Summary
Ammonium (NH4+) is toxic to root growth in most plants, even at moderate concentrations. Transcriptional regulation is one of the most important mechanisms in the response of plants to NH4+ ...toxicity, but the nature of the involvement of transcription factors (TFs) in this regulation remains unclear.
Here, RNA‐seq analysis was performed on Arabidopsis roots to screen for ammonium‐responsive TFs. WRKY46, the member of the WRKY transcription factor family most responsive to NH4+, was selected. We defined the role of WRKY46 using mutation and overexpression assays, and characterized the regulation of NUDX9 and indole‐3‐acetic acid (IAA)‐conjugating genes by WRKY46 via yeast one‐hybrid and electrophoretic mobility shift assays and chromatin immunoprecipitation‐quantitative real‐time polymerase chain reaction (ChIP‐qPCR).
Knockout of WRKY46 increased, while overexpression of WRKY46 decreased, NH4+‐suppression of the primary root. WRKY46 is shown to directly bind to the promoters of the NUDX9 and IAA‐conjugating genes (GH3.1, GH3.6, UGT75D1, UGT84B2) and to inhibit their transcription, thus positively regulating free IAA content and stabilizing protein N‐glycosylation, leading to an inhibition of NH4+ efflux in the root elongation zone (EZ).
We identify TF involvement in the regulation of NH4+ efflux in the EZ, and show that WRKY46 inhibits NH4+ efflux by negative regulation of NUDX9 and IAA‐conjugating genes.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
2.
Function of N-glycosylation in plants Nagashima, Yukihiro; von Schaewen, Antje; Koiwa, Hisashi
Plant science (Limerick),
September 2018, 2018-Sep, 2018-09-00, 20180901, Volume:
274
Journal Article
Peer reviewed
Open access
•N-linked complex glycans of plant glycoproteins have unique structures containing core α1,3-fucose and β1,2-xylose residues.•Some receptor-like kinases are under regulation of the ER quality control ...pathway.•Salt tolerance that involves coordinated cellulose biosynthesis activities at the plasma membrane is specifically affected by loss of complex N-glycan maturation.
Protein N-glycosylation is one of the major post-translational modifications in eukaryotic cells. In lower unicellular eukaryotes, the known functions of N-glycans are predominantly in protein folding and quality control within the lumen of the endoplasmic reticulum (ER). In multicellular organisms, complex N-glycans are important for developmental programs and immune responses. However, little is known about the functions of complex N-glycans in plants. Formed in the Golgi apparatus, plant complex N-glycans have structures distinct from their animal counterparts due to a set of glycosyltransferases unique to plants. Severe basal underglycosylation in the ER lumen induces misfolding of newly synthesized proteins, which elicits the unfolded protein response (UPR) and ER protein quality control (ERQC) pathways. The former promotes higher capacity of proper protein folding and the latter degradation of misfolded proteins to clear the ER. Although our knowledge on plant complex N-glycan functions is limited, genetic studies revealed the importance of complex N-glycans in cellulose biosynthesis and growth under stress.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Summary
The oligosaccharyltransferase (OT) complex catalyzes N‐glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known ...about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity‐tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super‐expression platform. Mass‐spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission‐electron microscopy showed that STT3a becomes incorporated into OT–ribosome super‐complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.
Significance Statement
A protein super‐expression system was established that is suitable for studying higher‐order structure and function of protein super‐complexes using Arabidopsis cell cultures. The system enabled the production and isolation of authentic oligosaccharyltransferase (OT) complexes assembled on a recombinant STT3a subunit, leading to identification and characterization of OT subunits and solving the three‐dimensional structure of plant OT–ribosome complexes.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
N‐glycosylation is one of the predominant modifications of eukaryotic proteins. It is catalyzed by oligosaccharyl transferase (OST), an eight‐subunit protein complex in the endoplasmic reticulum ...membrane. OST transfers the oligosaccharide from a lipid‐linked donor (LLO) to the Asn‐Xaa‐Ser/Thr sequon of nascent polypeptide, usually cotranslationally by partnering with the ribosome and the translocon. We and two other groups have recently determined high‐resolution cryo‐EM structures of the yeast and mammalian OST complexes. In this Structural Snapshot, we describe the molecular mechanism of eukaryotic OST and its interaction with the translocon.
In this Structural Snapshot, we provide an overview of the cryo‐EM structures of eukaryotic oligosaccharyl transferase (OST) solved recently by us and two other groups. We compare the cryo‐EM 3D density maps of the Saccharomyces cerevisiae OST in detergent digitonin at 3.5 Å resolution (left panel) or reconstituted in nanodisc at 3.3 Å resolution (right panel) and discuss the molecular mechanism of eukaryotic OST.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
GDP-D-mannose (GDP-D-Man) is an important intermediate in ascorbic acid (AsA) synthesis, cell wall synthesis, protein N-glycosylation, and glycosylphosphatidylinositol-anchoring in plants. Thus, the ...modulation of intracellular levels of GDP-D-Man could be important for maintaining various cellular processes. Here an Arabidopsis GDP-D-Man pyrophosphohydrolase, AtNUDX9 (AtNUDT9; At3g46200), which hydrolysed GDP-D-Man to GMP and mannose 1-phosphate, was identified. The Km and Vmax values for GDP-D-Man of AtNUDX9 were 376 ± 24 μM and 1.61 ± 0.15 μmol min−1 mg−1 protein, respectively. Among various tissues, the expression levels of AtNUDX9 and the total activity of GDP-D-Man pyrophosphohydrolase were the highest in the roots. The GDP-D-Man pyrophosphohydrolase activity was increased in the root of plants grown in the presence of ammonium. No difference was observed in the levels of AsA in the leaf and root tissues of the wild-type and knockout-nudx9 (KO-nudx9) plants, whereas a marked increase in N-glycoprotein levels and enhanced growth were detected in the roots of KO-nudx9 plants in the presence of ammonium. These results suggest that AtNUDX9 is involved in the regulation of GDP-D-Man levels affecting ammonium sensitivity via modulation of protein N-glycosylation in the roots.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
•Serum protein N-glycome is substantially altered in adults with T1DM.•N-glycome is not further influenced by the duration of the disease.•Complexity of N-glycome increases with higher HbA1c and ...smoking in T1DM.•Common N-glycosylation of new-onset and long-term T1DM may imply genetic interplay.•Changes unique to T1DM adults may reflect altered glucose metabolism and inflammation.
Previously we have shown that plasma protein N-glycosylation is changed in children at the onset of type 1 diabetes. In this study, we aim to identify N-glycan changes in adults with T1DM, compare them to those in children, and investigate their associations with disease duration, complications, glycaemic status, and smoking.
Serum protein N-glycans from 200 adults with type 1 diabetes and 298 healthy controls were analysed using ultra-high performance liquid chromatography and divided into 39 directly measured glycan groups from which 16 derived traits were calculated.
Compared to healthy controls, subjects with type 1 diabetes showed differences in 19 glycan groups and a decrease in monogalactosylated, an increase in digalactosylated, monosialylated, and antennary fucosylated derived traits, from which changes in monogalactosylation and seven directly measured traits overlapped with previously reported in children. Changes in four directly measured and two derived traits previously seen in children were not detected in adults. HbA1c was positively associated with sialylated and highly branched structures, whereas N-glycome was not influenced by disease duration or diabetic complications.
Our results suggest potential N-glycome involvement in different stages of type 1 diabetes, including processes underlying its development, the disease itself, as well as those occurring after disease establishment.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 ...(GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic “lipid-altered” tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
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•Structures of DPAGT1 with UDP-GlcNAc and tunicamycin reveal mechanisms of catalysis•DPAGT1 mutations in patients with glycosylation disorders modulate DPAGT1 activity•Structures, kinetics and biosynthesis reveal role of lipid in tunicamycin•Lipid-altered, tunicamycin analogues give non-toxic antibiotics against TB
Structural insights into tunicamycin’s toxic interactions with the human N-linked glycosylation pathway allows the identification of non-toxic antibiotics effective against tuberculosis in mice
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Free polymannose-type oligosaccharides (fOS) are processed by cytosolic enzymes to generate Man5GlcNAc which is transferred to lysosomes and degraded. Lysosomal fOS import was demonstrated in vitro ...but is poorly characterized in part due to lack of convenient substrates. As chitooligosaccharides (COS, oligomers β1,4-linked GlcNAc) block 3HMan5GlcNAc transport into lysosomes, we asked if COS are themselves transported and if so, can they be chemically modified to generate fluorescent substrates. We show that COS are degraded by lysosomal hydrolases to generate GlcNAc, and robust ATP-dependent transport of 3HCOS2/4 di and tetrasaccharides into intact rat liver lysosomes was observed only after blocking lysosomal 3HGlcNAc efflux with cytochalasin B. As oligosaccharides with unmodified reducing termini are the most efficient inhibitors of 3HCOS2/4 and 3HMan5GlcNAc transport, the non-reducing GlcNAc residue of COS2-4 was de-N-acetylated using Sinorhizobium meliloti NodB, and the resulting amine substituted with rhodamine B (RB) to yield RB-COS2-4. The fluorescent compounds inhibit 3HMan5GlcNAc transport and display temperature-sensitive, ATP-dependent transport into a sedimentable compartment that is ruptured with the lysosomotropic agent L-methyl methionine ester. Once in this compartment, RB-COS3 is converted to RB-COS2 further identifying it as the lysosomal compartment. RB-COS2/3 and 3HMan5GlcNAc transports are blocked similarly by competing sugars, and are partially inhibited by the vacuolar ATPase inhibitor bafilomycin and high concentrations of the P-type ATPase inhibitor orthovanadate. These data show that Man5GlcNAc, COS2/4 and RB-COS2/3 are transported into lysosomes by the same or closely related mechanism and demonstrate the utility of COS modified at their non-reducing terminus to study lysosomal oligosaccharide transport.
ABSTRACT
The ascorbic acid (AA)‐deficient Arabidopsis thaliana mutant vtc1‐1, which is defective in GDP‐mannose pyrophosphorylase (GMPase), exhibits conditional hypersensitivity to ammonium (NH4+), a ...phenomenon that is independent of AA deficiency. As GMPase is important for GDP‐mannose biosynthesis, a nucleotide sugar necessary for protein N‐glycosylation, it has been thought that GDP‐mannose deficiency is responsible for the growth defect in vtc1‐1 in the presence of NH4+. Therefore, the motivation for this work was to elucidate the growth and developmental processes that are affected in vtc1‐1 in the presence of NH4+ and to determine whether GDP‐mannose deficiency generally causes NH4+ sensitivity. Furthermore, as NH4+ may alter cytosolic pH, we investigated the responses of vtc1‐1 to pH changes in the presence and absence of NH4+. Using qRT‐PCR and staining procedures, we demonstrate that defective N‐glycosylation in vtc1‐1 contributes to cell wall, membrane and cell cycle defects, resulting in root growth inhibition in the presence of NH4+. However, by using mutants acting upstream of vtc1‐1 and contributing to GDP‐mannose biosynthesis, we show that GDP‐mannose deficiency does not generally lead to and is not the primary cause of NH4+ sensitivity. Instead, our data suggest that GMPase responds to pH alterations in the presence of NH4+.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Aims/hypothesis
We previously demonstrated that N-glycosylation of plasma proteins and IgGs is different in children with recent-onset type 1 diabetes compared with their healthy siblings. To search ...for genetic variants contributing to these changes, we undertook a genetic association study of the plasma protein and IgG N-glycome in type 1 diabetes.
Methods
A total of 1105 recent-onset type 1 diabetes patients from the Danish Registry of Childhood and Adolescent Diabetes were genotyped at 183,546 genetic markers, testing these for genetic association with variable levels of 24 IgG and 39 plasma protein N-glycan traits. In the follow-up study, significant associations were validated in 455 samples.
Results
This study confirmed previously known plasma protein and/or IgG N-glycosylation loci (candidate genes
MGAT3
,
MGAT5
and
ST6GAL1
, encoding beta-1,4-mannosyl-glycoprotein 4-beta-
N
-acetylglucosaminyltransferase, alpha-1,6-mannosylglycoprotein 6-beta-
N
-acetylglucosaminyltransferase and ST6 beta-galactoside alpha-2,6-sialyltransferase 1 gene, respectively) and identified novel associations that were not previously reported for the general European population. First, novel genetic associations of IgG-bound glycans were found with SNPs on chromosome 22 residing in two genomic intervals close to candidate gene
MGAT3
; these include core fucosylated digalactosylated disialylated IgG N-glycan with bisecting
N
-acetylglucosamine (GlcNAc) (
p
discovery
=7.65 × 10
−12
,
p
replication
=8.33 × 10
−6
for the top associated SNP rs5757680) and core fucosylated digalactosylated glycan with bisecting GlcNAc (
p
discovery
=2.88 × 10
−10
,
p
replication
=3.03 × 10
−3
for the top associated SNP rs137702). The most significant genetic associations of IgG-bound glycans were those with
MGAT3
. Second, two SNPs in high linkage disequilibrium (missense rs1047286 and synonymous rs2230203) located on chromosome 19 within the protein coding region of the complement C3 gene (
C3
) showed association with the oligomannose plasma protein N-glycan (
p
discovery
=2.43 × 10
−11
,
p
replication
=8.66 × 10
−4
for the top associated SNP rs1047286).
Conclusions/interpretation
This study identified novel genetic associations driving the distinct N-glycosylation of plasma proteins and IgGs identified previously at type 1 diabetes onset. Our results highlight the importance of further exploring the potential role of N-glycosylation and its influence on complement activation and type 1 diabetes susceptibility.
Graphical abstract
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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