Global Identification of Prokaryotic Glycoproteins Based on an Escherichia coli Proteome Microarray

Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA) were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB) was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA) staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.     

Analysis of glycosylation site occupancy reveals a role for Ost3p and Ost6p in site-specific N-glycosylation efficiency

Asparagine-linked glycosylation is the most common post-translational modification of proteins catalyzed in eukaryotes by the multiprotein complex oligosaccharyltransferase. Apart from the catalytic Stt3p, the roles of the subunits are ill defined. Here we describe functional investigations of the Ost3/6p components of the yeast enzyme. We developed novel analytical tools to quantify glycosylation site occupancy by enriching glycoproteins bound to the yeast polysaccharide cell wall, tagging glycosylated asparagines using endoglycosidase H glycan release, and detecting peptides and glycopeptides with LC-ESI-MS/MS. We found that the paralogues Ost3p and Ost6p were required for efficient glycosylation of distinct defined glycosylation sites. Our results describe a novel method for relative quantification of glycosylation occupancy in the genetically tractable yeast system and show that eukaryotic oligosaccharyltransferase isoforms have different activities toward protein substrates at the level of individual glycosylation sites.     

Inhibition of protein synthesis by TOR inactivation revealed a conserved regulatory mechanism of the BiP chaperone in Chlamydomonas

The target of rapamycin (TOR) kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates with highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here, we report that down-regulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis, since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP, such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function, as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification.     

The Arabidopsis mitochondrial membrane‐bound ubiquitin protease UBP27 contributes to mitochondrial morphogenesis

Mitochondria are essential organelles with dynamic morphology and function. Post‐translational modifications (PTMs), which include protein ubiquitination, are critically involved in animal and yeast mitochondrial dynamics. How PTMs contribute to plant mitochondrial dynamics is just beginning to be elucidated, and mitochondrial enzymes involved in ubiquitination have not been reported from plants. In this study, we identified an Arabidopsis mitochondrial localized ubiquitin protease, UBP27, through a screen that combined bioinformatics and fluorescent fusion protein targeting analysis. We characterized UBP27 with respect to its membrane topology and enzymatic activities, and analysed the mitochondrial morphological changes in UBP27T‐DNA insertion mutants and overexpression lines. We have shown that UBP27 is embedded in the mitochondrial outer membrane with an N_in–C_out orientation and possesses ubiquitin protease activities in vitro. UBP27 demonstrates similar sub‐cellular localization, domain structure, membrane topology and enzymatic activities with two mitochondrial deubiquitinases, yeast ScUBP16 and human HsUSP30, which indicated that these proteins are functional orthologues in eukaryotes. Although loss‐of‐function mutants of UBP27 do not show obvious phenotypes in plant growth and mitochondrial morphology, UBP27 overexpression can change mitochondrial morphology from rod to spherical shape and reduce the mitochondrial association of dynamin‐related protein 3 (DRP3) proteins, large GTPases that serve as the main mitochondrial fission factors. Thus, our study has uncovered a plant ubiquitin protease that plays a role in mitochondrial morphogenesis possibly through modulation of the function of organelle division proteins.     

Sweet new world: glycoproteins in bacterial pathogens

In eukaryotes, the combinatorial potential of carbohydrates is used for the modulation of protein function. However, despite the wealth of cell wall and surface-associated carbohydrates and glycoconjugates, the accepted dogma has been that prokaryotes are not able to glycosylate proteins. This has now changed and protein glycosylation in prokaryotes is an accepted fact. Intriguingly, in Gram-negative bacteria most glycoproteins are associated with virulence factors of medically significant pathogens. Also, important steps in pathogenesis have been linked to the glycan substitution of surface proteins, indicating that the glycosylation of bacterial proteins might serve specific functions in infection and pathogenesis and interfere with inflammatory immune responses. Therefore, the carbohydrate modifications and glycosylation pathways of bacterial proteins will become new targets for therapeutic and prophylactic measures. Here we discuss recent findings on the structure, genetics and function of glycoproteins of medically important bacteria and potential applications of bacterial glycosylation systems for the generation of novel glycoconjugates.     

Mitochondrial localization of AtOXA1, an arabidopsis homologue of yeast Oxa1p involved in the insertion and assembly of protein complexes in mitochondrial inner membrane

Components of some protein complexes present in the inner membrane of mitochondria are encoded in both nuclear and mitochondrial genomes, and correct sorting and assembly of these proteins is necessary for proper respiratory function. Recent studies in yeast suggest that Oxa1p, a protein conserved between prokaryotes and eukaryotes, is an essential factor for protein sorting and assembly into membranes. We previously identified AtOXA1, an Arabidopsis homologue of OXA1 by functional complementation of a yeast oxa1- mutant. In this study, we investigated the genomic organization of AtOXA1 and localization of the AtOXA1 protein. Characterization of the AtOXA1 genomic region indicated that the gene consists of 10 exons and is located on chromosome V. A database search also revealed another gene coding for a putative protein homologous to AtOXA1 on chromosome II. Transient expression of a green fluorescent protein (GFP) fusion in suspension-cultured tobacco cells showed that AtOXA1 is targeted into mitochondria by its N-terminal presequence. Antibodies raised against AtOXA1 recognized a 38-kDa intrinsic protein of the inner mitochondrial membrane. Thus, localization of AtOXA1 in the mitochondrial inner membrane, together with our previous complementation experiment in yeast, suggested that it is a functional homologue of Oxa1p.     

An oligosaccharyltransferase from Leishmania major increases the N‐glycan occupancy on recombinant glycoproteins produced in Nicotiana benthamiana

N‐glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central‐protein complex facilitating the N‐glycosylation of proteins in the lumen of the endoplasmic reticulum (ER). Not all potential N‐glycosylation sites are recognized in vivo and the site occupancy can vary in different expression systems, resulting in underglycosylation of recombinant glycoproteins. To overcome this limitation in plants, we expressed LmSTT3D, a single‐subunit oligosaccharyltransferase from the protozoan Leishmania major transiently in Nicotiana benthamiana, a well‐established production platform for recombinant proteins. A fluorescent protein‐tagged LmSTT3D variant was predominately found in the ER and co‐located with plant oligosaccharyltransferase subunits. Co‐expression of LmSTT3D with immunoglobulins and other recombinant human glycoproteins resulted in a substantially increased N‐glycosylation site occupancy on all N‐glycosylation sites except those that were already more than 90% occupied. Our results show that the heterologous expression of LmSTT3D is a versatile tool to increase N‐glycosylation efficiency in plants.     

ATAD3 proteins: brokers of a mitochondria–endoplasmic reticulum connection in mammalian cells

In yeast, a sequence of physical and genetic interactions termed the endoplasmic reticulum (ER)–mitochondria organizing network (ERMIONE) controls mitochondria–ER interactions and mitochondrial biogenesis. Several functions that characterize ERMIONE complexes are conserved in mammalian cells, suggesting that a similar tethering complex must exist in metazoans. Recent studies have identified a new family of nuclear‐encoded ATPases associated with diverse cellular activities (AAA+‐ATPase) mitochondrial membrane proteins specific to multicellular eukaryotes, called the ATPase family AAA domain‐containing protein 3 (ATAD3) proteins (ATAD3A and ATAD3B). These proteins are crucial for normal mitochondrial–ER interactions and lie at the heart of processes underlying mitochondrial biogenesis. ATAD3A orthologues have been studied in flies, worms, and mammals, highlighting the widespread importance of this gene during embryonic development and in adulthood. ATAD3A is a downstream effector of target of rapamycin (TOR) signalling in Drosophila and exhibits typical features of proteins from the ERMIONE‐like complex in metazoans. In humans, mutations in the ATAD3A gene represent a new link between altered mitochondrial–ER interaction and recognizable neurological syndromes. The primate‐specific ATAD3B protein is a biomarker of pluripotent embryonic stem cells. Through negative regulation of ATAD3A function, ATAD3B supports mitochondrial stemness properties.     

Fatty acylation of yeast glycoproteins proceeds independently of N-linked glycosylation

The relationship between protein glycosylation and fatty acylation of glycoproteins was studied in the wild-type and asparagine-linked glycosylation-deficient mutants (alg1 and alg2) of Saccharomyces cerevisiae. At the non-permissive temperature (37 degrees C), both mutant cells exhibited increased incorporation of [3H]palmitate into five polypeptides based on SDS-PAGE. In contrast, the wild-type yeast cells contained [3H]palmitate-labeled polypeptides of higher molecular weights, which were converted to the bands seen in the mutant cells upon treatment of the cell extract with endoglycosidase H prior to SDS-PAGE. In addition, labeling of the wild-type yeast cells with [3H]palmitate in the presence of tunicamycin revealed the incorporation of [3H]palmitate into the same five bands as found in the alg1 and alg2 mutants at the non-permissive temperature without tunicamycin. These results indicate that fatty acylation of glycoproteins proceeds independently of protein N-glycosylation in yeast cells.     

Structural analysis of <Emphasis Type="Italic">N</Emphasis>-/<Emphasis Type="Italic">O</Emphasis>-glycans assembled on proteins in yeasts

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.     

Glycoproteomic profile in wine: a 'sweet' molecular renaissance

Glycoproteins are believed to be important in several technological, oenological and allergological processes due to their physicochemical properties. The knowledge of the protein glycosylation status in wine will aid in the understanding of these processes. A multiplexed glycopeptide enrichment strategy in combination with tandem mass spectrometry was performed in order to analyze the glycoproteome of white wine. A total of 28 glycoproteins and 44 glycosylation sites were identified. The identified glycoproteins were from grape and yeast origin. In particular, several glycoproteins derived from grape, like invertase and pathogenesis-related (PR) proteins, and from the yeast, were found after the vinification process. Bioinformatic analysis revealed sequence similarity between the identified grape glycoproteins and known plant allergens. This study is an important step forward in order to investigate the implication of glycoproteins in several processes, like protein stabilization and potential allergenic cross-reactivity in wine.     

Vestiges of the Bacterial Signal Recognition Particle-Based Protein Targeting in Mitochondria

The main bacterial pathway for inserting proteins into the plasma membrane relies on the signal recognition particle (SRP), composed of the Ffh protein and an associated RNA component, and the SRP-docking protein FtsY. Eukaryotes use an equivalent system of archaeal origin to deliver proteins into the endoplasmic reticulum, whereas a bacteria-derived SRP and FtsY function in the plastid. Here we report on the presence of homologs of the bacterial Ffh and FtsY proteins in various unrelated plastid-lacking unicellular eukaryotes, namely Heterolobosea, Alveida, Goniomonas, and Hemimastigophora. The monophyly of novel eukaryotic Ffh and FtsY groups, predicted mitochondrial localization experimentally confirmed for Naegleria gruberi, and a strong alphaproteobacterial affinity of the Ffh group, collectively suggest that they constitute parts of an ancestral mitochondrial signal peptide-based protein-targeting system inherited from the last eukaryotic common ancestor, but lost from the majority of extant eukaryotes. The ability of putative signal peptides, predicted in a subset of mitochondrial-encoded N. gruberi proteins, to target a reporter fluorescent protein into the endoplasmic reticulum of Trypanosoma brucei, likely through their interaction with the cytosolic SRP, provided further support for this notion. We also illustrate that known mitochondrial ribosome-interacting proteins implicated in membrane protein targeting in opisthokonts (Mba1, Mdm38, and Mrx15) are broadly conserved in eukaryotes and nonredundant with the mitochondrial SRP system. Finally, we identified a novel mitochondrial protein (MAP67) present in diverse eukaryotes and related to the signal peptide-binding domain of Ffh, which may well be a hitherto unrecognized component of the mitochondrial membrane protein-targeting machinery.     

Effects of tunicamycin on protein glycosylation and development inVolvox carteri

Summary The involvement of protein glycosylation in regulation of the development of the multicellular green alga,Volvox carteri, was studied using the antibiotic, tunicamycin. Three specific developmental processes were found to be affected by the antibiotic: reproductive cell maturation; establishment of polar cellular organization during embryogenesis and release of progeny spheroids from the parental spheroids. Tunicamycin inhibited the transfer of GlcNAc-1-phosphate to dolichyl phosphate which is catalyzed byVolvox membrane preparations. Changes in the glycosylation of several secreted and cellular glycoproteins were observed when proteins were labelled with radioactive amino acids and sugars in the absence and presence of tunicamycin and then electrophoresed on sodium dodecylsulfate-polyacrylamide slab gels. The levels of a few secreted proteins were reduced in tunicamycin treated cultures and one protein band appeared exclusively in the treated cells. Tunicamycin treatment also altered the electrophoretic mobility of radio-iodinated surface macromolecules. Binding of concanavalin A by tunicamycin treatedVolvox spheroids was drastically reduced. It is there-fore likely that the aberrant development results from inhibition of protein glycosylation and the consequent changes in the structure of the cellular, secreted and surface glycoproteins.     

Proteomics reveals N-linked glycoprotein diversity in Caenorhabditis elegans and suggests an atypical translocation mechanism for integral membrane proteins

Protein glycosylation is one of the most common post-translational modifications in eukaryotes and affects various aspects of protein structure and function. To facilitate studies of protein glycosylation, we paired glycosylation site-specific stable isotope tagging of lectin affinity-captured N-linked glycopeptides with mass spectrometry and determined 1,465 N-glycosylated sites on 829 proteins expressed in Caenorhabditis elegans. The analysis shows the diversity of protein glycosylation in eukaryotes in terms of glycosylation sites and oligosaccharide structures attached to polypeptide chains and suggests the substrate specificity of oligosaccharyltransferase, a single multienzyme complex in C. elegans that incorporates an oligosaccharide moiety en bloc to newly synthesized polypeptides. In addition, topological analysis of 257 N-glycosylated proteins containing a putative single transmembrane segment that were identified based on the relative positions of glycosylation sites and transmembrane segments suggests that an atypical non-cotranslational mechanism translocates large N-terminal segments from the cytosol to the endoplasmic reticulum lumen in the absence of signal sequence function.     

Never say never again: protein glycosylation in pathogenic bacteria

In recent years, accumulating evidence for glycosylated bacterial proteins has overthrown an almost dogmatic belief that prokaryotes are not able to synthesize glycoproteins. Now it is widely accepted that eubacteria express glycoproteins. Although, at present, detailed information about glycosylation and structure-function relationships is available for only few eubacterial proteins, the variety of different components and structures observed already indicates that the variations in bacterial glycoproteins seem to exceed the rather limited display found in eukaryotes. Numerous virulence factors of bacterial pathogens have been found to be covalently modified with carbohydrate residues, thereby identifying these factors as true glycoproteins. In several bacterial species, gene clusters suggested to represent a general protein glycosylation system have been identified. In other cases, genes encoding highly specific glycosyltransferases have been found to be directly linked with virulence genes. These findings raise interesting questions concerning a potential role of glycosylation in pathogenesis. In this review, we will therefore focus on protein glycosylation in Gram-negative bacterial pathogens.     

A new method for quantitative analysis of cell surface glycoproteome

As the altered glycosylation expressions of cell surface proteins are associated with many diseases, glycoproteomics approach has been widely applied to characterization of surface glycosylation alteration. In general, the abundances of proteolytic glycopeptides derived from corresponding glycoproteins can be measured to determine the abundances of glycoproteins. However, this quantification strategy cannot distinguish whether the changes are results from changes of protein abundance or changes in glycosite occupancy. For the accurate and specific quantification of the cell surface glycosylation profile, we proposed a modified cell surface‐capturing strategy where the glycopeptides were submitted to LC‐MS/MS analysis directly for identification of glycoproteins and the non‐glycopeptides were isotopically labelled for quantification of glycoproteins. This strategy was applied to comparatively analyze cell surface glycoproteins of two human cell lines, i.e. Chang Liver and HepG2 cells. Totally 341 glycoproteins were identified with 82.4% specificity for cell membrane proteins and 33 glycoproteins were quantified with significant expression change between the two cell lines. The differential expressions of two selected proteins (EMMPRIN and BCAM) were validated by Western blotting. This method enables specific and accurate analysis of the cell surface glycoproteins and may have broad application in the field of biomarker and drug target discovery.     

Identification of Rabbit Oviductal Fluid Proteins Involved in Pre‐Fertilization Processes by Quantitative Proteomics

Oviductal fluid (ODF) proteins modulate and support reproductive processes in the oviduct. In the present study, proteins involved in the biological events that precede fertilization have been identified in the rabbit ODF proteome, isolated from the ampulla and isthmus of the oviduct at different time points within 8 h after intrauterine insemination. A workflow is used that integrates lectin affinity capture with stable‐isotope dimethyl labeling prior to nanoLC‐MS/MS analysis. In total, over 400 ODF proteins, including 214 lectin enriched glycoproteins, are identified and quantified. Selected data are validated by Western blot analysis. Spatiotemporal alterations in the abundance of ODF proteins in response to insemination are detected by global analysis. A subset of 63 potentially biologically relevant ODF proteins is identified, including extracellular matrix components, chaperones, oxidoreductases, and immunity proteins. Functional enrichment analysis reveals an altered peptidase regulator activity upon insemination. In addition to protein identification and abundance changes, N‐glycopeptide analysis further identifies 281 glycosites on 199 proteins. Taken together, these results show, for the first time, the evolving oviductal milieu early upon insemination. The identified proteins are likely those that modulate in vitro processes, including spermatozoa function.     

Disulfide Bond Formation: Sulfhydryl Oxidase ALR Controls Mitochondrial Biogenesis of Human MIA40

The conserved MIA pathway is responsible for the import and oxidative folding of proteins destined for the intermembrane space of mitochondria. In contrast to a wealth of information obtained from studies with yeast, the function of the MIA pathway in higher eukaryotes has remained enigmatic. Here, we took advantage of the molecular understanding of the MIA pathway in yeast and designed a model of the human MIA pathway. The yeast model for MIA consists of two critical components, the disulfide bond carrier Mia40 and sulfhydryl oxidase Erv1/ALR. Human MIA40 and ALR substituted for their yeast counterparts in the essential function for the oxidative biogenesis of mitochondrial intermembrane space proteins. In addition, the sulfhydryl oxidases ALR/Erv1 were found to be involved in the mitochondrial localization of human MIA40. Furthermore, the defective accumulation of human MIA40 in mitochondria underlies a recently identified disease that is caused by amino acid exchange in ALR. Thus, human ALR is an important factor that controls not only the ability of MIA40 to bind and oxidize protein clients but also the localization of human MIA40 in mitochondria.     

Bean homologs of the mammalian glucose-regulated proteins: induction by tunicamycin and interaction with newly synthesized seed storage proteins in the endoplasmic reticulum

Treatment of developing bean cotyledons with the inhibitor of N-glycosylation tunicamycin enhanced the synthesis of at least two polypeptides with molecular mass 78 kDa and 97 kDa. Pulse-chase experiments and subcellular fractionation indicated that these are endoplasmic reticulum (ER) residents. The 78 kDa protein is a major component of the ER protein fraction and, by N-terminal sequencing, was identified as a bean homolog of the mammalian 78 kDa glucose-regulated protein (GRP78). This is a molecular chaperone that is probably involved in the folding and oligomerization of several animal and yeast proteins in the ER. When newly synthesized storage glycoproteins phaseolin, phytohemagglutinin or alpha-amylase inhibitor were immunoprecipitated from an ER preparation of tunicamycin-treated tissue, the GRP78 homolog was always co-precipitated. Bound GRP78 homolog could be released by ATP treatment. These results suggest that, at least when glycosylation is inhibited, this protein plays a role in the early stages of the synthesis of vacuolar storage proteins.