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(Received for publication, June 27, 1995; and in revised form, November 22, 1995) From the
Rabbits injected with pure human placental transcobalamin
II-receptor (TC II-R) failed to thrive with no apparent tissue or organ
damage, but a 2-fold elevation of the metabolites, homocysteine,
methylmalonic acid, and the ligand, transcobalamin II, in their plasma.
Exogenously added transcobalamin
II-[
The plasma transport of absorbed dietary and biliary cobalamin
(Cbl: vitamin B Many acquired or
inherited causes of defective Cbl absorption and transport (7) and an autoimmune disorder, pernicious anemia, lead to the
development of Cbl deficiency. Although some aspects of pathophysiology
of development of Cbl deficiency due to acquired and inherited
disorders are known(7) , how altered immunity causes pernicious
anemia is not fully understood. Many patients with pernicious anemia
have circulating antibodies to gastric intrinsic factor or other
parietal cell surface and cytoplasmic antigens(8) . Due to
gastric mucosal atrophy, these patients fail to produce intrinsic
factor, a secretory glycoprotein essential for the absorption of Cbl
and thus develop Cbl deficiency due to malabsorption of the vitamin. In
contrast, no known autoimmune disorders leading to defective plasma
transport of Cbl involving functional loss of either transcobalamin II
or its cell surface receptor has been reported to date. However,
inherited disorders involving lack or defective expression of
transcobalamin II are known(9) , and these children generally
develop Cbl deficiency faster than those with absorption
defects(4) . Thus, the consequence of defective uptake of
plasma TC II-Cbl due to functional loss of TC II-R should also result
in the faster development of Cbl deficiency, since TC II-R-mediated
uptake of TC II-Cbl is the only mode of delivery of physiological
amounts of Cbl to all the tissues(10) . This hypothesis was
validated in rabbits that were injected with human TC II-R for the
purposes of raising polyclonal antibodies to TC II-R. The results of
the current study show that human TC II-R antibodies inhibit both in vivo and in vitro the binding of TC II-Cbl in
effect creating functional loss of TC II-R activity, thus suppressing
Cbl transport, development of intracellular Cbl deficiency, and the
noted failure to thrive. The following chemicals were purchased as indicated:
[
The ability of membrane extracts (0.1 M glycine HCl/KSCN and pH 5/EDTA extracts) and the harvested rabbit
serum to inhibit in vitro, the binding of TC
II-[ Affected and normal rabbit tissue total
membranes from kidney (5 µg of protein) and liver (150 µg of
protein) were subjected to nonreducing SDS-polyacrylamide gel
electrophoresis (7.5%), separated proteins transferred to
nitrocellulose membranes (90-min transfer time) and probed with
1000-fold diluted antiserum to TC II-R and
The binding of
Figure 1:
The photograph illustrates the relative
sizes of an affected (A) and a normal (N) rabbit
maintained for 6 weeks. The rabbits were weighed, anesthetized, and
photographed. The initial weight of rabbits were 2.8 kg (normal) and
3.0 kg (affected). After 6 weeks, the affected rabbit weighed 1.65 kg
and the normal rabbit 3.6 kg.
Figure 2:
Light micrographs of liver (A)
When the tissue membranes from affected rabbits were
treated with pH 5/EDTA buffer, the activity in all the three tissue
membranes rose only by a very modest amount of <5-10%,
indicating that the loss of binding sites was not due to occupancy by
the endogenous ligand (data not shown). The binding of TC II-Cbl to TC
II-R requires Ca
Figure 3:
In vitro inhibition of TC
II-[57Co]Cbl binding to pure TC II-R. Indicated amounts of
antiserum to TC II-R (
Figure 4:
Immunoblots of normal (N) and
affected (A) rabbit kidney and liver membranes. Indicated
tissue membranes from normal and affected rabbits were separated on
nonreducing SDS-polyacrylamide gel electrophoresis (7.5%) and subjected
to immunoblotting. Other details are provided under ``Materials
and Methods.''
In contrast to this observation,
when intrinsic factor-cobalamin receptor (25, 26) is
injected into rabbits, the rabbits do not become Cbl-deficient,
although they produced antibodies to IFCR, and this antiserum
inhibited, in vitro, the binding of
IF-[
Figure 5:
Binding of
Figure 6:
In vitro (panels A and C) and in vivo (panels B and D)
effects of TC II-R antiserum on TC II-R (panels A and B) and IFCR (panels C and D) activities in
the intestinal apical and basolateral membranes. Panel A,
isolated basolateral membranes (250 µg) and apical membranes (250
µg) from normal rabbits were incubated with (columns b and d) or without (columns a and c) TC II-R
antiserum (20 µl). Following 1-h incubation with TC II-R antiserum
at 5 °C, the membranes were pelleted down and washed with
phosphate-buffered saline. The pellet was then solubilized with Triton
X-100 (1%), and the detergent extract was used for TC II-R assay. Panel B, isolated basolateral membranes from normal (column f) and affected (column e) rabbits and apical
membranes from normal (column h) and affected (column
g) rabbits were assayed for TC II-[
Figure 7:
Panel A, inhibition of surface binding of
TC II-[
In conclusion, our
studies have shown that intracellular deficiency of Cbl can be induced
by preventing tissue uptake of TC II-Cbl by antiserum to TC II-R. This
experimental approach can be used for creating intracellular Cbl
deficiency in cells in culture to study the role of Cbl on cellular
proliferation and differentiation or in animals to study the
pathophysiology of hematological and/or neurological complications
known to occur in Cbl deficiency.
Volume 271,
Number 8,
Issue of February 23, 1996 pp. 4195-4200
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Co]cyanocobalamin bound very poorly
(2-5%) to the affected rabbit liver, kidney, and intestinal total
or intestinal basolateral membrane extracts relative to the binding by
membrane extracts from normal rabbit tissues. The activity was restored
to normal values following a wash of affected rabbit tissue membranes
with pH 3 buffer containing 200 mM potassium thiocyanate.
Immunoblot analysis of normal and affected rabbit kidney and liver
total membranes revealed similar amounts of 124-kDa TC II-R dimer
protein. The neutralized and dialyzed extract from the affected rabbit
membranes inhibited the binding of the ligand to pure TC II-R and the
harvested affected rabbit serum inhibited the uptake of TC
II-[
Co]cobalamin (Cbl) from the basolateral side
of human intestinal epithelial (Caco-2) cells and decreased the
utilization of [
Co]Cbl as coenzymes by the
Cbl-dependent enzymes. The loss of exogenously added ligand binding or
the binding of
I-protein A occurred with the intestinal
basolateral, but not the apical membranes. Based on these results, we
suggest that circulatory antibodies to TC II-R cause its in vivo functional inactivation, suppress Cbl uptake by multiple tissues,
and thus cause severe Cbl deficiency and the noted failure to thrive.
) (
)bound to plasma transporter,
transcobalamin II (TC II) occurs by receptor mediated endocytosis (1) via TC II-receptor (2) (TC II-R), which is
expressed as a noncovalent dimer of molecular mass of 124 kDa in all
tissue plasma membranes(3) . Disruption in the cellular uptake
of TC II-Cbl will ultimately result in intracellular Cbl deficiency and
decreased synthesis of coenzyme forms of Cbl, methyl-Cbl, and
adenosyl-Cbl(4) . This in turn will affect the enzymatic
conversion of homocysteine to methionine by methionine synthase and
methylmalonyl-CoA to succinyl-CoA by methylmalonyl-CoA mutase,
respectively. Thus, intracellular Cbl deficiency cause increased plasma
levels of homocysteine (HC) and methylmalonic acid (MMA), and their
measurements in plasma are indicative of intracellular functional
deficiency of Cbl (5, 6) .
Co]Cbl (15 µCi/ml, Amersham Corp.),
I-protein A (>30 µCi/µg, ICN Radiochemicals,
Irvine, CA), cellulose nitrate membranes (Schleicher and Schull).
Transcobalamin II used in TC II-R activity measurements was partially
purified from human plasma according to Lindemans et al.(11) . Intrinsic factor used in intrinsic factor-cobalamin
receptor assays was purified from rat gastric mucosa by affinity
chromatography of gastric mucosal extracts on Cbl-Sepharose column as
described earlier(12) . Antiserum to rabbit transcobalamin II
raised in goat was a gift from Dr. Robert H. Allen (University of
Colorado Health Science Center, Denver, CO).
Injections of TC II-R
New Zealand White rabbits weighing
3 kg were injected subcutaneously at multiple sites with pure human
placental TC II-R (3) (30 µg/rabbit) emulsified in
Freund's complete adjuvant. Ten days later, the rabbits received
a booster dose of TC II-R (30 µg/rabbit) emulsified in incomplete
adjuvant. The rabbits were bled through the ear vein, seven days later.
The rabbits were bled once more the following week. Twenty-four days
following the initial injections, the rabbits were pale and looked
listless. In the next 3-4 weeks, rabbits injected with TC II-R
started wasting and became progressively sick and were sacrificed at
the end of 6 weeks. Kidney, liver, and intestine were harvested and
stored at -70 °C.Serum Analysis
The serum obtained from the rabbits
was tested for the presence of antibodies to TC II-R by immunoblotting
human placental total membranes and for its ability to inhibit, in
vitro, the binding of TC II-[Co]Cbl to pure
placental TC II-R(3) . The serum from the affected and normal
rabbits was subjected to the following analysis: total
[
Co]Cbl binding ability was assessed by the
charcoal adsorption method(13) . The radioactive Cbl bound to
serum of normal and affected rabbits was immunoprecipitated with
antiserum to rabbit TC II to assess the serum TC II levels as described
previously(14) . Normal and affected animal serum MMA and HC
levels were determined by a single gas chromatography/mass spectrometry
run analysis(15) .
Cbl Receptor Activity and Protein Determination
TC
II-R activity in the Triton X-100 extracts of normal and affected
rabbit tissue total membranes or isolated intestinal apical and
basolateral membranes was determined as described earlier(3) .
In some experiments, prior to detergent extraction and ligand binding
assay, the total membranes were treated as follows. Total membrane
prepared was suspended and homogenized in 2 ml of (a) 10
mM Tris-HCl buffer, pH 7.5 or (b) 10 mM Tris-HCl adjusted to pH 5 and containing 5 mM EDTA or (c) 0.1 M glycine HCl buffer, pH 3, containing 200
mM KSCN and incubated for 1 h at room temperature. The
membranes were pelleted down by centrifugation, washed once with the
same respective buffers, re-pelleted down, and finally suspended and
homogenized in 10 mM Tris-HCl buffer, pH 7.5, and extracted
with Triton X-100 for 6 h. The intrinsic factor-cobalamin receptor
activities in the total or in the isolated apical and basolateral
membranes were determined using rat IF-[Co]Cbl
(2.5 pmol) as described earlier(16) . Protein concentration in
membrane and membrane extracts was determined by the method of
Bradford(17) .
Co]Cbl to pure human TC II-R was carried out
as described earlier(3) . The extracts were neutralized to pH
7.4 and dialyzed against 4 liters of 10 mM Tris-HCl buffer, pH
7.4, for 24 h, with one 2-liter exchange of the dialysis buffer at the
end of 12 h, prior to use.
I-protein A.
The bands were visualized by autoradiography and quantitated by the
AMBIS radioimaging system.
Isolation of Apical and Basolateral Membranes from Normal
and Affected Rabbit Intestine
The intestine from normal and
affected rabbits was harvested and flushed with 100 ml of ice-cold
saline, and the mucosa was scraped from the entire gut. The apical
membranes were isolated from rabbit intestine by the Ca aggregation method of Malathi et al.(18) as
described earlier(19) . The apical brush-border membrane was
enriched for the following apical markers, IFCR (10-fold, 15%
recovery),
-glutamyl transferase (12-fold, 10% recovery) and
contained <0.5-1% of NADPH-cytochrome c reductase and
-glucuronidase. The basolateral membranes from rabbit intestine
and kidney were isolated by the sucrose gradient centrifugation method
of Molitoris and Simon(20) . TC II-R activity was enriched
about 7-fold and Na/K
-ATPase was
enriched 10-fold with 15-20% yield, and the activity recovery of
apical markers, maltase and alkaline phosphatase, was <1-2%.
I-protein A was carried out as follows.
The isolated intestinal basolateral and apical membranes (250 µg of
protein) from affected and normal rabbits were incubated with
50-2000 pg of
I-Protein A (116 µCi/µg) in a
volume of 500 µl containing 10 mM Tris-HCl, pH 7.4,
containing 140 mM NaCl and 0.1 mM phenylmethylsulfonyl fluoride for 1 h at 22 °C. The reaction
mixture was microcentrifuged, and the resulting pellet was washed twice
with 2 ml of incubation buffer containing 1 mg of bovine serum
albumin/ml, and the resulting pellet was counted in a
counter.
TC II-[
Post-confluent
Caco-2 cells were grown on culture inserts as described
before(21) . Transepithelial resistance was measured according
to Fuller et al.(22) , and filter-grown cells that
demonstrated electrical resistance of 250-300 ohms/cmCo]Cbl Transport and
Cbl Utilization by Filter-grown Caco-2 Cells
were used. The ligand, human TC II-[Co]Cbl
(2 pmol), was presented on the basolateral side, and surface binding
was determined at 5 °C. Specific binding to TC II-R was determined
by subtracting the amount of ligand bound at the same temperature to
the basolateral side of cells that were incubated with TC II-R
antiserum (25 µl in 2 ml of basolateral medium) for 30 min, prior
to the addition of the ligand. Uptake and transport of Cbl was carried
out at 37 °C for 6 h by allowing the bound ligand to internalize.
After 6 h, the cells were scraped, washed with phosphate-buffered
saline, and extracted in the same buffer containing Triton X-100
(0.5%). The extract (1 ml) was subjected to size exclusion
chromatography on Sephadex G-150 to separate Cbl incorporated into
Cbl-dependent enzymes and that which was still bound to TC II according
to Rosenblatt et al.(23) .
Light Microscopy of Tissues from Normal and Affected
Rabbits
Tissues were fixed in neutral buffered formalin and cut
6 µm thick and stained with hematoxylin and eosin prior to light
microscopy.
Development of Cbl Deficiency in Rabbits Injected with
Transcobalamin II-Receptor
Following 3-4 weeks of
subcutaneous injection of human placental TC II-R, the affected rabbits
were listless, failed to thrive, and were wasting despite doubling of
the diet dose. By 6 weeks, the animals were sick and had lost nearly
50% of their initial body weight of 3 kg. The smaller size due to
weight loss of an affected rabbit and a normal rabbit maintained on the
same diet for 6 weeks is shown in Fig. 1. Despite the loss of
body weight, the affected rabbit tissues such as liver, intestine, and
the kidney showed normal morphology (Fig. 2) under light
microscopy. Liver sections (Fig. 2, panel A), including
portal triad, showed no consistent differences in the hepatic histology
between the affected and normal animals. Intestine of both normal and
affected animals showed normal morphology with long villi lined by tall
columnar cells with predominant brush borders (Fig. 2, panel
B). Renal cortex of both normal and affected rabbits showed normal
morphology with no differences in the glomeruli, tubule, and
interstitial tissue (Fig. 2, panel C). Since the
animals were extremely pale, they were suspected to be anemic and thus
they were tested for Cbl deficiency by measuring plasma levels of HC
and MMA. Compared with normal rabbit serum, MMA, HC, and unsaturated
Cbl binding due to TC II were elevated 2-fold in the affected rabbits (Table 1). The increase of both HC and MMA by 2-fold clearly
indicated that the rabbits had developed intracellular deficiency of
Cbl, and the increase in plasma TC II levels further indicated that the
development of Cbl deficiency could be due to decreased uptake of
plasma TC II-Cbl. These initial observations suggested that the noted
failure to thrive of these animals was not due to antibody induced
organ or tissue damage but instead could be due to the noted Cbl
deficiency. Recent studies have shown that in vitro, antiserum
to TC II-R inhibits the binding of TC II-[Co]Cbl
to pure TC II-R(3) , suggesting the possibility that
circulating TC II-R antiserum may also be functional, in vivo,
in blocking the binding of TC II-Cbl and hence its tissue uptake.
50, intestine (B) 100 and kidney (C)
50 sections (6 µm) stained with hematoxylin and
eosin.
Reversible Loss of TC II-R Activity in Affected Rabbit
Tissues
TC II-R activity was determined in the Triton X-100
extracts of the tissue total membranes of the normal and affected
rabbits (Table 2). Relative to normal rabbit tissue TC II-R
activity, there was dramatic decline (97-98%) of TC II-R activity
in affected rabbit tissues, such as kidney, intestine, and liver. In
contrast, the intrinsic factor-cobalamin receptor activity in either
intact membranes or the Triton X-100 extracts of both the normal and
affected kidney and intestine were the same. These results indicated
that the loss of exogenously added TC II-[Co]Cbl
binding activity is due specifically to loss of functional TC II-R and
may be due to occupancy of ligand binding sites by either the
endogenous ligand TC II-Cbl, known to be in excess in circulation (Table 1) or the circulating TC II-R antiserum, which is known to
inhibit, in vitro, the binding of exogenously added ligand (3) or to the loss of receptor protein from the cell surface.
In order to test these possibilities, the following experiments were
carried out.
and neutral pH and pH 5/EDTA
treatment dissociates the bound ligand from the receptor. However, when
the membranes were treated with glycine HCl buffer, pH 3, containing
200 mM KSCN, there was a dramatic increase in the binding of
TC II-[
Co]Cbl and 100% of the binding was
recovered in all the tissues tested (Table 2). These results
indicated that the recovery of ligand binding was not due to the
release of endogenous TC II-Cbl, but due to the release from the
membrane surface, the antibodies to TC II-R. Acidic pH buffers
containing chaotropic salt such as KSCN are known to dissociate the
immune complexes. However, in order to prove directly that the recovery
of receptor activity was actually due to the removal of TC II-R
antibody, the neutralized and dialyzed membrane eluant was titrated for
its ability to inhibit, in vitro, the binding of TC
II-[
Co]Cbl to pure human TC II-R (Fig. 3). The neutralized and dialyzed pH 3/KSCN extract was
able to inhibit ligand binding, and 50% inhibition was noted with 35
µl of the eluant compared with similar amount of inhibition of
ligand binding by only 2.5 µl of directly harvested TC II-R
antiserum from rabbit blood. This difference was due to the dilution of
the antibody in the membrane extract. In contrast, there was no
inhibition of binding with pH 5/EDTA membrane extract. These results
clearly indicate that the loss of TC II-R activity in the affected
rabbits was due to occupancy of the receptor ligand binding sites by TC
II-R antibody. Furthermore, immunoblot analysis (Fig. 4) of
liver and renal total membranes from normal and affected rabbits
revealed similar amounts of 124-kDa TC II-R, demonstrating that in
affected rabbit membranes, TC II-R was present, but was inactive in
ligand binding. It is interesting to note that the size of TC II-R
revealed in rabbit membranes is 124 kDa, the exact size of TC II-R
dimer revealed using the same antiserum against human (3) and
rat tissue (24) membranes. The recognition of a single protein
band of 124 kDa in tissue membranes across species demonstrated that
the antiserum contained antibody to a single membrane antigen and that
the observed effects of Cbl deficiency are due to functional loss of a
single membrane component, TC II-R.
), or the neutralized and dialyzed
extracts, 0.1 M glycine HCl buffer pH 3.0/KSCN () or the
pH 5/EDTA (
) treated were first incubated with diluted pure
receptor for 30 min at room temperature and then assayed for ligand
binding. The values reported represent an average of triplicate assays
performed at each concentration of the extracts or
antiserum.
Co]Cbl to IFCR(25, 26) .
However, the lack of effect of circulatory antibodies to IFCR in
causing Cbl deficiency may be due to the fact that the receptor which
is functionally active in the intestinal luminal or apical membranes
will not be in direct contact with the circulating antibodies. If this
hypothesis is true then the antiserum binding to TC II-R and its
subsequent functional inactivation in the affected rabbit intestine
should be a property of TC II-R expressed in the basolateral membranes
where it will be exposed to the circulation.
In Vivo Inactivation of the Basolaterally Expressed TC
II-R and in Vitro Inhibition of TC
II-[
Endogenous presence of TC II-R antibody bound to the
basolateral and apical surface membranes of the intestinal mucosa was
measured by determining the amount of Co]Cbl Uptake and
[
Co]Cbl Utilization from the
Basolateral Domain of Polarized Human Intestinal Epithelial
Cells
I-protein A binding
to these membranes (Fig. 5). The results show that saturable
binding of protein A occurred with the isolated mucosal basolateral but
not the apical membranes of the affected rabbit. No binding of
I-protein A occurred with either apical or basolateral
membranes of normal rabbit mucosa (data not shown). In addition, TC
II-R activity in the isolated apical and the basolateral membranes from
the normal and affected rabbit intestinal mucosa was also determined.
The results show (Fig. 6, panel A) that TC II-R
antiserum added in vitro inhibited the TC
II-[
Co]Cbl binding to both the apical (column d) and basolateral membranes (column b)
isolated from normal rabbit intestinal mucosa. The activity was
inhibited by >75-80% compared with the untreated apical (column c) and basolateral membranes (column a). On
the other hand, when the TC II-[
Co]Cbl binding
to the apical and basolateral membranes of affected rabbit intestine
was measured (Fig. 6, panel B), the ligand binding to
the basolateral (column e) but not the apical membrane (column g) was affected. The loss of ligand binding to the
basolateral membrane was completely restored following treatment of
basolateral (column f) membranes with pH 3/KSCN buffer.
Similar washing of the apical membranes from the affected rabbit had no
effect on the receptor activity (column h). The specificity of
both the in vitro and in vivo antibody effect on the
TC II-R activity was borne out by the observation that the antiserum in vitro (Fig. 6, panel C) or in vivo (Fig. 6, panel D) had no effect on the binding of
ligand, IF-[
Co]Cbl, to the apical or the
basolateral membranes. It is interesting to note that similar results
were noted with the isolated apical and basolateral membranes from the
normal and affected rabbit kidney (data not shown). In support of these
observations, direct evidence that antiserum to TC II-R by blocking
ligand binding sites also suppressed TC II-mediated Cbl transport was
obtained using polarized Caco-2 cells (Fig. 7). In these cells,
like in the intact intestinal mucosa (Fig. 6) and in the kidney
cortex(24) , TC II-R is expressed 8-fold higher in the
basolateral membranes (Fig. 7), and >90% of the surface
binding is due to TC II-R (panel A). TC II-R antiserum
inhibited not only the surface binding of TC
II-[
Co]Cbl, but also intracellular Cbl levels
and the utilization of Cbl by the Cbl-dependent enzymes (Fig. 7, panel B). Previously we have shown (23) that following
uptake of TC II-Cbl, Cbl liberated from within the lysosomes is
incorporated into intracellular Cbl-dependent enzymes, methionine
synthase and MMA CoA mutase, and that these enzymes eluted near void
volume on size exclusion chromatography earlier (V
/V
, 1.1) well separated
from Cbl still bound to TC II (V/V, 2.0). These results
have convincingly shown that cells exposed to TC II-R antiserum fail to
transport Cbl and thus become Cbl-deficient.
I-protein A to
the apical and basolateral membranes of the affected rabbits. Isolated
intestinal basolateral (
) and apical () membranes (250
µg of protein) from affected rabbits were incubated with different
concentrations of
I-protein A (50-2000 pg) for 1 h
at 22 °C. Other details are provided under ``Materials and
Methods.''
Co]Cbl
binding. The membranes were extracted with Triton X-100 (1%), and the
extract was assayed for TC II-R activity. Panel C, isolated
basolateral membranes (250 µg) and apical membranes (250 µg)
from normal rabbits were incubated with (columns a and c) or without (columns b and d) TC II-R
antiserum (20 µl). The TC II-R antiserum treated and untreated
membranes were then assayed for IF-[
Co]Cbl
binding. Panel D, basolateral membranes from normal (column f) and affected (column e) and apical
membranes from normal (column h) and affected (column
g) rabbits were assayed for IFCR activity. The values reported are
mean ± S.D. of duplicate assays performed using five separate
isolated apical and basolateral membrane preparations from two normal
and affected rabbits.
Co]Cbl and transport of
[
Co]Cbl in filter-grown Caco-2 cells treated on
the basolateral side with antiserum to human TC II-R. Panel B,
chromatography of cellular extract on Sephadex G-150 (1
60 cm). Solid and broken lines indicate distribution of
[Co]Cbl in untreated (solid lines) and
in antiserum-treated (broken lines)
cells.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
