Sequential tyrosine sulfation of CXCR4 by tyrosylprotein sulfotransferases

CXC-chemokine receptor 4 (CXCR4) is a G protein-coupled receptor for stromal cell-derived factor-1 (SDF-1/CXCL12). SDF-1-induced CXCR4 signaling is indispensable for embryonic development and crucial for immune cell homing and has been implicated in metastasis of numerous types of cancer. CXCR4 also serves as the major coreceptor for cellular entry of T-cell line-tropic (X4) HIV-1 strains. Tyrosine residues in the N-terminal tail of CXCR4, which are post-translationally sulfated, are implicated in the high-affinity binding of SDF-1 to CXCR4. However, the specific roles of three potential tyrosine sulfation sites are not well understood. We investigated the pattern and sequence of CXCR4 sulfation by using recombinant human tyrosylprotein sulfotransferases TPST-1 and TPST-2 to modify a peptide that corresponds to amino acids 1-38 of the receptor (CXCR4 1-38). We analyzed the reaction products with a combination of reversed-phase HPLC, proteolytic cleavage, and mass spectrometry. We found that CXCR4 1-38 is sulfated efficiently by both TPST enzymes, leading to a final product with three sulfotyrosine residues. Sulfates were added stepwise to the peptide, producing specific intermediates with one or two sulfotyrosines. The pattern of sulfation in these intermediates indicates that with both enzymes Tyr-21 is sulfated first, followed by Tyr-12 or Tyr-7. Using heteronuclear NMR spectroscopy, we demonstrated that the SDF-1 binding affinity of CXCR4 1-38 increases with the number of sulfotyrosines present, which suggests a potential physiological role for sulfation of all three sites in the N-terminus of CXCR4. These results provide a structural basis for understanding the role of post-translational tyrosine sulfation in SDF-1-induced CXCR4 signaling.     

HERG channel (dys)function revealed by dynamic action potential clamp technique

The human ether-a-go-go-related gene (HERG) encodes the rapid component of the cardiac delayed rectifier potassium current (I(Kr)). Per-Arnt-Sim domain mutations of the HERG channel are linked to type 2 long-QT syndrome. We studied wild-type and/or type 2 long-QT syndrome-associated mutant (R56Q) HERG current (I(HERG)) in HEK-293 cells, at both 23 and 36 degrees C. Conventional voltage-clamp analysis revealed mutation-induced changes in channel kinetics. To assess functional implication(s) of the mutation, we introduce the dynamic action potential clamp technique. In this study, we effectively replace the native I(Kr) of a ventricular cell (either a human model cell or an isolated rabbit myocyte) with I(HERG) generated in a HEK-293 cell that is voltage-clamped by the free-running action potential of the ventricular cell. Action potential characteristics of the ventricular cells were effectively reproduced with wild-type I(HERG), whereas the R56Q mutation caused a frequency-dependent increase of the action potential duration in accordance with the clinical phenotype. The dynamic action potential clamp approach also revealed a frequency-dependent transient wild-type I(HERG) component, which is absent with R56Q channels. This novel electrophysiological technique allows rapid and unambiguous determination of the effects of an ion channel mutation on the ventricular action potential and can serve as a new tool for investigating cardiac channelopathies.     

The Role of Dissolved Organic Carbon, Dissolved Organic Nitrogen, and Dissolved Inorganic Nitrogen in a Tropical Wet Forest Ecosystem

Although tropical wet forests play an important role in the global carbon (C) and nitrogen (N) cycles, little is known about the origin, composition, and fate of dissolved organic C (DOC) and N (DON) in these ecosystems. We quantified and characterized fluxes of DOC, DON, and dissolved inorganic N (DIN) in throughfall, litter leachate, and soil solution of an old-growth tropical wet forest to assess their contribution to C stabilization (DOC) and to N export (DON and DIN) from this ecosystem. We found that the forest canopy was a major source of DOC (232 kg C ha^–1 y^–1). Dissolved organic C fluxes decreased with soil depth from 277 kg C ha^–1 y^–1 below the litter layer to around 50 kg C kg C ha^–1 y^–1 between 0.75 and 3.5m depth. Laboratory experiments to quantify biodegradable DOC and DON and to estimate the DOC sorption capacity of the soil, combined with chemical analyses of DOC, revealed that sorption was the dominant process controlling the observed DOC profiles in the soil. This sorption of DOC by the soil matrix has probably led to large soil organic C stores, especially below the rooting zone. Dissolved N fluxes in all strata were dominated by mineral N (mainly NO₃⁻). The dominance of NO₃^– relative to the total amount nitrate of N leaching from the soil shows that NO₃^– is dominant not only in forest ecosystems receiving large anthropogenic nitrogen inputs but also in this old-growth forest ecosystem, which is not N-limited.     

Stabilization of recent soil carbon in the humid tropics following land use changes: evidence from aggregate fractionation and stable isotope analyses

Quantitative knowledge of stabilization- and decomposition processes is necessary to understand, assess and predict effects of land use changes on storage and stability of soil organic carbon (soil C) in the tropics. Although it is well documented that different soil types have different soil C stocks, it is presently unknown how different soil types affect the stability of recently formed soil C. Here, we analyze the main controls of soil C storage in the top 0.1 m of soils developed on Tertiary sediments and soils developed on volcanic ashes. Using a combination of fractionation techniques with ¹³C isotopes analyses we had the opportunity to trace origin and stability of soil carbon in different aggregate fractions under pasture and secondary forest. Soil C contents were higher in volcanic ash soils (47–130 g kg⁻¹) than in sedimentary soils (19–50 g kg⁻¹). Mean residence time (MRT) of forest-derived carbon in pastures increased from 37 to 57 years with increasing silt + clay content in sedimentary soils, but was independent from soil properties in volcanic ash soils. MRTs of pasture-derived carbon in secondary forests were considerably shorter, especially in volcanic ash soils, where no pasture-derived carbon could be detected in any of the four studied secondary forests. The implications of these results are that the MRT of recently incorporated organic carbon depends on clay mineralogy and is longer in soils dominated by smectite than non-crystalline minerals. Our results show that the presence of soil C stabilization processes, does not necessarily mean that recent incorporated soil C will also be effectively stabilized.     

Mannose-binding lectin does not explain the course and outcome of pregnancy in rheumatoid arthritis

Introduction  

Rheumatoid arthritis (RA) improves during pregnancy and flares after delivery. It has been hypothesized that high levels of the complement factor mannose-binding lectin (MBL) are associated with a favourable disease course of RA by facilitating the clearance of pathogenic immunoglobulin G (IgG) lacking galactose sugar moieties. During pregnancy, increased galactosylation of IgG and simultaneously increased MBL levels can be observed, with the latter being strictly related to maternal MBL genotypes. Therefore, increased MBL levels in concert with increased IgG galactosylation may be associated with pregnancy-induced improvement of RA. The objective of this study was to investigate whether MBL genotypes are associated with changes in RA disease activity and with changes in IgG galactosylation during pregnancy and in the postpartum period. We also studied the association between MBL genotypes and pregnancy outcomes in RA.     

T-helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus?

Introduction

A hallmark of systemic autoimmune diseases like systemic lupus erythematosus (SLE) is the increased expression of interferon (IFN) type I inducible genes, so-called IFN type I signature. Recently, T-helper 17 subset (Th17 cells), which produces IL-17A, IL-17F, IL-21, and IL-22, has been implicated in SLE. As CCR6 enriches for Th17 cells, we used this approach to investigate whether CCR6⁺ memory T-helper cells producing IL-17A, IL-17F, IL-21, and/or IL-22 are increased in SLE patients and whether this increase is related to the presence of IFN type I signature.

Methods

In total, 25 SLE patients and 15 healthy controls (HCs) were included. SLE patients were divided into IFN type I signature-positive (IFN⁺) (n = 16) and negative (IFN^-) (n = 9) patients, as assessed by mRNA expression of IFN-inducible genes (IFIGs) in monocytes. Expression of IL-17A, IL-17F, IL-21, and IL-22 by CD4⁺CD45RO⁺CCR6⁺ T cells (CCR6⁺ cells) was measured with flow cytometry and compared between IFN⁺, IFN^- patients and HCs.

Results

Increased percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ cells were observed in IFN⁺ patients compared with IFN^- patients and HCs. IL-17A and IL-17F expression within CCR6⁺ cells correlated significantly with IFIG expression. In addition, we found significant correlation between B-cell activating factor of the tumor necrosis family (BAFF)–a factor strongly correlating with IFN type I - and IL-21 producing CCR6⁺ cells.

Conclusions

We show for the first time higher percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ memory T-helper cells in IFN⁺ SLE patients, supporting the hypothesis that IFN type I co-acts with Th17 cytokines in SLE pathogenesis.     

Temporal variation in nitrous oxide (N2O) fluxes from an oil palm plantation in Indonesia: An ecosystem-scale analysis

The rapidly growing areal extent of oil palm (Elaeis guineensis Jacq.) plantations and their high fertilizer input raises concerns about their role as substantial N₂O sources. In this study, we present the first eddy covariance (EC) measurements of ecosystem-scale N₂O fluxes in an oil palm plantation and combine them with vented soil chamber measurements of point-scale soil N₂O fluxes. Based on EC measurements during the period August 2017 to April 2019, the studied oil palm plantation in the tropical lowlands of Jambi Province (Sumatra, Indonesia) is a high source of N₂O, with average emission of 0.32 ± 0.003 g N₂O-N m⁻² year⁻¹ (149.85 ± 1.40 g CO₂-equivalent m⁻² year⁻¹). Compared to the EC-based N₂O flux, average chamber-based soil N₂O fluxes (0.16 ± 0.047 g N₂O-N m⁻² year⁻¹, 74.93 ± 23.41 g CO₂-equivalent m⁻² year⁻¹) are significantly (~49%, p < 0.05) lower, suggesting that important N₂O pathways are not covered by the chamber measurements. Conventional chamber-based N₂O emission estimates from oil palm up-scaled to ecosystem level might therefore be substantially underestimated. We show that the dynamic gas exchange of the oil palm canopy with the atmosphere and the oil palms' response to meteorological and soil conditions may play an important but yet widely unexplored role in the N₂O budget of oil palm plantations. Diel pattern of N₂O fluxes showed strong causal relationships with photosynthesis-related variables, i.e. latent heat flux, incoming photosynthetically active radiation and gross primary productivity during day time, and ecosystem respiration and soil temperature during night time. At longer time scales (>2 days), soil temperature and water-filled pore space gained importance on N₂O flux variation. These results suggest a plant-mediated N₂O transport, providing important input for modelling approaches and strategies to mitigate the negative impact of N₂O emissions from oil palm cultivation through appropriate site selection and management.