Definition of the bacterial N-glycosylation site consensus sequence
2006; Springer Nature; Volume: 25; Issue: 9 Linguagem: Inglês
10.1038/sj.emboj.7601087
ISSN1460-2075
AutoresMichael Kowarik, N. Martin Young, Shin Numao, Benjamin L. Schulz, Isabelle Hug, Nico Callewaert, Dominic C. Mills, David C. Watson, Marcela Hernandez, John F. Kelly, Michael Wacker, Markus Aebi,
Tópico(s)Bacteriophages and microbial interactions
ResumoArticle13 April 2006free access Definition of the bacterial N-glycosylation site consensus sequence Michael Kowarik Michael Kowarik Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland Search for more papers by this author N Martin Young N Martin Young Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada Search for more papers by this author Shin Numao Shin Numao Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland Search for more papers by this author Benjamin L Schulz Benjamin L Schulz Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland Search for more papers by this author Isabelle Hug Isabelle Hug Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland Search for more papers by this author Nico Callewaert Nico Callewaert Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland The Zürich Glycomics Initiative (GlycoInit), Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, SwitzerlandPresent address: Cytos Biotechnology AG, Wagistrasse 25, 8952 Schlieren, Switzerland Search for more papers by this author Dominic C Mills Dominic C Mills Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland Search for more papers by this author David C Watson David C Watson Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada Search for more papers by this author Marcela Hernandez Marcela Hernandez Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, SwitzerlandPresent address: GlycoVaxyn AG, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland Search for more papers by this author John F Kelly John F Kelly Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada Search for more papers by this author Michael Wacker Michael Wacker Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, SwitzerlandPresent address: Unit for Molecular Glycobiology, Department for Molecular Biomedical Research, Ghent University and VIB, 9052 Ghent-Zwijnaarde, Belgium Search for more papers by this author Markus Aebi Corresponding Author Markus Aebi Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland Search for more papers by this author Michael Kowarik Michael Kowarik Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland Search for more papers by this author N Martin Young N Martin Young Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada Search for more papers by this author Shin Numao Shin Numao Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland Search for more papers by this author Benjamin L Schulz Benjamin L Schulz Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland Search for more papers by this author Isabelle Hug Isabelle Hug Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland Search for more papers by this author Nico Callewaert Nico Callewaert Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland The Zürich Glycomics Initiative (GlycoInit), Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, SwitzerlandPresent address: Cytos Biotechnology AG, Wagistrasse 25, 8952 Schlieren, Switzerland Search for more papers by this author Dominic C Mills Dominic C Mills Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland Search for more papers by this author David C Watson David C Watson Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada Search for more papers by this author Marcela Hernandez Marcela Hernandez Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, SwitzerlandPresent address: GlycoVaxyn AG, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland Search for more papers by this author John F Kelly John F Kelly Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada Search for more papers by this author Michael Wacker Michael Wacker Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, SwitzerlandPresent address: Unit for Molecular Glycobiology, Department for Molecular Biomedical Research, Ghent University and VIB, 9052 Ghent-Zwijnaarde, Belgium Search for more papers by this author Markus Aebi Corresponding Author Markus Aebi Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland Search for more papers by this author Author Information Michael Kowarik1, N Martin Young2, Shin Numao1, Benjamin L Schulz1, Isabelle Hug1, Nico Callewaert1,3, Dominic C Mills1, David C Watson2, Marcela Hernandez1, John F Kelly2, Michael Wacker1 and Markus Aebi 1 1Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland 2Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada 3The Zürich Glycomics Initiative (GlycoInit), Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland *Corresponding author. Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, 8093 Zürich, Switzerland. Tel.: +41 1 632 6413; Fax: +41 1 632 1375; E-mail: [email protected] The EMBO Journal (2006)25:1957-1966https://doi.org/10.1038/sj.emboj.7601087 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Campylobacter jejuni pgl locus encodes an N-linked protein glycosylation machinery that can be functionally transferred into Escherichia coli. In this system, we analyzed the elements in the C. jejuni N-glycoprotein AcrA required for accepting an N-glycan. We found that the eukaryotic primary consensus sequence for N-glycosylation is N terminally extended to D/E-Y-N-X-S/T (Y, X≠P) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were N-glycosylated when they were either artificially introduced or when they were present in non-C. jejuni proteins. We were able to produce recombinant glycoproteins with engineered N-glycosylation sites and confirmed the requirement for a negatively charged side chain at position −2 in C. jejuni N-glycoproteins. N-glycosylation of AcrA by the eukaryotic OST in Saccharomyces cerevisiae occurred independent of the acidic residue at the −2 position. Thus, bacterial N-glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence. Introduction N-linked protein glycosylation is an essential and conserved process occurring in the endoplasmic reticulum (ER) of eukaryotic organisms. It is important for protein folding, oligomerization, quality control, sorting, and transport of secretory and membrane proteins (Helenius and Aebi, 2004). The oligosaccharyltransferase (OST) catalyzes the transfer of the oligosaccharide from the lipid donor dolichylpyrophosphate to the acceptor protein. In yeast, eight different membrane proteins have been identified that constitute the complex in vivo (Kelleher and Gilmore, 2006). STT3 is thought to represent the catalytic subunit of the OST (Yan and Lennarz, 2002; Nilsson et al, 2003). It is the most conserved subunit in the OST complex (Burda and Aebi, 1999). Within recent years, it has been shown that the food-borne bacterial pathogen Campylobacter jejuni contains a general protein glycosylation system (Szymanski et al, 1999). The machinery required for glycosylation is encoded by 12 genes clustered in the so-called pgl locus (for protein glycosylation; Linton et al, 2005). The Pgl enzymes synthesize a heptasaccharide, GalNAc-α1,4-GalNAc-α1,4-(Glc-β1,3)-GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac, where Bac is 2,4-diacetamido-2,4,6-trideoxy-D-Glc (Young et al, 2002) on a lipid carrier undecaprenylpyrophosphate (Feldman et al, 2005). The heptasaccharide is transferred to asparagine side chains present in a tripeptide consensus sequence of the type N-X-S/T (where X can be any amino acid except proline) by the bacterial OST, PglB (Nita-Lazar et al, 2005). Some biological functions of protein glycosylation in bacteria have been identified. Disruption of N-glycosylation in C. jejuni diminishes not only immunogenicity of several glycoproteins but also host cell adherence and invasion in vitro, and colonization of mice and chicks (Szymanski et al, 2002; Hendrixson and DiRita, 2004; Karlyshev et al, 2004). The goal of this study was to elucidate the C. jejuni pgl system and to compare it to the eukaryotic system. It is known from eukaryotic N-glycosylation that biosynthesis, translocation and in particular folding of acceptor proteins strongly influence glycosylation (Pless and Lennarz, 1977; Chen and Helenius, 2000). Thus, we decided to address the substrate requirements for the bacterial N-glycosylation in vivo using native proteins in order to identify requirements that are not detained by short peptide acceptors used in vitro (Glover et al, 2005). From truncation and mutation experiments with the C. jejuni protein AcrA expressed in Escherichia coli strains bearing the pgl system together with analyses of native glycoproteins from C. jejuni, we found that bacterial N-glycoproteins require a negatively charged side chain at the −2 position to the glycosylated Asn for N-glycosylation to occur, resulting in the stringent acceptor sequence D/E-Y-N-X-S/T (Y, X≠P). This consensus pentapeptide was required but not sufficient for N-glycosylation in E. coli, whereas glycosylation site selection in yeast was independent of the negatively charged residue at the −2 position. Results What are the signals in the AcrA protein that trigger glycosylation? In order to analyze the structural requirements of the acceptor protein for bacterial N-glycosylation, we performed studies on the C. jejuni N-glycoprotein, AcrA (Parkhill et al, 2000; Wacker et al, 2002), a periplasmic lipoprotein. The N-glycosylation sites of this 350 amino acid residue protein are identified (Nita-Lazar et al, 2005), and the crystal structure of a homologue of this protein, MexA from Pseudomonas aeroginosa, had recently been determined (Akama et al, 2004; Higgins et al, 2004). The structure of MexA shows three ordered domains, a β barrel hybrid domain, a lipoyl domain, and a coiled coil domain. As the MexA and AcrA proteins share 29% sequence identity and 50% similarity, and also belong to the periplasmic efflux protein family of proteins predicted to exhibit similar structures (Johnson and Church, 1999), we reasoned that both proteins feature a similar overall fold. To find the minimal requirements in an acceptor protein for glycosylation, we aimed to identify the smallest domain of the AcrA protein that could be glycosylated. As was previously determined, the hypothetical coiled coil domain of AcrA contains three potential N-glycosylation sites, of which only the site at N123 is used (Nita-Lazar et al, 2005). We constructed a plasmid coding for the lipoyl and the glycosylation site containing coiled coil domains of AcrA (named ‘Lip’, corresponding to residues D60-D210 of the mature AcrA polypeptide sequence), fused it N terminally to the signal sequence of OmpA (Choi and Lee, 2004) and C terminally to a hexa-His tag. The Lip domain sequence was chosen based on the domain margins of the lipoyl domain part in the crystal structure of MexA and a sequence alignment of MexA and AcrA (Figure 1). We further constructed seven truncated forms of Lip, stepwise deleting the residues flanking the natural glycosylation site at N123 without affecting the lipoyl domain sequence in Lip (Figure 2A). The peptide stretches of the different truncation variants containing the glycosylation site at N123 tested in this way are indicated in brackets, according to their numbering in the native AcrA polypeptide (Figure 2A). In some recombinant proteins, we also replaced K131 with Gln (K131Q) to reduce the extent of proteolytic cleavage by protease specifically acting between positions K131 and S132 (data not shown). Figure 1.The structural homology of MexA and AcrA. (A) Crystal structure of the MexA protein (adapted from Higgins et al, 2004). Colors indicate the different domains. Red spheres show a space fill representation of the residues in MexA corresponding to the glycosylated N123 and N273 of AcrA based on the homology alignment (C). (B) Scheme of the secondary structure elements in MexA. Orange is the β barrel sandwich hybrid domain. It is connected to an unordered part of the protein containing the N and C termini. Blue: lipoyl domain. Green: coiled coil. Note the overall antiparallel organization of the polypeptide. Red circles indicate the residues in MexA, which align to N123 and N273 (C). (C) Polypeptide alignment of signal peptide processed MexA and AcrA amino acid sequences. The ruler is adjusted to the AcrA amino acid numbering. Colored lines indicate the sequences constituting the domains in MexA (see A, B). The dashed line designates the loop connecting the ascending and descending strands of the coiled coil in MexA. The black line below indicates the part of AcrA constituting the Lip sequence (see Figure 2); the red circles show the naturally glycosylated N123 and N273 of AcrA, the asterisk and the empty circle discriminate Asn residues in additionally introduced glycosylation sites that were active (N117, N145, N274) or inactive (N184, see Figure 3E). Filled triangles indicate glycosylation sites, which were glycosylated in the yeast expression system (see Figure 6). + shows the residue in the protease sensitive loop that was replaced by Gln (K131Q, see Figure 3). Black and gray shading shows identical and similar amino acid residues according to the BLOSUM62 substitution matrix. Note that the AcrA sequence corresponding to the coiled coil is longer than the homologous stretch of MexA. Download figure Download PowerPoint Figure 2.Glycosylation analysis of truncated forms of AcrA. (A) Sequence details of the hypothetical coiled coil domain (see Figure 1, the green domain) present in Lip and truncated forms thereof, which were tested as N-glycan acceptors in E. coli. The ruler indicates the amino acid numbering of the corresponding residues as in the AcrA sequence. Residues D95 and L167 belong to the predicted lipoyl domain and flank the sequence of the coiled coil that include the glycosylation at N123. The peptide stretches of the different Lip truncation variants containing the glycosylation site at N123 are indicated in brackets, according to the numbering in the AcrA sequence. According to this nomenclature, the full length, nontruncated Lip protein is Lip(K96-D166). Note that some residues were mutated due to cloning reasons or to render the protein more resistant to proteolytic cleavage (K131Q). (B) Characterization of the glycosylated lipoyl coiled coil domain of AcrA (Lip). SDS–PAGE analysis of four different Ni2+ affinity purified protein fractions by Coomassie Brilliant Blue staining (left panel), immunoblotting with anti-AcrA antiserum (middle left), R12 antiserum (middle right), or HRP-coupled SBA (right panel). Lip was expressed in presence (+, lanes 1, 5, 9, and 13) and absence (−, lanes 2, 6, 10, and 14) of a functional pgl locus, soluble AcrA only in its presence (+, lanes 3, 7, 11, and 15). Unglycosylated AcrA was purified from the cytoplasm (lanes 4, 8, 12, and 16; Nita-Lazar et al, 2005). Note that the glycosylated proteins are detected with the R12 antiserum and SBA. (C) Glycosylation analysis of truncated forms of Lip. Proteins were Ni2+ affinity purified from periplasmic extracts of Top10 cells expressing the corresponding proteins as indicated above the gel frame and analyzed by SDS–PAGE and immunoblotting. Top gel: Coomassie stained; middle gel: anti-AcrA; bottom: R12. (D) MALDI-MS/MS of m/z=3229.3 from the in-gel trypsinized protein band labeled with a star in panel C. The mass corresponds to the expected glycopeptide derived from the Lip(D121-A127) protein (GQTLFIIEQDQDFN123R). The inset shows the C. jejuni N-glycan attached to the peptide and the corresponding fragmentation pattern. Download figure Download PowerPoint The Lip and the Lip truncation mutants were expressed in glycosylation proficient E. coli Top 10 cells bearing a plasmid containing the wild-type pgl locus from C. jejuni (pACYCpgl). As a negative control for glycosylation, we used cells carrying a plasmid with an inactive PglB (pACYCpglmut; Wacker et al, 2002). Recombinant proteins were purified from periplasmic extracts by Ni2+ affinity chromatography and analyzed by SDS–PAGE, and subsequent Coomassie staining or immunoblotting (for a typical purification see Supplementary Figure S1). Ni2+ affinity purified fractions from cells expressing Lip showed a major protein at a mass of 17 kDa and an additional protein band at a mass of 18.5 kDa when the wild-type pgl locus was present in the cells (Figure 2B, lane 1). Both of the proteins contained the same N terminus (D60VII…, data not shown), and they reacted with anti-AcrA antiserum (lanes 5 and 6) as did a full-length cytoplasmically expressed, nonglycosylated (lanes 4 and 8) or periplasmically expressed, glycosylated AcrA protein (lanes 3 and 7). As the 18.5 kDa protein was not present in extracts from glycosylation deficient cells (lanes 2 and 6), we hypothesized that this additional protein may be the singly glycosylated form of Lip. To confirm this, we tested the proteins reactivity towards R12 antiserum (lanes 9–12). This serum was raised against C. jejuni whole-cell extracts and has been shown to detect preferentially C. jejuni N-glycoproteins (Wacker et al, 2002). We also used the GalNAc-specific lectin soybean agglutinin (SBA), which recognizes the terminal GalNAc residues present on the C. jejuni N-glycans (Young et al, 2002; Linton et al, 2005). The 18.5 kDa band reacted with R12 antiserum as well as with SBA (lanes 9 and 13). Fragmentation analysis of tryptic peptides derived from the 18.5 kDa protein by MALDI-TOF/TOF confirmed the presence of an N-linked C. jejuni glycan attached to the peptide DFN123R (not shown). The truncated forms of Lip expressed in glycosylation competent cells were analyzed in a similar manner. The decreasing size of the major protein species detected by SDS–PAGE in each elution fraction reflected the stepwise reduction of the proteins as expected (Figure 2C, lanes 2–6). With the exception of the Lip(F122-E138)-K131Q, all of the samples contained an additional slower mobility band. These proteins reacted with the anti-AcrA antiserum as well as with the R12 serum suggesting glycosylation. We performed MALDI-TOF/TOF analysis of in-gel trypsinized, R12-reactive protein derived from the Lip(D121-A127) expression. The MS/MS spectrum of m/z=3229.3 demonstrated fragmentation behavior indicative of the C. jejuni N-glycan structure (Figure 2D). A complete Y-ion series was observed down to the nonglycosylated peptide GQTLFIIEQDQDFNR (m/z=1824), which encompasses the Asn residue of the glycosylation site at N123 (underlined). These results demonstrated that, with the exception of Lip(F122-E138)-K131Q, all of the truncation variants were glycosylated. Comparing the primary sequence of the nonglycosylated Lip(F122-E138) protein and the glycosylated Lip(D121-A127) protein, we speculated that the residue D121 missing in the former construct is essential for glycosylation by PglB (see Figure 2A). To address the role of D121 in glycosylation, we mutated D121 to A in the Lip(D121-A127) protein and tested the resulting protein in the in vivo glycosylation assay (Figure 2C, lane 7). Lip(A121-A127) was not glycosylated showing that the Asp residue in the −2 position was essential for glycosylation. The D121A mutation disrupts glycosylation at N123 in AcrA To confirm the importance of the Asp at the −2 position for N-glycosylation, we analyzed the effect of different mutations in the full-length AcrA protein. For protein production and glycosylation, we used the strain CLM24 (Feldman et al, 2005) bearing the plasmid-borne pgl locus (Wacker et al, 2002) and a plasmid for the expression of soluble AcrA containing the PelB signal peptide for secretion and a C-terminal hexa-His tag (Nita-Lazar et al, 2005). The glycosylation status of the periplasmic AcrA was analyzed by SDS–PAGE and immunoblotting with anti-AcrA or R12 antisera. Cells containing the functional pgl locus produced three proteins reactive towards the anti-AcrA antiserum (Figure 3A, lane 2), which were respectively un-, mono- and diglycosylated forms of AcrA as shown by their reactivity towards the R12 antiserum (Figure 3A, R12, lane 2). Residual reactivity of the R12 antiserum towards nonglycosylated AcrA was also detected as it has been observed before (Wacker et al, 2002). The mutation N123Q inactivated the glycosylation site at N123 and resulted in only two protein bands in anti-AcrA immunoblots. The R12 antiserum detected monoglycosylated AcrA only (lane 3). The double mutant, AcrA-N123,273Q, produced a single band reactive towards anti-AcrA, but no signal was observed with R12 antiserum showing that glycosylation was absent despite the presence of a functional pgl machinery (lane 4). When a D121A mutation was made, only a single glycosylated AcrA protein was detected in glycosylation competent cells (lane 6). This result confirmed our finding that the Asp at the position −2 to the modified Asn represents a novel determinant for pgl system N-glycosylation. Replacement of D121A by a Glu residue rescued the glycosylation negative phenotype and resulted in un-, mono- and diglycosylated AcrA (lane 8). Hence, an amino acid with a negatively charged side chain at position −2 was sufficient for glycosylation at N123. Figure 3.SDS–PAGE analysis of N-glycosylation site mutants of AcrA. All top panels are immunoblots probed with anti-AcrA antiserum, bottom panels represent identical samples detected with the C. jejuni N-glycan specific R12 antiserum. + and − indicate the presence of pACYCpgl or pACYCpglmut in the cells. All samples contained a plasmid expressing soluble AcrA with an N-terminal signal peptide. The different point mutations in the AcrA protein are indicated. Numbers on the left of the gel frame show the electrophoretic mobility of the molecular weight marker. The numbers of N-glycans in the different glycoforms of AcrA are indicated at the right of the gel frames. (A) Analysis of the naturally used glycosylation sites N123 and N273. (B) Analysis of artificially introduced glycosylation sites at positions N117, N147, and N274, in the presence of active natural glycosylation sites. (C) Analysis of artificially introduced glycosylation sites at positions N117, N147, and N274, in the absence of other glycosylation sites. (D) Pro in position −1 of the natural glycosylated N123 abolishes glycosylation. (E) A sequon that was mutated into the putative lipoyl domain of AcrA was not active. Download figure Download PowerPoint Engineering additional glycosylation sites into AcrA We tested the activation of all three cryptic N-X-S/T sequences within the AcrA polypeptide for glycosylation by introducing an Asp residue at the −2 position. Accordingly, we mutated the residue in the −2 position of sites N117, N147, and N274 (F115, T145, and N272) of wild-type AcrA to Asp. The mutations were placed either into the wild-type AcrA or into the glycosylation inactive AcrA-N123,273Q. Expression of AcrA-F115D in glycosylation competent E. coli cells led to the detection of four proteins with anti-AcrA antiserum (Figure 3B, lane 4). The slowest migrating protein triggered the strongest signal of all four bands with the R12 antiserum indicating the presence of multiple C. jejuni N-glycans. Based on the electrophoretic mobilities in SDS–PAGE, we concluded that the F115D mutation had resulted in an AcrA protein with three glycosylation sites. The AcrA-T145D also generated triply glycosylated AcrA (lane 6). When both mutations were introduced in the same AcrA protein (AcrA-F115D-T145D), five proteins reactive towards the anti-AcrA antiserum were detected (lane 9). The protein with the slowest mobility triggered a strong signal with the R12 antiserum despite the weak staining observed with anti-AcrA antiserum, and it appeared larger than AcrA-T145D or AcrA-F115D on SDS–PAGE. Taken together, these data indicated that the AcrA-F115D-T145D protein was glycosylated at all four sites. A different result was obtained with the third cryptic site of AcrA, N274. Expression of AcrA-N272D did not trigger the detection of an additional glycosylation. In fact, the glycosylation pattern was similar to the one obtained with wild-type AcrA (compare lanes 8 and 2). Due to our assay, we are not able to judge if the site N273 or N274 was glycosylated in the AcrA-N272D protein. It is likely that glycosylation of the native site N273 inhibited modification of N274 – or vice versa – due to spatial restrictions. Next, we tested if the introduced N-glycosylation sites were used in absence of other sites within the same protein (Figure 3C). Therefore, all three point mutations were individually inserted into the AcrA-N123,273Q double mutant protein (lanes 1 and 2). Periplasmic extracts of glycosylation competent cells expressing either AcrA-N123,273Q-F115D (lane 4), AcrA-N123,273Q-T145D (lane 6), or AcrA-N123,273Q-N272D (lane 8) contained a singly glycosylated, anti-AcrA-reactive protein. We concluded that glycosylation reactions occurring on the same protein are self-sufficient. The absence of triply glycosylated AcrA-N272D represented an exception to this rule. Probably, two adjacent Asn residues participating in overlapping glycosylation sites cannot be modified within the same molecule. Proline at position −1 inhibits N-glycosylation A Pro residue between the Asn acceptor and the hydroxyamino acid of the N-X-S/T consensus sequon inactivates the consensus site (Bause et al, 1995; Nita-Lazar et al, 2005). We observed that a Pro residue between the acidic side chain residue at the −2 position and the Asn acceptor abolished glycosylation in E. coli as well (Figure 3D). Expression of AcrA-N273Q in glycosylating cells produced un- and monoglycosylated AcrA (lane 2), whereas the AcrA-N273Q-F122P protein was not glycosylated at all (lane 4). The bacterial consensus sequence is required but not sufficient for glycosylation We also identified a region in the AcrA protein where it was not possible to generate an active glycosylation site. The mutant AcrA-L184N-N186T was constructed such that it contained the glycosylation site (182DANVT186) in the sequence constituting the predicted lipoyl domain (see Figure 1). As shown in Figure 3E, there is no additional, glycosylated AcrA detectable when compared to the wild-type protein. Likewise, three periplasmic E. coli proteins that contained the consensus sequon in their native sequence could not be glycosylated in E. coli cells containing the pgl machinery (see Supplementary Figure S2). Thus, the consensus sequence alone is not sufficient for bacterial N-glycosylation. In contrast, N-glycosylation of a heterologous protein was achieved with a form of the Cholera toxin B subunit (CtxB) bearing the N-terminal signal sequence of the OmpA protein and a C-terminal hexa-His tag. A bacterial N-glycosylation consensus sequence was created by introducing the point mutation W88D, generating the sequence 88DNNKT92. When expressed in the presence of the functional pgl locus, CtxB-W88D produced an additional band (Figure 4A). This slower migrating protein was glycosylated as shown by its reaction with R12 antiserum. Although N-glycosylation was inefficient, these results demonstrated site-directed glycosylation of a heterologous protein in E. coli. Figure 4.Bacterial N-Glycosylation of a non-C. jejuni protein. (A) Periplasmic extracts derived from cells expressing wild-type CtxB and CtxB-W88D were analyzed by immunoblotting with anti-CtxB antiserum (left panel) and R12 (right panel). Expression was performed in SCM7 E. coli cells containing either the glycosylation competent (+) or incompetent (−) pgl machinery on a plasmid. (B) Ni2+ purified, in-gel trypsinized protein corresponding to the glycosylated CtxB-W88D (marked with an asterisk in (A)) was analyzed by NanoESI-MS/MS. The fragmentation spectrum of the doubly charged pseudomolecular ion at m/z=1134.5, correspon
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