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3
*
Division of Gastroenterology and Hepatology, Department of Medicine and
Biochemistry, Medical College of Wisconsin and Veterans Administration Medical Center, Milwaukee, WI 53226
3To whom correspondence and reprint requests should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONCobalamin Binding Protein...Cobalamin Receptors....Summary and Perspectives.REFERENCES |
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KEY WORDS: • cobalamin • import • intrinsic factor • transcobalamin II • receptor • epithelial cell
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONCobalamin Binding Protein...Cobalamin Receptors....Summary and Perspectives.REFERENCES |
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and 1999
, Seetharam and Li 1999
). Cellular import of exogenous (dietary) Cbl bound to
gastric intrinsic factor (IF) occurs during intestinal absorption; that
of endogenous (circulatory) Cbl bound to the plasma transporter,
transcobalamin II (TC II), occurs during plasma transport. Both of
these processes occur by receptor-mediated endocytosis
(Seetharam 1999
). After the lysosomal degradation of IF
and TC II, the exit of Cbl from lysosomes is critical for its further
sorting for either export to the circulation (when imported bound to
IF) or for its intracellular retention and utilization (when imported
bound to TC II) in its coenzyme forms. Cellular import of Cbl by IF and
TC II is best exemplified in a polarized epithelial cell in which both
import mechanisms operate; this is illustrated in Figure 1
. This review updates more recent work on the Cbl transporting ligands,
IF and TC II, and their cell surface receptors.
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Cobalamin Binding Protein Ligands. IF and TC II. |
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TOPABSTRACTINTRODUCTIONCobalamin Binding Protein...Cobalamin Receptors....Summary and Perspectives.REFERENCES |
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) and studies on the
development of monoclonal antibodies to IF and TC II
(Seetharam 1994
and references therein) show that both
of these proteins have separate sites for binding Cbl and their
respective cell surface receptors. The primary structure analyses of IF
and TC II (Li et al. 1993
, Platica et al. 1991
), as predicted from their cDNA clones, reveal six very
highly conserved regions that are implicated in Cbl binding; available
data indicate that the initial receptor binding regions of these
proteins may be localized in the nonconserved regions. In support of
this hypothesis, the receptor binding by IF has been localized to the
nonconserved residues (25–62) at the N-terminus of IF (Tang et al. 1992
). It is interesting to note that although IF and TC
II are localized to different chromosomes, IF to chromosome 11
(Hewitt et al.1991
) and TC II to chromosome 22
(Li et al. 1995
), they contain the same number of
similarly sized exons. Five out of six highly conserved hydrophobic
regions of IF and TC II are localized in different exons, and four out
of eight intron-exon boundaries are identical (Li et al. 1995
). This indicates that these two proteins have evolved from
a common ancestral gene through gene duplication, and their receptor
binding regions localized in the nonconserved regions evolved later.
As a result of its role in dietary Cbl transport, IF expression is
limited to gastric parietal and chief cells; in some species such as
dogs and opossum it is expressed in the pancreas (Seetharam 1999
). The molecular mechanisms involved in the cell or tissue
specific regulation of the IF gene are not fully understood. Expression
of a transgene containing the mouse IF promoter (-1029 to + 55)
demonstrated the presence of IF in parietal, but not chief cells, the
site of IF synthesis in mice (Lorenz and Gordon 1993
),
indicating that cis-trans interactions
that modulate IF transcription in parietal vs. chief cells are
different. In contrast, TC II gene expression occurs in many
tissues/cells (Li et al. 1994c
), but in a regulated
manner. The TC II gene lacks a TATA box and an initiator element, and
the weak promoter activity of TC II in epithelial and leukemic cells is
regulated positively by a distal GC-box and negatively by a
proximal GC/GC-box. Because both of these
cis-elements are bound by transcription factors
Sp1 and Sp3, cotransfection studies with Sp1 and Sp3 expression
plasmids have revealed that although Sp1 stimulated the transcriptional
activity, Sp3 suppressed the Sp1-mediated transactivation. These
studies suggested that tissue/cell specific regulation of the TC II
gene is controlled by the relative ratios of Sp1 and Sp3 that bind to a
proximal GC/GT box (Li et al. 1998
). On the basis of
studies using a 69-bp promoter fragment (Li and Seetharam 1998
), there is some evidence to suggest that physical
interaction between Sp1 and the members of the helix-loop-helix family
of transcription factors, USF1/USF2, that bind to the GC box and an
E-box, respectively, up-regulate transcription of the TC II
gene. Although not proven, such interactions may be responsible for the
elevated levels of plasma TC II noted in many forms of human cancer
(Seetharam and Li 1999
and references therein). It would
be interesting to test whether other members of the helix-loop-helix
family of transcription factors such as Myc/Max, Mad/Max, Max/Max, bind
to the E-box in vivo.
Cbl deficiency in children occurs because of a functional lack of IF
and TC II (Fenton and Rosenberg 1995
). In the most
common form of human TC II deficiency, lack of immunoreactive plasma TC
II is due to a lack of TC II synthesis, which in turn is due to a
significant reduction of TC II mRNA (Li et al. 1994a
, 1994b
and 1994c
). Many of the defective alleles contain nonsense
mutations; thus, it is likely that the reduction of TC II mRNA is due
to nonsense-mediated decay of the transcript. In addition, there is
evidence to suggest that null alleles of TC II could also arise due to
deletions and transcriptional defects (Li et al. 1994b
).
In juvenile pernicious anemia patients, Southern blotting of the
genomic DNA revealed normal restriction fragments, indicating that lack
of IF synthesis in these patients is not due to a gross alteration of
the IF gene (Hewitt et al.1991
). It is obvious that
additional studies are required to understand further the molecular
pathophysiology of Cbl deficiency due to defective expression of its
binding proteins.
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Cobalamin Receptors. IFCR/cubilin: a multipurpose receptor? |
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TOPABSTRACTINTRODUCTIONCobalamin Binding Protein...Cobalamin Receptors....Summary and Perspectives.REFERENCES |
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, further studies on intrinsic factor-cobalamin
receptor (IFCR) structure and function were difficult because of its
extremely low levels of expression in this tissue. However, because of
its high levels of expression in nonintestinal tissues, IFCR has been
isolated from the kidney, and several aspects of its synthesis,
processing and apical brush border expression have been studied using
rat renal cortical slices and polarized proximal tubular epithelial
cells [reviewed in Seetharam (1994)
and (1999)
,
Seethram et al. (1994)
]. In addition to its presence in
kidney, functional IFCR is also expressed at high levels in rat yolk
sac (Seethram et al. 1997
), and rat IFCR cDNA has been
isolated recently from yolk sac (Moestrup et al. 1998
).
The primary structure of IFCR as revealed by the cDNA predicted
sequence has shown that the earlier estimates of Mr of 230–280
kDa for IFCR were not accurate; a more accurate Mr of IFCR is ~460
kDa (Birn et al. 1997
, Moestrup et al. 1998
). Recent studies have shown that IFCR functions as a
multipurpose receptor by binding to megalin (Moestrup et al. 1998
), receptor-associated protein(Birn et al. 1997
) and apolipoprotein A-1 (Kozyraki et al. 1999
). Due to its unusual structure and its ability to form
noncovalent trimers (Lindblom et al. 1999
), it is
possible that IFCR may also bind to ligands that are yet to be
identified and may be involved in the general endocytic process in the
kidney.
IFCR contains three structural domains (Moestrup et al. 1998
), a N-terminal stretch of 110 amino acids that is
involved in hydrophobic membrane interactions, followed by eight
epidermal growth factor-like domains, and 27 modules, each
containing ~110 amino acids with a characteristic hydropathy pattern
predicted to form antiparallel ß-barrels. These modules, known as CUB
domains, contain IF-Cbl (CUB domains 5–8) and receptor associated
protein binding (CUB domains 13–14) regions (Kristiansen et al. 1999
). Because the CUB domains of IFCR represent nearly 85% of
its total mass of ~460 kDa and, more importantly, contain the region
of IF-Cbl binding, IFCR is now referred to as cubilin. Sequencing
of cubilin from 17 Finnish hereditary megaloblastic anemia 1 (MGA1)
patients has identified two independent disease-specific mutations.
One, a missense mutation changing a proline to leucine in CUB domain 8
that produced cubilin; the other was an in-frame insertion in the
intron interrupting CUB domain 6 (Aminoff et al. 1999
)
that did not produce cubilin. These two or any other mutations
were detected in the cubilin of MGA1 patients from Norway and Saudi
Arabia. These studies have suggested that MGA 1 may be caused by
mutations in the cubilin molecule itself or may involve other genes
mapping nearby and may produce many phenotypes. In a canine model with
selective inherited intestinal malabsorption of Cbl, the defect in
cubilin, although not defined, produces a transport incompetent
phenotype that is retained in the intracellular membranes and not
delivered to the apical brush border membrane (Fyfe et al. 1991
). To date, it is not known whether this phenotype exists
in the human disease.
Transcobalamin II-receptor (TC II-R).
Consistent with its role in mediating the import of circulatory Cbl to
all cells of the body, TC II-R expression has been detected in many
human (Bose et al. 1995b
) and rat tissues (Bose et al. 1995a
); however, its levels of expression vary, with the
highest in the kidney. Pure TC II-R is a single polypeptide of Mr
62 kDa containing ~27% carbohydrate and four intramolecular
disulfide bonds (Bose and Seetharam 1997a
). The
disruption of disulfide bonds formed, utilizing six buried and two
exposed half-cysteines, resulted in loss of ligand binding and in
the formation of an extended monomer with an apparent increase in the
molecular mass by 10 kDa to 72 kDa. The extended monomer formed within
the cells after treatment of polarized intestinal epithelial human
colon adenocarcinoma (Caco-2) cells with low concentrations of
sulphydral alkylating agents failed to be expressed in the
basolateral plasma membranes of these cells (Bose and Seetharam 1997a
), in which the native TC II-R of Mr 62 kDa is
predominantly expressed as a dimer and functions in the import of Cbl
to be utilized as Cbl-coenzymes (Bose et al. 1996b
and 1997
). These studies have indicated that intramolecular
disulfide bonds of TC II-R are important not only for acquisition
of the ligand binding property of TC II-R, but also for its
post-trans-Golgi trafficking to basolateral plasma membranes.
Although TC II-R is synthesized as a monomer of Mr 62 kDa, it
functions as a noncovalent dimer of Mr 124 kDa in the plasma membrane.
The dimerization of TC II-R is rather unusual in that it is
dependent upon cholesterol fatty acyl interactions of the membrane
bilayer (Bose et al. 1996a
). In tissues, TC II-R
dimer protein levels are between 8- and 10-fold higher than the TC
II-R monomer levels (Bose et al. 1995a
), indicating
that at steady state, the bulk of the TC II-R is present in the
tissue plasma membranes, with only 10% present in the intracellular
pool. In vitro (Bose et al. 1996a
) manipulation of
cholesterol levels in isolated tissue microsomal and plasma membranes
and in vivo studies (Bose et al. 1998
) using polarized
human intestinal epithelial Caco-2 cells, in which cholesterol delivery
to the plasma membranes was inhibited by treatment of cells with
brefeldin A, have shown that plasma membrane dimerization of TC
II-R requires at least 10 mol% of cholesterol, below which it
remains as a monomer. Membrane perturbations causing interconversion of
the TC II-R physical state and the immunobloting method designed to
detect TC II monomer and dimer forms have been reviewed recently
(Bose and Seetharam 1997b
).
The importance of TC II-R in Cbl import is borne out by the
observation that its functional inactivation by circulating receptor
antibody (Bose et al. 1996a
) in rabbits results in a
failure to thrive and the development of Cbl deficiency. During
postnatal development of rat tissue, TC II-R levels are unchanged
(Bose et al. 1995a
), indicating its role in cellular Cbl
import throughout the adult life of the rat; however, in
adrenalectomized rats, its levels are drastically reduced, resulting in
inhibition of plasma delivery of Cbl to tissues (Bose et al. 1995a
). These studies suggest that cortisone may have a role in
TC II-R regulation; additional studies should clarify whether
cortisone affects the half-life of TC II-R protein or its
transcript, or affects its transcription. At a cellular level, TC
II-R levels are up-regulated in proliferating leukemia cells
(Jacobsen et al. 1990
and references therein),
transplanted sarcomas (Collins and Hogenkemp 1997
) and
methionine-dependent P(60) glioma cells (Fiskerstrand et al. 1998
) that have characteristics of methionine-dependent
cancer cells. Taken together, these studies suggest that TC II-R
levels are regulated by a network of intracellular events that could
include intracellular levels of Cbl, more particularly, the
methyl-Cbl levels. When the demand for Cbl increases during rapid
cellular proliferation, cells may up-regulate TC II-R levels to
import more Cbl from the circulation to meet their increased Cbl need.
Some of the properties of IF and TC II receptors are summarized in
Table 1
.
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Summary and Perspectives. |
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TOPABSTRACTINTRODUCTIONCobalamin Binding Protein...Cobalamin Receptors....Summary and Perspectives.REFERENCES |
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FOOTNOTES |
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2 Manuscript received: 21 July 1999.
4 Abbreviations used: Caco-2, human colon
adenocarcinoma cells; Cbl, cobalamin; IF, intrinsic factor; IFCR,
intrinsic factor-cobalamin receptor; MGA, megaloblastic anemia; TC
II, transcobalamin II; TC II-R, transcobalamin II receptor.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONCobalamin Binding Protein...Cobalamin Receptors....Summary and Perspectives.REFERENCES |
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1. Aminioff M., Carter J. E., Chadwick R. B., Johnson C., Grasbeck R., Abdelaal M. A., Broch H., Jenner L. B., Verroust P. J., Moestrup S. K., de la Chapelle A., Krafe R. Mutations in CUBN, encoding the intrinsic factor-vitamin B12 receptor, cubilin, cause hereditary megaloblastic anemia 1. Nat. Genet. 1999;21:309-313[Medline]
2.
Birn H., Verroust P. J., Nexo E., Hager H., Jocobsen C., Christensen E. I., Moestrup S. K. Characterization of an epithelial 460 kDa protein that facilitates endocytosis of intrinsic factor-vitamin B12 and binds to receptor associated protein. J. Biol. Chem. 1997;272:26497-26504
3.
Bose S., Chapin S. J., Seetharam S., Feix J., Mostov K. E., Seetharam B. Brefeldin A (BFA) inhibits the basolateral membrane delivery and dimerization of transcobalamin in human intestinal epithelial Caco-2 cells. J. Biol. Chem. 1998;273:16163-16169
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Bose S., Dahms N. M., Seetharam S., Seetharam B. Bipolar functional expression of transcobalamin II receptor in human intestinal polarized Caco-2 cells. J. Biol. Chem. 1997;272:3538-3543
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Bose S., Feix J., Seetharam S., Seetharam B. Dimerization of transcobalamin II-receptor: requirement of a structurally ordered lipid bilayer. J. Biol. Chem 1996;271:11718-11725
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Bose S., Komorowski R. A., Seetharam S., Gilfix B., Rosenblatt D. S., Seetharam B. In vitro and in vivo inactivation of transcobalamin II-receptor by its antiserum. J. Biol. Chem. 1996;271:4195-4200
7. Bose S., Seetharam S., Hammond T. G., Seetharam B. Regulation of expression of transcobalamin II-receptor in the rat. Biochem. J. 1995;310:923-929
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Bose S., Seetharam B. Effect of disulfide bonds of transcobalamin II-receptor on its activity and basolateral targeting in human intestinal epithelial Caco-2 cells. J. Biol. Chem. 1997;272:20920-20928
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Collins D. A., Hogenkemp H.P.C. Transcobalamin II-receptor imaging via radiolabeled diethyl-triaminepentaacetate cobalamin analogs. J. Nucl. Med. 1997;38:717-723
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Fiskerstrand T., Reidel B., Uleland P. M., Seetharam B., Pezacka E. H., Gulati S., Bose S., Banerjee R., Berge R. K., Refsum H. Disruption of a regulatory system involving cobalamin distribution and function in a methionine-dependent human glioma cell line. J. Biol. Chem. 1998;273:20180-20184
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Fyfe J. C., Ramanujam K. S., Ramaswamy K., Patterson D. F., Seetharam B. Defective brush border expression of intrinsic factor cobalamin receptor in inherited selective intestinal cobalamin malabsorption. J. Biol. Chem. 1991;266:4489-4494
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