Veterans Affairs Medical Center, Long Beach, 90822; and University of California, Irvine, California 92697
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SLC19A2 is a membrane thiamine transporter expressed in a variety of human tissues, including the gastrointestinal tract. Little is currently known about the structure/function relationship of SLC19A2. We examined the effect of introducing mutations in SLC19A2 identical to those found in thiamine-responsive megaloblastic anemia syndrome (TRMA), on functional activity and membrane expression of the transporter. We also examined the effect of mutating the only conserved anionic residue (E138) in the transmembrane (TM) domains of the SLC19A2 and that of the putative glycosylation sites (N63, N314). Northern blot analysis showed SLC19A2 mRNA was expressed at the same level in HeLa cells transfected with wild-type or mutated SLC19A2. Introducing the clinically relevant mutations (D93H, S143F, G172D) or mutation at the conserved anionic residue (E138A) of SLC19A2 led to a significant (P < 0.01) inhibition of thiamine uptake. Mutations of the two potential N-linked glycosylation sites (N63Q, N314Q) of SLC19A2 did not affect functional activity; they did, however, lead to a noticeable reduction in apparent molecular weight of protein. Western blot analysis showed all proteins (except D93H) were expressed in the membrane (not the cytoplasmic) fraction of HeLa cells. These results provide direct confirmation that clinically relevant mutations in SLC19A2 observed in TRMA cause malfunctioning of the transporter and/or a defect in its translation/stability. Results also show conserved TM anionic residue of the SLC19A2 protein is critical for its function. Furthermore, native SLC19A2 is glycosylated, but this is not important for its function.
thiamine transport; site-directed mutagenesis; membrane transport; thiamine-responsive megaloblastic anemia syndrome
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
WATER-SOLUBLE VITAMIN
thiamine is involved as a cofactor in many critical cellular reactions,
and its deficiency leads to a variety of clinical abnormalities
including cardiovascular and neurological disorders (2, 26,
27). Human and mammalian cells cannot synthesize thiamine and
thus must obtain the vitamin from exogenous sources via transport
across the cell membrane. Previous physiological and biochemical
studies have shown the process of cellular uptake of thiamine across
the plasma membrane to be via a carrier-mediated mechanism(s). This
includes transport of the vitamin in absorptive epithelial cells of the
human small and large intestine (4, 8, 11, 19, 20).
Recently several groups have identified a new human gene,
SLC19A2, that encodes a thiamine transporter, and cDNAs from
human heart, skeletal muscle, fibroblast, and placenta have been
reported (3, 5, 15). Mutations in this gene are believed
to be the cause of thiamine-responsive megaloblastic anemia syndrome
(TRMA) (3, 6, 10, 17, 21, 24). This human thiamine
transporter is predicted to encode a multitransmembrane protein that,
when expressed in mammalian cells, specifically induces thiamine uptake
(5, 20). Recent studies in our laboratory have shown that
this gene is also expressed in different tissues of the human
gastrointestinal tract, raising the possibility that this transporter
may play a role in the normal human intestinal thiamine absorption
process (18). However, to date, there has been very little
known about the structure-function relationship of SLC19A2,
including the link between the reported mutations in the
SLC19A2 gene in TRMA and the function of the protein in
transporting thiamine. In this study, we used site-directed mutagenesis
and examined the importance of specific amino acid residues in the
SLC19A2 polypeptide on its function. Specifically, we
examined the effect of introducing mutations in SLC19A2
identical to those found in TRMA patients on thiamine uptake. We
extended our study further to understand the SLC19A2
functions on the basis of its putative secondary structure model (Fig.
1). In this context, we also examined the
effect of mutating the only conserved anionic amino acid residue in any
of the SLC19A2 transmembrane (TM) domains, or two putative
glycosylation sites on the ability of the protein to transport the
cationic thiamine. Mutants and wild-type SLC19A2 cDNA were
expressed in HeLa cells, and the level of expression and thiamine
uptake was determined and compared. Results showed that the clinically
relevant mutations in SLC19A2 (D93H, S143F, and G172D) cause
malfunctioning of the thiamine transporter and/or a defect in its
translation/stability. Results also showed that the conserved anionic
residue in the predicted fourth TM domain of the SLC19A2
protein is critical for its function. Furthermore, native
SLC19A2 appears to be glycosylated, but this glycosylation does not appear to be important for its function.
|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
[3H]thiamine (specific activity 555 GBq/mmol; radiochemical purity >98%) was purchased from American Radiolabeled Chemicals (St. Louis, MO). HeLa cells were purchased from American Type Tissue Culture Collection (Rockville, MD) and routinely cultured in MEM with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in 5% CO2. TRIzol reagent and Lipofectamine 2000 were purchased from Life Technologies (Rockville, MD). Polyvinylidene difluoride (PVDF) membrane was purchased from Bio-Rad (Hercules, CA). DNA oligonucleotide primers were ordered from Sigma Genosys (Woodlands, TX). Routine biochemicals, enzymes, and cell culture reagents were all of molecular biology quality and purchased from either Fisher Scientific (Tustin, CA) or Sigma (St. Louis, MO).
Functional Expression of the SLC19A2 in HeLa Cells
Vaccinia virus expression system. vTF7.3 vaccinia virus encoding T7RNA polymerase and pVOTE.1 plasmid vector were obtained through the generosity of Dr. Bernard Moss, National Institute of Allergy and Infectious Diseases, Bethesda, MD. Vaccinia is a complex DNA virus that replicates in the cytoplasm of most mammalian cells, encodes RNA polymerase and transcription factors, and accommodates large amounts of recombinant DNA (13, 14). Synthesis of T7 RNA polymerase by a recombinant vaccinia virus leads to a high-level of expression of genes placed next to a T7 promoter within a transfected plasmid or a second coinfecting virus (28). In the present study, the full-length SLC19A2 cDNA was inserted into the pVOTE.1 expression vector in such a way that the sense transcription of the cDNA is under the control of the T7 promoter in the plasmid. This made the use of transient vaccinia virus expression system for functional characterization of the wild-type and mutant SLC19A2 gene possible.
Site-directed mutagenesis.
Mutations in SLC19A2 were introduced by site-directed
mutagenesis (9) using the SLC19A2 cDNA
corresponding to the open reading frame (ORF) and cloned into the
mammalian expression vector pcDNA3.1. Mutations were made to introduce
the clinically relevant missense mutations reported in patients with
TRMA (D93H, S143F, and G172D), replace the only conserved anionic amino
acid residue located in the TMs of the SLC19A2 protein
(E138A; the 4th TM), the few negatively charged residues outside the TM
domains (E28A, E66A, D444A), or the two putative glycosylation sites
(N63Q and N314Q). Site-directed mutagenesis was performed using the
Quick-Change mutagenesis kit (Stratagene, La Jolla, CA) according to
manufacturer's instructions. T7-based expression vector pcDNA 3.1 containing the ORF of SLC19A2 was the target plasmid and a
pair of sense- and antisense-priming oligonucleotides encompassing the
desired changes were used for mutagenesis (Table
1). The final plasmids were sequenced by
dye termination cycle sequencing using an ABI PRISM 377 DNA sequencer
(Perkin-Elmer ABI) at the Nucleic Acid Facility at University of
California-Irvine to verify correct transfer of mutations and lack of
unwanted mutations. After sequence verification, the wild-type and
mutated SLC19A2 genes were cloned into the mammalian
expression vectors pVOTE.1 (for uptake studies) and pcDNA3.1/His (for
the protein expression/detection studies).
|
Transient transfection. Transient transfections were performed on subconfluent HeLa cells using Lipofectamine 2000 transfection reagent (Life Technologies, Gaithersburg, MD) following the manufacturer's procedure. The SLC19A2 in pcDNA 3.1/His contains the codons for a 6-histidine-tag as well as an epitope for an antibody. This allows easy identification and purification of the expressed protein. Cells were grown in flask culture, and the whole cell extracts were used to evaluate the level of SLC19A2 protein expression. The His-tagged proteins were purified from these extracts using a proband column (Invitrogen, Carlsbad, CA) and the manufacturer's instructions. Protein concentrations were determined using the Bio-Rad detection system on a Beckman DU640B spectrophotometer.
Western Blot Analysis
Immunoblotting was used to address the question of whether the His-tagged SLC19A2, and mutant proteins were inserted into the plasma membrane or not. For the purpose, the plasma membrane and cytoplasmic fractions were isolated from the whole cell extract of transfected HeLa cells following an established procedure (1, 25) and then were purified by proband column before applying them onto SDS-PAGE. Equal amounts of protein were loaded on 10% polyacrylamide gels (SDS-PAGE) and resolved at 200 V for 4 h in Tris-glycine SDS running buffer. Proteins were transferred onto a PVDF membrane at 35 mA for overnight at 4°C in wet transfer buffer. Membranes were blocked for 2 h at room temperature with PBS-Tween 20 (PBS-T) buffer containing 5% (wt/vol) nonfat dry milk powder. Membranes were then incubated overnight at 4°C with anti-Xpress antibody (1:5,000 in PBS-T buffer), and washed four times with PBS-T buffer. The secondary antibody (anti-mouse IgG-peroxidase conjugate diluted in 1:5,000 in PBS-T buffer) was applied for 2 h at room temperature followed by four washes with PBS-T buffer. Proteins were detected using enhanced chemiluminescence substrate (ECL kit; Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's instructions, and exposed to Kodak X-ray film for periods of 30 s to 5 min. Blots were rechecked for the presence of SLC19A2 protein by using specific polyclonal antibodies raised against a synthetic peptide of SLC19A2, as previously described by us (20).Northern Blotting
Total RNA was isolated from the wild-type and mutant SLC19A2 transfected HeLa cells using Trizol reagent as per the manufacturer's procedure. For Northern analysis, equal amounts of total RNA were loaded on a 1% agarose gel containing 2.2 M formaldehyde and transferred to nylon membranes by standard method. Blots were stained with ethidium bromide to estimate the quantitative loading variations. Hybridization was carried out as described previously (18). Full-length ORF SLC19A2 cDNA was randomly labeled with [32P]dCTP and used as a probe for detection. Final washing was performed in 0.2× SSC, 0.1% SDS for 15 min at 65°C. Membranes were exposed to Kodak X-ray films, and autoradiographs were analyzed. By the use of densitometry, data were normalized relative to humanThiamine Uptake Studies
Uptake studies were performed on confluent monolayers of HeLa cells incubated in Krebs-Ringer buffer (in mM): 133 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES, pH 7.4, at 37°C. [3H]thiamine (0.3 µCi/ml; 15.0 nM) was added to the incubation medium at the onset of the uptake experiment. Uptake was examined over a period of 3 min, i.e., initial rate (unpublished observations), and the reaction was terminated by the addition of 2 ml of ice-cold buffer followed by immediate aspiration. Cells were then rinsed twice with ice-cold buffer and digested with 1 ml of 1 N NaOH, neutralized with HCl, and then counted for radioactivity. Protein contents of cell digest were measured on parallel wells using a Bio-Rad kit (Richmond, Virginia).Statistical Analysis
All measurements were made in triplicate and each experiment was repeated three times with separate transfections. Uptake results presented in this paper are means ± SE of multiple separate uptake determinations and were expressed by femtomoles per milligrams of protein per unit of time. Statistical differences were analyzed by Student's t-test, with statistical significance set at 0.05 (P < 0.05) relative to simultaneously run controls. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of SLC19A2 in HeLa Cells: Direct Evidence for Functional Impairment of SLC19A2 in TRMA
In this study, we first validated the ability of the vaccinia virus for use in the expression of SLC19A2 in HeLa cells by transient transfection. This was performed by examining the initial rate (3 min; data not shown) of thiamine (15.0 nM) uptake in HeLa cells transfected with SLC19A2 and those of controls, i.e., cells transfected with pVOTE.1 lacking the SLC19A2 insert and nontransfected cells. Results showed significant (P < 0.01) induction in thiamine uptake in SLC19A2 transfected cells compared with controls (Fig. 2).
|
In another study, we examined the effect of introducing identical
missense mutations to those found in TRMA patients on transport function of SLC19A2. TRMA is a rare autosomal recessive
disease caused by mutations in the SLC19A2 gene (3, 6,
10, 17, 21, 24). Mutations were introduced as follows: D93H,
S143F, and G172D. Mutant or wild-type SLC19A2 were then
expressed in HeLa cells as described in MATERIALS AND
METHODS. Results showed that all mutations lead to a significant
(P < 0.01) inhibition in thiamine uptake compared with
wild-type SLC19A2 (Fig. 2). We also examined, using Northern
blot analysis, the level of mRNA expression of the different mutants in
transfected HeLa cells. Results showed a similar level of mRNA
expression of mutants and wild-type SLC19A2 (Fig.
3). Also, we determined whether the
mutated SLC19A2 protein is expressed in HeLa cells or not,
and whether the expression is at the cell membrane. Western blot
analysis using either anti-Xpress monoclonal antibody directed against an epitope in pcDNA 3.1/His or a specific polyclonal antibodies directed against the SLC19A2 protein were employed. Results
showed that the wild-type protein and two of the TRMA mutants (G172D and S142F) are expressed (~66 kDa) at a similar level in the cell membrane fraction of these cells (Fig. 4;
lanes 3 and 4); no expression was found in the
cytoplasmic fractions for either mutants. In contrast, the protein of
the D93H mutant was found neither in the cell membrane fraction nor the
cytoplasmic fraction of HeLa cells (Fig. 4; lanes 5 and
6).
|
|
Role of Anionic Amino Acids in SLC19A2 Function
Recent studies have shown that the transport of charged substrates is influenced by amino acid residues in the TM domain of their transporters that carry opposing charges (23). In this study, we examined the effect of mutating the only conserved anionic amino acid in any of the TM domains of SLC19A2 (E138A) on the ability of the carrier protein to transport the positively charged (cationic) thiamine molecule. We also mutated few anionic amino acid residues outside the TM domains of the SLC19A2 polypeptide for comparison (E28A, E66A, and D444A). Results showed mutating the only conserved anionic amino acid (E138A) lead to a significant (P < 0.01) inhibition in thiamine uptake (Fig. 2). In contrast, no inhibition in thiamine uptake was seen with the E28A, E66A, and D444A mutations (Fig. 2).In another study, we determined the level of expression of the message of the E138A mutant in HeLa cells by Northern blot analysis and compared the results to that of wild-type SLC19A2 in HeLa cells (Fig. 3). Results showed that the level of mRNA expression to be similar in both cases (Fig. 3; lanes 2 and 3). To determine whether the SLC19A2 protein in the E138A mutant is expressed in HeLa cells, we performed Western blot analysis as described earlier. Results showed that both the wild-type and the E138A mutant proteins are expressed (~66 kDa) in the membrane fraction of HeLa cells and that the level of expression is similar in the two cases (Fig. 4; lanes 2 and 7); no expression was found in the cytoplasm in either case (Fig. 4; lane 8).
SLC19A2 Glycosylation: Role in Function and Expression of the Thiamine Transporter at Cell Membrane
SLC19A2 has two potential N-glycosylation sites, one at N63 and the other at N314. It is not known, however, if either of these sites is actually glycosylated and if glycosylation affects function and/or membrane expression of the thiamine carrier protein. To investigate these issues, we mutated the potential N-glycosylated sites (N63Q, N314Q) simultaneously or sequentially and examined the effect of these mutations on thiamine uptake, expression of SLC19A2 at cell membrane, and mutant protein. Results showed that simultaneous or sequential mutations of the potential N-linked glycosylation sites of SLC19A2 have no significant functional consequences on thiamine uptake (Fig. 2). We also examined the effect of treating HeLa cells expressing the wild-type SLC19A2 with the glycosylation inhibitor tunicamycin (2 µM for 24 h) on thiamine uptake. Results showed that tunicamycin had no significant affect on thiamine uptake in the treated cells compared with control (Fig. 2).In another study, we examined whether the N63Q/E314Q mutations affect membrane expression of the SLC19A2 protein. This was performed using Western blot analysis as described in MATERIALS AND METHODS. Results showed that the N63Q/N314Q mutant is expressed in the cell membrane fraction of HeLa cells (Fig. 4; lane 11) and that the level of expression is similar to that of the wild-type; no expression was found in the cytoplasm in either case. However, the predominant band evident in the wild-type SLC19A2 transfected cells at ~66 kDa (Fig. 4; lane 10) was shifted to ~56 kDa after mutation (Fig. 4; lane 11). A similar reduction in the apparent molecular weight of the wild-type SLC19A2 protein was also observed in transfected cells treated with tunicamycin (Fig. 4; lane 12). However, cells transfected with the mutated potentially N-linked glycosylation sites individually show the 66 kDa-band to be either smeared or only slightly shifted from its original position (Fig. 4; lanes 9 and 10).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aim of this study was to gain some insight into the structure-function relationship of the recently cloned thiamine transporter SLC19A2. Specifically, we sought to provide direct evidence for a functional defect in SLC19A2-caused mutations found in TRMA patients, and to determine whether this is the result of a lack of expression of the protein at the cell membrane. We also sought to examine the effect of mutating the only conserved anionic amino acid residue in any of the SLC19A2 TM domains and that of the two putative glycosylation sites on the ability of the protein to transport the cationic thiamine and expression of the protein at the cell membrane. All cDNAs were then expressed in HeLa cells, and the level of their expression and thiamine uptake was determined and compared.
TRMA is an autosomal recessive disorder with features that include megaloblastic anemia, diabetes mellitus and sensory-neural deafness. Missense and non-sense mutations in SLC19A2 have both been reported in patients with this disorder. In this study, we focused only on the missense mutations identified in three different families from three different geographic locations. Mutations included a D93H mutation, a negatively charged residue (aspartic acid) that was mutated into a positively charged residue (histidine); a S143F mutation, a hydrophilic amino acid (serine) that was mutated to a hydrophobic amino acid (phenylalanine); and a G172D mutation, a nonpolar hydrophobic amino acid (glycine) that was mutated to a negatively charged hydrophilic amino acid (aspartic acid). All these mutations were located in exon two of the SLC19A2 gene. Our results showed that all mutants caused significant impairment in thiamine uptake following expression in HeLa cells. This impairment in thiamine uptake was not due to a difference in the level of expression of the SLC19A2 message, because similar levels of mRNA were found (by Northern blot analysis) for these mutants and the wild-type SLC19A2. However, protein levels of the mutants were found to vary as shown by the Western blot analysis. Mutants S142F and G172D were both found to be expressed in the cell membrane fraction of HeLa cells and at a comparable level to that of wild-type protein. In both cases, no expression of the protein was detected in the cytoplasmic fraction of the transfected HeLa cells. In contrast, mutant D93H was detected neither in the membrane nor in the cytoplasmic fractions of HeLa cells, indicating that either the transfected cells were unable to translate this mutant or there is a defect in the stability of this mutant protein. Findings that both S142F and G172D mutants were expressed in the cell membrane (not cytoplasmic) fraction of transfected cells suggest that the defect in thiamine uptake in these particular TRMA mutations is not the result of impaired trafficking of the SLC19A2 protein to the cell membrane; rather the defect appears to be in the function due to impairment of the thiamine carrier. These findings are interesting compared with the mutations found in the glucose transporter SGLT1 in patients with glucose-galactose malabsorption, another autosomal recessive disorder. In this disorder, many of the mutations lead to a defect in intracellular trafficking of the protein to the cell membrane (12).
Recent studies (23) have shown transport of charged substrates to be influenced by amino acid residues in the TM domain of their transporters that carry an opposing charge. SLC19A2 protein has only one conserved anionic amino acid residue in any of its TM domains located in the fourth TM stretch. The effect of mutating this amino acid residue was found to lead to a significant inhibition in uptake of the cationic thiamine. This inhibition was not due to a decrease in the level of message and protein expression of the mutated protein as shown by the results of the Northern and Western blot analysis, respectively. Rather, the effect appears to be mediated via impairment in the function of the thiamine carrier itself. This, raises the possibility that this amino acid residue may play a role in the transport function of the thiamine carrier. It is interesting to mention here that mutating anionic residues outside the TM domains of SLC19A2 (E28A, E66A, and D444A) did not affect the function of the thiamine transporter, thus providing support to this suggestion. Further studies are required to establish this possibility.
Glycosylation of proteins has been reported to play a role in folding and membrane targeting of that protein (7, 22). SLC19A2 protein has two potential glycosylation sites (5, 6) but nothing is known about their glycosylation status. Therefore, we examined the effect of simultaneous and sequential mutation of these sites on transport function and membrane expression of SLC19A2. Our findings showed that alteration of these sites cause a significant shift in the SLC19A2 protein from ~66 to ~56 kDa. This clearly indicates that the native SLC19A2 protein is glycosylated in living cells. Mutating the glycosylation sites, however, did not affect thiamine uptake. Similarly, such mutations did not affect the expression of the SLC19A2 protein at the cell membrane. The latter observation suggests that glycosylation of SLC19A2 is not important for intracellular trafficking/targeting of the protein to cell membrane. These findings were confirmed by the studies with the glycosylation inhibitor tunicamycin. In these studies, treatment of HeLa cells expressing the wild-type SLC19A2 with this inhibitor did not affect the transport function of the protein. It did, however, lead to a significant reduction in the apparent molecular weight of the protein to a level similar to that seen with the mutation approach.
In summary, our findings provide direct confirmation that the clinically relevant mutations in SLC19A2 in TRMA patients cause malfunctioning of the transporter per se or a defect in its translation/stability. Results also show that the conserved anionic residue in the predicted fourth TM domain of the SLC19A2 protein is critical for its function. Furthermore, native SLC19A2 appears to be glycosylated, but this glycosylation may not be important for its function.
![]() |
ACKNOWLEDGEMENTS |
---|
This study was supported by a Department of Veterans Affairs grant and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56061 and DK-58057.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: H. M. Said, Veterans Affairs Medical Center-151, Long Beach, CA 90822 (E-mail: hmsaid{at}uci.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 6, 2002;10.1152/ajpgi.00547.2001
Received 28 December 2001; accepted in final form 31 January 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abmayr, SM,
and
Workman JL.
Protocols in Molecular Biology, edited by Ausubel FM,
Brent R,
Kingston RE,
Moore DD,
Seidman JG,
Smith JA,
and Struhl K.. New York: Wiley, 1994, vol. 2, p. 12.1.1-12.1.7.
2.
Berdanier, CD.
Advanced Nutrition Micronutrients. New York: CRC, 1998.
3.
Diaz, GA,
Banikazemi M,
Oishi K,
Desnick RJ,
and
Gelb BD.
Mutations in a new gene encoding a thiamine transporter cause thiamine-responsive megaloblastic anaemia syndrome.
Nat Genet
22:
309-312,
1999[ISI][Medline].
4.
Dudeja, PK,
Tyagi S,
Kavilaveettil RJ,
Gill R,
and
Said HM.
Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles.
Am J Physiol Cell Physiol
281:
C786-C792,
2001
5.
Dutta, B,
Huang W,
Molero M,
Kekuda R,
Leibach FH,
Devoe LD,
Ganapathy V,
and
Prasad PD.
Cloning of the human thiamine transporter, a member of the folate transporter family.
J Biol Chem
274:
31925-31929,
1999
6.
Fleming, JC,
Tartaglini E,
Steinkamp MP,
Schorderet DF,
Cohen N,
and
Neufeld EJ.
The gene mutated in thiamine-responsive anaemia with diabetes and deafness (TRMA) encodes a functional thiamine transporter.
Nat Genet
22:
305-308,
1999[ISI][Medline].
7.
Gut, A,
Kappeler F,
Hyka N,
Balda MS,
Hauri HP,
and
Matter K.
Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins.
EMBO J
17:
1919-1929,
1998
8.
Hoyumpa, AM, Jr,
Strickland R,
Sheehan JJ,
Yarborough G,
and
Nichols S.
Dual system of intestinal thiamine transport in humans.
J Lab Clin Med
99:
701-708,
1982[ISI][Medline].
9.
Kunkel, TA.
Rapid and efficient site-specific mutagenesis without phenotypic selection.
Proc Natl Acad Sci USA
82:
488-492,
1985[Abstract].
10.
Labay, V,
Raz T,
Baron D,
Mandel H,
Williams H,
Barrett T,
Szargel R,
McDonald L,
Shalata A,
Nosaka K,
Gregory S,
and
Cohen N.
Mutations in SLC19A2 cause thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and deafness.
Nat Genet
22:
300-304,
1999[ISI][Medline].
11.
Laforenza, U,
Patrini C,
Alvisi C,
Faelli A,
Licandro A,
and
Rindi G.
Thiamine uptake in human intestinal biopsy specimens, including observations from a patient with acute thiamine deficiency.
Am J Clin Nutr
66:
320-326,
1997[Abstract].
12.
Lam, JT,
Martin MG,
Turk E,
Hirayama BA,
Bosshard NU,
Steinmann B,
and
Wright EM.
Missense mutations in SGLT1 cause glucose-galactose malabsorption by trafficking defects.
Biochim Biophys Acta
1453:
297-303,
1999[ISI][Medline].
13.
Moss, B.
Recombinant DNA virus vectors for vaccination.
Semin Immunol
5:
317-327,
1990.
14.
Moss, B.
Vaccinia virus: a tool for research and vaccine development.
Science
252:
1662-1667,
1991[ISI][Medline].
15.
Neufeld, EJ,
Mandel H,
Raz T,
Szargel R,
Yandava CN,
Stagg A,
Faure S,
Barrett T,
Buist N,
and
Cohen N.
Localization of the gene for thiamine-responsive megaloblastic anemia syndrome, on the long arm of chromosome 1, by homozygosity mapping.
Am J Hum Genet
61:
1335-1341,
1997[ISI][Medline].
16.
Oishi, K,
Hirai T,
Gelb BD,
and
Diaz GA.
SLC19A2: cloning and characterization of the murine thiamin transporter cDNA and genomic sequence, the orthologue of the human TRMA gene.
Mol Genet Metab
73:
149-159,
2001[ISI][Medline].
17.
Raz, T,
Labay V,
Baron D,
Szargel R,
Anbinder Y,
Barrett T,
Rabl W,
Viana MB,
Mandel H,
Baruchel A,
Cayuela JM,
and
Cohen N.
The spectrum of mutations, including four novel ones, in the thiamine-responsive megaloblastic anemia gene SLC19A2 of eight families.
Hum Mutat
16:
37-43,
2000[ISI][Medline].
18.
Reidling JC, Subramanian VS, Dudeja PK, and Said HM. Expression
and promoter analysis of SLC19A2 in human intestine. Biochem
Biophy Acta-Biomembranes. In press.
19.
Said, HM,
Ortiz A,
Kumar CK,
Chatterjee N,
Dudeja PK,
and
Rubin S.
Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2.
Am J Physiol Cell Physiol
277:
C645-C651,
1999
20.
Said, HM,
Ortiz A,
Subramanian VS,
Neufeld EJ,
Moyer MP,
and
Dudeja PK.
Mechanism of thiamine uptake by human colonocytes: studies with cultured colonic epithelial cell line NCM460.
Am J Physiol Gastrointest Liver Physiol
281:
G144-G150,
2001
21.
Scharfe, C,
Hauschild M,
Klopstock T,
Janssen AJM,
Heidemann PH,
Meitinger T,
and
Jaksch M.
A novel mutation in the thiamine responsive megaloblastic anaemia gene SLC19A2 in a patient with deficiency of respiratory chain complex I.
J Med Genet
37:
669-673,
2000
22.
Scheiffele, P,
Peranen J,
and
Simons K.
N-glycans as apical sorting signals in epithelial cells.
Nature
378:
96-98,
1995[ISI][Medline].
23.
Sharina, IG,
Zhao R,
Wang Y,
Babani S,
and
Goldman ID.
Mutational analysis of the functional role of conserved arginine and lysine residues in transmembrane domains of the murine reduced folate carrier.
Mol Pharmacol
59:
1022-1028,
2001
24.
Stagg, AR,
Fleming JC,
Baker MA,
Sakamoto M,
Cohen N,
and
Neufeld EJ.
Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts.
J Clin Invest
103:
723-729,
1999
25.
Sweet, DH,
Miller DS,
and
Pritchard JB.
Basolateral localization of organic cation transporter 2 in intact renal proximal tubules.
Am J Physiol Renal Physiol
279:
F826-F834,
2000
26.
Tanphaichirt V. Thiamine. In: Modern Nutrition in Health and
Disease, edited by Shils ME, Olsen JA, and Shike M. New York: Lea
and Febiger, 359-375, 1994.
27.
Victor, M,
Adams RD,
and
Collins GH.
The Wernicke-Korsakoff Syndrome and Related Neurological Disorders due to Alcoholism and Malnutrition. Philadelphia, PA: Davis, 1989.
28.
Ward, GA,
Stover CK,
Moss B,
and
Fuerst TR.
Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells.
Proc Natl Acad Sci USA
92:
6773-6777,
1995[Abstract].