Phylum‐wide general protein O‐glycosylation system of the Bacteroidetes

The human gut symbiont Bacteroides fragilis has a general protein O‐glycosylation system in which numerous extracytoplasmic proteins are glycosylated at a three amino acid motif. In B. fragilis, protein glycosylation is a fundamental and essential property as mutants with protein glycosylation defects have impaired growth and are unable to competitively colonize the mammalian intestine. In this study, we analysed the phenotype of B. fragilis mutants with defective protein glycosylation and found that the glycan added to proteins is comprised of a core glycan and an outer glycan. The genetic region encoding proteins for the synthesis of the outer glycan is conserved within a Bacteroides species but divergent between species. Unlike the outer glycan, an antiserum raised to the core glycan reacted with all Bacteroidetes species tested, from all four classes of the phylum. We found that diverse Bacteroidetes species synthesize numerous glycoproteins and glycosylate proteins at the same three amino acid motif. The wide‐spread conservation of this protein glycosylation system within the phylum suggests that this system of post‐translational protein modification evolved early, before the divergence of the four classes of Bacteroidetes, and has been maintained due to its physiological importance to the diverse species of this phylum.     

Porphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism

The type IX secretion system (T9SS) of Porphyromonas gingivalis secretes proteins possessing a conserved C-terminal domain (CTD) to the cell surface. The C-terminal signal is essential for these proteins to translocate across the outer membrane via the T9SS. On the surface the CTD of these proteins is cleaved prior to extensive glycosylation. It is believed that the modification on these CTD proteins is anionic lipopolysaccharide (A-LPS), which enables the attachment of CTD proteins to the cell surface. However, the exact site of modification and the mechanism of attachment of CTD proteins to the cell surface are unknown. In this study we characterized two wbaP (PG1964) mutants that did not synthesise A-LPS and accumulated CTD proteins in the clarified culture fluid (CCF). The CTDs of the CTD proteins in the CCF were cleaved suggesting normal secretion, however, the CTD proteins were not glycosylated. Mass spectrometric analysis of CTD proteins purified from the CCF of the wbaP mutants revealed the presence of various peptide/amino acid modifications from the growth medium at the C-terminus of the mature CTD proteins. This suggested that modification occurs at the C-terminus of T9SS substrates in the wild type P. gingivalis. This was confirmed by analysis of CTD proteins from wild type, where a 648 Da linker was identified to be attached at the C-terminus of mature CTD proteins. Importantly, treatment with proteinase K released the 648 Da linker from the CTD proteins demonstrating a peptide bond between the C-terminus and the modification. Together, this is suggestive of a mechanism similar to sortase A for the cleavage and modification/attachment of CTD proteins in P. gingivalis. PG0026 has been recognized as the CTD signal peptidase and is now proposed to be the sortase-like protein in P. gingivalis. To our knowledge, this is the first biochemical evidence suggesting a sortase-like mechanism in Gram-negative bacteria.     

OsMOGS is required for N‐glycan formation and auxin‐mediated root development in rice (Oryza sativa L.)

N‐glycosylation is a major modification of glycoproteins in eukaryotic cells. In Arabidopsis, great progress has been made in functional analysis of N‐glycan production, however there are few studies in monocotyledons. Here, we characterized a rice (Oryza sativa L.) osmogs mutant with shortened roots and isolated a gene that coded a putative mannosyl‐oligosaccharide glucosidase (OsMOGS), an ortholog of α‐glucosidase I in Arabidopsis, which trims the terminal glucosyl residue of the oligosaccharide chain of nascent peptides in the endoplasmic reticulum (ER). OsMOGS is strongly expressed in rapidly cell‐dividing tissues and OsMOGS protein is localized in the ER. Mutation of OsMOGS entirely blocked N‐glycan maturation and inhibited high‐mannose N‐glycan formation. The osmogs mutant exhibited severe defects in root cell division and elongation, resulting in a short‐root phenotype. In addition, osmogs plants had impaired root hair formation and elongation, and reduced root epidemic cell wall thickness due to decreased cellulose synthesis. Further analysis showed that auxin content and polar transport in osmogs roots were reduced due to incomplete N‐glycosylation of the B subfamily of ATP‐binding cassette transporter proteins (ABCBs). Our results demonstrate that involvement of OsMOGS in N‐glycan formation is required for auxin‐mediated root development in rice.     

Does the importance of the C-terminal residues in the maturation of RgpB from Porphyromonas gingivalis reveal a novel mechanism for protein export in a subgroup of Gram-Negative bacteria?

The mature 507-residue RgpB protein belongs to an important class of extracellular outer membrane-associated proteases, the gingipains, from the oral pathogen Porphyromonas gingivalis that has been shown to play a central role in the virulence of the organism. The C termini of these gingipains along with other outer membrane proteins from the organism share homologous sequences and have been suggested to function in attachment of these proteins to the outer membrane. In this report, we have created a series of truncated and site-directed mutants of the C terminus from a representative member of this class, the RgpB protease, to investigate its role in the maturation of these proteins. Truncation of the last two residues (valyl-lysine) from the C terminus is sufficient to create an inactive version of the protein that lacks the posttranslational glycosylation seen in the wild type, and the protein remains trapped behind the outer membrane. Alanine scanning of the last five residues revealed the importance of the C-terminal motif in mediating correct posttranslational modification of the protein. This result may have a wider implication in a novel secretory pathway in distinct members of the Cytophaga-Flavobacterium-Bacteroidetes phylum.     

The TPR domain of BepA is required for productive interaction with substrate proteins and the β‐barrel assembly machinery complex

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N‐terminal protease domain and a C‐terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone‐like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the β‐barrel assembly machinery (BAM) driving integration of β‐barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site‐directed in vivo photo‐cross‐linking was used to map the protein–protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane.     

Lethal mutations in the major homology region and their suppressors act by modulating the dimerization of the rous sarcoma virus capsid protein C‐terminal domain

An infective retrovirus requires a mature capsid shell around the viral replication complex. This shell is formed by about 1500 capsid protein monomers, organized into hexamer and pentamer rings that are linked to each other by the dimerization of the C‐terminal domain (CTD). The major homology region (MHR), the most highly conserved protein sequence across retroviral genomes, is part of the CTD. Several mutations in the MHR appear to block infectivity by preventing capsid formation. Suppressor mutations have been identified that are distant in sequence and structure from the MHR and restore capsid formation. The effects of two lethal and two suppressor mutations on the stability and function of the CTD were examined. No correlation with infectivity was found for the stability of the lethal mutations (D155Y‐CTD, F167Y‐CTD) and suppressor mutations (R185W‐CTD, I190V‐CTD). The stabilities of three double mutant proteins (D155Y/R185W‐CTD, F167Y/R185W‐CTD, and F167Y/I190V‐CTD) were additive. However, the dimerization affinity of the mutant proteins correlated strongly with biological function. The CTD proteins with lethal mutations did not dimerize, while those with suppressor mutations had greater dimerization affinity than WT‐CTD. The suppressor mutations were able to partially correct the dimerization defect caused by the lethal MHR mutations in double mutant proteins. Despite their dramatic effects on dimerization, none of these residues participate directly in the proposed dimerization interface in a mature capsid. These findings suggest that the conserved sequence of the MHR has critical roles in the conformation(s) of the CTD that are required for dimerization and correct capsid maturation. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

The prodomain of the Bordetella two‐partner secretion pathway protein FhaB remains intracellular yet affects the conformation of the mature C‐terminal domain

Two‐partner secretion (TPS) systems use β‐barrel proteins of the Omp85‐TpsB superfamily to transport large exoproteins across the outer membranes of Gram‐negative bacteria. The Bordetella FHA/FhaC proteins are prototypical of TPS systems in which the exoprotein contains a large C‐terminal prodomain that is removed during translocation. Although it is known that the FhaB prodomain is required for FHA function in vivo, its role in FHA maturation has remained mysterious. We show here that the FhaB prodomain is required for the extracellularly located mature C‐terminal domain (MCD) of FHA to achieve its proper conformation. We show that the C‐terminus of the prodomain is retained intracellularly and that sequences within the N‐terminus of the prodomain are required for this intracellular localization. We also identify sequences at the C‐terminus of the MCD that are required for release of mature FHA from the cell surface. Our data support a model in which the intracellularly located prodomain affects the final conformation of the extracellularly located MCD. We hypothesize that maturation triggers cleavage and degradation of the prodomain.     

The histone H1 C-terminal domain binds to the apoptotic nuclease, DNA fragmentation factor (DFF40/CAD) and stimulates DNA cleavage

The apoptotic nuclease, DNA fragmentation factor (DFF40/CAD), is primarily responsible for internucleosomal DNA cleavage during the terminal stages of programmed cell death. Previously, we demonstrated that histone H1 greatly stimulates naked DNA cleavage by this nuclease. Here, we investigate the mechanism of this stimulation with native and recombinant mouse and human histone H1 species. Using a series of truncation mutants of recombinant histone H1-0, we demonstrate that the H1 C-terminal domain (CTD) is responsible for activation of DFF40/CAD. We show further that the intact histone H1-0 CTD and certain synthetic CTD fragments bind to DFF40/CAD and confer upon it an increased ability to bind to DNA. Interestingly, we find that each of the six somatic cell histone H1 isoforms, whose CTDs differ significantly in primary sequence but not amino acid composition, equally activate DFF40/CAD. We conclude that the interactions identified here between the histone H1 CTD and DFF40/CAD target and activate linker DNA cleavage during the terminal stages of apoptosis.     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

N‐glycan maturation is crucial for cytokinin‐mediated development and cellulose synthesis in Oryza sativa

To explore the physiological significance of N‐glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N‐acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N‐glycan maturation and accumulated high‐mannose N‐glycans. Phenotypic analyses revealed that gnt1 shows defective post‐seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark‐induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A‐type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N‐glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system.     

Mass spectrometric characterization of the Campylobacter jejuni adherence factor CadF reveals post‐translational processing that removes immunogenicity while retaining fibronectin binding

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn‐binding is mediated by a 37 kDa outer membrane protein termed Campylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2‐DE and MS. Several spots were identified as products of the cadF gene. These included mass and pI variants of 34 and 30 kDa, as well as 24 kDa (CadF₂₄) and 22 kDa (CadF₂₂) mass variants. CadF variants were fully characterized by MALDI‐TOF MS and MALDI‐MS/MS. These data confirmed that CadF forms re‐folding variants resulting in spots with lower mass and varying pI that are identical at the amino acid sequence level and are not modified post‐translationally. CadF₂₂ and CadF₂₄, however, were characterized as N‐terminal, membrane‐associated polypeptides resulting from cleavage between serine¹⁹⁵ and leucine¹⁹⁶, and glycine²⁰¹ and phenylalanine²⁰², respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full‐length CadF (34 kDa), CadF₂₄, and the deleted C‐terminal OmpA domain (14 kDa; CadF₁₄) were created in Escherichia coli. Recombinant CadF variants were probed against patient sera and revealed that only full‐length CadF retained reactivity. Binding assays showed that CadF₂₄ retained Fn‐binding capability, while CadF₁₄ did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn‐binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni.     

A two‐component Kdo hydrolase in the inner membrane of Francisella novicida

Lipid A coats the outer surface of the outer membrane of Gram‐negative bacteria. In Francisella tularensis subspecies novicida lipid A is present either as the covalently attached anchor of lipopolysaccharide (LPS) or as free lipid A. The lipid A moiety of Francisella LPS is linked to the core domain by a single 2‐keto‐3‐deoxy‐D‐manno‐octulosonic acid (Kdo) residue. F. novicida KdtA is bi‐functional, but F. novicida contains a membrane‐bound Kdo hydrolase that removes the outer Kdo unit. The hydrolase consists of two proteins (KdoH1 and KdoH2), which are expressed from adjacent, co‐transcribed genes. KdoH1 (related to sialidases) has a single predicted N‐terminal transmembrane segment. KdoH2 contains 7 putative transmembrane sequences. Neither protein alone catalyses Kdo cleavage when expressed in E. coli. Activity requires simultaneous expression of both proteins or mixing of membranes from strains expressing the individual proteins under in vitro assay conditions in the presence of non‐ionic detergent. In E. coli expressing KdoH1 and KdoH2, hydrolase activity is localized in the inner membrane. WBB06, a heptose‐deficient E. coli mutant that makes Kdo₂‐lipid A as its sole LPS, accumulates Kdo‐lipid A when expressing the both hydrolase components, and 1‐dephospho‐Kdo‐lipid A when expressing both the hydrolase and the Francisella lipid A 1‐phosphatase (LpxE).     

Characterization of a novel intramolecular chaperone domain conserved in endosialidases and other bacteriophage tail spike and fiber proteins

Folding and assembly of endosialidases, the trimeric tail spike proteins of Escherichia coli K1-specific bacteriophages, crucially depend on their C-terminal domain (CTD). Homologous CTDs were identified in phage proteins belonging to three different protein families: neck appendage proteins of several Bacillus phages, L-shaped tail fibers of coliphage T5, and K5 lyases, the tail spike proteins of phages infecting E. coli K5. By analyzing a representative of each family, we show that in all cases, the CTD is cleaved off after a strictly conserved serine residue and alanine substitution prevented cleavage. Further structural and functional analyses revealed that (i) CTDs are autonomous domains with a high alpha-helical content; (ii) proteolytically released CTDs assemble into hexamers, which are most likely dimers of trimers; (iii) highly conserved amino acids within the CTD are indispensable for CTD-mediated folding and complex formation; (iv) CTDs can be exchanged between proteins of different families; and (v) proteolytic cleavage is essential to stabilize the native protein complex. Data obtained for full-length and proteolytically processed endosialidase variants suggest that release of the CTD increases the unfolding barrier, trapping the mature trimer in a kinetically stable conformation. In summary, we characterize the CTD as a novel C-terminal chaperone domain, which assists folding and assembly of unrelated phage proteins.     

Identification and characterization of site‐specific N‐glycosylation in the potassium channel Kv3.1b

The potassium ion channel Kv3.1b is a member of a family of voltage‐gated ion channels that are glycosylated in their mature form. In the present study, we demonstrate the impact of N‐glycosylation at specific asparagine residues on the trafficking of the Kv3.1b protein. Large quantities of asparagine 229 (N229)‐glycosylated Kv3.1b reached the plasma membrane, whereas N220‐glycosylated and unglycosylated Kv3.1b were mainly retained in the endoplasmic reticulum (ER). These ER‐retained Kv3.1b proteins were susceptible to degradation, when co‐expressed with calnexin, whereas Kv3.1b pools located at the plasma membrane were resistant. Mass spectrometry analysis revealed a complex type Hex₃HexNAc₄Fuc₁ glycan as the major glycan component of the N229‐glycosylated Kv3.1b protein, as opposed to a high‐mannose type Man₈GlcNAc₂ glycan for N220‐glycosylated Kv3.1b. Taken together, these results suggest that trafficking‐dependent roles of the Kv3.1b potassium channel are dependent on N229 site‐specific glycosylation and N‐glycan structure, and operate through a mechanism whereby specific N‐glycan structures regulate cell surface expression.     

Regulation of protease‐activated receptor‐2 expression in gingival fibroblasts and Jurkat T cells by Porphyromonas gingivalis

Periodontal disease destroys the tooth‐supporting tissues as a result of chronic inflammation elicited by bacterial accumulation on tooth surfaces. Porphyromonas gingivalis is a major periodontal pathogen, with a significant capacity to perturb connective tissue homeostasis and immune responses in the periodontium, attributed to its virulence factors, including a group of secreted cysteine proteases (gingipains). PAR‐2 (protease‐activated receptor‐2) is a G‐protein‐coupled receptor activated upon proteolytic cleavage, mediating intracellular signalling events related to infection and inflammation, such as cytokine production. GF (gingival fibroblasts) and T cells have central roles in periodontal inflammation, which can potentially be mediated by PAR‐2. The aims of this study were to investigate the effects of P. gingivalis on PAR‐2 gene expression in human GF and Jurkat T cells, using quantitative real‐time PCR, and to evaluate the involvement of gingipains. After 6 h of challenge with ascending concentrations of P. gingivalis, PAR‐2 expression was up‐regulated in both cell types by approximately 5‐fold, compared with the control. The P. gingivalis concentration required for maximal PAR‐2 induction was 4‐fold greater in GF than Jurkat T cells. Heat inactivation or chemical inhibition of cysteine proteases abolished the capacity of P. gingivalis to induce PAR‐2 expression in Jurkat T cells. In conclusion, P. gingivalis can induce PAR‐2 expression in GF and Jurkat T cells, potentially attributed to its gingipains. These findings denote that P. gingivalis may perturb the host immune and inflammatory responses by enhancing PAR‐2 expression, thus contributing to the pathogenesis of periodontal disease.     

The C‐terminal portion of the cleaved HT motif is necessary and sufficient to mediate export of proteins from the malaria parasite into its host cell

The malaria parasite exports proteins across its plasma membrane and a surrounding parasitophorous vacuole membrane, into its host erythrocyte. Most exported proteins contain a Host Targeting motif (HT motif) that targets them for export. In the parasite secretory pathway, the HT motif is cleaved by the protease plasmepsin V, but the role of the newly generated N‐terminal sequence in protein export is unclear. Using a model protein that is cleaved by an exogenous viral protease, we show that the new N‐terminal sequence, normally generated by plasmepsin V cleavage, is sufficient to target a protein for export, and that cleavage by plasmepsin V is not coupled directly to the transfer of a protein to the next component in the export pathway. Mutation of the fourth and fifth positions of the HT motif, as well as amino acids further downstream, block or affect the efficiency of protein export indicating that this region is necessary for efficient export. We also show that the fifth position of the HT motif is important for plasmepsin V cleavage. Our results indicate that plasmepsin V cleavage is required to generate a new N‐terminal sequence that is necessary and sufficient to mediate protein export by the malaria parasite.