Characterization of N-Linked Protein Glycosylation in Helicobacter pullorum

The first bacterial N-linked glycosylation system was discovered in Campylobacter jejuni, and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that pglB orthologues are present in a subset of species from the Deltaproteobacteria and Epsilonproteobacteria, including three Helicobacter species: H. pullorum, H. canadensis, and H. winghamensis. In contrast to C. jejuni, in which a single pglB gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these Helicobacter species contain two unrelated pglB genes (pglB1 and pglB2), neither of which is located within a larger locus involved in protein glycosylation. In complementation experiments, the H. pullorum PglB1 protein, but not PglB2, was able to transfer C. jejuni N-linked glycan onto an acceptor protein in Escherichia coli. Analysis of the characterized C. jejuni N-glycosylation system with an in vitro oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to H. pullorum, PglB1-dependent N glycosylation with a linear pentasaccharide was observed. This reaction required an acidic residue at the −2 position of the N-glycosylation sequon, as for C. jejuni. Attempted insertional knockout mutagenesis of the H. pullorum pglB2 gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied C. jejuni system.Glycosylation is one of the most common protein modifications, and eukaryotes glycosylate many of their secreted proteins with asparagine or N-linked glycans. This process is thought to have diverse roles in protein folding, quality control, protein secretion, and sorting (13). Eukaryotic glycosylation takes place at the luminal side of the endoplasmic reticulum (ER) membrane, where a preassembled oligosaccharide is transferred from a lipid carrier to asparagine residues within an N-X-S/T consensus sequence, where X can be any amino acid except proline (19). The coupling of glycan to the protein takes place cotranslationally as nascent polypeptide chains cross the ER membrane via a translocon apparatus (5). This reaction involves a protein complex of at least eight subunits (49), with the STT3 protein (50, 52) apparently acting as the central enzyme in the process of N-linked protein glycosylation (29, 48). The STT3 protein consists of an amino terminus with multiple membrane-spanning domains and a carboxy-terminal region containing the highly conserved WWDYG amino acid sequence motif (15).The first prokaryotic glycoproteins were described for archaeal species over 30 years ago (26), and for some time it was thought that protein glycosylation was a eukaryotic and archaeal, but not a bacterial, trait. However, there are now many examples of protein glycosylation in species from the domain Bacteria. For example, general O-linked protein glycosylation systems in which functionally diverse sets of proteins are glycosylated via a single pathway have recently been identified in Neisseria and Bacteroides spp. (8, 21, 44). The most-well-characterized bacterial species with respect to protein glycosylation is the enteropathogen Campylobacter jejuni, which encodes an O-linked system that glycosylates the flagellin protein of the flagellar filament along with the first described bacterial N-linked glycosylation system (39).The C. jejuni N-linked glycosylation pathway is encoded by genes from a single protein glycosylation, or pgl, locus (38). The glycosylation reaction is thought to occur at the periplasmic face of the bacterial inner membrane mediated by the product of the STT3 orthologue pglB (46). The C. jejuni heptasaccharide glycan is assembled on a lipid carrier in the cytoplasm through the action of glycosyltransferases encoded by the pglA, pglC, pglH, pglJ, and pglI genes (11, 12, 24, 31). This lipid-linked oligosaccharide (LLO) is then “flipped” into the periplasm by the pglK gene product, or “flippase” (1), and transferred by PglB onto an asparagine residue within an extended D/E-X-N-X-S/T sequon (19). Many C. jejuni periplasmic and surface proteins of diverse function are N glycosylated (51), yet the function of glycosylation remains elusive. Unlike in eukaryotes, this process occurs posttranslationally, and the surface location of the sequon in folded proteins appears to be required for glycosylation (20).The C. jejuni pgl gene locus can be transferred into Escherichia coli, and the corresponding gene products will function to transfer the heptasaccharide onto asparagine residues of coexpressed C. jejuni glycoproteins as well as non-C. jejuni proteins containing the appropriately located acceptor sequon (19, 46). When alternative lipid-linked glycans are present, such as those involved in lipopolysaccharide biosynthesis, glycans with diverse structure can also be transferred onto proteins (7). Although there are limitations, particularly with regard to the apparent structural requirement for an acetamido group on the C-2 carbon of the reducing end sugar (7, 47), this is still a significant advance toward tractable in vivo systems for glycoconjugate synthesis. The identification and characterization of further bacterial PglB proteins with potentially diverse properties would considerably expand the utility of such systems. Data from genome sequencing indicate that pglB orthologues are found in species closely related to C. jejuni, such as Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis (40), as well as in the more distantly related species Wolinella succinogenes (2). These species are members of the phylogenetic grouping known as the epsilon subdivision of the Proteobacteria, or Epsilonproteobacteria, consisting of the well-established genera Campylobacter, Helicobacter, Arcobacter, and Wolinella, which are often associated with human and animal hosts, as well as a number of newly recognized groupings of environmental bacteria often found in sulfidic environments (3). However, not all species of Epsilonproteobacteria contain pglB orthologues, and until recently, all characterized Helicobacter species lacked pglB genes.Given the considerable interest in exploiting bacterial protein glycosylation, especially the C. jejuni N-linked glycosylation system, for generating glycoconjugates of biotechnological and therapeutic potential, the functional characterization of newly discovered pglB orthologues is a priority. In this report we describe the application of an in vitro oligosaccharyltransferase assay to investigate N-linked glycosylation initially in C. jejuni, where the utility of this approach was demonstrated, and then in Helicobacter pullorum, demonstrating that one of the two H. pullorum PglB enzymes is responsible for N-linked protein glycosylation with a pentasaccharide glycan.     

Intracellular trafficking of recycling apolipoprotein E in Chinese hamster ovary cells

We have investigated apolipoprotein E (apoE) recycling in Chinese hamster ovary (CHO) cells, a peripheral cell that does not produce lipoproteins or express apoE. Using a pulse-chase protocol in which cells were pulsed with 125I-apoE-VLDL and chased for different periods, approximately 30% of the apoE internalized during the pulse was resecreted within a 4 h chase in a relatively lipid-free state. The addition of lysosomotropic agents or brefeldin A had no effect on apoE recycling. Unlike previous results with hepatocytes and macrophages, neither apoA-I nor upregulation of ABCA1 stimulated apoE recycling. However, cyclodextrin, which extracts cholesterol from plasma membrane lipid rafts, increased recycling. Confocal studies revealed that apoE, internalized during a 1 h pulse, colocalizes with early endosomal antigen-1, Rab5, Rab11a, and lysobisphosphatidic acid but not with lysosomal-associated membrane protein-1. Colocalization of apoE and Rab11a persisted even after cells had been chased for 1 h, suggesting a pool of apoE within the endosomal recycling compartment (ERC). Our data suggest that apoE recycling in CHO cells is linked to cellular cholesterol removal via the ERC and phospholipid-containing acceptors in a pathway alternative to the ABCA1-apoA-I axis.     

Function of the factor I modules (FIMS) of human complement component C6.

In order to elucidate the function of complement component C6, truncated C6 molecules were expressed recombinantly. These were either deleted of the factor I modules (FIMs) (C6des-748-913) or both complement control protein (CCP) modules and FIMs (C6des-611-913). C6des-748-913 exhibited approximately 60-70% of the hemolytic activity of full-length C6 when assayed for Alternative Pathway activity, but when measured for the Classical Pathway, C6des-748-914 was only 4-6% as effective as C6. The activity difference between C6 and C6des-748-913 for the two complement pathways can be explained by a greater stability of newly formed metastable C5b* when produced by the Alternative Pathway compared with that made by the Classical Pathway. The half-lives of metastable C5b* and the decay of (125)I-C5b measured from cells used to activate the Alternative Pathway were found to be about 5-12-fold longer than those same parameters derived from cells that had activated the Classical Pathway. (125)I-C5 binds reversibly to C6 in an ionic strength-dependent fashion, but (125)I-C5 binds only weakly to C6des-FIMs and not at all to C6des-CCP/FIMs. Therefore, although the FIMs are not required absolutely for C6 activity, these modules promote interaction of C6 with C5 enabling a more efficient bimolecular coupling ultimately leading to the formation of the C5b-6 complex.