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P M Rudd

Publications and source records attributed to P M Rudd.

At least 37 records · Page 2Linked to original sources
PubMed1999

Glycosylation differences between the normal and pathogenic prion protein isoforms.

Prion protein consists of an ensemble of glycosylated variants or glycoforms. The enzymes that direct oligosaccharide processing, and hence control the glycan profile for any given glycoprotein, are often exquisitely sensitive to other events taking place within the cell in which the glycoprotein is expressed. Alterations in the populations of sugars attached to proteins can reflect changes caused, for example, by developmental processes or by disease. Here we report that normal (PrP(C)) and pathogenic (PrP(Sc)) prion proteins (PrP) from Syrian hamsters contain the same set of at least 52 bi-, tri-, and tetraantennary N-linked oligosaccharides, although the relative proportions of individual glycans differ. This conservation of structure suggests that the conversion of PrP(C) into PrP(Sc) is not confined to a subset of PrPs that contain specific sugars. Compared with PrP(C), PrP(Sc) contains decreased levels of glycans with bisecting GlcNAc residues and increased levels of tri- and tetraantennary sugars. This change is consistent with a decrease in the activity of N-acetylglucosaminyltransferase III (GnTIII) toward PrP(C) in cells where PrP(Sc) is formed and argues that, in at least some cells forming PrP(Sc), the glycosylation machinery has been perturbed. The reduction in GnTIII activity is intriguing both with respect to the pathogenesis of the prion disease and the replication pathway for prions.

Animals↗
PubMed1999

Roles for glycosylation of cell surface receptors involved in cellular immune recognition.

The majority of cell surface receptors involved in antigen recognition by T cells and in the orchestration of the subsequent cell signalling events are glycoproteins. The length of a typical N-linked sugar is comparable with that of an immunoglobulin domain (30 A). Thus, by virtue of their size alone, oligosaccharides may be expected to play a significant role in the functions and properties of the cell surface proteins to which they are attached. A databank of oligosaccharide structures has been constructed from NMR and crystallographic data to aid in the interpretation of crystal structures of glycoproteins. As unambiguous electron density can usually only be assigned to the glycan cores, the remainder of the sugar is then modelled into the crystal lattice by superimposing the appropriate oligosaccharide from the database. This approach provides insights into the roles that glycosylation might play in cell surface receptors, by providing models that delineate potential close packing interactions on the cell surface. It has been proposed that the specific recognition of antigen by T cells results in the formation of an immunological synapse between the T cell and the antigen-presenting cell. The cell adhesion glycoproteins, such as CD2 and CD48, help to form a cell junction, providing a molecular spacer between opposing cells. The oligosaccharides located on the membrane proximal domains of CD2 and CD48 provide a scaffold to orient the binding faces, which leads to increased affinity. In the next step, recruitment of the peptide major histocompatibility complex (pMHC) by the T-cell receptors (TCRs) requires mobility on the membrane surface. The TCR sugars are located such that they could prevent non-specific aggregation. Importantly, the sugars limit the possible geometry and spacing of TCR/MHC clusters which precede cell signalling. We postulate that, in the final stage, the sugars could play a general role in controlling the assembly and stabilisation of the complexes in the synapse and in protecting them from proteolysis during prolonged T-cell engagement.

Animals↗
PubMed1999

Glycosylation of natural human neutrophil gelatinase B and neutrophil gelatinase B-associated lipocalin.

Gelatinase B is a matrix metalloproteinase (MMP-9) involved in tissue remodeling, development, cancer, and inflammation. Neutrophils produce three major forms of (pro)gelatinase B: 92 kDa monomers, homodimers, and complexes of gelatinase B covalently bound to neutrophil gelatinase B-associated lipocalin (NGAL). In contrast to the case for other proteinases, little information about the glycosylation of any natural human MMP is available. Here, both gelatinase B and NGAL were purified from human peripheral blood neutrophils, and the entire contents of the released N- and O-glycan pools were analyzed simultaneously using recently developed high-performance liquid chromatography-based technology. The results are discussed within the context of the domain structure of gelatinase B and a molecular model of NGAL based on data from this study and the three-dimensional nuclear magnetic resonance (NMR) structure of the protein. More than 95% of the N-linked glycans attached to both gelatinase B and NGAL were partially sialylated, core-fucosylated biantennary structures with and without outer arm fucose. The O-linked glycans, which were estimated to comprise approximately 85% of the total sugars on gelatinase B, mainly consisted of type 2 cores with Galbeta1,4GlcNAc (lactosamine) extensions, with or without sialic acid or outer arm fucose. This paper also contains the first report of O-linked glycans attached to NGAL. Although both proteins were isolated from neutrophils and contained O-linked glycans mainly with type 2 cores, the glycans attached to individual serine/threonine residue(s) in NGAL were significantly smaller than those on gelatinase B. In contrast to NGAL, gelatinase B contains a region rich in Ser, Thr, and Pro typical of O-glycosylated mucin-like domains.

Acute-Phase Proteins↗
PubMed1999

Sugar printing rheumatic diseases: a potential method for disease differentiation using immunoglobulin G oligosaccharides.

OBJECTIVE: To look for oligosaccharide structural variants of IgG that may be unique to specific rheumatic diseases. METHODS: Using normal-phase high-performance liquid chromatography technology, a comparison was made of the oligosaccharide pools released from serum IgG from patients with systemic lupus erythematosus (SLE) (n = 10), ankylosing spondylitis (AS) (n = 10), primary Sjögren's syndrome (n = 6), juvenile chronic arthritis (JCA) (n = 13), psoriatic arthritis (n = 9), rheumatoid arthritis (RA) (n = 5), and healthy control individuals (n = 19). RESULTS: The oligosaccharide pools were resolved into 13 peaks and the relative proportions of the peaks in each disease group was significantly different from that in healthy controls (P < 0.0001-0.05). A characteristic serum IgG oligosaccharide profile, or sugar print, for each of the rheumatic diseases was found. The sugar prints exhibited a range of glycosylation patterns whereby all RA (P < 0.0001) and JCA (P < 0.006) patients had predominantly agalactosyl structures, while SLE (P < 0.03-0.0001) and AS (P < 0.025-0.0001) patients had predominantly digalactosyl structures. CONCLUSION: The data suggest that each disease is associated with a specific mechanism that gives rise to alterations in the normal glycosylation pattern of IgG. Sugar printing of IgG is therefore a potential means for the differentiation of rheumatic diseases and may provide insight into disease pathogenesis.

Adult↗
PubMed1999

The glycan processing and site occupancy of recombinant Thy-1 is markedly affected by the presence of a glycosylphosphatidylinositol anchor.

Thy-1 is a cell surface glycoprotein containing three N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The effect of the anchor on its N-linked glyco-sylation was investigated by comparing the glycosylation of soluble recombinant Thy-1 (sThy-1) with that of recombinant GPI anchored Thy-1, both expressed in Chinese hamster ovary cells. The sThy-1 was produced in a variety of isoforms including some which lacked carbohydrate on all three sequons whereas the GPI anchored form appeared fully glycosylated like native Thy-1. This was surprising as the attachment of N-linked sugars occurs cotranslationally and it was not expected that the presence of a C-terminal GPI anchor signal sequence would affect sequon occupancy. Furthermore sThy-1 lacking glycosylation could be produced with the inhibitor tunicamycin but in contrast cell surface expression of unglycosylated GPI anchored Thy-1 could not be obtained. The GPI anchored form appeared less processed with almost 4-fold more oligo-mannose oligosaccharides than in sThy-1 and also with less sialylated and core fucosylated biantennary glycans. Possible mechanisms whereby the anchor or the anchor signal sequence, control site occupancy and maturation are discussed.

Animals↗
PubMed1999

Oligosaccharide analysis and molecular modeling of soluble forms of glycoproteins belonging to the Ly-6, scavenger receptor, and immunoglobulin superfamilies expressed in Chinese hamster ovary cells.

Most cell surface molecules are glycoproteins consisting of linear arrays of globular domains containing stretches of amino acid sequence with similarities to regions in other proteins. These conserved regions form the basis for the classification of proteins into superfamilies. Recombinant soluble forms of six leukocyte antigens belonging to the Ly-6 (CD59), scavenger receptor (CD5), and immunoglobulin (CD2, CD48, CD4, and Thy-1) superfamilies were expressed in the same Chinese hamster ovary cell line, thus providing an opportunity to examine the extent to which N-linked oligosaccharide processing might vary in a superfamily-, domain-, or protein-dependent manner in a given cell. While we found no evidence for superfamily-specific modifications of the glycans, marked differences were seen in the types of oligosaccharides attached to individual proteins within a given superfamily. The relative importance of local protein surface properties versus the overall tertiary structure of the molecules in directing this protein-specific variation was examined in the context of molecular models. These were constructed using the 3D structures of the proteins, glycan data from this study, and an oligosaccharide structural database. The results indicated that both the overall organization of the domains and the local protein structure can have a large bearing on site-specific glycan modification of cells in stasis. This level of control ensures that the surface of a single cell will display a diverse repertoire of glycans and precludes the presentation of multiple copies of a single oligosaccharide on the cell surface. The glycans invariably shield large regions of the protein surfaces although, for the glycoproteins examined here, these did not hinder the known active sites of the molecules. The models also indicated that sugars are likely to play a role in the packing of the native cell surface glycoproteins and to limit nonspecific protein-protein interactions. In addition, glycans located close to the cell membrane are likely to affect crucially the orientation of the glycoproteins to which they are attached.

Animals↗
PubMed1998

Oligosaccharides of recombinant mouse gelatinase B variants.

Gelatinase B (matrix metalloproteinase-9, MMP-9) contains three N-glycosylation sites and a Ser/Thr/Pro-rich type V collagen domain with repetitive attachment sites for O-linked sugars. Recombinant mouse gelatinase B was expressed in the yeast Pichia pastoris and the N-linked oligosaccharides of the truncated glycoprotein variants were analysed by in gel enzymatic release followed by mass spectrometry and normal phase HPLC. This technology, despite of the limiting amount of material, allowed the analysis of the formula of N- and O-linked sugars of the different glycoprotein variants. The 112/99- and 88-kDa gelatinase B forms each contained an oligomannose series (Man8GlcNAc2 to Man15GlcNAc2). Analysis of the hydrazine-released sugars showed that the O-linked oligosaccharides contained alpha1-2, alpha1-3 or alpha1-6 linked mannoses. These results were confirmed by lectin blot analysis of intact and glycosidase-treated enzyme variants.

Acetaldehyde↗
PubMed1998

Structural analysis of the CD5 antigen--expression, disulphide bond analysis and physical characterisation of CD5 scavenger receptor superfamily domain 1.

CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and a subset of B-cells. The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) superfamily, for which no three-dimensional structure has been obtained. Recombinant soluble CD5 domain 1 (CD5d1), the N-terminal SRCR domain, has been expressed in both chinese hamster ovary (CHO) cells and Pichia pastoris. CD5d1 was shown to be correctly folded by binding to the CD5 monoclonal antibody Leul. Circular dichroism and NMR analyses indicate that CD5d1 has a high beta-sheet content. CD5d1 from both CHO cells and P. pastoris have very similar properties. The disulphide bonding pattern was determined and is consistent with that found for the group-A SRCR domain of type-1 macrophage scavenger receptor and MARCO, the macrophage receptor with collagenous structure. Observations have been made of the role of glycosylation of CD5. P. pastoris expression provides large quantities of correctly folded recombinant CD5d1 for multidimensional NMR and for X-ray crystallographic studies. The whole extracellular region of CD5, expressed as a chimaera with rat CD4 domains 3 and 4 (cCD5d1-3-CD4d3+4), was studied by electron microscopy and carbohydrate analysis to gain an overview of the structure of the extracellular portion of intact CD5. Carbohydrate analysis identified N-linked glycans on CD5 domains 1 and 2, and sialylated O-linked glycans on the linker peptide between domains 1 and 2. Electron microscopy and carbohydrate analysis together suggest that the extracellular region of CD5 forms a rod-like structure with domain 1 distal from the cell surface and separated from domains 2 and 3 by an O-glycosylated peptide linker region.

Amino Acid Sequence↗
PubMed1998

Alpha-glucosidase inhibitors as potential broad based anti-viral agents.

N-Linked oligosaccharides play many roles in the fate and functions of glycoproteins. One function is to assist in the folding of proteins by mediating interactions of the lectin-like chaperone proteins calnexin and calreticulin with nascent glycoproteins. These interactions can be prevented by inhibitors of the alpha-glucosidases and this causes some proteins to be misfolded and retained within the endoplasmic reticulum. In human immunodeficiency virus (HIV) and hepatitis B virus (HBV) the misfolding of key viral envelope glycoproteins interferes with the viral life cycle. It has been demonstrated in an animal model of chronic HBV that glucosidase inhibitors can alter glycosylation and have anti-viral activity. As the mechanism of action of alpha-glucosidase inhibitors is the induction of misfolded or otherwise defective viral glycoproteins, such inhibitors may be useful therapeutics for many viruses, especially those which bud from the endoplasmic reticulum (where protein folding takes place). For example bovine viral diarrhea virus, a pestivirus akin to hepatitis C virus, is also extremely sensitive to glucosidase inhibition.

Animals↗
PubMed1998

Quantitation of the oligosaccharides of human serum IgG from patients with rheumatoid arthritis: a critical evaluation of different methods.

Several different chromatographic methods and a lectin-based assay have been compared for the quantitation of oligosaccharides released from immunoglobulin G (IgG). The analysis of a series of IgG samples purified from the serum of rheumatoid arthritis patients was carried out by these methods to evaluate the percentage of the glycoforms having 0, 1 or 2 galactose residues (G0, G1 and G2) in order to (a) identify the method that can be most widely used for quantitation, (b) accurately define the range of G0 values found in patients with rheumatoid arthritis, and (c) make available a series of characterised standards for distribution to clinical chemistry laboratories. The chromatographic methods involved: release of oligosaccharides by glycoamidase A after protease digestion followed by HPLC analysis of aminopyridine derivatives on reverse phase and normal phase columns; hydrazinolysis treatment with exoglycosidases (G0 mix) and Biogel P4 chromatography of 2-aminobenzamide (2-AB) derivatives; hydrazinolysis and weak anion exchange or normal phase HPLC of 2-AB derivatives; release of oligosaccharides by PNGase F and either Biogel P4 chromatography of 2-AB derivatives or HPAEC-PAD analysis of native oligosaccharides. The G0 values given by these methods compared favourably with each other and a dot blot assay of denatured IgG interaction with Ricinus communis agglutinin and Bandeiraea simplicifolialectin II. The HPLC and HPAEC methods give additional information that may be important in less routine assays.

Arthritis, Rheumatoid↗
PubMed1998

Mutant analysis reveals an alternative pathway for N-linked glycosylation in Drosophila melanogaster.

The mas-1 gene of Drosophila melanogaster encodes Golgi mannosidase I (MAS-1), and flies homozygous for small deletions of the gene are viable. They show but few abnormalities and those have a low penetrance [Kerscher, S., Albert, S., Wucherpfennig, D., Heisenberg, M. & Schneuwly, S. (1995) Dev. Biol. 168, 613-626]. Here we sequence the N-linked oligosaccharides associated with a reporter protein, and with membrane proteins prepared from wild type and MAS-1 null organisms. The results show that the null organisms synthesise the same range of oligosaccharides as wild type, albeit with different ratios. There is an accumulation of the Man8GlcNAc2 which is one of the substrates for the MAS-1 enzyme. This supports the suggestion of Kerscher et al. that the lack of severe phenotypic disturbances in the null organisms is due to the presence of an alternative pathway(s), but it also shows that this alternative pathway(s) does not entirely compensate for the normal pathway.

Animals↗
PubMed1998

The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fcα receptor interactions.

The human serum immunoglobulins IgG and IgA1 are produced in bone marrow and both interact with specific cellular receptors that mediate biological events. In contrast to IgA1, the glycosylation of IgG has been well characterized, and its interaction with various Fc receptors (Fc Rs) has been well studied. In this paper, we have analyzed the glycosylation of IgA1 and IgA1 Fab and Fc as well as three recombinant IgA1 molecules, including two N-glycosylation mutants. Amino acid sequencing data of the IgA1 Fc O-glycosylated hinge region indicated that O-glycans are located at Thr228, Ser230, and Ser232, while O-glycan sites at Thr225 and Thr236 are partially occupied. Over 90% of the N-glycans in IgA1 were sialylated, in contrast to IgG, where < 10% contain sialic acid. This paper contains the first report of Fab glycosylation in IgA1, and (in contrast to IgG Fab, which contains only N-linked glycans) both N- and O-linked oligosaccharides were identified. Analysis of the N-glycans attached to recombinant IgA1 indicated that the Cα 2 N-glycosylation site contained mostly biantennary glycans, while the tailpiece site, absent in IgG, contained mostly triantennary structures. Further analysis of these data suggested that processing at one Fc N-glycosylation site affects the other. Neutrophil Fcα R binding studies, using recombinant IgA1, indicated that neither the tailpiece region nor the N-glycans in the C alpha 2 domain contribute to IgA1-neutrophil Fcα R binding. This contrasts with IgG, where removal of the Fc N-glycans reduces binding to the Fcγ R. The primary sequence and disulfide bond pattern of IgA1, together with the crystal structures of IgG1 Fc and mouse IgA Fab and the glycan sequencing data, were used to generate a molecular model of IgA1. As a consequence of both the primary sequence and S-S bond pattern, the N-glycans in IgA1 Fc are not confined within the inter-α-chain space. The accessibility of the Cα 2 N-glycans provides an explanation for the increased sialylation and galactosylation of IgA1 Fc over that of IgG Fc N-glycans, which are confined in the space between the two Cγ 2 domains. This also suggests why in contrast to IgG Fc, the IgA1 N-glycans are not undergalactosylated in rheumatoid arthritis.

Amino Acid Sequence↗
PubMed1998

Genomic cloning and expression of three murine UDP-galactose: beta-N-acetylglucosamine beta1,3-galactosyltransferase genes.

Based on the detection of expressed sequence tags that are similar to known galactosyltransferase sequences, we have isolated three novel UDP-galactose:beta-N-acetylglucosamine beta1, 3-galactosyltransferase (beta3GalT) genes from a mouse genomic library. The three genes, named beta3GalT-I, -II, and -III, encode type II transmembrane proteins of 326, 422, and 331 amino acids, respectively. The three proteins constitute a distinct subfamily as they do not share any sequence identity with other eucaryotic galactosyltransferases. Also, the entire protein-coding region of the three beta3GalT genes was contained in a single exon, which contrasts with the genomic organization of the beta1,4- and alpha1, 3-galactosyltransferase genes. The three beta3GalT genes were mainly expressed in brain tissue. The expression of the full-length murine genes as recombinant baculoviruses in insect cells revealed that the beta3GalT enzymes share the same acceptor specificity for beta-linked GlcNAc, although they differ in their Km for this acceptor and the donor UDP-Gal. The identification of beta3GalT genes emphasizes the structural diversity present in the galactosyltransferase gene family.

Amino Acid Sequence↗
PubMed1998

Cytokine and protease glycosylation as a regulatory mechanism in inflammation and autoimmunity.

Cytokines are locally produced hormones that alert the innate and specific immune systems. Many cytokines induce, enhance and govern the traffic of leukocytes. An important mechanism in cell trafficking and migration through endothelial basement membranes and connective tissues is the cytokine-regulated production of matrix degrading proteases. The latter include the serine proteinases of plasminogen activation and metalloproteinases such as collagenases, stromelysins and gelatinases. Many cytokines and all known matrix proteinases are glycoproteins and thus occur as sets of glycoforms. The relation between structures and functions of these glycoproteins has already been probed extensively at the protein level but not yet at the carbohydrate level. Attached oligosaccharides target the cytokines and proteinases to specific cellular receptors and matrix binding sites. In addition, a number of cytokines possess lectin-like functions and may thus interact with carbohydrates of the host or parasites. These intermolecular interactions influence for instance the compartmentalisation, the cell- and tissue-specific distribution and the pharmacokinetics of cytokines and proteinases. Attempts were done to deduce structure-function rules for the intramolecular effects of carbohydrates on cytokines and matrix proteinases. The relatively voluminous N-linked sugars downmodulate the specific activities of enzymes and cytokines. Because in host stress reactions (infection, inflammation, trauma) N-linked glycosylation is less efficient, glycosylation may constitute an important regulatory mechanism in the cytokine network and in multi-enzyme cascades.

Animals↗
PubMed1998

Concepts and principles of O-linked glycosylation.

The biosynthesis, structures, and functions of O-glycosylation, as a complex posttranslational event, is reviewed and compared for the various types of O-glycans. Mucin-type O-glycosylation is initiated by tissue-specific addition of a GalNAc-residue to a serine or a threonine of the fully folded protein. This event is dependent on the primary, secondary, and tertiary structure of the glycoprotein. Further elongation and termination by specific transferases is highly regulated. We also describe some of the physical and biological properties that O-glycosylation confers on the protein to which the sugars are attached. These include providing the basis for rigid conformations and for protein stability. Clustering of O-glycans in Ser/Thr(/Pro)-rich domains allows glycan determinants such as sialyl Lewis X to be presented as multivalent ligands, essential for functional recognition. An additional level of regulation, imposed by exon shuffling and alternative splicing of mRNA, results in the expression of proteins that differ only by the presence or absence of Ser/Thr(/Pro)-rich domains. These domains may serve as protease-resistant spacers in cell surface glycoproteins. Further biological roles for O-glycosylation discussed include the role of isolated mucin-type O-glycans in recognition events (e.g., during fertilization and in the immune response) and in the modulation of the activity of enzymes and signaling molecules. In some cases, the O-linked oligosaccharides are necessary for glycoprotein expression and processing. In contrast to the more common mucin-type O-glycosylation, some specific types of O-glycosylation, such as the O-linked attachment of fucose and glucose, are sequon dependent. The reversible attachment of O-linked GlcNAc to cytoplasmic and nuclear proteins is thought to play a regulatory role in protein function. The recent development of novel technologies for glycan analysis promises to yield new insights in the factors that determine site occupancy, structure-function relationship, and the contribution of O-linked sugars to physiological and pathological processes. These include diseases where one or more of the O-glycan processing enzymes are aberrantly regulated or deficient, such as HEMPAS and cancer.

Animals↗