N-linked glycosylation enzymes in the diatom Thalassiosira oceanica exhibit a diel cycle in transcript abundance and favor for NXT-type sites

AbstractN-linked glycosylation is a posttranslational modification affecting protein folding and function. The N-linked glycosylation pathway in algae is poorly characterized, and further knowledge is needed to understand the cell biology of algae and the evolution of N-linked glycosylation. This study investigated the N-linked glycosylation pathway in Thalassiosira oceanica, an open ocean diatom adapted to survive at growth-limiting iron concentrations. Here we identified and annotated the genes coding for the essential enzymes involved in the N-linked glycosylation pathway of T. oceanica. Transcript levels for genes coding for calreticulin, oligosaccharyltransferase (OST), N-acetylglucosaminyltransferase (GnT1), and UDP-glucose glucosyltransferase (UGGT) under high- and low-iron growth conditions revealed diel transcription patterns with a significant decrease of calreticulin and OST transcripts under iron-limitation. Solid-phase extraction of N-linked glycosylated peptides (SPEG) revealed 118 N-linked glycosylated peptides from cells grown in high- and low-iron growth conditions. The identified peptides had 81% NXT-type motifs, with X being any amino acids except proline. The presence of N-linked glycosylation sites in the iron starvation-induced protein 1a (ISIP1a) confirmed its predicted topology, contributing to the biochemical characterization of ISIP1 proteins. Analysis of extensive oceanic gene databases showed a global distribution of calreticulin, OST, and UGGT, reinforcing the importance of glycosylation in microalgae.

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Production and analysis of a mammalian septin hetero-octamer complex

10.1101/2020.06.15.152751 ◽

2020 ◽

Author(s):

Barry T. DeRose ◽

Robert S. Kelley ◽

Roshni Ravi ◽

Bashkim Kokona ◽

Elias T. Spiliotis ◽

...

Keyword(s):

Cell Biology ◽

Biochemical Characterization ◽

Fluorescent Protein ◽

Mechanistic Study ◽

Central Position ◽

Protein Fusion ◽

Recombinant Production ◽

And Function ◽

Order Structures

AbstractThe septins are filament-forming proteins found in diverse eukaryotes from fungi to vertebrates, with roles in cytokinesis, shaping of membranes and modifying cytoskeletal organization. These GTPases assemble into rod-shaped soluble hetero-hexamers and hetero-octamers in mammals, which polymerize into filaments and higher order structures. While the cell biology and pathobiology of septins are advancing rapidly, mechanistic study of the mammalian septins is limited by a lack of recombinant hetero-octamer materials. We describe here the production and characterization of a recombinant mammalian septin hetero-octamer of defined stoichiometry, the SEPT2/SEPT6/SEPT7/SEPT3 complex. Using a fluorescent protein fusion to the complex, we observed filaments assembled from this complex. In addition, we used this novel tool to resolve recent questions regarding the organization of the soluble septin complex. Biochemical characterization of a SEPT3 truncation that disrupts SEPT3-SEPT3 interactions is consistent with SEPT3 occupying a central position in the complex while the SEPT2 subunits are at the ends of the rod-shaped octameric complexes. Consistent with SEPT2 being on the complex ends, we find that our purified SEPT2/SEPT6/SEPT7/SEPT3 hetero-octamer copolymerizes into mixed filaments with separately purified SEPT2/SEPT6/SEPT7 hetero-hexamer. We expect this new recombinant production approach to lay essential groundwork for future studies into mammalian septin mechanism and function.

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Molecular cloning and biochemical characterization of rabbit factor XI

Biochemical Journal ◽

10.1042/bj20020232 ◽

2002 ◽

Vol 367 (1) ◽

pp. 49-56 ◽

Cited By ~ 13

Author(s):

Dipali SINHA ◽

Mariola MARCINKIEWICZ ◽

David GAILANI ◽

Peter N. WALSH

Keyword(s):

Human Factor ◽

Biochemical Characterization ◽

Protein A ◽

Size Exclusion ◽

Factor Xi ◽

Sds Page ◽

Exclusion Chromatography ◽

Intracellular Process ◽

And Function

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.

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Arabidopsis thaliana β1,2-xylosyltransferase: an unusual glycosyltransferase with the potential to act at multiple stages of the plant N-glycosylation pathway

Biochemical Journal ◽

10.1042/bj20042091 ◽

2005 ◽

Vol 388 (2) ◽

pp. 515-525 ◽

Cited By ~ 42

Author(s):

Peter BENCÚR ◽

Herta STEINKELLNER ◽

Barbara SVOBODA ◽

Jan MUCHA ◽

Richard STRASSER ◽

...

Keyword(s):

Catalytic Activity ◽

Substrate Specificity ◽

Metal Ion ◽

Catalytic Domain ◽

Protein Characterization ◽

Reaction Products ◽

Glycosylation Sites ◽

Glycosylation Pathway ◽

Multiple Stages

XylT (β1,2-xylosyltransferase) is a unique Golgi-bound glycosyltransferase that is involved in the biosynthesis of glycoprotein-bound N-glycans in plants. To delineate the catalytic domain of XylT, a series of N-terminal deletion mutants was heterologously expressed in insect cells. Whereas the first 54 residues could be deleted without affecting the catalytic activity of the enzyme, removal of an additional five amino acids led to the formation of an inactive protein. Characterization of the N-glycosylation status of recombinant XylT revealed that all three potential N-glycosylation sites of the protein are occupied by N-linked oligosaccharides. However, an unglycosylated version of the enzyme displayed substantial catalytic activity, demonstrating that N-glycosylation is not essential for proper folding of XylT. In contrast with most other glycosyltransferases, XylT is enzymatically active in the absence of added metal ions. This feature is not due to any metal ion directly associated with the enzyme. The precise acceptor substrate specificity of XylT was assessed with several physiologically relevant compounds and the xylosylated reaction products were subsequently tested as substrates of other Golgi-resident glycosyltransferases. These experiments revealed that the substrate specificity of XylT permits the enzyme to act at multiple stages of the plant N-glycosylation pathway.

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Characterization of an Escherichia coli K12 mutant that is sensitive to chlorate when grown aerobically

Biochemical Journal ◽

10.1042/bj1760553 ◽

1978 ◽

Vol 176 (2) ◽

pp. 553-561 ◽

Cited By ~ 17

Author(s):

G Giordano ◽

L Grillet ◽

R Rosset ◽

J H Dou ◽

E Azoulay ◽

...

Keyword(s):

Escherichia Coli ◽

Biosynthetic Pathway ◽

Formate Dehydrogenase ◽

Biochemical Characterization ◽

Reductase Activity ◽

Growth Conditions ◽

Aerobic Growth ◽

Benzyl Viologen ◽

Genetic Lesion

Escherichia coli can normally grow aerobically in the presence of chlorate; however, mutants can be isolated that can no longer grow under these conditions. We present here the biochemical characterization of one such mutant and show that the primary genetic lesion occurs in the ubiquinone-8-biosynthetic pathway. As a consequence of this, under aerobic growth conditions the mutant is apparently unable to synthesize formate dehydrogenase, but can synthesize a Benzyl Viologen-dependent nitrate reductase activity. The nature of this activity is discussed.

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N-Glycosylation of Haloferax volcanii Flagellins Requires Known Agl Proteins and Is Essential for Biosynthesis of Stable Flagella

Journal of Bacteriology ◽

10.1128/jb.00731-12 ◽

2012 ◽

Vol 194 (18) ◽

pp. 4876-4887 ◽

Cited By ~ 54

Author(s):

Manuela Tripepi ◽

Jason You ◽

Sevcan Temel ◽

Özlem Önder ◽

Dustin Brisson ◽

...

Keyword(s):

Gradient Centrifugation ◽

Haloferax Volcanii ◽

Content Type ◽

Glycosylation Sites ◽

Cscl Density Gradient ◽

Domains Of Life ◽

Covalently Linked ◽

Glycosylation Pathway ◽

A Minor ◽

And Function

ABSTRACTN-glycosylation, a posttranslational modification required for the accurate folding and stability of many proteins, has been observed in organisms of all domains of life. Although the haloarchaeal S-layer glycoprotein was the first prokaryotic glycoprotein identified, little is known about the glycosylation of other haloarchaeal proteins. We demonstrate here that the glycosylation ofHaloferax volcaniiflagellins requires archaeal glycosylation (Agl) components involved in S-layer glycosylation and that the deletion of anyHfx. volcaniiaglgene impairs its swimming motility to various extents. A comparison of proteins in CsCl density gradient centrifugation fractions from supernatants of wild-typeHfx. volcaniiand deletion mutants lacking the oligosaccharyltransferase AglB suggests that when the Agl glycosylation pathway is disrupted, cells lack stable flagella, which purification studies indicate consist of a major flagellin, FlgA1, and a minor flagellin, FlgA2. Mass spectrometric analyses of FlgA1 confirm that its three predicted N-glycosylation sites are modified with covalently linked pentasaccharides having the same mass as that modifying its S-layer glycoprotein. Finally, the replacement of any of three predicted N-glycosylated asparagines of FlgA1 renders cells nonmotile, providing direct evidence for the first time that the N-glycosylation of archaeal flagellins is critical for motility. These results provide insight into the role that glycosylation plays in the assembly and function ofHfx. volcaniiflagella and demonstrate thatHfx. volcaniiflagellins are excellent reporter proteins for the study of haloarchaeal glycosylation processes.

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Identification of Residues Important for the Activity ofHaloferax volcaniiAglD, a Component of the Archaeal N-Glycosylation Pathway

Archaea ◽

10.1155/2010/315108 ◽

2010 ◽

Vol 2010 ◽

pp. 1-9 ◽

Cited By ~ 7

Author(s):

Lina Kaminski ◽

Jerry Eichler

Keyword(s):

Biochemical Characterization ◽

Haloferax Volcanii ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Natural Substrate ◽

Identify Amino Acid ◽

Glycosylation Pathway

InHaloferax volcanii, AglD adds the final hexose to the N-linked pentasaccharide decorating the S-layer glycoprotein. Not knowing the natural substrate of the glycosyltransferase, together with the challenge of designing assays compatible with hypersalinity, has frustrated efforts at biochemical characterization of AglD activity. To circumvent these obstacles, an in vivo assay designed to identify amino acid residues important for AglD activity is described. In the assay, restoration of AglD function in anHfx. volcanii aglDdeletion strain transformed to express plasmid-encoded versions of AglD, generated through site-directed mutagenesis at positions encoding residues conserved in archaeal homologues of AglD, is reflected in the behavior of a readily detectable reporter of N-glycosylation. As such Asp110 and Asp112 were designated as elements of the DXD motif of AglD, a motif that interacts with metal cations associated with nucleotide-activated sugar donors, while Asp201 was predicted to be the catalytic base of the enzyme.

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Detailed structural and biochemical characterization of the nexin-dynein regulatory complex

Molecular Biology of the Cell ◽

10.1091/mbc.e14-09-1367 ◽

2015 ◽

Vol 26 (2) ◽

pp. 294-304 ◽

Cited By ~ 34

Author(s):

Toshiyuki Oda ◽

Haruaki Yanagisawa ◽

Masahide Kikkawa

Keyword(s):

Biochemical Characterization ◽

Electron Tomography ◽

Three Dimensional ◽

Dimensional Structure ◽

Dynein Motor ◽

Cryo Electron Tomography ◽

Regulatory Complex ◽

And Function

The nexin-dynein regulatory complex (N-DRC) forms a cross-bridge between the outer doublet microtubules of the axoneme and regulates dynein motor activity in cilia/flagella. Although the molecular composition and the three-dimensional structure of N-DRC have been studied using mutant strains lacking N-DRC subunits, more accurate approaches are necessary to characterize the structure and function of N-DRC. In this study, we precisely localized DRC1, DRC2, and DRC4 using cryo–electron tomography and structural labeling. All three N-DRC subunits had elongated conformations and spanned the length of N-DRC. Furthermore, we purified N-DRC and characterized its microtubule-binding properties. Purified N-DRC bound to the microtubule and partially inhibited microtubule sliding driven by the outer dynein arms (ODAs). Of interest, microtubule sliding was observed even in the presence of fourfold molar excess of N-DRC relative to ODA. These results provide insights into the role of N-DRC in generating the beating motions of cilia/flagella.

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Copper binding by a unique family of metalloproteins is dependent on kynurenine formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2100680118 ◽

2021 ◽

Vol 118 (23) ◽

pp. e2100680118

Author(s):

Anastasia C. Manesis ◽

Richard J. Jodts ◽

Brian M. Hoffman ◽

Amy C. Rosenzweig

Keyword(s):

Biochemical Characterization ◽

Protein A ◽

Copper Ion ◽

Copper Homeostasis ◽

Metabolic Enzyme ◽

Copper Binding ◽

Methane Oxidizing Bacteria ◽

And Function

Some methane-oxidizing bacteria use the ribosomally synthesized, posttranslationally modified natural product methanobactin (Mbn) to acquire copper for their primary metabolic enzyme, particulate methane monooxygenase. The operons encoding the machinery to biosynthesize and transport Mbns typically include genes for two proteins, MbnH and MbnP, which are also found as a pair in other genomic contexts related to copper homeostasis. While the MbnH protein, a member of the bacterial diheme cytochrome c peroxidase (bCcP)/MauG superfamily, has been characterized, the structure and function of MbnP, the relationship between the two proteins, and their role in copper homeostasis remain unclear. Biochemical characterization of MbnP from the methanotroph Methylosinus trichosporium OB3b now reveals that MbnP binds a single copper ion, present in the +1 oxidation state, with high affinity. Copper binding to MbnP in vivo is dependent on oxidation of the first tryptophan in a conserved WxW motif to a kynurenine, a transformation that occurs through an interaction of MbnH with MbnP. The 2.04-Å-resolution crystal structure of MbnP reveals a unique fold and an unusual copper-binding site involving a histidine, a methionine, a solvent ligand, and the kynurenine. Although the kynurenine residue may not serve as a CuI primary-sphere ligand, being positioned ∼2.9 Å away from the CuI ion, its presence is required for copper binding. Genomic neighborhood analysis indicates that MbnP proteins, and by extension kynurenine-containing copper sites, are widespread and may play diverse roles in microbial copper homeostasis.

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Proximity interactome of LC3B in normal growth conditions

10.1101/2021.10.08.463639 ◽

2021 ◽

Author(s):

Marie Nollet ◽

Alexander Agrotis ◽

Fanourios Michailidis ◽

Arran David Dokal ◽

Vinothini Rajeeve ◽

...

Keyword(s):

Cell Biology ◽

Normal Growth ◽

Growth Conditions ◽

Biological Functions ◽

Normal Medium ◽

Outer Membranes ◽

Cellular Processes ◽

Proteomic Approach ◽

And Function

LC3 (Light Chain 3) is a key player of autophagy, a major stress-responsive proteolysis pathway promoting cellular homeostasis. It coordinates the formation and maturation of autophagosomes and recruits cargo to be further degraded upon autophagosome-lysosome fusion. To orchestrate its functions, LC3 binds to multiple proteins from the autophagosomes inner and outer membranes, but the full extent of these interactions is not known. Moreover, LC3 has been increasingly reported in other cellular locations than the autophagosome, with cellular outcome not fully understood and not all related to autophagy. Furthermore, novel functions of LC3 as well as autophagy can occur in cells growing in a normal medium thus in non-stressed conditions. A better knowledge of the molecule in proximity to LC3 in normal growth conditions will improve the understanding of LC3 function in autophagy and in other cell biology function. Using an APEX2 based proteomic approach, we have detected 407 proteins in proximity to the well-characterised LC3B isoform in non-stress conditions. These include known and novel LC3B proximity proteins, associated with various cell localisation and biological functions. Sixty-nine of these proteins contain a putative LIR (LC3 Interacting Region) including 41 not reported associated to autophagy. Several APEX2 hits were validated by co-immunoprecipitation and co-immunofluorescence. This study uncovers the LC3B global interactome and reveals novel LC3B interactors, irrespective of LC3B localisation and function. This knowledge could be exploited to better understand the role of LC3B in autophagy and non-autophagy cellular processes.

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Cyclic nucleotide phosphodiesterases in Drosophila melanogaster

Biochemical Journal ◽

10.1042/bj20050057 ◽

2005 ◽

Vol 388 (1) ◽

pp. 333-342 ◽

Cited By ~ 34

Author(s):

Jonathan P. DAY ◽

Julian A. T. DOW ◽

Miles D. HOUSLAY ◽

Shireen-A. DAVIES

Keyword(s):

Drosophila Melanogaster ◽

Cyclic Nucleotide ◽

Biochemical Characterization ◽

Genetic Model ◽

Renal Tubules ◽

Drosophila Genome ◽

Dual Specificity ◽

And Function ◽

High Degree

Cyclic nucleotide PDEs (phosphodiesterases) are important enzymes that regulate intracellular levels of cAMP and cGMP. In the present study, we identify and characterize novel PDEs in the genetic model, Drosophila melanogaster. The Drosophila genome encodes five novel PDE genes in addition to dunce. Predicted PDE sequences of Drosophila show highly conserved critical domains when compared with human PDEs. Thus PDE-encoding genes of D. melanogaster are CG14940-PDE1C, CG8279-PDE6β, CG5411-PDE8A, CG32648-PDE9 and CG10231-PDE11. Reverse transcriptase–PCRs of adult tissues reveal widespread expression of PDE genes. Drosophila Malpighian (renal) tubules express all the six PDEs: Drosophila PDE1, dunce (PDE4), PDE6, PDE8, PDE9 and PDE11. Antipeptide antibodies were raised against PDE1, PDE6, PDE9 and PDE11. Verification of antibody specificity by Western blotting of cloned and expressed PDE constructs allowed the immunoprecipitation studies of adult Drosophila lysates. Biochemical characterization of immunoprecipitated endogenous PDEs showed that PDE1 is a dual-specificity PDE (Michaelis constant Km for cGMP: 15.3±1 μM; Km cAMP: 20.5±1.5 μM), PDE6 is a cGMP-specific PDE (Km cGMP: 37±13 μM) and PDE11 is a dual-specificity PDE (Km cGMP: 6±2 μM; Km cAMP: 18.5±5.5 μM). Drosophila PDE1, PDE6 and PDE11 display sensitivity to vertebrate PDE inhibitors, zaprinast (IC50 was 71±39 μM for PDE1, 0.65±0.015 μM for PDE6 and 1.6±0.5 μM for PDE11) and sildenafil (IC50 was 1.3±0.9 μM for PDE1, 0.025±0.005 μM for PDE6 and 0.12±0.06 μM for PDE11). We provide the first characterization of a cGMP-specific PDE and two dual-specificity PDEs in Drosophila, and show a high degree of similarity in structure and function between human and Drosophila PDEs.

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