(Received for publication, February 12, 1996; and in revised form, March 19, 1996)
From the
The blood-brain barrier necessitates disparate macromolecular transport systems in the brain and central nervous system. We now report the discovery of a new member of the low density lipoprotein receptor (LDLR) family whose expression is highly restricted to the brain. The full-length cDNA specifying the chicken receptor (open reading frame, 2754 base pairs) as well as a cDNA for the major portion of its murine homologue have been obtained. The novel receptor shows the greatest similarity to the group of LDLR relatives with 8 ligand binding repeats, in chicken termed LR8 and in mammals, very low density lipoprotein receptors. Thus, in addition to 8 tandemly arranged ligand binding repeats, the five-domain receptor contains an O-linked sugar region and the internalization signal, Phe-Asp-Asn-Pro-Val-Tyr, typical for all LDLR gene family members. In chicken, the 6.5-kb receptor transcript is present at high levels in brain and at much lower levels in extraoocytic cells of the ovary; in mouse, the same transcript of 6.5 kb was detected in brain, but not in heart (the major site of very low density lipoprotein receptor expression), lung, liver, kidney, and ovary. An antibody directed against the predicted carboxyl terminus of the avian receptor detected a 130-kDa protein in brain extracts. The apparent size of the immunoreactive protein is compatible with extensive glycosylation of the 894-residue mature form of the receptor. The presence of this novel receptor in brain of a bird and a rodent suggests an important and evolutionary conserved function.
Since the molecular characterization of the low density
lipoprotein receptor (LDLR)()(1) , an ever
increasing number of related proteins have been discovered. The members
of the LDLR family are characterized by distinct functional domains
present in characteristic numbers. These modules are (i) the
``type A binding repeats'' of
40 residues each,
displaying a triple-disulfide-bond-stabilized negatively charged
surface; certain head-to-tail combinations of these repeats are
believed to specify ligand interactions(1) ; (ii) ``type B
repeats,'' also containing six cysteines each; (iii) modules of
50 residues with a consensus tetrapeptide, YWTD, found in the
epidermal growth factor precursor; (iv) a transmembrane domain, and (v)
the cytoplasmic region with (a) signal(s) for receptor internalization
via coated pits, containing the consensus tetrapeptide Asn-Pro-Xaa-Tyr
(NPXY).
The members of this protein family characterized to date are the LDLR (2) , the LDLR-related protein (LRP)(3) , gp330/megalin(4) , and the VLDL receptor(5) . At least in vitro, these receptors bind a large number of unrelated ligands, suggesting overlapping ligand specificities in vivo(6, 7, 8, 9, 10, 11) . One of these common ligands is the small intracellular ``receptor-associated protein'' (RAP), which acts as a chaperone preventing intracellular ligand-induced receptor aggregation(12, 13) . Interestingly, the most specific family member, the LDLR, which binds only apoB and apoE with high affinity, has the lowest affinity for RAP(14) . In the chicken, we have characterized 4 members of the LDLR family; two of these receptors mediate the massive transport of yolk precursors into growing oocytes. These are (i) the oocyte-specific LDLR relative with 8 binding repeats termed LR8(15) , a homologue of the mammalian VLDL receptors(16) , and (ii) the oocyte-specific LRP(17) . The other two receptors, expressed in somatic cells, are avian homologues of the mammalian LDLR (18) and of LRP, respectively(19) .
It has been proposed that apoE, which is highly expressed in the mammalian central nervous system and is a high affinity ligand for the above-mentioned receptors, may serve as mediator of local lipid transport in the brain. Such transport would be expected to be independent of systemic lipoprotein metabolism, due to separation via the blood-brain barrier(20) . To date, it has been assumed, but not proven experimentally, that in the central nervous system of mammals LDLR and/or LRP could function as corresponding receptor(s) for the uptake of apoE containing, possibly brain-specific, lipoproteins. However, brain abnormalities in humans with homozygous LDLR defects or in mice homozygous for the knock-out of the LDLR gene have not been observed.
Here we report the molecular characterization of LR8B, a new LDLR homologue with 8 ligand binding repeats from chicken and mouse. Since both species express this receptor predominantly in the brain, we propose that it represents a new candidate receptor for brain-specific transport processes.
In order to detect brain-specific members of the LDLR gene family in the chicken, several degenerate primer pairs corresponding to regions highly conserved among members of the gene family were used in RT-PCRs with mRNA from different chicken tissues. Sequence analysis of a 700-bp product obtained from brain mRNA and absent in all other tissues tested revealed, depending upon the domains used for comparison, 40-50% identity with chicken LR8 (16) and somatic LRP (19) . Thus, we used this fragment to screen a chicken brain cDNA library and obtained 2 independent overlapping clones (D1, D2) covering about 2.9 kb. D1 contained an internal EcoRI site (Fig. 1), and the resulting 2 fragments were subcloned independently. Using primers flanking the EcoRI site, we confirmed by RT-PCR that we had not missed a small EcoRI/EcoRI fragment. The 5`-end of the coding region was cloned by 5`-RACE yielding an additional 100 bp containing a single start codon.
Figure 1: Nucleotide and deduced amino acid sequence of the cDNA for chicken LR8B. Amino acid sequence numbering starts from the putative signal sequence cleavage site; negative numbers refer to the signal peptide. Cysteine residues are encircled. Potential O-linked glycosylation sites (threonine clusters) are marked by asterisks, and N-linked glycosylation sites by circles, respectively. In the O-linked sugar domain, single proline residues following clusters of threonines are boxed. The transmembrane domain is underlined. The internalization signal sequence is boxed in, and the internal EcoRI site (see text) is underlined.
The complete cDNA sequence contained one open reading frame of 2754 bp coding for a 918-residue protein (Fig. 1). The single ATG codon at the 5`-end of the cDNA most likely represents the translation initiation site, since it is in a sequence context fulfilling the rules of Kozak(27) . The initiator methionine is followed by a stretch of 23 predominantly hydrophobic residues defining a cleavable signal sequence (28) . The mature protein consists of five domains, starting with a domain of 8 cysteine-rich binding repeats of about 40 amino acids in length and 6 cysteines each. This structure is very reminiscent of the ligand binding domains of LR8 in chicken and the VLDL receptor in mammals, but differs significantly from that of LDLR containing only 7 such repeats. A ``linker'' region connecting repeats 4 and 5 in the LDLR (29) is found between repeats 5 and 6, and therefore in identical location as in the VLDL receptor and LR8(5, 16) . The remaining 4 domains of the protein are entirely analogous to those of the LDLR or of LR8+, the splice variant form of LR8 containing an O-linked sugar region(15) . The 8 ligand binding repeats are followed by the epidermal growth factor precursor domain consisting of 3 type B (3) repeats and five repeats containing the signature tetrapeptide, (Y/F)WXD. The third domain contains several threonine clusters and multiple serine residues flanked by a single proline within 4 residues on either side, sequences which have a high probability of containing O-linked glycans(30) . A hydrophobic region of 22 amino acids at residues 817-838 represents the putative transmembrane domain of the receptor. The intracellular domain contains a perfect internalization signal (FDNPVY)(31) . In summary, alignment of the amino acid sequence of the brain protein with that of LR8 (16) and mammalian VLDL receptors from different species (4, 5, 32, 33) establishes that this newly discovered member of the LDLR family is a novel 8-ligand binding repeat receptor.
Using D2 as probe for Northern blotting, we
re-evaluated the expression pattern found in the original RT-PCR
experiment. Indeed, the corresponding transcript had a size of 6.5
kb (Fig. 2), indicating untranslated 5`- and 3`-regions
totalling at least 3.5 kb, and was detected only in brain. Upon
prolonged exposure (3 days) of the blot, faint signals in granulosa
cell sheets as well as in cultured chicken embryo fibroblasts could be
detected. In contrast to the LDLR, transcript levels in fibroblasts
were not regulated by extracellular cholesterol(18) .
Figure 2:
Expression of LR8B in chicken tissues.
Total RNA (15 µg/lane) was isolated from the various indicated
chicken tissues and cells, denatured and separated by electrophoresis
on a 1.5% agarose gel. Fibroblasts: +, cultured in medium
supplemented with lipoprotein-deficient serum in the presence of 2
µg/ml mevinolin; -, cultured in medium supplemented with
fetal bovine serum in the presence of 25-OH-cholesterol (4 µg/ml).
Hybridization was performed with P-labeled D2 as probe.
The blot was standardized by rehybridizing with a labeled 1.3-kb cDNA
fragment of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Autoradiography was at -70 °C for 1 day
with an intensifying screen.
-DNA digested by BstEII was
used as size marker (in kb).
Polyclonal rabbit antibodies against a synthetic peptide
corresponding to the deduced carboxyl-terminal 14 amino acids of the
novel receptor recognized a protein with an apparent relative molecular
mass of 130 kDa in membrane extracts of brain, but not of liver
and muscle (Fig. 3A). The additional low molecular
weight bands in lanes 1-3 most likely represent
unidentified cross-reactive proteins. Upon affinity chromatography on
RAP-Sepharose of brain membrane extract, the same protein which reacts
with the antibody was present in the bound fraction (Fig. 3B, lane 1). This protein represents 1
of 6 bands reacting with a single chain antibody fragment,
ScFv7(26) , which recognizes the ligand binding domain of all
known members of the LDLR family (Fig. 3B, lane
2). The double band at about 95 kDa most likely corresponds to
very small amounts of the two splice variants of LR8 (15) present in brain and detectable after enrichment on the
RAP-matrix. The other 3 membrane proteins which bound to RAP-Sepharose
and reacted with ScFv7 might be yet unidentified proteins belonging to
the LDLR family. As control for the reciprocity of ScFv7 and RAP in
identification of LDLR family members, we blotted the RAP-Sepharose
eluate with
I-RAP (Fig. 3B, lane
3); no bands other than those detected with ScFv7 were visualized.
Figure 3:
Blotting analysis of chicken LR8B. A, Triton X-100 extracts from total chicken brain (lane
1), skeletal muscle (lane 2), and liver (lane 3)
were subjected to electrophoresis under nonreducing conditions on a
4.5-18% gradient SDS-polyacrylamide gel and transferred onto
nitrocellulose. Strips were incubated with anti-LR8B antibody (10
µg/ml). B, Triton X-100 extract from total chicken brain
was enriched on a RAP-Sepharose matrix as described under
``Experimental Procedures.'' Eluted proteins were subjected
to electrophoresis and blotted as above. Strips were incubated with
anti-LR8B antibody (lane 1, 10 µg/ml), ScFv7 (lane
2, 10 µg/ml), and I-RAP-GST (lane 3,
0.5 µg/ml; specific activity, 10
cpm/ng). Bound
anti-LR8B was visualized with Protein A-horseradish peroxidase and the
chemiluminescence system as described under ``Experimental
Procedures.'' For detection of ScFv7, the anti-myc-tag
antibody 9E10 and rabbit anti-mouse IgG-horseradish peroxidase were
used. Exposure time was 2 min for lanes 1-3 in A and lanes 1 and 2 in B and 24 h for lane 3. The positions of marker proteins are
shown.
To determine whether this newly characterized brain-specific member of the LDLR family is also expressed in mammals, we searched by PCR, performed exactly as described for the chicken receptor, for the corresponding murine mRNA. A 700-bp PCR-amplified product was 90% identical with the corresponding product from chicken brain. We used this fragment to screen a mouse brain cDNA library and sequenced the longest positive clone identified; it contained a continuous open reading frame of 1755 bp including the carboxyl terminus of the protein. Protein sequence alignment of the murine receptor portion with the corresponding region of chicken LR8B showed an overall identity of 73% (Fig. 4), with most of the differences observed in the putative O-linked sugar region. Northern blot analysis revealed that in the mouse the same transcript of 6.5 kb is found in brain, but not in heart, lung, liver, kidney, and ovary (Fig. 5).
Figure 4: Comparison of the partial sequences of murine and chicken LR8B. Numbering of the amino acid residues of chicken LR8B is identical with Fig. 1. Gaps have been introduced to optimize the alignment. Identical residues are boxed.
Figure 5:
Expression of LR8B in murine tissues.
Total RNA (15 µg/lane) prepared from the indicated mouse tissues
were denatured and separated by electrophoresis on a 1.5% agarose gel.
Hybridization was performed with a P-labeled 2-kb partial
cDNA clone of mouse LR8B. The blot was standardized by rehybridizing
with a labeled 1.3-kb cDNA fragment of rat glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Autoradiography was at -70 °C
for 1 day with an intensifying screen.
-DNA digested by BstEII was used as size marker (in
kb).
Since the cloning of the human LDLR(2) , an increasing number of related proteins have been discovered. Among them are LRP, megalin, and the VLDL receptor, to name only those sharing the very same structural modules that constitute the LDLR. The simultaneous discovery of potential ligands for these receptors expanded their possible functions far beyond those in lipoprotein metabolism (reviewed in (7) ).
We now describe a new member of this receptor
family in chicken and mouse, which we have discovered by an RT-PCR
screening approach. Following our suggestion(15) , we term the
new receptor LR8B, where LR stands for LDLR-relative, the number
specifies the number of ligand binding repeats, and the B indicates the
brain-specific expression of this receptor. This nomenclature appears
preferable over one that refers to ligands, which might be misleading
since the list of proposed ligands grows(14, 34) . The
new receptor shows the highest homology, both in modular structure and
primary sequence, to the so-called VLDL receptors. It has 8 ligand
binding repeats, and the linker, a short stretch between binding
repeats 4 and 5 in LDLRs(29) , is located at the same position
as in VLDL receptors, i.e. between repeats 5 and 6. The
presence of a perfect internalization signal, FDNPVY, in the
cytoplasmic domain of the receptor strongly suggests endocytic
competence(31) . Deduced from the sequence, the receptor might
be heavily glycosylated, in agreement with its apparent M of
130,000 on SDS gels versus a
calculated M
of
101,000.
An anti-peptide
antibody detected the 130-kDa protein in brain fractions, but not in
other tissues tested. Due to the expected binding of RAP to the new
receptor, we were able to partially purify the receptor by affinity
chromatography of brain membrane extracts on immobilized RAP. Deduced
from the extremely high conservation of the ligand binding domain with
that of LR8 and mammalian VLDL receptors, we assume that these
receptors potentially recognize a very similar set of ligands:
apoB(24) , apoE(35) ,
-macroglobulin(34) , lactoferrin, and, as
demonstrated here, RAP(14) .
With respect to a possible function of LR8B, we must consider that LR8B is the first member of the LDLR gene family predominantly expressed in brain of such diverse species as chicken and mouse. It is widely appreciated that in mammals apoE might serve as mediator of brain-specific lipid transport, which operates separated from the general circulation by the blood-brain barrier(20) . ApoE is synthesized by astrocytes, and apoE-containing lipoprotein particles are found in cerebrospinal fluid. The involvement of the LDLR and LRP, both expressed in mammalian brain(36, 37) , in apoE-mediated brain-specific lipid transfer is not defined, however. The fact that patients with a total absence of functional LDLRs lack abnormalities in the brain suggests that LDLR is not essential for such pathway(s). LRP may play a crucial role in the brain, since a certain proportion of mice homozygous for the disruption of the LRP gene do implant into the uterus, but die early showing failure of neuronal tube closure and incomplete expansion of brain vesicles(38, 39) . In addition to a role in normal brain physiology, LR8B, if expressed in man, may also be a new candidate for involvement in the pathophysiology of neurodegenerative disorders due to the association of the apoE4 allele with late onset Alzheimer's disease(40) . At first sight, the situation in chicken is different, as birds are not known to synthesize apoE(41, 42) . However, they do synthesize apoAI in brain (43) and, importantly, at sites of peripheral nerve injury and regeneration(44, 45) . In view of these observations, it has been proposed that apoAI in chickens may function in analogy to apoE in mammals(44, 45) . It remains to be tested whether chicken apoAI is the pendant to mammalian apoE in brain metabolism, or whether this novel receptor's physiological ligand is a yet unidentified gene product common to chicken and mammals.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X97001[GenBank].