Purification of glycopeptides of human ceruloplasmin and immunoglobulin D by high-pressure liquid chromatography

To facilitate structural studies of glycoproteins, reverse-phase high-pressure liquid chromatography (HPLC) methods have been developed for preparative isolation of glycopeptides and have been applied to human ceruloplasmin as an example of glycopeptides containing glucosamine (GlcN) and to human immunoglobulin D (IgD) for glycopeptides containing galactosamine (GalN). The use of RP-P columns and of trifluoroacetic acid and heptafluorobutyric acid as counterions was investigated. Various elution systems (both isocratic and programmed gradient) were used with n-propanol to assess the relative hydrophilicity of the peptides. The procedure developed for the GlcN glycopeptides of ceruloplasmin enabled purification of nine major chymotryptic peptides (ranging in size from 15 to 29 residues) and also of many minor peaks. These were characterized by amino acid and endgroup analysis, and the complete sequence of five was determined. These represent three different sites of GlcN attachment in the amino-terminal half of the ceruloplasmin chain. The procedures developed have enabled isolation of glycopeptides from ceruloplasmin having a single GlcN oligosaccharide attached; the latter are valuable for study of the structure and function of the carbohydrate groups. Separation of GalN glycopeptides from IgD was more difficult because of the high content of GalN in the hinge. Purification and sequence analysis was aided by partial removal of sugar by treatment with HF and by other methods. Four (or five) GalN oligosaccharides are attached to serine or threonine residues in the IgD hinge region, and all but one are in close proximity in the repeating sequence Ala-Thr-Thr-Ala-Pro-Ala-Thr-Thr.     

Requirement of helix P2.2 and nucleotide G1 for positioning the cleavage site and cofactor of the glmS ribozyme

The glmS ribozyme is a catalytic RNA that self-cleaves at its 5'-end in the presence of glucosamine 6-phosphate (GlcN6P). We present structures of the glmS ribozyme from Thermoanaerobacter tengcongensis that are bound with the cofactor GlcN6P or the inhibitor glucose 6-phosphate (Glc6P) at 1.7 A and 2.2 A resolution, respectively. The two structures are indistinguishable in the conformations of the small molecules and of the RNA. GlcN6P binding becomes apparent crystallographically when the pH is raised to 8.5, where the ribozyme conformation is identical with that observed previously at pH 5.5. A key structural feature of this ribozyme is a short duplex (P2.2) that is formed between sequences just 3' of the cleavage site and within the core domain, and which introduces a pseudoknot into the active site. Mutagenesis indicates that P2.2 is required for activity in cis-acting and trans-acting forms of the ribozyme. P2.2 formation in a trans-acting ribozyme was exploited to demonstrate that N1 of the guanine at position 1 contributes to GlcN6P binding by interacting with the phosphate of the cofactor. At neutral pH, RNAs with adenine, 2-aminopurine, dimethyladenine or purine substitutions at position 1 cleave faster with glucosamine than with GlcN6P. This altered cofactor preference provides biochemical support for the orientation of the cofactor within the active site. Our results establish two features of the glmS ribozyme that are important for its activity: a sequence within the core domain that selects and positions the cleavage-site sequence, and a nucleobase at position 1 that helps position GlcN6P.     

Immunochemical studies of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1 O-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates

There is no licensed vaccine for the prevention of shigellosis. Our approach to the development of a Shigella vaccines is based on inducing serum IgG antibodies to the O-specific polysaccharide (O-SP) domain of their lipopolysaccharides (LPS). We have shown that low molecular mass O-SP-core (O-SPC) fragments isolated from Shigella sonnei LPS conjugated to proteins induced significantly higher antibody levels in mice than the full length O-SP conjugates. This finding is now extended to the O-SPC of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1. The structures of O-SPC, containing core plus 1-4 O-SP repeat units (RUs), were analyzed by NMR and mass spectroscopy. The first RUs attached to the cores of S. flexneri 2a and 6 LPS were different from the following RUs in their O-acetylation and/or glucosylation. Conjugates of core plus more than 1 RU were necessary to induce LPS antibodies in mice. The resulting antibody levels were comparable to those induced by the full length O-SP conjugates. In S. dysenteriae type 1, the first RU was identical to the following RUs, with the exception that the GlcNAc was bound to the core in the β-configuration, while in all other RUs the GlcNAc was present in the α-configuration. In spite of this difference, conjugates of S. dysenteriae type 1 core with 1, 2, or 3 RUs induced LPS antibodies in mice with levels statistically higher than those of the full size O-SP conjugates. O-SPC conjugates are easy to prepare, characterize, and standardize, and their clinical evaluation is planned.     

Ring-opening mechanism revealed by crystal structures of NagB and its ES intermediate complex

Glucosamine 6-phosphate deaminase (NagB) catalyzes the conversion of d-glucosamine 6-phosphate (GlcN6P) to d-fructose 6-phosphate and ammonia. This reaction is the final step of N-acetylglucosamine utilization and decides its metabolic fate. The enzyme from Streptococcus mutans belongs to the monomeric subfamily of NagB. The crystal structure of the native SmuNagB (NagB from S. mutans) presented here, compared with the structures of its homologs BsuNagB (NagB from Bacillus subtilis) and EcoNagB (NagB from E. coli), implies a conformational change of the ‘lid’ motif in the activation of the monomeric NagB enzyme. We have also captured the enzyme-substrate intermediate complex of the NagB family at low pH, where a remarkable loss of the catalytic activity of SmuNagB was detected. The enzyme-substrate intermediate presents the initial step of the GlcN6P deaminase reaction. The structural evidence (1) supports the α-anomer of GlcN6P as the specific natural substrate of NagB; (2) displays the substrate-binding pocket at the active site; and (3) together with the site-directed mutagenesis studies, demonstrates the ring-opening mechanism of an Asn-His-Glu triad that performs the proton transfer from O1 to O5 to open the sugar ring.     

Acceptor substrate binding revealed by crystal structure of human glucosamine-6-phosphate N-acetyltransferase 1

Glucosamine-6-phosphate (GlcN6P) N-acetyltransferase 1 (GNA1) is a key enzyme in the pathway toward biosynthesis of UDP-N-acetylglucosamine, an important donor substrate for N-linked glycosylation. GNA1 catalyzes the formation of N-acetylglucosamine-6-phosphate (GlcNAc6P) from acetyl-CoA (AcCoA) and the acceptor substrate GlcN6P. Here, we report crystal structures of human GNA1, including apo GNA1, the GNA1-GlcN6P complex and an E156A mutant. Our work showed that GlcN6P binds to GNA1 without the help of AcCoA binding. Structural analyses and mutagenesis studies have shed lights on the charge distribution in the GlcN6P binding pocket, and an important role for Glu156 in the substrate binding. Hence, these findings have broadened our knowledge of structural features required for the substrate affinity of GNA1. STRUCTURED SUMMARY:     

Subcellular distributions of UDP-galactose: Polysaccharide transferase and UDP-glucose pyrophosphorylase involved in biosynthesis of prespore-specific acid mucopolsaccharide in Dictyostelium discoideum

During the development of a cell aggregate of Dictystelium discoideum into a fruiting body, an antigenic acid mucopolysaccharide is synthesized only in the prespore cells of a cell mass. In this study, the subcellular distributions of UCPgalactose: polysaccharide transferase and UDPglucose pyrophosphorylase involved in biosynthesis of the mucopolysaccharide were determined. The transferase was specifically localized in the smaller vesicles with lighter density than the prespore-specific vacuoles identifiable electronmicroscopically. In contrast to the enzyme, the antigenic mucopolysaccharide was exclusively localized in the prespore-specific vacuoles. Unlike the transferase, UDPglucose pyrophosphorylase was confined to the soluble fraction. The sucrose gradient profiles of the transferase activity in the 5000 × g supernatant gave two main peaks. When the profiles were compared among standing and migrating slugs and culminating cell mass, the difference in the profiles closely reflected the state of biosynthesis of the acid mucopolysaccharide in eac developmental stage.     

Mass Spectrometry-based Structural Analysis and Systems Immunoproteomics Strategies for Deciphering the Host Response to Endotoxin

One cause of sepsis is systemic maladaptive immune response of the host to bacteria and specifically, to Gram-negative bacterial outer-membrane glycolipid lipopolysaccharide (LPS). On the host myeloid cell surface, proinflammatory LPS activates the innate immune system via Toll-like receptor-4/myeloid differentiation factor-2 complex. Intracellularly, LPS is also sensed by the noncanonical inflammasome through caspase-11 in mice and 4/5 in humans. The minimal functional determinant for innate immune activation is the membrane anchor of LPS called lipid A. Even subtle modifications to the lipid A scaffold can enable, diminish, or abolish immune activation. Bacteria are known to modify their LPS structure during environmental stress and infection of hosts to alter cellular immune phenotypes. In this review, we describe how mass spectrometry-based structural analysis of endotoxin helped uncover major determinations of molecular pathogenesis. Through characterization of LPS modifications, we now better understand resistance to antibiotics and cationic antimicrobial peptides, as well as how the environment impacts overall endotoxin structure. In addition, mass spectrometry-based systems immunoproteomics approaches can assist in elucidating the immune response against LPS. Many regulatory proteins have been characterized through proteomics and global/targeted analysis of protein modifications, enabling the discovery and characterization of novel endotoxin-mediated protein translational modifications.     

Modifications of Bordetella bronchiseptica core lipopolysaccharide influence immune response without affecting protective activity

Bordetella bronchiseptica produces respiratory disease primarily in mammals including humans. Although a considerably amount of research has been generated regarding lipopolysaccharide (LPS) role during infection and stimulating innate and adaptive immune response, mechanisms involved in LPS synthesis are still unknown. In this context we searched in B. bronchiseptica genome for putative glycosyltransferases. We found possible genes codifying for enzymes involved in sugar substitution of the LPS structure. We decided to analyse BB3394 to BB3400 genes, closed to a previously described LPS biosynthetic locus in B. pertussis. Particularly, conservation of BB3394 in sequenced B. bronchiseptica genomes suggests the importance of this gene for bacteria normal physiology. Deletion of BB3394 abolished resistance to naive serum as described for other LPS mutants. When purified LPS was analyzed, differences in the LPS core structure were found. Particularly, a GalNA branched sugar substitution in the core was absent in the LPS obtained from BB3394 deletion mutant. Absence of GalNA in core LPS alters immune response in vivo but is able to induce protective response against B. bronchiseptica infection.     

Structure of the lipopolysaccharide core of Vibrio vulnificus type strain 27562

The structure of the lipopolysaccharide core of Vibrio vulnificus type strain 27562 is presented. LPS hydrolysis gave two oligosaccharides, OS-1 and OS-2, as well as lipid A. NMR spectroscopic data corresponded to the presence of one Kdo residue, one β-glucopyranose, three heptoses, one glyceric acid, one acetate, three PEtN, and one 5,7-diacylamido-3,5,7,9-tetradeoxynonulosonic acid residue (pseudaminic acid, Pse) in OS1. OS2 differed form OS 1 by the absence of glyceric acid, acetate, and Pse residues. Lipid A was analyzed for fatty acid composition and the following fatty acids were found: C14:0, C12:0-3OH, C16:0, C16:1, C14:0-3OH, C18:0, C18:1 in a ratio of 1:3:3:1:2.5:0.6:0.8.