Adrian R. Walmsley

Membrane topology influences N-glycosylation of the prion protein

The glycosylation state of the glycosyl‐phosphatidylinositol (GPI) anchored cellular prion protein (PrPC) can influence the formation of the disease form of the protein responsible for the neurodegenerative spongiform encephalopathies. We have investigated the role of membrane topology in the N‐glycosylation of PrP by expressing a C‐terminal transmembrane anchored form, PrP‐CTM, an N‐terminal transmembrane anchored form, PrP‐NTM, a double‐anchored form, PrP‐DA, and a truncated form, PrPΔGPI, in human neuroblastoma SH‐SY5Y cells. Wild‐type PrP, PrP‐ CTM and PrP‐DA were membrane anchored and present on the cell surface as glycosylated forms. In contrast, PrP‐NTM, although membrane anchored and localized at the cell surface, was not N‐glycosylated. PrPΔGPI was secreted from the cells into the medium in a hydrophilic form that was unglycosylated. The 4‐fold slower rate at which PrPΔGPI was trafficked through the cell compared with wild‐type PrP was due to the absence of the GPI anchor not the lack of N‐glycans. Retention of PrPΔGPI in the endoplasmic reticulum did not lead to its glycosylation. These results indicate that C‐terminal membrane anchorage is required for N‐glycosylation of PrP.

Tethering the N-terminus of the prion protein compromises the cellular response to oxidative stress

The role of the N‐terminal half of the prion protein (PrPC) in normal cellular function and pathology remains enigmatic. To investigate the biological role of the N‐terminus of PrP, we examined the cellular properties of a construct of murine PrP, PrP‐DA, in which the N‐terminus is tethered to the membrane by an uncleaved signal peptide and which retains the glycosyl‐phosphatidylinositol anchor. Human neuroblastoma SH‐SY5Y cells expressing PrP‐DA were more susceptible to hydrogen peroxide and copper induced toxicity than wtPrP expressing cells. The PrP‐DA expressing cells had an increased level of intracellular free radicals and reduced levels of superoxide dismutase and glutathione peroxidase as compared to the wtPrP expressing cells. The membrane topology, cell surface location, lipid raft localisation, intracellular trafficking and copper‐mediated endocytosis of PrP‐DA were not significantly different from wtPrP. However, cells expressing PrP‐DA accumulated an N‐terminal fragment that was resistant to proteinase K. The data presented here are consistent with the N‐terminal region of PrPC having a role in the cellular response to oxidative stress, and that tethering this region of the protein to the membrane compromises this function through the accumulation of a protease‐resistant N‐terminal fragment, similar to that seen in some forms of human prion disease.

alpha-cleavage of the prion protein occurs in a late compartment of the secretory pathway and is independent of lipid rafts

Endoproteolysis of the cellular prion protein (PrP(C)) modulates both the normal function of the protein and the pathogenesis of the neurodegenerative prion diseases. PrP(C) undergoes alpha-cleavage to generate the N-terminally truncated fragment C1. Utilizing various constructs of PrP(C) expressed in human neuroblastoma cells we investigated the subcellular compartment where alpha-cleavage occurs. C1 was detected at the cell surface and the generation of C1 occurred in mutants of PrP(C) incapable of Cu2+-mediated endocytosis. A transmembrane-anchored form that is not lipid raft-localised, as well as a secreted construct lacking the glycosyl-phosphatidylinositol membrane anchor, were also subject to alpha-cleavage. However, when this transmembrane-anchored form was modified with an endoplasmic reticulum retention motif, C1 was not formed. Inhibition of protein export from the Golgi by temperature block increased the amount of C1. Our data thus demonstrate that the alpha-cleavage of PrP(C) occurs predominantly in a raft-independent manner in a late compartment of the secretory pathway.

Purification of the Tn10-specified tetracycline efflux antiporter TetA in a native state as a polyhistidine fusion protein

The bacterial tetracycline‐resistance determinant from Tn 10 encodes a 43 kDa membrane protein, TetA, responsible for active efflux of tetracyclines. The tetA gene was cloned behind a T7 promoter/ac operator in a plasmid that provided fusion of TetA to a polyhis‐tidine‐carboxy terminal tail. A second plasmid provided a regulated T7 RNA polymerase. The specific activity of the TetA fusion protein was between 10–40% that of the wild‐type protein as assayed by tetracycline resistance in cells and by transport in membrane vesicles. The fusion protein, overproduced approximately 3–13‐fold, was purified by nickel chelation chromatography. Calculations from circular dichroism spectra of the purified protein solubilized in dodecylmaltoside gave an α‐helix content of 54–64%, close to the 68% predicted from the amino acid sequence by hydropathy analysis (12 membrane‐spanning helices) for the native protein in the membrane bilayer. Fluorescence studies showed binding activity of the purified protein to its substrate, the tetracycline analogue 13‐(cyclopentylthio)‐5‐hydroxy‐6‐α‐deoxyte‐tracycline. These findings suggested that the purified protein was in a native state.

Asparagine 394 in Putative Helix 11 of the Galactose-H+ Symport Protein (GalP) from Escherichia coli Is Associated with the Internal Binding Site for Cytochalasin B and Sugar

The galactose-H+ symport protein (GalP) of Escherichia coli is very similar to the human glucose transport protein, GLUT1, and both contain a highly conserved Asn residue in predicted helix 11 that is different in a cytochalasin B-resistant member of this sugar transport family (XylE). The role of the Asn394 residue (which is predicted to be in putative trans-membrane α-helix 11) in the structure/activity relationship of the d-galactose-H+ symporter (GalP) was therefore assessed by measuring the interaction of sugar substrates and the inhibitory antibiotics, cytochalasin B, and forskolin with the wild-type and Asn394 → Gln mutant proteins. Steady-state fluorescence quenching experiments show that the mutant protein binds cytochalasin B with a K d 37–53-fold higher than the wild type. This low affinity binding was not detected with equilibrium binding or photolabeling experiments. In contrast, the mutant protein binds forskolin with aK d similar to that of the wild type and is photolabeled by 3-125I-4-azido-phenethylamido-7-O-succinyl-desacetyl-forskolin. The mutant protein displays an increased amount of steady-state fluorescence quenching with the binding of forskolin, suggesting that the substitution of the Asn residue has altered the environment of a tryptophan, probably Trp395, in a conformationally active region of the protein. Time-resolved fluorescence measurements on the mutant protein provided association and dissociation rate constants (k 2 and k −2), describing the initial interaction of cytochalasin B to the inward-facing binding site (Ti), that are decreased (9-fold) and increased (4.9-fold) compared with the wild type. This yielded a dissociation constant (K 2) for cytochalasin B to the inward-facing binding site 44-fold higher than that of the wild type. The binding of forskolin gave values fork 2 and k −2 3.9- and 3.6-fold lower, respectively, yielding a K 2value for Ti similar to that of the wild type. The low overall affinity (high K d ) of the mutant protein for cytochalasin B is due mainly to a disruption in binding to the Ti conformation. It is proposed that Asn394forms either a direct binding interaction with cytochalasin B or is part of the immediate environment of the binding site and that Asn394 is in the immediate environment, but not part, of the forskolin binding site. The ability of the mutant protein to catalyze energized transport is only mildly impaired with 4.8- and 2.1-fold reduction inV max/K m values ford-galactose and d-glucose, respectively. In stark contrast, the overall K d describing binding of d-galactose and d-glucose to the inward-facing conformation of the mutant and their subsequent translocation across the membrane is substantially increased (64-fold for d-galactose and 163.3-fold for d-glucose). These data indicate that Asn394 is associated with both the cytochalasin B and internal sugar binding sites. This conclusion is also supported by data showing that the sugar specificity of the mutant protein has been altered for d-xylose. This work powerfully illustrates how comparisons of the aligned amino acid sequences of homologous membrane proteins of unknown structure and characterization of their phenotypes can be used to map substrate and ligand binding sites.

Distance of sequons to the C-terminus influences the cellular N-glycosylation of the prion protein.

Cell-specific differences in the utilization of the two N-glycosylation sequons (Asn180-Ile-Thr and Asn196-Phe-Thr) of the prion protein (PrP) have been proposed to influence the aetiology of the neurodegenerative prion diseases. As the N-glycosylation of PrP is ablated by deletion of the C-terminal glycosyl-phosphatidylinositol (GPI) anchor signal sequence, we have investigated the determinants for PrP sequon utilization in human neuronal cells using the novel approach of restoring N-glycosylation to secreted forms of PrP lacking a GPI anchor. N-glycosylation was restored to an efficiency comparable with that of GPI anchored PrP when the distance of the sequon to the C-terminus was increased so that it was sufficient to reach the active site of oligosaccharyltransferase before chain termination. Our findings indicate that sequon utilization in PrP is a co-translational process that precedes GPI anchor addition and, as such, will be greatly influenced by the dynamics of the translocon-oligosaccharyltransferase complex.

The mechanism of substrate and coenzyme binding to clostridial glutamate dehydrogenase during oxidative deamination

The binding of NAD+ and L-Glutamate to glutamate dehydrogenase (GDH) from Clostridium symbiosum has been investigated by stopped-flow fluorescence spectroscopy. The formation of the binary complexes produces little change in the protein fluorescence but formation of the ternary complex results in quenching of its fluorescence with a maximum value of 40%. This finding, coupled with the finding that a step prior to hydride transfer but subsequent to ternary complex formation is rate limiting, has enabled us to monitor the kinetics of ternary complex formation in detail. The ternary complex can be formed via the GDH-NAD+ or the GDH-L-Glu binary complexes, but the route via the GDH-NAD+ binary complex is the preferred pathway. The equilibrium and rate constants for the formation of the two binary complexes and the ternary complex formed via the two possible pathways have been determined. These studies have revealed an interaction between the coenzyme-binding site and the substrate-binding site, which lead to a decrease in the binding constant for the second substrate binding to the enzyme. The free energy coupling between the binary and ternary complexes is about 2.4-2.8 kJ.mol-1. We propose that there is a further isomerisation of the ternary complex, which is rate limiting for the steady-state turnover of the enzyme. Formation of this complex is characterised by an increased negative interaction, with a free energy coupling between these complexes of 6.3-11.6 kJ.mol-1.

Kinetic studies on the binding of 1,N6-etheno-NAD+ to glutamate dehydrogenase from Clostridium symbiosum

The mechanism of the binding of reduced coenzyme (NAD+) to clostridial glutamate dehydrogenase (GDH) was determined by transient kinetics. The fluorescent 1,N6-ethenoadenine analogue of NAD+ (epsilonNAD+) was used as a probe of nucleotide binary and ternary complex formation because the binding of NAD+ is optically silent. The kinetics of epsilonNAD+ binding were consistent with a 3-step binding process. The enzyme was found to oscillate between two conformational forms, termed E1 and E2, in the presence and absence of L-glutamate. However, L-glutamate shifted the equilibrium from 96.8% to 99% of the enzyme in the E1 form. The rapid-equilibrium binding of epsilonNAD+ to the E2 form was rate limited by a slow isomerisation of the ternary complex as the binary complex became saturated with epsilonNAD+. The L-glutamate binary complex had a greater affinity for the coenzyme (Kd = 11 microM) than the free enzyme (Km = 39 microM), indicative of a positive interaction of the substrate and coenzyme binding sites. Steady-state studies were also indicative of a positive interaction in the formation of the catalytic complex, with this complex having a Kd for epsilonNAD+ of 6.8 microM. Consequently, there is stabilization of successive complexes on the reaction pathway.

Dissection of discrete kinetic events in the binding of antibiotics and substrates to the galactose-H+ symport protein, GalP, of Escherichia coli.

GalP is the membrane protein responsible for H+-driven uptake of D-galactose intoEscherichia coli. It is suggested to be the bacterial equivalent of the mammalian glucose transporter, GLUT1, since these proteins share sequence homology, recognise and transport similar substrates and are both inhibited by cytochalasin B and forskolin. The successful over-production of GalP to 35–55% of the total inner membrane protein ofE. coli has allowed direct physical measurements on isolated membrane preparations. The binding of the antibiotics cytochalasin B and forskolin could be monitored from changes in the inherent fluorescence of GalP, enabling derivation of a kinetic mechanism describing the interaction between the ligands and GalP. The binding of sugars to GalP produces little or no change in the inherent fluorescence of the transporter. However, the binding of transported sugars to GalP produces a large increase in the fluorescence of 8-anilino-1-naphthalene sulphonate (ANS) excited via tryptophan residues. This has allowed a binding step, in addition to two putative translocation steps, to be measured. From all these studies a basic kinetic mechanism for the transport cycle under non-energised conditions has been derived. The ease of genetical manipulation of thegalP gene inE. coli has been exploited to mutate individual amino acid residues that are predicted to play a critical role in transport activity and/or the recognition of substrates and antibiotics. Investigation of these mutant proteins using the fluorescence measurements should elucidate the role of individual residues in the transport cycle as well as refine the current model.