Precision mapping of the human O‐GalNAc glycoproteome through SimpleCell technology

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc‐type O‐glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc‐transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O‐glycosylation (SimpleCells) that enables proteome‐wide discovery of O‐glycan sites using ‘bottom‐up’ ETD‐based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O‐glycoproteome with almost 3000 glycosites in over 600 O‐glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O‐glycosylation. The finding of unique subsets of O‐glycoproteins in each cell line provides evidence that the O‐glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O‐glycoproteome should facilitate the exploration of how site‐specific O‐glycosylation regulates protein function.     

Quantification of Na+,K+ pumps and their transport rate in skeletal muscle: Functional significance

During excitation, muscle cells gain Na⁺ and lose K⁺, leading to a rise in extracellular K⁺ ([K⁺]_o), depolarization, and loss of excitability. Recent studies support the idea that these events are important causes of muscle fatigue and that full use of the Na⁺,K⁺-ATPase (also known as the Na⁺,K⁺ pump) is often essential for adequate clearance of extracellular K⁺. As a result of their electrogenic action, Na⁺,K⁺ pumps also help reverse depolarization arising during excitation, hyperkalemia, and anoxia, or from cell damage resulting from exercise, rhabdomyolysis, or muscle diseases. The ability to evaluate Na⁺,K⁺-pump function and the capacity of the Na⁺,K⁺ pumps to fill these needs require quantification of the total content of Na⁺,K⁺ pumps in skeletal muscle. Inhibition of Na⁺,K⁺-pump activity, or a decrease in their content, reduces muscle contractility. Conversely, stimulation of the Na⁺,K⁺-pump transport rate or increasing the content of Na⁺,K⁺ pumps enhances muscle excitability and contractility. Measurements of [³H]ouabain binding to skeletal muscle in vivo or in vitro have enabled the reproducible quantification of the total content of Na⁺,K⁺ pumps in molar units in various animal species, and in both healthy people and individuals with various diseases. In contrast, measurements of 3-O-methylfluorescein phosphatase activity associated with the Na⁺,K⁺-ATPase may show inconsistent results. Measurements of Na⁺ and K⁺ fluxes in intact isolated muscles show that, after Na⁺ loading or intense excitation, all the Na⁺,K⁺ pumps are functional, allowing calculation of the maximum Na⁺,K⁺-pumping capacity, expressed in molar units/g muscle/min. The activity and content of Na⁺,K⁺ pumps are regulated by exercise, inactivity, K⁺ deficiency, fasting, age, and several hormones and pharmaceuticals. Studies on the α-subunit isoforms of the Na⁺,K⁺-ATPase have detected a relative increase in their number in response to exercise and the glucocorticoid dexamethasone but have not involved their quantification in molar units. Determination of ATPase activity in homogenates and plasma membranes obtained from muscle has shown ouabain-suppressible stimulatory effects of Na⁺ and K⁺.

Introduction: Transport and content of Na⁺ and K⁺ in skeletal muscle

The Na⁺,K⁺-ATPase (also known as the Na⁺,K⁺ pump) is the major translator of metabolic energy in the form of ATP to electrical and chemical gradients for the two most common ions in the body. These gradients enable the generation of action potentials, which are essential for muscle cell function. Evaluation of the physiological and clinical significance of the Na⁺,K⁺ pumps requires measuring the transmembrane fluxes of Na⁺ and K⁺ in intact muscles or cultured muscle cells. The simplest approach involves incubating intact muscles isolated from small animals in temperature-controlled and oxygenated buffers with electrolyte and glucose concentration comparable to that normally present in blood plasma. Initial studies used cut hemi- or quarter-diaphragm muscles from rats, mice, or guinea pigs for incubation because these muscles were considered thin enough to allow adequate oxygenation under these conditions (Gemmill, 1940). However, such preparations have numerous cut muscle ends, allowing large passive movements of Na⁺ and K⁺ and free access of Ca²⁺ to the cell interior. This unavoidably boosts the energy required for active transport of Na⁺, K⁺, and Ca²⁺, and leads to impaired cell survival. Thus, in cut muscles, the components of O₂ consumption and ⁴²K uptake attributable to the Na⁺,K⁺ pump (i.e., the fraction suppressible by the cardiac glycoside ouabain, which binds to and inhibits the Na⁺,K⁺-ATPase) have been severely overestimated. (The ouabain-suppressible components of O₂ consumption or ⁴²K uptake are measured in isolated muscles incubated without or with ouabain and calculated as the difference.) Such overestimation led to the assumption that in skeletal muscle, the Na⁺,K⁺ pumps mediate a large fraction of total energy turnover, suggesting that a major part of the thermogenic action of thyroid hormone is caused by an increased rate of active Na⁺,K⁺ transport (Asano et al., 1976). In contrast, in intact resting muscle preparations, only 2–10% of the total energy turnover is used for active Na⁺,K⁺ transport (Creese, 1968; for details, see Clausen et al., 1991). Even during maximum contractile work in human muscles, only a small fraction (2%) (Medbø and Sejersted, 1990) of total energy release is used for the Na⁺,K⁺ pumps. Thus, in skeletal muscle, the thermogenic action of the Na⁺,K⁺ pumps is modest.For the analysis of Na⁺,K⁺ transport in skeletal muscle, isolated intact limb muscles are used, primarily mammalian soleus, extensor digitorum longus (EDL), extensor digitorum brevis, or epitrochlearis muscles. These preparations can survive during incubation for many hours and can undergo repeated excitation. More recently, the isolated rat sternohyoid muscle, which also offers thin dimensions, cellular integrity, and simple handling, has been introduced (Mu et al., 2011).Measurement of muscle Na⁺ and K⁺ content requires extraction. In the past, this was done by digesting the muscle preparation in nitric acid, a sometimes risky procedure. More recently, this approach has been superseded by homogenization of the tissue in 0.3 M trichloroacetic acid, followed by centrifugation to sediment the proteins (Kohn and Clausen, 1971). The clear supernatant may then be diluted for flame photometric determination of Na⁺ and K⁺ or counting of the isotopes ²²Na or ⁴²K. Because ⁴²K has a short half-life (12.5 h) and is of limited availability, the K⁺ analogue ⁸⁶Rb is often used as a tracer for K⁺. For the quantification of Na⁺,K⁺ pump–mediated (i.e., ouabain-suppressible) transport of K⁺, ⁸⁶Rb gives the same results as ⁴²K (Clausen et al., 1987; Dørup and Clausen, 1994). For other flux measurements, however, the results obtained with ⁸⁶Rb differ appreciably from those obtained with ⁴²K. For example, the fractional loss of ⁸⁶Rb from intact resting rat soleus muscles is only 45% of that measured using ⁴²K. Moreover, two agents shown to stimulate the Na⁺,K⁺ pumps in isolated rat soleus muscle, salbutamol (Clausen and Flatman, 1977) and rat calcitonin gene–related peptide (CGRP) (Andersen and Clausen, 1993), both induce a highly significant stimulation of ⁸⁶Rb efflux from the same muscle (Dørup and Clausen, 1994). In contrast, these same agents induced a rapid but transient (20-min duration) inhibition of the fractional loss of ⁴²K, indicating that a large fraction of the ⁴²K lost from the cells is reaccumulated as a result of stimulation of the Na⁺,K⁺ pumps (Andersen and Clausen, 1993; Dørup and Clausen, 1994). Finally, in skeletal muscle, bumetanide, an inhibitor of the NaK2Cl cotransporter, produces no inhibition of ⁴²K uptake but clear-cut inhibition of ⁸⁶Rb uptake (see ​and22 in Dørup and Clausen, 1994). Thus, results obtained with ⁸⁶Rb must be verified with ⁴²K or flame photometric measurements of changes in intracellular Na⁺ and K⁺ content. The activity of the Na⁺,K⁺ pump depends on ATP supplied by glycolysis or oxidative phosphorylation. Excitability of isolated rat soleus or EDL muscles can be maintained in the presence of the electron transport inhibitor cyanide or during anoxia (Murphy and Clausen, 2007; Fredsted et al., 2012), whereas contractions are markedly suppressed by 2-deoxyglucose, which interferes with the production of glycolytic ATP. Thus, in keeping with the studies of Glitsch (2001), these data suggest that glycolytic ATP is a primary source of energy for the Na⁺,K⁺ pumps (see also Okamoto et al., 2001). More focused studies showed that Na⁺,K⁺-pump function is not adequately supported when cytoplasmic ATP is at the normal resting concentration of ∼8 mM, but that the addition of as little as 1 mM phosphoenol pyruvate produces a marked increase in Na⁺,K⁺-pump function that is supported by endogenous pyruvate kinase bound within the t-tubular triad (Dutka and Lamb, 2007). In anoxic rat EDL muscles, contractility could be restored by stimulating the Na⁺,K⁺ pumps with the β₂ agonists salbutamol or terbutaline, effects that were abolished by ouabain or 2-deoxyglucose (Fredsted et al., 2012). Glycolytic ATP furnishes energy for contractile activity under the critical condition of anoxia. Because the Na⁺,K⁺ pumps are essential for the maintenance of excitability and contractions, it would not be surprising if they were also kept going on glycolytic ATP.

Table 1.

Acute stimulation of the Na⁺,K⁺ pumps in skeletal muscle

Variation in fish community structure, richness, and diversity in 56 Danish lakes with contrasting depth, size, and trophic state: does the method matter?

Organisms inhabiting shallow near-shore waters are at risk of desiccation during water level fluctuations. Using laboratory experiments, we investigated the survival and behavioural defences of four freshwater amphipod species during substratum drying: three Ponto-Caspian invaders (Pontogammarus robustoides, Dikerogammarus haemobaphes and Dikerogammarus villosus) and the native Gammarus fossarum. We hypothesized that they would be able to survive air exposure events as well as to adjust their behaviour by following the decreasing water level and/or burying in the sediments. To test these hypotheses, we examined survival of each species on gradually drying sandy substratum as well as their horizontal and vertical migration behaviours. P. robustoides was most resistant to substratum drying and was the only species burying into the substratum. On the other hand, G. fossarum exhibited distinct horizontal migrations following the retreating waterline. These two species seem to be particularly well adapted to the drying environment. Defence mechanisms of D. haemobaphes and D. villosus were less efficient, though the former species also followed the retreating waterline to some extent. Our study demonstrates that exotic and native gammarids have several adaptations that enable them to invade and persist in habitats experiencing common water level fluctuations.     

Heritability of eleven metabolic phenotypes in Danish and Chinese twins: A cross‐population comparison

Objectives : A twin‐based comparative study on the genetic influences in metabolic endophenotypes in two populations of substantial ethnic, environmental, and cultural differences was performed. Design and Methods : Data on 11 metabolic phenotypes including anthropometric measures, blood glucose, and lipids levels as well as blood pressure were available from 756 pairs of Danish twins (309 monozygotic and 447 dizygotic twin pairs) with a mean age of 38 years (range: 18‐67) and from 325 pairs of Chinese twins (183 monozygotic and 142 dizygotic twin pairs) with a mean age of 40.5 years (range: 18‐69). Twin modeling was performed on full and nested models with the best fitting models selected. Results : Heritability estimates were compared between Danish and Chinese samples to identify differential genetic influences on each of the phenotypes. Except for hip circumference, all other body measures exhibited similar heritability patterns in the two samples with body weight showing only a slight difference. Higher genetic influences were estimated for fasting blood glucose level in Chinese twins, whereas the Danish twins showed more genetic regulation over most lipids phenotypes. Systolic blood pressure was more genetically controlled in Danish than in Chinese twins. Conclusions : Metabolic endophenotypes show disparity in their genetic determinants in populations under distinct environmental conditions.     

Vascular Disease and Risk Stratification for Ischemic Stroke and All-Cause Death in Heart Failure Patients without Diagnosed Atrial Fibrillation: A Nationwide Cohort Study

Background

Stroke and mortality risk among heart failure patients previously diagnosed with different manifestations of vascular disease is poorly described. We conducted an observational study to evaluate the stroke and mortality risk among heart failure patients without diagnosed atrial fibrillation and with peripheral artery disease (PAD) or prior myocardial infarction (MI).

Methods

Population-based cohort study of patients diagnosed with incident heart failure during 2000–2012 and without atrial fibrillation, identified by record linkage between nationwide registries in Denmark. Hazard rate ratios of ischemic stroke and all-cause death after 1 year of follow-up were used to compare patients with either: a PAD diagnosis; a prior MI diagnosis; or no vascular disease.

Results

39,357 heart failure patients were included. When compared to heart failure patients with no vascular disease, PAD was associated with a higher 1-year rate of ischemic stroke (adjusted hazard rate ratio [HR]: 1.34, 95% confidence interval [CI]: 1.08–1.65) and all-cause death (adjusted HR: 1.47, 95% CI: 1.35–1.59), whereas prior MI was not (adjusted HR: 1.00, 95% CI: 0.86–1.15 and 0.94, 95% CI: 0.89–1.00, for ischemic stroke and all-cause death, respectively). When comparing patients with PAD to patients with prior MI, PAD was associated with a higher rate of both outcomes.

Conclusions

Among incident heart failure patients without diagnosed atrial fibrillation, a previous diagnosis of PAD was associated with a significantly higher rate of the ischemic stroke and all-cause death compared to patients with no vascular disease or prior MI. Prevention strategies may be particularly relevant among HF patients with PAD.     

Urinary nucleic acid oxidation product levels show differential associations with pharmacological treatment in patients with type 2 diabetes

The relationship between RNA and DNA oxidation and pharmacological treatment has not been systematically investigated in patients with type 2 diabetes (T2D). We aimed to investigate the association between pharmacological treatments and levels of urinary markers of nucleic acid oxidation in T2D patients. Vejle Diabetes Biobank cohort data was nested into nationwide registry data. Multiple logistic regression was used to associate drug usage with risk of high (above median) RNA and DNA oxidation. Data from 2664 T2D patients (64% male, age range: 25–75) were included. Questionnaire-validated lipid lowering drug use was associated with low RNA oxidation (Odds ratio, OR 0.71, 95% CI: [0.59–0.87]). Insulin and non-specific antidiabetic drugs were associated with low DNA oxidation (insulin: OR 0.60, 95% CI [0.49–0.73]). Oral antidiabetics were associated with high DNA oxidation and RNA oxidation (OR 1.30, 95% CI [1.10–1.53] and OR 1.26, 95% CI [1.07–1.29]). Our findings indicate that diabetes-related drugs are associated with RNA and DNA oxidation and further studies are required to determine causality in T2D patients.     

Serum cytokines of the 20 Krad-irradiated S. mansoni cercariae vaccinated, primary and superinfected Cercopethicus aethiops aethiops

Appropriate animal models are necessary to better understand the immune response in schistosomiasis. Schistosoma mansoni infection was established using irradiated cercariae in Cercopithecus aethiops aethiops (Grivet monkey) to describe immune responses of the serum cytokines, IL-4, IL-10, IL-12, IFN- gamma, and TNF-alpha. Intraperitoneal irradiated cercariae immunization on three occasions resulted in some differences of cytokine production. In primary infection, IL-4 was significantly raised (p=0.03) in the immunized monkeys, and there was an insignificant increase (p>0.05) in IL-10. However, ova excretion did not influence the cytokines, except in the controls where both IL-4 and IL-10 were significantly increased (p<0.05). In the controls, IL-12 and INF-gamma levels were lower after ova excretion, but the inflammatory TNF-alpha increased (p=0.049) and these findings can be associated with more liver pathogenesis in the group. Thus, this work has indicated the potential importance of anti-schistosome vaccine studies on the grivet monkeys.