Báo cáo khoa học: Disruption of transport activity in a D93H mutant thiamine transporter 1, from a Rogers Syndrome family pdf

Using transient transfections into NIH3T3 cells of a D93H mutant THTR1 derived from a Rogers syndrome family, we determined the expression, post-translational modification, plasma mem-bra

Disruption of transport activity in a D93H mutant thiamine

transporter 1, from a Rogers Syndrome family

Dana Baron1, Yehuda G Assaraf2, Stavit Drori2and Ami Aronheim1

1

Department of Molecular Genetics, The Rappaport Institute for Research in the Medical Sciences and the B Rappaport Faculty

of Medicine, and2Department of Biology, The Technion-Israel Institute of Technology, Haifa, Israel

Rogers syndrome is an autosomal recessive disorder

result-ing in megaloblastic anemia, diabetes mellitus, and

sensori-neural deafness The gene associated with this disease

encodes for thiamine transporter 1 (THTR1), a member of

the SLC19 solute carrier family including THTR2 and the

reduced folate carrier (RFC) Using transient transfections

into NIH3T3 cells of a D93H mutant THTR1 derived

from a Rogers syndrome family, we determined the

expression, post-translational modification, plasma

mem-brane targeting and thiamine transport activity We also

explored the impact on methotrexate (MTX) transport

activity of a homologous missense D88H mutation in the

human RFC, a close homologue of THTR1 Western blot

analysis revealed that the D93H mutant THTR1 was

nor-mally expressed and underwent a complete N-glycosylation

However, while this mutant THTR1 was targeted to the

plasma membrane, it was completely devoid of thiamine

transport activity Consistently, introduction into MTX

transport null cells of a homologous D88H mutation in the hRFC did not result in restoration of MTX trans-port activity, thereby suggesting that D88 is an essential residue for MTX transport activity These results suggest that the D93H mutation does not interfere with trans-porter expression, glycosylation and plasma membrane targeting However, the substitution of this negatively charged amino acid (Asp93) by a positively charged residue (His) in an extremely conserved region (the bor-der of transmembrane domain 2/intracellular loop 2) in the SLC19 family, presumably inflicts deleterious struc-tural alterations that abolish thiamine binding and/or translocation Hence, this functional characterization

of the D93H mutation provides a molecular basis for Rogers syndrome

Keywords: Rogers syndrome; thiamine; transporter; muta-tions

Thiamine responsive megaloblastic anemia (TRMA) also

known as Rogers syndrome [1], is an early onset autosomal

recessive disorder that was described in only 20 families

all over the world Patients with Rogers syndrome are

diagnosed by the occurrence of multiple clinical

manifesta-tions including: megaloblastic anemia, diabetes mellitus and

sensorineural deafness [Online Mendelian Inheritance in

Man (OMIM) 249270, http://www.ncbi.nlm.nih.gov/

Omim/] The administration of high doses of thiamine or

alternatively the use of medications such as sulfonylureas

recovers insulin secretion [2–4] However, with disease

progression, insulin secretion gradually declines to levels that require insulin therapy On the other hand, in all TRMA patients, anemia is fully reversed following the administration of high doses of thiamine [5]

Fibroblasts isolated from Rogers syndrome patients, display only 5–10% of thiamine uptake as compared with fibroblasts derived from healthy individuals This uptake apparently occurs via a low-affinity, unsaturable thiamine route [6] These results are consistent with an entry of thiamine via passive diffusion and may explain the fact that high doses of thiamine are able to induce minimally sufficient thiamine levels in Rogers syndrome patients The gene responsible for Rogers syndrome, SLC19A2, was identified by positional cloning [7–9] SLC19A2 encodes for a thiamine transporter (THTR1), a member of the solute carrier family This family includes SLC19A1 that encodes for the reduced folate carrier (RFC) and SLC19A3 that encodes for thiamine transporter 2, THTR2 [10] THTR1 contains 497-amino acids and has 12 putative transmem-brane domains (TMD) [7–9] THTR1 is responsible for thiamine transport in a Na+independent manner and is stimulated by H+efflux gradient [11]

Although all Rogers syndrome patients exhibit similar clinical manifestations, multiple mutations were identified resulting in premature translation termination due to base pair insertions or deletions which result in frame-shift In addition, three missense mutations were described resulting

in single amino-acid substitutions Recently we have initiated

Correspondence to A Aronheim, Department of Molecular Genetics,

The B Rappaport Faculty of Medicine, 7th Efron St Bat-Galim,

The Technion-Israel Institute of Technology, Haifa 31096, Israel.

Fax: + 972 4 8295225, Tel.: + 972 4 8295226,

E-mail: aronheim@tx.technion.ac.il

Y G Assaraf, Department of Biology, The Technion-Israel

Institute of Technology, Haifa 32000, Israel.

Fax: + 972 4 8225153, Tel.: + 972 4 8293744,

E-mail: assaraf@tx.technion.ac.il

Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; MTX,

Methotrexate; THTR1, thiamine transporter 1; TRMA, thiamine

responsive megaloblastic anemia; hRFC, human reduced folate

carrier; TMD, transmembrane domain.

(Received 23 July 2003, revised 11 September 2003,

accepted 18 September 2003)

studies that are aimed at characterization of the impact

that these missense THTR1 mutations have on the

expres-sion, post-translational modification, plasma membrane

targeting and thiamine transport activity In a recent paper,

we described the molecular consequences of a single

amino-acid substitution identified in an Italian Rogers syndrome

family harboring a glycine to aspartate substitution at

position 172 of the THTR1 [12] Although this mutant

THTR1gene is properly transcribed and translated in vitro,

no protein could be detected in NIH3T3 cells transfected

with the mutant G172D THTR1 cultured at 37C Shifting

transfected cells to 28C resulted in a substantial expression

of the mutant G172D THTR1 protein thereby allowing

for biochemical and functional characterization [12] The

G172D mutant THTR1 failed to undergo a complete

N-glycosylation, on the glutamine 63 acceptor which is

found to be conserved in both THTR1 and THTR2 [12]

resulting in its cytoplasmic retention in the endoplasmic

reticulum This is in contrast to the other members of the

solute carrier family, including the hRFC, in which

N-glycosylation plays neither a role in its plasma membrane

localization nor in MTX transport activity [13] Here we

describe the characterization of another mutation identified

in a French Rogers family harboring an aspartate to

histidine substitution at position 93 (D93H) in THTR1 [14]

Unlike in a previous analysis of the D93H mutation [15],

here we show that the mutant protein was efficiently

expressed both in vitro and in vivo and upon

immunoflu-orescence studies this protein was properly targeted to the

plasma membrane Uptake studies with the D93H mutant

protein revealed no [3H]thiamine transport activity

Import-antly, this D93 region in THTR1 and the homologous D88

vicinity in the hRFC are extremely conserved among all

members of the solute carrier family (SLC19A1, SLC19A2

and SLC19A3) Indeed, a hRFC harboring the same D88H

mutation displayed no methotrexate transport activity This

is consistent with a previous report in which a D88V mutant

RFC showed no transport activity [16] We suggest that this

site (i.e D93 in THTR1 and D88 in hRFC) represents a

highly conserved region, which is absolutely essential for the

structure and function of all members of the solute carrier

family Hence, the present study constitutes the first

demonstration of a THTR1 mutation that does not interfere

with transporter expression, N-linked glycosylation and

plasma membrane targeting but completely disrupts

thi-amine transport activity

Experimental procedures

Cell cultures

NIH3T3 and Swiss 3T3 cells were cultured under

mono-layer conditions in a humidified atmosphere of 5% CO2at

37C Cells were grown in Dulbecco’s modified Eagle’s

medium (DMEM) containing 10% fetal bovine serum

(FBS), 2 mM L-glutamine and 100 unitsÆmL)1penicillin and

100 lgÆmL)1streptomycin

Transient and stable transfections

Transient NIH3T3 cell transfections were carried out in

60 mm plates using the LipofectAMINE reagent

(Gibco-BRL Inc.), whereas XtremeGENE reagent (Roche Inc.) was used for Swiss 3T3 cells Typically, for NIH3T3 cell transfections, 6 lg of plasmid DNA were used with 9 lL

of the LipofectAMINE reagent whereas for Swiss 3T3 cells,

2 lg of plasmid DNA were used along with 10 lL of XtremeGENE reagent according to the instructions of the manufacturer

Cell extract Whole cell extracts were isolated as previously described [17]

Isolation of THTR1 cDNA THTR1 cDNA was isolated as previously described [12] Mammalian expression plasmids

pCan-THTR1 expression plasmids; pCan is a pcDNA-based (Invitrogen Inc.) mammalian expression vector expressing proteins downstream of a Myc-epitope tag Protein expression is under the control of cytomegalovirus (CMV) immediate early promoter An oligonucleotide based PCR approach was used to amplify the human THTR1 cDNA that was fused, in frame, with the Myc-epitope tag using 5¢-EcoRI and 3¢-XhoI restriction sites The Myc tag and polylinker regions are composed of the following 17 amino acids: MVQKLISEEDLRIHRCR For fusion of Myc tag to the C-terminus of the THTR1, an oligonucleotide-based PCR approach was used to amplify the human THTR1 cDNA that was fused in frame, using 5¢-HindIII and 3¢-HindIII restriction sites with the Myc tag

in the 3¢-end The correct orientation was verified by XbaI digestion generating a 1100-bp fragment due to a unique XbaI site at position 377 within THTR1 cDNA and at the 3¢-end of the pCan expression vector’s multiple cloning site

GFP expression plasmid This plasmid encodes for the green fluorescent protein under the control of the CMV promoter (Life Technologies Inc.) Site-directed mutagenesis

The corresponding oligonucleotides were designed to yield

an aspartate 93 to histidine substitution and to generate a diagnostic AatII restriction site Site directed mutagenesis was performed using the QuikChange XL site directed mutagenesis kit according to the instructions of the manufacturer (Stratagene Inc.) Primers used for muta-genesis of THTR1 (D93H) were: Upstream oligo: 5¢-CCTGTGTTCCTTGCCACACACTACCTCCGTTA TAAACC-3¢ and downstream oligo: 5¢-GGTTTATAACG GAGGTAGTGTGTGGCAAGGAACACAGG-3¢ DNA sequencing was used to verify that THTR1 was free of any other mutations The primers for the introduction of the D88H mutation in the hRFC were: Upstream oligo: 5¢-GTGTTCCTGCTCACCCACTACCTGCGCTACAC G-3¢ and downstream oligo: 5¢-CGTGTAGCGCAGGT AGTGGGTGAGCAGGAACAC-3¢

In vitro translation studies

Coupled in vitro transcription and translation were

performed using the T7 TNT quick transcription and

translation kit according to the instructions of the

manu-facturer (Promega Inc.)

Tunicamycin treatment

Tunicamycin (Sigma Cat # T7765) was dissolved in

dimethyl sulfoxide to yield a stock solution of 10 mgÆmL)1

This N-glycosylation inhibitor was applied to monolayer

cells at a final concentration of 10 lgÆmL)1for 24 h prior

to cell harvesting and immunofluorescent staining

PeptideN-glycosidase F (PNGaseF) treatment

Aliquots of cell extracts (50 lg) were incubated with a

denaturing buffer containing sodium phosphate pH 7.5,

0.5% SDS and 1% 2-mercaptoethanol in the presence or

absence of 2000 U of PNGaseF (NEB Inc.) for 45 min at

37C

SDS/PAGE and Western blotting

Aliquots of protein extracts (50 lg) or 5 lL of a

pro-grammed rabbit reticulocyte lysate were resolved on a 10%

SDS/PAGE followed by immobilization onto Hybond

nitrocellulose membrane (Amersham Inc.) Western blots

were then blocked with a buffer containing: 10% low fat

milk in NaCl/Pifor 1 h and incubated with mouse anti-Myc

9E11 monoclonal IgG (Babco Ltd) followed by a secondary

goat anti-(mouse Ig) Ig conjugated to horse radish

peroxi-dase (HRP) Detection was performed with a

chemilumi-nescent substrate (SuperSignal, Pierce Inc.) and membranes

were exposed to X-ray films and autoradiogram with linear

exposures were obtained

Immunofluorescence

Swiss 3T3 cells were grown on 22-mm coverslips and

transfected as described above Forty-eight hours after

transfection, cells were fixed and immunostained as

des-cribed [12] Each coverslip contained at least 5· 104cells

and the efficiency of transfection varied between 10 and

20% About 90% of transfected cells exhibited red and

green staining and all of these cells displayed the staining

presented At least 100 transfected cells per slide from each

transfection were examined The immunofluorescence

ana-lysis, which was reproduced in five independent transfection

experiments, revealed an identical staining pattern

Assay of thiamine uptake

NIH3T3 cells transiently transfected with wild-type and

mutant D93H THTR1 and vector control were detached

with NaCl/Picontaining 1 mMEDTA 2 h prior to thiamine

transport measurements Cells were incubated in a 15-mL

tube (Greiner Ltd) at a density of 3· 105 cellsÆmL)1 in

serum-free DMEM lacking thiamine NIH3T3 cells were

centrifuged and resuspended in 100 lL of transport buffer

containing 0.6 l of [3H]thiamine (30 CiÆmmol)1;

Amer-sham) for 20 min at 37C as previously described [11] Cells were then washed twice with 10 mL of ice-cold NaCl/Piand lysed with 0.2 mL of 0.2MNaOH, 0.1% SDS at 65C for

30 min [18] The lysate was used to determine intracellular radioactivity in a liquid scintillation spectrometer

Stable transfections with wild-type and D88H mutant hRFC cDNA

A wild-type hRFC cDNA clone containing the entire coding region as well as a D88 mutant RFC cDNA prepared by site-directed mutagenesis (QuickChange XL kit, Stratagene) were directionally cloned into pcDNA3 at the BamHI/XhoI site (Ivitrogen) Mouse leukemia cells, L1210 and their MTX transport null cells, MTXrA, were used for transfections Exponentially growing cells (2· 107) were harvested by centrifugation and stably transfected by electroporation (1000 lF, 234 V) with 10 lg of an expres-sion vector (pcDNA3-hRFC1) harboring the normal hRFC

or the D88H mutant hRFC cDNA Following 24 h of growth at 37C, cells were exposed to 600–750 lgÆmL)1 active G-418 (Calbiochem-Novabiochem, San Diego, CA) Cells were then distributed into 96-well plates at

 2 · 104)4 · 104 cells per well and after 10–20 days of incubation at 37C, individual G418-resistant clones were picked and expanded for further studies Only clones expressing comparable RFC mRNA levels were used Assay of MTX transport

Antifolate transport measurements were performed as previously described [19] Briefly, exponentially growing cells were harvested, washed twice with Hepes/NaCl/Piat

pH 7.4 and suspended at a density of 2· 107cellsÆmL)1at

37C The uptake of [3H]MTX (specific activity 0.5 Ci mmol)1) was measured at an extracellular concentration of

2 lMin 1 mL of cell suspension at 37C Influx measure-ments were performed at 0.5, 1 and 2 min during which MTX transport rates were linear Uptake was terminated by adding 10 mL of ice-cold Hepes/NaCl/Pi, following which centrifugation and cell wash with another 10 mL ice-cold Hepes/NaCl/Pi was performed The final cell pellet was processed for liquid scintillation counting

Results

Expression of THTR1 and D93H THTR1 in NIH3T3 cells

A Rogers syndrome patient from a French family was found to harbor an aspartate 93 to histidine substitution (D93H) in THTR1 In order to study the effect of this mutation on THTR1 expression and function, we used site-directed mutagenesis to express D93H mutant THTR1 protein fused to an N-terminal Myc tag epitope NIH3T3 cells were transiently transfected with expression plasmids encoding for either the wild-type or the D93H mutant THTR1 Cell lysates isolated from transfected cells were subjected to SDS/PAGE followed by Western blot analysis using anti-Myc 9E11 monoclonal Igs (Fig 1A) A strong signal represented by a broadly migrating THTR1 protein with an average molecular mass of 68 kDa was detected in cell lysates derived from both the wild-type and D93H

mutant THTR1 transfected cells As expected, no THTR1

was observed in cell lysates derived from vector transfected

cells (Fig 1A) To test the effect of the Myc epitope tag

fused to the N-terminal of the protein we also designed

expression plasmids encoding for THTR1 fused to a Myc

tag epitope at the C-terminus Western blot analysis

performed with cell lysates derived from NIH3T3 cells

transfected with expression plasmids encoding for either the

wild-type THTR1 or the D93H mutant THTR1 fused to

Myc epitope at the C-terminus, did not reveal any

expression when probed with the anti-Myc Ig (Fig 1B)

To confirm whether THTR1-Myc and D93H mutant

THTR1-Myc can be properly transcribed and translated,

we used an in vitro system of coupled transcription and

translation (rabbit reticulocyte lysate) in the presence of

[35S]methionine In vitro translated, radiolabeled proteins

were then resolved on SDS/PAGE Both wild-type

THTR1-Myc and D93H mutant THTR1-THTR1-Myc were efficiently

transcribed and translated resulting in a sharp protein band

of 52 kDa (Fig 1C), consistent with the core molecular

mass of the unglycosylated transporter protein, as

previ-ously described [12] The small difference in the migration

between the N-terminal and C-terminal Myc-fusions is due

to the longer epitope tag of the latter In order to confirm

that these constructs contained a Myc epitope that can be

recognized by the anti-Myc antibody, we resolved the

in vitrotranslated protein products on SDS/PAGE followed

by Western blotting and probed them with the anti-Myc

antibody (Fig 1D) Both wild-type THTR1 and D93H mutant THTR1 proteins were detected as sharp protein bands of 52 kDa These results suggest that the Myc tag fusion in the C-terminus of THTR1 protein may interfere with the cellular expression and/or stability of the protein

In view of these results, all subsequent experiments were performed using expression plasmids encoding for the N-terminal Myc tag fusions of THTR1

The mutant D93H THTR1 isN-glycosylated The broad migration of the THTR1 proteins (Fig 1A) suggested that the well expressed D93H mutant protein apparently undergoes a normal N-glycosylation To test whether the slower migration of the D93H mutant THTR1

is indeed due to N-glycosylation, we treated transfected NIH3T3 cells with tunicamycin (10 lgÆmL)1), an estab-lished N-glycosylation inhibitor [12] Whole cell extracts derived from mutant D93H THTR1 transfected cells treated with tunicamycin exhibited the same migration pattern on SDS/PAGE when compared to cells transfected with the wild-type THTR1 after treatment with tunicamycin (Fig 2, compare lane 2 and 5) In order to assess whether N-glycosylation is the post-translational modification responsible for the markedly decreased electrophoretic mobility of the transfected wild-type and D93H mutant THTR1 proteins, we used peptide N-glycosidase F (PNGaseF) PNGaseF is an endoglycosidase, which

Fig 1 Expression of D93H mutant THTR1 and wild-type THTR1 proteins in NIH3T3 cell transfectants (A) NIH3T3 cells were transiently transfected either with wild-type or mutant THTR1 in which aspartate 93 was substituted by histidine (D93H) Cells transfected with an empty vector were used as a negative control (Vector) Total cell protein extracts were isolated and proteins (50 lg) were resolved on a 10% SDS/PAGE followed by Western blotting using an anti-Myc 9E11 monoclonal Ig and anti-(mouse IgG) Ig conjugated to HRP as a second antibody (Santa Cruz Inc.) following which chemiluminescence reaction was performed Molecular mass (MW) markers (ProSieve BioWhittaker Molecular Applications Inc.) are given in kDa (B) NIH3T3 cells were transiently transfected with wild-type or mutant D93H THTR1 constructs containing the Myc epitope tag in the C-terminus (THTR1-Myc, D93H-Myc) Wild-type THTR1 with the Myc tag in the N-terminus was used as a positive control Proteins (50 lg) were resolved on a 10% SDS/PAGE and THTR1 expression was followed as described in A (C and D) The indicated expression plasmids (with the Myc epitope tag at the C-terminus) were used in a coupled in vitro transcription-translation system using the T7 RNA polymerase in the presence of [ 35 S]methionine Proteins were then resolved on 10% SDS/PAGE followed by either autoradiography (C) or Western blotting probed with anti-cMyc Ig as described above (D).

cleaves the covalent bond between the innermost

N-acetyl-D-glucosamine and the acceptor asparagine, thereby resulting

in the complete removal of the N-glycan from glycosylated

proteins Treatment of total cell extracts from transfected

cells with PNGaseF resulted in an almost complete removal

of the carbohydrate, thereby revealing a sharp 52 kDa

protein band with a similar electrophoretic mobility for

both the core wild-type THTR1 protein and the in vitro

translated THTR1 or the mutant D93H THTR1 proteins

(Fig 2) Thus, these results strongly suggest that the slower

migration of both the wild-type and mutant D93H THTR1

proteins is largely due to an N-linked glycosylation These

results suggest that the D93H mutant THTR1 protein is

efficiently transcribed, translated and apparently undergoes

a complete N-glycosylation as compared to the wild-type

THTR1 protein

Mutant D93H THTR1 is targeted to the plasma membrane

We have previously shown that only the properly folded

THTR1 protein is targeted to the plasma membrane and

that only the fully glycosylated mature THTR1 protein

reaches its plasma membrane destination [12] We

deter-mined whether or not the D93H mutant THTR1 is indeed

properly glycosylated such that it will be properly trafficked

to the plasma membrane We used immunofluorescence

analysis with Swiss 3T3 cells in order to explore THTR1

protein localization We employed Swiss 3T3 cells for the

immunofluorescence analysis as these cells are larger in size

and exhibit a distinct cell morphology as compared with

NIH3T3 cells Swiss 3T3 cells were cotransfected with

expression plasmids encoding for wild-type or D93H

mutant THTR1 along with an expression plasmid encoding

for green fluorescence protein (GFP) (Fig 3) The GFP

expression plasmid is expected to cosegregate with THTR1

or mutant D93H THTR1 expression plasmids Therefore,

cells that exhibit green fluorescence in all cell compartments

assist in the localization of the wild-type or D93H mutant

THTR1 proteins (Fig 3A) Wild-type and D93H mutant THTR1 proteins were stained with anti-Myc 9E11 mono-clonal Ig followed by CY3-conjugated goat anti-mouse IgG

as a second antibody (Fig 3B) Co-staining with both green (GFP) and red fluorescence (THTR1) is shown (Fig 3C) Thus, colocalization is represented by a yellow stain Cells transfected with THTR1 expression plasmids exhi-bit perinuclear-endoplasmic reticulum (ER) staining, as well

as cytoplasmatic and plasma membrane staining as previ-ously reported (Fig 3, THTR1) [12] Similarly, the immu-nofluorescent localization pattern obtained with the D93H mutant THTR1 was indistinguishable from that observed with the wild-type THTR1 (Fig 3 compare D93H and wild-type THTR1)

To test whether the glycosylation of the mutant D93H THTR1 plays a role in the plasma membrane localization,

as compared with the wild-type THTR1, we treated transfected cells with tunicamycin (10 lgÆmL)1), 24 h prior

to immunofluorescence analysis As previously reported, treatment with tunicamycin resulted in a complete loss of plasma membrane localization of the THTR1 protein (Fig 3, THTR1 ± tunicamycin) [12] Cells transfected with the D93H mutant THTR1 and treated with tunicamycin displayed a similar lack of plasma membrane localization of the mutant transporter as well as a typical subcellular localization that was confined to the perinuclear ER membrane (Fig 3, D93H ± tunicamycin)

Transport in cells transfected with THTR1 or D93H THTR1 The fact that the D93H mutant THTR1 protein is expressed, fully glycosylated and sorted out to the plasma membrane as does the wild-type THTR1 protein raised the question of the functionality (i.e thiamine transport activity) of this mutant THTR1 To test the ability of the mutant THTR1 to transport thiamine, we transiently transfected NIH3T3 cells with expression plasmids repre-senting the empty vector as well as expression plasmids encoding for either the wild-type THTR1 or the D93H mutant THTR1 and measured [3H]thiamine uptake NIH3T3 cells transfected with an empty expression vector exhibited an endogenous background thiamine transport (Fig 4) Expectantly, NIH3T3 cells transfected with an expression vector harboring the wild-type THTR1 exhibited

a 2.5-fold increase in thiamine uptake as compared with cells transfected with the empty expression vector In contrast, cells transfected with the D93H mutant THTR1 showed thiamine uptake reflecting the background levels that were obtained with cells transfected with the empty vector (Fig 4) or untransfected cells (data not shown) These results establish that the mutant D93H THTR1 protein lacks thiamine transport activity Furthermore, this finding is in accord with Rogers syndrome patients carrying this D93H mutant protein which apparently lost thiamine transport activity

Aspartate 93 is conserved in all members

of the solute carrier SLC19 family Aspartate 93 is located in the border of TMD2 and the beginning of intracellular loop 2 that is composed of only eight amino acids [11] This region is extremely conserved in

Fig 2 The N-glycosylation pattern of the wild-type and D93H mutant

THTR1 proteins in NIH3T3 cells NIH3T3 cells were transfected as

described in Fig 1 legend Cell lysates were prepared and proteins

(50 lg) were resolved on a 10% SDS/PAGE and THTR1 expression

was followed as described in Fig 1 legend Where indicated, cells were

treated with tunicamycin (Tun.; 10 lgÆmL)1) for 24 h prior to cell

harvesting Cell lysates (50 lg protein) were treated with PNGaseF

where indicated and treated as described in experimental procedures

and subjected to 10% SDS/PAGE followed by Western blotting using

an anti-cMyc monoclonal antibody.

Fig 3 Sub-cellular localization of D93H mutant THTR1 proteins byimmunofluorescence Swiss 3T3 cells were cotransfected with a GFP expression plasmid, with the Myc-THTR1 or mutant D93H THTR1 expression plasmids as indicated Cells were treated (where indicated) with 10 lgÆmL)1 tunicamycin for 24 h prior to cell fixation and staining with anti-Myc 9E11 monoclonal Ig and secondary goat-anti-(mouse Cy3 Ig) Ig Cells were then analyzed using confocal microscopy The localization of GFP alone (green, A) or CY3-conjugated goat-anti-(mouse Ig) Ig staining alone (red, B) and costaining (orange/yellow, Panel C) is shown Orange/yellow stain indicates colocalization Cell transfections and treatments were as indicated: Wild-type THTR1 (THTR) and mutant D93H THTR1 (D93H) either untreated or treated with tunicamycin (10 lgÆmL)1) (+tun.).

all members of the solute carrier SLC19 family including

THTR1, THTR2 as well as RFC and across mammalian

species (i.e human, mouse, and hamster) (Table 1) The

substitution of aspartate by histidine reverses the net charge

at this position and this may strongly affect transporter

function (Table 1) In order to test whether or not the

conserved aspartic acid at position 93 is also essential for the

transport function of the hRFC, a THTR1 and THTR2

homologue, we used site-directed mutagenesis to substitute

the corresponding aspartic acid at position 88 by histidine

in the hRFC Methotrexate (i.e an established transport

substrate of RFC) transport null MTXrA murine leukemia

cells were stably transfected with the vehicle vector plasmid

and expression plasmids encoding for either the wild-type

or the D88H mutant RFC proteins Northern blot analysis

confirmed the similar levels of RFC mRNA in the

transfected cells with both the wild-type and D88H mutant

hRFC (data not shown) Cells were then tested for their

ability to take up [3H]methotrexate RFC transfected cells

exhibited 16-fold higher MTX transport as compared to

nontransfected cells or vector transfected cells (Fig 5) In

contrast, cells expressing the D88H mutant hRFC failed to

exhibit MTX uptake that was above the poor background

levels present in the transport null MTXrA cells and empty

vector-transfected cells (Fig 5) Based on the high

conser-vation of TMD2 and intracellular loop 2 between the

members of the SLC19 family, this D93H THTR1 mutation and D88H in the hRFC abolished transport for both thiamine and MTX, respectively, thereby, suggesting that this domain plays an important functional role in substrate binding and/or translocation

Discussion

Rogers syndrome, is a rare uni-gene autosomal recessive disease, currently identified in 20 families all over the world Rogers syndrome patients suffer from constant thiamine deficiency and display the three main disease manifestations including megaloblastic anemia, diabetes mellitus and sensorineural deafness Some of the non-developmental manifestations in Rogers syndrome can be overcome by treatment with high doses of thiamine thus suggesting that alterations in thiamine transporter activity may play a role in the pathogenesis Consistently, Rogers syndrome patients were found to harbor mutations in the SLC19A2, encoding for THTR1 [7–9] Different muta-tions in the THTR1 (SLC19A2) gene were described in all Rogers syndrome patients [14] There is no correlation between the type of mutation identified and the severity of disease symptoms We therefore initiated studies aimed at the characterization of missense mutations resulting in amino-acid substitutions Recently we have described one

of the three-missense mutations that occur in Rogers syndrome families [12] The mutation was identified in an Italian family, in which, glycine 172 is substituted by aspartate thereby resulting in an apparently missfolded THTR1 protein, that fails to undergo complete glycosy-lation, and is retained in the Golgi-ER compartment this resulted in the targeting of this mutant THTR1 protein to degradation [12]

We undertook the present study to determine the impact that the D93H THTR1 mutation from a French family with Rogers syndrome has on thiamine transport activity During the early stages of our research, we were interested

in selecting the appropriate Myc tag constructs (N- or C-terminal fusion) of the D93H mutant THTR1 We find here that insertion of a Myc-tag epitope at the N-terminus

Fig 4 [3H]Thiamine uptake in NIH3T3 cell transfectants expressing

the wild-type and mutant D93H THTR1 proteins [3H]Thiamine uptake

was determined in NIH3T3 cells expressing the indicated proteins Net

thiamine uptake was calculated by subtraction of the radioactivity

obtained at 37 C with that observed at 4 C Endogenous thiamine

transport in NIH3T3 was represented by cells transfected with an

empty vector The cells were incubated with 0.6 l M [3H]thiamine for

20 min in a transport buffer at pH 7.4 as described in Experimental

procedures.

Fig 5 [3H]Methotrexate transport in cell transfectants expressing the wild-type and D88H mutant hRFC proteins MTX transport null, MTXrA cells were stably transfected with pcDNA-based expression vectors harboring the wild-type and D88H mutant RFC genes Clonal cell lines stably expressing comparable levels of the wild-type and mutant RFC were used to determine [3H]MTX transport Exponen-tially growing cells were incubated with 2 l M [ 3 H]MTX for 30, 60 and

120 s at 37 C following which the initial rates of labeled radio MTX were determined as detailed in Experimental procedures.

Table 1 Sequence comparison of various members of the solute carrier

family Amino-acid alignment of the aspartate 93 in human THTR1

with various members of the SLC19 family The conserved aspartate 93

is indicated in bold Light shading indicates the amino acids showing

conservation in all members of the solute carrier family.

of the wild-type THTR1 retains normal expression, plasma

membrane trafficking, and thiamine transport activity,

whereas no detectable expression could be detected in

NIH3T3 cells transfected with a THTR1 harboring a

C-terminal Myc-tag Apparently, insertion of a six-histidine

tag to the N-terminal end of the wild-type THTR1 did not

interfere with transporter expression and thiamine transport

activity [15] This latter result is consistent with recent

studies that reported on the introduction of an N-terminal

Myc-tag to the human Na+-glucose cotransporter [20] and

the serotonin transporter [21] without interfering with their

expression and transport function The human THTR1 is

shorter by 94 amino acids than its close counterpart, the

hRFC, as it lacks the long C-terminus present in the latter It

is possible that the insertion of a highly charged Myc-tag

(i.e 9/17 amino acids are either negatively or positively

charged) to the C-terminus in the hTHTR1 may result in

protein missfolding that could be identified by the quality

control mechanism in the ER, thereby leading to rapid

transporter degradation This putative degradation may

gain support from the fact that the D93H-Myc mutant

THTR1 (i.e the Myc tag placed in the C-terminus) was

readily translated in vitro (Fig 1C,D) However, one should

keep in mind that not any epitope tagging at the C-terminal

end of the THTR1 may necessarily result in lack of protein

expression and/or rapid degradation; introduction of an

enhanced green fluorescent protein (EGFP) tag at the

C-terminus of THTR1 neither resulted in interference with

transporter expression nor with thiamine transport activity

[22] Consistently, either hemagglutinin (HA) or green

fluorescent protein (GFP) tagging at the C-terminus of the

human RFC did not interfere with transporter expression,

plasma membrane targeting and MTX transport activity

[23] Taken together, the tag of choice has to be carefully

taken into considerations We show here that the D93H

mutant THTR1, while fused to Myc epitope tag at the

N-terminal, is properly translated and targeted to the

plasma membrane Yet a previous study has shown that,

upon fusion of His and Xpress tags derived from pcDNA3.1

expression plasmid (Invitrogen Inc.) with D93H mutant

THTR1, this mutant protein is neither detected in the

cytosolic nor in the plasma membrane fractions of

trans-fected cells [15] According to the topological model

proposed by these authors [15], the N-terminal domain

including the six His-Xpress tags and the D93H residue

located in the intracellular loop between TMD2 and TMD3

are expected to come to a close proximity in the cytosolic

milieu This could result in the formation of

nonphysiolog-ical ion-pairs (i.e His-Asp) as the Xpress tag introduces as

much as five Asp residues; alternatively, His 93 could

augment and/or alter the metal-cation chelation capabilities

of the proximal six His domain Clearly, in both cases

transporter misfolding and rapid protein degradation could

be envisaged In fact, among all mutations introduced in the

His/Xpress tagged THTR1, only the D93H was

undetect-able [15] Support to the notion of His tag mediated protein

misfolding derives from several recent papers First,

intro-duction of a six His tag to lactate dehydrogenase [24] and

reverse transcriptase [25] resulted in misfolding and/or loss

of catalytic activity Second, introduction of a His tag can

also result in detrimental post-tanslational modifications;

for example, His tagging of the SH3 domains of Src tyrosine

kinase resulted in a spontaneous a-N-6 gluconoylation which led to a severe interference with its crystallization [26] Using transient transfections and immunofluorescent subcellular localization we find that the D93H mutant THTR1 is well expressed, undergoes an apparently com-plete N-linked glycosylation and is targeted to the plasma membrane However, despite this apparently normal expression, post-translational modification (i.e glycosyla-tion) and trafficking to the plasma membrane, the D93H mutant transporter did not exhibit any thiamine transport activity These results establish that the D93H THTR1 mutation disrupts thiamine uptake activity and may there-fore explain the thiamine deficiency in this French family with Rogers syndrome that clinically responds only to high doses of thiamine

We find here that the D93H mutant THTR1 as well as its D88H mutant hRFC counterpart lost thiamine and MTX transport activity, respectively The proposed topo-logical structure of THTR1 predicts that D93 is the first amino acid in the short intracellular loop 2 (IL2), whereas its D88 counterpart in the hRFC is embedded in the end

of TMD2 [13,23] An alignment of the deduced amino-acid sequence of the human THTR1 with that of the various members of the SLC19A family including rodent (i.e mouse and hamster) THTR1, human and rodent THTR2, as well as RFC reveals a striking conservation at the vicinity of D93 in THTR1, THTR2 and of D88 in its counterpart, RFC Specifically, not only is D93/D88 absolutely conserved across species in both THTR1, THTR2 and RFC, TMD2 and IL2 are extremely well conserved (Table 1) Consistently, we recently identified the very same D88H mutation (along with additional mutations in TMD2) in GW1843-resistant leukemia GW70/LF cells that displayed a markedly altered trans-port of antifolates and folates [27] Moreover, recent studies demonstrate that elimination of the negative charge at the D88 of the hRFC (e.g D88V) results in a complete loss of its MTX transport activity [16] It was further found that this negatively charged residue (i.e D88) is absolutely essential for folate (i.e leucovorin) and antifolate (i.e MTX) transport as it presumably forms an ion-pair with a positively charged residue (i.e R133) in TMD4 of the hRFC It was therefore concluded that this charge-pair presumably allows for a proper tertiary structure to occur that is absolutely essential for folate and antifolate transport Hence, it is possible that the conserved D93 residue in THTR1 also plays an important role in the formation of a certain tertiary structure that is crucial for thiamine binding and/or substrate translocation

Multiple amino-acid substitutions are known to disrupt RFC transport function [28] As the number of patients carrying THTR1 missense mutations is rather limited due

to the fact that Rogers syndrome is a relatively rare disorder [14], it would be interesting to test whether other substitutions in conserved residues of the orthologue hRFC can inactivate the thiamine transport function of THTR1 and THTR2 as well Therefore, further compar-ative mutagenesis studies between the three members of the solute carrier family should provide useful information regarding the specificity and functionality of these trans-porters

We thank Prof I.D Goldman for the MTXrA cells, Dr O Shenkar for

assistance with confocal microscopy and Ms A Cohen for technical

assistance.

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