Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages.

Most immunoglobulin (Ig) domains bear only a single highly conserved canonical intradomain, inter-ß-sheet disulfide linkage formed between Cys23-Cys104, and incorporation of rare noncanonical disulfide linkages at other locations can enhance Ig domain stability. Here, we exhaustively surveyed the sequence tolerance of Ig variable (V) domain framework regions (FRs) to noncanonical disulfide linkages. Starting from a destabilized VH domain lacking a Cys23-Cys104 disulfide linkage, we generated and screened phage-displayed libraries of engineered VHs, bearing all possible pairwise combinations of Cys residues in neighboring ß-strands of the Ig fold FRs. This approach identified seven novel Cys pairs in VH FRs (Cys4-Cys25, Cys4-Cys118, Cys5-Cys120, Cys6-Cys119, Cys22-Cys88, Cys24-Cys86, and Cys45-Cys100; the international ImMunoGeneTics information system numbering), whose presence rescued domain folding and stability. Introduction of a subset of these noncanonical disulfide linkages (three intra-ß-sheet: Cys4-Cys25, Cys22-Cys88, and Cys24-Cys86, and one inter-ß-sheet: Cys6-Cys119) into a diverse panel of VH, VL, and VHH domains enhanced their thermostability and protease resistance without significantly impacting expression, solubility, or binding to cognate antigens. None of the noncanonical disulfide linkages identified were present in the natural human VH repertoire. These data reveal an unexpected permissiveness of Ig V domains to noncanonical disulfide linkages at diverse locations in FRs, absent in the human repertoire, whose presence is compatible with antigen recognition and improves domain stability. Our work represents the most complete assessment to date of the role of engineered noncanonical disulfide bonding within FRs in Ig V domain structure and function.

Toward an experimental system for the examination of protein mannosylation in Actinobacteria.

The Actinobacterial species Cellulomonas fimi ATCC484 has long been known to secrete mannose-containing proteins, but a closer examination of glycoproteins associated with the cell has never been reported. Using ConA lectin chromatography and mass spectrometry, we have surveyed the cell-associated glycoproteome from C. fimi and collected detailed information on the glycosylation sites of 19 cell-associated glycoproteins. In addition, we have expressed a previously known C. fimi secreted cellulase, Celf_3184 (formerly CenA), a putative peptide prolyl-isomerase, Celf_2022, and a penicillin-binding protein, Celf_0189, in the mannosylation capable host, Corynebacterium glutamicum. We found that the glycosylation machinery in C. glutamicum was able to use the recombinant C. fimi proteins as substrates and that the glycosylation matched closely that found in the native proteins when expressed in C. fimi. We are pursuing this observation as a prelude to dissecting the biosynthetic machinery and biological consequences of this protein mannosylation.

A CHO stable pool production platform for rapid clinical development of trimeric SARS-CoV-2 spike subunit vaccine antigens.

Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time-consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non-clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP-toxicology and early-phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS-CoV-2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost-effective clinical evaluation of potentially life-saving vaccines.

Isolation and characterization of a VHH targeting the Acinetobacter baumannii cell surface protein CsuA/B.

Acinetobacter baumannii is a Gram-negative bacterial pathogen that exhibits high intrinsic resistance to antimicrobials, with treatment often requiring the use of last-resort antibiotics. Antibiotic-resistant strains have become increasingly prevalent, underscoring a need for new therapeutic interventions. The aim of this study was to use A. baumannii outer membrane vesicles as immunogens to generate single-domain antibodies (VHHs) against bacterial cell surface targets. Llama immunization with the outer membrane vesicle preparations from four A. baumannii strains (ATCC 19606, ATCC 17961, ATCC 17975, and LAC-4) elicited a strong heavy-chain IgG response, and VHHs were selected against cell surface and/or extracellular targets. For one VHH, OMV81, the target antigen was identified using a combination of gel electrophoresis, mass spectrometry, and binding studies. Using these techniques, OMV81 was shown to specifically recognize CsuA/B, a protein subunit of the Csu pilus, with an equilibrium dissociation constant of 17 nM. OMV81 specifically bound to intact A. baumannii cells, highlighting its potential use as a targeting agent. We anticipate the ability to generate antigen-specific antibodies against cell surface A. baumannii targets could provide tools for further study and treatment of this pathogen. KEY POINTS: •Llama immunization with bacterial OMV preparations for VHH generation •A. baumannii CsuA/B, a pilus subunit, identified by mass spectrometry as VHH target •High-affinity and specific VHH binding to CsuA/B and A. baumannii cells.

Characterizing the N- and O-linked glycans of the PGF-CTERM sorting domain-containing S-layer protein of Methanoculleus marisnigri.

The glycosylation of structural proteins is a widespread posttranslational modification in Archaea. Although only a handful of archaeal N-glycan structures have been determined to date, it is evident that the diversity of structures expressed is greater than in the other domains of life. Here, we report on our investigation of the N- and O-glycan modifications expressed by Methanoculleus marisnigri, a mesophilic methanogen from the Order Methanomicrobiales. Unusually, mass spectrometry (MS) analysis of purified archaella revealed no evidence for N- or O-glycosylation of the constituent archaellins, In contrast, the S-layer protein, identified as a PGF-CTERM sorting domain-containing protein encoded by MEMAR_RS02690, is both N- and O-glycosylated. Two N-glycans were identified by NMR and MS analysis: a trisaccharide α-GlcNAc-4-ß-GlcNAc3NGaAN-4-ß-Glc-Asn where the second residue is 2-N-acetyl, 3-N-glyceryl-glucosamide and a disaccharide ß-GlcNAc3NAcAN-4-ß-Glc-Asn, where the terminal residue is 2,3 di-N-acetyl-glucosamide. The same trisaccharide was also found N-linked to a type IV pilin. The S-layer protein is also extensively modified in the threonine-rich region near the C-terminus with O-glycans composed exclusively of hexoses. While the S-layer protein has a predicted PGF-CTERM processing site, no evidence of a truncated and lipidated C-terminus, the expected product of processing by an archaeosortase, was found. Finally, NMR also identified a polysaccharide expressed by M. marisnigri and composed of a repeating tetrasaccharide unit of [-2-ß-Ribf-3-α-Rha2OMe-3-α-Rha - 2-α-Rha-]. This is the first report of N- and O-glycosylation in an archaeon from the Order Methanomicrobiales.

High-level production of wild-type and oxidation-resistant recombinant alpha-1-antitrypsin in glycoengineered CHO cells.

Alpha-1-antitrypsin (A1AT) is a serine protease inhibitor which blocks the activity of serum proteases including neutrophil elastase to protect the lungs. Its deficiency is known to increase the risk of pulmonary emphysema as well as chronic obstructive pulmonary disease. Currently, the only treatment for patients with A1AT deficiency is weekly injection of plasma-purified A1AT. There is still today no commercial source of therapeutic recombinant A1AT, likely due to significant differences in expression host-specific glycosylation profile and/or high costs associated with the huge therapeutic dose needed. Accordingly, we aimed to produce high levels of recombinant wild-type A1AT, as well as a mutated protein (mutein) version for increased oxidation resistance, with N-glycans analogous to human plasma-derived A1AT. To achieve this, we disrupted two endogenous glycosyltransferase genes controlling core α-1,6-fucosylation (Fut8) and α-2,3-sialylation (ST3Gal4) in CHO cells using CRISPR/Cas9 technology, followed by overexpression of human α-2,6-sialyltransferase (ST6Gal1) using a cumate-inducible expression system. Volumetric A1AT productivity obtained from stable CHO pools was 2.5- to 6.5-fold higher with the cumate-inducible CR5 promoter compared to five strong constitutive promoters. Using the CR5 promoter, glycoengineered stable CHO pools were able to produce over 2.1 and 2.8 g/L of wild-type and mutein forms of A1AT, respectively, with N-glycans analogous to the plasma-derived clinical product Prolastin-C. Supplementation of N-acetylmannosamine to the cell culture media during production increased the overall sialylation of A1AT as well as the proportion of bi-antennary and disialylated A2G2S2 N-glycans. These purified recombinant A1AT proteins showed in vitro inhibitory activity equivalent to Prolastin-C and substitution of methionine residues 351 and 358 with valines rendered A1AT significantly more resistant to oxidation. The recombinant A1AT mutein bearing an improved oxidation resistance described in this study could represent a viable biobetter drug, offering a safe and more stable alternative for augmentation therapy.

Identification of a novel N-linked glycan on the archaellins and S-layer protein of the thermophilic methanogen, Methanothermococcus thermolithotrophicus.

Motility in archaea is facilitated by a unique structure termed the archaellum. N-Glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. The identity of some of these N-glycans has been determined, but archaea exhibit extensive variation in their glycans, meaning that further investigations can shed light not only on the specific details of archaellin structure and function, but also on archaeal glycobiology in general. Here we describe the structural characterization of the N-linked glycan modifications on the archaellins and S-layer protein of Methanothermococcus thermolithotrophicus, a methanogen that grows optimally at 65 °C. SDS-PAGE and MS analysis revealed that the sheared archaella are composed principally of two of the four predicted archaellins, FlaB1 and FlaB3, which are modified with a branched, heptameric glycan at all N-linked sequons except for the site closest to the N termini of both proteins. NMR analysis of the purified glycan determined the structure to be α-d-glycero-d-manno-Hep3OMe6OMe-(1-3)-[α-GalNAcA3OMe-(1-2)-]-ß-Man-(1-4)-[ß-GalA3OMe4OAc6CMe-(1-4)-α-GalA-(1-2)-]-α-GalAN-(1-3)-ß-GalNAc-Asn. A detailed investigation by hydrophilic interaction liquid ion chromatography-MS discovered the presence of several, less abundant glycan variants, related to but distinct from the main heptameric glycan. In addition, we confirmed that the S-layer protein is modified with the same heptameric glycan, suggesting a common N-glycosylation pathway. The M. thermolithotrophicus archaellin N-linked glycan is larger and more complex than those previously identified on the archaellins of related mesophilic methanogens, Methanococcus voltae and Methanococcus maripaludis This could indicate that the nature of the glycan modification may have a role to play in maintaining stability at elevated temperatures.

A novel glycan modifies the flagellar filament proteins of the oral bacterium Treponema denticola.

While protein glycosylation has been reported in several spirochetes including the syphilis bacterium Treponema pallidum and Lyme disease pathogen Borrelia burgdorferi, the pertinent glycan structures and their roles remain uncharacterized. Herein, a novel glycan with an unusual chemical composition and structure in the oral spirochete Treponema denticola, a keystone pathogen of periodontitis was reported. The identified glycan of mass 450.2 Da is composed of a monoacetylated nonulosonic acid (Non) with a novel extended N7 acyl modification, a 2-methoxy-4,5,6-trihydroxy-hexanoyl residue in which the Non has a pseudaminic acid configuration (L-glycero-L-manno) and is ß-linked to serine or threonine residues. This novel glycan modifies the flagellin proteins (FlaBs) of T. denticola by O-linkage at multiple sites near the D1 domain, a highly conserved region of bacterial flagellins that interact with Toll-like receptor 5. Furthermore, mutagenesis studies demonstrate that the glycosylation plays an essential role in the flagellar assembly and motility of T. denticola. To our knowledge, this novel glycan and its unique modification sites have not been reported previously in any bacteria.

Evidence that biosynthesis of the second and third sugars of the archaellin Tetrasaccharide in the archaeon Methanococcus maripaludis occurs by the same pathway used by Pseudomonas aeruginosa to make a di-N-acetylated sugar.

UNLABELLED: Methanococcus maripaludis has two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-ß-ManNAc3NAmA6Thr-1,4-ß-GlcNAc3NAcA-1,3-ß-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354 and mmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions in mmp0350, mmp0351, mmp0352, or mmp0353 were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the Δmmp0352 strain confirmed a glycan with only GalNAc, suggesting mmp0350 to mmp0353 were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The Δmmp0357 mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr). M. maripaludis mmp0350, mmp0351, mmp0352, mmp0353, and mmp0357 are proposed to be functionally equivalent to Pseudomonas aeruginosa wbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of the P. aeruginosa pathway with mmp0357 supports this hypothesis. IMPORTANCE: This work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins of Methanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for archaellum assembly. Pilins are modified with a different N-glycan consisting of the archaellin tetrasaccharide but with an additional hexose attached to the linking sugar. Mass spectrometry analysis of the pili of one mutant strain provided insight into how this different glycan might ultimately be assembled. This study includes a rare example of an archaeal gene functionally replacing a bacterial gene in a complex sugar biosynthesis pathway.

Preparation of legionaminic acid analogs of sialo-glycoconjugates by means of mammalian sialyltransferases.

Legionaminic acids are analogs of sialic acid that occur in several bacteria. The most commonly occurring form is Leg5Ac7Ac, which differs from Neu5Ac only at the C7 (acetamido) and C9 (deoxy) positions. While these differences greatly reduce the susceptibility of Leg compounds to sialidases, several sialyltransferases have been identified that can use CMP-Leg5Ac7Ac as a donor (Watson et al. 2011). We report the successful modification with Leg5Ac7Ac of a glycolipid, GM1a, and two glycoproteins, interferon-α2b and α1-antitrypsin, by means of two mammalian sialyltransferases, namely porcine ST3Gal1 and human ST6Gal1. The Leg5Ac7Ac form of GD1a was not recognized by the myelin-associated glycoprotein (MAG, Siglec-4), confirming the importance of the glycerol moiety in the interaction of sialo-glycans with Siglecs.

A Novel Antigen Design Strategy to Isolate Single-Domain Antibodies that Target Human Nav1.7 and Reduce Pain in Animal Models.

Genetic studies have identified the voltage-gated sodium channel 1.7 (Nav1.7) as pain target. Due to the ineffectiveness of small molecules and monoclonal antibodies as therapeutics for pain, single-domain antibodies (VHHs) are developed against the human Nav1.7 (hNav1.7) using a novel antigen presentation strategy. A 70 amino-acid peptide from the hNav1.7 protein is identified as a target antigen. A recombinant version of this peptide is grafted into the complementarity determining region 3 (CDR3) loop of an inert VHH in order to maintain the native 3D conformation of the peptide. This antigen is used to isolate one VHH able to i) bind hNav1.7, ii) slow the deactivation of hNav1.7, iii) reduce the ability of eliciting action potentials in nociceptors, and iv) reverse hyperalgesia in in vivo rat and mouse models. This VHH exhibits the potential to be developed as a therapeutic capable of suppressing pain. This novel antigen presentation strategy can be applied to develop biologics against other difficult targets such as ion channels, transporters and GPCRs.

Identification of genes involved in the biosynthesis of the third and fourth sugars of the Methanococcus maripaludis archaellin N-linked tetrasaccharide.

N-glycosylation is a protein posttranslational modification found in all three domains of life. Many surface proteins in Archaea, including S-layer proteins, pilins, and archaellins (archaeal flagellins) are known to contain N-linked glycans. In Methanococcus maripaludis, the archaellins are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-ß-ManNAc3NAmA6Thr-1,4-ß-GlcNAc3NAcA-1,3-ß-GalNAc, where Sug is the unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. In this study, four genes--mmp1084, mmp1085, mmp1086, and mmp1087--were targeted to determine their potential involvement of the biosynthesis of the sugar components in the N-glycan, based on bioinformatics analysis and proximity to a number of genes which have been previously demonstrated to be involved in the N-glycosylation pathway. The genes mmp1084 to mmp1087 were shown to be cotranscribed, and in-frame deletions of each gene as well as a Δmmp1086Δmmp1087 double mutant were successfully generated. All mutants were archaellated and motile. Mass spectrometry examination of purified archaella revealed that in Δmmp1084 mutant cells, the threonine linked to the third sugar of the glycan was missing, indicating a putative threonine transferase function of MMP1084. Similar analysis of the archaella of the Δmmp1085 mutant cells demonstrated that the glycan lacked the methyl group at the C-5 position of the terminal sugar, indicating that MMP1085 is a methyltransferase involved in the biosynthesis of this unique sugar. Deletion of the remaining two genes, mmp1086 and mmp1087, either singularly or together, had no effect on the structure of the archaellin N-glycan. Because of their demonstrated involvement in the N-glycosylation pathway, we designated mmp1084 as aglU and mmp1085 as aglV.

Simplifying glycan monitoring of complex antigens such as the SARS-CoV-2 spike to accelerate vaccine development.

Glycosylation is a key quality attribute that must be closely monitored for protein therapeutics. Established assays such as HILIC-Fld of released glycans and LC-MS of glycopeptides work well for glycoproteins with a few glycosylation sites but are less amenable for those with multiple glycosylation sites, resulting in complex datasets that are time consuming to generate and difficult to analyze. As part of efforts to improve preparedness for future pandemics, researchers are currently assessing where time can be saved in the vaccine development and production process. In this context, we evaluated if neutral and acidic monosaccharides analysis via HPAEC-PAD could be used as a rapid and robust alternative to LC-MS and HILIC-Fld for monitoring glycosylation between protein production batches. Using glycoengineered spike proteins we show that the HPAEC-PAD monosaccharide assays could quickly and reproducibly detect both major and minor glycosylation differences between batches. Moreover, the monosaccharide results aligned well with those obtained by HILIC-Fld and LC-MS.

A glyco-engineering approach for site-specific conjugation to Fab glycans.

Effective processes for synthesizing antibody-drug conjugates (ADCs) require: 1) site-specific incorporation of the payload to avoid interference with binding to the target epitope, 2) optimal drug/antibody ratio to achieve sufficient potency while avoiding aggregation or solubility problems, and 3) a homogeneous product to facilitate approval by regulatory agencies. In conventional ADCs, the drug molecules are chemically attached randomly to antibody surface residues (typically Lys or Cys), which can interfere with epitope binding and targeting, and lead to overall product heterogeneity, long-term colloidal instability and unfavorable pharmacokinetics. Here, we present a more controlled process for generating ADCs where drug is specifically conjugated to only Fab N-linked glycans in a narrow ratio range through functionalized sialic acids. Using a bacterial sialytransferase, we incorporated N-azidoacetylneuraminic acid (Neu5NAz) into the Fab glycan of cetuximab. Since only about 20% of human IgG1 have a Fab glycan, we extended the application of this approach by using molecular modeling to introduce N-glycosylation sites in the Fab constant region of other therapeutic monoclonal antibodies. We used trastuzumab as a model for the incorporation of Neu5NAz in the novel Fab glycans that we designed. ADCs were generated by clicking the incorporated Neu5NAz with monomethyl auristatin E (MMAE) attached to a self-immolative linker terminated with dibenzocyclooctyne (DBCO). Through this process, we obtained cetuximab-MMAE and trastuzumab-MMAE with drug/antibody ratios in the range of 1.3 to 2.5. We confirmed that these ADCs still bind their targets efficiently and are as potent in cytotoxicity assays as control ADCs obtained by standard conjugation protocols. The site-directed conjugation to Fab glycans has the additional benefit of avoiding potential interference with effector functions that depend on Fc glycan structure.

Virion glycosylation influences mycobacteriophage immune recognition.

Glycosylation of eukaryotic virus particles is common and influences their uptake, trafficking, and immune recognition. In contrast, glycosylation of bacteriophage particles has not been reported; phage virions typically do not enter the cytoplasm upon infection, and they do not generally inhabit eukaryotic systems. We show here that several genomically distinct phages of Mycobacteria are modified with glycans attached to the C terminus of capsid and tail tube protein subunits. These O-linked glycans influence antibody production and recognition, shielding viral particles from antibody binding and reducing production of neutralizing antibodies. Glycosylation is mediated by phage-encoded glycosyltransferases, and genomic analysis suggests that they are relatively common among mycobacteriophages. Putative glycosyltransferases are also encoded by some Gordonia and Streptomyces phages, but there is little evidence of glycosylation among the broader phage population. The immune response to glycosylated phage virions in mice suggests that glycosylation may be an advantageous property for phage therapy of Mycobacterium infections.

Preclinical evaluation of manufacturable SARS-CoV-2 spike virus-like particles produced in Chinese Hamster Ovary cells.

BACKGROUND: As the COVID-19 pandemic continues to evolve, novel vaccines need to be developed that are readily manufacturable and provide clinical efficacy against emerging SARS-CoV-2 variants. Virus-like particles (VLPs) presenting the spike antigen at their surface offer remarkable benefits over other vaccine antigen formats; however, current SARS-CoV-2 VLP vaccines candidates in clinical development suffer from challenges including low volumetric productivity, poor spike antigen density, expression platform-driven divergent protein glycosylation and complex upstream/downstream processing requirements. Despite their extensive use for therapeutic protein manufacturing and proven ability to produce enveloped VLPs, Chinese Hamster Ovary (CHO) cells are rarely used for the commercial production of VLP-based vaccines. METHODS: Using CHO cells, we aimed to produce VLPs displaying the full-length SARS-CoV-2 spike. Affinity chromatography was used to capture VLPs released in the culture medium from engineered CHO cells expressing spike. The structure, protein content, and glycosylation of spikes in VLPs were characterized by several biochemical and biophysical methods. In vivo, the generation of neutralizing antibodies and protection against SARS-CoV-2 infection was tested in mouse and hamster models. RESULTS: We demonstrate that spike overexpression in CHO cells is sufficient by itself to generate high VLP titers. These VLPs are evocative of the native virus but with at least three-fold higher spike density. In vivo, purified VLPs elicit strong humoral and cellular immunity at nanogram dose levels which grant protection against SARS-CoV-2 infection. CONCLUSIONS: Our results show that CHO cells are amenable to efficient manufacturing of high titers of a potently immunogenic spike protein-based VLP vaccine antigen.

Virus-like particles (VLPs) have a structure that is similar to viruses but they cannot cause infection or illness. If VLPs are injected into the body they produce an immune response similar to that seen following infection by a virus. This means that VLPs can be used as vaccines against viruses that cause illness in people. Many drugs, named biologics, are manufactured using living cells, including cells that were originally derived from Chinese Hamster Ovaries (CHO cells). We developed a simple method to produce VLPs similar to the SARS-CoV-2 virus in CHO cells. We show that vaccination of rodents with these VLPs prevents them from becoming ill following infection with SARS-CoV-2. These VLPs could become a part of an alternative, easily produced vaccine for the prevention of COVID-19 in humans.

Identification of genes involved in the acetamidino group modification of the flagellin N-linked glycan of Methanococcus maripaludis.

N-linked glycosylation of protein is a posttranslational modification found in all three domains of life. The flagellin proteins of the archaeon Methanococcus maripaludis are known to be modified with an N-linked tetrasaccharide consisting of N-acetylgalactosamine (GalNAc), a diacetylated glucuronic acid (GlcNAc3NAc), an acetylated and acetamidino-modified mannuronic acid with a substituted threonine group (ManNAc3NAmA6Thr), and a novel terminal sugar residue [(5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose]. To identify genes involved in biosynthesis of the component sugars of this glycan, three genes, mmp1081, mmp1082, and mmp1083, were targeted for in-frame deletion, based on their annotation and proximity to glycosyltransferase genes known to be involved in assembly of the glycan. Mutants carrying a deletion in any of these three genes remained flagellated and motile. A strain with a deletion of mmp1081 had lower-molecular-mass flagellins in Western blots. Mass spectrometry of purified flagella revealed a truncated glycan with the terminal sugar absent and the threonine residue and the acetamidino group missing from the third sugar. No glycan modification was seen in either the Δmmp1082 or Δmmp1083 mutant grown in complex Balch III medium. However, a glycan identical to the Δmmp1081 glycan was observed when the Δmmp1082 or Δmmp1083 mutant was grown under ammonia-limited conditions. We hypothesize that MMP1082 generates ammonia and tunnels it through MMP1083 to MMP1081, which acts as the amidotransferase, modifying the third sugar residue of the M. maripaludis glycan with the acetamidino group.

Cell surface glycoproteins from Thermoplasma acidophilum are modified with an N-linked glycan containing 6-C-sulfofucose.

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH 2 and 59°C. This extremophile is remarkable by the absence of a cell wall or an S-layer. Treating the cells with Triton X-100 at pH 3 allowed the extraction of all of the cell surface glycoproteins while keeping cells intact. The extracted glycoproteins were partially purified by cation-exchange chromatography, and we identified five glycoproteins by N-terminal sequencing and mass spectrometry of in-gel tryptic digests. These glycoproteins are positive for periodic acid-Schiff staining, have a high content of Asn including a large number in the Asn-X-Ser/Thr sequon and have apparent masses that are 34-48% larger than the masses deduced from their amino acid sequences. The pooled glycoproteins were digested with proteinase K and the purified glycopeptides were analyzed by NMR. Structural determination showed that the carbohydrate part was represented by two structures in nearly equal amounts, differing by the presence of one terminal mannose residue. The larger glycan chain consists of eight residues: six hexoses, one heptose and one sugar with an unusual residue mass of 226 Da which was identified as 6-deoxy-6-C-sulfo-D-galactose (6-C-sulfo-D-fucose). Mass spectrometry analyses of the peptides obtained by trypsin and chymotrypsin digestion confirmed the principal structures to be those determined by NMR and identified 14 glycopeptides derived from the main glycoprotein, Ta0280, all containing the Asn-X-Ser/Thr sequons. Thermoplasma acidophilum appears to have a "general" protein N-glycosylation system that targets a number of cell surface proteins.

Genetic and mass spectrometry analyses of the unusual type IV-like pili of the archaeon Methanococcus maripaludis.

The structure of pili from the archaeon Methanococcus maripaludis is unlike that of any bacterial pili. However, genetic analysis of the genes involved in the formation of these pili has been lacking until this study. Pili were isolated from a nonflagellated (ΔflaK) mutant and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist primarily of subunits with an apparent molecular mass of 17 kDa. In-frame deletions were created in three genes, MMP0233, MMP0236, and MMP0237, which encode proteins with bacterial type IV pilin-like signal peptides previously identified by in silico methodology as likely candidates for pilus structural proteins. Deletion of MMP0236 or MMP0237 resulted in mutant cells completely devoid of pili on the cell surface, while deletion of the third pilin-like gene, MMP0233, resulted in cells greatly reduced in the number of pili on the surface. Complementation with the deleted gene in each case returned the cells to a piliated state. Surprisingly, mass spectrometry analysis of purified pili identified the major structural pilin as another type IV pilin-like protein, MMP1685, whose gene is located outside the first pilus locus. This protein was found to be glycosylated with an N-linked branched pentasaccharide glycan. Deletion and complementation analysis confirmed that MMP1685 is required for piliation.

Identification of vascular breast tumor markers by laser capture microdissection and label-free LC-MS.

Blood vessels in tumors frequently show abnormal characteristics, such as tortuous morphology or leakiness, but very little is known about protein expression in tumor vessels. In this study, we have used laser capture microdissection (LCM) to isolate microvessels from clinical samples of invasive ductal carcinoma (IDC), the most common form of malignant breast cancer, and from patient-matched adjacent nonmalignant tissue. This approach eliminates many of the problems associated with the heterogeneity of clinical tumor tissues by controlling for differences in protein expression between both individual patients and different cell types. Proteins from the microvessels were trypsinized and the resulting peptides were quantified by a label-free nanoLC-MS method. A total of 86 proteins were identified that are overexpressed in tumor vessels relative to vessels isolated from the adjacent nonmalignant tissue. These proteins include well-known breast tumor markers such as Periostin and Tenascin C but also proteins with lesser-known or emerging roles in breast cancer and tumor angiogenesis (i.e., Serpin H1, Clic-1, and Transgelin 2). We also identified 40 proteins that were relatively under-expressed in IDC tumor vessels, including several components of the basement membrane whose lower expression could be responsible for weakening tumor vessels. Lastly, we show that a subset of 29 proteins, derived from our list of differentially expressed proteins, is able to predict survival in three publicly available clinical breast cancer microarray data sets, which suggests that this subset of proteins likely plays a functional role in cancer progression and outcome.