Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein.. Bind-ing of125I-labele
Trang 1folate binding protein
Henrik Birn1, Xiaoyue Zhai1, Jan Holm2, Steen I Hansen2, Christian Jacobsen3, Erik I Christensen1 and Søren K Moestrup3
1 Department of Cell Biology, Institute of Anatomy, University of Aarhus, Denmark
2 Department of of Clinical Chemistry, Hillerød Hospital, Denmark
3 Department of Medical Biochemistry, University of Aarhus, Denmark
Folate is a vitamin involved in essential biological
processes such as the synthesis of nucleic acid and
the metabolism of amino acids Humans are unable
to synthesize this vitamin and thus rely on intestinal
reabsorption Distribution into tissues thereafter is
dependent on specific uptake mechanisms involving
the reduced folate carrier and⁄ or the 30–40 kDa
folate binding proteins (FBPs) [1–3] At least three
isoforms of FBPs have been identified including both
glycosylphosphatidylinositol (GPI)-linked, membrane
associated folate receptors highly expressed in certain epithelial tissues and soluble proteins present in serum and other biological fluids [1,2] including the secretory fluids: milk, saliva, and semen [4–9] The significance of the soluble folate binders is largely unresolved, but it has been suggested that milk FBP may play a role in folate uptake [2], i.e by protect-ing against oxidation, by promotprotect-ing folate absorption
in the suckling animal [10–12], or protect against bacterial utilization of secreted folates [13] Based on
Keywords
absorption; endocytosis; intestine; kidney;
vitamins
Correspondence
H Birn, Department of Cell Biology,
Institute of Anatomy, University of Aarhus,
University Park Building 234, DK-8000
Aarhus C, Denmark
Fax: +45 86198664
Tel: +45 89423051
E-mail: hb@ana.au.dk
(Received 15 March 2005, revised 4 July
2005, accepted 11 July 2005)
doi:10.1111/j.1742-4658.2005.04857.x
Folate is an essential vitamin involved in a number of biological processes High affinity folate binding proteins (FBPs) exist both as glycosylphospha-tidylinositol-linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva and semen The function and significance of FBPs are unresolved, however, it has been suggested that they may facilitate folate uptake, e.g during suck-ling The present study shows that megalin, a large, multiligand endocytic receptor and member of the low-density lipoprotein-receptor family, is able
to bind and mediate cellular uptake of FBP Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein Bind-ing of125I-labeled folate binding protein (FBP) to sections of kidney proxi-mal tubule, known to express high levels of megalin, is inhibitable
by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin Immortalized rat yolk sac cells, represent-ing an established model for studyrepresent-ing megalin-mediated uptake, reveal
125I-labeled FBP uptake which is inhibited by receptor associated protein and by antimegalin antibodies Microinjection of 125I-labeled FBP into renal tubules in vivo shows proximal tubular uptake by endocytosis Megalin is expressed in several absorptive epithelia, including intestine and kidney proximal tubule, and thus the present findings provide a mechanism for intestinal and renal endocytic uptake of soluble FBP
Abbreviations
5-MTHF, 5-methyltetrahydrofolate; CPM, counts per minute; FBP, folate binding protein; GPI, glycosylphosphatidylinositol; LRP, low-density lipoprotein receptor related protein; RAP, receptor associated protein.
Trang 2studies of folate-FBP uptake in suckling rats it was
suggested that intestinal uptake of FBP-bound folate
resembles the endocytic absorption of other
macro-molecules in the neonate intestine [12], however, the
mechanism of this process has not been identified
We have previously characterized different receptors
involved in the endocytic uptake of proteins and
nutrients in the kidney and other tissues [14]
Meg-alin, an 600 kDa member of the low-density
lipoprotein-receptor family [15], is a multiligand,
endocytic receptor expressed in a number of
absorp-tive epithelia including kidney, yolk sac, choroid
plexus, and intestine [14,16,17] So far more than 40
different ligands have been identified representing a
wide variety of substance including lipoproteins,
hor-mones, carrier proteins, enzymes, and drugs Megalin
is involved in the endocytic uptake of a number of
vitamin carrier proteins [18], including retinol binding
protein [19], transcobalamin [20], and vitamin D
binding protein [21,22] and its role for the recovery
of vitamins and carrier proteins filtered in the renal
glomerulus is well established [18,23] In addition,
megalin seems important for normal expression of
the intestinal intrinsic factor-vitamin B12-receptor
cubilin [16,24] Inspired by these observations we
have examined a possible interaction between FBP
and megalin The presented data shows that megalin
is able to bind and mediate the endocytosis of
soluble FBP providing a potential mechanism for
intestinal and renal uptake of soluble FBP-bound
folate
Results
Megalin binds FBP Surface plasmon resonance analysis showed binding of purified, human and bovine milk apo-FBP to immo-bilized rabbit megalin Binding was also observed with bovine FBP saturated with folic acid, 5-methyl-tetrahydrofolate (5-MTHF), or methotrexate (Fig 1) Using a biaevaluation program Kd was estimated to
0.3–0.5 lm for apo-FBP and saturated FBP (Fig 1)
No binding was identified to low-density lipoprotein receptor related protein (LRP), the most closely related member of the LDL-receptor family [25]
To confirm binding of FBP to megalin in tissues we used autoradiography on sections of rat kidney cortex showing very high expression of megalin in kidney proximal tubule brush border membranes Incubation with 125I-labeled human or bovine milk FBP resulted
in accumulation of autoradiographic grains along the apical part of proximal tubule cross-sections similar to the localization of megalin (Fig 2) This was inhibited
by coincubation with excess unlabeled FBP or with receptor associated protein (RAP), an established inhibitor of the uptake of most ligands by megalin [14], suggesting specific binding to megalin
Endocytosis of FBP Microinjection of 125I-labeled bovine milk FBP in rat kidney nephrons in vivo followed by autoradiography
Fig 1 Binding of soluble FBP to immobilized megalin and LRP by surface plasmon resonance analysis (BIAcore) Soluble human or bovine milk FBP purified by a combination of ion-exchange and affinity chromatography was passed over the sensor chips with immobilized, purified megalin, or LRP and the SPR signal (RU) representing bound protein was recorded (A) SPR sensorgram showing binding of bovine milk apo-FBP, folic acid-FBP, 5-MTHF-FBP, and methotrexate-FBP to immobilized megalin Using the BIAEVALUATION program Kdwas estimated between 0.3 and 0.5 l M Similar binding of human apo-FBP was observed (not shown) (B) No binding of bovine milk apo-FBP, folic acid-FBP, 5-MTHF-acid-FBP, or methotrexate-FBP was observed to purified LRP.
Trang 3revealed uptake of FBP within proximal tubule
seg-ments (Fig 3), known to express high levels of
meg-alin In contrast, distal nephron segments and
collecting ducts revealed no significant labeling The
label could be identified in endocytic vesicles and
vacu-oles 15–30 min after microinjection (Fig 3B,C)
Megalin mediates cellular uptake of FBP
Megalin expressing, rat yolk sac BN-16 cells
internal-ized human milk 125I-labeled FBP (Fig 4) Total
uptake was calculated as the sum of degraded, non
tri-chloroacetic acid-precipitable label and cell associated
label The time course of uptake is comparable to
pre-vious published uptake of the vitamin B12 carrier
transcobalamin in similar cells [20] The total uptake
was significantly inhibited by RAP and antimegalin
antibodies, showing the involvement of megalin in uptake A small, however, significant inhibition with nonspecific IgG was observed at 4 h explained by low-affinity binding of immunoglobulin light chains to megalin [26] While degradation was clearly inhibited, the cell associated amount of label was less affected
by RAP
Discussion
FBP is present in different biological, secretory fluids, including saliva and semen, and in particular milk [4–9] The significance of these folate binders is largely unknown, but it has been suggested that milk FBP, shown to be resistant to gastric digestion [27], may play a role in folate uptake in the neonate intestine [10–12] The present study shows that megalin, a large,
Fig 2 Autoradiography showing RAP-inhibitable binding of soluble 125 I-labeled FBP to proximal tubules of rat kidney cortex Sections of per-fusion fixed rat kidney cortex were incubated with125I-labeled human or bovine milk FBP Binding to sections was identified by autoradiogra-phy following 36–47 days of exposure For inhibition studies sections were coincubated with either excess unlabeled FBP or RAP (50 lgÆmL)1) Labeled bovine (A) and human (C) milk FBP is concentrated along the apical part of proximal tubule cross-sections similar to the localization of megalin (insert, 2C) Binding to sections is inhibited by excess unlabeled bovine FBP (B, compare with A) and RAP (D, compare with C) suggesting binding to megalin Bars equal 10 lm.
Trang 4multiligand, endocytic receptor expressed in the
intes-tine and other absorptive epithelia binds and mediates
the internalization of FBP This is based on the
obser-vations that: (a) purified bovine and human milk FBP
binds to immobilized megalin when analysed by
sur-face plasmon resonance analysis The Kd can be
esti-mated to 0.3 lm comparable to the affinity of
several other ligands binding to megalin; (b) milk FBP
binds to proximal tubules in sections of rat kidney
cortex known to express high levels of megalin This
binding is inhibited by excess unlabeled FBP and by
RAP, a chaperone and known inhibitor of the uptake
of most ligands by megalin; (c) in vivo microinjection
of 125I-labeled milk FBP into rat kidney nephrons
reveal endocytic uptake in megalin-expressing proximal
tubule cells only; (d) BN-16 cells known to express
megalin and representing an established model for
megalin-mediated uptake, internalize 125I-labeled milk
FBP, and internalization is strongly inhibited by RAP
and antimegalin antibodies These findings suggest a
potential role for megalin in mediating internalization
of FBP-bound folate, providing a candidate
mecha-nism for both intestinal and renal tubular uptake of
soluble FBP
Megalin is expressed in the intestine [16,17] and
megalin-mediated endocytosis provides a mechanism
for the previously suggested [12] endocytic absorption
of FBP-bound folate during suckling It was noted
that in contrast to free folate, FBP-bound folate
is absorbed more avidly in the ileum than in the
jejunum correlating with the observed expression of
megalin in purified apical brush-border membranes
from distal, but not proximal, rat intestine [17] The
kinetics of FBP transport in neonatal goat intestinal brush border have revealed that Km¼ 0.39 lm for unsaturated FBP [11], comparable to our findings using isolated proteins Small differences in Kd were calculated comparing the response curves for apo-FBP, folic acid-apo-FBP, 5-methyltetrahydrofolate-apo-FBP, and methotrexate-FBP, with apo-FBP having the highest affinity compared to saturated FBP While this in line with the observation of [11], the differ-ences are small and probably within the methodological variation associated with surface plasmon resonance analysis
Renal proximal tubular epithelium expresses abun-dant megalin serving an important role mediating reabsorption of vitamin-carrier protein complexes fil-tered in the glomeruli thus preventing excessive urinary loss [18,23] These carrier complexes include retinol binding protein [19], transcobalamin [20], and vitamin
D binding protein [21,22] Although most folate in plasma is filtered in the renal glomerulus as unbound folate, a soluble FBP is present in plasma at a concen-tration of about 0.6 nm in humans [28] The molecular weight of 35 kDa suggests that this protein to a large extent is filtered, and FBP has been detected in human urine at a concentration of 0.3 nm [29] Assu-ming a glomerular filtration rate of 180 L⁄ 24 h and urinary excretion rate of 2 L⁄ 24 h it may be calculated that only < 1% of the plasma FBP actually filtered in the glomeruli is excreted suggesting efficient tubular reabsorption Thus, 108 nmoles or 48 lg of folate may be recovered daily by tubular uptake of FBP which may be important in individuals with very low-folate intake The present data provides a
mecha-Fig 3 Uptake of125I-labeled bovine milk FBP microinjected into rat nephrons in vivo Uptake of125I-labeled FBP is visualized by autoradio-graphy on sections from fixed kidney cortex Grains are located over proximal tubule profiles (A; PT) only revealing selective uptake in this part of the nephron characterized by heavy expression of megalin (Fig 2C, inset) No labeling is oberved in distal tubule profiles (A; DT) Labeling is concentrated in the subapical part of the proximal tubule cells localized close to vacuolar structures (arrows in B) which
by electron microscopy can be identified as apical, endocytic vesicles or vacuoles (C; E) MV, microvilli Bars equal 10 lm (A and B) and 0.5 lm (C).
Trang 5nism for this involving megalin-mediated endocytosis
similar to the uptake of other vitamin carrier proteins
Free folate is reabsorbed within the renal proximal
tubule by a mechanism dependent on GPI-anchored
FBP expressed in the luminal plasma membrane of
proximal tubule cells [30,31] However, in particular
at low plasma folate concentrations an additional
mechanism of tubular folate uptake may operate
[31] Megalin-mediated uptake of filtered folate bound
to plasma FBP may constitute such an alternative mechanism
Megalin-mediated uptake of FBP in BN-16 cells results in the degradation of FBP Most likely folate bound to FBP is released from the binder in the acidic environment of the endocytic compartment followed by transport across the vesicular membrane into the cyto-sol Similarly, the uptake of free folate mediated by GPI-anchored FBPs expressed in the apical membrane
is suggested to involve binding of folate to the GPI-linked FBPs, followed by endocytosis, release of folate from the receptor within an internal compartment and recycling of the receptor [32–34] Thus, it is possible that folate internalized either by megalin-mediated endocy-tosis of soluble FBP or by binding of free folates to membrane-associated folate receptors is released within the same endocytic compartment and may be further transported by a common pathway However, while the GPI-linked FBP is recycled to the plasma membrane, FBP bound to megalin seems to be degraded
In conclusion megalin is able to bind and internalize soluble FBP by endocytosis providing a potential mechanism for intestinal and renal uptake of soluble FBP-bound folate in milk or the ultrafiltrate Further studies are needed to determine the importance of folate uptake via this pathway
Experimental procedures
Purification of proteins High-affinity FBPs were purified from bovine and human milk by a combination of cation exchange chromatography and ligand (methotrexate) chromatography on a column desorbed with a pH-gradient [9,35,36] Bovine FBP was purified from cow’s whey powder and consisted of 222 amino-acid residues with a molecular mass of 30 kDa based
on amino-acid composition and carbohydrate content [37] Two FBPs were purified from Triton X-100 solubilized human raw milk obtained from voluntary donors with their full understanding and full consent One desorbed at pH 5 had a molecular mass of 30 kDa based on its amino-acid composition and sequence homology with bovine milk FBP [9], while one desorbed at pH 3 was GPI-linked and extre-mely hydrophobic only existing in a micellar form with Triton X-100 [36] Both FBPs had identical N-terminal sequence for 39 cycles and were immunologically identical [9,36] The enzyme phosphatidyl inositol specific phospholi-pase C cleaved the hydrophobic GPI residue of micellar FBP and converted it to soluble FBP [38] Human and bovine milk FBP was iodinated by the chloramine-T method [39] Receptor associated protein, an established inhibitor of the uptake of most ligands by megalin [14], was prepared
Fig 4 Uptake of soluble 125 I-labeled FBP in megalin expressing
BN-16 cells (percent of total added activity) Megalin expressing,
yolk sac BN-16 epithelial cells were grown to confluence and
incu-bated with 125 I-labeled human milk FBP [9000 counts per minute
(CPM)] Total uptake represents the sum of degraded
(non-trichloro-acetic acid-precipitable) and cell-associated activity For inhibition
studies cells were coincubated with either RAP (1 l M , A), sheep
anti-megalin IgG (200 lgÆmL)1, B), or nonspecific sheep IgG
(200 lgÆmL)1, B) Labeled FBP is internalized and degraded in
BN-16 cells (A) Uptake is significantly inhibited by RAP (A), and
anti-megalin IgG (B) A minor, however, significant inhibition with
nonspecific IgG was observed at 4 h Data represent mean ± SD
of four experiments In panel A the RAP induced difference in total
uptake was significant (P < 0.001, t-test) at all time points In (B),
significance is indicated by * (ANOVA followed by posthoc t-test
using Bonferroni correction at each time point).
Trang 6and used for purification of rabbit megalin by affinity
chromatography as described [40] LRP was purified from
solubilized human placental membranes as described [41]
Binding of milk FBP to megalin by surface
plasmon resonance analysis (SPR)
For the surface plasmon resonance analyses, the BIAcore
sensor chips (type CM5; Biosensor, Uppsala, Sweden) were
activated with a 1 : 1 mixture of 0.2 m
N-ethyl-N¢-(3-dimeth-ylaminopropyl) carbodiimide and 0.05 m
N-hydroxysuccini-mide in water according to the manufacturer Purified
megalin or LRP were immobilized on the sensor chip in
10 mm sodium acetate, pH 4.5, and the remaining binding
sites were blocked with 1 m ethanolamine, pH 8.5 The
resulting receptor densities were in the range of 23–40 fmol
receptor per mm2 A control flow cell was made by
perform-ing the activation and blockperform-ing procedures and by usperform-ing
immobilized receptor proteins reduced by injection of 0.5%
dithiothreitol in 6 m guanidine hydrochloride, 5 mm EDTA,
and 50 mm Tris, pH 8.0, into the flow cell Purified FBP,
with or without the addition of 2.5 lm folic acid, 5-MTHF
or methotrexate was dissolved in 10 mm Hepes, 150 mm
NaCl, 2 mm CaCl2, and 0.005% Tween 20, pH 7.4 Sample
and running buffers were identical The regeneration of
sen-sor chips after each analysis cycle was performed with 1.6 m
glycine-HCl buffer, pH 3.0 The BIAcore response is
expressed in relative response units and represents the
bind-ing response usbind-ing the native receptor corrected for the
response registered with the control flow cell Kdfor binding
was estimated using a biaevaluation program
Binding of milk125I-labeled FBP to tissue
cryosections
Binding of FBP to megalin in tissue was studied by
auto-radiography on 1 lm cryosections of rat kidney cortex, fixed
in 4% (v⁄ v) paraformaldehyde in 0.1 m sodium cacodylate
buffer, pH 7.4, by retrograde perfusion through the
abdom-inal aorta Sections were cut at 190–200 K using a Reichert
Ultracut S cryoultramicrotome and placed on gelatin coated
glass slides, preincubated in 0.01 m NaCl⁄ Pi, 0.05 m glycine,
0.15 m NaCl, 0.1% (w⁄ v) skimmed milk and 0.02 m NaN3
and incubated with125I-labeled human or bovine milk FBP
(2Æ 106CPMÆmL)1) in 0.01 m NaCl⁄ Pi, 0.05 m Tris buffer,
0.15 m NaCl, 1 mm CaCl2, 0,1% BSA and 0.02 NaN3 For
inhibition studies excess unlabeled bovine milk FBP (10 lm)
or receptor associated protein (RAP, 1.2 lm), was added to
the 125I-labeled FBP incubation buffer Sections were
washed, fixed in 1% (v⁄ v) glutaraldehyde in 0.1 m sodium
cacodylate buffer, pH¼ 7.4 and prepared for light
micro-scope autoradiography using Ilford emulsion After 8 days
of exposure the sections were developed and observed in a
Leica LMR microscope (Wetzlar, Germany)
Uptake of FBP in kidney proximal tubule and cultured yolk sac cells
To study uptake 125I-labeled bovine milk FBP was micro-injected into kidney proximal tubules from anaesthetized male Wistar rats (207–237 g) placed on a thermostatically controlled heated table A tracheostomy was performed, and the jugular vein was catherized and infused with saline, 3.8 mLÆh)1 The left kidney was exposed by flank incision, placed in a stabilized cup and covered with paraffin oil maintained at 37–38C Single surface proximal tubules were injected with 52 nL of 125I-labeled FBP in 0.15 m NaCl, 1 mm CaCl2and lissamine green and fixed by micro-injection of 1% glutaraldehyde 15–30 min after microinjec-tion with 125I-labeled FBP Small tissue blocks containing the microinfused tubules were postfixed, dehydrated and embedded into Epon 812 Sections were processed for light microscope or electron microscope autoradiography using Ilford emulsion K2 or L4, respectively, and observed in a Leica LMR microscope or Philips EM208 or CM100 elec-tron microscope (Eindhoven, the Netherlands) All animal experiments were carried out to minimize pain and discom-fort and in accordance with the provisions for the animal care license provided by the Danish National Animal Experiments Inspectorate
In addition, megalin-mediated uptake was studied in a rat yolk sac BN-16 epithelial cell-line previously shown to express megalin and representing an established model for megalin-mediated uptake [19,20,42] Cells were grown to confluence in 24 well cell culture plates using RPMI 1640 medium (Life Technologies, Gaithersburg, MD, USA) with 5% (v⁄ v) fetal bovine serum, 50 UÆmL)1 penicillin, and
50 lgÆmL)1 streptomycin (Bio-Whittaker, Wokingham, UK) At confluence cells were incubated with 125I-labeled human milk FBP ( 9000 CPM) in 0.5 mL RPMI 1640 with 0.1% fetal bovine serum at 37C Following 2, 4 or 8 h incu-bation, the medium was recovered, cells were washed once in warm medium, and harvested by trypsinization for 20 min
An equal volume of 1% (w⁄ v) BSA solution was added to the collected medium along with the washing medium and followed by precipitation with 10% (v⁄ v) trichloroacetic acid The activity of the precipitate, the supernatant, and the cells was counted separately in a Packard Cobra 5002 gamma-counter The degraded amount of 125I-labeled FBP
in the medium was estimated as non-trichloroacetic acid pre-cipitated activity in the medium corrected for the non-trichlo-roacetic acid precipitated activity in the medium of wells incubated for the same time without cells Total uptake of
125
I-labeled FBP was calculated as the sum of cell associated activity and degraded 125I-labeled FBP in the medium and expressed in percent of total activity added For inhibition studies cells were coincubated with either RAP (1 lm), purified sheep anti-rat megalin IgG (200 lgÆmL)1 [43]), or purified, nonspecific sheep IgG (200 lgÆmL)1, DAKO,
Trang 7Denmark) Antibodies were purified by protein A-agarose
affinity chromatography according to manufacturer’s
instructions (Pierce, Rockford, IL, USA) Data represent
mean ± SD of four experiments, and statistical analysis was
performed using unpaired t-test or ANOVA
Immunocytochemistry
Normal Wistar rats were fixed by retrograde fixation
through the abdominal aorta using 4% (v⁄ v)
paraformalde-hyde For light microscope immunocytochemistry semithin
cryosections were cut as described above Sections were
incubated 1 h with polyclonal sheep anti-rat megalin IgG
(1 : 50 000 [43]), in 10 mm NaCl⁄ Pi, 0.15 m NaCl, 0.1%
skimmed milk and 20 mm NaN3, followed by
HRP-conju-gated goat anti-sheep IgG, and visualization by incubation
with diaminobenzidine and 0.03% (v⁄ v) H2O2 for 10 min
All incubations were performed at room temperature and
sections were counterstained with Meiers before
examina-tion in the light microscope as described above
Acknowledgements
The work was supported in part by the Danish Medical
Research Council, the University of Aarhus, the
NOVO-Nordisk Foundation, Fonden til
Lægevidenska-bens Fremme, the Biomembrane Research Center, and
the Birn-Foundation The skillful technical assistance by
Pia K Nielsen, Hanne Sidelmann, and Inger
Kristoffer-sen is greatly appreciated The study was in part preKristoffer-sen-
presen-ted at the ASN Annual Meeting, Philadelphia, PA,
November 1–4, 2002 and at the 13th International
Sym-posium on Chemistry & Biology of Pteridines & Folates,
Egmond aan Zee, the Netherlands, June 20–24, 2005,
and published in part as abstract
References
1 Antony AC (1996) Folate receptors Annu Rev Nutr 16,
501–521
2 Henderson GB (1990) Folate-binding proteins Annu
Rev Nutr 10, 319–335
3 Matherly LH & Goldman DI (2003) Membrane
trans-port of folates Vitam Horm 66, 403–456
4 Ghitis J (1967) The folate binding in milk Am J Clin
Nutr 20, 1–4
5 Salter DN, Ford JE, Scott KJ & Andrews P (1972)
Iso-lation of the folate-binding protein from cow’s milk by
the use of affinity chromatography FEBS Lett 20, 302–
306
6 Selhub J, Arnold R, Smith AM & Piccano MF (1984)
Milk folate binding protein (FBP): a secretory protein
for folate? Nutr Res 4, 181–187
7 Verma RS & Antony AC (1992) Immunoreactive folate-binding proteins from human saliva Isolation and comparison of two distinct species Biochem J 286, 707–715
8 Holm J, Hansen SI & Høier Madsen M (1991) A high-affinity folate binding protein in human semen Biosci Report 11, 237–242
9 Svendsen I, Hansen SI, Holm J & Lyngbye J (1982) Amino acid sequence homology between human and bovine low molecular weight folate binding protein iso-lated from milk Carlsberg Res Comnun 47, 371–376
10 Colman N, Hettiarachchy N & Herbert V (1981) Detec-tion of a milk factor that facilitates folate uptake by intestinal cells Science 211, 1427–1429
11 Salter DN & Blakeborough P (1988) Influence of goat’s-milk folate-binding protein on transport of 5-methyltetrahydrofolate in neonatal-goat small intestinal brush-border-membrane vesicles Br J Nutr
59, 497–507
12 Mason JB & Selhub J (1988) Folate-binding protein and the absorption of folic acid in the small intestine of the suckling rat Am J Clin Nutr 48, 620–625
13 Ford JE (1974) Some observations on the possible nutri-tional significance of vitamin B12-and folate-binding proteins in milk Br J Nutr 31, 243–257
14 Christensen EI & Birn H (2002) Megalin and cubilin: multifunctional endocytic receptors Nat Rev Mol Cell Biol 3, 256–266
15 Saito A, Pietromonaco S, Loo AK & Farquhar MG (1994) Complete cloning and sequencing of rat gp330⁄ ’megalin’, a distinctive member of the low density lipoprotein receptor gene family Proc Natl Acad Sci USA 91, 9725–9729
16 Birn H, Verroust PJ, Nexø E, Hager H, Jacobsen C, Christensen EI & Moestrup SK (1997) Characterization
of an epithelial 460 kDa protein that facilitates endocy-tosis of intrinsic factor-vitamin B12and binds receptor-associated protein J Biol Chem 272, 26497–26504
17 Yammani RR, Seetharam S & Seetharam B (2001) Cubilin and megalin expression and their interaction in the rat intestine: effect of thyroidectomy Am J Physiol Endocrinol Metab 281, E900–E907
18 Christensen EI & Willnow TE (1999) Essential role of megalin in renal proximal tubule for vitamin homeo-stasis J Am Soc Nephrol 10, 2224–2236
19 Christensen EI, Moskaug JO, Vorum H, Jacobsen C, Gundersen TE, Nykjær A, Blomhoff R, Willnow TE & Moestrup SK (1999) Evidence for an essential role of megalin in transepithelial transport of retinol J Am Soc Nephrol 10, 685–695
20 Moestrup SK, Birn H, Fischer PB, Petersen CM, Verroust PJ, Sim RB, Christensen EI & Nexø E (1996) Megalin-mediated endocytosis of transcobalamin-vita-min-B12complexes suggests a role of the receptor in
Trang 8vitamin-B12homeostasis Proc Natl Acad Sci USA 93,
8612–8617
21 Nykjær A, Dragun D, Walther D, Vorum H, Jacobsen C,
Herz J, Melsen F, Christensen EI & Willnow TE (1999)
An endocytic pathway essential for renal uptake and
acti-vation of the steroid 25-(OH) vitamin D3 Cell 96, 507–
515
22 Nykjær A, Fyfe JC, Kozyraki R, Leheste JR, Jacobsen
C, Nielsen MS, Verroust PJ, Aminoff M, Chapelle A,
Moestrup SK, Ray R, Gliemann J & Christensen EI
(2001) Cubilin dysfunction causes abnormal metabolism
of the steroid hormone 25-(OH) vitamin D3 Proc Natl
Acad Sci USA 98, 13895–13900
23 Christensen EI & Birn H (2001) Megalin and cubilin:
synergistic endocytic receptors in renal proximal tubule
Am J Physiol Renal Physiol 280, F562–F573
24 Moestrup SK, Kozyraki R, Kristiansen M, Kaysen JH,
Rasmussen HH, Brault D, Pontillon F, Goda FO,
Christensen EI, Hammond TG & Verroust PJ (1998)
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 273, 5235–5242
25 Willnow TE, Nykjaer A & Herz J (1999) Lipoprotein
receptors: new roles for ancient proteins Nat Cell Biol
1, E157–E162
26 Birn H, Lebeolleux M, Moestrup SK, Ronco P,
Aucou-turier P & Christensen EI (2002) Receptor mediated
uptake of light chains in kidney proximal tubules In
Monoclonal Gammapathies and the Kidney(Aucouturier
P, Hermine P, Ronco P & Touchard G, eds) Klu¨wer,
the Netherlands
27 Salter DN & Mowlem A (1983) Neonatal role of milk
folate-binding protein: studies on the course of digestion
of goat’s milk folate binder in the 6-d-old kid Br J Nutr
50, 589–596
28 Holm J, Hansen SI & Lyngbye J (1980) High-affinity
binding of folate to a protein in serum of male subjects
Clin Chim Acta 100, 113–119
29 Hansen SI, Holm J & Lyngbye J (1980) High-affinity
protein binding of folate in urine IRCS Med Sci 8,
846–847
30 Selhub J, Emmanouel D, Stavropoulos T & Arnold R
(1987) Renal folate absorption and the kidney
folate binding protein I Urinary clearance studies
Am J Physiol 252, F750–F756
31 Birn H, Spiegelstein O, Christensen E, I & Finnell RH
(2005) Renal tubular reabsorption of folate mediated
by folate binding protein 1 J Am Soc Nephrol 16,
608–615
32 Hjelle JT, Christensen EI, Carone FA & Selhub J
(1991) Cell fractionation and electron microscope
stu-dies of kidney folate-binding protein Am J Physiol 260,
C338–C346
33 Birn H, Selhub J & Christensen EI (1993) Internalization and intracellular transport of folate-binding protein in rat kidney proximal tubule Am J Physiol 264, C302–C310
34 Birn H, Nielsen S & Christensen EI (1997) Internaliza-tion and apical to basolateral transport of folate in rat kidney proximal tubule Am J Physiol 272, F70–F78
35 Svendsen I, Martin B, Pedersen TG, Hansen SI, Holm J
& Lyngbye J (1979) Isolation and characterization of the folate-binding protein from cows milk Carlsberg Res Comnun 44, 89–99
36 Holm J, Hansen SI & Hoier-Madsen M (2002) A com-bination of cation exchange and ligand-affinity chroma-tography for purification of two molecular species of the folate binding protein in human milk, one equipped with a hydrophobic glycosyl phosphatidylinositol tail: characterization of hydrophobicity and electrical charge Biosci Report 22, 443–454
37 Svendsen I, Hansen SI, Holm J & Lyngbye J (1984) The complete amino acid sequence of the folate binding protein from cow’s milk Carlsberg Res Commun 49, 123–131
38 Hansen, SI & Holm, J (1992) Conversion of an appar-ent 100 kDa folate binding protein from human milk, choroid plexus and semen to a 25 kDa molecular species
by phosphatidylinositol-specific phospholipase C Biosci Report 12, 87–93
39 Greenwood FC, Hunter WM & Glover JS (1963) The preparation of I-131-labelled human growth hormone of high specific radioactivity Biochem J 89, 114–123
40 Moestrup SK, Nielsen S, Andreasen P, Jørgensen KE, Nykjær A, Roigaard H, Gliemann J & Christensen EI (1993) Epithelial glycoprotein-330 mediates endo-cytosis of plasminogen activator-plasminogen activator inhibitor type-1 complexes J Biol Chem 268, 16564– 16570
41 Moestrup SK, Kaltoft K, Sottrup-Jensen L & Gliemann
J (1990) The human alpha 2-macroglobulin receptor contains high affinity calcium binding sites important for receptor conformation and ligand recognition J Biol Chem 265, 12623–12628
42 Le Panse S, Galceran M, Pontillon F, Lelongt B, van
de Putte M, Ronco PM & Verroust PJ (1995) Immuno-functional properties of a yolk sac epithelial cell line expressing two proteins gp280 and gp330 of the inter-microvillar area of proximal tubule cells: inhibition of endocytosis by the specific antibodies Eur J Cell Biol
67, 120–129
43 Sahali D, Mulliez N, Chatelet F, Laurent Winter C, Citadelle D, Sabourin JC, Roux C, Ronco P & Verroust
P (1993) Comparative immunochemistry and ontogeny
of two closely related coated pit proteins The 280-kDa target of teratogenic antibodies and the 330-kDa target
of nephritogenic antibodies Am J Pathol 142, 1654– 1667