EDITORIAL FOCUS
Molecular mechanism of the intestinal biotin transport
process
Nabendu S.
Chatterjee1,
Chandira K.
Kumar1,
Alvaro
Ortiz1,
Stanley A.
Rubin2, and
Hamid M.
Said1
1 Medical Research Service,
Veterans Affairs Medical Center, Long Beach 90822, and Department of
Medicine and Physiology/Biophysics, University of California School of
Medicine, Irvine 92697; and
2 Veterans Affairs West Los
Angeles, Los Angeles 90073, and Department of Medicine, University of
California, Los Angeles, California 90024
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ABSTRACT |
Previous studies
have characterized different aspects of the cellular/membrane mechanism
and regulation of the intestinal uptake process of the water-soluble
vitamin biotin. Little, however, is known about the molecular
mechanisms of the uptake process. In this study, we have identified a
cDNA from rat small intestine that appears to be involved in biotin
transport. The open reading frame of this cloned cDNA consisted of
1,905 bases and was identical to that identified for the vitamin
transporter in placental tissue. Significant heterogeneity, however,
was found in the 5' untranslated region of this clone, with three
distinct variants (II, III, IV) being identified in the small
intestine; the placental variant (variant I), however, was not present
in the small gut. Variant II was found to be the predominant form
expressed in the rat small and large intestines. Functional identity of
the cloned intestinal cDNA was confirmed by stable expression in COS-7
cells, which showed a four- to fivefold increase in biotin uptake in
transfected COS-7 cells compared with controls. The induced biotin
uptake in transfected COS-7 cells was found to be
1)
Na+ dependent,
2) saturable as a function of
concentration with an apparent
Km of 8.77 µM
and a Vmax of
779.7 pmol · mg
protein
1 · 3 min
1, and
3) inhibited by unlabeled biotin and
pantothenic acid and their structural analogs. The distribution of
complementary mRNA transcripts of the cloned cDNA along the vertical
and longitudinal axes of the intestinal tract was also determined.
Results of this study describe the molecular characteristics of the
intestinal biotin absorption process and report the identification of a
cDNA that encodes a Na+-dependent
biotin uptake carrier that appears to exist in the form of multiple variants.
water-soluble vitamin; molecular transport mechanism; 5'
untranslated region; reverse transcriptase-polymerase chain
reaction
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INTRODUCTION |
THE WATER-SOLUBLE VITAMIN biotin is essential for
normal cellular functions, growth, and development (3, 5, 35). It acts
as a coenzyme for four carboxylases in pathways of fatty acid
biosynthesis, gluconeogenesis, and catabolism of several branched-chain
amino acids and odd-chain fatty acids (3, 5, 35). Recent studies have
identified additional cellular functions for biotin, which include
regulation of cellular cGMP levels (7, 38, 40). Biotin deficiency in
humans leads to a range of clinical abnormalities including
neurological disorders, growth retardation, and skin abnormalities (3,
5, 35, 40). Deficiency of biotin during pregnancy has also been shown
in several animal species to lead to embryonic growth retardation,
congenital malformation, and death (38). The incidence of biotin
deficiency and suboptimal levels has been reported with increased
frequency in recent years. Biotin deficiency has been reported to occur
in patients with inborn errors of biotin metabolism (3, 5, 35), in
patients on long-term therapy with anticonvulsant agents (7, 8), where
inhibition in intestinal biotin absorption is believed to be a
contributing factor (31), and in patients on long-term parenteral
nutrition (12, 13). Suboptimal biotin levels have also been reported
during pregnancy (14), in substantial numbers of alcoholics, where
impairment of the intestinal biotin absorption process is believed to
be a contributing factor (32), and in patients with inflammatory bowel
diseases (1, 37).
Humans and other mammals lack the ability to synthesize biotin and thus
must obtain the vitamin from exogenous sources through absorption in
the intestine. Therefore, the intestine plays a key role in determining
and regulating normal biotin body homeostasis, and thus understanding
the cellular and molecular mechanisms and regulation of the intestinal
absorption process of biotin is of physiological and nutritional
importance. Previous studies from our laboratory (11, 19-23,
25-30) and others (4) have characterized different aspects of the
mechanism and regulation of the intestinal biotin absorption process at
the tissue, cellular, and membrane levels. These studies have shown,
among other things, the involvement of a specialized,
Na+-dependent, carrier-mediated
mechanism for the biotin uptake process in the small intestine. This
mechanism was found to be capable of transporting the vitamin against a
concentration gradient across the intestinal brush-border membrane (20,
28-30) and was shared by the unrelated water-soluble vitamin,
pantothenic acid (19). A similar uptake mechanism was also found in
colonic epithelial cells and is believed to be responsible for the
absorption of the bacterially synthesized biotin in the large intestine
(19, 25). Studies in our laboratory have also shown that the intestinal biotin uptake process is regulated by biotin dietary levels (21) and by
specific intracellular regulatory pathways (19, 25). To date, however,
little is known about the molecular mechanism(s) of the intestinal
biotin absorption process. The serendipitous cloning of the so-called
Na+-dependent multivitamin
transporter (SMVT) from rat placenta (17), a transporter that also
appears to transport biotin, has assisted us in our effort to address
this issue. In this study, we describe the molecular characterization
of the small intestinal biotin uptake process and report the cloning of
a cDNA with multiple variants.
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MATERIALS AND METHODS |
Materials.
[3H]biotin (sp act
>30 Ci/mmol; radiochemical purity >98%) and all other radioactive
materials were obtained from Dupont NEN (Boston, MA) and Amersham
(Arlington Heights, IL). [14C]pantothenic acid
(sp act >55 mCi/mmol; radiochemical purity >98%) was obtained from
American Radiolabeled Chemicals (St. Louis, MO). All chemicals and
reagents used in this study were of analytical/molecular biology grade
and were obtained from commercial sources. Cellulose nitrate filters
(0.45 µm pore size) used in uptake studies with isolated villus and
crypt cells were purchased from Sartorius Filters (Hayward, CA). COS-7
cells were obtained from ATCC (Manassas, VA). DMEM, trypsin, fetal
bovine serum (FBS), and other cell culture materials were obtained from
Life Technologies (Grand Island, NY). Different kits used in this study
were purchased from commercial vendors and are identified in the text.
The oligonucleotides used in different experiments (Table
1) were synthesized by Genosys Biotechnologies (The Woodlands, TX). Restriction enzymes and the Vent
DNA polymerase were purchased from New England Biolabs (Beverly MA).
Adult male Sprague-Dawley rats were purchased from Harlan Sprague
Dawley (Indianapolis, IN) and were euthanized with
CO2. The use of rats in this study
was approved by the Institutional Review Board, and animal experiments
were conducted under appropriate regulatory guidelines.
cDNA cloning. Five micrograms of
poly(A)+ RNA isolated from rat
small intestine was primed with oligo(dT) primer (to synthesize the
first strand of cDNA) using a SuperScript preamplification system kit
(Life Technologies) as described by the manufacturer. Two specific
primers (primer 1F and primer 2R; Table 1) based on the rat SMVT cDNA
sequence (17) were used to search for cDNAs related to the intestinal
biotin transporter(s). PCR conditions were denaturation at
95°C for 1 min, annealing at 50°C for 2 min, and extension
at 76°C for 4 min. After a hot start, 35 cycles of PCR
were run, with 15 min of final extension following the last cycle. The
PCR products were electrophoresed through 0.7% agarose gel, purified,
then subcloned into the EcoR V site of the pBluescript II SK+ vector
(Stratagene, La Jolla, CA). The constructs were amplified in DH10B
electrocompetent Escherichia coli
cells (Life Technologies) and plated onto ampicillin-containing agar
plates. Plasmid DNA from several colonies was isolated using the Wizard
midiprep DNA purification system (Promega, Madison, WI), analyzed by
restriction digestion, and found to be identical. The identity of the
clone was then determined by sequencing, and the sequence was deposited in GenBank with accession number AF081204.
Because our primers were designed for the two extreme ends of the open
reading frame (ORF), we needed to determine the identity of the
5' and 3' UTR of our clone. This was done by rapid
amplification of cDNA ends (RACE) using a marathon cDNA amplification
kit (Clontech, Palo Alto, CA) as described by the vendor. Briefly, a
library of adapter-ligated double-stranded rat intestinal cDNA was
created using the above-mentioned kit and
poly(A)+ RNA from rat small
intestine. For 5' UTR-RACE, a downstream primer from the vicinity
of the ATG codon (primer 3R; Table 1) was designed, with restriction
sites based on the sequence of the cloned cDNA. RACE-PCR was performed
using the above-mentioned primer and an upstream, adapter-specific
primer supplied with the kit along with other reagents of an Advantage
cDNA polymerase mix (Clontech) using the manufacturer's protocol. The
5' RACE products ranging in size from 300 to 550 bp were gel
purified. The products were then digested with
EcoR I and
Spe I and subcloned into pBluescript II SK+ vector. The clones were
purified, analyzed by restriction digestion followed by Southern
blotting, and sequenced. Three distinct clones (named variants II, III,
and IV relative to SMVT that was considered variant I; see
RESULTS) were identified with sizes
of 522, 447, and 391 bp, respectively. The sequences of these 5'
UTR variants have been deposited in GenBank with accession numbers
AF143309, AF143310, and AF143311, respectively. As to the 3' UTR
of the cloned intestinal cDNA, this was identified using the same Marathon cDNA amplification kit described earlier and primer 4F described in Table 1. The resulting 1-kb fragment was subcloned into
the pBluescript II SK+ vector and sequenced.
All DNA sequencing was performed by commercial vendors (The University
of California, Irvine, CA, and Seqwright, Houston, TX). Nucleotide
sequences were analyzed using computer and web-based programs (DNA
Strider 1.2, Prosite database, and other related programs from ExPASy
web site with URL http://www.expasy.ch/).
Semiquantitative RT-PCR. RT was
performed as mentioned above using 5 µg
poly(A)+ RNA from different
tissues or cells. An aliquot of the synthesized cDNAs was amplified by
PCR. Details of the PCR conditions and the mRNA species detected have
been summarized in Table 2.
All PCR were performed within the linear range of amplification of the
corresponding mRNA species. The products were analyzed on 1.6% agarose
gels and their images captured using an Eagle Eye II system
(Stratagene, La Jolla, CA). The amplified RT-PCR products were
normalized to the amplified
-actin RT-PCR products using ImageQuant
software (Molecular Dynamics, Sunnyvale, CA).
Cell culture and uptake studies.
Wild-type and transfected (transfection was performed by
electroporation; Ref. 9) COS-7 cells were grown in DMEM
containing 10% FBS. G418 (1 mg/ml) antibiotic was added to the growth
medium of transfected COS-7 cells for selection and growth.
Subconfluent cells were subcultured and plated onto 12-well plates at a
concentration of 1 × 105
cells/well. All uptake studies were performed 2-5 days after confluence. Uptake was performed at 37°C in Krebs-Ringer phosphate buffer containing (in mM) 123 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES, pH 7.4. [3H]biotin (or
[14C]pantothenic acid)
was added to the incubation buffer at the beginning of the experiment
and uptake was terminated after 3 min of incubation (unless otherwise
specified) by the addition of 1 ml of ice-cold buffer followed by
immediate removal by aspiration. The monolayers were rinsed twice with
ice-cold buffer, digested with 1 ml of 1 N NaOH, neutralized by HCl,
harvested, and counted for radioactivity in a liquid scintillation
counter. Protein contents of cell digests were estimated on parallel
wells using a protein assay kit from Bio-Rad Laboratories (Hercules,
CA). Uptake data presented in this paper are means ± SE of multiple
separate experiments performed on at least two different occasions and
are expressed in picomoles or femtomoles per milligram of protein per
unit time. Statistical significance was set at the 5% level
(P < 0.05) as determined by
Student's t-test. Kinetic parameters
of biotin uptake, i.e., maximal velocity
(Vmax) and the
apparent Michaelis-Menten constant
(Km), were
calculated using the Lineweaver-Burk plot.
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RESULTS |
Cloning and characterization of an intestinal
cDNA. Using RT-PCR (see MATERIALS AND
METHODS) and primers designed based on the ORF of
SMVT (17), a product of 1.9 kb in size was identified from rat small
intestine. The sequence of this cDNA was found to be identical to the
ORF of SMVT (17). As to the 5' UTR, the result of our RACE-PCR
showed the existence of three distinct clones with sequences of 522, 447, and 391 bp (Fig.
1A).
Compared with the previously reported cDNA of the SMVT, these three
clones were each found to be uniquely different at the extreme 5'
UTR sequence, and all shared the absence of a 60-bp stretch in the middle of the 5' UTR sequence (Fig.
1B). These three newly identified sequences were named variants II, III, and IV, respectively, relative to the 5' UTR of SMVT that was considered variant I in this
report. No sequence corresponding to the 5' UTR of SMVT (i.e.,
variant I) was detected in the small intestine by our RT-PCR cloning
method. With regard to the 3' UTR, this region was found to be
identical to that of SMVT, with the exception of a 10-bp region missing in the cloned intestinal cDNA (Fig.
1C).


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Fig. 1.
Analysis of the 5' untranslated region (UTR) of cloned rat small
intestinal cDNA. A: nucleotide
sequence of different variants. Variants II-IV were identified in the
small intestine. Also shown is nucleotide sequence of 5' UTR of
placental clone, which is considered in this report as variant I,
adopted from GenBank (AF026554) for comparison. ATG initiation codon in
intestinal variants is numbered as 1. Shaded areas indicate
heterogeneity present at 5' UTR.
B: diagrammatic representation of
alignment of 5' UTR of rat intestinal and placental biotin
transporters. Shaded regions in extreme 5' end indicate
heterogeneity among different variants. Region from nt 232 to nt 291 (black) is present only in placenta (variant I) but is missing in other
intestinal variants (II, III, and IV).
C: comparison of 3' UTR of
cloned intestinal cDNA and placental
Na+-dependent multivitamin adapter
transporter (SMVT). * Identical regions between the 2 sequences.
Dashed line, 10-bp region that is missing from intestinal cloned
cDNA.
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Analysis of the nucleotide sequence showed that the ORF of the cloned
cDNA encodes a putative polypeptide of 634 amino acids with a
calculated molecular mass
(Mr) of 68,596 Da. The theoretical isoelectric point was estimated to be 9.4. With the
use of the Kyte-Doolittle algorithm (10) with a window of 11 amino
acids, 12 membrane-spanning domains were predicted with both amino and carboxy termini to be on the cytoplasmic side of the membrane (Fig.
2). The putative polypeptide was found to
carry a net positive charge of 5.4 at physiological pH, as indicated by
the program Protean of the Lasergene package (Dnastar, Madison, WI).
The instability index of the putative polypeptide was found to be
42.03, classifying it as unstable; also, the polypeptide contained 12 histidine residues and was rich in leucine, with 15% of its amino
acids being leucine. Examination of the predicted amino acid sequence
also revealed several putative posttranslational modification sites: 2 protein kinase C phosphorylation sites (T16 and S235), 1 protein kinase A phosphorylation site (S322), 3 N-glycosylation sites (N488, N497, and
N532), 11 O-glycosylation sites (T2, S5, T6, S10, T13, S14,
S491, T504, S509, T510, S512), and 13 N-myristoylation sites (G42, G183,
G193, G238, G271, G352, G361, G454, G458, G462, G472, G540, G548).

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Fig. 2.
Diagrammatic representation of a membrane model of cloned intestinal
cDNA as predicted by Kyte-Doolittle hydropathy algorithm (10). ,
Predicted N-glycosylation sites; *,
O-glycosylation sites; , protein
kinase C phosphorylation sites; and , protein kinase A/protein
kinase G phosphorylation site.
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Functional characteristics of the cloned
cDNA. Functional identity of the ORF of the cloned
intestinal cDNA was confirmed by stable transfection of the cDNA into
African green monkey kidney COS-7 cells (COS-7/cDNA) followed by
examination of
[3H]biotin uptake and
comparing the results to appropriate controls [the controls
included mock electroporated COS-7 cells (COS-7/wild) and COS-7 cells
transfected with an empty vector (COS-7/vector)]. Uptake of
[3H]biotin (5.2 nM)
was found to be four- to fivefold higher in COS-7/cDNA cells compared
with uptake by COS-7/vector cells; uptake by the latter cells was found
to be similar to that in the COS-7/wild cells [79.17 ± 2.67, 17.4 ± 0.49 (P < 0.01) and 19.08 ± 1.03 (P < 0.01)
fmol · mg
protein
1 · 3 min
1, respectively].
The level of mRNA transcripts corresponding to the cloned intestinal
cDNA in the different COS-7 cell subtypes was also determined using
RT-PCR. Results showed the presence of mRNA corresponding to the cloned
intestinal cDNA in COS-7/cDNA cells but not in control cells (Fig.
3).

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Fig. 3.
RT-PCR analysis of mRNA from different COS-7 cell subtypes. RT-PCR
products from different COS-7 cell subtypes were analyzed on an agarose
gel (see MATERIALS AND METHODS).
Lane 1, COS-7/wild type;
lane 2, COS-7/vector; and lane
lane 3, COS-7/cDNA. Primers used are
described in Table 1; PCR conditions used are described in Table 2.
Data from a representative experiment are shown. ORF, open reading
frame.
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In another study, we examined the effect of
Na+ removal from the incubation
medium on the induced biotin uptake in COS-7/cDNA cells. In this
experiment, Na+ was replaced
isosmotically with either K+,
Li+, choline, Tris, or mannitol.
The results showed significant (P < 0.01 for all) inhibition in biotin (5.2 nM) uptake when
Na+ was removed from the
incubation medium and regardless of what was used to replace it (79.17 ± 2.67, 6.88 ± 0.55, 21.35 ± 0.76, 15.15 ± 0.42, 2.25 ± 0.25, and 1.27 ± 0.10 fmol · mg
protein
1 · 3 min
1 in presence of
Na+,
K+,
Li+, choline, Tris, and mannitol,
respectively). In a related study, we examined the stoichiometry of the
coupling between biotin and Na+
using the "activation method" (36), as described by us previously (25). In this method, the initial rate of biotin (5.2 nM) uptake was
examined as a function of increasing the
Na+ concentration in the
incubation medium in the presence of 30 µg/ml valinomycin. Saturation
was observed as a function of increasing Na+ concentration
([Na+]) with a
Km for
Na+ of 18 mM (Fig.
4A).
Values of log[Na+]
were then plotted against logQ {logQ = log[v/(Vmax
v)], where v is the initial rate of biotin uptake at a
given Na+ concentration, and
Vmax is the
maximal uptake velocity (Fig. 4B)}. The Hill coefficient
was then calculated and found to be 1.3, suggesting a coupling ratio
between biotin and Na+ of 1:1.

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Fig. 4.
Uptake of biotin uptake by induced system in COS-7/cDNA cells as a
function of Na+ concentration in
incubation medium. Cells were incubated for 3 min at 37°C in
Krebs-Ringer buffer, pH 7.4, containing 30 µg/ml valinomycin.
Na+ was isosmotically replaced by
K+. Values are means ± SE of
4-5 separate uptake determinations.
A: initial rate of biotin (5.2 nM)
uptake as a function of Na+
concentration. B: Hill plot:
log[Na+] plotted
against logQ {logQ = log
[v/(Vmax v)], where v is initial rate of biotin uptake in presence
of a given Na+ concentration and
Vmax is maximal
uptake velocity}. y = 1.3x 1.57;
r = 0.986.
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We also determined the kinetic parameters of the induced biotin uptake
in COS-7/cDNA cells. This was performed by examining biotin uptake as a
function of concentration (1-100 µM) by COS-7/cDNA and
COS-7/vector cells. Uptake of
[3H]biotin by the
induced process was determined at each biotin concentration by
subtracting uptake by COS-7/vector cells from the uptake by COS-7/cDNA
cells (Fig. 5). Uptake of biotin by the induced process was found to be saturable as a function of
concentration with an apparent
Km and
Vmax (calculated
as described in materials and
methods) of 8.77 µM and 779.7 pmol · mg
protein
1 · 3 min
1, respectively.

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Fig. 5.
Uptake of biotin by induced system in COS-7/cDNA cells as a function of
biotin concentration in incubation medium. Cells were incubated for 3 min at 37°C in Krebs-Ringer buffer, pH 7.4, in presence of
different concentrations of biotin. Uptake by the induced system was
calculated as described in results.
Data are means ± SE of 6 separate uptake determinations.
Inset: Lineweaver-Burk plot of uptake
data.
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In another study, we examined the effect of the biotin structural
analogs desthiobiotin, thioctic acid, diaminobiotin, biotin methyl
ester, and biocytin on the induced
[3H]biotin (5.2 nM)
uptake in COS-7/cDNA cells. The results showed a
concentration-dependent inhibition in
[3H]biotin uptake by
desthiobiotin, thioctic acid, and diaminobiotin with inhibition
constant (Ki) values (determined
by the "Dixon" method) of 16.3, 59.7, and 114.1 µM,
respectively (Fig. 6). In contrast,
biocytin and biotin methyl ester (100 µM each) were found to have no
effect (69.04 ± 1.81, 67.36 ± 1.3, and 67.48 ± 0.46 fmol · mg
protein
1 · 3 min
1 for control and in the
presence of biocytin and biotin methyl ester, respectively).

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Fig. 6.
Dixon plot of effect of biotin structural analogs on
[3H]biotin uptake by
induced system in COS-7/cDNA. Cells were incubated for 3 min at
37°C in Krebs-Ringer buffer, pH 7.4. Different concentrations of
desthiobiotin (A), thioctic acid
(B) and diaminobiotin
(C) along with
[3H]biotin (5.2 nM)
were added to incubation medium at onset of experiment. Data are means
of 4-5 separate uptake determinations. V, rate of uptake.
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The effect of pantothenic acid and its structural analogs on the
induced uptake of
[3H]biotin (5.2 nM) in
COS-7/cDNA cells was also tested. The results showed a
concentration-dependent inhibition in
[3H]biotin uptake by
unlabeled pantothenic acid; with the use of the Dixon method, this
inhibition was found to be competitive in nature with a
Ki value of 4.9 µM (Fig. 7). Similarly, structural analogs of pantothenic acid, namely dl-pantoyltaurine,
D-pantethine, and pantothenyl alcohol (all at 100 µM)
caused significant inhibition in
[3H]biotin (5.2 nM)
uptake [73.08 ± 0.5, 44.94 ± 4.46 (P < 0.01), 42.78 ± 2.59 (P < 0.01), 30.97 ± 1.02 (P < 0.01), and 34 ± 1 (P < 0.01)
fmol · mg
protein
1 · 3 min
1 for control and in the
presence of DL-pantoyltaurine, D-pantethine, and pantothenyl alcohol, respectively]. In a related study, we examined the uptake of the
[14C]pantothenic acid
(1.82 µM) by COS-7/cDNA cells and compared the results to uptake by
control (COS-7/vector) cells. The results showed an approximately
fivefold higher pantothenic acid uptake in COS-7/cDNA cells compared
with control cells [32.6 ± 0.6 and 6.55 ± 0.12 (P < 0.01),
pmol · mg
protein
1 · 3 min
1, respectively].
The effect of Na+ removal from the
incubation medium on the induced pantothenic acid uptake by COS-7/cDNA
cells was also examined as described earlier. The results showed
significant (P < 0.01) inhibition for both K+ and
Li+ in pantothenic acid uptake on
Na+ removal from the incubation
medium (31.79 ± 0.57, 3.87 ± 1.2, 1.75 ± 0.77 pmol · mg
protein
1 · 3 min
1 in the presence of
Na+,
K+, and
Li+, respectively). We also
examined the effect of unlabeled biotin (100 µM) and its structural
analog thioctic acid (100 µM) on induced uptake of
[14C]pantothenic acid
(1.82 µM) by COS-7/cDNA cells. The results showed significant
(P < 0.01 for both) inhibition in
pantothenic acid uptake by both compounds (32.17 ± 0.54, 1.39 ± 0.13, and 1.26 ± 0.22 pmol · mg
protein
1 · 3 min
1 for control and in the
presence of unlabeled biotin and thioctic acid, respectively).

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Fig. 7.
Dixon plot of effect of unlabeled pantothenic acid on
[3H]biotin uptake by
induced system in COS-7/cDNA cells. Cells were incubated for 3 min at
37°C in Krebs-Ringer buffer, pH 7.4. [3H]biotin [0.5
µM ( ) and 5 µM ( )] and different concentrations of
pantothenic acid were added at onset of experiment. Data are means of
4-5 separate uptake determinations (y = 0.043x + 0.23;
r = 0.976 for 0.5 µM biotin and
y = 0.003x + 0.038;
r = 0.974 for 5 µM biotin).
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Tissue distribution of the different 5' UTR variants of the
cloned intestinal cDNA.
Distribution of the different variants (II-IV) of the cloned intestinal
cDNA and that of SMVT (variant I) along the length of the intestinal
tract was examined in this study using RT-PCR. The results showed that
variant II was the predominant form expressed in rat small and large
intestine, with the highest expression being in the small intestine
compared with the large intestine (Fig. 8).
Variant I was not detected in the small intestine but only in regions
of the large intestine (Fig. 8). Variant III was detected in the colon
and ileum but was absent from the duodenal/jejunal area (Fig. 8).
Variant IV was not detected in any region of the small and large
intestine under the PCR conditions used (35 cycles) in this study (Fig.
8); however, it was detected in the jejunal area only when more initial
template and higher amplification cycles (40 cycles) were used (data
not shown). In another study, we examined the distribution of mRNA
species of the predominant intestinal variant, i.e., variant II, in
other rat tissues. The result showed expression of this variant in many
other tissues in the following order (relative to
-actin): kidney = heart > liver > skeletal muscle > brain = stomach = lung (Fig.
9). This pattern of distribution of variant
II compared with the distribution of the ORF of SMVT in various rat
tissues reported previously (17) shows similarities and differences,
suggesting different patterns of distribution of the different variants
in different tissues.

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Fig. 8.
Distribution of different 5' UTR variants of cloned intestinal
cDNA along intestinal tract. RT-PCR products from different intestinal
tissues were analyzed on an agarose gel (see materials
and methods). Primers used are described in Table 1;
PCR conditions used are described in Table 2. Data from a
representative experiment are shown. Prox, proximal; dist,
distal.
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Fig. 9.
Distribution of variant II in different rat tissues. RT-PCR products
from different rat tissues were analyzed on an agarose gel (see
materials and methods). Primers used
are described in Table 1; PCR conditions used are described in Table 2.
Data from a representative experiment are shown. Sk, skeletal.
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Distribution of mRNA transcripts complementary to the
ORF of cloned cDNA along the intestinal vertical and longitudinal
axes. The distribution of mRNA transcripts
complementary to cloned cDNA along the vertical axis of the intestine,
i.e., villus vs. crypt cells, was determined by RT-PCR using primers
from the ORF and poly(A)+ RNA
extracted from rat jejunal villus and crypt cells isolated by the
Weiser method (39) as described by us before (24). Also examined was
Na+-dependent uptake of biotin
(5.2 nM) and D-glucose (0.171 µM) in the two cell types. The results showed that the level of mRNA transcripts complementary to the cloned cDNA (normalized to
-actin) was 2.6-fold higher in villus compared with crypt cells (Fig. 10). As to the
Na+-dependent uptake of biotin and
D-glucose, the results showed significantly (P < 0.01 for both)
higher uptake for both substrates in villus compared with crypt cells.
For biotin (5.2 nM), uptake was 191.33 ± 11.11 and 46.1 ± 4.26 pmol · mg
protein
1 · 2 min
1, respectively; for
glucose (0.17 µM), uptake was 3.31 ± 0.26 and 0.16 ± 0.06 pmol · mg
protein
1 · 2 min
1,
respectively.

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|
Fig. 10.
Distribution of mRNA transcripts complementary to the ORF of cloned
intestinal cDNA along vertical axis of small intestine. RT-PCR products
from villus and crypt cells were analyzed on an agarose gel (see
materials and methods). Primers used
are described in Table 1; PCR conditions used are described in Table 2.
Data from a representative experiment are shown.
|
|
For distribution of mRNA transcript complementary to the cloned cDNA
along the longitudinal axis of the rat intestinal tract as well as the
stomach and the liver, RT-PCR and
poly(A)+ RNA extracted from
specific tissues were used. The distribution was found to be similar
along the length of the small intestine and colon and was higher than
that in the liver and stomach (Fig. 11).

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[in this window]
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|
Fig. 11.
Distribution of mRNA transcripts complementary to the ORF of cloned
intestinal cDNA along longitudinal axis of intestinal tract as well as
in stomach and liver. RT-PCR products from different tissues were
analyzed on an agarose gel (see materials and
methods). Primers used are described in Table 1; PCR
conditions used are described in Table 2. Data from a representative
experiment are shown.
|
|
 |
DISCUSSION |
Compared with our current understanding of the mechanism and regulation
of the intestinal biotin absorption process at the tissue, cellular,
and membrane levels, little is known about the molecular
characteristics of the absorption process. Such information is crucial
for detailed understanding of the mechanism and regulation of the
absorption process of this essential micronutrient in the intestine
under normal physiological conditions and on how certain conditions
affect the absorption process. In this study, we used the method of
RT-PCR and knowledge obtained from the recently cloned placental SMVT
cDNA to address this issue. Our study has identified a cDNA clone that
has an ORF identical to that of SMVT but that displayed significant
heterogeneity at the 5' UTR. Three new variants (II, III, and IV)
were identified in the small intestine, with variant I (the placental
form) being absent in the small gut. The existence of multiple variants
suggests possible involvement of alternative splicing of the
transcript; it also suggests possible involvement of multiple promoters
in driving transcription of the cloned intestinal cDNA. The latter
suggestion is based on a substantial number of reports linking
multiplicity of promoters with heterogeneity at the 5' UTR (15,
18, 33). Of the different 5' UTR variants identified in the
intestine, variant II was found to be the predominant form and has a
higher expression in the small intestine compared with the large
intestine. This variant was also found to be expressed in other rat
tissues including the kidney and the heart. Although variant I was not
detected in the small intestine as mentioned earlier, some expression
was found in the large intestine.
Inspection of the deduced amino acid sequence of the cloned cDNA
indicates that the encoded protein carries a net positive charge of 5.4 at physiological pH, which may be important for the interaction of the
polypeptide with the negatively charged biotin molecule
(pKa of biotin is
4.51). Also, multiple histidine residues were found in the deduced
amino acid sequence of the predicted polypeptide, some of which may be
important for the normal function of the intestinal biotin uptake
process as suggested by previous studies in our laboratory with
group-specific reagents (22). Further studies with
site-directed mutagenesis are required to test this possibility.
To confirm the functional identity of the cloned intestinal cDNA as a
biotin transporter, we stably transfected COS-7 cells with this cDNA
and examined biotin transport activity. The results showed that,
compared with control cells, biotin transport in the transfected cell
(i.e., COS-7/cDNA cells) was significantly higher. The induced biotin
uptake in COS-7 cells was found to be
Na+ dependent, saturable as a
function of concentration
(Km of 8.77 µM), and inhibited by biotin structural analogs with a free carboxyl group in the valeric acid moiety but not by those analogs with a
blocked carboxyl group. Furthermore, the induced biotin uptake in
COS-7/cDNA cells was significantly inhibited by pantothenic acid and
its structural analogs. Moreover, uptake of pantothenic acid itself was
found to be significantly induced in COS-7/cDNA cells compared with
control cells. This induced uptake of pantothenic acid was also found
to be Na+ dependent and was
inhibited by unlabeled biotin and its structural analog thioctic acid.
These findings on biotin transport and the interaction with pantothenic
acid are all similar to those observed with intact intestinal
epithelial cells (19). This includes an apparent
Km of the induced biotin uptake in COS-7 cells
similar to that of the native rat intestine of 7.57 µM
(28). These observations support the conclusion that the
intestinal cDNA cloned in this study may be involved in the normal
intestinal absorption process of biotin. The ability of pantothenic
acid to interact with the biotin intestinal transport process has also
been observed in other cells and tissues such as the
blood-brain barrier and heart and was also demonstrated for
placental SMVT (2, 6, 16, 17, 34).
The distribution of mRNA transcripts complementary to the ORF of the
cloned intestinal cDNA along the vertical (villus vs. crypt) and
longitudinal (duodenum, jejunum, ileum, proximal colon, and distal
colon) axes of the intestinal tract was also investigated. A 2.6-fold
higher expression was found in villus compared with crypt cells. This
corresponds with the higher carrier-mediated biotin uptake observed in
this study in villus compared with crypt cells. As to the longitudinal
distribution of mRNA complementary to the cloned intestinal cDNA, a
discrepancy in expression compared with functional transport activity
was observed. While biotin transport activity is known to be higher in
the proximal small intestine compared with the ileum and the colon (19,
27, 30), a similar level of expression of mRNA complementary to the
cloned intestinal cDNA was found along the length of the small
intestine and colon. This paradox may suggest the involvement of a
cell-specific posttranslational event(s) that regulates the expression
of the functional protein in the different areas of the intestinal
tract. Further studies are needed to clarify this issue. The
identification of mRNA transcripts complementary to the cloned
intestinal cDNA in the colon corroborates the recent finding from our
laboratory (25) on the existence of a functional
Na+-dependent biotin
carrier-mediated uptake system in the colon that may be involved in the
absorption of biotin bacterially synthesized by the normal microflora
of the large intestine.
In summary, these studies describe the molecular characterization of
the intestinal biotin absorption process and report the identification
of a cDNA that appears to be involved in
Na+-dependent biotin uptake.
Furthermore, this cDNA appears to exist in the form of multiple
variants due to heterogeneity at the 5' UTR.
 |
ACKNOWLEDGEMENTS |
N. S. Chatterjee and C. K. Kumar contributed equally to this work.
 |
FOOTNOTES |
We extend our thanks to Dr. Toai Nguyen, Ana-Paula E. Duarte, Erica M. Miller, Qyhn Nhu Nguyen, and Scott Smith for excellent technical help.
This study was supported by grants from the Department of Veterans
Affairs and by National Institute of Diabetes and Digestive and Kidney
Diseases Grant DK-56061.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. M. Said,
Medical Research Service-151, Veterans Affairs Medical Center, Long
Beach, CA 90822 (E-mail: hmsaid{at}uci.edu).
Received 28 April 1999; accepted in final form 11 June 1999.
 |
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