Blood, 15 May 2001, Vol. 97, No. 10, pp. 3316-3318
CORRESPONDENCE
To the editor:
Cubilin and the hydrophobic intrinsic factor
receptor are distinct molecules
Cubilin is a 460-kd multiligand hydrophobic protein that binds
to intrinsic factor cobalamin (Cbl-IF) with a high affinity and that is
expressed in both kidney and ileal epithelial cells. Based on this
observation, it has been speculated that cubilin and intrinsic factor
cobalamin receptor (IFCR) are identical.1 Recently, Kristiansen et al showed that the P1297L mutation of cubilin
(FM1) associated with selective congenital cobalamin malabsorption (Imerslund-Gräsbeck disease, described in Finnish patients; also named megaloblastic anemia 1 [MGA1]) causes a 5-fold decrease of the
binding domain affinity for Cbl-IF.2 They concluded that
this decreased affinity explained the malabsorption of Cbl and the Cbl
deficiency related to the FM1 mutation of MGA1 patients. In our
opinion, the pathogenetic mechanism suggested by Kristiansen et al does
not explain the whole biologic phenotype of the Finnish patients. The
P1297L mutation of cubilin has been found in 16 of 17 Finnish patients
but not in patients from Norway or Kuwait. It has been located in the
same 6-cM region of chromosome 10 as the disease locus identified by
linkage analysis by Aminoff et al in Norwegian and Finnish
patients.3 We have shown in 4 French cases of
Imerslund-Gräsbeck disease that the binding activity of a
hydrophobic IFCR was dramatically decreased in both ileum and urine,
using a radioisotopic assay in which [57Co]Cbl-IF
bound to the receptor was precipitated by adsorption to
phenyl-Sepharose.4 In affected patients, the affinity of the IFCR for Cbl-IF was similar to that found in the ileums and the
urine of nonaffected healthy subjects. In collaboration with M. Aminoff, the study was extended to 10 Finnish patients, 11 first-degree
relatives and 13 healthy control subjects.5 We confirmed our previous finding, with a dramatic decrease of the IFCR
activity in all the urine specimens (640 times lower than in control
urine), whereas the affinity of IFCR for Cbl-IF remained unmodified,
with a Ka ranging from 1.2 to 4.8 (nmol/L)-1 in patients and from 1.4 to 6.4 (nmol/L)-1 in controls.5 Unlike what was found
for the hydrophobic IFCR, Aminoff et al have detected mutated cubilin
by Western blot of urine in affected Finnish patients, with the same
intensity and molecular size as that in controls,3 and
Kristiansen et al have shown now that the affinity of the mutated
protein for Cbl-IF was decreased 5-fold.2 The opposite
behavior of cubilin and IFCR in urines with regard to the excretion
level and the Cbl-IF affinity suggests, therefore, that cubilin and the
hydrophobic IFCR are distinct molecules. In a previous study, Moestrup
et al showed that cubilin binds to other proteins such as apoA-I, albumin, light chains of IgG, and RAP (receptor-associated protein) in
domains other than the Cub 5-8 IF-Cbl binding domain. They concluded
that the multiligand activity of cubilin may explain the proteinuria
observed in some of the affected patients.6 Considering
that the P1297L mutation of cubilin affects only its binding to
IF2 and has no consequences on its binding activity to
other proteins,2,6 this does not explain why 40% of the Finnish series had proteinuria while none of them, except a case with
another mutation, had an excretion of apoA-I in urine. The binding of
cubilin to the IFCR-Cbl-IF complex may be a molecular event needed for
the endocytosis of the complex. The IFCR and cubilin have a
distinct intracellular distribution. Indeed, intracellular cubilin is
located in the apical dense tubules,7 whereas the IFCR-IF-Cbl complex is targeted to the lysosomal pathway. Both molecules bind Cbl-IF with the same affinity and a similar pH-dependent pattern. But cubilin is weakly bound to the membrane via its N-terminal amphiphatic domain, as it can be released by ethylenediaminetetraacetic acid (EDTA) washing. In contrast, the 70-kd IFCR is very
hydrophobic, and EDTA washing increases activity rather than removing
it.9 In addition, the molecule purified from hog intestine
can be integrated through a phospholipid bilayer.10 Taken
together, these data suggest that the hydrophobic IFCR has a
transmembrane domain. The suggestion we made 2 years ago that cubilin
and IFCR may be distinct molecules is reinforced by the report by Xu et
al that the schnauzer dog model of MGA1 is not caused by a mutation of the CUBN gene nor by any gene within 50 cM of either side of
the CUBN locus.11 In conclusion, the
discovery by Moestrup and coworkers that cubilin has an IF-Cbl
binding activity by cubilin and its implication in
Imerslund-Gräsbeck disease represents a very significative and
convincing advance of our knowledge of Cbl cellular transport. But it
may be assumed that the pathogenetic concept discussed by Kristiansen
et al2 is only part of the mechanism and that cubilin and
the hydrophobic IFCR are distinct molecules that may act in concert.
Jean-Louis Guéant, Céline Chéry, and Farès Namour
Correspondence: Jean-Louis Guéant, Laboratoire de
Pathologie Cellulaire et Moleculair en Nutrition, Faculte de Medecine
de Nancy, BP 184, Vandoeuvre Cedex 54505, France
References
1.
Seetharam B, Christensen EI, Moestrup SK, Hammond TG, Verroust PJ.
Identification of rat yolk sac target protein of teratogenic antibodies, gp280, as intrinsic factor-cobalamin receptor.
J Clin Invest.
1997;99:2317-2322[Medline]
[Order article via Infotrieve].
2.
Kristiansen M, Aminoff M, Jacobsen C, et al.
Cubilin P1297L mutation associated with hereditary megaloblastic anemia 1 causes impaired recognition of intrinsic factor-vitamin B(12) by cubilin.
Blood.
2000;96:405-409[Abstract/Free Full Text].
3.
Aminoff M, Carter JE, Chadwick RB, et al.
Mutations in CUBN, encoding the intrinsic factor-vitamin B12 receptor, cubilin, cause hereditary megaloblastic anaemia 1.
Nat Genet.
1999;21:309-313[CrossRef][Medline]
[Order article via Infotrieve].
4.
Gueant JL, Saunier M, Gastin I, et al.
Decreased activity of intestinal and urinary intrinsic factor receptor in Grasbeck-Imerslund disease [published erratum appears in Gastroenterol. 1995;109:1722].
Gastroenterol.
1995;108:1622-1628[CrossRef][Medline]
[Order article via Infotrieve].
5.
Dugue B, Aminoff M, Aimone-Gastin I, Leppänen E, Gräsbeck R, Gueant J-L.
A urinary radioisotope-binding assay to diagnose Gräsbeck-Imerslund disease.
J Pediatr Gastroenterol Nutr.
1998;26:21-25[CrossRef][Medline]
[Order article via Infotrieve].
6.
Moestrup SK, Kozyraki R, Kristiansen M, et al.
The intrinsic factor-vitamin B12 receptor and target of teratogenic antibodies is a megalin-binding peripheral membrane protein with homology to developmental proteins.
J Biol Chem.
1998;273:5235-5242[Abstract/Free Full Text].
7.
Birn H, Verroust PJ, Nexo E, et al.
Characterization of an epithelial approximately 460-kDa protein that facilitates endocytosis of intrinsic factor-vitamin B12 and binds receptor-associated protein.
J Biol Chem.
1997;272:26497-26504[Abstract/Free Full Text].
8.
Alpers DH, Russell-Jones GJ.
Intrinsic factor, haptocorrin, and their receptor. In:
Banerjee R, ed.
Chemistry and Biochemistry of B12. New York, NY: John Wiley & Sons Inc; 1999:411-440.
9.
Gueant JL, Jokinen O, Schohn H, Monin B, Nicolas JP, Grasbeck R.
Purification of intrinsic factor receptor from pig ileum using as affinity medium human intrinsic factor covalently bound to Sepharose.
Biochim Biophys Acta.
1989;992:281-288[Medline]
[Order article via Infotrieve].
10.
Kouvonen I, Grasbeck R.
Topology of the hog intrinsic factor receptor in the intestine.
J Biol Chem.
1981;256:154-158[Abstract/Free Full Text].
11.
Xu D, Kozyraki R, Newman T, Fyfe J.
Genetic evidence of an accessory activity required specifically for cubilin brush-border expression and intrinsic factor-cobalamin absorption.
Blood.
1999;94:3604-3606[Abstract/Free Full Text].
Response:
Evidence for an accessory activity required for
cubilin expression
not for an alternative intrinsic factor cobalamin
receptor
We appreciate that Dr Guéant recognizes the importance of
our scientific work on the intrinsic factor cobalamin (IF-Cbl) receptor, cubilin. But it is very difficult for us to find scientific support for several of his statements, including the argument for the
existence of a distinct 70-kd IF-Cbl receptor and the hypothesis that
such a distinct protein should be affected in Finnish patients with
Imerslund-Gräsbeck (MGA1) having the P1297L cubilin mutation
causing Cbl deficiency.
In several papers by the groups of Seetharam,1,2
Fyfe,3 Verroust,4-6 and us,4-6
it has been definitely proven that the 460-kd cubilin protein is
identical to the high molecular IF-Cbl receptor (IFCR) protein. None of
these groups or other groups have ever reported the existence of an
alternative 70-kd receptor (as Dr Guéant also designates IFCR).
The introduction of the paper by Guéant and
colleagues7 on the 70-kd IF-Cbl-binding urinary protein
contains the following statements about the protein: "It is probably
produced by proteolytic cleavage of the receptor for IF-cobalamin
located in the epithelium of the kidney tubules. This urinary receptor
is likely to be a part of the same protein expressed in the ileum."
7(p25) Because of the high proteolytic activity in
the urine, we believe in this possibility and we can confirm
(unpublished data, November 1997) that cubilin released from the
tubules is partially degraded in the urine. Furthermore, we have shown
that CUB domains 5-8 of cubilin constitute the IF-Cbl binding region of
cubilin.8 The size of this region is about 65-70 kd
(glycosylated) and has a theoretical isoelectric point (amino acids
only) of 5.4, which is close to the estimated 4.8 value9
measured for the reported 70-kd protein in the urine. It is therefore a
great surprise for us that Dr Guéant now argues that his reported
70-kd protein should represent a distinct IF-Cbl receptor. It
would be relevant to determine the primary structure of the 70-kd
protein, or at least a part of it. In this way it would ultimately be
elucidated whether the 70-kd protein and cubilin are distinct proteins
or not. If the 70-kd protein is distinct from cubilin, it should be
characterized in terms of structure and function.
Dr Guéant claims that the low IF-Cbl binding activities that he
has measured in the urine of Finnish patients with
Imerslund-Gräsbeck are "opposite behavior" to our data on the
affinity decrease caused by the P1297L mutation. We do not agree.
First, the data are not opposite: they both show the same tendency,
namely, a decreased binding affinity in the patients. Second, we made a
qualitative analysis, not a quantitative measurement of cubilin in the
urine. For the Western blot we show in Aminoff et al,6 we
used a monoclonal antibody preferentially detecting nondegraded
cubilin. The intention of showing this blot was to support the genetic
data by demonstrating the existence of normal-size cubilin in the FM1
patients with the P1297L mutation and the absence of full-length
cubilin expression/excretion in the FM2 patient with the intronic
mutation in the cubilin gene. Using a polyclonal antibody (not
published) for the Western blot would show cubilin fragments as well.
Third, the very low IF-Cbl binding activities in the urine of the
patients, as Dr Guéant and coworkers have observed, may
also reflect a higher level of proteolytic inactivation of cubilin
caused by an increased content of protein including proteinases in the urine.
Dr Guéant cites us for a statement that the P1297L mutation only
affects the binding of IF-B12 and not other proteins
filtered in the urine. This is not correct: we have reported only that the binding site is distinct from the binding sites of apoA-I and
receptor-associated protein (RAP).8 The implication of the
P1297L mutation for protein excretion in the urine is not finally clarified.
Finally, Dr Guéant claims that a study10 on a canine
Imerslund-Gräsbeck disease model reinforces his theory that
cubilin and the putative 70-kd protein are distinct molecules. However, the conclusion of this interesting study by Xu et al10 is
not that there exists an alternative IF-Cbl receptor but that these affected dogs have a genetic defect in an accessory activity important for cubilin processing. The genetic data of the Xu et al
paper10 are very convincing, and in our recent Blood
paper11 we discuss the possibility that patients with
Imerslund-Gräsbeck in ethnic populations other than the Finnish
might have a genetic defect similar to that of the dogs.
Mette Kristiansen and Søren K. Moestrup
Correspondence: Søren K. Moestrup, Department of Medical
Biochemistry, University of Aarhus, Ole Worms Alle, Building 170, 8000 Aarhus C, Denmark
References
1.
Seetharam B, Bose S, Li N.
Cellular import of cobalamin (vitamin B-12).
J Nutr.
1999;29:1761-1764.
2.
Seetharam B, Christensen EI, Moestrup SK, Hammond TG, Verroust PJ.
Identification of rat yolk sac target protein of teratogenic antibodies, gp280 as intrinsic factor-cobalamin receptor.
J Clin Invest.
1997;9:2317-2322.
3.
Xu D, Fyfe JC.
Cubilin expression and posttranslational modification in the canine gastrointestinal tract.
Am J Physiol.
2000;279:G748-G756[Abstract/Free Full Text].
4.
Moestrup SK, Kozyraki R, Kristiansen M, et al.
The intrinsic factor-vitamin B12 receptor and target of teratogenic antibodies is a megalin-binding peripheral membrane protein with homology to developmental proteins.
J Biol Chem.
1998;273:5235-5245.
5.
Kozyraki R, Kristiansen M, Silahtaroglu A, et al.
The human intrinsic factor-vitamin-B12 receptor, cubilin: molecular characterization and chromosomal mapping of the gene to 10p within the autosomal recessive megaloblastic anemia (MGA1) region.
Blood.
1998;91:3593-3600[Abstract/Free Full Text].
6.
Aminoff M, Carter JE, Chadwick R, et al.
Mutations in CUBN, encoding the intrinsic factor-vitamin B12 receptor, cubilin causes hereditary megaloblastic anemia 1 (MGA1).
Nature Gen.
1999;21:309-313.
7.
Dugue B, Aminoff M, Aimone-Gastin I, Leppanen E, Gräsbeck R, Gueant JL.
A urinary radioisotope-binding assay to diagnose Grasbeck-Imerslund disease.
J Pediatr Gastroenterol Nutr.
1998;26:21-25.
8.
Kristiansen M, Kozyraki R, Jacobsen C, Nexø E, Verroust P, Moestrup SK.
Molecular dissection of the intrinsic factor-vitamin B12 receptor, cubilin, discloses regions important for membrane association and ligand binding.
J Biol Chem.
1999;274:20540-20544[Abstract/Free Full Text].
9.
Safi A, Saunier M, Gastin I, Alibaba Y, Dugué B, Gúeant JL.
Intrinsic factor covalently bound to Sepharose as affinity medium for the purification of a soluble intrinsic factor receptor from human urine.
J Chromatogr B Biomed Appl.
1995;664:253-259[Medline]
[Order article via Infotrieve].
10.
Xu D, Kozyraki R, Newman TC, Fyfe JC.
Genetic evidence of an accessory activity required specifically for cubilin brush-border expression and intrinsic factor-cobalamin absorption.
Blood.
1999;94:3604-3606.
11.
Kristiansen M, Aminoff M, Jacobsen C, de la Chapelle A, Krahe R, Verroust P, Moestrup SK.
The cubilin P1297L mutation associated with hereditary megaloblastic anaemia 1 (MGA1) causes impaired recognition of intrinsic factor-vitamin B12 by cubilin.
Blood.
2000;96:405-409.
