Structural insights from random mutagenesis of <it>Campylobacter jejuni</it> oligosaccharyltransferase PglB
<p>Abstract</p> <p>Background</p> <p>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 <it>Campylobacter jejuni</i...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2012-09-01
|
| Series: | BMC Biotechnology |
| Subjects: | |
| Online Access: | http://www.biomedcentral.com/1472-6750/12/67 |
| _version_ | 1818081166051246080 |
|---|---|
| author | Ihssen Julian Kowarik Michael Wiesli Luzia Reiss Renate Wacker Michael Thöny-Meyer Linda |
| author_facet | Ihssen Julian Kowarik Michael Wiesli Luzia Reiss Renate Wacker Michael Thöny-Meyer Linda |
| author_sort | Ihssen Julian |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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 <it>Campylobacter jejuni</it> in <it>Escherichia coli</it> 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.</p> <p>Results</p> <p>In this study capsular polysaccharides of the clinically relevant pathogen <it>Staphylococcus aureus</it> serotype 5 (CP5) were expressed in <it>Escherichia coli</it> and linked <it>in vivo</it> to a detoxified version of <it>Pseudomonas aeruginosa</it> exotoxin (EPA). 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 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 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 <it>C. lari</it> PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, 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.</p> <p>Conclusions</p> <p>This study provides novel structure-function relationships for the periplasmic domain of the <it>Campylobacter jejuni</it> N-oligosaccharyltransferase PglB and describes procedures for generating and screening oligosaccharyltransferase mutant libraries in an engineered <it>E. coli</it> system.</p> |
| first_indexed | 2024-12-10T19:01:53Z |
| format | Article |
| id | doaj.art-7b762831e1594418a0547138273cff37 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-10T19:01:53Z |
| publishDate | 2012-09-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-7b762831e1594418a0547138273cff372022-12-22T01:36:59ZengBMCBMC Biotechnology1472-67502012-09-011216710.1186/1472-6750-12-67Structural insights from random mutagenesis of <it>Campylobacter jejuni</it> oligosaccharyltransferase PglBIhssen JulianKowarik MichaelWiesli LuziaReiss RenateWacker MichaelThöny-Meyer Linda<p>Abstract</p> <p>Background</p> <p>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 <it>Campylobacter jejuni</it> in <it>Escherichia coli</it> 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.</p> <p>Results</p> <p>In this study capsular polysaccharides of the clinically relevant pathogen <it>Staphylococcus aureus</it> serotype 5 (CP5) were expressed in <it>Escherichia coli</it> and linked <it>in vivo</it> to a detoxified version of <it>Pseudomonas aeruginosa</it> exotoxin (EPA). 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 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 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 <it>C. lari</it> PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, 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.</p> <p>Conclusions</p> <p>This study provides novel structure-function relationships for the periplasmic domain of the <it>Campylobacter jejuni</it> N-oligosaccharyltransferase PglB and describes procedures for generating and screening oligosaccharyltransferase mutant libraries in an engineered <it>E. coli</it> system.</p>http://www.biomedcentral.com/1472-6750/12/67glycosylationoligosaccharyltransferasePglB<it>Campylobacter jejuni</it>random mutagenesisscreeningELISAdirected evolutionconjugate vaccine |
| spellingShingle | Ihssen Julian Kowarik Michael Wiesli Luzia Reiss Renate Wacker Michael Thöny-Meyer Linda Structural insights from random mutagenesis of <it>Campylobacter jejuni</it> oligosaccharyltransferase PglB BMC Biotechnology glycosylation oligosaccharyltransferase PglB <it>Campylobacter jejuni</it> random mutagenesis screening ELISA directed evolution conjugate vaccine |
| title | Structural insights from random mutagenesis of <it>Campylobacter jejuni</it> oligosaccharyltransferase PglB |
| title_full | Structural insights from random mutagenesis of <it>Campylobacter jejuni</it> oligosaccharyltransferase PglB |
| title_fullStr | Structural insights from random mutagenesis of <it>Campylobacter jejuni</it> oligosaccharyltransferase PglB |
| title_full_unstemmed | Structural insights from random mutagenesis of <it>Campylobacter jejuni</it> oligosaccharyltransferase PglB |
| title_short | Structural insights from random mutagenesis of <it>Campylobacter jejuni</it> oligosaccharyltransferase PglB |
| title_sort | structural insights from random mutagenesis of it campylobacter jejuni it oligosaccharyltransferase pglb |
| topic | glycosylation oligosaccharyltransferase PglB <it>Campylobacter jejuni</it> random mutagenesis screening ELISA directed evolution conjugate vaccine |
| url | http://www.biomedcentral.com/1472-6750/12/67 |
| work_keys_str_mv | AT ihssenjulian structuralinsightsfromrandommutagenesisofitcampylobacterjejuniitoligosaccharyltransferasepglb AT kowarikmichael structuralinsightsfromrandommutagenesisofitcampylobacterjejuniitoligosaccharyltransferasepglb AT wiesliluzia structuralinsightsfromrandommutagenesisofitcampylobacterjejuniitoligosaccharyltransferasepglb AT reissrenate structuralinsightsfromrandommutagenesisofitcampylobacterjejuniitoligosaccharyltransferasepglb AT wackermichael structuralinsightsfromrandommutagenesisofitcampylobacterjejuniitoligosaccharyltransferasepglb AT thonymeyerlinda structuralinsightsfromrandommutagenesisofitcampylobacterjejuniitoligosaccharyltransferasepglb |