Biology subjects

Goddard-Borger, E. D.

Publications and source records attributed to Goddard-Borger, E. D..

10 recordsLinked to original sources

bioRxiv2025

Uncovering bacterial pseudaminylation with pan-specific antibody tools

Pseudaminic acids (Pse) are a family of carbohydrates found within bacterial lipopolysaccharides, capsular polysaccharides and glycoproteins that are critical for the virulence of human pathogens. However, a dearth of effective tools for detecting and enriching Pse has restricted study to only the most abundant Pse-containing glycoconjugates. Here, we devise a synthesis of - and {beta}-O-pseudaminylated glycopeptides to generate pan-specific monoclonal antibodies (mAbs) that recognise - and {beta}-configured Pse and its C8 epimer (8ePse) presented within glycans or directly linked to polypeptide backbones. Structural characterisation reveals the molecular basis of Pse recognition across a range of diverse chemical contexts. Using these mAbs, we establish a glycoproteomic platform to provide unprecedented depth in mapping the Pse glycome of Helicobacter pylori, Campylobacter jejuni, and Acinetobacter baumannii strains. Finally, we demonstrate that the mAbs recognise diverse capsule types in multidrug-resistant Acinetobacter baumannii and enhance phagocytosis to eliminate infections in mice.

biochemistry↗

bioRxiv2024

The RBR E3 ubiquitin ligase HOIL-1 can ubiquitinate diverse non-proteinaceous substrates in vitro

HOIL-1 is a RING-between-RING (RBR)-family E3 ubiquitin ligase and component of the linear ubiquitin chain assembly complex (LUBAC). While most E3 ubiquitin ligases conjugate ubiquitin to protein lysine sidechains, HOIL-1 has been reported to ubiquitinate hydroxyl groups in protein serine and threonine sidechains and glucosaccharides, such as glycogen and its building block maltose, in vitro. However, HOIL-1 substrate specificity is currently poorly defined. Here we show that HOIL-1 is unable to ubiquitinate lysine but can efficiently ubiquitinate serine as well as a variety of model and physiologically relevant di- and monosaccharides in vitro. We identify a critical catalytic histidine residue, His510, in the flexible catalytic site of HOIL-1 that enables this O-linked ubiquitination and prohibits ubiquitin discharge onto lysine sidechains. Finally, we utilise HOIL-1s in vitro non-proteinaceous ubiquitination activity and an engineered, constitutively active HOIL-1 variant to produce preparative amounts of different ubiquitinated saccharides that can be used as tool compounds and standards in the rapidly emerging field of non-proteinaceous ubiquitination.

biochemistry↗

bioRxiv2024

The molecular basis of immunosuppression by soluble CD52 is defined by interactions of N-linked and O-linked glycans with HMGB1 Box B

AbstractHuman soluble CD52 is a short glycopeptide comprising 12 amino acids (GQNDTSQTSSPS) which functions as an immune regulator by sequestering the pro-inflammatory high mobility group box protein 1 (HMGB1) and supressing immune responses. Recombinant CD52 has been shown to act as a broad anti-inflammatory agent, dampening both adaptive and innate immune responses. This short glycopeptide is heavily glycosylated, with a complex sialylated N-linked glycan at N3 and reported O- linked glycosylation possible on several serine and threonine residues. Previously we demonstrated that specific glycosylation features of CD52 are essential for its immunosuppressive function, with terminal -2,3-linked sialic acids required for binding to the inhibitory SIGLEC-10 receptor leading to T-cell suppression. Using high resolution mass spectrometry, we have further characterised the N- and O-linked glycosylation of Expi293 recombinantly produced CD52 at a glycopeptide and released glycan level, accurately determining glycan heterogeneity of both N- and O-linked glycosylation, and localising the site of O-glycosylation to T8 with high confidence and direct spectral evidence. This detailed knowledge of CD52 glycosylation informed the construction of a model system, which we analysed by molecular dynamics simulations to understand the mechanism of recognition and define interactions between bioactive CD52, HMGB1 and the SIGLEC-10 receptor. Our results confirm the essential role of glycosylation, more specifically hyper-sialylation, in the function of CD52, and identify at the atomistic level specific interactions between CD52 glycans and the Box B domain of HMGB1 that determine recognition, and the stability of the CD52/HMGB1 complex. These insights will inform the development of synthetic CD52 as an immunotherapeutic agent.

molecular biology↗

bioRxiv2023

A broad-spectrum family GH13 α-glucosidase from Marinovum sp., a member of the Roseobacter clade

Glycoside hydrolases (GHs) are a diverse group of enzymes that catalyze the hydrolysis of glycosidic bonds. The Carbohydrate-Active enZymes (CAZy) classification organizes GHs into families based on sequence data and function, with fewer than 1% of the predicted proteins characterized biochemically. Consideration of genomic context can provide clues to infer possible enzyme activities for proteins of unknown function. We used the MultiGeneBLAST tool to discover a gene cluster in Marinovum sp., a member of the marine Roseobacter clade, that encodes homologues of enzymes belonging to the sulfoquinovose monooxygenase pathway for sulfosugar catabolism. This cluster lacks a gene encoding a classical family GH31 sulfoquinovosidase candidate, but which instead includes an uncharacterized family GH13 protein (MsGH13) that we hypothesized could be a non-classical sulfoquinovosidase. Surprisingly, recombinant MsGH13 lacks sulfoquinovosidase activity and is a broad spectrum -glucosidase that is active on a diverse array of -linked disaccharides, including: maltose, sucrose, nigerose, trehalose, isomaltose, and kojibiose. Using AlphaFold, a 3D model for the MsGH13 enzyme was constructed that predicted its active site shared close similarity with an -glucosidase from Halomonas sp. H11 of the same GH13 subfamily that shows narrower substrate specificity.

biochemistry↗

bioRxiv2023

Sulfolactaldehyde dehydrogenase of Rhizobium leguminosarum: Kinetic and structural analysis

Sulfolactate (SL) is a short-chain organosulfonate that is an important reservoir of sulfur in the biosphere. SL is produced by oxidation of sulfolactaldehyde (SLA), which in turn derives from sulfoglycolysis of the sulfosugar sulfoquinovose, or through oxidation of 2,3-dihydroxypropanesulfonate. Oxidation of SLA is catalyzed by SLA dehydrogenases belonging to the aldehyde dehydrogenase superfamily. We report that SLA dehydrogenase RlGabD from the sulfoglycolytic bacterium Rhizobium leguminsarum SRDI565 can use both NAD+ and NADP+ as cofactor to oxidize SLA, and indicatively operates through a rapid equilibrium ordered mechanism. We report the cryo-EM structure of RlGabD bound to NADH, revealing a tetrameric quaternary structure and supporting proposal of organosulfonate binding residues in the active site, and a catalytic mechanism. Sequence based homology searches identified SLA dehydrogenase homologs in a range of putative sulfoglycolytic gene clusters in bacteria predominantly from the phyla Actinobacteria, Firmicutes, and Proteobacteria. This work provides a structural and biochemical view of SLA dehydrogenases to complement our knowledge of SLA reductases, and provide detailed insights into a critical step in the organosulfur cycle. Graphical abstractSulfolactate is an important species in the biogeochemical sulfur cycle. Herein we report the 3D cryo-EM structure and kinetics of its biosynthetic enzyme, sulfolactaldehyde dehydrogenase. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/532361v4_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1f3d1d3org.highwire.dtl.DTLVardef@548615org.highwire.dtl.DTLVardef@12efc0eorg.highwire.dtl.DTLVardef@1a6145c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Identification of levoglucosan degradation pathways in bacteria and sequence similarity network analysis

Levoglucosan is produced in the pyrolysis of cellulose and starch, including from bushfires or the burning of biofuels, and is deposited from the atmosphere across the surface of the earth. We describe two levoglucosan degrading Paenarthrobacter spp. (Paenarthrobacter nitrojuajacolis LG01 and Paenarthrobacter histidinolovorans LG02) that were isolated from soil by metabolic enrichment using levoglucosan as the sole carbon source. Genome sequencing and proteomics analysis revealed the expression of a series of genes encoding known levoglucosan degrading enzymes, levoglucosan dehydrogenase (LGDH, LgdA), 3-keto-levoglucosan {beta}-eliminase (LgdB1) and glucose 3-dehydrogenase (LgdC), along with an ABC transporter cassette and an associated solute binding protein. However, no homologues of 3-ketoglucose dehydratase (LgdB2) were evident, while the expressed genes contained a range of putative sugar phosphate isomerases/xylose isomerases with weak similarity to LgdB2. Sequence similarity network analysis of genome neighbours of LgdA revealed that homologues of LgdB1 and LgdC are generally conserved in a range of bacteria in the phyla Firmicutes, Actinobacteria and Proteobacteria. One group of sugar phosphate isomerase/xylose isomerase homologues (named LgdB3) was identified with limited distribution that is mutually exclusive with LgdB2, and we propose that they may fulfill a similar function. LgdB1, LgdB2 and LgdB3 adopt similar predicted 3D folds, suggesting overlapping function in processing intermediates in LG metabolism. Our findings highlight diversity within the LGDH pathway, through which bacteria utilize levoglucosan as a nutrient source.

microbiology↗

bioRxiv2020

Structural basis of substrate recognition and catalysis by fucosyltransferase 8

Fucosylation of the inner-most N-acetyl-glucosamine (GlcNAc) of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can have a dramatic impact on the activity and half-life of glycoproteins. These effects are relevant to understanding the invasiveness of some cancers, the development of monoclonal antibody therapeutics, and to a congenital disorder of glycosylation. The acceptor substrate preferences of FUT8 are well characterised and provide a framework for understanding N-glycan maturation in the Golgi, however the structural basis for these substrate preferences and the mechanism through which catalysis is achieved remains unknown. Here, we describe several structures of mouse and human FUT8 in the apo state and in complex with guanosine diphosphate (GDP), a mimic of the donor substrate, and a glycopeptide acceptor substrate. These structures provide insights into: a unique conformational change associated with donor substrate binding; common strategies employed by fucosyltransferases to coordinate GDP; features that define acceptor substrate preferences; and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also reveal how FUT8 dimerisation plays an important role in defining the acceptor substrate binding site. Collectively, this information significantly builds on our understanding of the core-fucosylation process.

biochemistry↗

bioRxiv2019

A sulfoglycolytic Entner-Doudoroff pathway in Rhizobium leguminosarum bv. trifolii SRDI565

Rhizobia are nitrogen fixing bacteria that engage in symbiotic relationships with plant hosts but can also persist as free-living bacteria with the soil and rhizosphere. Here we show that free living Rhizobium leguminosarum SRDI565 can grow on the sulfosugar sulfoquinovose (SQ) using a sulfoglycolytic Entner-Doudoroff (sulfo-ED) pathway resulting in production of sulfolactate (SL) as the major metabolic end-product. Comparative proteomics supports the involvement of a sulfo-ED operon encoding an ABC transporter cassette, sulfo-ED enzymes and an SL exporter. Consistent with an oligotrophic lifestyle, proteomics data revealed little change in expression of the sulfo-ED proteins during growth on SQ versus mannitol, a result confirmed through biochemical assay of sulfoquinovosidase activity in cell lysates (data are available via ProteomeXchange with identifier PXD015822). Metabolomics analysis showed that growth on SQ involves gluconeogenesis to satisfy metabolic requirements for glucose-6-phosphate and fructose-6-phosphate. Metabolomics analysis also revealed the unexpected production of small amounts of sulfofructose and 2,3-dihydroxypropanesulfonate, which are proposed to arise from promiscuous activities of the glycolytic enzyme phosphoglucose isomerase and a non-specific aldehyde reductase, respectively. This work shows that rhizobial metabolism of the abundant sulfosugar SQ may contribute to persistence of the bacteria in the soil and to mobilization of sulfur in the pedosphere.

microbiology↗

bioRxiv2019

Trefoil factors share a lectin activity that defines their role in mucus

The trefoil factors (TFFs) are disulfide-rich mucosal peptides that protect the epithelium by promoting cell migration and increasing the viscoelasticity of the mucosa. Here we show that all TFFs are divalent lectins that recognise the GlcNAc--1,4-Gal disaccharide, which terminates type-III mucin-like O-glycans. Structural, mutagenic and biophysical data support a model of mucus viscoelasticity that features non-covalent cross-linking of glycoproteins by TFFs.

biochemistry↗

bioRxiv2018

POFUT2-mediated O-glycosylation of MIC2 is dispensable for Toxoplasma gondii tachyzoites

Toxoplasma gondii is a ubiquitous obligate intracellular eukaryotic parasite that causes congenital birth defects, disease of the immunocompromised and blindness. Protein glycosylation plays an important role in the infectivity and evasion of immune response of many eukaryotic parasites and is also of great relevance to vaccine design. Here, we demonstrate that MIC2, the motility-associated adhesin of T. gondii, has highly glycosylated thrombospondin repeat domains (TSR). At least seven C-linked and three O-linked glycosylation sites exist within MIC2, with >95% occupancy at O-glycosylation sites. We demonstrate that the addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homologue (TgPOFUT2) encoded by TGGT1_273550. While POFUT2 homologues are important for stabilizing motility associated adhesins and host infection in other apicomplexan parasites, in T. gondii loss of TgPOFUT2 has only a modest impact on MIC2 levels and the wider proteome. Consistent with this, both plaque formation and tachyzoite infectivity are broadly similar in the presence or absence of TgPOFUT2. These findings demonstrate that TgPOFUT2 O-glycosylates MIC2 and that this glycan is dispensable in T. gondii tachyzoites.

microbiology↗