The non-ELR CXC chemokine encoded by human cytomegalovirus UL146 genotype 5 contains a C-terminal ß-hairpin and induces neutrophil migration as a selective CXCR2 agonist.

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. The UL146 gene exists as 14 diverse genotypes among clinical isolates, which encode 14 different CXC chemokines. One genotype (vCXCL1GT1) is a known agonist for CXCR1 and CXCR2, while two others (vCXCL1GT5 and vCXCL1GT6) lack the ELR motif considered crucial for CXCR1 and CXCR2 binding, thus suggesting another receptor targeting profile. To determine the receptor target for vCXCL1GT5, the chemokine was probed in a G protein signaling assay on all 18 classical human chemokine receptors, where CXCR2 was the only receptor being activated. In addition, vCXCL1GT5 recruited ß-arrestin in a BRET-based assay and induced migration in a chemotaxis assay through CXCR2, but not CXCR1. In contrast, vCXCL1GT1 stimulated G protein signaling, recruited ß-arrestin and induced migration through both CXCR1 and CXCR2. Both vCXCL1GT1 and vCXCL1GT5 induced equally potent and efficacious migration of neutrophils, and ELR vCXCL1GT4 and non-ELR vCXCL1GT6 activated only CXCR2. In contrast to most human chemokines, the 14 UL146 genotypes have remarkably long C-termini. Comparative modeling using Rosetta showed that each genotype could adopt the classic chemokine core structure, and predicted that the extended C-terminal tail of several genotypes (including vCXCL1GT1, vCXCL1GT4, vCXCL1GT5, and vCXCL1GT6) forms a novel ß-hairpin not found in human chemokines. Secondary NMR shift and TALOS+ analysis of vCXCL1GT1 supported the existence of two stable ß-strands. C-terminal deletion of vCXCL1GT1 resulted in a non-functional protein and in a shift to solvent exposure for tryptophan residues likely due to destabilization of the chemokine fold. The results demonstrate that non-ELR chemokines can activate CXCR2 and suggest that the UL146 chemokines have unique C-terminal structures that stabilize the chemokine fold. Increased knowledge of the structure and interaction partners of the chemokine variants encoded by UL146 is key to understanding why circulating HCMV strains sustain 14 stable genotypes.

Circulating Concentrations of C-Type Natriuretic Peptides Increase with Sacubitril/Valsartan Treatment in Healthy Young Men.

BACKGROUND: C-type natriuretic peptide (CNP) is a cardioprotective peptide with high affinity for the ectoenzyme neutral endopeptidase (neprilysin). We aimed to determine whether angiotensin receptor-neprilysin inhibitor treatment acutely affects circulating concentrations of bioactive CNP and its molecular amino-terminal precursor (NT-proCNP). METHODS: We included 9 and 10 healthy young men in 2 randomized crossover trials with sacubitril/valsartan vs control (Trial 1) and sacubitril/valsartan and sitagliptin vs sitagliptin (Trial 2). The participants were randomized to a single dose of sacubitril/valsartan (194/206 mg) or control at the first visit 30 min prior to a standardized meal intake. We obtained blood samples at 12 time points over 5 h and measured plasma concentrations of NT-proCNP in both trials and CNP in Trial 2. RESULTS: NT-proCNP concentrations increased 3.5 h after sacubitril/valsartan treatment, and at 4.5 h concentrations were 42% and 65% higher compared with control in Trial 1 and Trial 2, respectively. The total area under the curve (tAUC)15-270 min was 22% higher (P = 0.007) in Trial 1 and 17% higher with treatment (P = 0.017) in Trial 2. Concentrations of bioactive CNP followed a similar temporal pattern with an increase of 93% at 4.5 h and a 31% higher tAUC15-270 min compared with control (P = 0.001) in Trial 2. CONCLUSIONS: Sacubitril/valsartan augments circulating concentrations of both bioactive CNP and NT-proCNP in healthy young men. The increase in bioactive CNP is most likely caused by de novo synthesis and secretion rather than diminished breakdown through neprilysin inhibition.ClinicalTrials.gov registration number NCT03717688.

Discovery of O-glycans on atrial natriuretic peptide (ANP) that affect both its proteolytic degradation and potency at its cognate receptor.

Atrial natriuretic peptide (ANP) is a peptide hormone that in response to atrial stretch is secreted from atrial myocytes into the circulation, where it stimulates vasodilatation and natriuresis. ANP is an important biomarker of heart failure where low plasma concentrations exclude cardiac dysfunction. ANP is a member of the natriuretic peptide (NP) family, which also includes the B-type natriuretic peptide (BNP) and the C-type natriuretic peptide. The proforms of these hormones undergo processing to mature peptides, and for proBNP, this process has previously been demonstrated to be regulated by O-glycosylation. It has been suggested that proANP also may undergo post-translational modifications. Here, we conducted a targeted O-glycoproteomics approach to characterize O-glycans on NPs and demonstrate that all NP members can carry O-glycans. We identified four O-glycosites in proANP in the porcine heart, and surprisingly, two of these were located on the mature bioactive ANP itself. We found that one of these glycans is located within a conserved sequence motif of the receptor-binding region, suggesting that O-glycans may serve a function beyond intracellular processing and maturation. We also identified an O-glycoform of proANP naturally occurring in human circulation. We demonstrated that site-specific O-glycosylation shields bioactive ANP from proteolytic degradation and modifies potency at its cognate receptor in vitro Furthermore, we showed that ANP O-glycosylation attenuates acute renal and cardiovascular ANP actions in vivo The discovery of novel glycosylated ANP proteoforms reported here significantly improves our understanding of cardiac endocrinology and provides important insight into the etiology of heart failure.

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates ß1-Adrenergic Receptor N-terminal Cleavage.

The ß1-adrenergic receptor (ß1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for ß-adrenergic antagonists, such as ß-blockers, relatively little is yet known about its regulation. We have shown previously that ß1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates ß1AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of ß1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the ßAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of ß1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases.

Augmented Reality for Subsurface Utility Engineering, Revisited.

Civil engineering is a primary domain for new augmented reality technologies. In this work, the area of subsurface utility engineering is revisited, and new methods tackling well-known, yet unsolved problems are presented. We describe our solution to the outdoor localization problem, which is deemed one of the most critical issues in outdoor augmented reality, proposing a novel, lightweight hardware platform to generate highly accurate position and orientation estimates in a global context. Furthermore, we present new approaches to drastically improve realism of outdoor data visualizations. First, a novel method to replace physical spray markings by indistinguishable virtual counterparts is described. Second, the visualization of 3D reconstructions of real excavations is presented, fusing seamlessly with the view onto the real environment. We demonstrate the power of these new methods on a set of different outdoor scenarios.

Neprilysin Inhibition Increases Glucagon Levels in Humans and Mice With Potential Effects on Amino Acid Metabolism.

CONTEXT: Inhibitors of the protease neprilysin (NEP) are used for treating heart failure, but are also linked to improvements in metabolism. NEP may cleave proglucagon-derived peptides, including the glucose and amino acid (AA)-regulating hormone glucagon. Studies investigating NEP inhibition on glucagon metabolism are warranted. OBJECTIVE: This work aims to investigate whether NEP inhibition increases glucagon levels. METHODS: Plasma concentrations of glucagon and AAs were measured in eight healthy men during a mixed meal with and without a single dose of the NEP inhibitor/angiotensin II type 1 receptor antagonist, sacubitril/valsartan (194 mg/206 mg). Long-term effects of sacubitril/valsartan (8 weeks) were investigated in individuals with obesity (n = 7). Mass spectrometry was used to investigate NEP-induced glucagon degradation, and the derived glucagon fragments were tested pharmacologically in cells transfected with the glucagon receptor (GCGR). Genetic deletion or pharmacological inhibition of NEP with or without concomitant GCGR antagonism was tested in mice to evaluate effects on AA metabolism. RESULTS: In healthy men, a single dose of sacubitril/valsartan significantly increased postprandial concentrations of glucagon by 228%, concomitantly lowering concentrations of AAs including glucagonotropic AAs. Eight-week sacubitril/valsartan treatment increased fasting glucagon concentrations in individuals with obesity. NEP cleaved glucagon into 5 inactive fragments (in vitro). Pharmacological NEP inhibition protected both exogenous and endogenous glucagon in mice after an AA challenge, while NEP-deficient mice showed elevated fasting and AA-stimulated plasma concentrations of glucagon and urea compared to controls. CONCLUSION: NEP cleaves glucagon, and inhibitors of NEP result in hyperglucagonemia and may increase postprandial AA catabolism without affecting glycemia.

An atlas of O-linked glycosylation on peptide hormones reveals diverse biological roles.

Peptide hormones and neuropeptides encompass a large class of bioactive peptides that regulate physiological processes like anxiety, blood glucose, appetite, inflammation and blood pressure. Here, we execute a focused discovery strategy to provide an extensive map of O-glycans on peptide hormones. We find that almost one third of the 279 classified peptide hormones carry O-glycans. Many of the identified O-glycosites are conserved and are predicted to serve roles in proprotein processing, receptor interaction, biodistribution and biostability. We demonstrate that O-glycans positioned within the receptor binding motifs of members of the neuropeptide Y and glucagon families modulate receptor activation properties and substantially extend peptide half-lives. Our study highlights the importance of O-glycosylation in the biology of peptide hormones, and our map of O-glycosites in this large class of biomolecules serves as a discovery platform for an important class of molecules with potential opportunities for drug designs.

Sacubitril/Valsartan Augments Postprandial Plasma Concentrations of Active GLP-1 When Combined With Sitagliptin in Men.

CONTEXT: Combined inhibition of neprilysin and dipeptidyl peptidase 4 (DPP-4) has been shown to augment plasma concentrations of glucagon-like peptide-1(GLP-1) in animal models, but whether this occurs in humans is unknown. OBJECTIVE: To investigate the effects of inhibition of neprilysin by sacubitril/valsartan alone or in combination with a DPP-4 inhibitor (sitagliptin) on plasma concentrations of GLP-1 in healthy men. DESIGN: Two open-labeled crossover studies were performed in human subjects. SETTING: General community. PARTICIPANTS: Nine and 10 healthy young men were included in study 1 and study 2, respectively. INTERVENTION: Study participants received a standardized meal (34% carbohydrates, 45% fat, 21% protein; total caloric content, 2106 kJ) combined with a prior dose of either sacubitril/valsartan (194/206 mg) or control in study 1 and in study 2, with a prior dose of sitagliptin (2 ×100 mg, given ∼10 hours apart) either alone or with sacubitril/valsartan (194/206 mg). MAIN OUTCOME MEASURES: Plasma concentrations of total and intact GLP-1. RESULTS: Sacubitril/valsartan increased postprandial plasma concentrations of total GLP-1 by 67% [total area under the curve (tAUC)0-240min: 3929 ± 344 vs 2348 ± 181 minutes × pmol/L, P = 0.0023] and increased concentrations of intact GLP-1 plasma concentrations more than sitagliptin alone (tAUC0-240min: 1021 ± 114 vs 660 ± 80 minutes × pmol/L, P = 0.01). Plasma concentrations of glucose, insulin, and GIP were not significantly (P > 0.10) changed upon sacubitril/valsartan treatment. CONCLUSIONS: Sacubitril/valsartan combined with a DPP-4 inhibitor led to markedly higher concentrations of intact GLP-1 than DPP-4 inhibition alone, supporting a role for both neprilysin and DPP-4 in the metabolism of GLP-1 in humans, a finding that may have therapeutic implications.

Cardiac procholecystokinin expression during haemodynamic changes in the mammalian heart.

Cardiac myocytes express the cholecystokinin gene (CCK) at propeptide level. We recently reported that cardiac CCK expression is acutely regulated by isoprenaline in a porcine model. The regulation of CCK expression after myocardial infarction, in exercise, and in severe heart failure is, however, unknown. Cardiac tissue was obtained from healthy new-born and adolescent farm pigs. Myocardial infarction was induced by coronary artery occlusion in adult minipigs. Healthy male subjects performed a 3-hour exercise test, and patients with severe heart failure referred for right heart catheterization were included. Extracts of porcine cardiac tissue and human plasma were analysed with specific proCCK radioimmunoassays. Cardiac proCCK expression shifted from the right atrium in new-born piglets to include the left atrium in adolescent pigs. Regional proCCK expression in the adolescent pig heart was mainly confined to the atria without different expression in sinus node tissue. In adult minipigs with myocardial infarction, no changes in overall left ventricular function or proCCK expression were observed after 8 weeks. In healthy adults, proCCK in circulation increased markedly during exercise in parallel with pro-B-type natriuretic peptide. Finally, patients with severe heart failure displayed markedly increased proCCK - but not CCK - concentrations in plasma. Taken together, our data shows that regional proCCK expression reflects haemodynamic changes in the mammalian heart. The data supports the notion that cardiac CCK expression resembles that of cardiac natriuretic peptides in atria. The ventricular content of proCCK, however, differs from natriuretic peptides and suggests a distinct secretory pathway in ventricular cardiomyocytes.

Chromogranin A in the mammalian heart: expression without secretion.

AIM: To investigate whether chromogranin A (CgA) is secreted from the heart into circulation. MATERIALS & METHODS: Porcine cardiac tissue was analyzed for the presence of CgA-derived glycopeptides using a global O-glycoproteomic strategy. Blood was sampled from the femoral vein, right atrium, coronary sinus and the left atrium from patients with predominantly atrial disease. The local concentration of proatrial natriuretic peptide and CgA was measured with immunoassays. RESULTS: We identified CgA-derived glycopeptides exclusively in the atrial tissue. Proatrial natriuretic peptide is secreted from the heart (coronary sinus [795 pmol/l] vs left atrium [678 pmol/l]; p < 0.01) whereas no CgA gradient across the heart could be established (p = 0.6366). CONCLUSION: The cardiac atria express but do not secrete CgA into circulation in patients with atrial disease.

Atrial natriuretic peptides in plasma.

Measurement of cardiac natriuretic peptides in plasma has gained a diagnostic role in the assessment of heart failure. Plasma measurement is though hampered by the marked instability of the hormones, which has led to the development of analyses that target N-terminal fragments from the prohormone. These fragments are stable in plasma and represent surrogate markers of the actual natriuretic hormone. Post-translational processing of the precursors, however, is revealing itself to be a complex event with new information still being reported on proteolysis, covalent modifications, and amino acid derivatizations. In this mini-review, we summarize measurement of the principal cardiac hormone, e.g. atrial natriuretic peptide (ANP) and its precursor fragments. We also highlight some of the analytical pitfalls and problems and the concurrent clinical "proof of concept". We conclude that biochemical research into proANP-derived peptides is still worthy of attention and that new biological insight may change our chemical perception of the markers.

Cardiac C-type natriuretic peptide gene expression and plasma concentrations in neonatal piglets.

BACKGROUND: C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family. Cardiac ANP and BNP expressions are firmly established, whereas CNP expression in the mammalian heart remains controversial. In the present report, we used a porcine model of the neonatal period with high expressions of cardiac ANP and BNP in order to elucidate the cardiac CNP expression profile. METHODS: Plasma and cardiac tissue were obtained from newborn piglets during the first 72 h of life. The chamber-specific CNP mRNA contents were quantified by real-time PCR analysis. The proCNP concentrations in plasma and cardiac tissue extracts were quantified by a porcine-specific radioimmunoassay. RESULTS: Cardiac CNP mRNA contents (n=24) were low compared to sites of known expression, where porcine seminal vesicle CNP mRNA contents were 200-fold higher. In addition, plasma proCNP concentrations in the newborn piglets (n=44) were exceedingly low compared to proANP concentrations (5.3 pmol/L (3.2-8.6) vs. 3438 pmol/L (2790-5418), p<0.0001). The proCNP concentrations in atrial tissue extracts were barely detectable (≤0.06 pmol/g) (n=2) compared to ventricular proANP (130 pmol/g (101-159)) and atrial proANP (12,303 pmol/g (10,623-15,412)). CONCLUSION: Our data show that the heart is not a major source of circulating proCNP in neonatal piglets.