N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP

N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of P2X(3) receptor (Asn(139), Asn(170), Asn(194) and Asn(290)) and performed comparative analysis of the role of N-glycans on protein stability, plasma membrane delivery, trimer formation and inward currents. We have found that in transiently transfected HEK293 cells, Asn(170) is apparently the most important site for receptor stability, since its mutation causes a primary loss in protein content and indirect failure in membrane expression, oligomeric association and inward current responses. Even stronger effects are obtained when mutating Thr(172) in the same glycosylation consensus. Asn(194) and Asn(290) are the most dispensable, since even their simultaneous mutation does not affect any tested receptor feature. All double mutants containing Asn(170) mutation or the Asn(139)/Asn(290) double mutant are instead almost unable to assemble into a functional trimeric structure. The main emerging finding is that the inability to assemble into trimers might account for the impaired function in P2X(3) mutants where residue Asn(170) is replaced. These results improve our knowledge about the role of N-glycosylation in proper folding and oligomeric association of P2X(3) receptor.     

ATP binding at human P2X1 receptors. Contribution of aromatic and basic amino acids revealed using mutagenesis and partial agonists

P2X receptors comprise a family of ATP-gated ion channels with the basic amino acids Lys-68, Arg-292, and Lys-309 (P2X(1) receptor numbering) contributing to agonist potency. In many ATP-binding proteins aromatic amino acids coordinate the binding of the adenine group. There are 20 conserved aromatic amino acids in the extracellular ligand binding loop of at least 6 of the 7 P2X receptors. We used alanine replacement mutagenesis to determine the effects of individual conserved aromatic residues on the properties of human P2X(1) receptors expressed in Xenopus oocytes. ATP evoked concentration-dependent (EC(50) approximately 1 microm) desensitizing currents at wild-type receptors and for the majority of mutants there was no change (10 residues) or a <6-fold decrease in ATP potency (6 mutants). Mutants F195A and W259A failed to form detectable channels at the cell surface. F185A and F291A produced 10- and 160-fold decreases in ATP potency. The partial agonists 2',3'-O-(4-benzoyl)-ATP (BzATP) and P(1),P(5)-di(adenosine 5')-pentaphosphate (Ap(5)A) were tested on a range of mutants that decreased ATP potency to determine whether this resulted predominantly from changes in agonist binding or gating of the channel. At K68A and K309A receptors BzATP and Ap(5)A had essentially no agonist activity but antagonized, or for R292A potentiated, ATP responses. At F185A receptors BzATP was an antagonist but Ap(5)A no longer showed affinity for the receptor. These results suggest that residues Lys-68, Phe-185, Phe-291, Arg-292, and Lys-309 contribute to ligand binding at P2X(1) receptors, with Phe-185 and Phe-291 coordinating the binding of the adenine ring of ATP.     

Cysteine substitution mutagenesis and the effects of methanethiosulfonate reagents at P2X2 and P2X4 receptors support a core common mode of ATP action at P2X receptors

The agonist binding site of ATP-gated P2X receptors is distinct from other ATP-binding proteins. Mutagenesis on P2X(1) receptors of conserved residues in mammalian P2X receptors has established the paradigm that three lysine residues, as well as FT and NFR motifs, play an important role in mediating ATP action. In this study we have determined whether cysteine substitution mutations of equivalent residues in P2X(2) and P2X(4) receptors have similar effects and if these mutant receptors can be regulated by charged methanethiosulfonate (MTS) compounds. All the mutants (except the P2X(2) K69C and K71C that were expressed, but non-functional) showed a significant decrease in ATP potency, with >300-fold decreases for mutants of the conserved asparagine, arginine, and lysine residues close to the end of the extracellular loop. MTS reagents had no effect at the phenylalanine of the FT motif, in contrast, cysteine mutation of the threonine was sensitive to MTS reagents and suggested a role of this residue in ATP action. The lysine-substituted receptors were sensitive to the charge of the MTS reagent consistent with the importance of positive charge at this position for coordination of the negatively charged phosphate of ATP. At the NFR motif the asparagine and arginine residues were sensitive to MTS reagents, whereas the phenylalanine was either unaffected or showed only a small decrease. These results support a common site of ATP action at P2X receptors and suggest that non-conserved residues also play a regulatory role in agonist action.     

Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors

P2X receptors are ATP-gated cation channels and assembled as homotrimers or heterotrimers from seven cloned subunits. Each subunit contains two transmembrane domains connected by a large extracellular loop. We have previously shown that replacement of two conserved residues, K68 and F291, by cysteine residues leads to disulfide cross-linking between neighbouring P2X₁ subunits. Since mutation of these residues results in a reduced ATP potency and cysteine cross-linking is prevented in the presence of ATP, we suggested an inter-subunit ATP binding site. To investigate whether the proximity of these residues is preserved in other P2X subtypes, we tested for spontaneous cystine formation between the corresponding P2X₂ (K69C, F289C), P2X₃ (K63C, F280C), and P2X₄ (K67C, F294C) mutants upon pairwise expression in Xenopus laevis oocytes. Non-reducing SDS-PAGE analysis of the purified receptors revealed a specific dimer formation between P2X₂K69C and P2X₂F289C mutants. Likewise, co-expression of P2X₁K68C and P2X₂F289C, but not P2X₁F291C and P2X₂K69C, mutants resulted in dimer formation between the respective subunits. Cross-linked P2X_1/2 heteromers showed strongly reduced or absent function that was selectively recovered upon treatment with DTT. Cross-linking was less efficient between P2X₃ or P2X₄ mutants but could be enhanced by the short cysteine-reactive cross-linker MTS-2-MTS. These results show that the spatial proximity and/or orientation of residues analogous to positions K68 and F291 in P2X₁ are preserved in P2X₂ receptors and at one of two possible interfaces in heteromeric P2X_1/2 receptors but appears to be redundant for P2X₃ and P2X₄ receptor function. EBSA Satellite Meeting: Ion channels, Leeds, July 2007.     

Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure

The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-beta-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-beta-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.     

N-glycan-mediated quality control in the endoplasmic reticulum is required for the expression of correctly folded delta-opioid receptors at the cell surface

A great majority of G protein-coupled receptors are modified by N-glycosylation, but the functional significance of this modification for receptor folding and intracellular transport has remained elusive. Here we studied these phenomena by mutating the two N-terminal N-glycosylation sites (Asn(18) and Asn(33)) of the human delta-opioid receptor, and expressing the mutants from the same chromosomal integration site in stably transfected inducible HEK293 cells. Both N-glycosylation sites were used, and their abolishment decreased the steady-state level of receptors at the cell surface. However, pulse-chase labeling, cell surface biotinylation, and immunofluorescence microscopy revealed that this was not because of intracellular accumulation. Instead, the non-N-glycosylated receptors were exported from the endoplasmic reticulum with enhanced kinetics. The results also revealed differences in the significance of the individual N-glycans, as the one attached to Asn(33) was found to be more important for endoplasmic reticulum retention of the receptor. The non-N-glycosylated receptors did not show gross functional impairment, but flow cytometry revealed that a fraction of them was incapable of ligand binding at the cell surface. In addition, the receptors that were devoid of N-glycans showed accelerated turnover and internalization and were targeted for lysosomal degradation. The results accentuate the importance of protein conformation-based screening before export from the endoplasmic reticulum, and demonstrate how the system is compromised when N-glycosylation is disrupted. We conclude that N-glycosylation of the delta-opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.     

On the role of the first transmembrane domain in cation permeability and flux of the ATP-gated P2X2 receptor

P2X receptors are a family of seven ligand-gated ion channels (P2X1-P2X7) that open in the presence of ATP. We used alanine-scanning mutagenesis and patch clamp photometry to study the role of the first transmembrane domain of the rat P2X2 receptor in cation permeability and flux. Three alanine-substituted mutants did not respond to ATP, and 19 of the 22 functional receptors resembled the wild-type receptor with regard to the fraction of the total ATP-gated current carried by calcium or the permeability of calcium relative to cesium. The remaining three mutants showed modest changes in calcium dynamics. Two of these occurred at sites (Gly30 and Phe44) that are unlikely to interact with permeating cations in a meaningful way. The third was a conserved tyrosine (Tyr43) that may form an inter-pore binding site for calcium. The data suggest that, with the possible exception of Tyr43, the first transmembrane domain contributes little to the permeation properties of the P2X2 receptor.     

Mutagenesis studies of conserved proline residues of human P2X receptors for ATP indicate that proline 272 contributes to channel function

Proline residues can play a major role in the secondary structure of proteins. In the extracellular ATP binding loop of P2X receptors there are four totally conserved proline residues (P2X1 receptor numbering; P93, P166, P228 and P272) and three less conserved residues P196 (six of seven isoforms), P174 and P225 (five of seven isoforms). We have mutated individual conserved proline residues in the human P2X1 receptor and determined their properties. Mutants were expressed in Xenopus oocytes and characterized using a two-electrode voltage clamp. Mutants P166A, P174A, P196A, P225A and P228A had no effect on ATP potency compared with wild-type and P93A had a fourfold decrease in ATP potency. The P272A, P272D and P272K receptor mutants were expressed at the cell surface; however, these mutants were non-functional. In contrast, P272I, P272G and P272F produced functional channels, with either no effect or a 2.5- or 6.5-fold increase in ATP potency, respectively. At P272F receptors the apparent affinity of the ATP analogue antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP was increased by 12.5-fold. These results suggest that individual proline residues are not essential for normal P2X receptor function and that the receptor conformation around P272 contributes to ATP binding at the receptor.     

Unique functional properties of a sensory neuronal P2X ATP-gated channel from zebrafish

We report here the structural and functional characterization of an ionotropic P2X ATP receptor from the lower vertebrate zebrafish (Danio rerio). The full-length cDNA encodes a 410-amino acid-long channel subunit zP2X(3), which shares only 54% identity with closest mammalian P2X subunits. When expressed in XENOPUS: oocytes in homomeric form, ATP-gated zP2X(3) channels evoked a unique nonselective cationic current with faster rise time, faster kinetics of desensitization, and slower recovery than any other known P2X channel. Interestingly, the order of agonist potency for this P2X receptor was found similar to that of distantly related P2X(7) receptors, with benzoylbenzoyl ATP (EC(50) = 5 microM) > ATP (EC(50) = 350 microM) = ADP > alpha,beta-methylene ATP (EC(50) = 480 microM). zP2X(3) receptors are highly sensitive to blockade by the antagonist trinitrophenyl ATP (IC(50) < 5 nM) but are weakly sensitive to the noncompetitive antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid. zP2X(3) subunit mRNA is exclusively expressed at high levels in trigeminal neurons and Rohon-Beard cells during embryonic development, suggesting that neuronal P2X receptors mediating fast ATP responses were selected early in the vertebrate phylogeny to play an important role in sensory pathways.     

The role of positively charged amino acids in ATP recognition by human P2X(1) receptors

P2X receptors for ATP are a family of ligand-gated cation channels. There are 11 conserved positive charges in the extracellular loop of P2X receptors. We have generated point mutants of these conserved residues (either Lys --> Arg, Lys --> Ala, Arg --> Lys, or Arg --> Ala) in the human P2X(1) receptor to determine their contribution to the binding of negatively charged ATP. ATP evoked concentration-dependent (EC(50) approximately 0.8 microm) desensitizing responses at wild-type (WT) P2X(1) receptors expressed in Xenopus oocytes. Suramin produced a parallel rightward shift in the concentration response curve with an estimated pK(B) of 6.7. Substitution of amino acids at positions Lys-53, Lys-190, Lys-215, Lys-325, Arg-202, Arg-305, and Arg-314 either had no effect or only a small change in ATP potency, time course, and/or suramin sensitivity. Modest changes in ATP potency were observed for mutants at K70R and R292K/A (20- and 100-fold decrease, respectively). Mutations at residues K68A and K309A reduced the potency of ATP by >1400-fold and prolonged the time course of the P2X(1) receptor current but had no effect on suramin antagonism. Lys-68, Lys-70, Arg-292, and Lys-309 are close to the predicted transmembrane domains of the receptor and suggest that the ATP binding pocket may form close to the channel vestibule.     

Contribution of conserved polar glutamine, asparagine and threonine residues and glycosylation to agonist action at human P2X1 receptors for ATP

The role of conserved polar glutamine, asparagine and threonine residues in the large extracellular loop, and glycosylation, to agonist action at human P2X1 receptors was tested by generating alanine substitution mutants. For the majority of mutants (Q56A, Q95A, T104A, T109A, Q112A, Q114A, T146A, N153A, T158A, N184A, N191A, N242A, N300A) alanine substitution had no effect on ATP potency. The mutants Q95A, Q112A, Q114A and T158A showed changes in efficacy for the partial agonists BzATP and Ap5A, suggesting that these polar residues may contribute to the gating of the channel. The mutants T186A, N204A and N290A had six-, three- and 60-fold decreases in ATP potency, respectively. For T186A and N290A, the partial agonists BzATP and Ap5A were no longer agonists but still bind to the receptor as shown by the ability to modulate the response to co-applied ATP. N153, N184 and N242 are glycosylated in the endoplasmic reticulum and N300 acquires complex glycosylation in the golgi. These results aid in refining a model for ATP binding at the P2X1 receptor where the residues F185T186, and the conserved triplet N290F291R292, are likely to play a role in ATP action at the receptor.     

Functional characterization of a P2X receptor from Schistosoma mansoni

The cloning and characterization of a P2X receptor (schP2X) from the parasitic blood fluke Schistosoma mansoni provides the first example of a non-vertebrate ATP-gated ion channel. A number of functionally important amino acid residues conserved throughout vertebrate P2X receptors, including 10 extracellular cysteines, aromatic and positively charged residues involved in ATP recognition, and a consensus protein kinase C site in the amino-terminal tail, are also present in schP2X. Overall, the amino acid sequence identity of schP2X with human P2X(1-7) receptors ranges from 25.8 to 36.6%. ATP evoked concentration-dependent currents at schP2X channels expressed in Xenopus oocytes with an EC(50) of 22.1 microM. 2',3'-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) was a partial agonist (maximum response 75.4 +/- 4.4% that of ATP) with a higher potency (EC(50) of 3.6 microM) than ATP. Suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid blocked schP2X responses to 100 microm ATP with IC(50) values of 9.6 and 0.5 microM, respectively. Ivermectin (10 microM) potentiated currents to both ATP and Bz-ATP by approximately 60% with a minimal effect on potency (EC(50) of 18.2 and 1.6 microM, respectively). The relative permeability of schP2X expressed in HEK293 cells to various cations was determined under bi-ionic conditions. schP2X has a relatively high calcium permeability (P(Ca)/P(Na) = 3.80 +/- 0.29) and an estimated minimum pore diameter similar to that of vertebrate P2X receptors. SchP2X provides a useful comparative model for the better understanding of human P2X receptor function and may also provide an alternative drug target for treatment of schistosomiasis.     

P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels.

P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross-linking and blue native PAGE. P2X1 and P2X3 were N-terminally tagged with six histidine residues to allow for non-denaturing receptor isolation from cRNA-injected, [35S]methionine-labeled oocytes. The His-tag did not change the electrophysiological properties of the P2X1 receptor. His-P2X1 was found to carry four N-glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3, 3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) cross-linked digitonin-solubilized His-P2X1 and His-P2X3 quantitatively to homo-trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl-beta-D-maltoside migrated entirely as non-covalently linked homo-trimers, whereas the alpha2 beta gamma delta nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His-P2X1 was also identified as a homo-trimer. If n-octylglucoside was used for P2X receptor solubilization, homo-hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure. Keywords: blue native PAGE/cross-linking/P2X receptor/quaternary structure.     

Cysteine scanning mutagenesis (residues Glu52-Gly96) of the human P2X1 receptor for ATP: mapping agonist binding and channel gating

P2X receptors are ATP-gated cation channels. The x-ray structure of a P2X4 receptor provided a major advance in understanding the molecular basis of receptor properties. However, how agonists are coordinated, the extent of the binding site, and the contribution of the vestibules in the extracellular domain to ionic permeation have not been addressed. We have used cysteine-scanning mutagenesis to determine the contribution of residues Glu(52)-Gly(96) to human P2X1 receptor properties. ATP potency was reduced for the mutants K68C, K70C, and F92C. The efficacy of the partial agonist BzATP was also reduced for several mutants forming the back of the proposed agonist binding site. Molecular docking in silico of both ATP and BzATP provided models of the agonist binding site consistent with these data. Individual cysteine mutants had no effect or slightly increased antagonism by suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate. Mutants at the entrance to and lining the upper vestibule were unaffected by cysteine-reactive methanethiosulfonate (MTS) reagents, suggesting that it does not contribute to ionic permeation. Mutants that were sensitive to modification by MTS reagents were predominantly found either around the proposed ATP binding pocket or on the strands connecting the binding pocket to the transmembrane region and lining the central vestibule. In particular, ATP sensitivity and currents were increased by a positively charged MTS reagent at the G60C mutant at the interface between the central and extracellular vestibule. This suggests that dilation of the base of the central vestibule contributes to gating of the receptor.     

Desensitization masks nanomolar potency of ATP for the P2X1 receptor

ATP-gated P2X1 receptors feature fast activation and fast desensitization combined with slow recovery from desensitized states. Here, we exploited a non-desensitizing P2X2/P2X1 chimera that includes the entire P2X1 ectodomain (Werner, P., Seward, E. P., Buell, G. N., and North, R. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15485-15490) to obtain a macroscopic representation of intrinsic receptor kinetics without bias arising from the overlap of channel activation and desensitization. From the stationary currents made amenable to analysis by this chimera, an EC50 for ATP of 3.3 nM was derived, representing a >200- and >7000-fold higher ATP potency than observed for the parental P2X1 and P2X2A receptors, respectively. Also, other agonists activated the P2X2/P2X1 chimera with nanomolar EC50 values ranging from 3.5 to 73 nM in the following rank order: 2-methylthio-ATP, 2',3'-O-(4-benzoylbenzoyl)-ATP, alpha,beta-methylene-ATP, adenosine 5'-O-(3-thiotriphosphate). Upon washout, the P2X2/P2X1 chimera deactivated slowly with a time constant (ranging from 63 to 2.5 s) that is inversely related to the EC50 value for the corresponding agonist. This suggests that deactivation time courses reflect unbinding rates, which by themselves define agonist potencies. The P2X2/P2X1 chimera and the P2X1 receptor possess virtually identical sensitivity to inhibition by the P2X1 receptor-selective antagonist NF279, a suramin analog. These results suggest that the P2X1 ectodomain confers nanomolar ATP sensitivity, which, within the wild-type P2X1 receptor, is obscured by desensitization such that only a micromolar ATP potency can be deduced from peak current measurements, representing an amalgam of activation and desensitization.     

Clarification of the role of N-glycans on the common beta-subunit of the human IL-3, IL-5 and GM-CSF receptors and the murine IL-3 beta-receptor in ligand-binding and receptor activation

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 are related cytokines that play key roles in regulating the differentiation, proliferation, survival and activation of myeloid blood cells. The cell surface receptors for these cytokines are composed of cytokine-specific alpha-subunits and a common beta-receptor (betac), a shared subunit that is essential for receptor signaling in response to GM-CSF, IL-3 and IL-5. Previous studies have reached conflicting conclusions as to whether N-glycosylation of the betac-subunit is necessary for functional GM-CSF, IL-3 and IL-5 receptors. We sought to clarify whether betac N-glycosylation plays a role in receptor function, since all structural studies of human betac to date have utilized recombinant protein lacking N-glycosylation at Asn(328). Here, by eliminating individual N-glycans in human betac and the related murine homolog, beta(IL-3), we demonstrate unequivocally that ligand-binding and receptor activation are not critically dependent on individual N-glycosylation sites within the beta-subunit although the data do not preclude the possibility that N-glycans may exert some sort of fine control. These studies support the biological relevance of the X-ray crystal structures of the human betac domain 4 and the complete ectodomain, both of which lack N-glycosylation at Asn(328).     

Functional and biochemical evidence for heteromeric ATP-gated channels composed of P2X1 and P2X5 subunits.

The mammalian P2X receptor gene family encodes two-transmembrane domain nonselective cation channels gated by extracellular ATP. Anatomical localization data obtained by in situ hybridization and immunocytochemistry have shown that neuronal P2X subunits are expressed in specific but overlapping distribution patterns. Therefore, the native ionotropic ATP receptors diversity most likely arises from interactions between different P2X subunits that generate hetero-multimers phenotypically distinct from homomeric channels. Rat P2X1 and P2X5 mRNAs are localized within common subsets of peripheral and central sensory neurons as well as spinal motoneurons. The present study demonstrates a functional association between P2X1 and P2X5 subunits giving rise to hybrid ATP-gated channels endowed with the pharmacology of P2X1 and the kinetics of P2X5. When expressed in Xenopus oocytes, hetero-oligomeric P2X1+5 ATP receptors were characterized by slowly desensitizing currents highly sensitive to the agonist alpha,beta-methylene ATP (EC50 = 1.1 microM) and to the antagonist trinitrophenyl ATP (IC50 = 64 nM), observed with neither P2X1 nor P2X5 alone. Direct physical evidence for P2X1+5 co-assembly was provided by reciprocal subunit-specific co-purifications between epitope-tagged P2X1 and P2X5 subunits transfected in HEK-293A cells.     

The C-terminal N-glycosylation sites of the human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, adn -VI) are necessary for the expression of full enzyme activity

The alpha1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to approximately 40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the Km values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved C-terminal N-glycosylation sites for expression of full enzyme activity.     

N-Glycans on the Nipah Virus Attachment Glycoprotein Modulate Fusion and Viral Entry as They Protect against Antibody Neutralization

Nipah virus (NiV) is the deadliest known paramyxovirus. Membrane fusion is essential for NiV entry into host cells and for the virus'' pathological induction of cell-cell fusion (syncytia). The mechanism by which the attachment glycoprotein (G), upon binding to the cell receptors ephrinB2 or ephrinB3, triggers the fusion glycoprotein (F) to execute membrane fusion is largely unknown. N-glycans on paramyxovirus glycoproteins are generally required for proper protein conformational integrity, transport, and sometimes biological functions. We made conservative mutations (Asn to Gln) at the seven potential N-glycosylation sites in the NiV G ectodomain (G1 to G7) individually or in combination. Six of the seven N-glycosylation sites were found to be glycosylated. Moreover, pseudotyped virions carrying these N-glycan mutants had increased antibody neutralization sensitivities. Interestingly, our results revealed hyperfusogenic and hypofusogenic phenotypes for mutants that bound ephrinB2 at wild-type levels, and the mutant''s cell-cell fusion phenotypes generally correlated to viral entry levels. In addition, when removing multiple N-glycans simultaneously, we observed synergistic or dominant-negative membrane fusion phenotypes. Interestingly, our data indicated that 4- to 6-fold increases in fusogenicity resulted from multiple mechanisms, including but not restricted to the increase of F triggering. Altogether, our results suggest that NiV-G N-glycans play a role in shielding virions against antibody neutralization, while modulating cell-cell fusion and viral entry via multiple mechanisms.