Wolfgang Egge-Jacobsen

Summary: Functional modules within protein sequences are often extracted by consensus sequence patterns representing a linear motif; however, other functional regions may only be described by combined features such as amino acid composition, profiles of amino acid properties and randomly distributed short sequence motifs. If only a small number of functional examples are well characterized, the researcher needs a tool to extract similar sequences for further investigation. Availability and Implementation: We provide the web application ‘SAPA tool’, which allows the user to search with combined properties, ranks the extracted target regions by an integrated score, estimates false discovery rates by using decoy sequences and provides them as a sequence file or spreadsheet. Source code, user manual and the web application implemented in Perl, HTML, CSS and JavaScript and running on Apache are freely available at http://sapa-tool.uio.no/sapa/ Contact:josef.maier@istls.de or w.m.egge-jacobsen@imbv.uio.no Supplementary information: Supplementary data are available at Bioinformatics online.

The infectious liver disease hepatitis C is caused by the small, enveloped, positive single-strand RNA hepatitis C virus (HCV). The HCV genome encodes for a single polyprotein precursor of ∼3010 amino acid residues. Host and cellular proteases co- and posttranslational process the precursor creating six nonstructural (NS) proteins and four structural components. Properly folded forms of the envelope proteins E1 and E2 form the associated E1–E2 complex. This complex represents a significant antigenic component at the viral surface that can interact with several target cell receptors. Extent and type of glycosylation is an important factor for virulence and escape from the immune system. Detailed characterization of the glycosylated sites is helpful for the understanding of different phenotypes as well as for the development of E1/E2-related treatments of HCV infection. Here, we have investigated in detail the O-linked glycosylation of the HCV envelope protein E2 expressed in and isolated from human embryonic kidney (HEK 293) cells. Using nano-liquid chromatography and tandem mass spectrometry approaches, we clearly have identified six residues for O-linked glycosylation within isolated glycopeptides (Ser393, Thr396, Ser401, Ser404, Thr473 and Thr518), carrying mainly Core 1 and Core 2 mucin-type structures. Based on our data, Thr385 is probably glycosylated as well. In addition, we could show that Ser479 within the hyper variable region (HVR) I is not O-glycosylated. For most of these sites, different degrees of microheterogeneity could be verified. Concerning HCV E2, this is the first case of experimentally proven O-linked glycosylation in detail via mass spectrometry.

Findings from a number of studies suggest that the PilA pilin proteins may play an important role in the pathogenesis of disease caused by species within the genusFrancisella. As such, a thorough understanding of PilA structure and chemistry is warranted. Here, we definitively identified the PglA protein-targeting oligosaccharyltransferase by virtue of its necessity for PilA glycosylation inFrancisella tularensisand its sufficiency for PilA glycosylation inEscherichia coli. In addition, we used mass spectrometry to examine PilA affinity purified fromFrancisella tularensissubsp.tularensisandF. tularensissubsp.holarcticaand demonstrated that the protein undergoes multisite,O-linked glycosylation with a pentasaccharide of the structure HexNac-Hex-Hex-HexNac-HexNac. Further analyses revealed microheterogeneity related to forms of the pentasaccharide carrying unusual moieties linked to the distal sugar via a phosphate bridge. Type A and type B strains ofFrancisellasubspecies thus express anO-linked protein glycosylation system utilizing core biosynthetic and assembly pathways conserved in other members of the proteobacteria. As PglA appears to be highly conserved inFrancisellaspecies,O-linked protein glycosylation may be a feature common to members of this genus.

Different classes of glycans are implicated as mediators of apical protein sorting in the secretory pathway of epithelial cells, but recent research indicates that sorting to the apical and basolateral surfaces may occur before completion of glycan synthesis. We have previously shown that a proteoglycan (PG) core protein can obtain different glycosaminoglycan (GAG) structures in the apical and basolateral secretory routes (Tveit H, Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells. J Biol Chem. 280:29596–29603) of epithelial Madin–Darby canine kidney (MDCK) cells. We have now also determined the detailed N-glycan structures acquired by a single glycoprotein species in the same apical and basolateral secretory pathways. For this purpose, rat growth hormone (rGH) with two N-glycan sites (rGH-2N) inserted into the rGH portion (NAS and NFT) was fused to green fluorescent protein (GFP) and expressed in MDCK cells. Immunoisolated rGH variants were analyzed for site occupancy and N-glycan structure by mass spectrometry. The extent of NAS and NFT site occupancy was different, but comparable for rGH-2N secreted apically and basolaterally. Microheterogeneity existed for the glycans attached to each N-glycan site, but no major differences were observed in the apical and basolateral pathways. Transfer of the GAG modification domain from the PG serglycin to the fusion site of rGH-2N and GFP allowed polymerization of GAG chains onto the novel protein variant and influenced the microheterogeneity of the N-glycans toward more acidic glycans, but did not alter the relative site occupancy. In conclusion, no major differences were observed for N-glycan structures obtained by the expressed model proteins in the apical and basolateral secretory pathways of epithelial MDCK cells, but insertion of a GAG attachment domain shifted the N-glycans to more acidic structures.

Background: Surface proteins are a key to a deeper understanding of the behaviour of Gram-positive bacteria interacting with the human gastro-intestinal tract. Such proteins contribute to cell wall synthesis and maintenance and are important for interactions between the bacterial cell and the human host. Since they are exposed and may play roles in pathogenicity, surface proteins are interesting targets for drug design. Results: Using methods based on proteolytic "shaving" of bacterial cells and subsequent mass spectrometry-based protein identification, we have identified surface-located proteins in Enterococcus faecalis V583. In total 69 unique proteins were identified, few of which have been identified and characterized previously. 33 of these proteins are predicted to be cytoplasmic, whereas the other 36 are predicted to have surface locations (31) or to be secreted (5). Lipid-anchored proteins were the most dominant among the identified surface proteins. The seemingly most abundant surface proteins included a membrane protein with a potentially shedded extracellular sulfatase domain that could act on the sulfate groups in mucin and a lipid-anchored fumarate reductase that could contribute to generation of reactive oxygen species. Conclusions: The present proteome analysis gives an experimental impression of the protein landscape on the cell surface of the pathogenic bacterium E. faecalis. The 36 identified secreted (5) and surface (31) proteins included several proteins involved in cell wall synthesis, pheromone-regulated processes, and transport of solutes, as well as proteins with unknown function. These proteins stand out as interesting targets for further investigation of the interaction between E. faecalis and its environment.

Protein glycosylation is an important element of biologic systems because of its significant effects on protein properties and functions. Although prominent within all domains of life, O-linked glycosylation systems modifying serine and threonine residues within bacteria and eukaryotes differ substantially in target protein selectivity. In particular, well-characterized bacterial systems have been invariably dedicated to modification of individual proteins or related subsets thereof. Here we characterize a general O-linked glycosylation system that targets structurally and functionally diverse groups of membrane-associated proteins in the Gram-negative bacterium Neisseria gonorrhoeae , the etiologic agent of the human disease gonorrhea. The 11 glycoproteins identified here are implicated in activities as varied as protein folding, disulfide bond formation, and solute uptake, as well as both aerobic and anaerobic respiration. Along with their common trafficking within the periplasmic compartment, the protein substrates share quasi-related domains bearing signatures of low complexity that were demonstrated to encompass sites of glycan occupancy. Thus, as in eukaryotes, the broad scope of this system is dictated by the relaxed specificity of the glycan transferase as well as the bulk properties and context of the protein-targeting signal rather than by a strict amino acid consensus sequence. Together, these findings reveal previously unrecognized commonalities linking O-linked protein glycosylation in distantly related life forms.

Summary: Neisseria gonorrhoeae expresses an O‐linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a ‘top‐down’ mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O‐acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N‐linked glycosylation system that adds N‐acetylgalactosamine onto undecaprenylpyrophosphate‐linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O‐linked di‐ and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid‐linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O‐ and N‐linked pathways can be combined in novel glycoengineering strategies.