(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-[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.
The plasma transport of absorbed dietary and biliary cobalamin
(Cbl: vitamin B) (
)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) .
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:
[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).
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-[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.
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 I-protein A.
The bands were visualized by autoradiography and quantitated by the
AMBIS radioimaging system.
The binding of 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.
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)
50, intestine (B)
100 and kidney (C)
50 sections (6 µm) stained with hematoxylin and
eosin.
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 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.
Figure 3:
In vitro inhibition of TC
II-[57Co]Cbl binding to pure TC II-R. Indicated amounts of
antiserum to 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.
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-[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.
Figure 5:
Binding of 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.''
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-[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.
Figure 7:
Panel A, inhibition of surface binding of
TC II-[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.
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.