BACKGROUND
n-linked glycosylation is of fundamental importance for the biological activity of many eukaryotic proteins.
bacterial oligosaccharyltransferases  perform a similar type of posttranslational modification in prokaryotic cells and thus hold great promise for producing proteins with defined glycosylation patterns in simple and cost-efficient expression systems such as escherichia coli <cit> .
the ost of campylobacter jejuni  has been functionally expressed in e. coli <cit>  and has been shown to have a relaxed specificity towards its lipid-linked substrate.
therefore it can be exploited, e.g., for the production of conjugate vaccines where polysaccharide antigens of bacterial pathogens are covalently coupled to carrier proteins  <cit> .
polysaccharide-protein conjugates  belong to the most effective measures for combating life-threatening infectious diseases  <cit> .
diverse o-specific polysaccharides of the outer lipopolysaccharide layer of gram-negative bacteria can be efficiently coupled to carrier proteins containing a surface-exposed d/e-x1-n-x2-t/s acceptor sequon by pglbcj, provided that they contain an n-acetyl sugar at the reducing end  <cit> .
examples are the o <dig>  o9a, o <dig> and o <dig> antigens of e. coli, the o <dig> antigen of shigella dysenteriae as well as the o <dig> antigen of pseudomonas aeruginosa <cit> .
production of conjugate vaccines by in vivo conjugation in e. coli is much simpler than with conventional chemical conjugation technologies, and it has already been demonstrated that glycoconjugates can be produced at a larger scale in fed-batch culture with yields of 18– <dig> mg l- <dig> of purified product  <cit> .
recently, it was found that capsular polysaccharides of medically important gram-positive bacterial pathogens such as staphylococcus aureus can also be transferred to acceptor proteins by pglbcj .
pglbcj is a monomeric protein of  <dig> amino acids and comprises  <dig> predicted transmembrane helixes in the n-terminal two thirds of the polypeptide and a globular domain at the c-terminus which is soluble and exposed to the periplasm  <cit> .
the latter domain of pglbcj was crystallized by maita et al.
<cit> .
it harbors the highly conserved w457wdyg <dig> motif which is also found in the stt <dig> subunit of eukaryotic ost  <cit> .
when w <dig> and d <dig> were both replaced by alanine residues, pglbcj was rendered inactive  <cit> .
the crystal structure of the soluble domain of pglbcj revealed a kinked helix opposite of the wwdyg motif which contained the conserved residues m <dig>  i <dig> and v <dig>  subsequently termed “miv” motif of bacterial ost  <cit> .
mutational studies confirmed the importance of i <dig> for activity  <cit> .
pglb of campylobacter lari, a bacterial ost with 56% amino acid identity to pglbcj, also glycosylates recombinant proteins in the periplasm when over-expressed in e. coli <cit> .
the crystal structure of full-length pglbc.lari was elucidated recently  <cit> .
the structure contained a divalent metal co-factor as well as a bound synthetic d-q-n-a-t-p-nitro phenylalanine acceptor peptide.
it revealed that both the transmembrane and the soluble periplasmic domains of pglbc.lari participate in substrate binding and catalysis  <cit> .
the strictly conserved residues w463-w464-d <dig> of pglbc.lari were shown to form a strong network of hydrogen bonds with the hydroxyl group of the threonine residue at position + <dig> relative to the glycosylated asparagine  of the acceptor peptide  <cit> .
this explains the strict requirement for either s or t in the d/e-x1-n-x2-s/t  consensus sequence of bacterial n-glycosylation sites  <cit> .
furthermore, the crystal structure indicated that conserved residues d <dig>  r <dig>  d <dig>  d <dig>  e <dig>  r <dig> and r <dig> of pglbc.lari are ligands of the acceptor peptide and/or the metal cofactor.
amino acid changes at d <dig>  d <dig>  e <dig> of pglbc.lari lead to partial or complete loss of activity  <cit> .
the relevance of d <dig>  within the transmembrane domain of pglbcj had been demonstrated previously   <cit> .
a role of the miv motif in substrate binding is also supported by the pglbc.lari structure because the position of the side chain of i <dig>  is in agreement with a hydrophobic interaction with the methyl group of the + <dig> t residue of the acceptor peptide, presumably leading to enhanced affinity for d-x1-n-x2-t acceptor sequons compared to d-x1-n-x2-s as previously shown by chen et al.
<cit> .
from the full-length structure of pglbc.lari it was deduced that binding of acceptor proteins occurs from one side of the protein while undecaprenyl-pyrophosphate-linked oligosaccharide precursors access the active site from the other side  <cit> .
an elongated, flexible loop of the transmembrane domain containing the essential amino acid e <dig> was suggested to play a central role in catalysis  <cit> .
an in silico glycan docking experiment indicated that conserved y <dig>  may interact with the n-acetyl group of the innermost sugar of the transferred oligosaccharide  <cit> .
in this study we aimed at elucidating residues of the periplasmic domain of pglbcj which are important for the formation of engineered glycoconjugates.
therefore, we established methodologies for the construction and screening of ost variant libraries in e. coli.
two mutant libraries generated by either error-prone pcr mutagenesis of the periplasmic domain or cassette mutagenesis of specific active site residues were analyzed for inactive, but pglb polypeptide producing variants.
the obtained results were discussed in the context of the recently published crystal structure of the c. lari ost.
RESULTS
elisa detection of glycoconjugates
n-linked glycosylation in recombinant e. coli is a powerful method for obtaining conjugates of polysaccharide antigens and immunogenic carrier proteins that can be used as safe and efficient vaccines against bacterial pathogens.
a method for detection of glycoproteins in the periplasm of e.coli which is suitable for medium throughput screening has not yet been described, but is required for the analysis of pglb mutant libraries.
therefore, we developed an enzyme-linked immunosorbent assay  in 96-well format and compared the new analytical method to the commonly used western blot analysis.
in this study we used a plasmid combination facilitating the biosynthesis of conjugates of staphylococcus aureus capsular polysaccharides type  <dig>  and the carrier protein pseudomonas aeruginosa exotoxoid a   .
in strains co-expressing cp <dig> from plasmid pgvxn <dig> and epa with engineered glycosylation sites from plasmid pgvxn <dig>  while at the same time pglbcj was provided from the low copy number plasmid  pgvxn <dig>  epa-cp <dig> glycoconjugates were obtained.
a ladder of protein bands reacting with anti-cp <dig> antibodies was detected in periplasmic extracts of cells grown in shake flask by western blot analysis .
similar protein ladders have been observed previously when o-polysaccharide-protein conjugates were produced in recombinant e. coli <cit> .
random polysaccharide chain lengths result during o-antigen biosynthesis by the combined action of the enzymes wzy and wzz  <cit> .
an alternative medium copy number expression vector for pglbcj, pgvxn112kan  also enabled glycosylation of epa, even though glycoprotein levels were lower .
when periplasmic proteins of control strains harboring the same combinations of epa and cp <dig> producing plasmids and expressing mutated, inactive pglb  were analyzed, no specific bands were detected .
however, strong bands with the expected size of unglycosylated epa  were detected in periplasmic extracts of both wild-type pglb and mutated  pglb strains when blots were analyzed with anti-epa antibodies .
a ladder of proteins of a similar size range as the ladder observed in anti-cp <dig> blots was also seen for wild-type pglb samples on this blot .
anti-cp <dig> specific bands below the size of epa were most likely due to glycosylated degradation products of epa.
such degradation products were also observed when engineered epa was glycosylated with shigella o <dig> polysaccharides  <cit> .
next we tested whether glycosylation using this system can also be reliably detected by elisa, as this method could be used for the analysis of a larger number of clones.
the elisa setup with the best signal-to-noise ratio for periplamic extracts of e. coli comprised anti-epa capturing antibodies and anti-cp <dig> detection antibodies .
elisa absorbance values were reproducibly and significantly higher for samples from wild-type pglb strains compared to samples from isogenic control strains expressing inactive pglb .
background signals  were higher when the low copy number pglb expression plasmid was used than when the medium copy number, pact <dig> derived plasmid was used .
the latter plasmid was used for the construction of pglb mutant libraries.
in later experiments where mutants were re-analyzed in replicates, a second batch of anti-cp <dig> antibodies with a higher degree of purity was used, which lead to a reduction of background signals to ±  <dig>  , independent of the type of pglb expression plasmid.
the elisa results for shake flask cultures indicated that the method is suitable for detecting and quantifying glycoproteins in e. coli clones expressing different variants of pglb.
the coefficient of variation of elisa absorbance values was below 5% of the mean value for periplasmic extracts of strains expressing either wild-type or inactive pglb.
the lower levels of glycoproteins produced by the strain with medium copy number plasmid pgvxn112kan were reflected in lower values of elisa absorbance .
screening system for ost mutant libraries
based on the positive results with the sandwich elisa assay for shake flask samples, we developed a 96-well cultivation and screening method for glycoprotein production in e. coli.
screening of library clones was performed in 96-well format using deep well plates  for cultivation followed by sucrose-lysozyme extraction of periplasmic proteins and relative quantification of epa-cp <dig> glycoconjugates by sandwich elisa.
a test plate was inoculated with  <dig> clones of the epa-cp <dig> producing, wild-type pglb strain  and with  <dig> clones of the negative control strain expressing inactive pglb mut .
the elisa signals of all wells containing the wild-type pglb strain were clearly separated from those wells which contained the inactive control strain .
the average absorbance  was  <dig>   and  <dig>   for wild-type and inactive pglb, respectively.
the reproducibility and sensitivity of the dwp-elisa screening assay was considered sufficiently high for detecting inactivating mutations in pglb mutant libraries.
random mutagenesis of pglbcj by error-prone pcr
procedures for random mutagenesis of pglbcj were also developed in the course of this study.
the periplasmic domain  was randomly mutated by error-prone pcr  using mutagenic dna polymerase mutazyme ii.
the pglb plasmid library was transformed into the expression strain which co-expressed epa and cp <dig> polysaccharides.
on average  <dig>  amino acid changes were introduced within the periplasmic domain of pglb  as shown when  <dig> randomly picked clones were sequenced.
analysis of  <dig> clones with the dwp-elisa screening system showed that  <dig>  ±  <dig> %  of variants were inactive, i.e.
exhibited a background-corrected elisa signal which was < 20% of the average signal of wt control wells.
elisa data for a representative 96-well eppcr library are shown in figure  <dig> .
the signals of pglb mut control samples  always grouped with inactive clones on the right-hand part of sorted elisa data, while the wild-type controls  grouped with active clones .
sequencing of  <dig> inactive clones without prior analysis of pglb expression showed that 27% of the clones harbored mutations with expected negative effects on expression and/or activity of pglb, i.e., amino acid substitutions within the wwdyg motif , mutations to a stop codon , frame shift mutations , amino acid substitutions within the miv motif  and changes to potentially structure-disrupting proline residues .
the overall frequency of clones with one, two and ≥  <dig> amino acid changes was  <dig> %,  <dig> % and  <dig> %, respectively.
the proportion of wild-type pglb clones was  <dig> % while  <dig> % harbored silent mutations in the periplasmic domain.
for elucidation of structure-function relationships only inactivating mutations which do not abolish expression of pglb are of interest.
the vectors used for expression of pglb and construction of mutant libraries yield recombinant protein with a hemagglutinin tag epitope  fused to the c-terminal end .
several hundred further eppcr clones with inactive phentotype were screened for production of full-length pglb by anti-ha western blot analysis.
growth, induction and cell lysis was performed in 96-deep well plates.
for validation of the screening method, several clones with one amino acid change were compared to stop codon mutants as well as to wild-type and inactive control strains on one blot.
no strong anti-ha specific bands were detected for stop codon mutants and the host strain without pglb plasmid .
in the case of wild-type pglb and inactive pglb w458a-d459a , two strong bands were detected.
although the position of the upper band was below the expected  <dig> kda we assumed this band to represent full-length pglb.
due to specific procedures required for obtaining blots of membrane proteins with sufficient quality, i.e., denaturation at 60°c instead of 95°c, membrane proteins often do not migrate at similar positions as soluble marker proteins in sds-polyacrylamide gels  <cit> .
for the homologous membrane-bound ost of c. lari, which was expressed with a c-terminal ha tag, the anti-ha specific bands on western blots were also migrating at lower positions than the theoretical molecular mass .
the lower anti-ha specific bands on our blot may represent the c-terminal, globular domain of pglb generated by limited proteolysis during expression.
the theoretical molecular mass of the periplasmic domain including the c-terminal ha tag is  <dig> kda.
for quantification of relative expression levels of pglb by image analysis only the upper band was used.
pglb expression in eppcr variants with one amino acid change ranged from not detectable to wild-type levels .
when  <dig> inactive clones of eppcr libraries were screened by anti-ha western blot analysis, it was found that the majority  had lost the ability to produce any pglb, while  <dig>  exhibited weak expression and only  <dig>  produced pglb at similar levels as the wild-type strain.
all clones showing strong pglb-specific bands and  <dig> clones with weak pglb production were sequenced.
the glycosylation deficiency of the pglb positive clones was re-evaluated by dwp-elisa and could be confirmed in 63% of the cases .
in the rescreening experiment, the majority of the remaining pglb positive clones exhibited elisa signals between  <dig> to 50% of wild-type controls  while  <dig> % were identified as false negatives .
single amino acid substitutions leading to a reproducible lack of glycosylation  without abolishing production of pglb are summarized in table  <dig>  in several cases, the same residues like those hit in single mutants were found to be mutated in pglb-positive clones with two amino acid changes .
four inactivating amino acid substitutions were identified in two or more individual clones, indicating that the diversity of the eppcr mutant library was covered adequately.
the wwdyg motif was hit in  <dig> single mutant clones and in four clones with two amino acid changes .
a second region where inactivating mutations clustered was r <dig> to g <dig>  d <dig> and g <dig> were mutated in several clones with strong expression of pglb .
random mutagenesis of pglbcj by eppcr was restricted to the region encoding the periplasmic domain .
all clones reproducibly exhibited elisa signals for epa-cp <dig> which were ≤ 20% of the wild-type pglb control strain .
only results for clones which expressed detectable amounts of pglb in dwp are shown .
selected variant plasmids were isolated by mini-prep, re-transformed into the expression strain e. coli clm <dig>  and re-analyzed in triplicate with the dpw-elisa and anti-ha screening systems.
mutation v432f reduced background-corrected epa-cp <dig> signals by 89% without any negative effect on expression of pglb .
change of g <dig> to either s or n abolished formation of glycoconjugates completely in spite of wild-type levels of pglb detected by anti-ha western blot .
in addition to mutations y460n and g461d within the wwdyg motif, four other amino acid changes lead to a complete loss of glycosylation activity: r465h, d469l, d475f and d475y.
lack of glycosylation in these cases cannot be explained solely by reduced expression of the ost.
there were still substantial amounts of pglb-ha detectable by anti ha western blot .
in the case of variants a682d and a528v, the strongly reduced yields of epa-cp <dig> are most likely related to a concomitant reduction in pglb expression levels .
as a control, an eppcr variant with an amino acid substitution with no effect, y645c, was also analyzed within the same dwp-elisa assay.
for this clone, no significant difference in elisa signals and expression levels of pglb to the wild-type control was observed .
y <dig> resides in a non-conserved region of pglb distal to the active site.
d <dig> and g <dig> are highly conserved in bacterial pglb homologues .
in the crystal structure of pglbc.
lari the corresponding amino acids  are part of a flexible loop which is localized just above the amide nitrogen atom of the acceptor peptide where the covalent linkage to the reducing sugar of the undecyprenyl-linked oligosaccharide substrate occurs .
to further confirm the importance of this loop we changed the conserved residue h <dig> , which is located in only  <dig>  Å distance to the acceptor nitrogen atom , to glutamine, an amino acid which occurs in several distantly related pglbcj homologues .
for comparison, the adjacent, non-conserved residue l <dig>  with an upward orientation of the side chain in the c. lari pglb crystal structure was mutated to alanine .
the pglb h479q variant was expressed at wild-type levels, with anti-ha band intensities reaching 97 ± 23% of wild-type pglb .
however, the yield of glycoprotein was reduced more than five-fold as determined by sandwich elisa .
by contrast, we did not observe any significant difference between variant l480a and wild-type pglbcj in epa-cp <dig> and pglb levels .
in agreement with its relevance for activity, h <dig> was also found to be mutated in an inactive, pglb-positive eppcr clone with three amino acid changes .
the eppcr experiment did not yield any variants with reproducibly increased glycosylation efficiency; however, numerous variants with neutral single or double mutations were found, e.g., v470m, d705y and k603e-s664r all of which exhibited epa-cp <dig> elisa signals similar to wild-type pglb.
in contrast to residues mutated in inactive variants, only non-conserved residues were mutated in the latter cases.
cassette mutagenesis of i <dig> and v <dig>
opposite of the strictly conserved wwdyg motif of n-oligosaccharyltransferases, three conserved hydrophobic residues are found in bacterial enzymes which comprise the so-called “miv” motif  <cit>  .
m <dig> was hit once in the eppcr experiment, but no inactivating mutations were recovered for i <dig> or v <dig>  in order to elucidate the plasticity and structural requirements at these sites, random cassette mutagenesis was performed.
randomization was achieved with mutagenic primers bearing ndt degenerate codons  instead of wild-type att and gtg at positions i <dig> and v <dig>  respectively.
ndt mutagenesis introduces codons for l, i, v, f, y, g, s, c, d, n, h, and r at targeted positions in gene sequences and combines a high degree of chemical diversity with a relatively small library size  <cit> .
after confirming the success of ndt mutagenesis by sequencing of random clones, the pglb plasmid library was re-transformed in the expression strain e. coli clm <dig>  and screened with the dwp-elisa system.
sorted elisa data for a representative 96-well library are shown in figure  <dig> .
none of the library clones exhibited increased epa-cp <dig> elisa signals compared to the top signal from wild-type clones of the same 96-well screening plate.
the majority of clones was inactive .
only  <dig> out of  <dig> screened library clones produced signals within the range of wild-type control clones .
three of the active clones corresponded to the quick change template and two clones carried silent mutations introduced with the ndt primers, i.e.
control mutation f572: ttt > ttc and v575: gtg > gtt due to the ndt degenerate codon.
in the other active clones several single and double amino acid substitutions were identified which are listed in table  <dig>  one particular substitution, i571v, was found in three clones with one amino acid change and in two clones with two changes.
with the exception of i571c, hydrophobic side chains were replaced by other hydrophobic side chains in active variants .
in comparison to amino acids bearing hydroxyl, amine, amide or carboxyl functional groups in their side chains, cystein is non-polar/neutral.
elisa absorbance measured against air.
average values and standard deviations of at least n =  <dig> replicate mini-well cultures.
* significant difference to wt and mut, p value of student’s t-test <  <dig> .
the relative levels of epa-cp <dig> and pglb were analyzed for each type of pglb variant in replicate dwp mini-cultures after re-transformation of the sequenced variant plasmids .
no significant difference to wild-type pglb was found for variants i571v and i571v-v575i, respectively .
in the case of i571v -v575l, a moderate, but significant reduction of epa-cp <dig> biosynthesis was observed which was not due to reduced expression of pglb .
the level of epa-cp <dig> detected by elisa was reduced by a factor of three in variant i571f-v575l without any reduction of pglb expression, indicating that an f at position  <dig> has a negative effect on ost activity .
the double mutant i571c-v575i was characterized by a parallel, moderate reduction in the levels of both epa-cp <dig> and pglb .
the alignment of a set of diverse, non-redundant amino acid sequences of bacterial pglb homologues indicates that i <dig> and v <dig> are strictly conserved in ε-proteobacteria, but not in other species .
however, also in non-ε-proteobacteria mostly hydrophobic amino acids occur at the corresponding positions .
isoleucine and leucine at position pglbcj v <dig> were found in active variants of the cassette mutagenesis experiment and also occur in natural pglb sequences .
the spatial orientation of the equivalent residues i <dig> and v <dig> of pglbc.
lari towards the bound acceptor peptide is shown in figure 6b.
the aliphatic side chains of both amino acids are in close proximity to the methyl group of the threonine residue at the n +  <dig> position of the d-q-n-a-t-p-nitro phenylalanine acceptor peptide .
discussion
the use of bacterial ost in recombinant e. coli facilitates in vivo production of conjugates of polysaccharide antigens of pathogenic bacteria and immunogenic carrier proteins  <cit> .
recombinant e. coli equipped with the oligosaccharyltransferase pglb of c. jejuni is the most advanced system for the biotechnological production of n-linked conjugate vaccines.
in order to gain a better understanding of residues of pglbcj required for the glycosylation reaction we studied the effect of random mutations on the formation of glycoconjugates composed of pseudomonas aeruginosa exotoxoid and staphylococcus aureus capsular polysaccharide cp <dig>  therefore, we established methods for constructing eppcr mutant libraries of recombinant pglb and for the screening of glycoprotein-producing e. coli in 96-well format.
growth and induction of pglb library clones in deep well plates followed by sandwich elisa analysis of the periplasmic extracts allowed a reliable distinction of active and inactive ost variants.
the screening of an eppcr library where the periplasmic domain of pglbcj had been mutagenized facilitated the identification of previously unknown residues which are relevant for ost activity.
apart from confirming the importance of the well-known, strictly conserved w457wdyg <dig> residues of pglbcj, the eppcr experiment revealed that the region v <dig> to g <dig> immediately adjacent to this motif is also highly important .
in the partial crystal structure of pglbcj and the recently revealed full-length structure of pglbc.lari, these amino acids form a flexible loop in close proximity to the side chains of tryptophan and aspartic acid residues of the wwdyg motif  <cit> .
due to the spatial orientation of the bound acceptor peptide and the assumed reaction mechanism of pglb  <cit> , this loop is believed to interact with sugar moieties of the glycan substrate.
the highly conserved residue g <dig>  which was hit several times in inactive eppcr mutants, could function as a helix breaker before the ascending helix starting at k <dig>  thereby providing the loop d475-g <dig>  with sufficient flexibility for interaction with the oligo-/polysaccharide .
the inactivating mutations at g <dig> identified in our eppcr experiment presumably induce structural changes which disturb the interaction of loop residues such as h <dig> and d <dig> with the glycan substrate.
in most homologous sequences which do not bear a glycine at this position, a helix-breaking glycine or proline residue is present at an adjacent position .
our results further confirm the importance of the strictly conserved aspartic acid residue at position  <dig>  for activity of pglbcj as proposed by maita et al.
<cit> .
in a study using radio-labeled undecaprenyl-pyrophosphate linked oligosacharides of c. jejuni at limiting concentrations and a synthetic ac-d-q-n-a-t-p-nitro phenylalanine acceptor peptide in excess, mutations d475e and d475a decreased glycosylation rates of pglbcj by a factor of four and six, respectively  <cit> .
the negative effect of the mutations was strongest at low glycan substrate concentrations  <cit> .
together with the reduced activity of a k478a mutant reported by the same authors these results support the importance of the flexible loop d475-g <dig> and its probable involvement in glycan binding as suggested by our data.
judged from its distance to the acceptor nitrogen atom , the carboxylate group of d <dig> might be involved in a hydrogen bond network coordinating the c <dig> n-acetyl group of the innermost sugar of the transferred glycan.
similarly to eukaryotic ost, pglbcj is believed to require an n-acetyl sugar at the reducing end for efficient transfer of poly- and oligosaccharides to acceptor proteins  <cit> .
the side chain of the strictly conserved tyrosine residue at position  <dig>  is in close proximity to d <dig>  .
for c. lari y <dig> an interaction with the n-acetyl group of the innermost sugar has been suggested by others based on in silico glycan docking  <cit> .
the importance of the loop d475-g <dig> is further backed by the phenotype of the h479q variant generated in this study.
the side chain of the equivalent residue h <dig> in pglbc.lari is spatially very close to the nucleophilic amide nitrogen of the bound d-q-n-a-t-p-nitro phenylalanine acceptor peptide .
with few exceptions glutamine is the only other amino acid occurring at this position in bacterial pglb homologues .
the side chain of glutamine has a similar length as that of histidine and also contains an amino functionality.
in pglbcj glutamine seems to be able to substitute for the function of histidine at position  <dig> only to a limited degree.
interestingly, removal of the aliphatic side chain of the adjacent, non-conserved residue l <dig> by mutation to alanine had no effect on activity at all.
this is in agreement with the spatial orientation of the side chain of the equivalent residue l <dig> in pglbc.lari pointing away from the acceptor peptide .
in only one inactive clone with strong expression of pglb an amino acid change was found which mapped to a region outside of w <dig> to g <dig>  the mutation v432f strongly reduced the glycoprotein specific elisa signals, but the variant was not completely inactive as epa-cp <dig> formation was still detectable.
judged from the position of the equivalent residue v <dig> in the crystal structure of pglbc.lari, the bulkier and less flexible phenyl side chain in the variant enzyme might interfere with the binding of d-x1-n-x2-t/s acceptor peptides.
in particular, steric hindrance of the side chain of the glutamine residue at the − <dig> position in d-q-n-x-t/s sequons could play a role .
in the engineered epa used in this study as acceptor protein two glycosylation sites had been introduced in surface exposed loops, d-n-n-n-s and d-q-n-r-t. it would be interesting to study whether glycosylation of the d-q-n-r-t site is more affected than that of the other site and whether a change of − <dig> q to amino acids with shorter side chains could compensate for the negative effect of pglbcj v432f.
v <dig> is not strictly conserved in pglb homologues; however, in ε-proteobacteria a short “vf” motif is present at this position in most species .
the so-called miv motif of bacterial n-oligosaccharyltransferases  which is assumed to interact with the acceptor peptide was hit only once in the eppcr experiment.
the side chains of the equivalent residues i <dig> and v <dig> of pglbc.lari are very close to the side chains of the wwdyg motif in the crystal structure .
therefore, we specifically randomized these residues in pglbcj using degenerate primers and screened the resulting focused library.
our results indicate that only few other, chemically similar amino acids are tolerated at these positions.
wild-type isoleucine and valine at position  <dig> exhibited the same phenotype.
presumably, both side chains allow for sufficient hydrophobic interaction with the methyl group of + <dig> t of the acceptor sequon as suggested for pglbc.lari <cit> .
the strong preference for hydrophobic residues at i <dig> as well as v <dig> in active variants indicates that a hydrophobic surface promotes binding of the polypeptide chain of the acceptor protein.
in addition to i <dig> and v <dig>  numerous other surface-exposed amino acids of the acceptor peptide binding pocket are also hydrophobic in pglbc.lari: i <dig>  m <dig>  y <dig>  v <dig>  y <dig>  y <dig> and v <dig>  <cit> .
with the exception of m <dig> and v <dig> these residues are conserved in pglbcj .
an elongated hydrophobic patch containing the wwdyg motif in its center was suggested to interact with acceptor polypeptides in the archaeal n-oligosaccharyltransferase of pyrococcus furiosus <cit> .
directed evolution, comprising multiple rounds of random mutagenesis, randomization of active site residues and recombination of beneficial mutations, has proven to be a powerful method for tuning enzymes to specific needs, provided that suitable screening systems are available  <cit> .
theoretically, it should be possible to evolve bacterial osts for enhanced activity towards non-native polysaccharide substrates, although no such studies have yet been reported in the literature.
we did not detect any pglb variants with increased yields of glycoprotein in our libraries.
most likely, the number of screened eppcr clones was too low to find improved variants, which are expected to be very rare.
amino acid substitutions at the miv motif were at best neutral, which indicates that it is difficult to further optimize interactions of d-x1-n-x2-s/t acceptor peptides with pglb.
however, we cannot exclude the possibility that protein glycosylation in the applied e. coli system was limited by other factors than ost activity, e.g., biosynthesis of undecaprenyl-pyrophosphate linked polysaccharide precursors.
furthermore, the crystal structure of full-length pglbc.lari strongly suggests that residues of the membrane domain are also involved in the catalytic mechanism  <cit> .
it might well be that an increase in catalytic activity is not possible without simultaneous changes both in the periplasmic and membrane domains of pglbcj.
a general problem of enzyme engineering is the destabilizing effect of introduced mutations.
in the case of pglbcj this was reflected in a high proportion of amino acid substitutions having detrimental effects on production and stable maintenance of the pglb polypeptide.
as conserved positions of pglbcj such as i <dig> and v <dig> were found to be particularly sensitive to changes, future mutagenesis strategies should focus on non-conserved residues in the vicinity of the active site.
in addition, randomization at specific positions might be restricted to amino acids found in natural sequences as proposed by jochens and bornscheuer  <cit> , thereby reducing the number of unwanted instable variants and thus the screening effort.
CONCLUSIONS
in summary, this study adds important information to structure-function relationships of bacterial ost and provides methods for generating and screening ost mutant libraries in e. coli strains capable of producing engineered glycoproteins.

 n-linked glycosylation is of fundamental importance for the biological activity of many eukaryotic proteins. bacterial oligosaccharyltransferases perform a similar type of posttranslational modification in prokaryotic cells and thus hold great promise for producing proteins with defined glycosylation patterns in simple and cost-efficient expression systems such as escherichia coli. the ost of campylobacter jejuni has been functionally expressed in e. coli and has been shown to have a relaxed specificity towards its lipid-linked substrate. therefore it can be exploited, e.g., for the production of conjugate vaccines where polysaccharide antigens of bacterial pathogens are covalently coupled to carrier proteins. polysaccharide-protein conjugates belong to the most effective measures for combating life-threatening infectious diseases. diverse o-specific polysaccharides of the outer lipopolysaccharide layer of gram-negative bacteria can be efficiently coupled to carrier proteins containing a surface-exposed d/e-x1-n-x2-t/s acceptor sequon by pglbcj, provided that they contain an n-acetyl sugar at the reducing end. examples are the o o9a, o and o antigens of e. coli, the o antigen of shigella dysenteriae as well as the o antigen of pseudomonas aeruginosa. production of conjugate vaccines by in vivo conjugation in e. coli is much simpler than with conventional chemical conjugation technologies, and it has already been demonstrated that glycoconjugates can be produced at a larger scale in fed-batch culture with yields of 18– mg l- of purified product. recently, it was found that capsular polysaccharides of medically important gram-positive bacterial pathogens such as staphylococcus aureus can also be transferred to acceptor proteins by pglbcj. pglbcj is a monomeric protein of amino acids and comprises predicted transmembrane helixes in the n-terminal two thirds of the polypeptide and a globular domain at the c-terminus which is soluble and exposed to the periplasm. the latter domain of pglbcj was crystallized by maita et al.. it harbors the highly conserved w457wdyg motif which is also found in the stt subunit of eukaryotic ost. when w and d were both replaced by alanine residues, pglbcj was rendered inactive. the crystal structure of the soluble domain of pglbcj revealed a kinked helix opposite of the wwdyg motif which contained the conserved residues m i and v subsequently termed “miv” motif of bacterial ost. mutational studies confirmed the importance of i for activity. pglb of campylobacter lari, a bacterial ost with 56% amino acid identity to pglbcj, also glycosylates recombinant proteins in the periplasm when over-expressed in e. coli. the crystal structure of full-length pglbc.lari was elucidated recently. the structure contained a divalent metal co-factor as well as a bound synthetic d-q-n-a-t-p-nitro phenylalanine acceptor peptide. it revealed that both the transmembrane and the soluble periplasmic domains of pglbc.lari participate in substrate binding and catalysis. the strictly conserved residues w463-w464-d of pglbc.lari were shown to form a strong network of hydrogen bonds with the hydroxyl group of the threonine residue at position + relative to the glycosylated asparagine of the acceptor peptide. this explains the strict requirement for either s or t in the d/e-x1-n-x2-s/t consensus sequence of bacterial n-glycosylation sites. furthermore, the crystal structure indicated that conserved residues d r d d e r and r of pglbc.lari are ligands of the acceptor peptide and/or the metal cofactor. amino acid changes at d d e of pglbc.lari lead to partial or complete loss of activity. the relevance of d within the transmembrane domain of pglbcj had been demonstrated previously. a role of the miv motif in substrate binding is also supported by the pglbc.lari structure because the position of the side chain of i is in agreement with a hydrophobic interaction with the methyl group of the + t residue of the acceptor peptide, presumably leading to enhanced affinity for d-x1-n-x2-t acceptor sequons compared to d-x1-n-x2-s as previously shown by chen et al.. from the full-length structure of pglbc.lari it was deduced that binding of acceptor proteins occurs from one side of the protein while undecaprenyl-pyrophosphate-linked oligosaccharide precursors access the active site from the other side. an elongated, flexible loop of the transmembrane domain containing the essential amino acid e was suggested to play a central role in catalysis. an in silico glycan docking experiment indicated that conserved y may interact with the n-acetyl group of the innermost sugar of the transferred oligosaccharide. in this study we aimed at elucidating residues of the periplasmic domain of pglbcj which are important for the formation of engineered glycoconjugates. therefore, we established methodologies for the construction and screening of ost variant libraries in e. coli. two mutant libraries generated by either error-prone pcr mutagenesis of the periplasmic domain or cassette mutagenesis of specific active site residues were analyzed for inactive, but pglb polypeptide producing variants. the obtained results were discussed in the context of the recently published crystal structure of the c. lari ost.  elisa detection of glycoconjugates n-linked glycosylation in recombinant e. coli is a powerful method for obtaining conjugates of polysaccharide antigens and immunogenic carrier proteins that can be used as safe and efficient vaccines against bacterial pathogens. a method for detection of glycoproteins in the periplasm of e.coli which is suitable for medium throughput screening has not yet been described, but is required for the analysis of pglb mutant libraries. therefore, we developed an enzyme-linked immunosorbent assay in 96-well format and compared the new analytical method to the commonly used western blot analysis. in this study we used a plasmid combination facilitating the biosynthesis of conjugates of staphylococcus aureus capsular polysaccharides type and the carrier protein pseudomonas aeruginosa exotoxoid a. in strains co-expressing cp from plasmid pgvxn and epa with engineered glycosylation sites from plasmid pgvxn while at the same time pglbcj was provided from the low copy number plasmid pgvxn epa-cp glycoconjugates were obtained. a ladder of protein bands reacting with anti-cp antibodies was detected in periplasmic extracts of cells grown in shake flask by western blot analysis. similar protein ladders have been observed previously when o-polysaccharide-protein conjugates were produced in recombinant e. coli. random polysaccharide chain lengths result during o-antigen biosynthesis by the combined action of the enzymes wzy and wzz. an alternative medium copy number expression vector for pglbcj, pgvxn112kan also enabled glycosylation of epa, even though glycoprotein levels were lower. when periplasmic proteins of control strains harboring the same combinations of epa and cp producing plasmids and expressing mutated, inactive pglb were analyzed, no specific bands were detected. however, strong bands with the expected size of unglycosylated epa were detected in periplasmic extracts of both wild-type pglb and mutated pglb strains when blots were analyzed with anti-epa antibodies. a ladder of proteins of a similar size range as the ladder observed in anti-cp blots was also seen for wild-type pglb samples on this blot. anti-cp specific bands below the size of epa were most likely due to glycosylated degradation products of epa. such degradation products were also observed when engineered epa was glycosylated with shigella o polysaccharides. next we tested whether glycosylation using this system can also be reliably detected by elisa, as this method could be used for the analysis of a larger number of clones. the elisa setup with the best signal-to-noise ratio for periplamic extracts of e. coli comprised anti-epa capturing antibodies and anti-cp detection antibodies. elisa absorbance values were reproducibly and significantly higher for samples from wild-type pglb strains compared to samples from isogenic control strains expressing inactive pglb. background signals were higher when the low copy number pglb expression plasmid was used than when the medium copy number, pact derived plasmid was used. the latter plasmid was used for the construction of pglb mutant libraries. in later experiments where mutants were re-analyzed in replicates, a second batch of anti-cp antibodies with a higher degree of purity was used, which lead to a reduction of background signals to ± , independent of the type of pglb expression plasmid. the elisa results for shake flask cultures indicated that the method is suitable for detecting and quantifying glycoproteins in e. coli clones expressing different variants of pglb. the coefficient of variation of elisa absorbance values was below 5% of the mean value for periplasmic extracts of strains expressing either wild-type or inactive pglb. the lower levels of glycoproteins produced by the strain with medium copy number plasmid pgvxn112kan were reflected in lower values of elisa absorbance. screening system for ost mutant libraries based on the positive results with the sandwich elisa assay for shake flask samples, we developed a 96-well cultivation and screening method for glycoprotein production in e. coli. screening of library clones was performed in 96-well format using deep well plates for cultivation followed by sucrose-lysozyme extraction of periplasmic proteins and relative quantification of epa-cp glycoconjugates by sandwich elisa. a test plate was inoculated with clones of the epa-cp producing, wild-type pglb strain and with clones of the negative control strain expressing inactive pglb mut. the elisa signals of all wells containing the wild-type pglb strain were clearly separated from those wells which contained the inactive control strain. the average absorbance was and for wild-type and inactive pglb, respectively. the reproducibility and sensitivity of the dwp-elisa screening assay was considered sufficiently high for detecting inactivating mutations in pglb mutant libraries. random mutagenesis of pglbcj by error-prone pcr procedures for random mutagenesis of pglbcj were also developed in the course of this study. the periplasmic domain was randomly mutated by error-prone pcr using mutagenic dna polymerase mutazyme ii. the pglb plasmid library was transformed into the expression strain which co-expressed epa and cp polysaccharides. on average amino acid changes were introduced within the periplasmic domain of pglb as shown when randomly picked clones were sequenced. analysis of clones with the dwp-elisa screening system showed that  ±  % of variants were inactive, i.e. exhibited a background-corrected elisa signal which was < 20% of the average signal of wt control wells. elisa data for a representative 96-well eppcr library are shown in figure. the signals of pglb mut control samples always grouped with inactive clones on the right-hand part of sorted elisa data, while the wild-type controls grouped with active clones. sequencing of inactive clones without prior analysis of pglb expression showed that 27% of the clones harbored mutations with expected negative effects on expression and/or activity of pglb, i.e., amino acid substitutions within the wwdyg motif, mutations to a stop codon, frame shift mutations, amino acid substitutions within the miv motif and changes to potentially structure-disrupting proline residues. the overall frequency of clones with one, two and ≥  amino acid changes was %, % and %, respectively. the proportion of wild-type pglb clones was % while % harbored silent mutations in the periplasmic domain. for elucidation of structure-function relationships only inactivating mutations which do not abolish expression of pglb are of interest. the vectors used for expression of pglb and construction of mutant libraries yield recombinant protein with a hemagglutinin tag epitope fused to the c-terminal end. several hundred further eppcr clones with inactive phentotype were screened for production of full-length pglb by anti-ha western blot analysis. growth, induction and cell lysis was performed in 96-deep well plates. for validation of the screening method, several clones with one amino acid change were compared to stop codon mutants as well as to wild-type and inactive control strains on one blot. no strong anti-ha specific bands were detected for stop codon mutants and the host strain without pglb plasmid. in the case of wild-type pglb and inactive pglb w458a-d459a, two strong bands were detected. although the position of the upper band was below the expected kda we assumed this band to represent full-length pglb. due to specific procedures required for obtaining blots of membrane proteins with sufficient quality, i.e., denaturation at 60°c instead of 95°c, membrane proteins often do not migrate at similar positions as soluble marker proteins in sds-polyacrylamide gels. for the homologous membrane-bound ost of c. lari, which was expressed with a c-terminal ha tag, the anti-ha specific bands on western blots were also migrating at lower positions than the theoretical molecular mass. the lower anti-ha specific bands on our blot may represent the c-terminal, globular domain of pglb generated by limited proteolysis during expression. the theoretical molecular mass of the periplasmic domain including the c-terminal ha tag is kda. for quantification of relative expression levels of pglb by image analysis only the upper band was used. pglb expression in eppcr variants with one amino acid change ranged from not detectable to wild-type levels. when inactive clones of eppcr libraries were screened by anti-ha western blot analysis, it was found that the majority had lost the ability to produce any pglb, while exhibited weak expression and only produced pglb at similar levels as the wild-type strain. all clones showing strong pglb-specific bands and clones with weak pglb production were sequenced. the glycosylation deficiency of the pglb positive clones was re-evaluated by dwp-elisa and could be confirmed in 63% of the cases. in the rescreening experiment, the majority of the remaining pglb positive clones exhibited elisa signals between to 50% of wild-type controls while % were identified as false negatives. single amino acid substitutions leading to a reproducible lack of glycosylation without abolishing production of pglb are summarized in table in several cases, the same residues like those hit in single mutants were found to be mutated in pglb-positive clones with two amino acid changes. four inactivating amino acid substitutions were identified in two or more individual clones, indicating that the diversity of the eppcr mutant library was covered adequately. the wwdyg motif was hit in single mutant clones and in four clones with two amino acid changes. a second region where inactivating mutations clustered was r to g d and g were mutated in several clones with strong expression of pglb. random mutagenesis of pglbcj by eppcr was restricted to the region encoding the periplasmic domain. all clones reproducibly exhibited elisa signals for epa-cp which were ≤ 20% of the wild-type pglb control strain. only results for clones which expressed detectable amounts of pglb in dwp are shown. selected variant plasmids were isolated by mini-prep, re-transformed into the expression strain e. coli clm and re-analyzed in triplicate with the dpw-elisa and anti-ha screening systems. mutation v432f reduced background-corrected epa-cp signals by 89% without any negative effect on expression of pglb. change of g to either s or n abolished formation of glycoconjugates completely in spite of wild-type levels of pglb detected by anti-ha western blot. in addition to mutations y460n and g461d within the wwdyg motif, four other amino acid changes lead to a complete loss of glycosylation activity: r465h, d469l, d475f and d475y. lack of glycosylation in these cases cannot be explained solely by reduced expression of the ost. there were still substantial amounts of pglb-ha detectable by anti ha western blot. in the case of variants a682d and a528v, the strongly reduced yields of epa-cp are most likely related to a concomitant reduction in pglb expression levels. as a control, an eppcr variant with an amino acid substitution with no effect, y645c, was also analyzed within the same dwp-elisa assay. for this clone, no significant difference in elisa signals and expression levels of pglb to the wild-type control was observed. y resides in a non-conserved region of pglb distal to the active site. d and g are highly conserved in bacterial pglb homologues. in the crystal structure of pglbc. lari the corresponding amino acids are part of a flexible loop which is localized just above the amide nitrogen atom of the acceptor peptide where the covalent linkage to the reducing sugar of the undecyprenyl-linked oligosaccharide substrate occurs. to further confirm the importance of this loop we changed the conserved residue h, which is located in only Å distance to the acceptor nitrogen atom, to glutamine, an amino acid which occurs in several distantly related pglbcj homologues. for comparison, the adjacent, non-conserved residue l with an upward orientation of the side chain in the c. lari pglb crystal structure was mutated to alanine. the pglb h479q variant was expressed at wild-type levels, with anti-ha band intensities reaching 97 ± 23% of wild-type pglb. however, the yield of glycoprotein was reduced more than five-fold as determined by sandwich elisa. by contrast, we did not observe any significant difference between variant l480a and wild-type pglbcj in epa-cp and pglb levels. in agreement with its relevance for activity, h was also found to be mutated in an inactive, pglb-positive eppcr clone with three amino acid changes. the eppcr experiment did not yield any variants with reproducibly increased glycosylation efficiency; however, numerous variants with neutral single or double mutations were found, e.g., v470m, d705y and k603e-s664r all of which exhibited epa-cp elisa signals similar to wild-type pglb. in contrast to residues mutated in inactive variants, only non-conserved residues were mutated in the latter cases. cassette mutagenesis of i and v opposite of the strictly conserved wwdyg motif of n-oligosaccharyltransferases, three conserved hydrophobic residues are found in bacterial enzymes which comprise the so-called “miv” motif. m was hit once in the eppcr experiment, but no inactivating mutations were recovered for i or v in order to elucidate the plasticity and structural requirements at these sites, random cassette mutagenesis was performed. randomization was achieved with mutagenic primers bearing ndt degenerate codons instead of wild-type att and gtg at positions i and v respectively. ndt mutagenesis introduces codons for l, i, v, f, y, g, s, c, d, n, h, and r at targeted positions in gene sequences and combines a high degree of chemical diversity with a relatively small library size. after confirming the success of ndt mutagenesis by sequencing of random clones, the pglb plasmid library was re-transformed in the expression strain e. coli clm and screened with the dwp-elisa system. sorted elisa data for a representative 96-well library are shown in figure. none of the library clones exhibited increased epa-cp elisa signals compared to the top signal from wild-type clones of the same 96-well screening plate. the majority of clones was inactive. only out of screened library clones produced signals within the range of wild-type control clones. three of the active clones corresponded to the quick change template and two clones carried silent mutations introduced with the ndt primers, i.e. control mutation f572: ttt > ttc and v575: gtg > gtt due to the ndt degenerate codon. in the other active clones several single and double amino acid substitutions were identified which are listed in table one particular substitution, i571v, was found in three clones with one amino acid change and in two clones with two changes. with the exception of i571c, hydrophobic side chains were replaced by other hydrophobic side chains in active variants. in comparison to amino acids bearing hydroxyl, amine, amide or carboxyl functional groups in their side chains, cystein is non-polar/neutral. elisa absorbance measured against air. average values and standard deviations of at least n =  replicate mini-well cultures. * significant difference to wt and mut, p value of student’s t-test < . the relative levels of epa-cp and pglb were analyzed for each type of pglb variant in replicate dwp mini-cultures after re-transformation of the sequenced variant plasmids. no significant difference to wild-type pglb was found for variants i571v and i571v-v575i, respectively. in the case of i571v -v575l, a moderate, but significant reduction of epa-cp biosynthesis was observed which was not due to reduced expression of pglb. the level of epa-cp detected by elisa was reduced by a factor of three in variant i571f-v575l without any reduction of pglb expression, indicating that an f at position has a negative effect on ost activity. the double mutant i571c-v575i was characterized by a parallel, moderate reduction in the levels of both epa-cp and pglb. the alignment of a set of diverse, non-redundant amino acid sequences of bacterial pglb homologues indicates that i and v are strictly conserved in ε-proteobacteria, but not in other species. however, also in non-ε-proteobacteria mostly hydrophobic amino acids occur at the corresponding positions. isoleucine and leucine at position pglbcj v were found in active variants of the cassette mutagenesis experiment and also occur in natural pglb sequences. the spatial orientation of the equivalent residues i and v of pglbc. lari towards the bound acceptor peptide is shown in figure 6b. the aliphatic side chains of both amino acids are in close proximity to the methyl group of the threonine residue at the n +  position of the d-q-n-a-t-p-nitro phenylalanine acceptor peptide. discussion the use of bacterial ost in recombinant e. coli facilitates in vivo production of conjugates of polysaccharide antigens of pathogenic bacteria and immunogenic carrier proteins. recombinant e. coli equipped with the oligosaccharyltransferase pglb of c. jejuni is the most advanced system for the biotechnological production of n-linked conjugate vaccines. in order to gain a better understanding of residues of pglbcj required for the glycosylation reaction we studied the effect of random mutations on the formation of glycoconjugates composed of pseudomonas aeruginosa exotoxoid and staphylococcus aureus capsular polysaccharide cp therefore, we established methods for constructing eppcr mutant libraries of recombinant pglb and for the screening of glycoprotein-producing e. coli in 96-well format. growth and induction of pglb library clones in deep well plates followed by sandwich elisa analysis of the periplasmic extracts allowed a reliable distinction of active and inactive ost variants. the screening of an eppcr library where the periplasmic domain of pglbcj had been mutagenized facilitated the identification of previously unknown residues which are relevant for ost activity. apart from confirming the importance of the well-known, strictly conserved w457wdyg residues of pglbcj, the eppcr experiment revealed that the region v to g immediately adjacent to this motif is also highly important. in the partial crystal structure of pglbcj and the recently revealed full-length structure of pglbc.lari, these amino acids form a flexible loop in close proximity to the side chains of tryptophan and aspartic acid residues of the wwdyg motif. due to the spatial orientation of the bound acceptor peptide and the assumed reaction mechanism of pglb, this loop is believed to interact with sugar moieties of the glycan substrate. the highly conserved residue g which was hit several times in inactive eppcr mutants, could function as a helix breaker before the ascending helix starting at k thereby providing the loop d475-g with sufficient flexibility for interaction with the oligo-/polysaccharide. the inactivating mutations at g identified in our eppcr experiment presumably induce structural changes which disturb the interaction of loop residues such as h and d with the glycan substrate. in most homologous sequences which do not bear a glycine at this position, a helix-breaking glycine or proline residue is present at an adjacent position. our results further confirm the importance of the strictly conserved aspartic acid residue at position for activity of pglbcj as proposed by maita et al.. in a study using radio-labeled undecaprenyl-pyrophosphate linked oligosacharides of c. jejuni at limiting concentrations and a synthetic ac-d-q-n-a-t-p-nitro phenylalanine acceptor peptide in excess, mutations d475e and d475a decreased glycosylation rates of pglbcj by a factor of four and six, respectively. the negative effect of the mutations was strongest at low glycan substrate concentrations. together with the reduced activity of a k478a mutant reported by the same authors these results support the importance of the flexible loop d475-g and its probable involvement in glycan binding as suggested by our data. judged from its distance to the acceptor nitrogen atom, the carboxylate group of d might be involved in a hydrogen bond network coordinating the c n-acetyl group of the innermost sugar of the transferred glycan. similarly to eukaryotic ost, pglbcj is believed to require an n-acetyl sugar at the reducing end for efficient transfer of poly- and oligosaccharides to acceptor proteins. the side chain of the strictly conserved tyrosine residue at position is in close proximity to d. for c. lari y an interaction with the n-acetyl group of the innermost sugar has been suggested by others based on in silico glycan docking. the importance of the loop d475-g is further backed by the phenotype of the h479q variant generated in this study. the side chain of the equivalent residue h in pglbc.lari is spatially very close to the nucleophilic amide nitrogen of the bound d-q-n-a-t-p-nitro phenylalanine acceptor peptide. with few exceptions glutamine is the only other amino acid occurring at this position in bacterial pglb homologues. the side chain of glutamine has a similar length as that of histidine and also contains an amino functionality. in pglbcj glutamine seems to be able to substitute for the function of histidine at position only to a limited degree. interestingly, removal of the aliphatic side chain of the adjacent, non-conserved residue l by mutation to alanine had no effect on activity at all. this is in agreement with the spatial orientation of the side chain of the equivalent residue l in pglbc.lari pointing away from the acceptor peptide. in only one inactive clone with strong expression of pglb an amino acid change was found which mapped to a region outside of w to g the mutation v432f strongly reduced the glycoprotein specific elisa signals, but the variant was not completely inactive as epa-cp formation was still detectable. judged from the position of the equivalent residue v in the crystal structure of pglbc.lari, the bulkier and less flexible phenyl side chain in the variant enzyme might interfere with the binding of d-x1-n-x2-t/s acceptor peptides. in particular, steric hindrance of the side chain of the glutamine residue at the − position in d-q-n-x-t/s sequons could play a role. in the engineered epa used in this study as acceptor protein two glycosylation sites had been introduced in surface exposed loops, d-n-n-n-s and d-q-n-r-t. it would be interesting to study whether glycosylation of the d-q-n-r-t site is more affected than that of the other site and whether a change of − q to amino acids with shorter side chains could compensate for the negative effect of pglbcj v432f. v is not strictly conserved in pglb homologues; however, in ε-proteobacteria a short “vf” motif is present at this position in most species. the so-called miv motif of bacterial n-oligosaccharyltransferases which is assumed to interact with the acceptor peptide was hit only once in the eppcr experiment. the side chains of the equivalent residues i and v of pglbc.lari are very close to the side chains of the wwdyg motif in the crystal structure. therefore, we specifically randomized these residues in pglbcj using degenerate primers and screened the resulting focused library. our results indicate that only few other, chemically similar amino acids are tolerated at these positions. wild-type isoleucine and valine at position exhibited the same phenotype. presumably, both side chains allow for sufficient hydrophobic interaction with the methyl group of + t of the acceptor sequon as suggested for pglbc.lari. the strong preference for hydrophobic residues at i as well as v in active variants indicates that a hydrophobic surface promotes binding of the polypeptide chain of the acceptor protein. in addition to i and v numerous other surface-exposed amino acids of the acceptor peptide binding pocket are also hydrophobic in pglbc.lari: i m y v y y and v. with the exception of m and v these residues are conserved in pglbcj. an elongated hydrophobic patch containing the wwdyg motif in its center was suggested to interact with acceptor polypeptides in the archaeal n-oligosaccharyltransferase of pyrococcus furiosus. directed evolution, comprising multiple rounds of random mutagenesis, randomization of active site residues and recombination of beneficial mutations, has proven to be a powerful method for tuning enzymes to specific needs, provided that suitable screening systems are available. theoretically, it should be possible to evolve bacterial osts for enhanced activity towards non-native polysaccharide substrates, although no such studies have yet been reported in the literature. we did not detect any pglb variants with increased yields of glycoprotein in our libraries. most likely, the number of screened eppcr clones was too low to find improved variants, which are expected to be very rare. amino acid substitutions at the miv motif were at best neutral, which indicates that it is difficult to further optimize interactions of d-x1-n-x2-s/t acceptor peptides with pglb. however, we cannot exclude the possibility that protein glycosylation in the applied e. coli system was limited by other factors than ost activity, e.g., biosynthesis of undecaprenyl-pyrophosphate linked polysaccharide precursors. furthermore, the crystal structure of full-length pglbc.lari strongly suggests that residues of the membrane domain are also involved in the catalytic mechanism. it might well be that an increase in catalytic activity is not possible without simultaneous changes both in the periplasmic and membrane domains of pglbcj. a general problem of enzyme engineering is the destabilizing effect of introduced mutations. in the case of pglbcj this was reflected in a high proportion of amino acid substitutions having detrimental effects on production and stable maintenance of the pglb polypeptide. as conserved positions of pglbcj such as i and v were found to be particularly sensitive to changes, future mutagenesis strategies should focus on non-conserved residues in the vicinity of the active site. in addition, randomization at specific positions might be restricted to amino acids found in natural sequences as proposed by jochens and bornscheuer, thereby reducing the number of unwanted instable variants and thus the screening effort.  in summary, this study adds important information to structure-function relationships of bacterial ost and provides methods for generating and screening ost mutant libraries in e. coli strains capable of producing engineered glycoproteins. 
in addition to inactive variants with amino acid changes within the previously known, strictly conserved wwdyg motif of n-oligosaccharyltransferases, inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme c. lari pglb. this study provides novel structure-function relationships for the periplasmic domain of the campylobacter jejuni n-oligosaccharyltransferase pglb and describes procedures for generating and screening oligosaccharyltransferase mutant libraries in an engineered e. coli system. protein glycosylation is of fundamental importance in many biological systems. a particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins. we investigated which amino acids of the periplasmic domain of pglb are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. in addition, we investigated functional requirements in the so-called miv motif of bacterial n-oligosaccharyltransferases. the discovery of n-glycosylation in bacteria and the functional expression of the n-oligosaccharyltransferase pglb of campylobacter jejuni in escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. amino acid residues i and v which had been postulated to interact with the acceptor peptide, were subjected to cassette saturation mutagenesis. a random mutant library was generated by error-prone pcr and screened for inactivating amino acid substitutions. with the exception of i571c only hydrophobic residues were found in active variants.
 protein glycosylation is of fundamental importance in many biological systems. the discovery of n-glycosylation in bacteria and the functional expression of the n-oligosaccharyltransferase pglb of campylobacter jejuni in escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. a particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins.  in this study capsular polysaccharides of the clinically relevant pathogen staphylococcus aureus serotype were expressed in escherichia coli and linked in vivo to a detoxified version of pseudomonas aeruginosa exotoxin. we investigated which amino acids of the periplasmic domain of pglb are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. a random mutant library was generated by error-prone pcr and screened for inactivating amino acid substitutions. in addition to inactive variants with amino acid changes within the previously known, strictly conserved wwdyg motif of n-oligosaccharyltransferases, inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme c. lari pglb. the importance of the conserved loop residue h for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved l had no effect. in addition, we investigated functional requirements in the so-called miv motif of bacterial n-oligosaccharyltransferases. amino acid residues i and v which had been postulated to interact with the acceptor peptide, were subjected to cassette saturation mutagenesis. with the exception of i571c only hydrophobic residues were found in active variants. variant i571v performed equally well as the wild type, cysteine at the same position reduced glycoprotein yield slightly, while a change to phenylalanine reduced activity by a factor of three.  this study provides novel structure-function relationships for the periplasmic domain of the campylobacter jejuni n-oligosaccharyltransferase pglb and describes procedures for generating and screening oligosaccharyltransferase mutant libraries in an engineered e. coli system. glycosylationoligosaccharyltransferasepglbcampylobacter jejunirandom mutagenesisscreeningelisadirected evolutionconjugate vaccine 