Involvement of sialic acid in the regulation of γ--aminobutyric acid uptake activity of γ-aminobutyric acid transporter 1

The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.     

Effect of sodium lithium and proton concentrations on the electrophysiological properties of the four mouse GABA transporters expressed in Xenopus oocytes

Mouse GABA transporters belong to the family of Na(+) and Cl(-) dependent neurotransmitter transporter. GABA transport, by these family members, was shown to be electrogenic and driven by sodium ions. It was demonstrated that, as in several other transporters, sodium binding and release by GAT1, GAT3 and BGT-1, the canine homolog of GAT2, resulted in the appearance of presteady-state currents. In this work we show that each of the four GABA transporters exhibit unique presteady-state currents when expressed in Xenopus oocytes. The properties of the presteady-state currents correspond to the transporters affinities to Na(+). At 100 mM GAT1 exhibited symmetric presteady-state currents at all imposed potentials, whereas GAT2 exhibited asymmetric presteady-state currents exclusively at negative imposed potentials, GAT3 or GAT4 exhibited presteady-state currents predominantly at positive imposed potentials. GABA uptake by GAT2 and GAT4 was much more sensitive to external pH than GAT1 and GAT3. Reducing the external Na(+) concentration rendered the GABA uptake activity by GAT1 and GAT3 to be sensitive to pH. Lowering the external pH reduced the Na(+) affinity of GAT1. Substitution of the external Na(+) to Li(+) resulted in the appearance of leak currents exclusively at negative potentials in Xenopus oocyte expressing GAT1 and GAT3. Low Na(+) concentrations inhibited the leak currents of GAT1 but Na(+) had little effect on the leak currents of GAT3. Washing of occluded Na(+) in GAT1 enhanced the leak currents. Similarly addition of GABA in the presence of 80 mM Li(+), that presumably accelerated the release of the bound Na(+), also induced the leak currents. Conversely, addition of GABA to GAT3 expressing oocytes, in the presence of 80 mM Li(+), inhibited the leak currents.     

Role of Cl- in electrogenic Na+-coupled cotransporters GAT1 and SGLT1

We have investigated the functional role of Cl(-) in the human Na(+)/Cl(-)/gamma-aminobutyric acid (GABA) and Na(+)/glucose cotransporters (GAT1 and SGLT1, respectively) expressed in Xenopus laevis oocytes. Substrate-evoked steady-state inward currents were examined in the presence and absence of external Cl(-). Replacement of Cl(-) by gluconate or 2-(N-morpholino)ethanesulfonic acid decreased the apparent affinity of GAT1 and SGLT1 for Na(+) and the organic substrate. In the absence of substrate, GAT1 and SGLT1 exhibited charge movements that manifested as pre-steady-state current transients. Removal of Cl(-) shifted the voltage dependence of charge movements to more negative potentials, with apparent affinity constants (K(0.5)) for Cl(-) of 21 and 115 mm for SGLT1 and GAT1, respectively. The maximum charge moved and the apparent valence were not altered. GAT1 stoichiometry was determined by measuring GABA-evoked currents and the unidirectional influx of (36)Cl(-), (22)Na(+), or [(3)H]GABA. Uptake of each GABA molecule was accompanied by inward movement of 2 positive charges, which was entirely accounted for by the influx of Na(+) in the presence or absence of Cl(-). Thus, the GAT1 stoichiometry was 2Na(+):1GABA. However, Cl(-) was transported by GAT1 because the inward movement of 2 positive charges was accompanied by the influx of one Cl(-) ion, suggesting unidirectional influx of 2Na(+):1Cl(-):1GABA per transport cycle. Activation of forward Na(+)/Cl(-)/GABA transport evoked (36)Cl(-) efflux and was blocked by the inhibitor SKF 89976A. These data suggest a Cl(-)/Cl(-) exchange mechanism during the GAT1 transport cycle. In contrast, Cl(-) was not transported by SGLT1. Thus, in both GAT1 and SGLT1, Cl(-) modulates the kinetics of cotransport by altering Na(+) affinity, but does not contribute to net charge transported per transport cycle. We conclude that Cl(-) dependence per se is not a useful criterion to classify Na(+) cotransporters.     

GAT1 (GABA:Na+:Cl-) cotransport function. Steady state studies in giant Xenopus oocyte membrane patches.

Neurotransmitter transporters are reported to mediate transmembrane ion movements that are poorly coupled to neurotransmitter transport and to exhibit complex "channel-like" behaviors that challenge the classical "alternating access" transport model. To test alternative models, and to develop an improved model for the Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporter, GAT1, we expressed GAT1 in Xenopus oocytes and analyzed its function in detail in giant membrane patches. We detected no Na+- or Cl--dependent currents in the absence of GABA, nor did we detect activating effects of substrates added to the trans side. Outward GAT1 current ("reverse" transport mode) requires the presence of all three substrates on the cytoplasmic side. Inward GAT1 current ("forward" transport mode) can be partially activated by GABA and Na+ on the extracellular (pipette) side in the nominal absence of Cl-. With all three substrates on both membrane sides, reversal potentials defined with specific GAT1 inhibitors are consistent with the proposed stoichiometry of 1GABA:2Na+:1Cl-. As predicted for the "alternating access" model, addition of a substrate to the trans side (120 mM extracellular Na+) decreases the half-maximal concentration for activation of current by a substrate on the cis side (cytoplasmic GABA). In the presence of extracellular Na+, the half-maximal cytoplasmic GABA concentration is increased by decreasing cytoplasmic Cl-. In the absence of extracellular Na+, half-maximal cytoplasmic substrate concentrations (8 mM Cl-, 2 mM GABA, 60 mM Na+) do not change when cosubstrate concentrations are reduced, with the exception that reducing cytoplasmic Cl- increases the half-maximal cytoplasmic Na+ concentration. The forward GAT1 current (i.e., inward current with all extracellular substrates present) is inhibited monotonically by cytoplasmic Cl- (Ki, 8 mM); cytoplasmic Na+ and cytoplasmic GABA are without effect in the absence of cytoplasmic Cl-. In the absence of extracellular Na+, current-voltage relations for reverse transport current (i.e., outward current with all cytoplasmic substrates present) can be approximated by shallow exponential functions whose slopes are consistent with rate-limiting steps moving 0.15-0.3 equivalent charges. The slopes of current-voltage relations change only little when current is reduced four- to eightfold by lowering each cosubstrate concentration; they increase twofold upon addition of 100 mM Na+ to the extracellular (pipette) side.     

Identification of a lithium interaction site in the gamma-aminobutyric acid (GABA) transporter GAT-1

The sodium- and chloride-dependent electrogenic gamma-aminobutyric acid (GABA) transporter GAT-1, which transports two sodium ions together with GABA, is essential for synaptic transmission by this neurotransmitter. Although lithium by itself does not support GABA transport, it has been proposed that lithium can replace sodium at one of the binding sites but not at the other. To identify putative lithium selectivity determinants, we have mutated the five GAT-1 residues corresponding to those whose side chains participate in the sodium binding sites Na1 and Na2 of the bacterial leucine-transporting homologue LeuT(Aa). In GAT-1 and in most other neurotransmitter transporter family members, four of these residues are conserved, but aspartate 395 replaces the Na2 residue threonine 354. At varying extracellular sodium, lithium stimulated sodium-dependent transport currents as well as [3H]GABA uptake in wild type GAT-1. The extent of this stimulation was dependent on the GABA concentration. In mutants in which aspartate 395 was replaced by threonine or serine, the stimulation of transport by lithium was abolished. Moreover, these mutants were unable to mediate the lithium leak currents. This phenotype was not observed in mutants at the four other positions, although their transport properties were severely impacted. Thus at saturating GABA, the site corresponding to Na2 behaves as a low affinity sodium binding site where lithium can replace sodium. We propose that GABA participates in the other sodium binding site, just like leucine does in the Na1 site, and that at limiting GABA, this site determines the apparent sodium affinity of GABA transport.     

Differential effect of pH on sodium binding by the various GABA transporters expressed in Xenopus oocytes

Mouse GABA transporters belong to the family of Na(+)- and Cl(-)-dependent neurotransmitter transporters. The four GABA transporters exhibit unique presteady-state currents when expressed in Xenopus oocytes. The properties of the presteady-state currents correspond to their different affinities to Na(+). In the presence of 20 microM GABA and at pH 7.5, the half-maximal uptake activity was 47, 120, 25 and 35 mM Na(+) for GAT1, GAT2, GAT3 and GAT4, respectively. The appearance of presteady-state currents at positive or negative imposed potentials was in correlation with the affinity to Na(+). Changing the external pH differentially affected the GABA uptake and the presteady-state activities of the various GABA transporters. It is suggested that protons compete with Na(+) on its binding site; however, the proton binding is not productive and is unable to drive GABA uptake.     

Using lithium to probe sequential cation interactions with GAT1

Li(+) interacts with the Na(+)/Cl(-)-dependent GABA transporter, GAT1, under two conditions: in the absence of Na(+) it induces a voltage-dependent leak current; in the presence of Na(+) and GABA, Li(+) stimulates GABA-induced steady-state currents. The amino acids directly involved in the interaction with the Na(+) and Li(+) ions at the so-called "Na2" binding site have been identified, but how Li(+) affects the kinetics of GABA cotransport has not been fully explored. We expressed GAT1 in Xenopus oocytes and applied the two-electrode voltage clamp and (22)Na uptake assays to determine coupling ratios and steady-state and presteady-state kinetics under experimental conditions in which extracellular Na(+) was partially substituted by Li(+). Three novel findings are: 1) Li(+) reduced the coupling ratio between Na(+) and net charge translocated during GABA cotransport; 2) Li(+) increased the apparent Na(+) affinity without changing its voltage dependence; 3) Li(+) altered the voltage dependence of presteady-state relaxations in the absence of GABA. We propose an ordered binding scheme for cotransport in which either a Na(+) or Li(+) ion can bind at the putative first cation binding site (Na2). This is followed by the cooperative binding of the second Na(+) ion at the second cation binding site (Na1) and then binding of GABA. With Li(+) bound to Na2, the second Na(+) ion binds more readily GAT1, and despite a lower apparent GABA affinity, the translocation rate of the fully loaded carrier is not reduced. Numerical simulations using a nonrapid equilibrium model fully recapitulated our experimental findings.     

Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase.

Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.     

Residues in the extracellular loop 4 are critical for maintaining the conformational equilibrium of the gamma-aminobutyric acid transporter-1

We mutated residues Met345 and Thr349 in the rat gamma-aminobutyric acid transporter-1 (GAT-1) to histidines (M345H and T349H). These two residues are located four amino acids apart at the extracellular end of transmembrane segment 7 in a region of GAT-1 that we have previously suggested undergoes conformational changes critical for the transport process. The two single mutants and the double mutant (M345H/T349H) were expressed in Xenopus laevis oocytes, and their steady-state and presteady-state kinetics were examined and compared with wild type GAT-1 by using the two-electrode voltage clamp method. Oocytes expressing M345H showed a decrease in apparent GABA affinity, an increase in apparent affinity for Na+, a shift in the charge/voltage (Q/Vm) relationship to more positive membrane potentials, and an increased Li+-induced leak current. Oocytes expressing T349H showed an increase in apparent GABA affinity, a decrease in apparent Na+ affinity, a profound shift in the Q/Vm relationship to more negative potentials, and a decreased Li+-induced leak current. The data are consistent with a shift in the conformational equilibrium of the mutant transporters, with M345H stabilized in an outward-facing conformation and T349H in an inward-facing conformation. These data suggest that the extracellular end of transmembrane domain 7 not only undergoes conformational changes critical for the translocation process but also plays a role in regulating the conformational equilibrium between inward- and outward-facing conformations.     

A metabonomic study of inhibition of GABA uptake in the cerebral cortex

GABAergic activity is regulated by rapid, high affinity uptake of GABA from the synapse. Perturbation of GABA reuptake has been implicated in neurological disease and inhibitors of GABA transporters (GAT) have been used therapeutically but little detail is known about the ramifications of GAT inhibition on brain neurochemistry. Here, we incubated Guinea pig cortical tissue slices with [3-¹³C]pyruvate and major, currently available GABA uptake inhibitors. Metabolic fingerprints were generated from these experiments using ¹³C/¹H NMR spectroscopy. These fingerprints were analyzed using multivariate statistical approaches and compared with an existing library of fingerprints of activity at GABA receptors. This approach identified five distinct clusters of metabolic activity induced by blocking GABA uptake. Inhibition of GABA uptake via GAT1 produced patterns similar to activity at mainstream GABAergic synapses in particular those containing α1-subunits but still statistically separable. This indicated that inhibition of GABA uptake, an indirect method of activating GABA receptors, produces different effects to direct receptor activation or to exogenous GABA. The mechanism of inhibitor function also produced different outcomes, with the channel blocker SKF 89976A yielding a unique metabolic response. Blocking GAT1 and GAT3 simultaneously induces a large metabolic response consistent with induction of tonic inhibition via high affinity GABA receptors. Blocking BGT produces patterns similar to activity at less common receptors such as those containing α5 subunits. This approach is useful for determining where in the spectrum of GABAergic responses a particular GABA transport inhibitor is effective.     

Novel Properties of a Mouse γ-Aminobutyric Acid Transporter (GAT4)

We expressed the mouse gamma-aminobutyric acid (GABA) transporter GAT4 (homologous to rat/ human GAT-3) in Xenopus laevis oocytes and examined its functional and pharmacological properties by using electrophysiological and tracer uptake methods. In the coupled mode of transport (Na+/ Cl-/GABA cotransport), there was tight coupling between charge flux and GABA flux across the plasma membrane (2 charges/GABA). Transport was highly temperature-dependent with a temperature coefficient (Q10) of 4.3. The GAT4 turnover rate (1.5 s(-l); -50 mV, 21 degrees C) and temperature dependence suggest physiological turnover rates of 15-20 s(-1). No uncoupled current was observed in the presence of Na+. In the absence of external Na+, GAT4 exhibited two distinct uncoupled currents. (i) A Cl- leak current (ICl(leak)) was observed when Na+ was replaced with choline or tetraethylammonium. The reversal potential of (ICl(leak)) followed the Cl- Nernst potential. (ii) A Li+ leak current (ILi(leak)) was observed when Na+ was replaced with Li+. Both leak currents were inhibited by Na+, and both were temperature-independent (Q10 approximately 1). The two leak modes appeared not to coexist, as Li+ inhibited (ICl(leak)). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na+/Cl-/GABA cotransport, ILi(leak), and ICl(leak), (Ki approximately 30 microM), and the voltage-induced presteady-state charge movements (Ki approximately 440 microM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jicroumps revealed that slow Na+ binding to the transporter is followed by rapid GABA-induced translocation of the ligands across the plasma membrane. Thus, Na+ binding and associated conformational changes constitute the rate-limiting steps in the transport cycle.     

The role of external loop regions in serotonin transport. Loop scanning mutagenesis of the serotonin transporter external domain

Chimeric transporters were constructed in which the predicted external loops of the serotonin transporter (SERT) were replaced one at a time with a corresponding sequence from the norepinephrine transporter (NET). All of the chimeric transporters were expressed at levels equal to or greater than those of wild type SERT, but the transport and binding activity of the mutants varied greatly. In particular, mutants in which the NET sequence replaced external loops 4 or 6 of SERT had transport activity 5% or less than that of wild type, and the loop 5 replacement was essentially inactive. In some of these mutants, binding of a high affinity cocaine analog was less affected than transport, suggesting that the mutation had less effect on the initial binding steps in transport than on subsequent conformational changes. The more severely affected mutants also displayed an altered response to Na(+). In contrast to the dramatic reduction in transport and binding, the specificity of ligand binding was essentially unchanged. Chimeric transporters did not gain affinity for dopamine, a NET substrate, or desipramine, an inhibitor, at the expense of affinity for serotonin or paroxetine, a selective SERT inhibitor. The results suggest that external loops are not the primary determinants of substrate and inhibitor binding sites. However, they are not merely passive structures connecting transmembrane segments but rather active elements responsible for maintaining the stability and conformational flexibility of the transporter.     

GABA reverse transport by the neuronal cotransporter GAT1: influence of internal chloride depletion

The role of intracellular ions on the reverse GABA transport by the neuronal transporter GAT1 was studied using voltage-clamp and [(3)H]GABA efflux determinations in Xenopus oocytes transfected with heterologous mRNA. Reverse transport was induced by intracellular GABA injections and measured in terms of the net outward current generated by the transporter. Changes in various intracellular ionic conditions affected the reverse current: higher concentrations of Na(+) enhanced the ratio of outward over inward transport current, while a considerable decrease of the outward current and a parallel reduction of the transporter-mediated GABA efflux were observed after treatments causing a diminution of the intracellular Cl(-) concentration. Particularly interesting was the impairment of the reverse transport observed after depletion of internal Cl(-) generated by the activity of a coexpressed K(+)-Cl(-) exporter KCC2. This finding suggests that reverse GABA transport may be physiologically regulated during early neuronal development, similarly to the functional alterations seen in GABA receptors caused by KCC2 activity.     

Myrosinases TGG1 and TGG2 from Arabidopsis thaliana contain exclusively oligomannosidic N-glycans

In all eukaryotes N-glycosylation is the most prevalent protein modification of secretory and membrane proteins. Although the N-glycosylation capacity and the individual steps of the N-glycan processing pathway have been well studied in the model plant Arabidopsis thaliana, little attention has been paid to the characterization of the glycosylation status of individual proteins. We report here the structural analysis of all N-glycans present on the endogenous thioglucoside glucohydrolases (myrosinases) TGG1 and TGG2 from A. thaliana. All nine glycosylation sites of TGG1 and all four glycosylation sites of TGG2 are occupied by oligomannosidic structures with Man₅GlcNAc₂ as the major glycoform. Analysis of the oligomannosidic isomers from wild-type plants and mannose trimming deficient mutants by liquid chromatography with porous graphitic carbon and mass spectrometry revealed that the N-glycans from both myrosinases are processed by Golgi-located α-mannosidases.     

Truncation of the C terminus of the rat brain Na(+)-Ca(2+) exchanger RBE-1 (NCX1.4) impairs surface expression of the protein.

The C terminus of the rat brain Na(+)-Ca(2+) exchanger (RBE-1; NCX1. 4) (amino acids 875-903) is modeled to contain the last transmembrane alpha helix (amino acids 875-894) and an intracellular extramembraneous tail of 9 amino acids (895-903). Truncation of the last 9 C-terminal amino acids, Glu-895 to stop, did not significantly impair functional expression in HeLa or HEK 293 cells. Truncation, however, of 10 amino acids (Leu-894 to stop; mutant C10) reduced Na(+) gradient-dependent Ca(2+) uptake to 35-39% relative to the wild type parent exchanger, and further truncation of 13 or more amino acids resulted in expression of trace amounts of transport activity. Western analysis indicated that Na(+)-Ca(2+) exchanger protein was produced whether transfection was carried out with functional or non-functional mutants. Immunofluorescence studies of HEK 293 cells expressing N-Flag epitope-tagged wild type and mutant Na(+)-Ca(2+) exchangers revealed that transport activity in whole cells correlated with surface expression. All cells expressing the wild type exchanger or C9 exhibited surface expression of the protein. Only 39% of the cells expressing C10 exhibited surface expression, and none was detected in cells transfected with non-functional mutants C13 and C29. Since functional and non-functional mutants were glycosylated, the C terminus is not mandatory to translocation into the endoplasmic reticulum (ER). Endoglycosidase H digestion of [(35)S]methionine-labeled protein derived from wild type Na(+)-Ca(2+) exchanger and from C10 indicated that resistance to the digestion was acquired after 1 and 5 h of chase, respectively. C29 did not acquire detectable resistance to endoglycosidase H digestion even after 10 h of chase. Taken together, these results suggest that the "cellular quality control machinery" can tolerate the structural change introduced by truncation of the C terminus up to Ser-893 albeit with reduced rate of ER-->Golgi transfer and reduced surface expression of the truncated protein. Further truncation of C-terminal amino acids leads to retention of the truncated protein in the ER, no transfer to the Golgi, and no surface expression.     

N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.     

Dual effect of 1-deoxymannojirimycin on the mannose uptake and on the N-glycan processing of the human colon cancer cell line HT-29

1-Deoxymannojirimycin (dMM), a specific alpha-mannosidase I inhibitor, completely blocks the conversion of Man9-8GlcNAc2 into Man7-5-GlcNAc2 in both differentiated and undifferentiated human adenocarcinoma HT-29 cells. Besides this well known effect on N-glycan trimming, we describe here a novel effect of this inhibitor on the D-[2-3H]mannose uptake that is exclusively observed in differentiated intestinal cells, i.e. cells that display a functional apical brush border membrane. This inhibition of D-[2-3H]mannose uptake was shown to be dose-dependent and reversible. Moreover, using microsomal fractions we showed that this effect depends only on the integrity of the brush border and is unrelated to the classical inhibitory effect of dMM on N-glycan processing. Furthermore, another N-glycan trimming inhibitor 1-deoxynojirimycin, an epimer of dMM, did not interfere with D-[2-3H]mannose uptake. This observation was in good agreement with the specificity of the effect induced by dMM. These results demonstrate a novel effect of dMM on highly differentiated intestinal cells and suggest that a carrier-mediated mannose transport could exist in those cells. Such an interaction between cell morphology and the biological effect of dMM should lead to a careful use of drugs acting on N-glycan processing.     

N-glycosylation of the Xenopus laevis ClC-5 protein plays a role in cell surface expression, affecting transport activity at the plasma membrane

Mutations in the gene encoding ClC-5 lead to X-linked hypercalciuric nephrolithiasis (XLHN), characterized by proteinuria, hypercalciuria, and phosphaturia. In renal proximal tubule cells, ClC-5 was identified as an important player in endocytosis, which ensures reabsorption of filtered protein. However, the recent finding that ClC-5 is a Cl(-)/H(+) antiporter and not a Cl(-) channel as long thought points to the lack of understanding of its functional role. Also, little biochemical data are available about ClC-5 and its post-translational modifications have not been investigated. Here, we examined the role of N-glycosylation of xClC-5 in the Xenopus oocyte expression system by comparing wild-type (WT) xClC-5 and N-glycosylation site mutants. We found that xClC-5 is N-glycosylated on asparagines 169 and 470, which are the only N-glycosylated sites. xClC-5 mutants have an increased susceptibility to polyubiquitination and proteasomal degradation; however, without a notable impact on the expression level. Using a cross-linking reagent, we showed that xClC-5 assembles into protein complexes, independent of its N-glycosylation. Voltage-clamp measurements showed a reduced conductance in the presence of tunicamycin and with xClC-5 N-glycosylation site mutants. Using immunocytochemistry, we localized xClC-5 mainly in intracellular compartments, and found that its cell surface pool is reduced in the absence of N-glycans. We further examined the plasma membrane retrieval of WT and mutant xClC-5 in the presence of Brefeldin A (BFA), and found that the non-glycosylated mutant was retrieved more than five times faster than the WT protein. We conclude that N-glycosylation enhances cell surface expression of xClC-5, increasing its plasma membrane transport activity.     

A glutamine residue conserved in the neurotransmitter:sodium:symporters is essential for the interaction of chloride with the GABA transporter GAT-1

Neurotransmitter:sodium symporters are crucial for efficient synaptic transmission. The transporter GAT-1 mediates electrogenic cotransport of GABA, sodium, and chloride. The presence of chloride enables the transporter to couple the transport of the neurotransmitter to multiple sodium ions, thereby enabling its accumulation against steep concentration gradients. Here we study the functional impact of mutations of the putative chloride-binding residues on transport by GAT-1, with the emphasis on a conserved glutamine residue. In contrast to another putative chloride coordinating residue, Ser-331, where mutation to glutamate led to chloride-independent GABA transport, the Q291E mutant was devoid of any transport activity, despite substantial expression at the plasma membrane. Low but significant transport activity was observed with substitution mutants with small side chains such as Q291S/A/G. Remarkably, when these mutations were combined with the S331E mutation, transport was increased significantly, even though the activity of the S331E single mutant was only ～25% of that of wild type GAT-1. Transport by these double mutants was largely chloride-independent. Like mutants of other putative chloride coordinating residues, the apparent affinity of the active Gln-291 single mutants for chloride was markedly reduced along with a change their anion selectivity. In addition to the interaction of the transporter with chloride, Gln-291 is also required at an additional step during transport. Electrophysiological analysis of the Q291N and Q291S mutants, expressed in Xenopus laevis oocytes, is consistent with the idea that this additional step is associated with the gating of the transporter.     

A hypomorphic allele of the first N-glycosylation gene, ALG7, causes mitochondrial defects in yeast

The modification of proteins at asparagine residues with oligosaccharides (N-glycans) plays critical roles in diverse cell functions. N-glycans originate from a common lipid-linked oligosaccharide (LLO) precursor whose synthesis is initiated by the Dol-P-dependent GlcNAc-1-P transferase (GPT) encoded by an essential ALG7 gene. To identify cellular processes affected by ALG7 and N-glycosylation, we replaced the genomic copy of ALG7 with its hypomorphic allele in two genetically distinct haploid yeast cells. We show that ALG7 knockdown gave rise to an unexpected phenotype of mitochondrial dysfunction. The alg7 mutants did not grow on glycerol and DNA arrays revealed the absence of mitochondrial genes' expression. Accordingly, the alg7 mutants displayed no detectable mtDNA and respiratory activity. Both mutants exhibited diminished abundance of LLO and under-glycosylation of carboxypeptidase Y (CPY). Moreover, another N-glycosylation mutant with a LLO defect, alg6, was respiratory deficient. Collectively, our studies provide evidence that the dysregulation of N-glycosylation in haploid yeast cells leads to mitochondrial dysfunction.