Thrombospondin Type 1 Repeats Research Articles

Structure and function of Semaphorin-5A glycosaminoglycan interactions

Integration of extracellular signals by neurons is pivotal for brain development, plasticity, and repair. Axon guidance relies on receptor-ligand interactions crosstalking with extracellular matrix components. Semaphorin-5A (Sema5A) is a bifunctional guidance cue exerting attractive and inhibitory effects on neuronal growth through the interaction with heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAGs), respectively. Sema5A harbors seven thrombospondin type-1 repeats (TSR1-7) important for GAG binding, however the underlying molecular basis and functions in vivo remain enigmatic. Here we dissect the structural basis for Sema5A:GAG specificity and demonstrate the functional significance of this interaction in vivo. Using x-ray crystallography, we reveal a dimeric fold variation for TSR4 that accommodates GAG interactions. TSR4 co-crystal structures identify binding residues validated by site-directed mutagenesis. In vitro and cell-based assays uncover specific GAG epitopes necessary for TSR association. We demonstrate that HS-GAG binding is preferred over CS-GAG and mediates Sema5A oligomerization. In vivo, Sema5A:GAG interactions are necessary for Sema5A function and regulate Plexin-A2 dependent dentate progenitor cell migration. Our study rationalizes Sema5A associated developmental and neurological disorders and provides mechanistic insights into how multifaceted guidance functions of a single transmembrane cue are regulated by proteoglycans.

A Novel Variant on the Thrombospondin Type-1 Repeat 2 Domain of ADAMTS13 in a Parturient with Suspected Hereditary Thrombotic Thrombocytopenic Purpura and Unusually High ADAMTS13 Activity.

A Novel Variant on the Thrombospondin Type-1 Repeat 2 Domain of ADAMTS13 in a Parturient with Suspected Hereditary Thrombotic Thrombocytopenic Purpura and Unusually High ADAMTS13 Activity.

Destabilization of vitelline membrane outer layer protein 1 homolog (VMO1) by C-mannosylation.

C-mannosylation is a rare type of protein glycosylation whereby a single mannose is added to the first tryptophan in the consensus sequence Trp-Xaa-Xaa-Trp/Cys (in which Xaa represents any amino acid). Its consensus sequence is mainly found in proteins containing a thrombospondin type-1 repeat (TSR1) domain and in type I cytokine receptors. In these proteins, C-mannosylation affects protein secretion, intracellular localization, and protein stability; however, the role of C-mannosylation in proteins that are not type I cytokine receptors and/or do not contain a TSR1 domain is less well explored. In this study, we focused on human vitelline membrane outer layer protein 1 homolog (VMO1). VMO1, which possesses two putative C-mannosylation sites, is a 21-kDa secreted protein that does not contain a TSR1 domain and is not a type I cytokine receptor. Mass spectrometry analyses revealed that VMO1 is C-mannosylated at Trp105 but not at Trp44 . Although C-mannosylation does not affect the extracellular secretion of VMO1, it destabilizes the intracellular VMO1. In addition, a structural comparison between VMO1 and C-mannosylated VMO1 showed that the modification of the mannose changes the conformation of three loops in VMO1. Taken together, our results demonstrate the first example of C-mannosylation for protein destabilization of VMO1.

SCO-spondin-derived peptide NX210 rescues neurons from cerebral ischemia/reperfusion injury through modulating the Integrin-β1 mediated PI3K/Akt pathway

Ischemic stroke is a common condition with high morbidity and mortality, causing irreversible neuronal damage and seriously affecting neurological function. There has been no ideal effective treatment so far. The NX210 peptide is derived from the thrombospondin type 1 repeat (TSR) sequence of SCO-spondin, and has been reported to exert various neurogenic properties. This study investigated whether NX210 had therapeutic effects and possible underlying mechanisms against cerebral ischemia/reperfusion (I/R). Therefore, primary embryonic rat cortical neurons and Sprague–Dawley (SD) rats that were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion/reperfusion (MCAO/R) injuries, respectively, were treated with or without NX210. We found that NX210 reduced OGD/R-induced cell viability loss and cytotoxicity. NX210 decreased cerebral infarct volume and brain edema, ameliorated neurological dysfunction, attenuated oxidative stress damage, and diminished neuronal apoptosis in MCAO/R rats. Furthermore, western blot analysis shown that treatment with NX210 up-regulated the expression of Integrin-β1, phosphorylated-PI3K (p-PI3K) and phosphorylated-Akt (p-Akt). The Integrin-β1 specific inhibitor, ATN-161, was used to identify pathways involved. The anti-oxidation activities and anti-apoptosis of NX210 was reversed by treatment with ATN-161. Overall, our results indicated that NX210 prevents oxidative stress and neuronal apoptosis in cerebral I/R via upregulation of the Integrin-β1/PI3K/Akt signaling pathway. These results indicated that NX210 may be a promising therapeutic candidate for ischemic stroke.

O-fucosylation stabilizes the TSR3 motif in thrombospondin-1 by interacting with nearby amino acids and protecting a disulfide bond

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.

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-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.

RTN4/NoGo-receptor binding to BAI adhesion-GPCRs regulates neuronal development

RTN4-binding proteins were widely studied as "NoGo" receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65Å crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.

Involvement of DPY19L3 in Myogenic Differentiation of C2C12 Myoblasts.

DPY19L3 has been identified as a C-mannosyltransferase for thrombospondin type-1 repeat domain-containing proteins. In this study, we focused on the role of DPY19L3 in the myogenic differentiation of C2C12 mouse myoblast cells. We carried out DPY19L3 gene depletion using the CRISPR/Cas9 system. The result showed that these DPY19L3-knockout cells could not be induced for differentiation. Moreover, the phosphorylation levels of MEK/ERK and p70S6K were suppressed in the DPY19L3-knockout cells compared with that of parent cells, suggesting that the protein(s) that is(are) DPY19L3-mediated C-mannosylated and regulate(s) MEK/ERK or p70S6K signaling is(are) required for the differentiation.

Peters plus syndrome mutations affect the function and stability of human β1,3-glucosyltransferase

Peters Plus Syndrome (PTRPLS OMIM #261540) is a severe congenital disorder of glycosylation where patients have multiple structural anomalies, including Peters anomaly of the eye (anterior segment dysgenesis), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable additional abnormalities. PTRPLS patients and some Peters Plus-like (PTRPLS-like) patients (who only have a subset of PTRPLS phenotypes, have mutations in the gene encoding β1,3-glucosyltransferase [B3GLCT]). B3GLCT catalyzes the transfer of glucose to O-linked fucose on thrombospondin type-1 repeats. Most B3GLCT substrate proteins belong to the ADAMTS superfamily and play critical roles in extracellular matrix. We sought to determine whether the PTRPLS or PTRPLS-like mutations abrogated B3GLCT activity. B3GLCT has two putative active sites, one in the N-terminal region and the other in the C-terminal glycosyltransferase domain. Using sequence analysis and in vitro activity assays, we demonstrated that the C-terminal domain catalyzes transfer of glucose to O-linked fucose. We also generated a homology model of B3GLCT and identified D421 as the catalytic base. PTRPLS and PTRPLS-like mutations were individually introduced into B3GLCT, and the mutated enzymes were evaluated using in vitro enzyme assays and cell-based functional assays. Our results demonstrated that PTRPLS mutations caused loss of B3GLCT enzymatic activity and/or significantly reduced protein stability. In contrast, B3GLCT with PTRPLS-like mutations retained enzymatic activity, although some showed a minor destabilizing effect. Overall, our data supports the hypothesis that loss of glucose from B3GLCT substrate proteins is responsible for the defects observed in PTRPLS patients, but not for those observed in PTRPLS-like patients.

Role of O ‐linked glucose‐fucose disaccharide modification of thrombospondin type I repeats in protein folding and embryo development

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, resulting from functional reduction of ADAMTS9 in extraembryonic tissue. 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. We propose that hydrocephalus in B3glct mutants results from the combined effects of the mutation on ADAMTS9 and ADAMTS20 in ependymal cells and Sco-Spondin (SSPO) in the subcommissural organ. 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.

Hydrocephalus in mouse B3glct mutants is likely caused by defects in multiple B3GLCT substrates in ependymal cells and subcommissural organ.

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.

Regulation of N-glycosylation and secretion of Isthmin-1 by its C-mannosylation

BackgroundC-mannosylation is a type of protein glycosylation. Human Isthmin-1 (ISM1) is a 52-kDa secreted protein with a thrombospondin type 1 repeat (TSR) domain, containing two consensus C-mannosylation sequences at Trp223 and Trp226. In this study, we sought to examine the role of C-mannosylation in the secretion of ISM1. MethodsWe established and cultured an ISM1-overexpressing HT1080 cell line and purified recombinant ISM1 for analysis from the conditioned medium by LC-MS/MS. Subcellular localization of ISM1 was observed by confocal fluorescence microscopy. ResultsWe found that ISM1 is C-mannosylated at Trp223 and Trp226 in the TSR domain. To determine the functions of the C-mannosylation of ISM1, we established a C-mannosylation-defective mutant ISM1-overexpressing HT1080 cell line and measured its secretion of ISM1. The secretion of ISM1 decreased significantly in this mutant ISM1-overexpressing line compared with wild-type cells. Furthermore, ISM1 was N-glycosylated only in these C-mannosylation-defective cells. ConclusionsISM1 is C-mannosylated in its TSR domain, and the status of the C-mannosylation of ISM1 affects its N-glycosylation. General significanceThe C-mannosylation of ISM1 regulates its N-glycosylation status.

Requirement for C-mannosylation to be secreted and activated a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4)

BackgroundC-mannosylation is a unique type of glycosylation. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a multidomain extracellular metalloproteinase that contains several potential C-mannosylation sites. Although some ADAMTS family proteins have been reported to be C-mannosylated proteins, whether C-mannosylation affects the activation and protease activity of these proteins is unclear. MethodsWe established wild-type and mutant ADAMTS4-overexpressing HT1080 cell lines. Recombinant ADAMTS4 was purified from the conditioned medium of the wild-type ADAMTS4-overexpressing cells, and the C-mannosylation sites of ADAMTS4 were identified by LC-MS/MS. The processing, secretion, and intracellular localization of ADAMTS4 were examined by immunoblot and immunofluorescence analyses. ADAMTS4 enzymatic activity was evaluated by assessing the cleavage of recombinant aggrecan. ResultsWe identified that ADAMTS4 is C-mannosylated at Trp404 in the metalloprotease domain and at Trp523, Trp526, and Trp529 in the thrombospondin type 1 repeat (TSR). The replacement of Trp404 with Phe affected ADAMTS4 processing, without affecting secretion and intracellular localization. In contrast, the substitution of Trp523, Trp526, and Trp529 with Phe residues suppressed ADAMTS4 secretion, processing, intracellular trafficking, and enzymatic activity. ConclusionsOur results demonstrated that the C-mannosylation of ADAMTS4 plays important roles in protein processing, intracellular trafficking, secretion, and enzymatic activity. General significanceBecause C-mannosylation appears to regulate many ADAMTS4 functions, C-mannosylation may also affect other members of the ADAMTS superfamily.

O-Fucosylation of ADAMTSL2 is required for secretion and is impacted by geleophysic dysplasia-causing mutations

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.

Identification of scavenger receptors and thrombospondin-type-1 repeat proteins potentially relevant for plastid recognition in Sacoglossa.

Functional kleptoplasty is a photosymbiotic relationship, in which photosynthetically active chloroplasts serve as an intracellular symbiont for a heterotrophic host. Among Metazoa, functional kleptoplasty is only found in marine sea slugs belonging to the Sacoglossa and recently described in Rhabdocoela worms. Although functional kleptoplasty has been intensively studied in Sacoglossa, the fundamentals of the specific recognition of the chloroplasts and their subsequent incorporation are unknown. The key to ensure the initiation of any symbiosis is the ability to specifically recognize the symbiont and to differentiate a symbiont from a pathogen. For instance, in photosymbiotic cnidarians, several studies have shown that the host innate immune system, in particular scavenger receptors (SRs) and thrombospondin‐type‐1 repeat (TSR) protein superfamily, is playing a major role in the process of recognizing and differentiating symbionts from pathogens. In the present study, SRs and TSRs of three Sacoglossa sea slugs, Elysia cornigera, Elysia timida, and Elysia chlorotica, were identified by translating available transcriptomes into potential proteins and searching for receptor specific protein and/or transmembrane domains. Both receptors classes are highly diverse in the slugs, and many new domain arrangements for each receptor class were found. The analyses of the gene expression of these three species provided a set of species‐specific candidate genes, that is, SR‐Bs, SR‐Es, C‐type lectins, and TSRs, that are potentially relevant for the recognition of kleptoplasts. The results set the base for future experimental studies to understand if and how these candidate receptors are indeed involved in chloroplast recognition.

How Do O‐Fucose Glycans Stabilize Thrombospondin Type 1 Repeats?

Thrombospondin Type 1 Repeats (TSRs) are small cysteine‐rich domains found in multiple cell‐surface and secreted proteins. TSRs containing the consensus sequence Cxx(S/T)C are typically modified on the serine or threonine with an O‐linked Glcβ1‐3Fuc disaccharide. The O‐fucose is added by Protein O‐fucosyltransferase 2 (POFUT2), which is then elongated by β1‐3glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in early embryonic lethality, and human mutations in B3GLCT cause Peters Plus Syndrome (PPS), a Congenital Disorder of Glycosylation. POFUT2 and B3GLCT are localized in the endoplasmic reticulum (ER) ‐ the folding compartment for proteins in the secretory pathway, and POFUT2 only adds fucose to properly folded TSRs. Thus, POFUT2 appears to function as a folding sensor for TSRs, and both POFUT2 and B3GLCT have been proposed to assist in the folding of TSR‐containing proteins. Interestingly, loss of POFUT2 causes secretion defects for most TSR‐containing proteins, while loss of B3GLCT only effects secretion of a subset of these proteins. The mechanisms behind why POFUT2 is required for section of most TSR‐containing proteins but B3GLCT is required for only a few is not understood. We hypothesize that this unique disaccharide on TSRs is interacting with amino acids in close proximity to the sugars thereby stabilizing the folded state of TSRs and ultimately the protein as a whole, and that the fucose has the major stabilizing effect, while the glucose is only required for some TSRs. To test this hypothesis, we designed a Reductive Unfolding Assay to monitor the effects of the sugars on the stability of TSRs. We have identified amino acids affected by the presence of the fucose or glucose using NMR and molecular dynamics simulations. We have mutated several of these amino acids to determine if they reduce the stabilizing effects of the sugars using the reductive unfolding assay. One such mutant, P547A in TSR3 from human thrombospondin‐1, decreases the sugar‐mediated stabilization of TSR3, providing evidence that the disaccharide stabilizes the folded state by interacting with certain amino acids in close proximity. We are examining the stabilizing effects of the fucose and glucose on several other TSRs as well. These results provide further evidence that POFUT2 and B3GLCT act as quality control enzymes inside the ER.Support or Funding InformationThis work was supported by NIH grant HD090156.

Analyzing the Effects of Peters Plus Syndrome Mutations on Human β1,3‐glucosyltransferase

Peters plus syndrome (PPS) is a severe autosomal recessive congenital disorder of glycosylation characterized by multiple structural anomalies. PPS patients have classic phenotypes, including Peters anomaly (an ocular anterior segment malformation), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable other systemic abnormalities, including cleft lip/palate, and anomalies in heart, skeletal and genitourinary system. PPS patients have mutations in the β1,3‐glucosyltransferase (B3GLCT) gene, which encodes an enzyme that catalyzes the addition of a glucose to an O‐linked fucose on thrombospondin type‐1 repeats (TSRs), forming an unusual Glucoseβ1‐3Fucose (GlcFuc) disaccharide on TSRs. Protein O‐fucosyltransferase 2 (POFUT2) is the enzyme that adds the O‐fucose to a serine or threonine in the consensus sequence C1XX(S/T)C2 found in TSRs, where C1 and C2 are the first two of six conserved cysteines in TSRs. Currently 65 human proteins are predicted to contain TSRs, and 49 out of these proteins contain the consensus sequence for this GlcFuc disaccharide modification. Half of these proteins belong to ADAMTS/ADAMTS‐like family of extracellular matrix proteins and have critical roles in mediating angiogenesis, re‐structuring extracellular matrix, and developmental processes. Previous results demonstrate that secretion of several ADAMTS/L proteins, in particular ADAMTS20 TSR2‐8, is dependent on the presence of POFUT2 and B3GLCT. Since both POFUT2 and B3GLCT are localized in endoplasmic reticulum and only modify folded TSRs, this leads to the proposal that both POFUT2 and B3GLCT assist the folding of TSRs, modulating the secretion of TSR‐containing proteins. Many identified PPS mutations in B3GLCT are splicing mutations upstream of the catalytic domain and are predicted to result in non‐functional proteins. To analyze the missense and truncation PPS mutations downstream of the catalytic domain, we introduced these mutations separately into a plasmid encoding human B3GLCT and analyzed for their ability to rescue secretion of ADAMTS20 TSR2‐8 from B3GLCT‐null cells. Our results showed that these PPS missense and truncation mutants not only abolished their ability to rescue ADAMTS20 TSR2‐8 secretion, but also greatly reduced mutant protein levels compared to wild type B3GLCT within the cells. Mutant enzymes harboring mutations associated with Peters Plus Spectrum, which is a milder form of PPS with patients who have a subset of PPS phenotypes, all rescued ADAMTS20 TSR2‐8 secretion and had the same protein levels within the cells as wild type B3GLCT. On‐going experiments are comparing the enzymatic activity and protein stability of mutants versus wild type enzyme. Overall, by analyzing the impact of PPS and Peters Plus Spectrum mutations on B3GLCT, we are aiming to discover novel therapeutic targets and advancing the development of novel treatments to alleviate the symptoms of PPS as well as other congenital disorders of glycosylation. This project is supported by NIH grant HD096030.

C-mannosylation supports folding and enhances stability of thrombospondin repeats.

Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both C-mannosylated and non-C-mannosylated thrombospondin type 1 repeats (TSRs) of netrin receptor UNC-5. In absence of C-mannosylation, UNC-5 TSRs could only be obtained at low temperature and a significant proportion displayed incorrect intermolecular disulfide bridging, which was hardly observed when C-mannosylated. Glycosylated TSRs exhibited higher resistance to thermal and reductive denaturation processes, and the presence of C-mannoses promoted the oxidative folding of a reduced and denatured TSR in vitro. Molecular dynamics simulations supported the experimental studies and showed that C-mannoses can be involved in intramolecular hydrogen bonding and limit the flexibility of the TSR tryptophan-arginine ladder. We propose that in the endoplasmic reticulum folding process, C-mannoses orient the underlying tryptophan residues and facilitate the formation of the tryptophan-arginine ladder, thereby influencing the positioning of cysteines and disulfide bridging.

A Plasmodium falciparum C-mannosyltransferase is dispensable for parasite asexual blood stage development

C-mannosylation was recently identified in the thrombospondin-related anonymous protein (TRAP) from Plasmodium falciparum salivary gland sporozoites. A candidate P. falciparum C-mannosyltransferase (PfDPY-19) was demonstrated to modify thrombospondin type 1 repeat (TSR) domains in vitro, exhibiting a different acceptor specificity than their mammalian counterparts. According to the described minimal acceptor of PfDPY19, several TSR domain-containing proteins of P. falciparum could be C-mannosylated in vivo. However, the relevance of this protein modification for the parasite viability remains unknown. In the present study, we used CRISPR/Cas9 technology to generate a PfDPY19 null mutant, demonstrating that this glycosyltransferase is not essential for the asexual blood development of the parasite. PfDPY19 gene disruption was not associated with a growth phenotype, not even under endoplasmic reticulum-stressing conditions that could impair protein folding. The data presented in this work strongly suggest that PfDPY19 is unlikely to play a critical role in the asexual blood stages of the parasite, at least under in vitro conditions.

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 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.