Publications by authors named "Jerry Eichler"

97 Publications

The prokaryotic ubiquitin-like protein presents poor cleavage sites for proteasomal degradation.

Cell Rep 2021 Jul;36(4):109428

Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. Electronic address:

In an event reminiscent of eukaryotic ubiquitination, the bacterial prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS) marks target proteins for proteasomal degradation by covalently attaching Pup, the bacterial tagging molecule. Yet, ubiquitin is released from its conjugated target following proteasome binding, whereas Pup enters the proteasome and remains conjugated to the target. Here, we report that although Pup can be degraded by the bacterial proteasome, it lacks favorable 20S core particle (CP) cleavage sites and is thus a very poor 20S CP substrate. Reconstituting the PPS in vitro, we demonstrate that during pupylated protein degradation, Pup can escape unharmed and remain conjugated to a target-derived degradation fragment. Removal of this degradation fragment by Dop, a depupylase, facilitates Pup recycling and re-conjugation to a new target. This study thus offers a mechanistic model for Pup recycling and demonstrates how a lack of protein susceptibility to proteasome-mediated cleavage can play a mechanistic role in a biological system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2021.109428DOI Listing
July 2021

Revisiting N-glycosylation in Halobacterium salinarum: Characterizing a dolichol phosphate- and glycoprotein-bound tetrasaccharide.

Glycobiology 2021 Jul 26. Epub 2021 Jul 26.

Department of Life Sciences, Ben-Gurion University of the Negev, Beersheva, Israel.

Although Halobacterium salinarum provided the first example of N-glycosylation outside the Eukarya, much regarding such post-translational modification in this halophilic Archaea remains either unclear or unknown. The composition of an N-linked glycan decorating both the S-layer glycoprotein and archaellins offers one such example. Originally described some 40 years ago, reports from that time on have presented conflicted findings regarding the composition of this glycan, as well as differences between the protein-bound glycan and that version of the glycan attached to the lipid upon which it is assembled. To clarify these points, liquid chromatography-electrospray ionization mass spectrometry was employed here to revisit the composition of this glycan both when attached to selected asparagine residues of target proteins and when bound to the lipid dolichol phosphate upon which the glycan is assembled. Such efforts revealed the N-linked glycan as corresponding to a tetrasacchride comprising a hexose, a sulfated hexuronic acid, a hexuronic acid and a second sulfated hexuronic acid. When attached to dolichol phosphate but not to proteins, the same tetrasaccharide is methylated on the final sugar. Moreover, in the absence of the oligosaccharyltransferase AglB, there is an accumulation of the dolichol phosphate-linked methylated and disulfated tetrasacchride. Knowing the composition of this glycan at both the lipid- and protein-bound stages, together with the availability of gene deletion approaches for manipulating Halobacterium salinarum, will allow delineation of the N-glycosylation pathway in this organism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/glycob/cwab080DOI Listing
July 2021

A possible universal role for mRNA secondary structure in bacterial translation revealed using a synthetic operon.

Nat Commun 2020 09 24;11(1):4827. Epub 2020 Sep 24.

Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel.

In bacteria, translation re-initiation is crucial for synthesizing proteins encoded by genes that are organized into operons. The mechanisms regulating translation re-initiation remain, however, poorly understood. We now describe the ribosome termination structure (RTS), a conserved and stable mRNA secondary structure localized immediately downstream of stop codons, and provide experimental evidence for its role in governing re-initiation efficiency in a synthetic Escherichia coli operon. We further report that RTSs are abundant, being associated with 18%-65% of genes in 128 analyzed bacterial genomes representing all phyla, and are selectively depleted when translation re-initiation is advantageous yet selectively enriched so as to insulate translation when re-initiation is deleterious. Our results support a potentially universal role for the RTS in controlling translation termination-insulation and re-initiation across bacteria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-18577-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518266PMC
September 2020

Evolutionary considerations of the oligosaccharyltransferase AglB and other aspects of N-glycosylation across Archaea.

Mol Phylogenet Evol 2020 12 1;153:106951. Epub 2020 Sep 1.

Dept. of Life Sciences, Ben Gurion University of the Negev, Beersheva 84105, Israel. Electronic address:

Various biological markers in members of the TACK and Asgard archaeal super-phyla show Eukarya-like traits. These include the oligosaccharyltransferase, responsible for transferring glycans from the lipid carrier upon which they are assembled onto selected asparagine residues of target proteins during N-glycosylation. In Archaea, oligosaccharyltransferase activity is catalyzed by AglB. To gain deeper insight into AglB and N-glycosylation across archaeal phylogeny, bioinformatics approaches were employed to address variability in AglB sequence motifs involved in enzyme activity, construct a phylogenetic tree based on AglB sequences, search for archaeal homologues of non-catalytic subunits of the multimeric eukaryal oligosaccharyltransferase complex and predict the presence of aglB-based clusters of glycosylation-related genes in the Euryarchaeota and the DPANN, TACK and Asgard super-phyla. In addition, site-directed mutagenesis and mass spectrometry were employed to study the natural variability in the WWDXG motif central to oligosaccharyltransferase activity seen in archaeal AglB. The results clearly distinguish AglB from members of the DPANN super-phylum and the Euryarchaeota from the same enzyme in members of the TACK and Asgard super-phyla, which showed considerable similarity to its eukaryal homologue Stt3. The results thus support the evolutionary proximity of Eukarya and the TACK and Asgard archaea.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ympev.2020.106951DOI Listing
December 2020

Agl22 and Agl23 are involved in the synthesis and utilization of the lipid-linked intermediates in the glycosylation pathways of the halophilic archaeaon Haloarcula hispanica.

Mol Microbiol 2020 11 10;114(5):762-774. Epub 2020 Aug 10.

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

Like both eukaryotes and bacteria, archaea can decorate proteins with N- and O-linked glycans. Whereas pathways and roles of N-glycosylation have been studied in several model archaeal organisms, little is known of O-glycosylation. To explore commonalities and variations of these two versions of glycosylation, we used Haloarcula hispanica as a model. Our previous work showed that H. hispanica S-layer glycoproteins are modified by an N-linked glucose-α-(1, 2)-[sulfoquinovosamine-β-(1, 6)-]galactose trisaccharide and an O-linked glucose-α-(1, 4)-galactose disaccharide. Here, we found that H. hispanica membrane contains C60 dolichol phosphate (DolP) as a lipid carrier for glycosylation. As revealed by bioinformatics, gene deletion and phenotype analysis, gene HAH_1571, renamed agl22, encodes a predicted glucosyltransferase that transfers glucose from glucose-DolP onto galactose-DolP to form the glucose-α-(1, 4)-galactose-DolP precursor of the N-glycosylation. Gene HAH_2016, renamed agl23, encodes a putative flippase-associated protein responsible for flipping of hexose-DolPs across the membrane to face the exterior. Our results also suggested that the synthesis of the N- and O-linked glycans onto target protein occurs on the outer surface of the cell using hexose-DolPs as sugar donors. Deletion mutant showed that N- and O-glycosylation are required for growth in the defined medium mimicking the natural habitat of H. hispanica.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/mmi.14577DOI Listing
November 2020

N-glycosylation in Archaea-New roles for an ancient posttranslational modification.

Authors:
Jerry Eichler

Mol Microbiol 2020 11 26;114(5):735-741. Epub 2020 Jul 26.

Department of Life Sciences, Ben-Gurion University of the Negev, Beersheva, Israel.

Genome analysis points to N-glycosylation as being an almost universal posttranslational modification in Archaea. Although such predictions have been confirmed in only a limited number of species, such studies are making it increasingly clear that the N-linked glycans which decorate archaeal glycoproteins present diversity in terms of both glycan composition and architecture far beyond what is seen in the other two domains of life. In addition to continuing to decipher pathways of N-glycosylation, recent efforts have revealed how Archaea exploit this variability in novel roles. As well as encouraging glycoprotein synthesis, folding and assembly into properly functioning higher ordered complexes, N-glycosylation also provides Archaea with a strategy to cope with changing environments. Archaea can, moreover, exploit the apparent species-specific nature of N-glycosylation for selectivity in mating, and hence, to maintain species boundaries, and in other events where cell-selective interactions are required. At the same time, addressing components of N-glycosylation pathways across archaeal phylogeny offers support for the concept of an archaeal origin for eukaryotes. In this MicroReview, these and other recent discoveries related to N-glycosylation in Archaea are considered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/mmi.14569DOI Listing
November 2020

Modifying Post-Translational Modifications: A Strategy Used by Archaea for Adapting to Changing Environments?: Manipulating the Extent, Position, or Content of Post-Translational Modifications May Help Archaea Adapt to Environmental Change.

Authors:
Jerry Eichler

Bioessays 2020 03 29;42(3):e1900207. Epub 2020 Jan 29.

Department of Life Sciences, Ben Gurion University of the Negev, Beersheva, 84105, Israel.

In concert with the selective pressures affecting protein folding and function in the extreme environments in which they can exist, proteins in Archaea have evolved to present permanent molecular adaptations at the amino acid sequence level. Such adaptations may not, however, suffice when Archaea encounter transient changes in their surroundings. Post-translational modifications offer a rapid and reversible layer of adaptation for proteins to cope with such situations. Here, it is proposed that Archaea further augment their ability to survive changing growth conditions by modifying the extent, position, and, where relevant, the composition of different post-translational modifications, as a function of the environment. Support for this hypothesis comes from recent reports describing how patterns of protein glycosylation, methylation, and other post-translational modifications of archaeal proteins are altered in response to environmental change. Indeed, adjusting post-translational modifications as a means to cope with environmental variability may also hold true beyond the Archaea.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bies.201900207DOI Listing
March 2020

Specificity of protein-DNA interactions in hypersaline environment: structural studies on complexes of Halobacterium salinarum oxidative stress-dependent protein hsRosR.

Nucleic Acids Res 2019 09;47(16):8860-8873

Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 8410510, Israel.

Interactions between proteins and DNA are crucial for all biological systems. Many studies have shown the dependence of protein-DNA interactions on the surrounding salt concentration. How these interactions are maintained in the hypersaline environments that halophiles inhabit remains puzzling. Towards solving this enigma, we identified the DNA motif recognized by the Halobactrium salinarum ROS-dependent transcription factor (hsRosR), determined the structure of several hsRosR-DNA complexes and investigated the DNA-binding process under extreme high-salt conditions. The picture that emerges from this work contributes to our understanding of the principles underlying the interplay between electrostatic interactions and salt-mediated protein-DNA interactions in an ionic environment characterized by molar salt concentrations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145548PMC
September 2019

-Glycosylation Is Important for Archaellin Expression, Archaellum Assembly and Cell Motility.

Front Microbiol 2019 18;10:1367. Epub 2019 Jun 18.

Department of Life Sciences, Ben Gurion University of the Negev, Beersheba, Israel.

are halophilic archaea that display directional swimming in response to various environmental signals, including light, chemicals and oxygen. In , the building blocks (archaellins) of the archaeal swimming apparatus (the archaellum) are -glycosylated. However, the physiological importance of archaellin -glycosylation remains unclear. Here, a tetrasaccharide comprising a hexose and three hexuronic acids decorating the five archaellins was characterized by mass spectrometry. Such analysis failed to detect sulfation of the hexuronic acids, in contrast to earlier reports. To better understand the physiological significance of archaellin -glycosylation, a strain deleted of , encoding the archaeal oligosaccharyltransferase, was generated. In this Δ strain, archaella were not detected and only low levels of archaellins were released into the medium, in contrast to what occurs with the parent strain. Mass spectrometry analysis of the archaellins in Δ cultures did not detect -glycosylation. Δ cells also showed a slight growth defect and were impaired for motility. Quantitative real-time PCR analysis revealed dramatically reduced transcript levels of archaellin-encoding genes in the mutant strain, suggesting that -glycosylation is important for archaellin transcription, with downstream effects on archaellum assembly and function. Control of AglB-dependent post-translational modification of archaellins could thus reflect a previously unrecognized route for regulating motility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2019.01367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591318PMC
June 2019

Protein glycosylation.

Authors:
Jerry Eichler

Curr Biol 2019 04;29(7):R229-R231

Department of Life Sciences, Ben Gurion University of the Negev, Beersheva 84105, Israel. Electronic address:

Eichler introduces the diversity of protein glycosylations and the roles of these modifications in regulating protein function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2019.01.003DOI Listing
April 2019

Halobacterium salinarum.

Authors:
Jerry Eichler

Trends Microbiol 2019 07 4;27(7):651-652. Epub 2019 Mar 4.

Department of Life Sciences, Ben Gurion University of the Negev, Beersheva, Israel. Electronic address:

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tim.2019.02.005DOI Listing
July 2019

Sialic Acid-Like Sugars in Archaea: Legionaminic Acid Biosynthesis in the Halophile sp. PV6.

Front Microbiol 2018 7;9:2133. Epub 2018 Sep 7.

Department of Life Sciences, Ben-Gurion University of the Negev, Beersheva, Israel.

N-glycosylation is a post-translational modification that occurs in all three domains. In Archaea, however, N-linked glycans present a degree of compositional diversity not observed in either Eukarya or Bacteria. As such, it is surprising that nonulosonic acids (NulOs), nine-carbon sugars that include sialic acids, pseudaminic acids, and legionaminic acids, are routinely detected as components of protein-linked glycans in Eukarya and Bacteria but not in Archaea. In the following, we report that the N-linked glycan attached to the S-layer glycoprotein of the haloarchaea sp. PV6 includes an -formylated legionaminic acid. Analysis of the sp. PV6 genome led to the identification of sequences predicted to comprise the legionaminic acid biosynthesis pathway. The transcription of pathway genes was confirmed, as was the co-transcription of several of these genes. In addition, the activities of LegI, which catalyzes the condensation of 2,4-di--acetyl-6-deoxymannose and phosphoenolpyruvate to generate legionaminic acid, and LegF, which catalyzes the addition of cytidine monophosphate (CMP) to legionaminic acid, both heterologously expressed in , were demonstrated. Further genome analysis predicts that the genes encoding enzymes of the legionaminic acid biosynthetic pathway are clustered together with sequences seemingly encoding components of the N-glycosylation pathway in this organism. In defining the first example of a legionaminic acid biosynthesis pathway in Archaea, the findings reported here expand our insight into archaeal N-glycosylation, an almost universal post-translational modification in this domain of life.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2018.02133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137143PMC
September 2018

The 3-D structure of VNG0258H/RosR - A haloarchaeal DNA-binding protein in its ionic shell.

J Struct Biol 2018 11 12;204(2):191-198. Epub 2018 Aug 12.

Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 8410510, Israel. Electronic address:

Protein-DNA interactions are highly dependent on salt concentration. To gain insight into how such interactions are maintained in the highly saline cytoplasm of halophilic archaea, we determined the 3-D structure of VNG0258H/RosR, the first haloarchaeal DNA-binding protein from the extreme halophilic archaeon Halobactrium salinarum. It is a dimeric winged-helix-turn-helix (wHTH) protein with unique features due to adaptation to the halophilic environment. As ions are major players in DNA binding processes, particularly in halophilic environments, we investigated the solution structure of the ionic envelope and located anions in the first shell around the protein in the crystal using anomalous scattering. Anions that were found to be tightly bound to residues in the positively charged DNA-binding site would probably be released upon DNA binding and will thus make significant contribution to the driving force of the binding process. Unexpectedly, ions were also found in a buried internal cavity connected to the external medium by a tunnel. Our structure lays a solid groundwork for future structural, computational and biochemical studies on complexes of the protein with cognate DNA sequences, with implications to protein-DNA interactions in hyper-saline environments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jsb.2018.08.008DOI Listing
November 2018

Comparative Analysis of Surface Layer Glycoproteins and Genes Involved in Protein Glycosylation in the Genus Haloferax.

Genes (Basel) 2018 Mar 20;9(3). Epub 2018 Mar 20.

School of Molecular and Cell Biology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

Within the genus, both the surface (S)-layer protein, and the glycans that can decorate it, vary between species, which can potentially result in many different surface types, analogous to bacterial serotypes. This variation may mediate phenotypes, such as sensitivity to different viruses and mating preferences. Here, we describe S-layer glycoproteins found in multiple strains and perform comparative genomics analyses of major and alternative glycosylation clusters of isolates from two coastal sites. We analyze the phylogeny of individual glycosylation genes and demonstrate that while the major glycosylation cluster tends to be conserved among closely related strains, the alternative cluster is highly variable. Thus, geographically- and genetically-related strains may exhibit diverse surface structures to such an extent that no two isolates present an identical surface profile.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/genes9030172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867893PMC
March 2018

Biogenesis of Asparagine-Linked Glycoproteins Across Domains of Life-Similarities and Differences.

ACS Chem Biol 2018 04 26;13(4):833-837. Epub 2018 Feb 26.

Department of Biology and Department of Chemistry , Massachusetts Institute of Technology , Cambridge , Massachusetts , United States.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.8b00163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338551PMC
April 2018

Gene deletions leading to a reduction in the number of cyclopentane rings in Sulfolobus acidocaldarius tetraether lipids.

FEMS Microbiol Lett 2018 01;365(1)

Department of Life Sciences, Ben Gurion University of the Negev, Beersheva 84105, Israel.

The cell membrane of (hyper)thermophilic archaea, including the thermoacidophile Sulfolobus acidocaldarius, incorporates dibiphytanylglycerol tetraether lipids. The hydrophobic cores of such tetraether lipids can include up to eight cyclopentane rings. Presently, nothing is known of the biosynthesis of these rings. In this study, a series of S. acidocaldarius mutants deleted of genes currently annotated as encoding proteins involved in sugar/polysaccharide processing were generated and their glycolipids were considered. Whereas the glycerol-dialkyl-glycerol tetraether core of a S. acidocaldarius tetraether glycolipid considered here mostly includes four cyclopentane rings, in cells where the Saci_0421 or Saci_1201 genes had been deleted, species containing zero, two or four cyclopentane rings were observed. At the same time, in cells lacking Saci_0201, Saci_0275, Saci_1101, Saci_1249 or Saci_1706, lipids containing mostly four cyclopentane rings were detected. Although Saci_0421 and Saci_1201 are not found in proximity to other genes putatively involved in lipid biosynthesis, homologs of these sequences exist in other Archaea containing cyclopentane-containing tetraether lipids. Thus, Saci_0421 and Saci_1201 represent the first proteins described that somehow contribute to the appearance of cyclopentane rings in the core moiety of the S. acidocaldarius glycolipid considered here.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/femsle/fnx250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827618PMC
January 2018

Stereochemical Divergence of Polyprenol Phosphate Glycosyltransferases.

Trends Biochem Sci 2018 01 25;43(1):10-17. Epub 2017 Nov 25.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA; Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA. Electronic address:

In the three domains of life, lipid-linked glycans contribute to various cellular processes ranging from protein glycosylation to glycosylphosphatidylinositol anchor biosynthesis to peptidoglycan assembly. In generating many of these glycoconjugates, phosphorylated polyprenol-based lipids are charged with single sugars by polyprenol phosphate glycosyltransferases. The resultant substrates serve as glycosyltransferase donors, complementing the more common nucleoside diphosphate sugars. It had been accepted that these polyprenol phosphate glycosyltransferases acted similarly, given their considerable sequence homology. Recent findings, however, suggest that matters may not be so simple. In this Opinion we propose that the stereochemistry of sugar addition by polyprenol phosphate glycosyltransferases is not conserved across evolution, even though the GT-A fold that characterizes such enzymes is omnipresent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tibs.2017.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741494PMC
January 2018

Assembling Glycan-Charged Dolichol Phosphates: Chemoenzymatic Synthesis of a Haloferax volcanii N-Glycosylation Pathway Intermediate.

Bioconjug Chem 2017 09 30;28(9):2461-2470. Epub 2017 Aug 30.

Department of Life Sciences, Ben Gurion University of the Negev , Beersheva 8410501, Israel.

N-glycosylation, the covalent attachment of glycans to select protein target Asn residues, is a post-translational modification performed by all three domains of life. In the halophilic archaea Haloferax volcanii, in which understanding of this universal protein-processing event is relatively well-advanced, genes encoding the components of the archaeal glycosylation (Agl) pathway responsible for the assembly and attachment of an N-linked pentasaccharide have been identified. As elsewhere, the N-linked glycan is assembled on phosphodolichol carriers before transfer to target Asn residues. However, as little is presently known of the Hfx. volcanii Agl pathway at the protein level, the seemingly unique ability of Archaea to use dolichol phosphate (DolP) as the glycan lipid carrier, rather than dolichol pyrophosphate used by eukaryotes, remains poorly understood. With this in mind, a chemoenzymatic approach was taken to biochemically study AglG, one of the five glycosyltransferases of the pathway. Accordingly, a novel regio- and stereoselective reduction of naturally isolated polyprenol gave facile access to S-dolichol via asymmetric transfer hydrogenation under very mild conditions. This compound was used to generate glucose-charged DolP, a precursor of the N-linked pentasaccharide, as well as DolP-glucose-glucuronic acid and DolP-glucuronic acid. AglG, purified from Hfx. volcanii membranes in hypersaline conditions, like those encountered in situ, was subsequently combined with uridine diphosphate (UDP)-glucuronic acid and DolP-glucose to yield DolP-glucose-glucuronic acid. The in vitro system for the study of AglG activity developed here represents the first such tool for studying halophilic glycosyltransferases and will allow for a detailed understanding of archaeal N-glycosylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.bioconjchem.7b00436DOI Listing
September 2017

Cell Surface Glycosylation Is Required for Efficient Mating of .

Front Microbiol 2017 5;8:1253. Epub 2017 Jul 5.

School of Molecular and Cell Biology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv UniversityTel Aviv, Israel.

Halophilic archaea use a fusion-based mating system for lateral gene transfer across cells, yet the molecular mechanisms involved remain unknown. Previous work implied that cell fusion involves cell-cell recognition since fusion occurs more efficiently between cells from the same species. Long believed to be restricted only to Eukarya, it is now known that cells of all three domains of life perform N-glycosylation, the covalent attachment of glycans to select target asparagine residues in proteins, and that this post-translational modification is common for archaeal cell surface proteins. Here, we show that differences in glycosylation of the surface-layer glycoprotein, brought about either by changing medium salinity or by knocking out key glycosylation genes, reduced mating success. Thus, different glycosylation patterns are likely to underlie mating preference in halophilic archaea, contributing to speciation processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2017.01253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496957PMC
July 2017

The Mitochondrial Protein Atlas: A Database of Experimentally Verified Information on the Human Mitochondrial Proteome.

J Comput Biol 2017 Sep 20;24(9):906-916. Epub 2017 Jun 20.

Department of Life Sciences, Ben-Gurion University of the Negev , Beersheva, Israel .

Given its central role in various biological systems, as well as its involvement in numerous pathologies, the mitochondrion is one of the best-studied organelles. However, although the mitochondrial genome has been extensively investigated, protein-level information remains partial, and in many cases, hypothetical. The Mitochondrial Protein Atlas (MPA; URL: lifeserv.bgu.ac.il/wb/jeichler/MPA ) is a database that provides a complete, manually curated inventory of only experimentally validated human mitochondrial proteins. The MPA presently contains 911 unique protein entries, each of which is associated with at least one experimentally validated and referenced mitochondrial localization. The MPA also contains experimentally validated and referenced information defining function, structure, involvement in pathologies, interactions with other MPA proteins, as well as the method(s) of analysis used in each instance. Connections to relevant external data sources are offered for each entry, including links to NCBI Gene, PubMed, and Protein Data Bank. The MPA offers a prototype for other information sources that allow for a distinction between what has been confirmed and what remains to be verified experimentally.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/cmb.2017.0011DOI Listing
September 2017

Sweet New Roles for Protein Glycosylation in Prokaryotes.

Trends Microbiol 2017 08 21;25(8):662-672. Epub 2017 Mar 21.

Department of Biosciences, University of Oslo, 0316 Oslo, Norway.

Long-held to be a post-translational modification unique to Eukarya, it is now clear that both Bacteria and Archaea also perform protein glycosylation, namely the covalent attachment of mono- to polysaccharides to specific protein targets. At the same time, many of the roles assigned to this protein-processing event in eukaryotes, such as guiding protein folding/quality control, intracellular trafficking, dictating cellular recognition events and others, do not apply or are even irrelevant to prokaryotes. As such, protein glycosylation must serve novel functions in Bacteria and Archaea. Recent efforts have begun to elucidate some of these prokaryote-specific roles, which are addressed in this review.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tim.2017.03.001DOI Listing
August 2017

Lipid sugar carriers at the extremes: The phosphodolichols Archaea use in N-glycosylation.

Biochim Biophys Acta Mol Cell Biol Lipids 2017 Jun 19;1862(6):589-599. Epub 2017 Mar 19.

Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.

N-glycosylation, a post-translational modification whereby glycans are covalently linked to select Asn residues of target proteins, occurs in all three domains of life. Across evolution, the N-linked glycans are initially assembled on phosphorylated cytoplasmically-oriented polyisoprenoids, with polyprenol (mainly C undecaprenol) fulfilling this role in Bacteria and dolichol assuming this function in Eukarya and Archaea. The eukaryal and archaeal versions of dolichol can, however, be distinguished on the basis of their length, degree of saturation and by other traits. As is true for many facets of their biology, Archaea, best known in their capacity as extremophiles, present unique approaches for synthesizing phosphodolichols. At the same time, general insight into the assembly and processing of glycan-bearing phosphodolichols has come from studies of the archaeal enzymes responsible. In this review, these and other aspects of archaeal phosphodolichol biology are addressed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbalip.2017.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5424534PMC
June 2017

N-Glycosylation Is Important for Proper Haloferax volcanii S-Layer Stability and Function.

Appl Environ Microbiol 2017 03 2;83(6). Epub 2017 Mar 2.

Department of Life Sciences, Ben Gurion University of the Negev, Beersheva, Israel

N-Glycosylation, the covalent linkage of glycans to select Asn residues of target proteins, is an almost universal posttranslational modification in archaea. However, whereas roles for N-glycosylation have been defined in eukarya and bacteria, the function of archaeal N-glycosylation remains unclear. Here, the impact of perturbed N-glycosylation on the structure and physiology of the haloarchaeon was considered. Cryo-electron microscopy was used to examine right-side-out membrane vesicles prepared from cells of a parent strain and from strains lacking genes encoding glycosyltransferases involved in assembling the N-linked pentasaccharide decorating the surface layer (S-layer) glycoprotein, the sole component of the S-layer surrounding cells. Whereas a regularly repeating S-layer covered the entire surface of vesicles prepared from parent strain cells, vesicles from the mutant cells were only partially covered. To determine whether such N-glycosylation-related effects on S-layer assembly also affected cell function, the secretion of a reporter protein was addressed in the parent and N-glycosylation mutant strains. Compromised S-layer glycoprotein N-glycosylation resulted in impaired transfer of the reporter past the S-layer and into the growth medium. Finally, an assessment of S-layer glycoprotein susceptibility to added proteases in the mutants revealed that in cells lacking AglD, which is involved in adding the final pentasaccharide sugar, a distinct S-layer glycoprotein conformation was assumed in which the N-terminal region was readily degraded. Perturbed N-glycosylation thus affects S-layer glycoprotein folding. These findings suggest that could adapt to changes in its surroundings by modulating N-glycosylation so as to affect S-layer architecture and function. Long held to be a process unique to eukaryotes, it is now accepted that bacteria and archaea also perform N-glycosylation, namely, the covalent attachment of sugars to select asparagine residues of target proteins. Yet, while information on the importance of N-glycosylation in eukaryotes and bacteria is available, the role of this posttranslational modification in archaea remains unclear. Here, insight into the purpose of archaeal N-glycosylation was gained by addressing the surface layer (S-layer) surrounding cells of the halophilic species Relying on mutant strains defective in N-glycosylation, such efforts revealed that compromised N-glycosylation affected S-layer integrity and the transfer of a secreted reporter protein across the S-layer into the growth medium, as well as the conformation of the S-layer glycoprotein, the sole component of the S-layer. Thus, by modifying N-glycosylation, cells can change how they interact with their surroundings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AEM.03152-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5335521PMC
March 2017

AglM and VNG1048G, Two Haloarchaeal UDP-Glucose Dehydrogenases, Show Different Salt-Related Behaviors.

Life (Basel) 2016 Aug 3;6(3). Epub 2016 Aug 3.

Department of Life Sciences, Ben Gurion University, Beersheva 84105, Israel.

Haloferax volcanii AglM and Halobacterium salinarum VNG1048G are UDP-glucose dehydrogenases involved in N-glycosylation in each species. Despite sharing >60% sequence identity and the ability of VNG1048G to functionally replace AglM in vivo, these proteins behaved differently as salinity changed. Whereas AglM was active in 2-4 M NaCl, VNG1048G lost much of its activity when salinity dropped below 3 M NaCl. To understand the molecular basis of this phenomenon, each protein was examined by size exclusion chromatrography in 2 M NaCl. Whereas AglM appeared as a dodecamer, VNG1048G was essentially detected as a dodecamer and a dimer. The specific activity of the VNG1048G dodecamer was only a sixth of that of AglM, while the dimer was inactive. As such, not only was the oligomeric status of VNG1048G affected by lowered salinity, so was the behavior of the individual dodecamer subunits. Analyzing surface-exposed residues in homology models of the two UDP-glucose dehydrogenases revealed the more acidic and less basic VNG1048G surface, further explaining the greater salt-dependence of the Hbt. salinarum enzyme.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5041007PMC
http://dx.doi.org/10.3390/life6030031DOI Listing
August 2016

N-glycosylation in the thermoacidophilic archaeon Sulfolobus acidocaldarius involves a short dolichol pyrophosphate carrier.

FEBS Lett 2016 09 12;590(18):3168-78. Epub 2016 Sep 12.

Department of Life Sciences, Ben Gurion University of the Negev, Beersheva, Israel.

N-glycosylation is a post-translational modification that occurs across evolution. In the thermoacidophilic archaea Sulfolobus acidocaldarius, glycoproteins are modified by an N-linked tribranched hexasaccharide reminiscent of the N-glycans assembled in Eukarya. Previously, hexose-bearing dolichol phosphate was detected in a S. acidocaldarius Bligh-Dyer lipid extract. Here, we used a specialized protocol for extracting lipid-linked oligosaccharides to detect a dolichol pyrophosphate bearing the intact hexasaccharide, as well as its biosynthetic intermediates. Furthermore, evidence for N-glycosylation of two S. acidocaldarius proteins by the same hexasaccharide and its derivatives was collected. These findings thus provide novel insight into archaeal N-glycosylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.12341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039095PMC
September 2016

Structural characterization of the N-linked pentasaccharide decorating glycoproteins of the halophilic archaeon Haloferax volcanii.

Glycobiology 2016 07 10;26(7):745-756. Epub 2016 Feb 10.

Université de Lille, CNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F 59000 Lille, France.

N-Glycosylation is a post-translational modification performed in all three domains of life. In the halophilic archaea Haloferax volcanii, glycoproteins such as the S-layer glycoprotein are modified by an N-linked pentasaccharide assembled by a series of Agl (archaeal glycosylation) proteins. In the present study, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy were used to define the structure of this glycan attached to at least four of the seven putative S-layer glycoprotein N-glycosylation sites, namely Asn-13, Asn-83, Asn-274 and Asn-279. Such approaches detected a trisaccharide corresponding to glucuronic acid (GlcA)-β1,4-GlcA-β1,4-glucose-β1-Asn, a tetrasaccharide corresponding to methyl-O-4-GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, and a pentasaccharide corresponding to hexose-1,2-[methyl-O-4-]GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, with previous MS and radiolabeling experiments showing the hexose at the non-reducing end of the pentasaccharide to be mannose. The present analysis thus corrects the earlier assignment of the penultimate sugar as a methyl ester of a hexuronic acid, instead revealing this sugar to be a methylated GlcA. The assignments made here are in good agreement with what was already known of the Hfx. volcanii N-glycosylation pathway from previous genetic and biochemical efforts while providing new insight into the process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/glycob/cww014DOI Listing
July 2016

N-Linked Glycans Are Assembled on Highly Reduced Dolichol Phosphate Carriers in the Hyperthermophilic Archaea Pyrococcus furiosus.

PLoS One 2015 22;10(6):e0130482. Epub 2015 Jun 22.

Department of Biochemistry, Duke University Medical Center, Durham NC 27710, United States of America.

In all three domains of life, N-glycosylation begins with the assembly of glycans on phosphorylated polyisoprenoid carriers. Like eukaryotes, archaea also utilize phosphorylated dolichol for this role, yet whereas the assembled oligosaccharide is transferred to target proteins from dolichol pyrophosphate in eukaryotes, archaeal N-linked glycans characterized to date are derived from a dolichol monophosphate carrier, apart from a single example. In this study, glycan-charged dolichol phosphate from the hyperthermophile Pyrococcus furiosus was identified and structurally characterized. Normal and reverse phase liquid chromatography-electrospray ionization mass spectrometry revealed the existence of dolichol phosphate charged with the heptasaccharide recently described in in vitro studies of N-glycosylation on this species. As with other described archaeal dolichol phosphates, the α- and ω-terminal isoprene subunits of the P. furiosus lipid are saturated, in contrast to eukaryal phosphodolichols that present only a saturated α-position isoprene subunit. Interestingly, an additional 1-4 of the 12-14 isoprene subunits comprising P. furiosus dolichol phosphate are saturated, making this lipid not only the longest archaeal dolichol phosphate described to date but also the most highly saturated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130482PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476749PMC
April 2016

Archaeal S-layer glycoproteins: post-translational modification in the face of extremes.

Front Microbiol 2014 26;5:661. Epub 2014 Nov 26.

Department of Life Sciences, Ben Gurion University of the Negev , Beersheva, Israel.

Corresponding to the sole or basic component of the surface (S)-layer surrounding the archaeal cell in most known cases, S-layer glycoproteins are in direct contact with the harsh environments that characterize niches where Archaea can thrive. Accordingly, early work examining archaeal S-layer glycoproteins focused on identifying those properties that allow members of this group of proteins to maintain their structural integrity in the face of extremes of temperature, pH, and salinity, as well as other physical challenges. However, with expansion of the list of archaeal strains serving as model systems, as well as growth in the number of molecular tools available for the manipulation of these strains, studies on archaeal S-layer glycoproteins are currently more likely to consider the various post-translational modifications these polypeptides undergo. For instance, archaeal S-layer glycoproteins can undergo proteolytic cleavage, both N- and O-glycosylation, lipid-modification and oligomerization. In this mini-review, recent findings related to the post-translational modification of archaeal S-layer glycoproteins are considered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2014.00661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245038PMC
December 2014

Deciphering a pathway of Halobacterium salinarum N-glycosylation.

Microbiologyopen 2015 Feb 2;4(1):28-40. Epub 2014 Dec 2.

Department of Life Sciences, Ben Gurion University of the Negev, Beersheva, Israel.

Genomic analysis points to N-glycosylation as being a common posttranslational modification in Archaea. To date, however, pathways of archaeal N-glycosylation have only been described for few species. With this in mind, the similarities of N-linked glycans decorating glycoproteins in the haloarchaea Haloferax volcanii and Halobacterium salinarum directed a series of bioinformatics, genetic, and biochemical experiments designed to describe that Hbt. salinarum pathway responsible for biogenesis of one of the two N-linked oligosaccharides described in this species. As in Hfx. volcanii, where agl (archaeal glycosylation) genes that encode proteins responsible for the assembly and attachment of a pentasaccharide to target protein Asn residues are clustered in the genome, Hbt. salinarum also contains a group of clustered homologous genes (VNG1048G-VNG1068G). Introduction of these Hbt. salinarum genes into Hfx. volcanii mutant strains deleted of the homologous sequence restored the lost activity. Moreover, transcription of the Hbt. salinarum genes in the native host, as well as in vitro biochemical confirmation of the predicted functions of several of the products of these genes provided further support for assignments made following bioinformatics and genetic experiments. Based on the results obtained in this study, the first description of an N-glycosylation pathway in Hbt. salinarum is offered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mbo3.215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335974PMC
February 2015
-->