University of Groningen Exploring and exploiting bacterial protein glycosylation systems Yakovlieva, Liubov

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(1)University of Groningen. Exploring and exploiting bacterial protein glycosylation systems Yakovlieva, Liubov DOI: 10.33612/diss.173544104 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.. Document Version Publisher's PDF, also known as Version of record. Publication date: 2021 Link to publication in University of Groningen/UMCG research database. Citation for published version (APA): Yakovlieva, L. (2021). Exploring and exploiting bacterial protein glycosylation systems. University of Groningen. https://doi.org/10.33612/diss.173544104. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.. Download date: 21-07-2021.

(2) CHAPTER 5 Insight into the oligomeric states of bacterial NGTs and the H. influenzae adhesin fragment This Chapter describes preliminary studies of the structures of bacterial N-glycosyltransferases and the H. influenzae adhesin fragment. Using native gel electrophoresis and size-exclusion chromatography, evidence for multimeric assemblies of ApNGT, HiNGT and HMW1ct is presented. A discussion of the exact architecture of these protein complexes as well as their relevance to the glycosylation mechanism is provided.. The work described in this Chapter was performed in collaboration with A. Jeucken, D. J. Slotboom and D. J. Scheffers..

(3) CHAPTER 5. Protein glycosylation is one of the most ubiquitous modifications in nature. Whereas it is a generally conserved process across domains of life (Eukaryotes, Prokaryotes, Archaea), differences exist in monosaccharide composition of the glycans, variety of glycosidic linkages and glycosylation machinery.1 In recent years, protein glycosylation of bacteria has gained increased attention, as (surface) glycoproteins frequently mediate host-pathogen interactions or promote bacterial survival in the host.2 Multiple protein glycosyltransferases involved in the synthesis of these virulent proteins have been identified that don’t operate via a standard membrane-associated glycosylation process. Among these examples are the hyperglycosylation of high-molecular weight adhesin proteins by NGTs (discussed in Chapter 3), hyperglycosylation of autotransporters Ag43,3 TibA,4 and AIDA5 by TibC and other associated GTs, arginine rhamnosylation of elongation factor P by EarP (discussed in Chapter 7), and arginine GlcNAcylation of host proteins by bacterial effector proteins NleB and SseK.6 These protein glycosylation systems constitute promising novel antibiotic targets, as they are bacteria-specific and do not have equivalents in humans. Therefore, the insight into the mechanism of these unusual glycosyltransferases is a first step towards development of inhibitors of bacterial protein glycosylation. The initial evidence of a multimeric structure of ApNGT (involved in hyperglycosylation of the adhesin protein, Chapter 3) was obtained upon performing native MS measurements as part of the investigation into its mechanism of action. This inspired the thorough studies into the multimerism of ApNGT, HiNGT and the acceptor protein HMW1ct as described in this Chapter. Using several techniques, such as blue native PAGE, size-exclusion chromatography (SEC) and SEC combined with multi-angle laser light scattering (MALLS), it was elucidated that ApNGT exists as a dimer and HiNGT is primarily a trimer in solution. HMW1ct was concluded to be mainly monomeric, although a small degree of multimerization was also observed.. 128.

(4) OLIGOMERIC STATES OF NGTS AND HMW1CT. Native protein mass spectrometry allows to measure proteins in their nondenatured state and is therefore advantageous for observing the formation of protein-protein complexes and oligomeric structures. When the ApNGT enzyme was analyzed with native MS, it was observed that next to the expected (monomeric) molecular weight (Mw) also a significant amount of dimer was present (data not shown). This was an unexpected finding, as this enzyme was previously designated as a monomer.7 To further investigate these findings, a standard blue native PAGE experiment was performed on ApNGT, HiNGT and HMW1ct, both on individual proteins and their mixtures. In this experiment the denaturing step is omitted which allows proteins to migrate through the gel in their native (multimeric) state. In order to eliminate the migration discrepancies between samples due to the differences in charge, gel electrophoresis is performed in the presence of a blue anionic dye (hence the “blue” native PAGE). The results are depicted in Figure 1. As can be seen in lanes 1 and 2 of Figure 1, the HMW1ct protein and its glycosylated version Glc-HMW1ct (a mixture of 7-10Glc) are separated into two different fractions of lower (~20-40 kDa) and higher (~120-150 kDa) molecular weight. The calculated molecular weight of these proteins is between 34-36 kDa and therefore the band of lower Mw on the gel represents the monomeric state, whereas the smear of higher Mw indicates higher multimeric state of these proteins. The glycosyltransferase ApNGT (lane 3) migrated almost exactly at the height corresponding to ~146 kDa of the protein ladder which indicates that the multimeric state of ApNGT is a dimer. Some smearing was observed below the main band which can correspond to the presence of monomeric species (~70 kDa). The band of HiNGT (lane 4), on the other hand, appeared lower, between the bands corresponding to 146 kDa and 66 kDa of the protein ladder, which suggests a monomeric state for HiNGT. Importantly, on SDS-PAGE gel (denaturing conditions) both ApNGT and HiNGT appear as monomeric bands of ~75 kDa (data not shown).. 129.

(5) CHAPTER 5. Figure 1. Blue native PAGE experiment. L: protein ladder. EarP-SH and EFP-SH proteins were not part of this investigation and were used in this experiment as controls.. The mixtures of NGTs and their substrate HMW1ct or product GlcHMW1ct were also analyzed (lanes 7-10, Figure 1) to determine whether any complex formation could be observed. However, no new bands of higher molecular weight were detected, rather, the protein mixtures were separated into individual bands that can be correlated back to bands 1-4. Importantly, when two control proteins (EarP-SH and EFP-SH, previously reported to be monomeric)8-10 were used in this experiment (lanes 5 and 6), their apparent Mw appeared to be lower than calculated. This was especially pronounced in the case of EFP-SH (lane 6), that has a calculated Mw exactly the same as HMW1ct protein (lane 1), but runs lower on the gel. This observed discrepancy may be explained by the influence of the protein shape on its migration through the gel. EFP-SH protein adopts a loose L-shaped form,9 whereas the HMW1ct protein is predicted to be a more structured beta-barrel.11 The experimental observations of the apparent multimeric structures of HMW1ct, ApNGT and HiNGT were verified by performing size exclusion chromatography (SEC) analysis. The elution peaks of the analyzed proteins are presented in Figure 2. In the HMW1ct elution profile (Figure 2A) several peaks were observed: minor peaks at 9.1 and 10.4 mL and a major peak at 12.7 mL. According to SEC protein standards, the major peak at 12.7 mL corresponds to proteins with a Mw of ~150 kDa (Table 2 in Experimental procedures). Fractions collected from each of the peaks were analyzed by SDS-PAGE and were shown to contain HMW1ct in each peak (data not shown). In contrast, when EFP-SH was analyzed as a control, which has the same calculated Mw as HMW1ct, its 130.

(6) OLIGOMERIC STATES OF NGTS AND HMW1CT elution peak appeared later, at 14.9 mL (Figure 2B), corresponding to a Mw of ~44-66 kDa (based on protein standards, Table 2). For ApNGT and HiNGT the elution profiles looked relatively similar, with one major peak appearing at 13.3 mL (ApNGT, Figure 2C) and 12.4 mL (HiNGT, Figure 2D) which corresponds to a Mw of ~150 kDa or above for both proteins. When a mixture of ApNGTHMW1ct (1:2 molar ratio) was analyzed, a new peak at 11.7 mL appeared, together with a minor peak at 8.8 mL (Figure 2E). Both peaks contained ApNGT and HMW1ct (SDS-PAGE analysis). This elution profile was different from the individual runs of HMW1ct and ApNGT, and could indicate the formation of higher Mw complexes between these two proteins. In contrast, the mixture of HiNGT-HMW1ct (1:2 molar ratio) did not result in the appearance of new peaks, instead a single peak at 12.4 mL was observed (Figure 2F), which resulted from the overlap of the HMW1ct and the HiNGT SEC-peaks.. 131.

(7) CHAPTER 5. Figure 2. Size-exclusion chromatography elution profiles of A: HMW1ct, B: EFP-SH, C: ApNGT, D: HiNGT, E: ApNGT and HMW1ct mixture F: HiNGT and HMW1ct mixture.. 132.

(8) OLIGOMERIC STATES OF NGTS AND HMW1CT From the SEC analysis of the NGT and HMW1ct proteins it was concluded that both enzymes and substrate protein adopt oligomeric states of ~150 kDa molecular weight. Whereas this is in line with blue native PAGE analysis for ApNGT, it is not for HiNGT and HMW1ct. These observed discrepancies can be explained by the differences in architecture of the oligomeric protein standards and proteins of interest (globular vs non-globular). It was concluded that correlating SEC elution volumes of NGTs and HMW1ct to protein standards only gives an approximation of the molecular mass. Therefore, to unambiguously determine the oligomeric state of the proteins of interest, size exclusion chromatography was coupled with multi angle laser light scattering (SEC-MALLS) analysis. This technique allows the direct measurement of the absolute molecular mass of the protein in a specific SEC elution peak by measuring the differences in scattered light compared to the control (buffer solution). The physical principles and use of SEC-MALLS for the determination of oligomeric protein states are discussed elsewhere.12 In this way, the Mw of ApNGT (Figure 3A) was experimentally determined to be ~140 kDa, which is in line with previous results obtained with native MS, blue native PAGE and SEC and corresponds to a homodimer. Strikingly, the SEC peak of HiNGT contained masses in the range of ~200 to 150 kDa, which corresponds to a homotrimer and homodimer, respectively (Figure 3B). For HMW1ct, the main oligomeric state was determined to be monomeric (~34 kDa), whereas the Mw of the small early SEC peak (multimeric) was not determined (Figure 3C).. 133.

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(15). . . . !!#!. . . " Figure 3. Size-exclusion chromatography coupled with multi-angle laser light scattering analysis of A: ApNGT, B: HiNGT, C: HMW1ct. * - oligomeric SEC fraction of HMW1ct with unidentified Mw. (LS = light scattering, UV = absorption at 280 nm, dRI = difference in refractive index between the protein solution and buffer solution).. 134.

(16) OLIGOMERIC STATES OF NGTS AND HMW1CT. In this Chapter the first results are presented from the investigation of the multimeric structure of bacterial N-glycosyltransferases (ApNGT and HiNGT) and the HMW1ct adhesin protein. Based on the results of several techniques employed in this Chapter, the oligomeric state of ApNGT is designated as a homodimer and HiNGT primarily exists as homotrimer. It appears that HMW1ct exists primarily as a monomer with a minor fraction of an unidentified oligomeric state. Whereas multiple reports exist on multimeric bacterial adhesin proteins involved in self-aggregation and are reviewed elsewhere,13,14 the examples of bacterial oligomeric GTs are scarce. One example is the homododecameric Oheptosyltransferase TibC15 that is responsible for hyperglycosylation of the TibA autotransporter. The crystal structure of TibC revealed that the dodecameric architecture adopts a ring structure with a “gear-like shape”. The authors proposed that this multimeric structure of TibC was responsible for the “screwpropelling” mechanism important for processive glycosylation.15 Whereas the experiments described above reveal that HMW1ct primarily exists as a monomer structure, a small multimeric fraction was identified as well both in blue native PAGE experiment and in SEC(-MALLS). Previously, another Haemophilus influenzae adhesin, called Hap adhesin, was shown to adopt higher-order oligomeric states.16 The mechanism of oligomerization was shown to take place via its C-terminal self-associating autotransporter (SAAT) domain that adopts a beta-barrel structure. The authors propose a model wherein the Asp/Glu and Asn residues in the loops form stacked ladders in-between monomers. This results in the formation of megaDalton adhesin protein structures, necessary to promote bacterial selfaggregation in the host. Interestingly, the C-terminal domain of HMW1A, called HMW1ct, investigated in this Chapter is also predicted to adopt the beta-barrel architecture (Figure 4A, B).. 135.

(17) CHAPTER 5. Figure 4. I-TASSSER generated structure of HMW1ct. A: Front view. B: Top view. C: Clusters of Asn-Asp/Glu ladders in HMW1ct structure (Asn is shown in red, Asp/Glu in blue).. Similar to the SAAT domain of the Hap adhesin, the role of the Cterminal part of HMW is to tether the HMW adhesin protein to the bacterial cell wall where it promotes adherence to the host cell. Multiple Asn and Asp/Glu residues are present in the exposed loops of the HMW1ct structure (Figure 4C) and potentially could form oligomers via the model proposed for Hap adhesin.16 Whereas the adhesion function of HMW adhesin proteins is well-established, its role in autoaggregation and biofilm formation has not been explored. Interestingly, antibody-based labelling of the nontypeable H. influenzae biofilms revealed the presence of HMW proteins in the extracellular matrix.17 This suggests a possible second role for HMW adhesin proteins, namely selfaggregation of the cells via formation of oligomeric structures. Taken together, the data presented in this Chapter reveals the unprecedented multimeric states of cytoplasmic bacterial protein Nglycosyltransferases. Additionally, a tendency to form oligomeric states was also observed for HMW1ct, which is the C-terminal fragment of a surface adhesin protein. It will be highly insightful to investigate the architecture of ApNGT, 136.

(18) OLIGOMERIC STATES OF NGTS AND HMW1CT HiNGT and HMW1ct as well as their complexes via protein crystallography or cryo-EM analysis. The functional relevance of the higher oligomeric GT states for the mechanism of glycosylation and processive behavior can be further investigated by disrupting the formation of the oligomers via mutagenesis of the interacting residues to yield monomers. Further structural studies will provide more handles for manipulation and inhibition of this glycosylation system from clinically relevant H. influenzae..

(19) Dr. Aike Jeucken is acknowledged for performing the SEC analysis. Prof. Dirk J. Slotboom is acknowledged for performing the SEC-MALLS analysis. Prof. DirkJan Scheffers is acknowledged for his help with the blue native PAGE experiment.. 137.

(20) CHAPTER 5. Expression and purification of HiNGT, ApNGT and HMW1ct HiNGT-His6 was generated from the HMW1C gene extracted from the genomic plasmid isolated from H. influenzae R2846 (provided by Prof. Arnold Smith) and incorporated in a pET24 plasmid. HiNGT-His6 (pET24), His6-HMW1ct (pET45) and ApNGT-His6 (pET24) were overexpressed from BL21 E. coli cells. Cell cultures for protein expression were grown in TB (Terrific Broth) with 100 µg/mL of appropriate antibiotic. Protein overexpression was induced by addition of 1 mM final concentration isopropyl β-thiogalactopyranoside (IPTG) at OD600 0.6-0.8. Proteins were purified via Ni-affinity chromatography. Detailed experimental procedures of protein expression and purification are described in Chapter 3. Expression and purification of Glc-HMW1ct Glc-HMW1ct was produced via co-expression of ApNGT-His6 and His6HMW1ct in E. coli BL21 DE3. Culture growth, protein co-overexpression and purification were carried out in the same way as described in the previous section. The separation of ApNGT and glycosylated HMW1ct was performed via anion exchange on FPLC ÄKTA Pure system. Detailed experimental procedures of Glc-HMW1ct expression and purification are described in Chapter 3. Expression and purification of EarP-SH and EFP-SH pBADSUMO plasmids harbouring earp or efp gene (from Pseudomonas aeruginosa PAO1, synthesized and cloned by GenScript) were used produce proteins in E. coli TOP10 cells. Cell cultures for protein expression were grown in Terrific Broth (TB) with 100 µg/mL ampicillin and induced with L-Ara at OD600 0.6-0.8. Proteins were purified via Ni-affinity chromatography. Detailed experimental procedures for protein expression and purification are described in Chapter 7.. 138.

(21) OLIGOMERIC STATES OF NGTS AND HMW1CT Blue native PAGE Table 1. Reagents and buffers:. Sample buffer. 0.5% Coomassie G-250, 42.5% glycerol. Cathode buffer. 50 mM Tricine, 15 mM BisTris. Dark blue Cathode buffer. 50 mM Tricine, 15 mM BisTris, 0.02% Coomassie G-250. Light blue Cathode buffer. 1xDark blue cathode buffer + 9xcathode buffer.. Anode buffer. 50 mM BisTris, pH 7. Gels. Biorad, native precast gradient gels (4-15%). Destaining solution. 50% EtOH, 10% AcOH, aqueous. The gel electrophoresis system was washed thoroughly with water and soaked overnight to remove traces of SDS. The wells of precast gel were rinsed with the dark blue cathode buffer (1 mL, twice) and buffer was left in the wells. Protein samples (10 μL) were loaded into the wells and dark blue cathode buffer was poured into the inner chamber. Anode buffer was poured into the outside buffer chamber. Gels were run at 120 V. Once protein bands migrated halfway through the gel, the dark blue cathode buffer was replaced with light blue cathode buffer. The run was continued at 120 V for another ~2 h until the dye front disappeared. The gels were destained in the destaining solution for 10-15 min and subsequently hydrated in water. Size exclusion chromatography analysis Protein samples were injected onto a Superdex 200 10/300 gel filtration column (GE Healthcare) which was pre-equilibrated with gel filtration buffer (50 mM HEPES, 100 mM NaCl, pH 7). Runs were performed with system flow of 0.5 mL/min at 4 °C. The volume of elution peaks of analyzed protein samples was compared to SEC profile of protein standards (Table 2).. 139.

(22) CHAPTER 5 Table 2. Protein standards, their Mw and elution volume in SEC (confirmed by SDSPAGE analysis of the elution fractions).. Protein. Mw. Elution volume. Thyroglobulin. 669 kDa. ~8-10 mL. Ferritin. 440 kDa. ~11 mL. Aldolase. 158 kDa. n/d1. Bovine serum albumin. 66 kDa. ~14.5 mL. Ovalbumin. 44 kDa. 15.6 mL. Chymotrypsin A. 25 kDa. 18 mL. Ribonuclease. 13 kDa. n/d2. 1–. protein peak was absent in the SEC elution profile. 2-. protein bands from the elution peaks were not visible on the SDS-PAGE gel. Size exclusion chromatography coupled to multi-angle laser light scattering analysis For the SEC-MALLS analysis, first, the preparative SEC was performed as described above to ensure monodispersity of the samples. Equilibration buffer: 20 mM MOPS, 150 mM NaCl pH 7.5. After the preparative SEC the collected peak fraction was injected on a Superdex 200 column equilibrated with SEC buffer with flow of 0.5 mL/min. The system was equipped with light scattering detector (miniDAWN TREOS, Wyatt technologies). The Mw was calculated using ASTRA software package (Wyatt Technologies). 140.

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