(Received for publication, July 11, 1994; and in revised form, January 27, 1995)
From the
Antiserum raised to purified 62-kDa human placental
transcobalamin II receptor (TC II-R) has been used to study its
synthesis and membrane expression. The antiserum immunoprecipitated a
45-kDa protein from the cell-free translation using human kidney mRNA
and recognized a single 124-kDa band on immunoblotting of placental and
other human tissue membranes, and quantitation of the blots revealed
high levels of TC II-R expression in the human kidney followed by
placenta, intestine, and liver. Triton X-100 extraction of placental
membranes resulted in the complete (100%) solubilization of the
receptor, and immunoblotting of the Triton X-100-soluble fraction
revealed a single band of 62 kDa. Lipid extraction of placental
membranes with a mixture of chloroform:methanol (2:1) followed by
immunoblotting revealed a single band of molecular mass 62 kDa. The
molecular mass of the pure Triton X-100-bound receptor increased on
SDS-polyacrylamide gel electrophoresis from 62 to 124 kDa upon its
insertion in liposomes prepared using egg phosphatidylcholine and
cholesterol. Chemical cross-linking of native membrane- or lipid
vesicle-bound TC II-R or detergent-soluble extracts of the membrane
with I-TC II-cobalamin revealed that both the 124- and
62-kDa forms of the receptor were active in ligand binding. Based on
these results we suggest that TC II-R is synthesized as a single
polypeptide of 45 kDa, and following its maturation (involving N- and O-glycosylation) the 62-kDa mature receptor is
expressed in plasma membranes as a noncovalent dimer of 124 kDa. The
dimerization of TC II-R in the plasma membranes is due to its
interactions with annular lipids.
The cellular delivery of cobalamin (Cbl(): vitamin
B
) is mediated by transcobalamin II (TC II), a 45 kDa
non-glycoprotein plasma transporter of Cbl(1) . Cobalamin
complexed to TC II is endocytosed by a specific plasma membrane
receptor (TC II-R)(2) . The high affinity saturable binding of
TC II
Cbl complex to plasma membranes of liver (3) ,
kidney(4, 5) , and placenta (6, 7) has been demonstrated, suggesting that the TC
II-R is expressed in many tissues. A highly pure TC II-R from human
placenta has been obtained and reported to be a 50-55-kDa
glycoprotein containing about 33% carbohydrate (8) based on
sucrose density centrifugation and amino sugar analysis. There are
reports (9, 10, 11) based on animal studies
that kidney avidly takes up more TC II
Cbl than other tissues,
including liver. These studies have suggested the possibility that the
renal uptake of Cbl bound to TC II occurs either directly from the
circulation or indirectly via passage through liver and thus may be the
most important organ to take up Cbl bound to TC II. It has been
suggested that the kidney in many species acts as the storage organ of
Cbl(5, 12) . Cobalamin stored in the kidney is thought
to be delivered back into the circulation in times oftissue need and
excreted in urine once the renal threshold of storage is
reached(13, 14) . The bi-directional movement of Cbl
in the kidney is thought to be mediated by TC II and in support of this
hypothesis are our recent findings demonstrating the secretion of TC II
in both apical and basolateral directions in renal epithelial cells (15) . Despite the importance of TC II-R in maintaining Cbl
flux across plasma membranes by mediating the uptake and tissue
exchange of Cbl, the details of its synthesis and membrane expression
TC II-R in human is not known. The current work was undertaken to
address these issues.
The results of the current study show that human TC II-R is synthesized as a single polypeptide of molecular mass 45 kDa, glycosylated to contain both N- and O-linked sugars and the resulting mature receptor of molecular mass 62 kDa is expressed in the plasma membranes as a functional noncovalent dimer. Furthermore our results also show that the dimerization of TC II-R is due to its interactions in the membrane with annular lipids.
Human placenta was obtained from unidentified donors who had
normal vaginal delivery at the Doyne Hospital, Milwaukee, WI. The
placenta was washed with normal saline, cut into small pieces, and
stored at -70 °C until use. Pieces of human liver, kidney,
and intestine were obtained from the autopsy specimens of unidentified
patients. The tissue pieces were stored at -70 °C until use.
For the preparation of total RNA and mRNA, a surgical piece of human
kidney frozen at -70 °C was obtained as a gift from the
Nephrology Unit of Froedtert Memorial Lutheran Hospital, Milwaukee, WI.
The following reagents and chemicals were purchased commercially as
indicated: [Co]cyanocobalamin (specific
activity, 15 µCi/µg) and carrier free Na
I
(Amersham Corp.); rabbit serum (Life Technologies, Inc.);
Trans
S-label (>1000 Ci/mmol, containing 70% methionine,
15% cysteine, 7% methionine sulfone, 3% cysteic acid, and the rest
other sulfur compounds) (DuPont NEN); protein A, lactoperoxidase,
neuraminidase from Clostridium perifringens, and cholesterol
(Sigma); endo-
-N-acetylglucosaminidase H from Streptomyces plicatus, peptide:N-glycosidase from Flavobacterium meningosepticum, and O-glycosidase
from Diplococcus pneumoniae (Boehringer Mannheim);
disuccinimidyl suberate and iodogen (Pierce); and egg
phosphatidylcholine (Avanti Polar Lipids). Pure human TC II was a gift
from the late Dr. Charles A. Hall (Nutrition Assessment Laboratories,
VA Hospital, Albany, NY). Antiserum to rat renal intrinsic factor
receptor (16) and human TC II (17) was prepared as
described earlier.
Figure 1: SDS-PAGE of purified human placental transcobalamin II receptor. Purified receptor (5 µg) was subjected to SDS-PAGE (7.5%) and visualized with silver nitrate stain.
Figure 2: Cell-free translation and immunoprecipitation of TC II-R. Human mRNA from kidney was translated in the reticulocyte translation system, and the translated proteins were extracted with Triton X-100 and immunoprecipitated in the presence (lane 2) and absence (lane 3) of excess cold endogenous TC II-R. Other details of translation, extraction, and immunoprecipitation are provided under ``Materials and Methods.''
Confirmation that the antiserum raised in rabbits
to the 62-kDa band is indeed specific for TC II-R was obtained when
incubation of the receptor with graded amounts of antiserum was able to
inhibit the binding of TC II[
Co]Cbl to the
pure receptor (Fig. 3). About 100% inhibition was observed (Fig. 3) with 20 µl of undiluted antiserum. Using the same
volume, antiserum to the ligand, human TC II, or rat intrinsic
factor-cobalamin receptor did not demonstrate any inhibition. These
results show that TC II-R antibody, like the antibody raised against
intrinsic factor-cobalamin receptor(27) , is a blocking
antibody and is specific for TC II-R. In addition, lack of inhibition
by antiserum to the ligand, TC II, and intrinsic factor receptor
further suggest that TC II-R antiserum is specific.
Figure 3:
Immunoblocking of the binding of TC II
[
Co]Cbl to purified TC II-R. Pure TC II-R
(0.5 µg) was preincubated with the indicated amounts of antiserum
to human TC II-R (
), TC II (
), and rat intrinsic factor-Cbl
receptor (
) for 1 h at 22 °C. TC
II
[
Co]Cbl (1.5 pmol) was then added and
assayed for ligand binding. The activity is expressed as the percentage
of TC II-R activity in a sample incubated without
serum.
In order to further test the specificity of the antiserum, immunoblotting studies were carried out with human placental membranes (Fig. 4). Although a single band was revealed in these membranes, surprisingly the molecular mass of this band was 124 kDa under nonreducing (Fig. 4, lane 1) and 144 kDa (Fig. 4, lane 2) under reducing conditions, respectively, and not 62 kDa. The specificity of this reaction was confirmed when the diluted antiserum treated with human kidney membranes (see later) failed to recognize the same bands (lanes 3 and 4). These results suggested that the antiserum to TC II-R is specific for TC II-R and that the receptor exists possibly as a noncovalent dimer in the placental membranes. In addition, the apparent increase in molecular mass of membrane bound mature TC II-R by 20 kDa upon reduction suggested that the receptor does not contain disulfide-linked polypeptides but contains intramolecular disulfide bonds which upon reduction may alter the compactness of the membrane receptor thus altering it's mobility. Similarly the apparent molecular mass of the pure TC II-R increased from 62 to 72 kDa upon reductive alkylation (data not shown). This result is consistent with the idea that pure TC II-R does not contain disulfide linked subunits and that the intramolecular disulfide bonds of the receptor remain intact during it's transport to and following insertion in the plasma membrane. Similar observations of decreased mobility on SDS-PAGE following reduction of the cation-independent mannose 6-phosphate receptor(28) , and other proteins (29) have been noted and has been attributed to the presence of intramolecular disulfide bond clusters.
Figure 4: Western blotting of human placental membranes. Placental membranes (10 µg of protein) were incubated with (lanes 2 and 4) and without (lanes 1 and 3) 2-mercaptoethanol (2.5%) for 1 h at 22 °C and then subjected to SDS-PAGE (7.5%). The separated proteins were transferred to nylon membrane and probed with unabsorbed (lanes 1 and 2) or absorbed (lanes 3 and 4) anti-serum. The diluted antiserum was absorbed with total human kidney membranes.
Since the immunoblotting of placental membranes revealed a single band of 124 kDa, it is likely that TC II-R exists as a homodimer in these membranes. In addition, the dimerization of TC II-R appears to be noncovalent and is a property of the membrane bound receptor. In order to verify whether the TC II-R exists as a homodimer in other tissue membranes and to evaluate the relative distribution of TC II-R in other human tissue membranes, immunoblotting studies using total membranes from human placenta, intestine, kidney, and liver (Fig. 5) were carried out. A single band of 124 kDa was revealed in all the tissue membranes. Although the immune cross-reactive band was easily visible using 10 µg of membrane protein from placenta, intestine, and kidney, 50 µg of liver membrane protein was needed to visualize the band. Quantitation of these bands revealed that the relative distribution of TC II-R was highest in the kidney (100%) followed by placenta (28%), intestine (18%), and liver (2%). Although on a protein basis, TC II-R levels in the liver were lower relative to kidney or placenta or intestine, significant amounts of TC II-R will also be expressed in the liver, considering its larger mass. The high levels of TC II-R in these human tissues is consistent with the flux of Cbl bound to TC II that cross plasma membranes of these tissues in order to maintain the special functions or characteristics of these organs such as Cbl storage (liver), plasma Cbl uptake, storage, and either excretion or tissue exchange (kidney), maternal-fetal transfer (placenta), and rapid proliferation and differentiation of absorptive enterocytes (intestine).
Figure 5:
Immunoblotting of human tissue total
membranes. Total membranes prepared from the indicated tissues were
obtained by centrifuging a 10% homogenate (prepared in 10 mM Tris-HCl buffer, pH 7.4, containing 140 mM NaCl and 0.1
mM PMSF) at 100,000 g for 1 h. Indicated
amounts of membrane protein were subjected to SDS-PAGE (7.5%), and the
separated proteins were transferred to a nylon membrane (transfer time
90 min), probed with TC II-R antiserum and
I-protein A,
and subjected to autoradiography.
Figure 6:
Immunoblotting of placental membranes
treated with Triton X-100 or chloroform:methanol mixture. Placental
membrane protein (10 µg, lanes 1 and 7), Triton
X-100-soluble fraction (10 µg of protein, lanes 2 and 8), Triton X-100-insoluble pellet fraction (protein: 75, 150,
and 300 µg, respectively, in lanes 4 and 10, 5 and 11, and 6 and 12) and chloroform:methanol
delipidated membranes (10 µg of protein, lanes 3 and 9) were subjected SDS-PAGE (7.5%). The separated proteins were
blotted to nitrocellulose membranes for 90 (A) or 45 (B) min and probed with TC II-R antiserum and I-protein A. Other details of membrane treatment are
provided under ``Materials and
Methods.''
Direct
evidence of lipid involvement in the dimerization of TC II-R was
obtained when the receptor was inserted in lipid vesicles. The receptor
associated with the lipid vesicles revealed on SDS-PAGE a higher
molecular mass of around 124 kDa, twice the size of the Triton X-100
micellar bound receptor (Fig. 7A). Upon immunoblotting,
TC II-R associated with the lipid vesicles (Fig. 7B, lane
3) had a molecular mass of 124 kDa, similar to TC II-R present in
the native renal (lane 1) and placental (lane 2)
membranes. These results have confirmed the observation that
delipidation and relipidation result in the formation of monomer or
dimer, respectively, and that the dimerization of TC II-R in native
membranes is due to association with plasma membrane lipids. The TC
II-R expressed in the plasma membranes of several rat tissues and in
cultured cells is also a dimer with a molecular mass of 124 kDa,
suggesting that dimerization of TC II-R occurs in all the
tissue/cellular plasma membranes of other species.
Figure 7:
SDS-PAGE and immunoblot analysis of
purified TC II-R associated with egg PC and cholesterol vesicles. Egg
PC/cholesterol liposomes (molar ratio 1:0.25) were prepared by the
cholate dialysis method. During dialysis, TC II-R (10 µg) was also
present. After dialysis the receptor-lipid fraction was centrifuged for
2 h at 100,000 g. The lipid pellet was reconstituted
in Tris-buffered saline and subjected for SDS-PAGE and either stained
for protein with silver nitrate (A) or used for immunoblotting (B). A: lane 1, pure receptor (5 µg) and lane 2, liposomal bound TC II-R (3 µg). B, total
membrane protein from human kidney (5 µg), human placenta (10
µg) and liposomal bound TC II-R (5 µg) were used for SDS-PAGE
and immunoblotting.
The
functional significance of expression of the dimeric form of TC II-R in
plasma membranes is not known. Cross-linking of I-TC II
Cbl (Fig. 8) to the native membrane (Fig. 8A,
lane 1) or its detergent extract (Fig. 8A, lane 4)
or lipid vesicle-bound TC II-R (Fig. 8B, lane 5) or the
pure TC II-R (Fig. 8B, lane 8) show that both the
dimeric and monomeric form of the receptor are functional in ligand
binding. The specificity of ligand binding is borne out by the
observation that prior incubation with 100-fold molar excess of cold TC
II
Cbl abolished the binding of
I-TC II
Cbl to
TC II-R fractions (lanes 2, 3, 6, and 7). Based on
the size of the cross-linked products, the membrane-bound dimer binds 2
mol of the ligand, and it is thus possible that the expression of the
dimeric form of TC II-R may help in facilitating rapid clearance of
circulatory TC II
Cbl known to occur in
vivo(30) .
Figure 8:
Cross-linking of membrane-bound and
membrane free TC II-R. A, placental membranes (lanes 1 and 2) or Triton X-100 extract (lanes 3 and 4) were cross-linked with I-TC II in the absence (lanes 1 and 4) and presence (lanes 2 and 3) of 100-fold molar excess of TC II
Cbl. B, TC
II-R bound to egg PC-cholesterol liposomes (lanes 5 and 6) or pure TC II-R (lanes 7 and 8) was
cross-linked with
I-TC II
Cbl in the absence (lanes 5 and 8) and presence (lanes 6 and 7) of 100-fold molar excess of TC II
Cbl. The
cross-linked products were analyzed of SDS-PAGE (5%) and visualized by
autoradiography. Other details are provided under ``Materials and
Methods.''
Earlier studies have shown that TC II-R is a very hydrophobic protein capable of binding 1.25 times its own weight of Triton X-100(8) . Our studies suggest further that the dimerization is the result of strong lipid-protein interaction within the lipid microenvironment of the plasma membrane. It is interesting to note that the molecular mass of TC II-R from placental microsomes is 62 kDa (data not shown). This observation suggests that the receptor is either a monomer in the ER, or the interaction between the two monomers in the ER membrane is not of sufficient strength that it can be dissociated by treatment with SDS. If the receptor is indeed a true monomer in the ER membrane, the ensuing folding alterations that occur during the processing (following its exit from ER) might expose the hydrophobic surfaces of the receptor to favor a much stronger monomer-monomer interaction in the plasma membrane. On the other hand, the qualitative and quantitative differences in the lipids of the ER and plasma membrane may also be responsible for the presence of monomer or dimer in these membranes, respectively. Further studies are needed to elucidate the structural elements of TC II-R important in the formation of noncovalent dimers in native plasma membranes and the specific lipids that help mediate this event.
In conclusion, the current study has examined some of the molecular properties of TC II-R and its expression in human tissue membranes. These studies have provided insights into the role of membrane lipids in the formation of noncovalent homodimers of TC II-R in human tissue plasma membranes. With the availability of nanomole amounts of the receptor and mono-specific antiserum, further studies on its structure and regulation of expression are now possible.