fibrillin influence microfibril assembly and or protein interactions in the ECM environment.

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) domains 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 beta 3glucosyltransferase (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.

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.

We propose that Protein O‐fucosyltransferase 2 (POFUT2) and β3‐glucosyltransferase (B3GLCT) function to ensure efficient folding of forty‐nine proteins containing tandemly repeated Thrombospondin Type 1 Repeats (TSRs). In the endoplasmic reticulum (ER), POFUT2 and B3GLCT recognize properly folded TSRs and act sequentially to add the unusual O‐linked Glucoseβ1‐3Fucose disaccharide to either a serine or threonine residue located within the POFUT2 consensus sequence (C‐X‐X‐S/T‐C). Once all TSRs are modified the target protein exits the ER. In crystal structures the disaccharide forms H‐bonds and van der Waals interactions with underlying amino acids in the TSR, raising the possibility that the disaccharide functions as a surrogate amino acid to stabilize the correctly folded TSR. Results from in vitro folding/refolding assays provide evidence that the disaccharide stabilizes the TSR fold and accelerates the overall rate of protein folding. Consistent with this prediction, loss of POFUT2 or B3GLCT in cell culture impairs trafficking of POFUT2/B3GLCT targets. To begin to evaluate the function of the disaccharide in vivo, we generated mouse loss of function mutations in Pofut2 and B3glct. Loss of Pofut2 causes early embryo lethality due to a block in gastrulation. In contrast, B3glct animals survive, but have craniofacial and skeletal abnormalities, defects in pigmentation, soft tissue syndactyly, and develop hydrocephalus. The mouse B3glct phenotype shares similarities to Peters Plus syndrome in humans, caused by recessive mutations in B3GLCT. The distinct differences in the effects of these mutations during mouse development support a model whereby the O‐linked fucose is essential for folding and secretion of all POFUT2/B3GLCT target proteins and that B3GLCT‐mediated extension to the glucose‐fucose disaccharide is only required by a subset of sensitive targets.Support or Funding InformationThese studies were supported by funding from NIH R01 HD090156 and NIH R01 HD096030 and 2019 Hydrocephalus Association Innovator Award.Model for how POFUT2/B3GLCT mediated addition of the O‐linked glucose‐fucose disaccharide promotes efficient folding of target proteins containing the Thrombospondin Type 1 Repeats (TSR) fold. Addition of fucose prevents reentry of the TSR to the folding cycle and stabilizes the fold. Extension with glucose provides further stability to the TSR. Once the target protein is fully modified it exits the endoplasmic reticulum (ER).Figure 1

Peters plus syndrome, characterized by defects in eye and skeletal development with isolated cases of ventriculomegaly/hydrocephalus, is caused by mutations in the β3-glucosyltransferase (B3GLCT) gene. In the endoplasmic reticulum, B3GLCT adds glucose to O-linked fucose on properly folded thrombospondin type 1 repeats (TSRs). The resulting glucose-fucose disaccharide is proposed to stabilize the TSR fold and promote secretion of B3GLCT substrates, with some substrates more sensitive than others to loss of glucose. Mouse B3glct mutants develop hydrocephalus at high frequency. In this study, we demonstrated that B3glct mutant ependymal cells had fewer cilia basal bodies and altered translational polarity compared to controls. Localization of mRNA encoding A Disintegrin and Metalloproteinase with ThromboSpondin type 1 repeat 20 (ADAMTS20) and ADAMTS9 suggested that reduced function of these B3GLCT substrates contributed to ependymal cell abnormalities. In addition, we showed that multiple B3GLCT substrates (Adamts3, Adamts9 and Adamts20) are expressed by the subcommissural organ, that subcommissural organ-spondin ((SSPO) also known as SCO-spondin) TSRs were modified with O-linked glucose-fucose and that loss of B3GLCT reduced secretion of SSPO in cultured cells. In the B3glct mutant, intracellular levels of SSPO were reduced and BiP levels increased, suggesting a folding defect. Secreted SSPO colocalized with BiP, raising the possibility that abnormal extracellular assembly of SSPO into Reissner's fiber also contributed to impaired CSF flow in mutants. Combined, these studies underscore the complexity of the B3glct mutant hydrocephalus phenotype and demonstrate that impaired cerebrospinal fluid (CSF) flow likely stems from the collective effects of the mutation on multiple processes.

ADAMTSL2 mutations cause an autosomal recessive connective tissue disorder, geleophysic dysplasia 1 (GPHYSD1), which is characterized by short stature, small hands and feet, and cardiac defects. ADAMTSL2 is a matricellular protein previously shown to interact with latent transforming growth factor-β binding protein 1 and influence assembly of fibrillin 1 microfibrils. ADAMTSL2 contains seven thrombospondin type-1 repeats (TSRs), six of which contain the consensus sequence for O-fucosylation by protein O-fucosyltransferase 2 (POFUT2). O-fucose–modified TSRs are subsequently elongated to a glucose β1-3-fucose (GlcFuc) disaccharide by β1,3-glucosyltransferase (B3GLCT). B3GLCT mutations cause Peters Plus Syndrome (PTRPLS), which is characterized by skeletal defects similar to GPHYSD1. Several ADAMTSL2 TSRs also have consensus sequences for C-mannosylation. Six reported GPHYSD1 mutations occur within the TSRs and two lie near O-fucosylation sites. To investigate the effects of TSR glycosylation on ADAMTSL2 function, we used MS to identify glycan modifications at predicted consensus sequences on mouse ADAMTSL2. We found that most TSRs were modified with the GlcFuc disaccharide at high stoichiometry at O-fucosylation sites and variable mannose stoichiometry at C-mannosylation sites. Loss of ADAMTSL2 secretion in POFUT2−/− but not in B3GLCT−/− cells suggested that impaired ADAMTSL2 secretion is not responsible for skeletal defects in PTRPLS patients. In contrast, secretion was significantly reduced for ADAMTSL2 carrying GPHYSD1 mutations (S641L in TSR3 and G817R in TSR6), and S641L eliminated O-fucosylation of TSR3. These results provide evidence that abnormalities in GPHYSD1 patients with this mutation are caused by loss of O-fucosylation on TSR3 and impaired ADAMTSL2 secretion.

•Protein O-fucosyltransferases 1 and 2 (POFUT1 and POFUT2) are ER-localized and modify EGF repeats and TSRs.•Both POFUT1 and POFUT2 are exquisitely selective for properly folded substrates.•O-Fucose on Notch EGF repeats directly participates in intermolecular interactions with Notch ligands.•O-Fucose glycans on both EGF repeats and TSRs interact with underlying amino acids, stabilizing the folded domains.•POFUT1 and POFUT2 participate in non-canonical ER quality control pathways for folding of EGF repeats and TSRs, respectively.

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 β3-glucosyltransferase (B3GLCT) gene. Protein O-fucosyltransferase 2 (POFUT2) and B3GLCT work sequentially to add an O-linked glucose β1-3fucose 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.

Multiciliated cells of the airways, brain ventricles, and female reproductive tract provide the motive force for mucociliary clearance, cerebrospinal fluid circulation, and ovum transport. Despite their clear importance to human biology and health, the molecular mechanisms underlying multiciliated cell differentiation are poorly understood. Prior studies implicate the distal appendage/transition fiber protein CEP164 as a central regulator of primary ciliogenesis; however, its role in multiciliogenesis remains unknown. In this study, we have generated a novel conditional mouse model that lacks CEP164 in multiciliated tissues and the testis. These mice show a profound loss of airway, ependymal, and oviduct multicilia and develop hydrocephalus and male infertility. Using primary cultures of tracheal multiciliated cells as a model system, we found that CEP164 is critical for multiciliogenesis, at least in part, via its regulation of small vesicle recruitment, ciliary vesicle formation, and basal body docking. In addition, CEP164 is necessary for the proper recruitment of another distal appendage/transition fiber protein Chibby1 (Cby1) and its binding partners FAM92A and FAM92B to the ciliary base in multiciliated cells. In contrast to primary ciliogenesis, CEP164 is dispensable for the recruitment of intraflagellar transport (IFT) components to multicilia. Finally, we provide evidence that CEP164 differentially controls the ciliary targeting of membrane-associated proteins, including the small GTPases Rab8, Rab11, and Arl13b, in multiciliated cells. Altogether, our studies unravel unique requirements for CEP164 in primary versus multiciliogenesis and suggest that CEP164 modulates the selective transport of membrane vesicles and their cargoes into the ciliary compartment in multiciliated cells. Furthermore, our mouse model provides a useful tool to gain physiological insight into diseases associated with defective multicilia.

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.

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.

Mesoderm development (MESD) is a 224 amino acid mouse protein that acts as a molecular chaperone for the low-density lipoprotein receptor (LDLR) family. Here, we provide evidence that the region 45-184 of MESD is essential and sufficient for this function and suggest a model for its mode of action. NMR studies reveal a beta-alpha-beta-beta-alpha-beta core domain with an alpha-helical N-terminal extension that interacts with the beta sheet in a dynamic manner. As a result, the structural ensemble contains open (active) and closed (inactive) forms, allowing for regulation of chaperone activity through substrate binding. The mutant W61 R, which is lethal in Drosophila, adopts only the open state. The receptor motif recognized by MESD was identified by in vitro-binding studies. Furthermore, in vivo functional evidence for the relevance of the identified contact sites in MESD is provided.

Low‐density lipoprotein receptor family mediated endocytosis in the mouse visceral endoderm. Photo shows a whole mount view of a E7.5 wild‐type embryo that was incubated with 50nM receptor associated protein (RAP) tagged with AlexaFluor 488 to visualize endocytosis in the visceral endoderm. Embryos were then fixed and counterstained with Alexa 594‐Phalloidin to mark cell boundaries (color‐corrected to magenta in Photoshop) and DAPI to mark nuclei (blue). From Lighthouse et al., Developmental Dynamics 240:577–588, 2011.