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 ß3-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.

Clinical History and Detectable Troponin Concentrations below the 99th Percentile for Risk Stratification of Patients with Chest Pain and First Normal Troponin.

Decision-making is challenging in patients with chest pain and normal high-sensitivity cardiac troponin T (hs-cTnT; <99th percentile; <14 ng/L) at hospital arrival. Most of these patients might be discharged early. We investigated clinical data and hs-cTnT concentrations for risk stratification. This is a retrospective study including 4476 consecutive patients presenting to the emergency department with chest pain and first normal hs-cTnT. The primary endpoint was one-year death or acute myocardial infarction, and the secondary endpoint added urgent revascularization. The number of primary and secondary endpoints was 173 (3.9%) and 252 (5.6%). Mean hs-cTnT concentrations were 6.9 ± 2.5 ng/L. Undetectable (<5 ng/L) hs-cTnT (n = 1847, 41%) had optimal negative predictive value (99.1%) but suboptimal sensitivity (90.2%) and discrimination accuracy (AUC = 0.664) for the primary endpoint. Multivariable analysis was used to identify the predictive clinical variables. The clinical model showed good discrimination accuracy (AUC = 0.810). The addition of undetectable hs-cTnT (≥ or <5 ng/L; HR, hazard ratio = 3.80; 95% CI, confidence interval 2.27-6.35; p = 0.00001) outperformed the clinical model alone (AUC = 0.836, p = 0.002 compared to the clinical model). Measurable hs-cTnT concentrations (between detection limit and 99th percentile; per 0.1 ng/L, HR = 1.13; CI 1.06-1.20; p = 0.0001) provided further predictive information (AUC = 0.844; p = 0.05 compared to the clinical plus undetectable hs-cTnT model). The results were reproducible for the secondary endpoint and 30-day events. Clinical assessment, undetectable hs-cTnT and measurable hs-cTnT concentrations must be considered for decision-making after a single negative hs-cTnT result in patients presenting to the emergency department with acute chest pain.

Long-Term Prognostic Value of Cognitive Impairment on Top of Frailty in Older Adults after Acute Coronary Syndrome.

Frailty is a marker of poor prognosis in older adults after acute coronary syndrome. We investigated whether cognitive impairment provides additional prognostic information. The study population consisted of a prospective cohort of 342 older (>65 years) adult survivors after acute coronary syndrome. Frailty (Fried score) and cognitive function (Pfeiffer's Short Portable Mental Status Questionnaire-SPMSQ) were assessed at discharge. The endpoints were mortality or acute myocardial infarction at 8.7-year median follow-up. Patient distribution according to SPMSQ results was: no cognitive impairment (SPMSQ = 0 errors; n = 248, 73%), mild impairment (SPMSQ = 1-2 errors; n = 52, 15%), and moderate to severe impairment (SPMSQ ≥3 errors; n = 42, 12%). A total of 245 (72%) patients died or had an acute myocardial infarction, and 216 (63%) patients died. After adjustment for clinical data, comorbidities, and Fried score, the SPMSQ added prognostic value for death or myocardial infarction (per number of errors; HR = 1.11, 95%, CI 1.04-1.19, p = 0.002) and death (HR = 1.11, 95% 1.03-1.20, p = 0.007). An SPMSQ with ≥3 errors identified the highest risk subgroup. Geriatric conditions (SPSMQ and Fried score) explained 19% and 43% of the overall chi-square of the models for predicting death or myocardial infarction and death, respectively. Geriatric assessment after acute coronary syndrome should include both frailty and cognitive function. This is particularly important given that cognitive impairment without dementia can be subclinical and thus remain undetected.

Randomized Comparison of Exercise Intervention Versus Usual Care in Older Adult Patients with Frailty After Acute Myocardial Infarction.

BACKGROUND: Older adult patients with frailty are rarely involved in rehabilitation programs after myocardial infarction. Our aim was to investigate the benefits of exercise intervention in these patients. METHODS: A total of 150 survivors after acute myocardial infarction, ≥70 years and with pre-frailty or frailty (Fried scale ≥1 points), were randomized to control (n = 77) or intervention (n = 73) groups. The intervention consisted of a 3-month exercise program, under physiotherapist supervision, followed by an independent home-based program. The main outcome was frailty (Fried scale) at 3 months and 1 year. Secondary endpoints were clinical events (mortality or any readmission) at 1 year. RESULTS: Mean age was 80 years (range = 70-96). In the intervention group, 44 (60%) out of 73 patients participated in the program and 23 (32%) completed it. Overall, there was a decrease in the Fried score in the intervention group at 3 months, with no effect at 1 year. However, in the intention-to-treat analysis, such change did not achieve statistical significance (P = 0.110). Only treatment comparisons made among the subgroups that participated in (P = 0.033) and completed (P = 0.018) the program achieved statistical significance. There were no differences in clinical events. Worse Fried score trajectory along follow-up increased mortality risk (hazard ratio [HR] = 2.38, 95% confidence interval [CI] 1.24-4.55, P = 0.009) CONCLUSIONS: Recruitment and retention for a physical program in older adult patients with frailty after myocardial infarction was challenging. Frailty status improved in the subgroup that participated in the program, although this benefit was attenuated after shifting to a home-based program. A better frailty trajectory might influence midterm prognosis. (ClinicalTrials.govNCT02715453).

Frailty Tools for Assessment of Long-term Prognosis After Acute Coronary Syndrome.

OBJECTIVE: To evaluate the 5 components of the Fried frailty phenotype (self-reported unintentional weight loss, physical activity questionnaire, gait speed, grip strength, and self-reported exhaustion) for long-term outcomes in elderly survivors of acute coronary syndrome. METHODS: A total of 342 consecutive patients (from October 1, 2010, to February 1, 2012) were included. The 5 components of the Fried score and albumin concentration, as malnutrition index, were assessed before hospital discharge. Patients were followed up until April 2020 (median follow-up, 8.7 years). The end point was postdischarge all-cause mortality. RESULTS: Mean ± SD age was 77±7 years and mean ± SD Fried score was 2.0±1.1 points. A total of 216 (63%) patients died. After adjusting for clinical covariates, the Fried phenotype was associated with mortality (per points, hazard ratio [HR], 1.35; 95% CI, 1.17 to 1.57; P<.001). Among Fried components, physical activity (HR, 2.21; 95% CI, 1.34 to 3.65; P=.002) and gait speed (HR, 1.77; 95% CI, 1.29 to 2.43; P<.001) were the deficits independendtly associated with mortality. Albumin level provided further prognostic information (per increase in g/dL; HR, 0.63, 95% CI, 0.45 to 0.88; P=.007). The model adding the components of the Fried score and albumin level to the clinical model showed the highest risk reclassification (integrated discrimination improvement, 0.040; 95% CI, 0.018 to 0.075; P=.001; continuous net reclassification improvement, 0.291; 95% CI, 0.132 to 0.397; P=.001) in comparison with the model using clinical covariates alone. CONCLUSION: Frailty assessment using the Fried phenotype has prognostic value for long-term mortality in elderly survivors of acute coronary syndrome. Physical activity and gait speed are the predictive components of the Fried score. Albumin level provides incremental prognostic information.

Ejection Fraction by Echocardiography for a Selective Use of Magnetic Resonance After Infarction.

Background Cardiac magnetic resonance (CMR) permits robust risk stratification of discharged ST-segment-elevation myocardial infarction patients, but its indiscriminate use in all cases is not feasible. We evaluated the utility of left ventricular ejection fraction (LVEF) by echocardiography for a selective use of CMR after ST-segment-elevation myocardial infarction. Methods Echocardiography and CMR were performed in 1119 patients discharged for ST-segment-elevation myocardial infarction included in a multicenter registry. The prognostic power of CMR beyond echocardiography-LVEF was assessed using adjusted C statistic, net reclassification improvement index, and integrated discrimination improvement index. Results During a 4.8-year median follow-up, 136 (12%) first major adverse cardiac events (MACE) occurred (47 cardiovascular deaths and 89 readmissions for acute heart failure). In the entire group, CMR-LVEF (but not echocardiography-LVEF) independently predicted MACE occurrence. The MACE rate significantly increased only in patients with CMR-LVEF<40% (≥50%: 7%, 40%-49%: 9%, <40%: 27%, P<0.001). Most patients displayed echocardiography-LVEF≥50% (629, 56%), and they had a low MACE rate (57/629, 9%). In patients with echocardiography-LVEF<50% (n=490, 44%), the MACE rate was also low in those with CMR-LVEF≥40% (24/278, 9%) but significantly increased in patients with CMR-LVEF<40% (55/212, 26%; P<0.001). Compared with echocardiography-LVEF, CMR-LVEF significantly improved MACE prediction in the group of patients with echocardiography-LVEF<50% (C statistic, 0.80 versus 0.72; net reclassification improvement index, 0.73; integrated discrimination improvement index, 0.10) but not in those with echocardiography-LVEF≥50% (C statistic 0.66 versus 0.66; net reclassification improvement index, 0.17; integrated discrimination improvement index, 0.01). Conclusions A straightforward strategy based on a selective use of CMR for risk prediction in ST-segment-elevation myocardial infarction patients with echocardiography-LVEF<50% can provide insights into patient care. The cost-effectiveness of this approach, as well as the direct implications in clinical management, should be further explored.

Undetectable high-sensitivity troponin in combination with clinical assessment for risk stratification of patients with chest pain and normal troponin at hospital arrival.

BACKGROUND: Undetectable high-sensitivity cardiac troponin (hs-cTn) in a single determination upon admission may rule out acute coronary syndrome. We investigated undetectable hs-cTnT (

Noninvasive Imaging Estimation of Myocardial Iron Repletion Following Administration of Intravenous Iron: The Myocardial-IRON Trial.

Background Intravenous ferric carboxymaltose (FCM) improves symptoms, functional capacity, and quality of life in heart failure and iron deficiency. The mechanisms underlying these effects are not fully understood. The aim of this study was to examine changes in myocardial iron content after FCM administration in patients with heart failure and iron deficiency using cardiac magnetic resonance. Methods and Results Fifty-three stable heart failure and iron deficiency patients were randomly assigned 1:1 to receive intravenous FCM or placebo in a multicenter, double-blind study. T2* and T1 mapping cardiac magnetic resonance sequences, noninvasive surrogates of intramyocardial iron, were evaluated before and 7 and 30 days after randomization using linear mixed regression analysis. Results are presented as least-square means with 95% CI. The primary end point was the change in T2* and T1 mapping at 7 and 30 days. Median age was 73 (65-78) years, with N-terminal pro-B-type natriuretic peptide, ferritin, and transferrin saturation medians of 1690 pg/mL (1010-2828), 63 ng/mL (22-114), and 15.7% (11.0-19.2), respectively. Baseline T2* and T1 mapping values did not significantly differ across treatment arms. On day 7, both T2* and T1 mapping (ms) were significantly lower in the FCM arm (36.6 [34.6-38.7] versus 40 [38-42.1], P=0.025; 1061 [1051-1072] versus 1085 [1074-1095], P=0.001, respectively). A similar reduction was found at 30 days for T2* (36.3 [34.1-38.5] versus 41.1 [38.9-43.4], P=0.003), but not for T1 mapping (1075 [1065-1085] versus 1079 [1069-1089], P=0.577). Conclusions In patients with heart failure and iron deficiency, FCM administration was associated with changes in the T2* and T1 mapping cardiac magnetic resonance sequences, indicative of myocardial iron repletion. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT03398681.

Entrenamiento de la musculatura inspiratoria y la electroestimulación muscular funcional en el tratamiento de la insuficiencia cardiaca con función sistólica conservada: estudio TRAINING-HF / Inspiratory muscle training and functional electrical stimulation for treatment of heart failure with preserved ejection fraction: the TRAINING-HF trial

Introducción y objetivos: No se dispone de tratamientos farmacológicos que demuestren reducir la morbimortalidad asociada en pacientes con insuficiencia cardiaca y función sistólica conservada (IC-FEc). El objetivo del presente estudio fue evaluar si en pacientes con IC-FEc, el entrenamiento de la musculatura inspiratoria (EMI), la electroestimulación muscular funcional (EMF) o la combinación de ambas (EMI + EMF) puede mejorar la capacidad funcional, calidad de vida, parámetros de disfunción diastólica o biomarcadores a las 12 y 24 semanas. Métodos: Un total de 61 pacientes estables con IC-FEc (clase funcional de la New York Heart Association II-III) se aleatorizaron (1:1:1:1) a recibir un programa de 12 semanas de EMI, EMF, o EMI + EMF frente a tratamiento médico estándar (control). El objetivo primario fue evaluar el cambio en el consumo máximo de oxígeno. Los objetivos secundarios fueron los cambios en la calidad de vida, parámetros ecocardiográficos y biomarcadores. Se utilizó un modelo lineal mixto para comparar los cambios entre los diferentes grupos. Resultados: La edad media fue 74 +/- 9 años y la proporción de mujeres fue del 58%. El test de consumo máximo de oxígeno fue de 9,9 +/- 2,5ml/min/kg. A las 12 semanas, con respecto al grupo control, el incremento medio de consumo máximo de oxígeno fue de 2,98, 2,93 y 2,47 para EMI, EMF y EMI + EMF, respectivamente (p < 0,001). Este incremento se mantuvo a las 24 semanas (1,95, 2,08 y 1,56, respectivamente; p < 0,001). Resultados similares se observaron en la puntuación del cuestionario de calidad de vida (p < 0,001). Conclusiones: En los pacientes con IC-FEc e importante reducción de la capacidad funcional, tanto el EMI como la EMF se asocian con una marcada mejoría de la capacidad funcional y la calidad de vida

Introduction and objectives: Despite the prevalence of heart failure with preserved ejection fraction (HFpEF), there is currently no evidence-based effective therapy for this disease. This study sought to evaluate whether inspiratory muscle training (IMT), functional electrical stimulation (FES), or a combination of both (IMT + FES) improves 12- and 24-week exercise capacity as well as left ventricular diastolic function, biomarker profile, and quality of life in HFpEF. Methods: A total of 61 stable symptomatic patients (New York Heart Association functional class II-III) with HFpEF were randomized (1:1:1:1) to receive a 12-week program of IMT, FES, or IMT + FES vs usual care. The primary endpoint of the study was to evaluate change in peak exercise oxygen uptake at 12 and 24 weeks. Secondary endpoints were changes in quality of life, echocardiogram parameters, and prognostic biomarkers. We used a mixed-effects model for repeated-measures to compare endpoints changes. Results: Mean age and peak exercise oxygen uptake were 74 +/- 9 years and 9.9 +/- 2.5mL/min/kg, respectively. The proportion of women was 58%. At 12 weeks, the mean increase in peak exercise oxygen uptake (mL/kg/min) compared with usual care was 2.98, 2.93, and 2.47 for IMT, FES, and IMT + FES, respectively (P < .001) and this beneficial effect persisted after 24 weeks (1.95, 2.08, and 1.56; P < .001). Significant increases in quality of life scores were found at 12 weeks (P < .001). No other changes were found. Conclusions: In HFpEF patients with low aerobic capacity, IMT and FES were associated with a significant improvement in exercise capacity and quality of life

Inspiratory Muscle Training and Functional Electrical Stimulation for Treatment of Heart Failure With Preserved Ejection Fraction: The TRAINING-HF Trial.

INTRODUCTION AND OBJECTIVES: Despite the prevalence of heart failure with preserved ejection fraction (HFpEF), there is currently no evidence-based effective therapy for this disease. This study sought to evaluate whether inspiratory muscle training (IMT), functional electrical stimulation (FES), or a combination of both (IMT + FES) improves 12- and 24-week exercise capacity as well as left ventricular diastolic function, biomarker profile, and quality of life in HFpEF. METHODS: A total of 61 stable symptomatic patients (New York Heart Association II-III) with HFpEF were randomized (1:1:1:1) to receive a 12-week program of IMT, FES, or IMT + FES vs usual care. The primary endpoint of the study was to evaluate change in peak exercise oxygen uptake at 12 and 24 weeks. Secondary endpoints were changes in quality of life, echocardiogram parameters, and prognostic biomarkers. We used a mixed-effects model for repeated-measures to compare endpoints changes. RESULTS: Mean age and peak exercise oxygen uptake were 74 ± 9 years and 9.9 ± 2.5mL/min/kg, respectively. The proportion of women was 58%. At 12 weeks, the mean increase in peak exercise oxygen uptake (mL/kg/min) compared with usual care was 2.98, 2.93, and 2.47 for IMT, FES, and IMT + FES, respectively (P < .001) and this beneficial effect persisted after 6 months (1.95, 2.08, and 1.56; P < .001). Significant increases in quality of life scores were found at 12 weeks (P < .001). No other changes were found. CONCLUSIONS: In HFpEF patients with low aerobic capacity, IMT and FES were associated with a significant improvement in exercise capacity and quality of life. This trial was registered at ClinicalTrials.gov (Identifier: NCT02638961)..

Intrarenal venous flow in cardiorenal syndrome: a shining light into the darkness.

The aim of this case report is to assess the potential role of intrarenal Doppler ultrasonography as a non-invasive method to evaluate intrarenal venous flow (IRVF) in acute heart failure (AHF) and concomitant renal dysfunction. We report a case of an 81-year-old woman with valvular heart disease (previous mitral valve replacement) that presented with acutely decompensated heart failure and concomitant worsening renal function (WRF). In addition to complete physical examination, laboratory analysis, and echocardiography, IRVF was assessed at baseline and 48 h after the administration of diuretic treatment. At admission, physical examination and echocardiography revealed signs of intravascular congestion (jugular venous distension and severely dilated inferior vena cava). In addition, a significant increase in serum creatinine from 1.23 to 1.81 mg/dL was noted without signs of hypoperfusion at clinical evaluation. At baseline, intrarenal Doppler ultrasonography showed a monophasic IRVF pattern indicating a severely elevated interstitial renal pressure. After aggressive decongestion, a dynamic behaviour was found in IRVF changing from monophasic to biphasic pattern in parallel with an improvement in clinical parameters and renal function (serum creatinine changed from 1.81 to 1.44 mg/dL). In this case of a patient with AHF and WRF, IRVF changed after aggressive decongestion in agreement with clinical evolution. According to these findings, this technique could provide valuable information for identifying patients with a 'congestion kidney failure' phenotype. Further studies are needed confirming this observation and evaluating the potential role of this technique for guiding decongestive therapy in patients with AHF and WRF.

Changes in myocardial iron content following administration of intravenous iron (Myocardial-IRON): Study design.

Treatment with intravenous ferric carboxymaltose (FCM) has been shown to improve symptoms, functional capacity, and quality of life in patients with heart failure and iron deficiency. However, the underlying mechanisms for these beneficial effects remain undetermined. The aim of this study is to quantify cardiac magnetic resonance changes in myocardial iron content after administration of intravenous FCM in patients with heart failure and iron deficiency and contrast them with parameters of heart failure severity. This is a multicenter, double-blind, randomized study. Fifty patients with stable symptomatic heart failure, left ventricular ejection fraction <50%, and iron deficiency will be randomly assigned 1:1 to receive intravenous FCM or placebo. Intramyocardial iron will be evaluated by T2* and T1 mapping cardiac magnetic resonance sequences before and at 7 and 30 days after FCM. After 30 days, patients assigned to placebo will receive intravenous FCM in case of persistent iron deficiency. The main endpoint will be changes from baseline in myocardial iron content at 7 and 30 days. Secondary endpoints will include the correlation of these changes with left ventricular ejection fraction, functional capacity, quality of life, and cardiac biomarkers. The results of this study will add important knowledge about the effects of intravenous FCM on myocardial tissue and cardiac function. We hypothesize that short-term (7 and 30 days) myocardial iron content changes after intravenous FCM, evaluated by cardiac magnetic resonance, will correlate with simultaneous changes in parameters of heart failure severity. The study is registered at http://www.clinicaltrials.gov (NCT03398681).

Inhibitors against Fungal Cell Wall Remodeling Enzymes.

Fungal ß-1,3-glucan glucanosyltransferases are glucan-remodeling enzymes that play important roles in cell wall integrity, and are essential for the viability of pathogenic fungi and yeasts. As such, they are considered possible drug targets, although inhibitors of this class of enzymes have not yet been reported. Herein we report a multidisciplinary approach based on a structure-guided design using a highly conserved transglycosylase from Sacharomyces cerevisiae, that leads to carbohydrate derivatives with high affinity for Aspergillus fumigatus Gel4. We demonstrate by X-ray crystallography that the compounds bind in the active site of Gas2/Gel4 and interact with the catalytic machinery. The topological analysis of noncovalent interactions demonstrates that the combination of a triazole with positively charged aromatic moieties are important for optimal interactions with Gas2/Gel4 through unusual pyridinium cation-π and face-to-face π-π interactions. The lead compound is capable of inhibiting AfGel4 with an IC50 value of 42 µm.

Inspiratory Muscle Training and Functional Electrical Stimulation for Treatment of Heart Failure With Preserved Ejection Fraction: Rationale and Study Design of a Prospective Randomized Controlled Trial.

Heart failure with preserved ejection fraction (HFpEF) has become the most prevalent form of heart failure in developed countries. Regrettably, there is no evidence-based effective therapy for HFpEF. We seek to evaluate whether inspiratory muscle training, functional electrical stimulation, or a combination of both can improve exercise capacity as well as left ventricular diastolic function, biomarker profile, quality of life (QoL), and prognosis in patients with HFpEF. A total of 60 stable symptomatic patients with HFpEF (New York Heart Association class II-III/IV) will be randomized (1:1:1:1) to receive a 12-week program of inspiratory muscle training, functional electrical stimulation, a combination of both, or standard care alone. The primary endpoint of the study is change in peak exercise oxygen uptake; secondary endpoints are changes in QoL, echocardiogram parameters, and prognostic biomarkers. As of March 21, 2016, thirty patients have been enrolled. Searching for novel therapies that improve QoL and autonomy in the elderly with HFpEF has become a health care priority. We believe that this study will add important knowledge about the potential utility of 2 simple and feasible physical interventions for the treatment of advanced HFpEF.

A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2.

Protein O-fucosyltransferase 2 (POFUT2) is an essential enzyme that fucosylates serine and threonine residues of folded thrombospondin type 1 repeats (TSRs). To date, the mechanism by which this enzyme recognizes very dissimilar TSRs has been unclear. By engineering a fusion protein, we report the crystal structure of Caenorhabditis elegans POFUT2 (CePOFUT2) in complex with GDP and human TSR1 that suggests an inverting mechanism for fucose transfer assisted by a catalytic base and shows that nearly half of the TSR1 is embraced by CePOFUT2. A small number of direct interactions and a large network of water molecules maintain the complex. Site-directed mutagenesis demonstrates that POFUT2 fucosylates threonine preferentially over serine and relies on folded TSRs containing the minimal consensus sequence C-X-X-S/T-C. Crystallographic and mutagenesis data, together with atomic-level simulations, uncover a binding mechanism by which POFUT2 promiscuously recognizes the structural fingerprint of poorly homologous TSRs through a dynamic network of water-mediated interactions.

Rational Design of Glycomimetic Compounds Targeting the Saccharomyces cerevisiae Transglycosylase Gas2.

The transglycosylase Saccharomyces cerevisiae Gas2 (ScGas2) belongs to a large family of enzymes that are key players in yeast cell wall remodeling. Despite its biologic importance, no studies on the synthesis of substrate-based compounds as potential inhibitors have been reported. We have synthesized a series of docking-guided glycomimetics that were evaluated by fluorescence spectroscopy and saturation-transfer difference (STD) NMR experiments, revealing that a minimum of three glucose units linked via a ß-(1,3) linkage are required for achieving molecular recognition at the binding donor site. The binding mode of our compounds is further supported by STD-NMR experiments using the active site-mutants Y107Q and Y244Q. Our results are important for both understanding of ScGas2-substrate interactions and setting up the basis for future design of glycomimetics as new antifungal agents.

Deciphering the Non-Equivalence of Serine and Threonine O-Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti-MUC1 Antibody.

The structural features of MUC1-like glycopeptides bearing the Tn antigen (α-O-GalNAc-Ser/Thr) in complex with an anti MUC-1 antibody are reported at atomic resolution. For the α-O-GalNAc-Ser derivative, the glycosidic linkage adopts a high-energy conformation, barely populated in the free state. This unusual structure (also observed in an α-S-GalNAc-Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α-O-GalNAc-Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non-equivalence of Ser and Thr O-glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti-MUC1 antibodies.

Detection of tumor-associated glycopeptides by lectins: the peptide context modulates carbohydrate recognition.

Tn antigen (α-O-GalNAc-Ser/Thr) is a convenient cancer biomarker that is recognized by antibodies and lectins. This work yields remarkable results for two plant lectins in terms of epitope recognition and reveals that these receptors show higher affinity for Tn antigen when it is incorporated in the Pro-Asp-Thr-Arg (PDTR) peptide region of mucin MUC1. In contrast, a significant affinity loss is observed when Tn antigen is located in the Ala-His-Gly-Val-Thr-Ser-Ala (AHGVTSA) or Ala-Pro-Gly-Ser-Thr-Ala-Pro (APGSTAP) fragments. Our data indicate that the charged residues, Arg and Asp, present in the PDTR sequence establish noteworthy fundamental interactions with the lectin surface as well as fix the conformation of the peptide backbone, favoring the presentation of the sugar moiety toward the lectin. These results may help to better understand glycopeptide-lectin interactions and may contribute to engineer new binding sites, allowing novel glycosensors for Tn antigen detection to be designed.

Serine versus threonine glycosylation with α-O-GalNAc: unexpected selectivity in their molecular recognition with lectins.

The molecular recognition of several glycopeptides bearing Tn antigen (α-O-GalNAc-Ser or α-O-GalNAc-Thr) in their structure by three lectins with affinity for this determinant has been analysed. The work yields remarkable results in terms of epitope recognition, showing that the underlying amino acid of Tn (serine or threonine) plays a key role in the molecular recognition. In fact, while Soybean agglutinin and Vicia villosa agglutinin lectins prefer Tn-threonine, Helix pomatia agglutinin shows a higher affinity for the glycopeptides carrying Tn-serine. The different conformational behaviour of the two Tn biological entities, the residues of the studied glycopeptides in the close proximity to the Tn antigen and the topology of the binding site of the lectins are at the origin of these differences.