Protein O-glycosylation in Streptomyces

Previously, a protein O-mannosyl transferase (Pmt, SCO3154) and a polyprenol phosphate mannose synthase (Ppm1, SCO1423) were found to be required for the glycosylation of PstS, a phosphate binding protein, in the bacterium Streptomyces coelicolor. Bacteria in this genus are prolific producers of antibiotics and are often phenotypically resistant to multiple antibiotics. S. coelicolor pmt and ppm1 deficient mutants were hypersusceptible to cell-wall active antibiotics, suggesting that the protein modification could be required for cell wall and membrane homeostasis. The aim of this project was to investigate the S. coelicolor glycoproteome in order to better understand the physiological role of protein O-glycosylation in this model actinobacterium. Glycoproteins were detected in, and enriched from the membrane and culture filtrate proteomes of the S. coelicolor parent strain, J1929 and these were absent from the glycosylation deficient pmt (DT1025) and ppm1 (DT3017) mutants. Liquid chromatography coupled to mass spectrometry was used to characterise the membrane glycoproteome from the S. coelicolor parent strain, J1929 and 37 new glycoproteins were identified. Glycopeptides were modified on Ser/Thr residues with up to 3 hexoses; consistent with previous observations that the glycoprotein PstS is modified with a trihexose. The S. coelicolor glycoprotein glycans were shown to consist of Hex₂ and Hex₃ oligosaccharides. A carbohydrate linkage analysis led to the observation of 2-substituted, 4-substituted and terminal mannose residues, suggesting presence of (1->2) and (1->4) linkages in S. coelicolor glycoprotein glycans. The S. coelicolor glycoproteome comprises glycoproteins with various biological roles including solute binding, transport and cell wall biosynthesis. The genes encoding two S. coelicolor glycoproteins with putative roles in cell wall biosynthesis, an L, D transpeptidase (SCO4934) and a D-Ala-D-Ala, carboxypeptidase (SCO4847) were disrupted. Both mutants were hypersusceptible to β-lactam antibiotics, while the sco4847 mutant was hypersusceptible to lysozyme. These findings suggest that both proteins could be required for cell wall biosynthesis. As the phenotypes of the knockout mutants are reminiscent of the glycosylation deficient strains, we propose that glycosylation might be required for enzyme function

Protein O-glycosylation in Streptomyces

Previously, a protein O-mannosyl transferase (Pmt, SCO3154) and a polyprenol phosphate mannose synthase (Ppm1, SCO1423) were found to be required for the glycosylation of PstS, a phosphate binding protein, in the bacterium Streptomyces coelicolor. Bacteria in this genus are prolific producers of antibiotics and are often phenotypically resistant to multiple antibiotics. S. coelicolor pmt and ppm1 deficient mutants were hypersusceptible to cell-wall active antibiotics, suggesting that the protein modification could be required for cell wall and membrane homeostasis. The aim of this project was to investigate the S. coelicolor glycoproteome in order to better understand the physiological role of protein O-glycosylation in this model actinobacterium. Glycoproteins were detected in, and enriched from the membrane and culture filtrate proteomes of the S. coelicolor parent strain, J1929 and these were absent from the glycosylation deficient pmt (DT1025) and ppm1 (DT3017) mutants. Liquid chromatography coupled to mass spectrometry was used to characterise the membrane glycoproteome from the S. coelicolor parent strain, J1929 and 37 new glycoproteins were identified. Glycopeptides were modified on Ser/Thr residues with up to 3 hexoses; consistent with previous observations that the glycoprotein PstS is modified with a trihexose. The S. coelicolor glycoprotein glycans were shown to consist of Hex₂ and Hex₃ oligosaccharides. A carbohydrate linkage analysis led to the observation of 2-substituted, 4-substituted and terminal mannose residues, suggesting presence of (1->2) and (1->4) linkages in S. coelicolor glycoprotein glycans. The S. coelicolor glycoproteome comprises glycoproteins with various biological roles including solute binding, transport and cell wall biosynthesis. The genes encoding two S. coelicolor glycoproteins with putative roles in cell wall biosynthesis, an L, D transpeptidase (SCO4934) and a D-Ala-D-Ala, carboxypeptidase (SCO4847) were disrupted. Both mutants were hypersusceptible to β-lactam antibiotics, while the sco4847 mutant was hypersusceptible to lysozyme. These findings suggest that both proteins could be required for cell wall biosynthesis. As the phenotypes of the knockout mutants are reminiscent of the glycosylation deficient strains, we propose that glycosylation might be required for enzyme function

The characterisation of a galactokinase from Streptomyces coelicolor

Promiscuous galactokinases (GalKs), which catalyse the ATP dependent phosphorylation of galactose in nature, have been widely exploited in biotechnology for the rapid synthesis of diverse sugar-1-phosphates. This work focuses on the characterisation of a bacterial GalK from Streptomyces coelicolor (ScGalK), which was overproduced in Escherichia coli and shown to phosphorylate galactose. ScGalK displayed a broad substrate tolerance, with activity towards Gal, GalN, Gal3D, GalNAc, Man and L-Ara. Most interestingly, ScGalK demonstrated a high activity over a broad pH and temperature range, suggesting that the enzyme could be highly amenable to multi-enzyme systems

Characterisation of the Streptomyces coelicolor glycoproteome reveals glycoproteins important for cell wall biogenesis

In prokaryotes, the role of protein glycosylation is poorly understood due to our limited understanding of their glycoproteomes. In some Actinobacteria, defects in protein O-glycosylation have been shown to retard growth and result in hypersensitivity to cell wall-targeting antibiotics, suggesting that this modification is important for maintaining cell wall structure. Here, we have characterized the glycoproteome in Streptomyces coelicolor and shown that glycoproteins have diverse roles, including those related to solute binding, ABC transporters, and cell wall biosynthesis. We have generated mutants encoding two putative cell wall-active glycoproteins and shown them to be hypersensitive to cell wall-targeting antibiotics. These findings strongly suggest that both glycoproteins are required for maintaining cell wall integrity and that glycosylation affects enzyme function.The physiological role of protein O-glycosylation in prokaryotes is poorly understood due to our limited knowledge of the extent of their glycoproteomes. In Actinobacteria, defects in protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation have been shown to significantly retard growth (Mycobacterium tuberculosis and Corynebacterium glutamicum) or result in increased sensitivities to cell wall-targeting antibiotics (Streptomyces coelicolor), suggesting that protein O-glycosylation has an important role in cell physiology. Only a single glycoprotein (SCO4142, or PstS) has been identified to date in S. coelicolor. Combining biochemical and mass spectrometry-based approaches, we have isolated and characterized the membrane glycoproteome in S. coelicolor. A total of ninety-five high-confidence glycopeptides were identified which mapped to thirty-seven new S. coelicolor glycoproteins and a deeper understanding of glycosylation sites in PstS. Glycosylation sites were found to be modified with up to three hexose residues, consistent with what has been observed previously in other Actinobacteria. S. coelicolor glycoproteins have diverse roles and functions, including solute binding, polysaccharide hydrolases, ABC transporters, and cell wall biosynthesis, the latter being of potential relevance to the antibiotic-sensitive phenotype of pmt mutants. Null mutants in genes encoding a putative d-Ala-d-Ala carboxypeptidase (SCO4847) and an l,d-transpeptidase (SCO4934) were hypersensitive to cell wall-targeting antibiotics. Additionally, the sco4847 mutants displayed an increased susceptibility to lysozyme treatment. These findings strongly suggest that both glycoproteins are required for maintaining cell wall integrity and that glycosylation could be affecting enzyme function

Chemical Bioconjugation of Proteins in an Undergraduate Lab: One-Pot Oxidation and Derivatization of the N-Terminus

A laboratory experiment introducing the concept of chemical bioconjugation of proteins to undergraduate students in a therapeutically relevant context was developed. Initially, students installed an aldehyde functionality into a protein via the oxidation of the N-terminal threonine residue of the cholera toxin subunit B (CTB) protein, which was followed by subsequent modification via hydrazone addition under mild conditions with a chromophore bearing a distinct UV–vis-absorption peak. Students determined the yield of the reaction to be ca. 11% by HPLC coupled to UV–vis spectroscopy and developed key skills such as preparation of stock solutions, chemical manipulation of proteins, and analysis via HPLC. The reported experiment can be readily adapted for use with other proteins and may contribute to enhancing constructive alignment in interdisciplinary degree programs at the chemistry–biology interface

Biocatalytic Transfer of Pseudaminic Acid (Pse5Ac7Ac) Using Promiscuous Sialyltransferases in a Chemoenzymatic Approach to Pse5Ac7Ac-Containing Glycosides

Pseudaminic acid (Pse5Ac7Ac) is a nonmammalian sugar present on the cell surface of a number of bacteria including Pseudomonas aeruginosa, Campylobacter jejuni, and Acinetobacter baumannii. However, the role Pse5Ac7Ac plays in host–pathogen interactions remains underexplored, particularly compared to its ubiquitous sialic acid analogue Neu5Ac. This is primarily due to a lack of access to difficult to prepare Pse5Ac7Ac glycosides. Herein, we describe the in vitro biocatalytic transfer of an activated Pse5Ac7Ac donor onto glycosyl acceptors, enabling the enzymatic synthesis of Pse5Ac7Ac-containing glycosides. In a chemoenzymatic approach, chemical synthesis initially afforded access to a late-stage Pse5Ac7Ac biosynthetic intermediate, which was subsequently converted to the desired CMP-glycosyl donor in a one-pot two-enzyme process using biosynthetic enzymes. Finally, screening a library of 13 sialyltransferases (SiaT) with the unnatural substrate enabled the identification of a promiscuous inverting SiaT capable of turnover to afford β-Pse5Ac7Ac-terminated glycosides.</p

Profiling substrate promiscuity of wild-type sugar kinases for multifluorinated monosaccharides

Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluoro galactose, giving insight into changes in the active site that may account for the specificity of the enzyme towards certain substrate analogues

Educational paper: Abusive Head Trauma Part I. Clinical aspects

Abusive Head Trauma (AHT) refers to the combination of findings formerly described as shaken baby syndrome. Although these findings can be caused by shaking, it has become clear that in many cases there may have been impact trauma as well. Therefore a less specific term has been adopted by the American Academy of Pediatrics. AHT is a relatively common cause of childhood neurotrauma with an estimated incidence of 14–40 cases per 100,000 children under the age of 1 year. About 15–23% of these children die within hours or days after the incident. Studies among AHT survivors demonstrate that approximately one-third of the children are severely disabled, one-third of them are moderately disabled and one-third have no or only mild symptoms. Other publications suggest that neurological problems can occur after a symptom-free interval and that half of these children have IQs below the 10th percentile. Clinical findings are depending on the definitions used, but AHT should be considered in all children with neurological signs and symptoms especially if no or only mild trauma is described. Subdural haematomas are the most reported finding. The only feature that has been identified discriminating AHT from accidental injury is apnoea. Conclusion: AHT should be approached with a structured approach, as in any other (potentially lethal) disease. The clinician can only establish this diagnosis if he/she has knowledge of the signs and symptoms of AHT, risk factors, the differential diagnosis and which additional investigations to perform, the more so since parents seldom will describe the true state of affairs spontaneously