Glycoproteomics of a Single Protein: Revealing Tens of Thousands of Myozyme Glycoforms by Hybrid HPLC-MS Approaches.

Characterization of highly glycosylated biopharma-ceuticals by mass spectrometry is challenging because of the huge chemical space of coexistent glycoforms present. Here, we report the use of an array of HPLC-mass spectrometry-based approaches at different structural levels of released glycan, glycopeptide, and hitherto unexplored intact glycoforms to scrutinize the biopharmaceutical Myozyme, containing the highly complex lysosomal enzyme recombinant acid α-glucosidase. The intrinsic heterogeneity of recombinant acid α-glucosidase glycoforms was unraveled using a novel strong anion exchange HPLC-mass spectrometry approach involving a pH-gradient of volatile buffers to facilitate chromatographic separation of glycoforms based on their degree of sialylation, followed by the acquisition of native mass spectra in an Orbitrap mass spectrometer. Upon considering the structures of 60 different glycans attached to seven glycosylation sites in the intact protein, the large set of interdependent data acquired at different structural levels was integrated using a set of bioinformatic tools and allowed the annotation of intact glycoforms unraveling more than 1,000,000 putative intact glycoforms. Detectable isoforms also included several mannose-6-phosphate variants, which are essential for directing the drug toward its target, the lysosomes. Finally, for the first time, we sought to validate the intact glycoform annotations by integrating experimental data on the enzymatically dissected proteoforms, which reduced the number of glycoforms supported by experimental evidence to 42,104. The latter verification clearly revealed the strengths but also intrinsic limitations of this approach for fully characterizing such highly complex glycoproteins by mass spectrometry.

Structure-function relationship of a novel fucoside-binding fruiting body lectin from Coprinopsis cinerea exhibiting nematotoxic activity.

Lectins are non-immunoglobulin-type proteins that bind to specific carbohydrate epitopes and play important roles in intra- and inter-organismic interactions. Here, we describe a novel fucose-specific lectin, termed CML1, which we identified from fruiting body extracts of Coprinopsis cinerea. For further characterization, the coding sequence for CML1 was cloned and heterologously expressed in Escherichia coli. Feeding of CML1-producing bacteria inhibited larval development of the bacterivorous nematode Caenorhabditis tropicalis, but not of C. elegans. The crystal structure of the recombinant protein in its apo-form and in complex with H type I or Lewis A blood group antigens was determined by X-ray crystallography. The protein folds as a sandwich of 2 antiparallel ß-sheets and forms hexamers resulting from a trimer of dimers. The hexameric arrangement was confirmed by small-angle X-ray scattering (SAXS). One carbohydrate-binding site per protomer was found at the dimer interface with both protomers contributing to ligand binding, resulting in a hexavalent lectin. In terms of lectin activity of recombinant CML1, substitution of the carbohydrate-interacting residues His54, Asn55, Trp94, and Arg114 by Ala abolished carbohydrate-binding and nematotoxicity. Although no similarities to any characterized lectin were found, sequence alignments identified many non-characterized agaricomycete proteins. These results suggest that CML1 is the founding member of a novel family of fucoside-binding lectins involved in the defense of agaricomycete fruiting bodies against predation by fungivorous nematodes.

Exploring the Chemical Space of Protein Glycosylation in Noncovalent Protein Complexes: An Expedition along Different Structural Levels of Human Chorionic Gonadotropin by Employing Mass Spectrometry.

Modern analytical approaches employing high-resolution mass spectrometry (MS) facilitate the generation of a vast amount of structural data of highly complex glycoproteins. Nevertheless, systematic interpretation of this data at different structural levels remains an analytical challenge. The glycoprotein utilized as a model system in this study, human chorionic gonadotropin (hCG), exists as a heterodimer composed of two heavily glycosylated subunits. In order to unravel the multitude of glycoforms of recombinant hCG (drug product Ovitrelle), we combine established techniques, such as released glycan and glycopeptide analysis, with novel approaches employing high-performance liquid chromatography-mass spectrometry (HPLC-MS) to characterize protein subunits and native MS to analyze the noncovalent hCG complex. Starting from the deconvoluted mass spectrum of dimeric hCG comprising about 50 signals, it was possible to explore the chemical space of hCG glycoforms and elucidate the complexity that hides behind just 50 signals. Systematic, stepwise integration of data obtained at the levels of released glycans, glycopeptides, and subunits using a computational annotation tool allowed us to reveal 1031 underlying glycoforms. Additionally, critical quality attributes such as sialylation and core fucosylation were compared for two batches of Ovitrelle to assess the potential product variability.

How many human proteoforms are there?

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

Exploring sample preparation and data evaluation strategies for enhanced identification of host cell proteins in drug products of therapeutic antibodies and Fc-fusion proteins.

Manufacturing of biopharmaceuticals involves recombinant protein expression in host cells followed by extensive purification of the target protein. Yet, host cell proteins (HCPs) may persist in the final drug product, potentially reducing its quality with respect to safety and efficacy. Consequently, residual HCPs are closely monitored during downstream processing by techniques such as enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS). The latter is especially attractive as it provides information with respect to protein identities. Although the applied HPLC-MS/MS methodologies are frequently optimized with respect to HCP identification, acquired data is typically analyzed using standard settings. Here, we describe an improved strategy for evaluating HPLC-MS/MS data of HCP-derived peptides, involving probabilistic protein inference and peptide detection in the absence of fragment ion spectra. This data analysis workflow was applied to data obtained for drug products of various biotherapeutics upon protein A affinity depletion. The presented data evaluation strategy enabled in-depth comparative analysis of the HCP repertoires identified in drug products of the monoclonal antibodies rituximab and bevacizumab, as well as the fusion protein etanercept. In contrast to commonly applied ELISA strategies, the here presented workflow is process-independent and may be implemented into existing HPLC-MS/MS setups for drug product characterization and process development. Graphical abstract.

A Simple Strategy to Eliminate Hexosylation Bias in the Relative Quantification of N-Glycosylation in Biopharmaceuticals.

N-glycosylation may affect the safety and efficacy of biopharmaceuticals and is thus monitored during manufacturing. Mass spectrometry of the intact protein is increasingly used to reveal co-existing glycosylation variants. However, quantification of N-glycoforms via this approach may be biased by single hexose residues as introduced by glycation or O-glycosylation. Herein, we describe a simple strategy to reveal actual N-glycoform abundances of therapeutic antibodies, involving experimental determination of glycation levels followed by computational elimination of the "hexosylation bias". We show that actual N-glycoform abundances may significantly deviate from initially determined values. Indeed, glycation may even obscure considerable differences in N-glycosylation patterns of drug product batches. Our observations may thus have implications for biopharmaceutical quality control. Moreover, we solve an instance of the problem of isobaricity, which is fundamental to mass spectrometry.

MoFi: A Software Tool for Annotating Glycoprotein Mass Spectra by Integrating Hybrid Data from the Intact Protein and Glycopeptide Level.

Hybrid mass spectrometry (MS) is an emerging technique for characterizing glycoproteins, which typically display pronounced microheterogeneity. Since hybrid MS combines information from different experimental levels, it crucially depends on computational methods. Here, we describe a novel software tool, MoFi, which integrates hybrid MS data to assign glycans and other post-translational modifications (PTMs) in deconvoluted mass spectra of intact proteins. Its two-stage search algorithm first assigns monosaccharide/PTM compositions to each peak and then compiles a hierarchical list of glycan combinations compatible with these compositions. Importantly, the program only includes those combinations which are supported by a glycan library as derived from glycopeptide or released glycan analysis. By applying MoFi to mass spectra of rituximab, ado-trastuzumab emtansine, and recombinant human erythropoietin, we demonstrate how integration of bottom-up data may be used to refine information collected at the intact protein level. Accordingly, our software reveals that a single mass frequently can be explained by a considerable number of glycoforms. Yet, it simultaneously ranks proteoforms according to their probability, based on a score which is calculated from relative glycan abundances. Notably, glycoforms that comprise identical glycans may nevertheless differ in score if those glycans occupy different sites. Hence, MoFi exposes different layers of complexity that are present in the annotation of a glycoprotein mass spectrum.

A Generic HPLC Method for Absolute Quantification of Oxidation in Monoclonal Antibodies and Fc-Fusion Proteins Using UV and MS Detection.

Oxidation of biopharmaceuticals may affect their bioactivity, serum half-life, and (bio)chemical stability. The Fc domain of IgG monoclonal antibodies (mAbs) contains two methionine residues which are susceptible to oxidation. Here, we present a middle-down approach employing the cysteine protease IdeS under reducing conditions to obtain three mAb subunits of approximately 25 kDa: Fc/2, Fd', and LC. These subunits were separated by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) and detected by UV spectroscopy as well as Orbitrap mass spectrometry (MS), as well as MS upon all-ion fragmentation (AIF-MS). We evaluated the feasibility of three strategies for absolute quantification of oxidation in the Fc region of hydrogen peroxide-stressed Rituximab, using a single, commercially available software platform both for data acquisition and evaluation: UV spectroscopy, full-scan MS, and monitoring of product ions obtained by AIF-MS. UV spectroscopy showed the lowest limits of quantification (LOQ) (0.96 ng µL-1) and featured the lowest relative process standard deviation (Vx0%) of 7.2% compared to MS and AIF-MS with LOQs of 1.24-4.32 ng µL-1 and relative process standard deviations of 9.0-14%, respectively. Our approach is generic in that it allows monitoring and quantification of oxidation in the Fc regions of fully human and humanized IgG1 mAbs as well as of Fc-fusion proteins. This is exemplified by limits of detection of 1.2%, 1.0%, and 1.2% of oxidation in drug products containing the biopharmaceuticals Rituximab, Adalimumab, and Etanercept, respectively. The presented method is an attractive alternative to conventional time-intensive peptide mapping which is prone to artificial oxidation due to extensive sample preparation.

Methylated glycans as conserved targets of animal and fungal innate defense.

Effector proteins of innate immune systems recognize specific non-self epitopes. Tectonins are a family of ß-propeller lectins conserved from bacteria to mammals that have been shown to bind bacterial lipopolysaccharide (LPS). We present experimental evidence that two Tectonins of fungal and animal origin have a specificity for O-methylated glycans. We show that Tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) agglutinates Gram-negative bacteria and exerts toxicity toward the model nematode Caenorhabditis elegans, suggesting a role in fungal defense against bacteria and nematodes. Biochemical and genetic analysis of these interactions revealed that both bacterial agglutination and nematotoxicity of Lb-Tec2 depend on the recognition of methylated glycans, namely O-methylated mannose and fucose residues, as part of bacterial LPS and nematode cell-surface glycans. In addition, a C. elegans gene, termed samt-1, coding for a candidate membrane transport protein for the presumptive donor substrate of glycan methylation, S-adenosyl-methionine, from the cytoplasm to the Golgi was identified. Intriguingly, limulus lectin L6, a structurally related antibacterial protein of the Japanese horseshoe crab Tachypleus tridentatus, showed properties identical to the mushroom lectin. These results suggest that O-methylated glycans constitute a conserved target of the fungal and animal innate immune system. The broad phylogenetic distribution of O-methylated glycans increases the spectrum of potential antagonists recognized by Tectonins, rendering this conserved protein family a universal defense armor.

Identification of the galactosyltransferase of Cryptococcus neoformans involved in the biosynthesis of basidiomycete-type glycosylinositolphosphoceramide.

The pathogenic fungus Cryptococcus neoformans synthesizes a complex family of glycosylinositolphosphoceramide (GIPC) structures. These glycosphingolipids (GSLs) consist of mannosylinositolphosphoceramide (MIPC) extended by ß1-6-linked galactose, a unique structure that has to date only been identified in basidiomycetes. Further extension by up to five mannose residues and a branching xylose has been described. In this study, we identified and determined the gene structure of the enzyme Ggt1, which catalyzes the transfer of a galactose residue to MIPC. Deletion of the gene in C. neoformans resulted in complete loss of GIPCs containing galactose, a phenotype that could be restored by the episomal expression of Ggt1 in the deletion mutant. The entire annotated open reading frame, encoding a C-terminal GT31 galactosyltransferase domain and a large N-terminal domain of unknown function, was required for complementation. Notably, this gene does not encode a predicted signal sequence or transmembrane domain. The demonstration that Ggt1 is responsible for the transfer of a galactose residue to a GSL thus raises questions regarding the topology of this biosynthetic pathway and the function of the N-terminal domain. Phylogenetic analysis of the GGT1 gene shows conservation in hetero- and homobasidiomycetes but no homologs in ascomycetes or outside of the fungal kingdom.

Nematotoxicity of Marasmius oreades agglutinin (MOA) depends on glycolipid binding and cysteine protease activity.

Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a Galα1,3Gal/GalNAc-specific lectin from the fairy ring mushroom that consists of an N-terminal ricin B-type lectin domain and a C-terminal dimerization domain. The latter domain shows structural similarity to catalytically active proteins, suggesting that, in addition to its carbohydrate-binding activity, MOA has an enzymatic function. Here, we demonstrate toxicity of MOA toward the model nematode Caenorhabditis elegans. This toxicity depends on binding of MOA to glycosphingolipids of the worm via its lectin domain. We show further that MOA has cysteine protease activity and demonstrate a critical role of this catalytic function in MOA-mediated nematotoxicity. The proteolytic activity of MOA was dependent on high Ca(2+) concentrations and favored by slightly alkaline pH, suggesting that these conditions trigger activation of the toxin at the target location. Our results suggest that MOA is a fungal toxin with intriguing similarities to bacterial binary toxins and has a protective function against fungivorous soil nematodes.

Burkavidin: a novel secreted biotin-binding protein from the human pathogen Burkholderia pseudomallei.

The avidin-biotin technology has many applications, including molecular detection; immobilization; protein purification; construction of supramolecular assemblies and artificial metalloenzymes. Here we present the recombinant expression of novel biotin-binding proteins from bacteria and the purification and characterization of a secreted burkavidin from the human pathogen Burkholderia pseudomallei. Expression of the native burkavidin in Escherichia coli led to periplasmic secretion and formation of a biotin-binding, thermostable, tetrameric protein containing an intra-monomeric disulphide bond. Burkavidin showed one main species as measured by isoelectric focusing, with lower isoelectric point (pI) than streptavidin. To exemplify the potential use of burkavidin in biotechnology, an artificial metalloenzyme was generated using this novel protein-scaffold and shown to exhibit enantioselectivity in a rhodium-catalysed hydrogenation reaction.

Chemo-genetic optimization of DNA recognition by metallodrugs using a presenter-protein strategy.

The mode of action of precious metal anticancer metallodrugs is generally believed to involve DNA as a target. However, the poor specificity of such drugs often requires high doses and leads to undesirable side-effects. With the aim of improving the specificity of a ruthenium piano-stool complex towards DNA, we employed a presenter protein strategy based on the biotin-avidin technology. Guided by the X-ray structure of the assembly of streptavidin and a biotinylated piano-stool, we explored the formation of metallodrug-mediated ternary complexes with the presenter protein and DNA. The assemblies bound more strongly to telomere G-quadruplexes than to double-stranded DNA; chemo-genetic modifications (varying the complex or mutating the protein) modulated binding to these targets. We suggest that rational targeting of small molecules by presenter proteins could be exploited to bind metallodrugs to preferred macromolecular targets.

Expression, Purification, and Functional Characterization of Tectonin 2 from Laccaria bicolor: A Six-Bladed Beta-Propeller Lectin Specific for O-Methylated Glycans.

Tectonins are conserved defense proteins of innate immune systems featuring a ß-propeller fold. Tectonin 2 from Laccaria bicolor, Lb-Tec2, is the first fungal representative of the tectonin superfamily that has been described. In-depth characterization revealed a specificity for O-methylated glycans and identified a unique sequence motif and binding site architecture underlying this unusual specificity. This chapter provides information on how to produce and purify recombinant Lb-Tec2, characterize its interaction with O-methylated glycans and demonstrate its biological function.

Towards middle-up analysis of polyclonal antibodies: subclass-specific N-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS.

In recent years, advanced HPLC-MS strategies based on intact protein ("top-down") or protein subunit ("middle-up/middle-down") analysis have been implemented for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up/middle-down analysis for polyclonal IgGs exhibiting extensive sequence variability. Specifically, we addressed IgGs from mouse, representing an important model system in immunological investigations. To obtain Fc/2 portions as conserved subunits of IgGs, we made use of the bacterial protease SpeB. For this purpose, we initially determined SpeB cleavage sites in murine IgGs. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry. This enabled simultaneous relative quantification of IgG subclasses and their N-glycosylation variants, both of which influence IgG effector functions. To assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. The study revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts, respectively. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine, and may thus be implemented for quality control of functional antibodies.

Dilute-and-shoot analysis of therapeutic monoclonal antibody variants in fermentation broth: a method capability study.

Monoclonal antibodies (mAbs) are widely applied as highly specific and efficient therapeutic agents for various medical conditions, including cancer, inflammatory and autoimmune diseases. As protein production in cellular systems inherently generates a multitude of molecular variants, manufacturing of mAbs requires stringent control in order to ensure safety and efficacy of the drugs. Moreover, monitoring of mAb variants in the course of the fermentation process may allow instant tuning of process parameters to maintain optimal cell culture conditions. Here, we describe a fast and robust workflow for the characterization of mAb variants in fermentation broth. Sample preparation is minimal in that the fermentation broth is shortly centrifuged before dilution and HPLC-MS analysis in a short 15-min gradient run. In a single analysis, N-glycosylation and truncation variants of the expressed mAb are identified at the intact protein level. Simultaneously, absolute quantification of mAb content in fermentation broth is achieved. The whole workflow features excellent robustness as well as retention time and peak area stability. Additional enzymatic removal of N-glycans enables determination of mAb glycation levels, which are subsequently considered in relative N-glycoform quantification to correct for isobaric galactosylation. Several molecular attributes of the expressed therapeutic protein may thus be continuously monitored to ensure the desired product profile. Application of the described workflow in an industrial environment may therefore substantially enhance in-process control in mAb production, as well as targeted biosimilar development.

Crystal Structures of Fungal Tectonin in Complex with O-Methylated Glycans Suggest Key Role in Innate Immune Defense.

Innate immunity is the first line of defense against pathogens and predators. To initiate a response, it relies on the detection of invaders, where lectin-carbohydrate interactions play a major role. O-Methylated glycans were previously identified as non-self epitopes and conserved targets for defense effector proteins belonging to the tectonin superfamily. Here, we present two crystal structures of Tectonin 2 from the mushroom Laccaria bicolor in complex with methylated ligands, unraveling the molecular basis for this original specificity. Furthermore, they revealed the formation of a ball-shaped tetramer with 24 binding sites distributed at its surface, resembling a small virus capsid. Based on the crystal structures, a methylation recognition motif was identified and found in the sequence of many tectonins from bacteria to human. Our results support a key role of tectonins in innate defense based on a distinctive and conserved type of lectin-glycan interaction.

Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals.

Robust manufacturing processes resulting in consistent glycosylation are critical for the efficacy and safety of biopharmaceuticals. Information on glycosylation can be obtained by conventional bottom-up methods but is often limited to the glycan or glycopeptide level. Here, we apply high-resolution native mass spectrometry (MS) for the characterization of the therapeutic fusion protein Etanercept to unravel glycoform heterogeneity in conditions of hitherto unmatched mass spectral complexity. Higher spatial resolution at lower charge states, an inherent characteristic of native MS, represents a key component for the successful revelation of glycan heterogeneity. Combined with enzymatic dissection using a set of proteases and glycosidases, assignment of specific glycoforms is achieved by transferring information from subunit to whole protein level. The application of native mass spectrometric analysis of intact Etanercept as a fingerprinting tool for the assessment of batch-to-batch variability is exemplified and may be extended to demonstrate comparability after changes in the biologic manufacturing process.

Membrane cholesterol and sphingomyelin, and ostreolysin A are obligatory for pore-formation by a MACPF/CDC-like pore-forming protein, pleurotolysin B.

The mushroom Pleurotus ostreatus has been reported to produce the hemolytic proteins ostreolysin (OlyA), pleurotolysin A (PlyA) and pleurotolysin B (PlyB). The present study of the native and recombinant proteins dissects out their lipid-binding characteristics and their roles in lipid binding and membrane permeabilization. Using lipid-binding studies, permeabilization of erythrocytes, large unilamellar vesicles of various lipid compositions, and electron microscopy, we show that OlyA, a PlyA homolog, preferentially binds to membranes rich in sterol and sphingomyelin, but it does not permeabilize them. The N-terminally truncated Δ48PlyB corresponds to the mature and active form of native PlyB, and it has a membrane attack complex-perforin (MACPF) domain. Δ48PlyB spontaneously oligomerizes in solution, and binds weakly to various lipid membranes but is not able to perforate them. However, binding of Δ48PlyB to the cholesterol and sphingomyelin membranes, and consequently, their permeabilization is dramatically promoted in the presence of OlyA. On these membranes, Δ48PlyB and OlyA form predominantly 13-meric oligomers. These are rosette-like structures with a thickness of ∼9 nm from the membrane surface, with 19.7 nm and 4.9 nm outer and inner diameters, respectively. When present on opposing vesicle membranes, these oligomers can dimerize and thus promote aggregation of vesicles. Based on the structural and functional characteristics of Δ48PlyB, we suggest that it shares some features with MACPF/cholesterol-dependent cytolysin (CDC) proteins. OlyA is obligatory for the Δ48PlyB permeabilization of membranes rich in cholesterol and sphingomyelin.

Inhibition of Aspergillus fumigatus conidia binding to extracellular matrix proteins by sialic acids: a pH effect?

Infection by Aspergillus fumigatus, which causes the life-threatening disease invasive aspergillosis, begins with the inhalation of conidia that adhere to and germinate in the lung. Previous studies have shown that A. fumigatus conidia express high levels of the negatively charged 9-carbon sugar sialic acid, and that sialic acid appears to mediate the binding of A. fumigatus conidia to basal lamina proteins. However, despite the ability of sialic acid to inhibit adherence of A. fumigatus conidia, the exact mechanism by which this binding occurs remains unresolved. Utilizing various free sialic acids and other carbohydrates, sialic acid derivatives, sialoglycoconjugates, glycoproteins, alpha-keto acid related compounds and amino acids we have found that the binding of A. fumigatus conidia to type IV collagen and fibrinogen was inhibited by (i) glycoproteins (in a sialic acid-independent manner), and (ii) free sialic acids, glucuronic acid and alpha-keto acid related compounds. However, inhibition by the latter was found to be the result of a shift in pH from neutral (pH 7.4) to acidic (less than pH 4.6) induced by the relatively high concentrations of free sialic acids, glucuronic acid and alpha-keto acid related compounds used in the binding assays. This suggests that previous reports describing inhibition of A. fumigatus conidia binding by free sialic acid may actually be due to a pH shift similar to that shown here. As previously reported, we found that A. fumigatus conidia express only N-acetylneuraminic acid, the most common sialic acid found in nature. However, A. fumigatus appears to do so by an alternative mechanism to that seen in other organisms. We report here that A. fumigatus (i) does not incorporate sialic acid obtained from the environment, (ii) does not synthesize and incorporate sialic acid from exogenous N-acetylmannosamine, and (iii) lacks homologues of known sialic acid biosynthesizing enzymes.