High level in vivo mucin-type glycosylation in Escherichia coli
Open accessAbstract
BACKGROUND: Increasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins. Since a considerable part of therapeutic proteins are glycoproteins, the lack of the post-translational attachment of sugar moieties in standard E. coli expression strains represents a major caveat, thus limiting the use of E. coli based cell factories. The establishment of an E. coli expression system capable of protein glycosylation could potentially facilitate the production of therapeutics with a putative concomitant reduction of production costs. RESULTS: The previously established E. coli strain expressing the soluble form of the functional human-derived glycosyltransferase polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) was further modified by co-expressing the UDP-GlcNAc 4-epimerase WbgU derived from Plesiomonas shigelloides. This enables the conversion of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm. Initially, the codon-optimised gene wbgU was inserted into a pET-derived vector and a Tobacco Etch Virus (TEV) protease cleavable polyhistidine-tag was translationally fused to the C- terminus of the amino acid sequence. The 4-epimerase was subsequently expressed and purified. Following the removal of the polyhistidine-tag, WbgU was analysed by circular dichroism spectroscopy to determine folding state and thermal transitions of the protein. The in vitro activity of WbgU was validated by employing a modified glycosyltransferase assay. The conversion of UDP-GlcNAc to UDP-GalNAc was shown by capillary electrophoresis analysis. Using a previously established chaperone pre-/co- expression platform, the in vivo activity of both glycosyltransferase GalNAc-T2 and 4-epimerase WbgU was assessed in E. coli, in combination with a mucin 10-derived target protein. Monitoring glycosylation by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), the results clearly indicated the in vivo glycosylation of the mucin-derived acceptor peptide. CONCLUSION: In the present work, the previously established E. coli- based expression system was further optimized and the potential for in vivo O-glycosylation was shown by demonstrating the transfer of sugar moieties to a mucin-derived acceptor protein. The results offer the possibility to assess the practical use of the described expression platform for in vivo glycosylations of important biopharmaceutical compounds in E. coli.
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Written from the paper’s full text.
TL;DR
The authors engineered *E. coli* SHuffle® T7 Express to co-express the human glycosyltransferase GalNAc-T2, the UDP-GlcNAc 4-epimerase WbgU, and a MUC10-derived target protein, enabling in vivo mucin-type O-glycosylation. LC–MS analysis showed up to 24 GalNAc moieties attached to the truncated target protein, demonstrating high-level in vivo O-glycosylation in *E. coli*.
Key findings
- Co-expression of GalNAc-T2, WbgU, and T7Muc10 in *E. coli* SHuffle® T7 Express with chaperones Erv1p/PDI produced in vivo O-glycosylation; intact mass analysis detected up to 24 attached GalNAc moieties, each adding 203.2 Da, increasing the average mass by roughly 4877 Da.
- The N-terminally truncated T7Muc10 (amino acids 21–159) had an average molecular mass of 14,230 Da instead of the expected 16,531 Da, and up to 24 of its 57 potential O-glycosylation sites were glycosylated.
- Glycosylation sites were identified in the EA2 repeat at positions 3, 7, 11, and 12, and in tandem EA2 at positions 2, 3, 6, 7, 11, and 12; EAN modifications were found at positions 2, 6, 7, and 11, plus a single modified serine.
- WbgU was expressed and purified (41 kDa WbgUTEV6H; tag-free WbgU at 40 kDa), showed α-helical CD minima at 208 and 222 nm, and had thermal transition points of 42.55 ± 0.04 °C, increasing to 43.07 ± 0.04 °C and 51.29 ± 0.13 °C in the presence of UDP-GlcNAc and UDP-GalNAc, respectively.
- In vitro, WbgU converted UDP-GlcNAc to UDP-GalNAc: at 0.15 mM substrates the ratio was 30:70 UDP-GalNAc:UDP-GlcNAc; at 1.5 mM, about 30% of UDP-GlcNAc and 57% of UDP-GalNAc were converted. Adding WbgU with UDP-GalNAc increased specific activity of rhGalNAc-T2 and HisDapGalNAc-T2 by about 26% and 40%, respectively.
Methods
- *E. coli* SHuffle® T7 Express strains carrying pMJS9 (Erv1p/PDI chaperones) and pET-derived plasmids were used to express WbgU, GalNAc-T2, and T7Muc10, a MUC10-derived target containing six EA2 repeats, two EAN repeats, and 60 putative glycosylation sites (49 threonines, 11 serines).
- WbgU was purified by Ni–NTA affinity chromatography, TEV protease cleavage, and a second affinity step; characterization included SDS-PAGE, immunoblot, N-terminal Edman sequencing, circular dichroism spectroscopy, and capillary electrophoresis for substrate conversion.
- Glycosyltransferase activity was measured with a modified malachite-green phosphate release assay using EA2 peptide acceptor and either UDP-GalNAc or UDP-GlcNAc with WbgU. In vivo glycosylation was analyzed by LC–ESI–MS/MS after trypsin digestion, by UPLC-ESI-QTOF-MS for intact mass, and by HPLC-ESI-LTQ-OT-MS/MS after Asp-N digestion.
Limitations
- Stated: comparison of glycosyltransferase activities with or without WbgU is difficult because the ongoing epimerization reaction makes the UDP-GlcNAc:UDP-GalNAc ratio during the assay unknown.
- Stated: whether the N-terminal truncation of T7Muc10 was due to cleavage by proteases in the *E. coli* cytoplasm during expression or proteolytic degradation during purification remains unclear.
- Evident from the text: the work demonstrates glycosylation of an artificial MUC10-derived target protein rather than a full-length biopharmaceutical, and the authors note that the platform’s value for pharmaceutically relevant targets remains to be verified.
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Claims the paper makes
22 of 46 cited sources checked · 17 supported · 0 flagged · accuracy not yet measured
- Findingown data
Significantly higher activities were detected for rhGalNAc-T2 compared to HisDapGalNAc-T2.
“In general, significantly higher activities were detected for rhGalNAc-T2 as compared to HisDapGalNAc-T2 (Fig. 3b), thus confirming previously published results [6].”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [6] Lauber J, Handrick R, Leptihn S, Durre P, Gaisser S. Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli. Microb Cell Fact. 2015;14:3. source
The Results (Analysis of HisDapGalNAcT2 enzymatic activity; page unavailable) report higher specific activity for rhGalNAcT2 with both tested substrates, at approximately twice that of HisDapGalNAcT2. The paper does not report a statistical significance test for these differences, so the word “significantly” is not established if intended in its statistical sense. (LLM, conf=0.82)
Experimental — accuracy not yet measured
- Premisecitation
An active human GalNAcT2 derivative comprising amino acids 52-571 with a translationally fused N-terminal HisDap tag has been isolated from a SHuffle® T7 E. coli strain pre-/co-expressing chaperones.
“As a first step to establish an E. coli-based expression platform enabling the glycosylation of full-length biopharmaceuticals in vivo, an active human GalNAcT2 derivative comprising amino acids 52–571 with a translationally fused N-terminal HisDap (diamino peptidase) tag has been isolated from a SHuffle® T7 E. coli strain pre-/co-expressing chaperones [6].”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [6] Lauber J, Handrick R, Leptihn S, Durre P, Gaisser S. Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli. Microb Cell Fact. 2015;14:3. source
- Findingown data
The in vitro activity of WbgU in combination with GalNAc-T2 was demonstrated using a modified glycosyltransferase assay.
“The in vitro activity of WbgU in combination with GalNAc-T2 was demonstrated using a modified glycosyltransferase assay.”
Quote in the paperSubstantial claim - Findingown data
WbgU converts UDP-GlcNAc to UDP-GalNAc in vitro.
“The conversion of UDP-GlcNAc to UDP-GalNAc was shown by capillary electrophoresis analysis.”
Quote in the paperSubstantial claim - Findingown data
WbgU expressed in E. coli SHuffle T7 Express is functional.
“These results indicate the successful expression of functional WbgU of P. shigelloides in E. coli SHuffle®T7 Express.”
Quote in the paperSubstantial claim
- Findingown data
Intact mass analysis revealed an average molecular mass of T7Muc10 of 14,230 Da instead of the expected 16,531 Da.
“The intact mass analysis revealed an average molecular mass of T7Muc10 of 14,230 Da, instead of the expected 16,531 Da calculated from its theoretical amino acid sequence (Fig. 7a).”
Quote in the paperSubstantial claim - Findingown data
Soluble expression of WbgUTEV6H (41 kDa) was best in Terrific Broth medium without IPTG induction and with pre-/co-expression of Erv1p and PDI.
“Soluble expression of WbgUTEV6H (41 kDa) was found to be best in Terrific Broth (TB) medium without Isopropyl-β-d-thiogalactopyranosid (IPTG) induction and with the pre-/co-expression of the redox folding helper proteins Erv1p and PDI encoded by pMJS9 under the control of an arabinose promotor (data not shown).”
Quote in the paperNot a substantial claim - Findingown data
A single modified serine residue was identified at position 5 of the tandem EA2 sequence, adjacent to the asparagine-glycine linker.
“Interestingly, a single modified serine residue was also identified. This was detected at position 5 of the tandem EA2 sequence, adjacent to the asparagine- glycine linker (Additional file 1: Table S1).”
Quote in the paperNot a substantial claim - Findingown data
Addition of WbgU increased the specific activity of rhGalNAc-T2 and HisDapGalNAc-T2 by about 26% and 40%, respectively, in the presence of UDP-GalNAc.
“In the presence of UDP-GalNAc sugar substrate, the addition of WbgU increased the specific activity of rhGalNAc-T2 and HisDapGalNAc-T2 by about 26 and 40%, respectively.”
Quote in the paperNot a substantial claim - Findingown data
Fractions containing pre-purified mucin derivatives were subjected to affinity chromatography and the concentrated eluates contained T7Muc10 with sufficient purity for further analysis.
“Fractions containing pre-purified mucin derivatives were subjected to affinity chromatography and the concentrated eluates contained T7Muc10 with sufficient purity for further analysis (Fig. 6b, lanes E1 and E2).”
Quote in the paperSubstantial claim - Findingown data
Different glycosylation levels of EA2- and EAN-peptides ranging from zero to four attached GalNAc moieties were detected in the MS data.
“Different glycosylation levels of EA2- and EAN-peptides ranging from zero to four attached GalNAc moieties were detected in the MS data (Table 2).”
Quote in the paperSubstantial claim - Findingown data
In vitro, samples with both WbgU and UDP-GlcNAc showed comparable activities to samples with UDP-GalNAc in the described assay.
“In general, samples with both WbgU and UDP-GlcNAc showed comparable activities to samples with UDP-GalNAc, confirming the functionality of the 4-epimerase and the glycosyltransferases in the described assay in vitro.”
Quote in the paperSubstantial claim - Findingown data
The 4-epimerase WbgU was successfully expressed as judged by immunoblot analysis using polyhistidine-tag targeting antibodies.
“The 4-epimerase was successfully expressed as judged by immunoblot analysis using polyhistidine-tag targeting antibodies.”
Quote in the paperSubstantial claim - Findingown data
Measured activities tended to be higher when WbgU and GalNAc-T2 were used in combination with UDP-GalNAc.
“Measured activities tended to be higher when WbgU and GalNAc-T2 were used in combination with UDP-GalNAc.”
Quote in the paperNot a substantial claim - Findingown data
Expression of GalNAc-T2 (60 kDa) and WbgU (40 kDa) was detected in both insoluble and soluble fractions.
“The expression of GalNAc-T2 (60 kDa) and WbgU (40 kDa) was detected in both insoluble and soluble fractions (GalNAc-T2; Fig. 5b green arrows; WbgU, Fig. 5c blue arrows).”
Quote in the paperNot a substantial claim - Findingown data
No activity was detected for both glycosyltransferases in the presence of UDP-GlcNAc without WbgU.
“No activity was detected for both glycosyltransferases in the presence of UDP-GlcNAc without WbgU.”
Quote in the paperSubstantial claim - Findingown data
LC-ESI-MS/MS analysis indicated the presence of GalNAc-modified threonines in T7Muc10.
“The detected peptides were assigned and the results indicate the presence of GalNAc-modified threonines in T7Muc10 (Table 1).”
Quote in the paperSubstantial claim - Findingown data
Soluble WbgU was estimated at around 65% of total WbgU, compared to 35% in the insoluble fraction.
“Based on densitometric analysis of the Western Blot the amount of soluble WbgU was estimated at around 65% (Fig. 5c lane 7) in comparison to the corresponding fraction of 35% in the insoluble fraction (Fig. 5c lane 3).”
Quote in the paperNot a substantial claim - Findingown data
Monosaccharide modifications were found at positions 2, 3, 6, 7, 11 and 12 of the tandem EA2 sequence.
“Evaluation of the MS/MS spectra revealed monosaccharide modifications at positions 2, 3, 6, 7, 11 and 12 of the tandem EA2.”
Quote in the paperSubstantial claim - Findingown data
Expressed GalNAc-T2 in soluble fractions was about 33% compared to 67% in insoluble fractions for both E. coli strains.
“However, expressed GalNAc-T2 in the soluble fractions was less prominent (about 33% as compared to 67% in the insoluble fractions for both E. coli strains) (Fig. 5b).”
Quote in the paperNot a substantial claim - Findingown data
The presence of functional GalNAc-T2, WbgU and soluble expressed MUC10 derivative was demonstrated by immunoblot analysis.
“The presence of functional GalNAc-T2, WbgU and soluble expressed MUC10 derivative was demonstrated by immunoblot analysis.”
Quote in the paperSubstantial claim - Findingown data
At 0.15 mM UDP-sugar substrates, the WbgU reaction reaches a ratio of 30:70 UDP-GalNAc:UDP-GlcNAc.
“When 0.15 mM UDP-sugar substrates were used, the results showed a ratio of 30:70 of UDP-GalNAc:UDP-GlcNAc (Fig. 3a).”
Quote in the paperNot a substantial claim - Findingown data
The apparent molecular mass of T7Muc10 increased significantly in the presence of WbgU.
“Interestingly, the apparent molecular mass of T7Muc10 increased significantly in the presence of WbgU (Fig. 5d orange arrows)”
Quote in the paperSubstantial claim - Findingown data
Monosaccharide modifications were found at positions 2, 6, 7 and 11 of the EAN sequence.
“Monosaccharide modifications were also found at positions 2, 6, 7 and 11 of the EAN sequence.”
Quote in the paperNot a substantial claim - Findingown data
Positions 3, 7, 11 and 12 of the repetitive EA2 subsequence PTtDSTtPAPttK in T7Muc10 were identified as possible glycosylation sites.
“Positions 3, 7, 11 and 12 of the repetitive EA2 subsequence PTtDSTtPAPttK in T7Muc10 were identified as possible glycosylation sites (Table 1).”
Quote in the paperSubstantial claim - Findingown data
Co-expression of GalNAc-T2 and WbgU under the T7 promoter in the presence of Erv1p/PDI in E. coli SHuffle®T7 Express with IPTG and l-arabinose induction was demonstrated by immunoblot analysis.
“The successful co-expression of GalNAc-T2 and WbgU—under the control of the T7 promotor in the presence of the two chaperones Erv1p/PDI in E. coli SHuffle®T7 Express with IPTG and l-arabinose induction—was demonstrated by immunoblot analysis with Anti-GalNT2 antibodies and polyclonal Anti-WbgU serum.”
Quote in the paperSubstantial claim - Findingown data
The presence of truncated T7Muc10 was confirmed by N-terminal Edman sequencing of amino acids 1 to 5.
“The presence of truncated recombinantly expressed T7Muc10 has been confirmed by determining amino acids 1 to 5 using N-terminal Edman sequencing.”
Quote in the paperNot a substantial claim - Findingown data
Specific activities with UDP-GalNAc as a sugar substrate were roughly comparable to the values detected for the combination of WbgU and UDP-GlcNAc.
“Specific activities with UDP-GalNAc as a sugar substrate were roughly comparable to the values detected for the combination of WbgU and UDP-GlcNAc.”
Quote in the paperNot a substantial claim - Findingown data
LC-MS analysis of potentially glycosylated T7Muc10 revealed different glycosylation stages, ranging from zero up to 24 attached sugar moieties.
“LC–MS analysis of potentially glycosylated T7Muc revealed different glycosylation stages, ranging from zero up to 24 attached sugar moieties (Fig. 7b).”
Quote in the paperSubstantial claim - Findingown data
In vivo glycosylation of the mucin-derived acceptor peptide was indicated by LC-ESI-MS.
“Monitoring glycosylation by liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS), the results clearly indicated the in vivo glycosylation of the mucin-derived acceptor peptide.”
Quote in the paperSubstantial claim- Rests on an assumption: The E. coli cytoplasm contains sufficient UDP-GlcNAc to serve as substrate for WbgU to generate UDP-GalNAc in vivo.
- Rests on an assumption: The observed glycosylation of T7Muc10 is specifically mediated by the co-expressed GalNAc-T2 and WbgU, not by endogenous E. coli enzymes.
- Findingown data
With 1.5 mM UDP-GlcNAc, about 30% substrate conversion by WbgU was detected.
“In samples containing 1.5 mM UDP-GlcNAc, a substrate conversion of about 30% was detected”
Quote in the paperSubstantial claim - Findingown data
No sugar moieties attached to T7Muc10 that correlate with galactose residues were detected.
“Although UDP-galactose has been postulated as an alternative sugar substrate for GalNAc-T2 [21, 30], no sugar moieties attached to T7Muc10, which correlate with galactose residues were detected.”
Quote in the paperNot a substantial claim - Findingown data
No molecular shift of T7Muc10 was detected when GalNAc-T2 and WbgU were not co-expressed.
“No molecular shift of T7Muc10 was detected when GalNAc-T2 and WbgU were not co-expressed.”
Quote in the paperSubstantial claim - Findingown data
The potential glycosylation of T7Muc10 was verified by ESI-MS/MS.
“The potential glycosylation of T7Muc10 was subsequently verified by ESI–MS/MS.”
Quote in the paperSubstantial claim - Findingown data
Threonines at position 2, 3, 6, 7, 11 and 12 of the tandem EA2, correlating with threonines at position 2, 6, 7 and 11 of the EAN sequence, and a single serine residue at position 100 in the truncated T7Muc10, were identified as potential glycosylation sites in vivo.
“Threonines at position 2, 3, 6, 7, 11 and 12 of the tandem EA2, correlating with threonines at position 2, 6, 7 and 11 of the EAN sequence, and a single serine residue at position 100 in the truncated T7Muc10, were identified as potential glycosylation sites in vivo.”
Quote in the paperSubstantial claim - Findingown data
High level in vivo O-glycosylation of a MUC10 derived protein was demonstrated using the E. coli-based expression platform presented in this work.
“High level in vivo O-glycosylation of a MUC10 derived protein was demonstrated using the E. coli-based expression platform presented in this work.”
Quote in the paperSubstantial claim- Rests on an assumption: Only 57 of the 60 putative glycosylation target sites in T7Muc10 are possible target sites.
- Findingown data
Putative glycosylation sites were postulated for di- or triglycosylated EA2 and EAN sequences based on MS1 measurements.
“Putative glycosylation sites were postulated for di- or triglycosylated EA2 and EAN sequences based on MS1 measurements.”
Quote in the paperNot a substantial claim - Findingnone given
The synthetic T7Muc10 target protein used in the present work does not contain cysteine residues.
“The synthetic T7Muc10 target protein used in the present work does not contain cysteine residues.”
Quote in the paperSubstantial claim - Findingown data
ESI-MS/MS results showed different glycosylation patterns with varying target sites and different amounts of monosaccharide moieties.
“The results showed the presence of different glycosylation patterns with varying target sites and different amounts of monosaccharide moieties.”
Quote in the paperSubstantial claim - Findingown data
The data presented in this work indicate that truncated T7Muc10 is glycosylated at different positions of the 57 possible target sites, in various degrees and combinations, leading to a multitude of different glycopeptides.
“The data presented in this work indicates the truncated T7Muc10 glycosylation at different positions of the 57 possible target sites, in various degrees and combinations, leading to a multitude of different glycopeptides; thus demonstrating a high level mucin-type O-glycosylation.”
Quote in the paperSubstantial claim- Rests on an assumption: Only 57 of the 60 putative glycosylation target sites in T7Muc10 are possible target sites.
- Findingown data
A maximum of 4 glycosylated sites in a peptide EA2/EAN repeat were detected.
“A maximum of 4 glycosylated sites in a peptide EA2/EAN repeat were detected.”
Quote in the paperNot a substantial claim - Findingown data
O-glycosylation sites were modified in different combinations, and to various degrees, by the co-expression of the target protein, human GalNAc-T2, 4-epimerase WbgU, and redox folding helper proteins PDI and Erv1p in E. coli SHuffle® T7 Express.
“O-glycosylation sites were modified in different combinations, and to various degrees, by the co-expression of the target protein, the human glycosyltransferase GalNAc-T2, the 4-epimerase WbgU, and the redox folding helper proteins PDI and Erv1p in E. coli SHuffle® T7 Express.”
Quote in the paperSubstantial claim - Findingown data
These putative glycosylation sites were confirmed by subsequent MS2 analysis.
“These sites were confirmed by subsequent MS2 analysis.”
Quote in the paperSubstantial claim - Interpretationown data
Some peptides with the same molecular mass and equal glycosylation level were detected at different retention times, indicating different localizations of the attached GalNAc moieties.
“Some peptides with the same molecular mass and consequently equal glycosylation level were detected at different retention times indicating different localizations of the attached GalNAc moieties (Table 2).”
Quote in the paperNot a substantial claim - Interpretationown data
The 14,230 Da mass corresponds to a truncated version of T7Muc10 (AS 21 to 159), lacking the first 20 N-terminal amino acids, with a mass deviation of 68 ppm.
“This result corresponds to a truncated version of T7Muc10 (AS 21 to 159), lacking the first 20 N-terminal amino acids measured with a mass deviation of 68 ppm.”
Quote in the paperSubstantial claim - Interpretationown data
The increase in apparent molecular mass of T7Muc10 in the presence of WbgU suggests modification by glycosylation.
“suggesting a modification by glycosylation.”
Quote in the paperSubstantial claim- Rests on an assumption: The observed glycosylation of T7Muc10 is specifically mediated by the co-expressed GalNAc-T2 and WbgU, not by endogenous E. coli enzymes.
- Interpretationown data
The diffuse, slightly smeared protein signal between 33 and 60 kDa detected by Western Blot analysis indicates the presence of putative glycosylated T7Muc10.
“The diffuse, slightly smeared protein signal between 33 and 60 kDa detected by Western Blot analysis (Fig. 6, lanes E1 and E2) indicates the presence of putative glycosylated T7Muc10.”
Quote in the paperSubstantial claim - Premisecitation
The absence of necessary activated sugar substrate UDP-GalNAc in laboratory E. coli strains requires co-expression of an additional UDP-GlcNAc 4-epimerase to generate UDP-GalNAc from UDP-GlcNAc.
“The absence of the necessary activated sugar substrate (UDP-GalNAc) in laboratory E. coli strains requires the co-expression of an additional UDP-GlcNAc 4-epimerase to generate UDP-GalNAc from UDP-GlcNAc [9, 20].”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [9] Henderson GE, Isett KD, Gerngross TU. Site-specific modification of recombinant proteins: a novel platform for modifying glycoproteins expressed in E. coli. Bioconjug Chem. 2011;22:903–12. source
- cites [20] Merritt JH, Ollis AA, Fisher AC, DeLisa MP. Glycans-by-design: engineering bacteria for the biosynthesis of complex glycans and glycoconjugates. Biotechnol Bioeng. 2013;110:1550–64. source
- Premisecitation
In vivo O-glycosylation of a glutathione S-transferase-mucin peptide fusion in an E. coli SHuffle® T7 express strain co-expressing an N-terminal truncated version of GalNAc-T2, in combination with the UDP-GlcNAc 4-epimerase WbpP from Pseudomonas aeruginosa, has been indicated based on Western blot analysis using horseradish peroxidase-conjugated Vicia villosa lectin.
“The in vivo O-glycosylation of a glutathione S-transferase-mucin peptide fusion in an E. coli SHuffle® T7 express strain co-expressing an N-terminal truncated version of GalNAc-T2, in combination with the UDP-GlcNAc 4-epimerase WbpP from Pseudomonas aeruginosa has been indicated based on Western blot analysis using horseradish peroxidase-conjugated Vicia villosa lectin [9].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [9] Henderson GE, Isett KD, Gerngross TU. Site-specific modification of recombinant proteins: a novel platform for modifying glycoproteins expressed in E. coli. Bioconjug Chem. 2011;22:903–12. source
- Interpretationown data
The increase in thermal transition point in samples containing UDP-GalNAc may indicate a stabilizing effect in the presence of the sugar substrate.
“We concluded that the increase detected in samples containing UDP-GalNAc may indicate a stabilizing effect in the presence of the sugar substrate.”
Quote in the paperNot a substantial claim - Interpretationown data
The higher apparent molecular mass of T7Muc10 indicates potential dimerization.
“indicating a potential dimerization of the protein (Fig. 5d red arrows).”
Quote in the paperNot a substantial claim - Interpretationown data
The transfer of sugar moieties to a mucin-derived acceptor protein demonstrates the potential for in vivo O-glycosylation in the optimized E. coli-based expression system.
“In the present work, the previously established E. coli- based expression system was further optimized and the potential for in vivo O-glycosylation was shown by demonstrating the transfer of sugar moieties to a mucin-derived acceptor protein.”
Quote in the paperSubstantial claim- Rests on an assumption: The E. coli cytoplasm contains sufficient UDP-GlcNAc to serve as substrate for WbgU to generate UDP-GalNAc in vivo.
- Rests on an assumption: The observed glycosylation of T7Muc10 is specifically mediated by the co-expressed GalNAc-T2 and WbgU, not by endogenous E. coli enzymes.
- Interpretationown data
Co-expressing WbgU from Plesiomonas shigelloides in E. coli enables conversion of UDP-GlcNAc to UDP-GalNAc in the bacterial cytoplasm.
“This enables the conversion of uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm.”
Quote in the paperSubstantial claim - Premisecitation
At substrate saturation (1.5 mM), reaction velocities are different with the respective starting substrate.
“At substrate saturation (1.5 mM), reaction velocities are different with the respective starting substrate [17].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [17] Kowal P, Wang PG. New UDP-GlcNAc C4 epimerase involved in the biosynthesis of 2-acetamino-2-deoxy-l-altruronic acid in the O-antigen repeating units of Plesiomonas shigelloides O17†. Biochemistry. 2002;41:15410–4. source
- Interpretationreasoning
The observed putative dimerization of T7Muc10, indicated by the 33 kDa protein band in SDS-PAGE analysis, may be due to interactions different from disulfide bonds.
“As a result, the observed putative dimerization of the protein, indicated by the 33 kDa protein band in SDS-PAGE analysis, may be due to different interactions.”
Quote in the paperNot a substantial claim - Interpretationown data
These results are consistent with the presence of GalNAc residues attached to the glycosylated mucin.
“These results are consistent with the presence of GalNAc residues attached to the glycosylated mucin.”
Quote in the paperSubstantial claim - Interpretationown data
The data demonstrate a high level of mucin-type O-glycosylation.
“The data presented in this work indicates the truncated T7Muc10 glycosylation at different positions of the 57 possible target sites, in various degrees and combinations, leading to a multitude of different glycopeptides; thus demonstrating a high level mucin-type O-glycosylation.”
Quote in the paperSubstantial claim- Rests on an assumption: Only 57 of the 60 putative glycosylation target sites in T7Muc10 are possible target sites.
- Classified as probably not a substantial claim. 36
- May combine two claims. 15
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationE. coli SHuffle®T7 Express is useful for the expression of complex proteins containing disulfide bonds.
“The usefulness of E. coli SHuffle®T7 Express for the expression of complex proteins containing disulfide bonds has already been demonstrated [6, 22, 23].”
Quote in the paperNot a substantial claim2 of 3 sources checked- cites [6] Lauber J, Handrick R, Leptihn S, Durre P, Gaisser S. Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli. Microb Cell Fact. 2015;14:3. source
- cites [22] Robinson MP, Ke N, Lobstein J, Peterson C, Szkodny A, Mansell TJ, Tuckey C, Riggs PD, Colussi PA, Noren CJ, et al. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria. Nat Commun. 2015;6:8072. source
- cites [23] Yao M, Wang J, Wu L, Min L, Li W-Y, Zhu L-Y, Meng E, Zhang D-Y. An efficient strategy for the expression of Jingzhaotoxin-III in Escherichia coli. Biotechnol Biotechnol Equip. 2017;31:821–7. source
Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing). May combine two claims.
PremisecitationAn enhancement of conformational stability has been postulated for human UDP-galactose 4′-epimerase GALE when binding UDP-glucose, and the authors hypothesized that natural ligands modulate GALE stability in vivo.
“Similarly, an enhancement of the conformational stability has been postulated for the human UDP-galactose 4′-epimerase GALE when binding the substrate UDP-glucose and the authors hypothesized that natural ligands modulate GALE stability in vivo [18].”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [18] Pey AL, Padín-Gonzalez E, Mesa-Torres N, Timson DJ. The metastability of human UDP-galactose 4′-epimerase (GALE) is increased by variants associated with type III galactosemia but decreased by substrate and cofactor binding. Arch Biochem Biophys. 2014;562:103–14. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe released phosphate is optically quantified using malachite green reagents.
“The released phosphate is optically quantified using malachite green reagents [19].”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [19] Wu ZL. Phosphatase-coupled universal kinase assay and kinetics for first-order-rate coupling reaction. PLoS ONE. 2011;6:e23172. source
Classified as probably not a substantial claim.
PremisecitationO-glycosylation of eukaryotic target sites using a truncated form of human GalNAc-T2 has been shown without altering the target peptide sequence.
“In contrast, O-glycosylation of eukaryotic target sites using a truncated form of the human glycosyltransferase GalNAc-T2 has been shown without altering the target peptide sequence [6, 9, 14, 15].”
Quote in the paperSubstantial claim2 of 4 sources checked- cites [6] Lauber J, Handrick R, Leptihn S, Durre P, Gaisser S. Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli. Microb Cell Fact. 2015;14:3. source
- cites [9] Henderson GE, Isett KD, Gerngross TU. Site-specific modification of recombinant proteins: a novel platform for modifying glycoproteins expressed in E. coli. Bioconjug Chem. 2011;22:903–12. source
- cites [14] Pratt MR, Hang HC, Ten Hagen KG, Rarick J, Gerken TA, Tabak LA, Bertozzi CR. Deconvoluting the functions of polypeptide N-α-acetylgalactosaminyltransferase family members by glycopeptide substrate profiling. Chem Biol. 2004;11:1009–16. source
- cites [15] Fritz TA, Raman J, Tabak LA. Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-2. J Biol Chem. 2006;281:8613–9. source
Classified as probably not a substantial claim.
PremisecitationThe assay is based on the detection of inorganic phosphate, which is released during the sugar transfer reaction and subsequent hydrolysis of UDP catalyzed by a phosphatase.
“The assay is based on the detection of inorganic phosphate, which is released during the sugar transfer reaction and subsequent hydrolysis of UDP catalyzed by a phosphatase.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [19] Wu ZL. Phosphatase-coupled universal kinase assay and kinetics for first-order-rate coupling reaction. PLoS ONE. 2011;6:e23172. source
The Introduction and Discussion describe detecting inorganic phosphate released by a phosphatase-coupled assay. But this paper’s assay measures phosphate released from ADP produced by kinase reactions, not from UDP following a sugar-transfer reaction. The Introduction mentions a glycosyltransferase assay only as a similar assay. (LLM, conf=0.90)
Experimental — accuracy not yet measured
Classified as probably not a substantial claim.
PremisecitationThe activity of the purified glycosyltransferase has been demonstrated using the mucin peptide derivative EA2 and filgrastim as acceptor substrates in vitro.
“The activity of the purified glycosyltransferase has been demonstrated using the mucin peptide derivative EA2 and filgrastim (Granulocyte-colony stimulating factor, G-CSF) as acceptor substrates in vitro [6].”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [6] Lauber J, Handrick R, Leptihn S, Durre P, Gaisser S. Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli. Microb Cell Fact. 2015;14:3. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationGlycosylation of the synthetic peptide EA2 derived from Rattus norvegicus submandibular apomucin 10 (MUC10) has been demonstrated in various in vitro assays in the presence of soluble GalNAc-T2.
“As an example, glycosylation of the synthetic peptide EA2 (PTTDSTTPAPTTK)—derived from Rattus norvegicus submandibular apomucin 10 (MUC10)—has been demonstrated in various in vitro assays in the presence of soluble GalNAc-T2 [6, 14, 15].”
Quote in the paperNot a substantial claim2 of 3 sources checked- cites [6] Lauber J, Handrick R, Leptihn S, Durre P, Gaisser S. Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli. Microb Cell Fact. 2015;14:3. source
- cites [14] Pratt MR, Hang HC, Ten Hagen KG, Rarick J, Gerken TA, Tabak LA, Bertozzi CR. Deconvoluting the functions of polypeptide N-α-acetylgalactosaminyltransferase family members by glycopeptide substrate profiling. Chem Biol. 2004;11:1009–16. source
- cites [15] Fritz TA, Raman J, Tabak LA. Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-2. J Biol Chem. 2006;281:8613–9. source
The Experimental Procedures (page unavailable) describe an in vitro enzyme-activity assay using EA2, and Table 3 identifies EA2 as a peptide substrate of human GalNAc-T2. The paper gives the EA2 sequence, but the passages in our copy do not identify it as derived from Rattus norvegicus submandibular apomucin 10 (MUC10). (LLM, conf=0.84)
Experimental — accuracy not yet measured
Classified as probably not a substantial claim.
PremisecitationUDP-GlcNAc is not a substrate for GalNAc-T2.
“As expected from previously published results [21], UDP-GlcNAc is not a substrate for GalNAc-T2.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [21] Wandall HH, Hassan H, Mirgorodskaya E, Kristensen AK, Roepstorff P, Bennett EP, Nielsen PA, Hollingsworth MA, Burchell J, Taylor-Papadimitriou J, Clausen H. Substrate specificities of three members of the human UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3. J Biol Chem. 1997;272:23503–14. source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationUsing porcine submaxillary mucin (PSM) as an example, N-glycosylation is followed by dimerization of PSM via disulfide bond formation between the C-terminal domains.
“Using porcine submaxillary mucin (PSM) as an example, N-glycosylation is followed by the dimerization of PSM via disulfide bond formation between the C-terminal domains [25–27].”
Quote in the paperNot a substantial claim1 of 3 sources checked- cites [25] Perez-Vilar J, Hill RL. The carboxyl-terminal 90 residues of porcine submaxillary mucin are sufficient for forming disulfide-bonded dimers. J Biol Chem. 1998;273:6982–8. source
- cites [26] Perez-Vilar J, Eckhardt AE, DeLuca A, Hill RL. Porcine submaxillary mucin forms disulfide-linked multimers through its amino-terminal D-domains. J Biol Chem. 1998;273:14442–9. source
- cites [27] Perez-Vilar J, Hill RL. Identification of the half-cystine residues in porcine submaxillary mucin critical for multimerization through the D-domains. Roles of the CGLCG motif in the D1- and D3-domains. J Biol Chem. 1998;273:34527–34. source
The Discussion (page unavailable) states that PSM forms disulfide-linked dimers through its carboxyl-terminal domain in the endoplasmic reticulum, where N-glycosylation also occurs. It does not establish that N-glycosylation precedes dimerization, so the claimed sequence is unsupported. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationUDP-galactose has been postulated as an alternative sugar substrate for GalNAc-T2.
“Although UDP-galactose has been postulated as an alternative sugar substrate for GalNAc-T2 [21, 30], no sugar moieties attached to T7Muc10, which correlate with galactose residues were detected.”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites [21] Wandall HH, Hassan H, Mirgorodskaya E, Kristensen AK, Roepstorff P, Bennett EP, Nielsen PA, Hollingsworth MA, Burchell J, Taylor-Papadimitriou J, Clausen H. Substrate specificities of three members of the human UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3. J Biol Chem. 1997;272:23503–14. source
- cites [30] Gomez H, Rojas R, Patel D, Tabak LA, Lluch JM, Masgrau L. A computational and experimental study of O-glycosylation. Catalysis by human UDP-GalNAc polypeptide: GalNAc transferase-T2. Org Biomol Chem. 2014;12:2645–55. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationNon-covalent dimerization-probably by hydrophobic interactions-of domains of the colonic human MUC2 mucin containing cysteines has been identified.
“Furthermore, non-covalent dimerization—probably by means of hydrophobic interactions—of domains of the colonic human MUC2 mucin containing cysteines has been identified [28].”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [28] Ambort D, van der Post S, Johansson ME, Mackenzie J, Thomsson E, Krengel U, Hansson GC. Function of the CysD domain of the gel-forming MUC2 mucin. Biochem J. 2011;436:61–70. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationPrevious in vitro studies with GalNAc-T2 have identified Thr7 in the synthetic EA2 peptide sequence as a preferred glycosylation site.
“Previous in vitro studies with GalNAc-T2 have identified Thr7 in the synthetic EA2 peptide sequence [14, 15] and Thr9 in the MUC5AC peptide as preferred glycosylation sites [31].”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites [14] Pratt MR, Hang HC, Ten Hagen KG, Rarick J, Gerken TA, Tabak LA, Bertozzi CR. Deconvoluting the functions of polypeptide N-α-acetylgalactosaminyltransferase family members by glycopeptide substrate profiling. Chem Biol. 2004;11:1009–16. source
- cites [15] Fritz TA, Raman J, Tabak LA. Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-2. J Biol Chem. 2006;281:8613–9. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe authors have recently demonstrated the presence of up to three GalNAc residues attached by GalNAc-T2 to the EA2 peptide in vitro.
“We have recently demonstrated the presence of up to three GalNAc residues attached by GalNAc-T2 to the EA2 peptide in vitro [6].”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [6] Lauber J, Handrick R, Leptihn S, Durre P, Gaisser S. Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli. Microb Cell Fact. 2015;14:3. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationFor MUC5AC and MUC5B, higher molecular weight aggregates have been detected using SDS-PAGE under both reducing and non-reducing conditions.
“For MUC5AC and MUC5B, higher molecular weight aggregates have been detected using SDS-PAGE, under both reducing and non-reducing conditions [29].”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [29] Backstrom M, Ambort D, Thomsson E, Johansson ME, Hansson GC. Increased understanding of the biochemistry and biosynthesis of MUC2 and other gel-forming mucins through the recombinant expression of their protein domains. Mol Biotechnol. 2013;54:250–6. source
The CysD Domains section reports higher-molecular-mass aggregates in SDS gel electrophoresis of recombinant MUC5AC and MUC5B CysD domains. The claim omits that domain-specific qualification and could be read as referring to the full mucins. The passage also explicitly applies 'both reducing and non-reducing conditions' to the predominantly monomeric migration, rather than separately specifying the conditions under which aggregates appeared. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationSubsequently, O-glycosylation and N-terminal multimerization of the dimers occur, mediated by disulfide bonds between the N-termini.
“Subsequently, O-glycosylation and N-terminal multimerization of the dimers [25–27] occur, mediated by disulfide bonds between the N-termini [27].”
Quote in the paperNot a substantial claim1 of 3 sources checked- cites [25] Perez-Vilar J, Hill RL. The carboxyl-terminal 90 residues of porcine submaxillary mucin are sufficient for forming disulfide-bonded dimers. J Biol Chem. 1998;273:6982–8. source
- cites [26] Perez-Vilar J, Eckhardt AE, DeLuca A, Hill RL. Porcine submaxillary mucin forms disulfide-linked multimers through its amino-terminal D-domains. J Biol Chem. 1998;273:14442–9. source
- cites [27] Perez-Vilar J, Hill RL. Identification of the half-cystine residues in porcine submaxillary mucin critical for multimerization through the D-domains. Roles of the CGLCG motif in the D1- and D3-domains. J Biol Chem. 1998;273:34527–34. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationPrevious in vitro studies with GalNAc-T2 have identified Thr9 in the MUC5AC peptide as a preferred glycosylation site.
“Previous in vitro studies with GalNAc-T2 have identified Thr7 in the synthetic EA2 peptide sequence [14, 15] and Thr9 in the MUC5AC peptide as preferred glycosylation sites [31].”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [31] Tetaert D, Ten Hagen KG, Richet C, Boersma A, Gagnon J, Degand P. Glycopeptide N-acetylgalactosaminyltransferase specificities for O-glycosylated sites on MUC5AC mucin motif peptides. Biochem J. 2001;357:313–20. source
May combine two claims.
Findingown dataWith 1.5 mM UDP-GalNAc, 57% was converted during the reaction time, indicating faster conversion of UDP-GalNAc than UDP-GlcNAc.
“whereas 57% of 1.5 mM UDP-GalNAc was converted during the reaction time, indicating a faster conversion of UDP-GalNAc (Fig. 3a).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataFirst attempts to capture T7Muc10 using Ni-NTA affinity chromatography failed due to unspecific binding of host cell proteins and weak interaction between protein and column.
“First attempts to capture T7Muc10 using Ni–NTA affinity chromatography failed due to unspecific binding of host cell proteins and weak interaction between protein and column (data not shown).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataEach attached moiety adds 203.2 Da in mass, consistent with the presence of GalNAc.
“Each attached moiety adds 203.2 Da in mass, which is consistent with the presence of GalNAc.”
Quote in the paperSubstantial claim- Rests on an assumption: The mass spectrometric identification of GalNAc modifications is not confounded by other modifications of the same mass.
May combine two claims.
Findingown dataCD scans identified characteristic minima at 208 and 222 nm, typical for α-helical structures, indicating folding of WbgU.
“Characteristic minima at 208 and 222 nm, typical for α-helical structures, were identified in CD scans indicating the folding of WbgU (Fig. 2a).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataThe slight difference in CD scans at 20 °C and 37 °C potentially indicates some reduction of secondary structures at the higher temperature.
“The slight difference in the scans measured at 20 °C and 37 °C potentially indicates some reduction of secondary structures at the higher temperature.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataUp to 24 of the 57 potential O-glycosylation sites of N-terminally truncated T7Muc10 (AS 21-159) were glycosylated, increasing the average molecular mass by roughly 4877 Da.
“Up to 24 of the 57 potential O-glycosylation sites of the N-terminally truncated T7Muc10 (AS 21–159) were glycosylated, increasing the average molecular mass by roughly 4877 Da.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingown dataTag-free WbgU migrated at 40 kDa under reducing denaturing conditions and was no longer detected using Anti-His antibodies.
“The tag-free protein migrated at 40 kDa under reducing denaturing conditions in the SDS-gel and was no longer detected using Anti-His antibodies (Fig. 1a, b, lanes F1 and F2).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataAdding WbgU and UDP-GlcNAc instead of UDP-GalNAc, the in vitro activity of WbgU was demonstrated based on the release of phosphate in the glycosylation reaction, which indicates the conversion of UDP-GlcNAc to UDP-GalNAc.
“Adding WbgU and UDP-GlcNAc instead of UDP-GalNAc, the in vitro activity of WbgU was demonstrated based on the release of phosphate in the glycosylation reaction, which indicates the conversion of UDP-GlcNAc to UDP-GalNAc.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThermal transition points of WbgU were 42.55 ± 0.04 °C, increasing to 43.07 ± 0.04 °C and 51.29 ± 0.13 °C in the presence of UDP-GlcNAc and UDP-GalNAc, respectively.
“The thermal transition points at 42.55 ± 0.04 °C, increasing to 43.07 ± 0.04 °C, and 51.29 ± 0.13 °C in the presence of UDP-GlcNAc and UDP-GalNAc, respectively, were determined based on the sigmoid graphs displayed in Fig. 2b.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Findingown dataEvaluation of the tandem MS data provided information about the glycosylation sites targeted by the glycosyltransferase and modified threonines.
“Evaluation of the tandem MS data provided information about the glycosylation sites targeted by the glycosyltransferase and modified threonines (displayed as lower-case letters in bold type).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataT7Muc10 was detected at an apparent molecular mass of 32 kDa, higher than its expected 16.5 kDa.
“The detected molecular weight of 32 kDa for T7Muc10 was higher than the expected 16.5 kDa”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe soluble expression of both GalNAc-T2 and WbgU was confirmed.
“The functionality of GalNAc-T2 and WbgU was also demonstrated in vitro, and the soluble expression of both enzymes was confirmed.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe functionality of GalNAc-T2 and WbgU was demonstrated in vitro.
“The functionality of GalNAc-T2 and WbgU was also demonstrated in vitro, and the soluble expression of both enzymes was confirmed.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
InterpretationreasoningThe estimated high isoelectric point of T7Muc10 (pI = 10.1) allowed for an indirect anion exchange chromatography step.
“However, the estimated high isoelectric point of the protein (pI = 10.1) allowed for an indirect anion exchange chromatography step (AIEX), thus avoiding the addition of salt and an additional dialysis step to separate the majority of host cell proteins from T7Muc10.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
PremisecitationIn vivo glycosylation has been shown.
“In vivo glycosylation has also been shown [9].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [9] Henderson GE, Isett KD, Gerngross TU. Site-specific modification of recombinant proteins: a novel platform for modifying glycoproteins expressed in E. coli. Bioconjug Chem. 2011;22:903–12. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationWbgU was chosen based on the well-characterized properties of the enzyme.
“WbgU was chosen based on the well-characterized properties of the enzyme [17].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [17] Kowal P, Wang PG. New UDP-GlcNAc C4 epimerase involved in the biosynthesis of 2-acetamino-2-deoxy-l-altruronic acid in the O-antigen repeating units of Plesiomonas shigelloides O17†. Biochemistry. 2002;41:15410–4. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationWbgU exhibits the highest activity at 37 °C.
“However, it has already been shown by Kowal and Wang, that WbgU exhibits the highest activity at 37 °C [17].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [17] Kowal P, Wang PG. New UDP-GlcNAc C4 epimerase involved in the biosynthesis of 2-acetamino-2-deoxy-l-altruronic acid in the O-antigen repeating units of Plesiomonas shigelloides O17†. Biochemistry. 2002;41:15410–4. source
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductreasoningComparison of activities determined in the assay, whether in the presence of WbgU or not, is difficult due to the ongoing epimerization reaction.
“However, the comparison of the activities determined in this assay, whether in the presence of WbgU or not, is rather difficult to determine—due to the ongoing epimerization reaction.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataThe 4-epimerase and the glycosyltransferases are functional in the described in vitro assay.
“In general, samples with both WbgU and UDP-GlcNAc showed comparable activities to samples with UDP-GalNAc, confirming the functionality of the 4-epimerase and the glycosyltransferases in the described assay in vitro.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe authors have described a slower conversion rate of UDP-GlcNAc than that of UDP-GalNAc based on time course experiments using multiple enzyme dilutions.
“The authors have described a slower conversion rate of UDP-GlcNAc than that of UDP-GalNAc based on time course experiments using multiple enzyme dilutions [17].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [17] Kowal P, Wang PG. New UDP-GlcNAc C4 epimerase involved in the biosynthesis of 2-acetamino-2-deoxy-l-altruronic acid in the O-antigen repeating units of Plesiomonas shigelloides O17†. Biochemistry. 2002;41:15410–4. source
Classified as probably not a substantial claim.
PremisecitationDetailed analysis of the glycosylated target mucin peptide with eight potential glycosylation sites has not been shown.
“However, the detailed analysis of the glycosylated target mucin peptide with eight potential glycosylation sites has not been shown [9].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [9] Henderson GE, Isett KD, Gerngross TU. Site-specific modification of recombinant proteins: a novel platform for modifying glycoproteins expressed in E. coli. Bioconjug Chem. 2011;22:903–12. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationIn mammals, mucins form homooligomers via inter-molecular disulfide bonds between cysteine-rich domains at both ends of the proteins.
“In mammals, mucins form homooligomers via inter-molecular disulfide bonds between cysteine-rich domains at both ends of the proteins [24].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [24] McGuckin MA, Linden SK, Sutton P, Florin TH. Mucin dynamics and enteric pathogens. Nat Rev Microbiol. 2011;9:265–78. source
Classified as probably not a substantial claim.
PremisecitationLC/MS analysis of an anti-TNF-α antibody fragment fusion to the 15 amino acid mucin-derived small peptide CDNKPAPGSTAPPAA revealed an increase in molecular weight of 203 Da, consistent with a single GalNAc moiety.
“The analysis of an anti-TNF-α antibody fragment fusion to the 15 amino acid mucin-derived small peptide CDNKPAPGSTAPPAA by LC/MS revealed an increase in molecular weight of 203 Da, consistent with the presence of a single GalNAc moiety [9].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [9] Henderson GE, Isett KD, Gerngross TU. Site-specific modification of recombinant proteins: a novel platform for modifying glycoproteins expressed in E. coli. Bioconjug Chem. 2011;22:903–12. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationIn the cited paper, Km values of 0.13 mM are reported for both substrates.
“In the cited paper Km values of 0.13 mM are reported for both substrates.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [17] Kowal P, Wang PG. New UDP-GlcNAc C4 epimerase involved in the biosynthesis of 2-acetamino-2-deoxy-l-altruronic acid in the O-antigen repeating units of Plesiomonas shigelloides O17†. Biochemistry. 2002;41:15410–4. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationIn a previously published expression system, an N-terminally truncated GalNAc T2 under the T7 promoter was expressed with the 4-epimerase from P. aeruginosa including its endogenous promoter in E. coli SHuffle®T7 Express without IPTG induction.
“In the expression system published previously, an N-terminally truncated version of GalNAc T2, inserted under the control of the T7 promoter, has been expressed in combination with the 4-epimerase derived from P. aeruginosa including its endogenous promoter in E. coli SHuffle®T7 Express without IPTG induction [9].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [9] Henderson GE, Isett KD, Gerngross TU. Site-specific modification of recombinant proteins: a novel platform for modifying glycoproteins expressed in E. coli. Bioconjug Chem. 2011;22:903–12. source
Probably not a substantial claim: may not pass S3 (load-bearing).
InterpretationcitationThese results correlate with data previously published for the His-tagged 4-epimerase WbgU recombinantly expressed in E. coli BL21(DE3).
“These results also correlate with data previously published for the His-tagged 4-epimerase WbgU recombinantly expressed in E. coli BL21(DE3) [17].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [17] Kowal P, Wang PG. New UDP-GlcNAc C4 epimerase involved in the biosynthesis of 2-acetamino-2-deoxy-l-altruronic acid in the O-antigen repeating units of Plesiomonas shigelloides O17†. Biochemistry. 2002;41:15410–4. source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenThe constructs were verified by restriction digests and the inserted fragments were confirmed by DNA sequence analysis.
“The constructs were verified by restriction digests and the inserted fragments were confirmed by DNA sequence analysis.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S2 (checkable).
Interpretationown dataThe results emphasize the potential of the presented glycosylation platform for in vivo glycosylation of commercially relevant biopharmaceuticals.
“Furthermore, the results emphasize the potential of the presented glycosylation platform for in vivo glycosylation of commercially relevant biopharmaceuticals.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationGalNAc-T2 has been classified as an early transferase, preferring mono- or un-glycosylated protein substrates.
“GalNAc-T2 has been classified as an early transferase, preferring mono- or un-glycosylated protein substrates [14].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [14] Pratt MR, Hang HC, Ten Hagen KG, Rarick J, Gerken TA, Tabak LA, Bertozzi CR. Deconvoluting the functions of polypeptide N-α-acetylgalactosaminyltransferase family members by glycopeptide substrate profiling. Chem Biol. 2004;11:1009–16. source
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataThe construct pET23a(+)_wbgUTEV6H was verified by restriction digests and DNA sequence analysis.
“The resulting construct pET23a(+)_wbgUTEV6H was verified by restriction digests and DNA sequence analysis.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
The E. coli cytoplasm contains sufficient UDP-GlcNAc to serve as substrate for WbgU to generate UDP-GalNAc in vivo.
“This enables the conversion of uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm.”
The paper relies on this without support.
The observed glycosylation of T7Muc10 is specifically mediated by the co-expressed GalNAc-T2 and WbgU, not by endogenous E. coli enzymes.
“Interestingly, the apparent molecular mass of T7Muc10 increased significantly in the presence of WbgU (Fig. 5d orange arrows) suggesting a modification by glycosylation.”
The paper relies on this without support.
The mass spectrometric identification of GalNAc modifications is not confounded by other modifications of the same mass.
“Each attached moiety adds 203.2 Da in mass, which is consistent with the presence of GalNAc.”
The paper relies on this without support.
Only 57 of the 60 putative glycosylation target sites in T7Muc10 are possible target sites.
“the truncated T7Muc10 glycosylation at different positions of the 57 possible target sites”
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
CD spectrum temperatures disagree between methods and results
MinorThe methods say the spectra were recorded at 25 °C, whereas the Fig. 2 caption identifies spectra recorded at 20 °C and 37 °C.
“Spectra of the filtered solution (NanoSep® MF 0.2 mM, Pall) containing 0.6 mg/mL WbgU in 20 mM sodium phosphate buffer (pH 7.75) were recorded in a 715 CD-spectropolarimeter (Jasco, Hachioji, Japan) at 25 °C.”
“a Far UV (195–260 nm) spectra of WbgU at 20 °C (black line) and 37 °C (red line) indicating the folding status of the 4-epimerase in 20 mM sodium phosphate buffer at pH 7.75.”
Opposite GalNAc linkage configurations are reported
MinorThe background states that this mucin-type reaction forms an α-linked GalNAc residue, but the Fig. 7 caption describes the attached residues as β-linked.
“Each of the up to 24 O-linked β-N-acetylgalactosamines results in a mass increment of 203.2 Da”
“O-glycosylation of mucins is initiated by the transfer of GalNAc—a reaction catalyzed by UDP-N-Acetylgalactosaminyltransferases (GalNAc-Ts)—forming the GalNAcα1-O-serine or threonine linkage [16].”
Cites
Mucin dynamics and enteric pathogens
McGuckin, M. A., Lindén, S. K., Sutton, P., & Florin, T. H. (2011) · Nature Reviews Microbiology · doi:10.1038/nrmicro2538 · Cited by 1,428 · see citing works
Biopharmaceutical benchmarks 2014
Walsh, G. (2014) · Nature Biotechnology · doi:10.1038/nbt.3040 · Cited by 1,008 · see citing works
Post-translational modifications in the context of therapeutic proteins
Walsh, G., & Jefferis, R. (2006) · Nature Biotechnology · doi:10.1038/nbt1252 · Cited by 1,007 · see citing works
N-Linked Glycosylation in Campylobacter jejuni and Its Functional Transfer into E. coli
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- Authors
- Mueller, P., Gauttam, R., Raab, N., Handrick, R., Wahl, C., Leptihn, S., Zorn, M., Kussmaul, M., Scheffold, M., Eikmanns, B., Elling, L., & Gaisser, S.
- Affiliations
- University of Applied Sciences Biberach (DE)
- Universität Ulm (DE)
- RWTH Aachen University (DE)
- Boehringer Ingelheim (Germany) (DE)
- Corresponding author
- Gaisser, Sabine
- ORCID
- Venue
- Microbial Cell Factories
- Published
- 2018-10-26
- Volume
- 17
- Issue
- 1
- Pages
- 168-168
- ISSN
- 1475-2859 (ISSN-L)
- PMC
- PMC6202839
- OpenAlex
- W2898512685
- Semantic Scholar
- f935003d37811b98402a08377a4316f54ee7022d
- Cited by
- 22
- References
- 35
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 3 · 2023: 3 · 2022: 2 · 2021: 7 · 2020: 2 · 2019: 3
- FWCI
- 1.07
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Microbial Cell Factories (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- eScholarship (California Digital Library) (submittedVersion, other-oa)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- eScholarship (California Digital Library) (submittedVersion)
- RWTH Publications (RWTH Aachen) (submittedVersion, other-oa)
- RWTH Publications (RWTH Aachen)
- Funders
- Ministerium für Wissenschaft, Forschung und Kunst Baden-Württemberg
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Physical Sciences › Chemistry › Organic Chemistry › Carbohydrate Chemistry and Synthesis
- Keywords
- Escherichia coli expression, UDP-GalNAc, mucin-type glycosylation, chaperone co-expression, recombinant protein glycosylation, Plesiomonas shigelloides
- MeSH terms
- Polypeptide N-acetylgalactosaminyltransferase, Amino Acid Sequence, Carbohydrate Epimerases, Circular Dichroism, Escherichia coli, Glycosylation, Mucins, Peptides, N-Acetylgalactosaminyltransferases
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-28.
Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-28.
Cited sources are checked by gpt-6-sol.
Statistics and arithmetic are recomputed, not judged.
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