Anti-cysteine/spacer antibodies that open ADAMTS13 are a common feature in iTTP

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by an autoantibody-mediated deficiency of ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats member 13). In healthy individuals, ADAMTS13 has a folded conformation where the central spacer domain interacts with the C-terminal CUB domains. We recently showed that ADAMTS13 adopts an open conformation in iTTP and that patient immunoglobulin G's (IgGs) can open ADAMTS13. Anti-ADAMTS13 autoantibodies in iTTP patients are directed against the different ADAMTS13 domains but almost all patients have autoantibodies binding to the cysteine/spacer (CS) domains. In this study, we investigated whether the autoantibodies against the CS and CUB domains can disrupt the S-CUB interaction of folded ADAMTS13 thereby opening ADAMTS13. Therefore, we purified anti-CS and anti-CUB autoantibodies from 13 acute iTTP patients by affinity chromatography. The successfully purified anti-CS (10/13 patients) and anti-CUB (4/13 patients) autoantibody fractions were further tested in our ADAMTS13 conformation ELISA to study whether they could open ADAMTS13. Interestingly, all purified anti-CS fractions (10/10 patients) were able to open ADAMTS13. On the other hand, only half of the purified anti-CUB fractions (2/4 patients) opened ADAMTS13. Our finding highlights that anti-CS autoantibodies that open ADAMTS13 are a common feature of the autoimmune response in iTTP.

Congenital ADAMTS-13 deficiency presenting as life-threatening thrombosis during pregnancy

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterised by thrombocytopenia, microangiopathic haemolytic anaemia and microvascular thrombosis. Congenital TTP accounting for less than 5% of all TTP cases can have a late presentation in adulthood mostly triggered by predisposing factors such as infection, pregnancy and inflammation. We present a case of a 23-year-old woman who presented to us in the postpartum period with mesenteric artery thrombosis with infarcts and later was diagnosed as a case of TTP based on congenital a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS-13) deficiency detected on ADAMTS-13 levels and gene sequencing. She was successfully managed initially with therapeutic plasma exchanges and is now on prophylactic fortnightly fresh frozen plasma infusions at 15 mL/kg body weight and continues to be in remission.

Current and Future Perspectives on ADAMTS13 and Thrombotic Thrombocytopenic Purpura

Thrombotic thrombocytopenic purpura (TTP) is a rare, relapsing, and life-threatening disorder with an annual incidence of 10 cases per million people. TTP is a thrombotic microangiopathy characterized by severe thrombocytopenia, microangiopathic hemolytic anemia, and organ ischemia. The disease is caused by a severe deficiency of the enzyme ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13), which can either be acquired, mainly by autoantibodies targeting ADAMTS13, or congenital due to mutations in the ADAMTS13 gene. Thanks to the establishment of national registries worldwide, fundamental and translational research, major advances have been made on the diagnosis, treatment, and fundamental understanding of TTP, since the description of the first TTP case almost 100 years ago. The introduction of therapeutic plasma exchange in the 1970s has significantly improved patient survival, but novel diagnostic assays, targeted treatments (rituximab, caplacizumab, recombinant ADAMTS13), and the unraveling of both ADAMTS13 function and TTP pathophysiology should help to further improve the patients' quality of life. However, differential diagnosis of TTP remains challenging and still a lot of questions remain unanswered to completely understand this rare and devastating disease.

The thrombospondin module 1 domain of the matricellular protein CCN3 shows an atypical disulfide pattern and incomplete CWR layers

The members of the CCN (Cyr61/CTGF/Nov) family are a group of matricellular regulatory proteins that are essential to a wide range of functional pathways in cell signalling. Through interacting with extracellular matrix components and growth factors via one of their four domains, the CCN proteins are involved in critical biological processes such as angiogenesis, cell proliferation, bone development, fibrogenesis and tumorigenesis. Here, the crystal structure of the thrombospondin module 1 (TSP1) domain of CCN3 (previously known as Nov) is presented, which shares a similar three-stranded fold with the thrombospondin type 1 repeats of thrombospondin-1 and spondin-1, but with variations in the disulfide connectivity. Moreover, the CCN3 TSP1 domain lacks the typical π-stacked ladder of charged and aromatic residues on one side of the domain that is seen in other TSP1 domains. Using conservation analysis among orthologous domains, it is shown that a charged cluster in the centre of the domain is the most conserved site and this cluster is predicted to be a potential functional epitope for heparan sulfate binding. This variant TSP1 domain has also been used to revise the sequence determinants of TSP1 domains and to derive improved Pfam sequence profiles for the identification of novel TSP1 domains in more than 10 000 proteins across diverse phyla.

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.

C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence

C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase–deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.

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.

Protein O- and C-Glycosylation pathways in Toxoplasma gondii and Plasmodium falciparum

Apicomplexan parasites are amongst the most prevalent and morbidity-causing pathogens worldwide. They are responsible for severe diseases in humans and livestock and are thus of great public health and economic importance. Until the sequencing of apicomplexan genomes at the beginning of this century, the occurrence of N- and O-glycoproteins in these parasites was much debated. The synthesis of rudimentary and divergent N-glycans due to lineage-specific gene loss is now well established and has been recently reviewed. Here, we will focus on recent studies that clarified classical O-glycosylation pathways and described new nucleocytosolic glycosylations in Toxoplasma gondii, the causative agents of toxoplasmosis. We will also review the glycosylation of proteins containing thrombospondin type 1 repeats by O-fucosylation and C-mannosylation, newly discovered in Toxoplasma and the malaria parasite Plasmodium falciparum. The functional significance of these post-translational modifications has only started to emerge, but the evidence points towards roles for these protein glycosylation pathways in tissue cyst wall rigidity and persistence in the host, oxygen sensing, and stability of proteins involved in host invasion.