Characterisation of intestinal peptide transporter of the Antarctic haemoglobinless teleost Chionodraco hamatus
1 Laboratory of General Physiology, Department of Biological and
Environmental Science and Technology, University of Lecce, strada prov. le
Lecce-Monteroni, I-73100 Lecce, Italy
2 Institute of Nutritional Sciences, Physiology and Biochemistry of
Nutrition, Technical University of Munich, Hochfeldweg 2, D-85350
Freising-Weihenstephan, Germany
* Author for correspondence (e-mail: m.maffia{at}physiology.unile.it)
Accepted 18 November 2002
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Summary |
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Functional expression of H+/peptide cotransport was successfully performed in Xenopus laevis oocytes after injection of poly(A)+ RNA (mRNA) isolated from icefish intestinal mucosa. Injection of mRNA stimulated D-Phe-L-Ala uptake in a dose-dependent manner and an excess of glycyl-L-glutamine inhibited this transport. H+/peptide cotransport in the Antarctic teleost BBMV exhibited a marked difference in temperature optimum with respect to the temperate teleost Anguilla anguilla, the maximal activity rate occurring at approximately 0°C for the former and 25°C for the latter. Temperature dependence of icefish and eel intestinal mRNA-stimulated uptake in the heterologous system (oocytes) was comparable.
Key words: brush-border membrane vesicle, Xenopus laevis oocyte, PepT1, H+/peptide cotransport, fish, intestine, Antarctic fish
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Introduction |
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Peptide transport has also been well demonstrated in teleosts where, as in
higher vertebrates, peptide uptake occurs by carrier-mediated mechanisms and
is highly stimulated by an inside-negative trans-membrane electric potential
(Thamotharan et al., 1996;
Maffia et al., 1997
).
Furthermore, an inwardly directed trans-membrane H+ gradient has
resulted coupled to peptide transport, either alone
(Thamotharan et al., 1996
) or
in cooperation with the membrane potential
(Maffia et al., 1997
;
Thamotharan et al., 1996
).
Proton/glycyl-sarcosine (Gly-Sar) cotransport has been described in BBM of
absorbing cells of tilapia Oreochromis mossambicus intestine and
rockfish Sebastes caurinus intestine and pyloric ceca
(Thamotharan et al., 1996
).
Moreover, H+/glycyl-glycine, H+/glycyl-L-proline and
H+/D-phenyl-L-alanine cotransport has been described in eel
Anguilla anguilla intestinal BBM (Verri et al.,
1992
,
2000
;
Maffia et al., 1997
).
To extend information on peptide transporters in lower vertebrates, we have
investigated the presence of such a carrier in a very particular family of
teleost, Channichthyidae (icefish). The members of this family have been
ecologically confined for the last 13 million years to the low-temperature
Antarctic waters, developing an extremely high degree of stenothermy and
endemism (Eastman, 1993). In
particular we investigated the presence of a H+/peptide
cotransport, characterising several of its functional properties in brush
border membrane vesicles (BBMV) isolated from C. hamatus intestine
and making a functional comparison with the peptide transporter of eel
intestine. Icefish and eel dipeptide transporters were functionally expressed
in Xenopus laevis oocytes, and part of the icefish dipeptide
transporter nucleotide sequence was compared to the mammalian intestinal
PepT1-type transporter by RT-PCR approach. Finally, the effects of temperature
on transporter functionality were investigated by measuring the
temperature-dependent substrate uptake.
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Materials and methods |
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Isolation of poly(A)+ RNA
Total RNA was isolated from the scraped mucosa of the whole icefish and eel
intestine, using TRIzol reagent (Life Technologies, Gaithersburg, MD, USA)
(Chomzynski and Saachi, 1987). Poly(A)+ RNA (mRNA) was purified by
chromatography using oligo(dT)-cellulose affinity column
(Sambrook et al., 1989).
Reverse transcription-polymerase chain reaction (RT-PCR)
1 µg poly(A)+ RNA samples from icefish and eel intestinal
mucosa were subjected to RT-PCR using the GeneAmp RNA-PCR kit (PE Applied
Biosystems, Foster City, CA, USA) according to the manufacturer's protocol.
Briefly, reverse transcription was performed for 12 min at 42°C in the
presence of oligo(dT)16 primer and the resulting cDNA was subjected
to PCR using degenerated primers based on the human (PepT1, complete cds;
Liang et al., 1995) and eel
(AY167576 GenBank) intestinal peptide transporter sequence (forward primer:
5'-GACTCGTGGCTSGGRARGTTC-3', starting at nucleotide 279, and
reverse primer: 5'-CCAGTCCAKCCAG-TGCKCCCTCT-3', starting at
nucleotide 848).
PCR amplification was performed for 35 cycles with 95°C denaturation for 1 min, 48°C annealing for 1 min and 72°C extension for 1 min, followed by a final synthesis at 72°C for 7 min. RT-PCR products were separated by 1% agarose gel electrophoresis, stained by ethidium bromide (1 mg l-1) and visualised under UV light using the Gel-Doc System (Bio-Rad Laboratories, Hercules, CA, USA). The PCR product was subcloned into TOPO TA cloning vector (Invitrogen, CA, USA), and subjected to sequencing. Sequence alignment was done using the Clustal W program (EMBL-EBI).
Transport activity in intestinal brush border membrane vesicles
(BBMV)
Preparation of BBMV
BBMV were prepared from the whole intestine of the fishes as previously
described (Maffia et al.,
1996; Verri et al.,
2000
). Protein concentration was measured with the Bio-Rad protein
assay kit, using lyophilised bovine serum albumin as a standard. Intra- and
extravesicular buffers had the same ionic strength, anion concentration and
osmolarity.
Measurements of fluorescence quenching
BBM intravesicular acidification was assessed by monitoring the
fluorescence quenching of the pH-sensitive dye Acridine Orange as previously
detailed (Verri et al., 2000).
BBMVs were prepared in a buffer containing (in mmol l-1): 100 KCl,
100 mannitol, 2 Hepes, adjusted to pH 7.4 with Tris. To start the experiment,
20 µl of BBMVs (250 µg of protein) were injected into 1980 µl of
cuvette buffer containing 3 µmol l-1 Acridine Orange, 5 µmol
l-1 valinomycin, 0.5% ethanol, 100 mmol l-1 mannitol, 2
mmol l-1 Hepes, adjusted to pH 7.4 with Tris, and either KCl,
choline chloride or dipeptide. When present, peptides replaced mannitol
iso-osmotically. To obtain faster re-equilibration of the transient
transmembrane H+ asymmetry, 20 µl of 3 mol l-1 KCl
solution were added into the cuvette at the time indicated. Fluorescence
signals were recorded by a Perkin-Elmer LS-50B spectrofluorometer equipped
with an electronic stirring system and a thermostatically controlled cuvette
holder and managed by the Perkin-Elmer Fluorescence Data Manager software for
PC (Perkin-Elmer Ltd, Buckinghamshire, UK). Excitation and emission
wavelengths were 498 and 530 nm, respectively, and both slit widths were set
to 5 nm. Each experiment was repeated at least three times using membranes
prepared from different animals. Within a single experiment, each data point
represents 3-5 replicate measurements.
For diethylpyrocarbonate (DEP) inhibition of proton accumulation BBMVs were
incubated for 1 h at 0°C in buffer containing (in mmol l-1) 280
mannitol, 20 K2HPO4/KH2PO4, pH
6.4, either in the presence (100 mmol l-1 ethanol stock solution)
or in the absence (ethanol only) of 2 mmol l-1 diethylpyrocarbonate
(DEP). In both cases, the final ethanol concentration did not exceed 1%. Then,
to eliminate excess DEP, which affects the Acridine Orange fluorescence
signal, DEP-treated and untreated BBMVs were diluted in 35 ml of buffer
containing (in mmol l-1) 100 mannitol, 100 KCl, 2 Hepes, adjusted
to pH 7.4 with Tris, and centrifuged at 50000 g for 30 min. This
washing procedure was repeated twice. To start the experiment, BBMVs (20
µl, 250 µg of protein) loaded with (in mmol l-1) 100
mannitol, 100 KCl and 2 Hepes, adjusted to pH 7.4 with Tris, were injected
into 1980 µl of cuvette buffer containing 3 µmol l-1 Acridine
Orange, 5 µmol l-1 valinomycin, 0.5% ethanol and either (in mmol
l-1) 100 choline chloride, 80 mannitol and 2 Hepes, adjusted to pH
7.4 with Tris, plus 20 Gly-L-Pro. The net peptide-dependent H+
fluxes were obtained by subtracting H+ flux in the absence of
peptide (control) from the total H+ flux in the presence of peptide
(Gly-L-Pro), expressed as F% mg-1 protein
min-1, where
F% is the fluorescence quenching.
For the temperature-dependence experiments, icefish and eel BBMVs (20 µl, 250 µg of protein), prepared in the same buffer as above, were injected into 1980 µl of cuvette buffer containing 3 µmol l-1 Acridine Orange, 5 µmol l-1 valinomycin, 0.5% ethanol, 100 mmol l-1 mannitol, 2 mmol l-1 Hepes, adjusted to pH 7.4 with Tris, and 100 mmol l-1 choline chloride or 100 mmol l-1 choline chloride and 20 mmol l-1 Gly-L-Pro. The Gly-L-Pro dependent proton uptake and passive diffusion (choline chloride) were measured at increasing temperatures: -2, 0, 2, 4, 8, 12, 18, 24 and 30°C for C. hamatus and 1, 5, 9, 12, 18, 25, 30 and 40°C for A. anguilla.
Kinetic analysis
BBMV (20 µl, 250 µg of protein), prepared in a buffer containing (in
mmol l-1): 100 mannitol, 100 KCl, 2 Hepes, adjusted to pH 7.4 with
Tris, were injected into 1980 µl of an incubation buffer containing 3
µmol l-1 Acridine Orange, 5 µmol l-1 valinomycin,
0.5% ethanol, 100 mmol l-1 choline chloride, 100 mmol
l-1 mannitol, 2 mmol l-1 Hepes, adjusted to pH 7.4 with
Tris and increasing Gly-L-Pro concentrations from 0.5 to 20 mmol
l-1, iso-osmotically compensated by decreasing mannitol
concentrations. Kinetic parameters were determined by non-linear regression
analysis, based on the Marquardt algorithm
(Marquardt, 1963) by using the
software package Statgraphics (STSC, Rockville, MD, USA). Carrier-mediated
Gly-L-Pro-dependent H+ influx kinetics were determined by a
curve-fitting procedure using the iterative non-linear regression method based
on the following MichaelisMenten type equation:
F%=(
F%x[S]) /
(Km,app+[S]), where
F% was
peptide-dependent H+ influx, [S] extravesicular peptide
concentration and Km,app the concentration that yielded
one half
F%max.
Transport activity in Xenopus laevis oocytes
Oocytes and injections
Mature females X. leavis frogs were anaesthetised by partial
immersion in a solution of ethyl-3-aminobenzoate (MS-222; 1.5 g
l-1). Oocytes at stages V and VI were removed through a small
incision in the abdomen and separated from the ovarian lobes using fine
forceps. Following collagenase treatment for 1 h, defolliculated oocytes were
allowed to recover in Barth's medium
(Colman, 1984) overnight at
18°C. Oocytes were injected into the vegetal pole with 40 nl of either
mRNA solution (1 ng nl-1) or RNAse-free water using a World
Precision Instrument nanoliter injector (Sarasota, FL, USA). Oocytes were then
maintained at 4 or 18°C in daily changes of Barth's medium for up to 3
days.
Uptake measurements in oocytes
X. laevis oocytes, injected with 40 nl of either water or
poly(A)+ mRNA (1 ng nl-1) prepared from icefish and eel
intestinal mucosa cells, were incubated either at 4°C or 18°C for 3
days. Then dipeptide (D-Phe-L-Ala) uptake was measured, at 0°C and
18°C, in a pH 6.5 buffer. 5-10 oocytes were used for each experimental
point. Uptake measurement was initiated by placing the oocytes in 200 µl of
the uptake medium (100 mmol l-1 NaCl, 2 mmol l-1 KCl, 1
mmol l-1 MgCl2 and 1 mmol l-1
CaCl2, buffered with 5 mmol l-1 MES/Tris, pH 6.5)
containing [3H]-D-Phe-L-Ala (10 µCi ml-1). After
incubation the uptake solution was removed and the oocytes were washed three
times with 3 ml of ice-cold buffer (100 mmol l-1 NaCl, 2 mmol
l-1 KCl, 1 mmol l-1 MgCl2, and 1 mmol
l-1 CaCl2 and was buffered with 5 mmol l-1
MES/Tris, pH 6.5). Each single oocyte was then placed into a scintillation
vial, dissolved in 250 µl of 1% SDS, followed by the addition of 1 ml of
Ready-Safe scintillation fluid (Beckman, Fullerton, CA, USA) and counted (1
min per oocyte) using liquid scintillation spectrometry.
In the dose-dependence study of the expression of D-Phe-L-Ala uptake, oocytes were injected with either 40 nl of water or increasing amounts of icefish intestinal mucosal cells mRNA (13, 19, 23 and 40 ng) and then incubated at 18°C for 3 days. 5 oocytes were used for each uptake determination. D-Phe-L-Ala uptake in the oocytes was determined at 18°C for 1 h after 3 days of incubation at 18°C. The composition of the uptake medium and the concentration of radiolabel were the same as for the uptake experiments.
In the competition experiments oocytes were injected with 40 nl of either water or mRNA (1 ng nl-1) isolated from icefish and eel intestinal mucosa cells. 5 oocytes were used for each uptake determination. Uptake of peptide was measured at 18°C for 1 h after 3 days of incubation at 18°C. The composition of the uptake medium was the same as for the uptake experiments. The concentrations of [3H]-D-Phe-L-Ala and glycyl-L-glutamine (Gly-Gln) were 2 mmol l-1 (10 µCi ml-1) and 10 mmol l-1, respectively.
In the temperature dependence experiments oocytes were injected with 40 nl of poly(A)+ mRNA (1 ng nl-1) isolated from eel and icefish intestine and then incubated at 18°C for 3 days. 5 oocytes were used for each uptake determination. D-Phe-L-Ala uptake was evaluated by pre-incubating the oocytes for 30 min at different temperatures (0, 12, 18, 25°C) and then measuring the peptide uptake at 18°C for 1 h. The composition of the uptake medium was the same as described above. The concentration of D-Phe-L-Ala was 2 mmol l-1 (10 µCi ml-1).
Statistics
Each experiment was repeated at least three times using membranes prepared
from different animals. Within a single experiment, each data point represents
3-5 replicate measurements. Data points reported in the figures are given as
means ± standard error (S.E.M.). Error bars are shown wherever they
exceed the size of the symbols.
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Results |
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Peptide-dependent H+ uptake in BBMV
The functional expression of a peptide transporter in the icefish
intestine, working at low temperatures, was confirmed by the observation of a
peptide-dependent proton influx in isolated BBMV, followed by monitoring
Acridine Orange fluorescence quenching at 0°C
(Fig. 2A). As shown in this
figure, the addition of vesicles to the cuvette, in shortcircuited conditions
([K+]i=[K+]=100 mmol l-1, plus
valinomycin), did not produce any significant change in AO fluorescence
quenching, either in the absence (trace a) or in the presence (trace b) of 20
mmol l-1 glycyl-L-proline (Gly-L-Pro) in the extravesicular medium.
However, when a transmembrane electrical potential was imposed
([K+]i=100 mmol l-1, [K+]=1 mmol
l-1 plus valinomycin), a transient fluorescence quenching was
observed due to intravesicular acidification (trace c), which was further
enhanced in the presence of 20 mmol l-1 Gly-L-Pro (trace d).
Specific peptide-dependent H+ influx was determined by subtracting
from this value the fluorescence quenching in the absence of peptide (trace d
trace c). As shown in Fig.
2B, the icefish transporter is able to recognise different
dipeptides. When specific peptide-dependent H+ influx was measured
at saturating concentrations of three different dipeptides, Gly-L-Pro
exhibited higher maximal velocities with respect to glycyl-L-alanine
(Gly-L-Ala) and D-phenylalanyl-L-alanine (D-Phe-L-Ala). Several kinetic
features of the icefish peptide transporter were determined by monitoring
Gly-L-Pro-dependent H+ influx, at 0°C, in the presence of
increasing peptide concentrations (Fig.
3C). Non-linear regression analysis of the experimental data
yielded calculated values at 0°C for
Vmax=29.06±1.27 F% mg-1
protein min-1 and Km,app=0.806±0.161
mmol l-1. The linear relationship observed in a
WoolfAugustinssonHofstee plot
(Segel, 1975
) (inset to
Fig. 3C) strengthened the
hypothesis for the presence of a single carrier system for Gly-L-Pro
transporter. The hypothesis of the presence of a peptide transporter in
intestinal icefish BBMV is further strengthened by the significant inhibition
of Gly-L-Pro dependent proton influx that occurs in the presence of DEP, a
reactive agent specific for hystidyl-residues that inhibits mammalian peptide
transporters (Fei et al.,
1997
; Miyamoto et al.,
1986
; Terada et al.,
1996
) (Fig.
2D).
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Peptide uptake in mRNA-injected oocytes
Measurement of peptide uptake in mRNA-injected oocytes
(Fig. 3A) revealed no
expression of the intestinal peptide transporter in oocytes pre-incubated for
3 days at 4°C, compared with a marked expression in those pre-incubated at
18°C (i.e. the physiological temperature of X. laevis). Injection
of increasing concentrations of poly(A)+ mRNA prepared from C.
hamatus enterocytes into X. laevis oocytes led to a
dose-dependent stimulation of peptide transport
(Fig. 3B), as shown by the
[3H]-D-Phe-L-Ala oocyte uptake activity after 3 days of incubation
at 18°C. Intestinal peptide transporters such as hPepT1 are characterised
by their ability to transport a variety of di-/tripeptides. Unlabeled peptide
(glycyl-glutamine, Gly-Gln) competes with radiolabeled D-Phe-L-Ala for the
uptake process in mRNA-injected oocytes
(Fig. 3C), demonstrating the
competition between different dipeptides for the icefish intestinal peptide
transporter heterologously expressed in X. laevis oocytes.
Temperature dependence of the peptide transport system in the
homologous (BBMV) and heterologous (oocyte) systems
Total dipeptide uptake in icefish and eel BBMVs is shown in
Fig. 4 (upper traces) together
with passive diffusion (lower traces). Passive diffusion was almost constant
at temperatures up to 10°C for the icefish and 25°C for the eel, then
started to decrease toward zero. The net, carrier-mediated dipeptide uptake
measured in BBMV and oocytes is shown in
Fig. 5, where differences in
temperature dependence, relative to the expression system under consideration,
can be seen.
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Discussion |
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In the present work we have investigated the presence and characteristics
of a peptide transport system in a member of the Channichthyidae (teleosts,
sub-order Notothenioids), a family whose members possess unique
eco-physiological characteristics deriving from their 13 million-year-long
confinement in the cold waters of the Antarctic seas. The most dramatic
physiological peculiarities of these animals include sub-zero temperature
survival while avoiding freezing, the absence of red blood cells, aglomerular
kidneys, etc., but whether they have any `metabolic' adaptation to the low
temperatures is still under debate
(Eastman, 1993;
Hardewig et al., 1998
;
Somero, 1995
;
Giardina et al., 1998
;
Guderley, 1998
), and their
exact feeding and predation behaviours, as well as mechanisms of food
digestion, absorption and assimilation, are still quite unknown. Adaptation of
membrane transport processes is a common component of the wide range of
physiological and adaptive reactions to cold shown by ectothermic organisms.
As well as the generality of poikilotherm enzymatic processes, substance
transport through the cell membrane is impaired by the low environmental
temperatures, which can directly affect protein stability and functionality
and the physical state of the lipid microenvironment.
As for cold-adapted transporters, the functional and structural
characterisation of some carrier proteins belonging to Antarctic fish (Maffia
et al., 1996,
2000
,
2001
;
Maffia and Pellegrino, 1999
;
Storelli et al., 1998
) and
other vertebrates (Tibbits et al.,
1992
; Xue et al.,
1999
; Elias et al.,
2001
; Dode et al.,
2001
) has been recently undertaken. Although many functional
characteristics of these transporters are similar to those of their
warm-adapted homologues, in some cases they display distinctive adaptive
features to cold, such as low-temperature adapted kinetic parameters
(Kcat, Ea, Km), a
narrow range of temperature optimum, etc.
(Maffia et al., 1996
;
Storelli et al., 1998
;
Tibbits et al., 1992
), the
reasons for which are still unknown at the molecular level. In addition to the
sodium-glucose cotransporter already described in Antarctic Notothenioids
(Maffia et al., 1996
), we have
now extended our research on cold-adapted carrier proteins to a proton
oligopeptide transporter (POT) (Paulsen
and Skurray, 1994
) in the intestine of the Antarctic
haemoglobinless teleost Chionodraco hamatus. A functional comparison
was performed on the same transporter of the temperate teleost Anguilla
anguilla. Reverse transcription of mRNA extracted from icefish and eel
intestinal mucosa cells and subsequent amplification by PCR using degenerate
primers derived from eel and human (PepT1) H+/peptide transporters
led to an identification of 570 bp cDNA in both fishes
(Fig. 1A). Partial nucleotide
sequence of the resulting cDNA of icefish revealed 73% similarity to hPepT1,
and it presumably encodes an amino acid sequence with 74% similarity to
hPepT1. These data constitute a first evidence for the presence of a
PepT1-related mRNA product in the mRNA pool isolated from C. hamatus
intestinal mucosa, thus suggesting the possible functional expression of a
PepT1-related protein in the absorbing epithelium of the icefish. The
hypothesis was confirmed by the observation of a dipeptide uptake activity in
intestinal BBMV, whose main characteristics resemble those of the well-known
human low-affinity, high-capacity transporter and eel and tilapia transporters
(Fig. 2). Also dipeptide uptake
by the icefish transporter was voltage-dependent and proton-coupled
(Fig. 2A), with a relatively
low substrate specificity, as it has generally been found for the PepT1
isoforms studied to date (Fig.
2B; Ganapathy et al.,
1994
; Liang et al.,
1995
; Thamotharan et al.,
1996
; Maffia et al.,
1997
; Verri et al.,
2000
; Meredith and Boyd,
2000
). Proton influx shows a hyperbolic function with substrate
concentration typical of a carrier-mediated process
(Fig. 2C), and the
corresponding WoolfAugustinssonHofstee plot
(Segel, 1975
) (inset to
Fig. 2C) confirmed that
Gly-L-Pro-dependent H+ influx in the icefish intestinal BBMV occurs
by a single carrier, whose kinetic parameters at 0°C are
Vmax=29.06±1.27
F% mg-1
protein min-1 and Km,app=0.806±0.161
mmoll-1. The finding that treatment of BBMVs with 2
mmoll-1 DEP, a known inhibitor of proton-peptide cotransport
(Fei et al., 1997
;
Miyamoto et al., 1986
;
Terada et al., 1996
), inhibits
approximately 95% of peptide-dependent H+ influx in icefish
intestine (Fig. 2D), further
confirms the identification of the transporter as a hPepT1-related isoform.
DEP inhibition suggests that histidyl residues in C. hamatus peptide
transporter are located at, or near, critical catalytic site(s). In higher
vertebrates, specific DEP-reactive residues, i.e. histidyl residues 57 and 121
(Fei et al., 1997
;
Terada et al., 1996
), are
crucial for the transporter function. These histidyl residues are suggested to
be involved in H+ binding and translocation. Whereas mutation of
histidine 57 in both rat and human PepT1 transporters completely blocked
transport, mutation of histidine 121 appeared to abolish transport activity
only in the human PepT1, and not in the rat PepT1
(Fei et al., 1997
;
Terada et al., 1996
). Although
we possess only preliminary data on the secondary and tertiary structure of
the transporter protein, our studies reveal that active side histidyl residues
in the icefish transporter are essential for function, as judged by loss of
proton transport capability on DEP treatment
(Verri et al., 2000
).
The expression of the POT in oocytes of Xenopus laevis gave us the
possibility of analysing the relationship between transport activity and the
lipid milieu in which the transporter is embedded (i.e. the cell membrane).
The expression of the proton-peptide cotransporter in the heterologous system
was carried out at two different incubation temperatures by injecting
enterocyte mRNA in the oocytes and maintaining them at 4°C or 18°C for
3 days. Then the transport activity was measured in oocytes from the two pools
at the environmental temperatures of the two species: 0 and 18°C. A first
result arising from this trial, was the absence of any measurable transport
activity in the oocytes incubated at 4°C. On the other hand, in the
oocytes incubated at 18°C, it was possible to detect at 0°C a low but
significant transport activity for the icefish transporter only, whereas
noticeable transport activity was detected at 18°C for both the icefish
and eel transporters (Fig. 3A).
These results suggest an `intrinsic' capacity of the transport protein of the
Antarctic species to actively perform substrate transport at 0°C, with
respect to the eel. Furthermore, the absence of detectable transport activity
in oocytes incubated at 4°C suggests that frog oocytes face physiological
difficulties in expressing the heterologous transporter in a functionally
active form at this low temperature. An explanation of these phenomena could
be the necessity of the cell membrane to possess an optimal fluid state for a
perfect positioning and functioning of its transporters, receptors, channels,
etc. But the cell biochemical machinery of the frog is also markedly impaired
at 4°C, while at 18°C (i.e. its environmental temperature) processes
such as transcription, translation, post-translational modifications, membrane
positioning can occur normally and efficiently. Injection of increasing
concentrations of poly(A+) mRNA prepared from C. hamatus
enterocytes into X. laevis oocytes produced a dose-dependent peptide
transport activity (Fig. 3B),
as shown by the [3H]-D-Phe-L-Ala oocyte uptake activity after 3
days of incubation at 18°C. The [3H]-D-Phe-L-Ala uptake in
water-injected oocytes can be explained by a simple diffusion mechanism and/or
the presence of an endogenous transport activity in the frog oocyte membrane.
Icefish peptide transporter appears to share the same low substrate
specificity as the mammalian protein PepT1
(Meredith and Boyd, 2000),
characterised by its ability to transport a variety of di-/tripeptides. The
capacity of unlabeled peptide (glycyl-glutamine, Gly-Gln) to compete with
radiolabeled D-Phe-L-Ala for the uptake process in mRNA-injected oocytes
(Fig. 3C) demonstrates the
competition between different dipeptides on the icefish intestinal peptide
transporter heterologously expressed in X. laevis oocytes. Substrate
competition in water-injected oocytes demonstrates that at least part of the
uptake that normally occurs in the frog oocyte depends on active
transport.
More detailed characterisation of the temperature effects on transport
activity of the peptide transport system in homologous (BBMV) and heterologous
(oocyte) systems was carried out by measuring passive diffusion and active
transport at different temperatures (Figs
4,
5). Passive diffusion was
constant up to 10°C in icefish BBMV and 25°C in eel BBMV
(Fig. 4), then decreased toward
zero with further temperature increments. Membrane passive diffusion is a
process that, in the absence of any particular specificity of the diffusion
systems (i.e. highly specific channels;
Maffia and Acierno, in press),
is quite independent from the temperature within a range that permits the cell
membrane to retain a sufficient degree of integrity. The differences shown by
our data are in good agreement with the different physicochemical profiles of
the membranes of the two fishes, adapted to Antarctic and temperate waters,
respectively (Acierno et al.,
1996
; Maffia and Acierno, in
press
). This temperature specificity appears more evident when
passive diffusion is subtracted from total transport
(Fig. 5). In this case, as
expected, the temperature range of physiological activity of the transporter
is narrower, in agreement with the complex temperature requirements of a
trans-membrane carrier protein and the specificity of proteo-lipid
interactions (Maffia and Acierno, in
press
; Sharpe et al.,
2002
). The structure of a protein that spans the entire cell
membrane is markedly affected by the physicochemical state of the lipid
bilayer, which provides the required degree of plasticity to permit the
conformational protein changes necessary for substrate translocation.
Interestingly, within this narrow temperature range, the optimum temperature
for transport corresponds with the respective temperatures to which the two
fish are adapted (icefish) or acclimated (eel). Indirect confirmation of this
behaviour is provided by the active transport activity measured for the same
transporters expressed in X. laevis oocytes. The increased activity
up to 25°C (i.e. the acclimation temperature of the frog) suggests that
the narrow optimal temperature range is basically derived from the general
efficiency of the membrane, rather than being a specific thermodynamic effect
on the carrier protein itself. In any event, both the intrinsic structural
characteristics (i.e. amino acid sequence) and specific proteo-lipid membrane
interactions can contribute, alternatively or in parallel, to the adaptive
features displayed by temperature-adapted enzymes and transporters.
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Acknowledgments |
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References |
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