Congenital disorder of glycosilation PMM2-CDG

Congenital glycosylation disorders represent a group of genetically determined diseases which violate the synthesis and addition of glycans to glycoproteins and glycolipids, and also the synthesis of glycosylphosphatidyl inositol. The most common defects are the defects of protein N-glycosylation. Jaken syndrome, a congenital disorder of PMM2-CDG glycosylation, is the most commonly diagnosed type (about 800 cases worldwide). However, there are only a few descriptions of clinical cases in the Russian literature. The article presents a clinical observation of a child with this type of congenital glycosylation disorder due to a defect in phosphomannomtase 2 (PMM2 gene). The diagnose was based on the combination of clinical, laboratory and instrumental data: a characteristic phenotype, hyperinsulinism, delayed physical and psychomotor development, neurological manifestations, coagulopathy, liver damage, exudative enteropathy, abnormal forms of transferrin, PMM2 gene mutations associated with Jaken’s syndrome. For the first time the authors described positive clinical and laboratory dynamics due to the inclusion of D-mannose to the therapy for this type of congenital glycosylation disorder.

726. APX001 (Fosmanogepix) Is Effective in an Immunosuppressed Mouse Model of Rhizopus oryzae Infection

Background Mucormycosis is a life-threatening infection that predominantly occurs in immunocompromised hosts. The antifungal APX001A (manogepix) inhibits Gwt1, an enzyme required for the conserved glycosylphosphatidyl inositol (GPI) post-translational modification in eukaryotes. We previously reported the activity of APX001 (fosmanogepix, the prodrug of APX001A) against Rhizopus delemar (minimum effective concentration [MEC] = 0.25 µg/mL). Here we assessed the activity against R. oryzae, which has an elevated MEC value. Methods R. oryzae 99–892 MIC and MEC values were 0.125 µg/mL and 4.0 µg/mL for isavuconazole (ISAV) and APX001A, respectively. ICR mice were immunosuppressed with cyclophosphamide (200 mg/kg) and cortisone acetate (500 mg/kg) on Days -2, +3, and +8 relative to intratracheal infection with 2.5 × 105 cells of R. oryzae 99–892. For survival studies, treatment with 104 mg/kg APX001 was compared with ISAV (110 mg/kg TID). Oral treatment started on Day +1 through Day +7, relative to infection for survival studies, and through Day +4 for tissue fungal burden studies (assessed by conidial equivalent [CE] using qPCR). Placebo mice received vehicle control. To extend the half-life of APX001, mice were administered 50 mg/kg of the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) 2 h prior to APX001 administration. Results APX001 and ISAV equally prolonged median survival time of mice (n = 20) vs. placebo (12 and 14 days for APX001 and ISAV, respectively, vs. 8 days for placebo). Furthermore, APX001 and ISAV treatment both resulted in 30% 21-day survival vs. 0% survival of placebo mice (P < 0.05 by log-rank test). Both drug treatments resulted in ~1.5 log10 reduction in lung and brain CE vs. placebo-treated mice (n = 10, P < 0.005 by Wilcoxon rank-sum test). Conclusion Despite a higher MEC value, APX001 showed significant efficacy against R. oryzae that was as protective as ISAV in immunosuppressed mice. Given the previously reported activity of APX001 against a strain of R. delemar with a lower MEC value,APX001 has now been shown to be efficacious against both species of Rhizopus, which together are responsible for ~60–70% of isolates causing lethal mucormycosis. Thus, continued investigation of APX001 against mucormycosis is warranted. Disclosures All authors: No reported disclosures.

A molecular dynamics model for glycosylphosphatidyl-inositol anchors: “flop down” or “lollipop”?

Computational model for GPI anchors tested in DMPC and POPC bilayers. The free anchor rarely occurs as an erected “lollipop-like” conformation, it rather “flops down” onto the bilayer surface. Yet an attached protein (here green fluorescent protein) exhibits extensive orientational flexibility due to the phospho-ethanolamine linker.

Frequency of and reasons for paroxysmal nocturnal haemoglobinuria screening in patients with unexplained anaemia

Referral to hematology for anemia is common. In paroxysmal nocturnal hemoglobinuria (PNH), cells deficient in the glycosylphosphatidyl inositol (GPI) anchor are lysed by complement. Eculizumab improves overall survival and quality of life while reducing hemolysis, transfusion requirements, and thrombosis. We evaluated the frequency of screening for PNH in patients with unexplained anemia. Key clinical features, laboratory data, and investigations were recorded for patients referred for anemia since 2010, without a specific cause found. PNH testing was done by flow cytometry. 540 patients had: anemia not yet diagnosed (NYD, n=318 (including unexplained iron deficiency, n=92; DAT-negative hemolysis, n=9)); anemia of chronic disease, n=173; and pancytopenia NYD, n=49. 82.4% had LDH testing done; 85.0% total bilirubin; 78.7% reticulocyte counts; and 40.6% haptoglobin level; 131 (24.2%) had possible hemolysis. PNH testing was done in 56 (10.4%). Those screened for PNH were more likely to have: younger age (P=0.04); a history of thrombosis (P<0.001); undergone a BMBx (P<0.001); received RBC transfusions (P=0.0018); or evidence of DAT-negative hemolysis (P<0.001). In summary, PNH was tested for in a minority of patients with unexplained anemia (10.4%) despite potential indicators of hemolysis in 24.2%. Increased screening could identify patients who would benefit from treatment and should be considered.

Biology of tissue factor pathway inhibitor

Recent studies of the anticoagulant activities of the tissue factor (TF) pathway inhibitor (TFPI) isoforms, TFPIα and TFPIβ, have provided new insight into the biochemical and physiological mechanisms that underlie bleeding and clotting disorders. TFPIα and TFPIβ have tissue-specific expression patterns and anticoagulant activities. An alternative splicing event in the 5′ untranslated region allows for translational regulation of TFPIβ expression. TFPIα has 3 Kunitz-type inhibitor domains (K1, K2, K3) and a basic C terminus, whereas TFPIβ has the K1 and K2 domains attached to a glycosylphosphatidyl inositol–anchored C terminus. TFPIα is the only isoform present in platelets, whereas endothelial cells produce both isoforms, secreting TFPIα and expressing TFPIβ on the cell surface. TFPIα and TFPIβ inhibit both TF–factor VIIa–dependent factor Xa (FXa) generation and free FXa. Protein S enhances FXa inhibition by TFPIα. TFPIα produces isoform-specific inhibition of prothrombinase during the initiation of coagulation, an anticoagulant activity that requires an exosite interaction between its basic C terminus and an acidic region in the factor Va B domain. Platelet TFPIα may be optimally localized to dampen initial thrombin generation. Similarly, endothelial TFPIβ may be optimally localized to inhibit processes that occur when endothelial TF is present, such as during the inflammatory response.

N-Glycosylation instead of cholesterol mediates oligomerization and apical sorting of GPI-APs in FRT cells

Sorting of glycosylphosphatidyl-inositol–anchored proteins (GPI-APs) in polarized epithelial cells is not fully understood. Oligomerization in the Golgi complex has emerged as the crucial event driving apical segregation of GPI-APs in two different kind of epithelial cells, Madin–Darby canine kidney (MDCK) and Fisher rat thyroid (FRT) cells, but whether the mechanism is conserved is unknown. In MDCK cells cholesterol promotes GPI-AP oligomerization, as well as apical sorting of GPI-APs. Here we show that FRT cells lack this cholesterol-driven oligomerization as apical sorting mechanism. In these cells both apical and basolateral GPI-APs display restricted diffusion in the Golgi likely due to a cholesterol-enriched membrane environment. It is striking that N-glycosylation is the critical event for oligomerization and apical sorting of GPI-APs in FRT cells but not in MDCK cells. Our data indicate that at least two mechanisms exist to determine oligomerization in the Golgi leading to apical sorting of GPI-APs. One depends on cholesterol, and the other depends on N-glycosylation and is insensitive to cholesterol addition or depletion.