Production of gastric intrinsic factor, transcobalamin, and
haptocorrin in opossum kidney cells
N.
Brada,
M. M.
Gordon,
J.-S.
Shao,
J.
Wen, and
D. H.
Alpers
Division of Gastroenterology, Department of Internal Medicine,
Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
Opossum kidney epithelial cells were
shown previously to synthesize and secrete two cobalamin (Cbl)-binding
proteins, presumed to be haptocorrin (Hc) and transcobalamin II (TCII).
The present study examines the hypothesis that renal tubular cells also
produce intrinsic factor (IF), and this production provides an
explanation for the presence of IF in urine. By using antisera raised
against human IF and against TCII, the presence of TCII was confirmed, and that of IF discovered in the media of opossum kidney (OK) cells in
culture. The apparent molecular weight of IF and TCII was 68 and 43 kDa, respectively. Immunoreactivity on Western blot of the putative IF
protein was blocked by recombinant human IF. When proteins secreted
into the media were separated electrophoretically under nondenaturing
conditions after binding with [57Co]Cbl, a broad major
band migrated at a relative front independently of recombinant IF or
TCII, and probably represents Hc, as the Cbl binding is blocked by
cobinamide. Small amounts of bound [57Co]Cbl migrated in
the position of both IF and TCII, when cobinamide was present. The
presence of IF and TCII in OK cells was confirmed by immunohistology.
Specific reactivity for IF (blocked by recombinant IF) was found in
proximal tubules of opossum kidney, but not in other portions of the
nephron, confirming the ability of anti-human IF antiserum to detect
opossum IF. A 732-bp fragment of IF, nearly identical in sequence to
rat IF, was isolated by RT-PCR from opossum kidney mRNA, and Western
blot confirmed the presence of IF protein. The presence of IF was also
documented in rat kidney by isolation of an RT-PCR fragment,
immunocytochemistry, and Western blot. IF should be added to the list
of renal (proximal) tubular antigens that are shared by other epithelia.
proximal tubule
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INTRODUCTION |
GASTRIC INTRINSIC FACTOR
(IF) is a protein expressed in most adult mammals in gastric
mucosa, but also in many other tissues (2). Within the
stomach, more than one cell type may express IF, even in the same
species (19, 38). In some species (e.g., dog) the
pancreatic duct cells provide the major source of IF (3, 4, 23,
40). In these ectopic locations, IF secretion appears to respond
to the regulatory signals appropriate for that organ, e.g.,
cholecystokinin for pancreatic duct stimulation (39). IF
is expressed in some fetal tissues, e.g., amniotic fluid
(42), although it is largely gone by parturition
(12). The amniotic IF is fetal in origin
(15), probably derives from fetal gastric mucosa (1,
30), and mediates cobalamin (Cbl) absorption by the fetal
intestine (1). In addition, the anlage of the amniotic sac, the yolk sac, also produces cubilin, the IF-cobalamin (IF-Cbl) receptor (28, 33, 37), providing another means by which amniotic fluid Cbl could be absorbed.
The kidney is another tissue that expresses cubilin, and in which IF
has been found bound to its brush border receptor (31). IF
has been found in rat and human urine, but the source was not known,
although it was assumed that it was filtered from the serum (31,
42). The role of cubilin in the renal brush border has been
thought to include recovery of IF filtered into the urine. IF in the
serum has been detected only in the rat, but the level is low
(31). The epithelial cell of the proximal tubule of the kidney shares membrane antigens with many other tissues, including bile
ducts, intestinal villi, epididymal tubules, allantochorionic cells,
and exocrine cells of the pancreatic, salivary, and lacrimal glands
(7, 27); however, gastric antigens have not been
identified previously in the proximal tubule. The present study
examines the hypothesis that urinary IF may derive from the proximal
tubule, not from the serum. IF was identified in the media of opossum kidney (OK) cells, a cell line with characteristics of epithelium of
the proximal tubule (21), and in native opossum
and rat kidney. Previous identification without the use of immunologic
identification suggested transcobalamin II (TCII) and haptocorrin (Hc)
as the only two Cbl-binding proteins produced by OK cells
(32). The present finding extends the range of
tissue-sharing antigens with the proximal tubule epithelium, and could
provide an explanation for some of the IF found in urine and renal
brush border.
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MATERIALS AND METHODS |
Materials.
OK cells were obtained from the American Type Culture Collection (ATCC;
CRL 1840). Cells were grown on T-75 flasks or on polypropylene filters
(Transwell, Corning Costar, Cambridge, MA) in Eagle's modified minimal
medium (Cellgro, Mediatech, Herndon, VA) with the addition of 10% FCS.
Medium was changed every other day, and cells were grown 7 days
postconfluence (~10 days after plating) for analysis of proteins in
the medium. Fresh media without FCS were added 48 h before removal
for analysis. Fresh opossum kidney, pancreas, and stomach was
generously provided by Drs. Nathaniel Soper and Robert Underwood, Dept
of Surgery, Washington University Medical School. Rat kidney was
removed from Sprague-Dawley rats (Sasco, Omaha, NE) after anesthesia
with methoxyflurane and saline perfusion via the thoracic aorta.
[57Co]Cbl (300 µCi/µg) was obtained from ICN (Costa
Mesa, CA). Cobinamide and porcine Hc were obtained from Sigma (St.
Louis, MO).
Sample preparation and Western blotting.
Total RNA was prepared from frozen OK cell pellets, or from freshly
isolated opossum kidney, pancreas, and stomach, or from rat kidney, by
using an Rneasy Midi Kit (Qiagen, Valencia, CA). Media were removed,
dialyzed, and either concentrated 40-fold by using Centriprep (Amicon,
Beverly, MA), or concentrated 24-fold on Cbl-Sepharose columns (Sigma).
The concentrated media (400 µl) were incubated with 100 µl of a
50% suspension of Cbl immobilized on 4% beaded agarose (Sigma) for
1 h at 4°C. The agarose was then washed 2× with 100 mM
Tris · HCl, pH 7.5, with 2 mM sodium azide (13).
The agarose pellet was then resuspended in 100 µl of disruption buffer (62.5 mM Tris · HCl, pH 6.8, 2% sodium dodecyl sulfate, 1.55 g/l of dithiothreitol, and 2.5% glycerol) and released by boiling
for 5 min. Concentrated media samples were applied to 7.5% or 10%
polyacrylamide gels (22), in either the presence or
absence of the denaturing agent, SDS.
Opossum and rat kidney and opossum gastric mucosa (2.5g) were
homogenized in 2.5 ml of PBS, pH 7.4, containing 1% N-P40, 12 mM
sodium deoxycholate, and 0.1% sodium dodecyl sulfate, with 10%
protease inhibitor cocktail for mammalian cell extracts (Sigma) added
just before homogenization. After incubation on ice for 1 h,
homogenates were spun in the microfuge at 4°C, and the supernatant was decanted and frozen until use. One milliliter of extract with ~60
mg of protein was incubated with 0.2 ml of washed Cbl-Sepharose beads
(Sigma) for 1 h at 4°C, washed ×2 with Tris, pH 7.15, and resuspended in 2% SDS, 100 mM dithiothreitol (DTT), and 50 mM Tris, pH
6.8. After boiling and centrifugation, the agarose supernatant was
concentrated to 60 µl, reboiled, and loaded on a 10% SDS gel.
Western blots were developed as previously described by using antiserum
(1:3,000) raised against recombinant IF produced in the baculovirus
system (13, 14). In some experiments the antiserum against
IF (1:3,000 dilution) was incubated overnight at 4°C with 6.25 µg
of recombinant IF. This mixture was added to an Immobilon-P membrane
(Millipore, Bedford, MA), containing the separated and transferred
proteins from the OK cell media. Recombinant IF and TCII produced in
Pichia pastoris (43) were used as binding
protein standards. Antiserum against human TCII used at 1:2,500
dilution was obtained from Robert Allen, (Univ. of Colorado, Denver,
CO). Antiserum against the rat IF-Cbl receptor and porcine Hc have been
described previously (18, 36).
Nondenatured gel electrophoresis.
OK cells were grown until 7 days postconfluence. Fresh media without
FCS were added 48 h before removal for analysis. Media was
concentrated 40-fold as described above before analysis. Twenty nanograms of porcine Hc (Sigma), 4.6 ng of recombinant IF or TCII, or
14 µg of total protein in samples of concentrated media were incubated with labeled Cbl in the presence and absence of a 5,300-fold (Hc and IF), a 5,500-fold (TCII), and a 10,500-fold (media) molar excess of cobinamide to block Cbl binding to Hc (but not to IF or
TCII). Protein-bound radioactivity was separated in a nondenaturing 7.5% acrylamide gel system identical to standard SDS-PAGE, except that
no SDS was included in either the sample, stacking gel, or separating
gel. After the dye reached the bottom of the gel, the unfixed gel was
subjected to autoradiography.
Immunocytochemistry.
This analysis was carried out by using the ABC biotinylation system
(Vector Laboratories, Burlingame, CA) as described previously (38). Fixed tissues were embedded in paraffin and
sectioned at 5-µm thickness. The slides were deparaffinized,
rehydrated, and treated with 1% H2O2 in
methanol. Slides were then blocked in PBS containing 5% BSA and 10%
normal goat serum. Dilutions of the primary IF or TCII antiserum were
used at 1:200 and 1:100, respectively, and the slides were incubated at
37°C for 60 min. The second antibody, goat anti-rabbit biotinylated
IgG, was added at a dilution of 1:200 for 20 min at 37°C, after which
the slides were washed in PBS and incubated with Vectastain ABC
(reagents A and B at 20 µl/ml for 30 min at 37°C).
3,3'-Diaminobenzidine tetrahydrochloride [Sigma Fast DAB tablet (0.7 mg/ml) + H2O2 (20 mg/ml)] was added to
the slide for 3-5 min or until color was evident. Normal rabbit
serum as primary antiserum was used as a control, and showed no
reaction. Recombinant IF for blocking reactivity was added at a
concentration of 1-2 µg/slide.
RT-PCR.
The forward primers used corresponded to bp 180-209
(-CCCAAACCCCAGCATCCTGATTGCCATGAA-) and the reverse primer to bp
780-812 (-CGGAATTTCCCCTGCTTAATCTCCTTGAGTATC-) from rat IF
(8), yielding an expected fragment of 732 bp. Reaction
products were cloned into the vector pCR 2.1 TOPO (Invitrogen,
Carlsbad, CA) and sequenced by using an Applied Biosystems Sequencer.
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RESULTS |
Identification of Cbl-binding proteins after SDS electrophoresis.
OK cell media and homogenates were bound to Cbl-Sepharose, boiled to
release the proteins, and electrophoresed on denaturing SDS gels.
Figure 1 shows that cell lysates and
media both contain a protein that reacts with IF antiserum. The
secreted protein shows the expected size of ~68 kDa, whereas the
intracellular protein ran with an apparent relative mass
(Mr) of 55 kDa. The IF-Cbl receptor was
detected only in the cell lysate (Fig. 1). TCII immunoreactivity in
cell lysate and media corresponded with a protein of ~48 kDa,
identical in mobility to recombinant human TCII (Fig.
2). No immunoreactive protein was found
by using antiserum raised against porcine Hc. To confirm the presence
of IF, media was concentrated without initial passage over a Cbl
affinity column. Once again a protein of ~70 kDa was identified by
Western blot (Fig. 3). When the blot was
performed on conditioned media with antibody against IF and with
purified recombinant human IF added, IF immunoreactivity was completely
blocked (Fig. 3). Western blots on media not concentrated by affinity
columns, and using antiserum raised against human TCII or against
porcine Hc, revealed no immunoreactive bands (data not shown).

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Fig. 1.
Western blot of intrinsic factor (IF) and its receptor in
media and lysates of opossum kidney (OK) cells. Media were recovered
after 48-h incubation in the absence of FCS and concentrated 24-fold.
Cells were scraped from a T-75 flask, and the pellet homogenized in 0.5 ml of radioimmunoprecipitation assay buffer. Ten percent SDS-PAGE gels
were used without stacking gels (Sigma, St. Louis, MO). Ten microliters
of the cell lysates and concentrated media were applied to the gels.
Western blots were performed as described in MATERIALS AND
METHODS. Left: rabbit antiserum against rat
IF-cobalamin complex (Cbl) receptor (CR; 1:2,000). Right:
rabbit antiserum against human IF (1:3,000).
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Fig. 2.
Western blot of trancobalamin (TC) II in OK cell media
and lysate. Samples were obtained and analyzed as described in Fig. 1.
Rabbit antiserum against human TCII was used (1:2,500). Human
recombinant TCII (0.1 µg protein) was used as a standard.
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Fig. 3.
Western blot of IF in OK cell media. Media were recovered
after 48-h incubation in the absence of FCS and were concentrated
40-fold. Left: two samples of OK cell media demonstrate 1 immunoreactive band identical with that of the major band of
recombinant human IF (0.1 µg protein) added for comparison. The blot
was incubated for 1 h at room temperature with antibody against IF
(1:3,000 dilution). Right: the membrane was incubated with
antiserum plus 6.25 µg of recombinant human IF (total vol 20 ml). No
immunoreactive bands were detected. Antiserum against human recombinant
IF was incubated overnight at 4°C with 6.25 µg of recombinant human
IF to assess the absence of reactivity (final dilution 1:3,000).
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Identification of Cbl-binding proteins after nondenaturing
electrophoresis.
Concentrated recombinant and media proteins with bound
[57Co]Cbl were separated on a gel without any SDS (Fig.
4). Porcine Hc migrated as a broad band
in the top half of the gel (lane 1), and the labeled Cbl was
completely displaced by excess cobinamide (lane 2).
Recombinant human IF showed a band that ran coincident with the Hc peak
(lane 3), and Cbl binding to IF was not altered by excess
cobinamide (lane 4). In the media one broad major band ran
with a relative front (Rf) similar to porcine Hc and accounted for over 90% of the total binding activity in the media (lane 5). The diffuse nature of the band is consistent with behaviour of
Hc in other systems, due to its extensive glycosylation, and probably
represents opossum Hc. When conditioned media was incubated with
cobinamide, one band of radioactivity remained (lane 6), unlike the incubation with porcine Hc alone (lane 1). This
band corresponded with the Rf of IF, and probably represents opossum IF
secreted into the OK cell media. Recombinant TCII labeled with Cbl runs
closer to the electrophoretic front (lane 7), and retains its label in the presence of cobinamide (lane 8). A labeled
protein of similar Rf was seen in conditioned media with and without
cobinamide (lanes 5 and 6) and accounts for only a small
percent (6%) of Cbl binding activity in the media. The absence of SDS
in the gel allows determination of Cbl binding, but did not provide
sufficiently concentrated bands for detection of IF and TCII by Western
blotting.

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Fig. 4.
Identification of cobalamin binding proteins in OK cell
media on nondenaturing polyacrylamide gel. Polyacrylamide
electrophoresis in 7.5% gels was performed to separate Cbl-binding
proteins in concentrated media from OK cells as described in
MATERIALS AND METHODS. Purified recombinant (human IF,
TCII) and native [porcine haptocorrin (Hc)] binding proteins and
conditioned media from OK cells were mixed with labeled Cbl in the
presence and absence of a molar excess of cobinamide (Cob; 5,300-fold
for Hc and IF, 5,500-fold for TCII, and 10,500-fold for media). The
affinity column-concentrated media (14 µg protein) and purified
Cbl-binding proteins (4.6 µg protein) were separated on a
nondenaturing acrylamide gel, as described in MATERIALS AND
METHODS. The migration of porcine Hc alone and with added Cob is
shown in lanes 1 and 2. Lanes
3-8 show binding of IF, OK cell media, and TCII without
(lanes 3, 5, 7) and with (lanes 4, 6, 8) added
Cob. Note that Cob completely blocks the binding of
[57Co]Cbl (lane 2) but does not block binding
to IF or TCII (lanes 4 and 8). Note the wide radioactive
band in the media [relative front (Rf) 0.16-0.40, by using
the fast-moving free Cbl band as the leading edge, lane 5]
that is largely blocked by Cob (lane 6). However, a band of
radioactivity persists at the Rf of IF (0.3). The minor faster moving
band with Rf of 0.64 corresponds with TCII, and this shows up faintly
in the media as well (lane 6). The leading band of
radioactivity corresponds with free Cbl.
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Immunocytochemistry.
Figure 5 shows that OK cells stained
positively for both IF (A) and TCII (B), whereas
no staining was seen with normal rabbit serum (D). The
immunoreactivity was weaker for IF than for TCII, consistent with the
2-fold greater Cbl binding of TCII vs. IF in OK cell media (Fig. 4).
The stain was seen in the cytoplasm and appeared more concentrated near
the apical surface of the cells. Recombinant IF was added to the media
for 1 h at 37°C to produce a pattern that might be expected when
proximal tubular cells absorb IF from the urinary lumen. In this
instance (Fig. 5C) the stain was found much more on the
apical surface of the cells, and the cytoplasm was more deeply stained
than by endogenous IF alone (A).

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Fig. 5.
Immunocytochemical identification of IF and TC in OK
cells. The cells were grown on Transwell filters until 7 days
postconfluence. A: antiserum against human IF (1:200).
B: antiserum against human TC (1:200). C:
antiserum against human IF (1:200) after incubation of the cells with
recombinant human IF (17 µg/ml). D: normal rabbit serum
(1:200).
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Previous studies failed to detect immunoreactive IF in OK cells
(32), and the reaction in the present study was also weak. Figure 6A demonstrated
strongly positive IF staining in proximal collecting tubules (PCT)
present in opossum kidney PCT. The distal tubules were not reactive
(arrows). This staining was blocked completely by the addition of
recombinant human IF (Fig. 6B) showing that opossum IF is
readily detected by anti-human IF antibody. Thus the weak response in
OK cells (Fig. 5) is probably related to limited production by those
cells and not to decreased antibody reactivity. Addition of recombinant
human IF blocked reactivity in rat kidney as well (compare B
and D with A and C, Fig.
7). However, in the rat kidney the distal
tubules (arrows) also contained IF, although much less than the
proximal tubules (arrowheads). In the intact kidneys of both species,
the immunoreactive precipitate could identify IF either endogenously
produced or IF endocytosed from the lumen, or both. The absence of
immunostaining in the apical membranes favors endogenous IF as the
source of positive staining.

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Fig. 6.
Immunocytochemical identification of IF in normal opossum
kidney. A: antiserum against human IF was applied at 1:200
dilution. Note the restriction of IF reactivity to the proximal
collecting tubule. Rat kidney, but not opossum kidney, shows decreased
IF reactivity in the distal tubule. B: recombinant human IF
at 1.7 µg/ml was added to the antiserum. Note the absence of
immunoreactivity. PCT, proximal collecting tubules; DCT, distal tubule.
Magnification ×125.
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Fig. 7.
Immunocytochemical identification of IF in normal rat
kidneys. A and C: antiserum against IF alone as
in Fig. 6. Note immunostain in distal tubules (arrows) as well as
proximal tubules (arrowheads), although the staining in proximal
tubules is greater. B and D: recombinant IF was
added as in Fig. 6, and immunostaining was blocked (×250, A
and B; ×500, C and D).
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Identification of endogenous renal IF.
RT-PCR was performed to demonstrate that IF was indeed produced
endogenously. RT-PCR reactions produced a product of the appropriate size (732 bp) by using mRNA from opossum kidney and pancreas, but not
from stomach, under the conditions used (Fig.
8A). The sequence of 190 internal amino acids obtained showed 99% identity (188/190) with that
of rat IF, with the exception of C527T, resulting in amino acid change
F164S, and G688A, corresponding to D218N, according to the numbering
assignments of the rat-cDNA clone (8). RT-PCR reactions
with the same primers produced a similar- sized fragment by using mRNA
from OK cells (data not shown) and from rat kidney (Fig.
8B). In the rat, the stomach contains the largest amount of
IF mRNA (Fig. 8B); however, IF mRNA was clearly seen in rat
kidney.

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Fig. 8.
RT-PCR-derived fragments from opossum and rat tissue
mRNA. A: the opossum kidney and pancreas contained the
predicted 732-bp fragment, but opossum stomach did not, under the
conditions used. Sequencing 1 fragment from each tissue revealed near
identity with rat IF sequence (8). B: the rat
kidney fragment was identical in size to that from the more abundant
gastric one. Sequence from both fragments revealed rat IF.
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Western blots were performed on affinity column-concentrated extracts
of opossum stomach and kidney and rat kidney (Fig.
9). In the opossum stomach, faintly
positive bands were seen, corresponding to apparent
Mr of 62, 70, and 95 kDa (Fig. 9A).
Three bands were also seen in opossum and rat kidney extracts (Fig.
9B), consistent with a similar pattern seen previously in
rat stomach (23). The two upper bands in opossum and rat
kidney were identical in size with those in opossum stomach, but the
smallest band was somewhat smaller, and a doublet was found in the
opossum kidney.

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Fig. 9.
Western blots of extracts from opossum and rat tissues.
A: opossum stomach; B: opossum and rat kidney.
Tissue extracts were obtained and concentrated by using Cbl-Sepharose
as described in MATERIALS AND METHODS. Antibody against
human recombinant IF was used at 1:3,000 dilution.
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DISCUSSION |
The production of IF by (proximal) renal tubular cells provides an
explanation for the presence of some IF in the urine. In fact, all
three Cbl-binding proteins (IF, TCII, and Hc) appear to be produced by
these cells, and all have been found in human urine (42).
Consistent with this finding, the Cbl-binding proteins are
preferentially secreted into the apical media by OK cells; however,
they also appear in the basolateral media (32). About one-half of the Cbl binding activity in human urine was blocked by
cobinamide and might represent Hc (42). Although the
contribution of IF to the total Cbl binding in OK cell media was low,
the data on Cbl binding in human urine suggest a larger contribution
from IF.
It has been suggested that some of the IF in urine might derive from
filtration of serum IF. Two IF reactive polypeptides (62 and 50 kDa)
were found in rat serum, but intravenously administered [125I]IF was taken up rapidly by an uncharacterized
receptor in the liver, and was not reported to appear in urine to
account for the 80-170 pg of IF secreted daily in urine
(31). Although IF was detected by immunoblot in the rat
renal cytosol and Golgi, it was suggested that serum was the origin of
the urinary IF (31). Serum IF might result from absorbed
IF that escapes lysosomal hydrolysis after endocytosis in the ileum.
However, intact IF does not appear to survive transcytosis across
intestinal cells in culture (14, 32). Alternatively, IF
might reach the blood by direct secretion from gastric mucosal cells
(19) or from the proximal and distal tubule of the kidney.
The present data showing IF mRNA, cytosolic staining in rat kidney, and
Western blot identification in kidney extracts are consistent with the cytosolic and Golgi distribution of IF reported previously
(31).
Several explanations have been offered to account for the variation
between ileal and urinary IF-Cbl receptor activity in patients with
Grasbeck-Imerslund disease, a syndrome characterized by Cbl deficiency,
the cause of which is decreased Cbl absorption across the intestinal
mucosa, although IF production is normal (17). Urine has
been used as a source of receptor fragments for a radioisotope binding
assay to detect the IF-Cbl receptor (8, 18). The values
for IF-Cbl binding to a receptor fragment in the urine have been low in
cases of Grasbeck-Imerslund disease, a syndrome in which Cbl absorption
from the intestine is decreased. Yet, two recent case reports show an
elevated ileal level of the IF-Cbl receptor (11). In
addition, increased IF has been reported in the urine of some Finnish
patients with Grasbeck-Imerslund syndrome (10). This
result was interpreted as confirmation of the low IF-Cbl receptor
activity in those patients. The present data are consistent with the
interpretation that varying levels of IF in the urine, originating from
the kidney itself, might combine with Cbl and inhibit binding of
labeled IF-Cbl to urinary protein, thus producing unexpected values for
the IF-Cbl binding assay.
Both IF and TCII sequences are well preserved between rodents and
humans (2, 24). Amplification of IF gene expression occurs
after weaning in the rat and is not observed in the distal tubule
(41). Opossums hold a position on the phylogenetic tree midway between rodents and chickens (5, 26). Analysis of cloned sequences of many proteins reveal identity of 65-91% with rat and mouse sequences, and 54-81% with human ones (6, 24, 29, 35). One would expect that some cross-reaction with
antiserum raised against human proteins should be detected. The portion of the opossum IF mRNA isolated is nearly identical to rat IF. Moreover, IF immunoreactivity in the opossum kidney is blocked by
recombinant human IF. Thus it is likely that the small amount of
immunoreactive IF secreted by OK cells reflects the small amount of Cbl
binding activity in conditioned OK media that comigrates with
recombinant IF. Opossum IF binds better to its own receptor than does
rat IF (34), suggesting differences in the two IF-Cbl receptors. The differences from other species for opossum TCII might be
greater than for IF, as conservation of TCII primary structure may not
be so great. For example, not all species have a structure for TCII
that allows binding to Quso under the same conditions as for human TC
(20).
The present data show, surprisingly, that OK cells expressed IF as well
as TCII, in addition to Hc. The antiserum against porcine Hc did not
identify an opossum counterpart. The same negative result has been
found by using antiserum against human Hc (32). However,
the majority of Cbl binding capacity secreted from OK cells would
appear to represent Hc, as it migrates on native gels with a broad Rf
that does not correspond to the Rf of TCII, and overlaps the Rf of IF.
The presence of the IF-Cbl receptor suggests that OK cells were
sufficiently differentiated to express the expected membrane antigens
shared with other tissues. The Mr of the
receptor was only about one-half (110 kDa) that of the receptor
isolated from opossum kidney (34). Proteolytic degradation
might have occurred during the processing of cells to demonstrate the
OK cell receptor. IF was found at the expected
Mr by Western blotting in denaturing gels. In
nondenaturing gels Cbl binding activity was found in OK cell
conditioned media at the Rf of recombinant IF, when Hc binding of Cbl
was blocked by cobinamide. PCR from RNA isolated from opossum kidney,
pancreas, and OK cells detected an internal sequence nearly identical
to rat IF. In addition, IF mRNA was found by PCR in rat kidney, and
Western blots on opossum stomach and kidney and on rat kidney extracts
identified the pattern of three IF-reactive proteins reported
previously in rat gastric extracts (23). Therefore, IF
should be added to the list of antigens expressed by the proximal
tubule, but which are not kidney specific (7, 27).
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ACKNOWLEDGEMENTS |
This work was supported in part by National Institute of Diabetes
and Digestive and Kidney Diseases Grant DK-33487.
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FOOTNOTES |
Address for reprint requests and other correspondence: D. H. Alpers, Washington Univ. School of Medicine, Dept. of Medicine, Box
8124, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail:
dalpers{at}im.wustl.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.
Received 27 October 1999; accepted in final form 10 August 2000.
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