From the Max-Planck-Institute of Molecular Cell
Biology and Genetics, Pfotenhauerstrasse 108 and
§ Medical Clinic and Polyclinic I TU-Dresden,
Fetscherstrasse 74, D-01307 Dresden, Germany
Received for publication, October 18, 2002, and in revised form, December 26, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Prominin/CD133 is a 115/120-kDa integral membrane
glycoprotein specifically associated with plasma membrane protrusions
in epithelial and non-epithelial cells including neuroepithelial and
hematopoietic stem cells. Here we report the identification as well as
molecular and cell biological characterization of mouse, rat, and human
prominin-2, a 112-kDa glycoprotein structurally related to prominin
(referred to as prominin-1). Although the amino acid identity between
prominin-2 and prominin-1 is low (<30%), their genomic organization
is strikingly similar, suggesting an early gene duplication event. Like
prominin-1, prominin-2 exhibits a characteristic membrane topology with
five transmembrane segments and two large glycosylated extracellular
loops. Upon its ectopic expression in Chinese hamster ovary cells as a
green fluorescent protein fusion chimera, prominin-2 was also found to
be associated with plasma membrane protrusions, as revealed by its
co-localization with prominin-1, suggesting a related role. Consistent
with this, prominin-2 shows a similar tissue distribution to
prominin-1, being highly expressed in the adult kidney and detected all
along the digestive tract as well as in various other epithelial
tissues. However, in contrast to prominin-1, prominin-2 was not
detected in the eye, which perhaps explains why a loss-of function
mutation in the human prominin-1 gene causes retinal
degeneration but no other obvious pathological signs. Finally, we
present evidence for the existence of a family of pentaspan membrane
proteins, the prominins, which are conserved in evolution.
Prominin-1 is a 115-kDa glycoprotein originally described to be
expressed on the apical surface of mouse neuroepithelial stem cells and
several other epithelia including kidney brush border membranes (1).
Remarkably, within the apical plasma membrane domain, prominin-1 is
selectively associated with microvilli and other related plasma
membrane protrusions rather than with the planar portion of the
membrane (1, 2). This specific subcellular localization does not depend
on an epithelial phenotype because prominin-1 is selectively found in
plasma membrane protrusions when ectopically expressed in fibroblasts
(1). The retention of prominin-1 in these plasma membrane protrusions
involves a novel cholesterol-based lipid microdomain, referred to as
Lubrol raft, in which prominin-1 interacts specifically with
cholesterol (3).
The human orthologue of mouse prominin-1 (CD133; prominin (mouse)-like
1 (PROML1)) has been originally identified as an antigenic marker
(AC133 antigen) on a subset of hematopoietic stem cells (Refs. 4 and 5
and for review see Ref. 6). Human and murine prominin-1 share a
similar, if not identical, cellular distribution and subcellular
localization (7, 8).1 They
show an average 60% amino acid identity and display a characteristic membrane topology that is unique among the multispan transmembrane proteins, with an N-terminal extracellular domain, five transmembrane segments flanking two short cytoplasmic loops and two large
glycosylated extracellular loops (over 250 residues each), and a
cytoplasmic C-terminal domain (9-11).
Although the precise physiological function of prominin-1 is
incompletely understood, a clue about its role comes from studying human pathology. In a consanguineous pedigree from India, individuals afflicted with retinal degeneration are homozygous carriers of a
frameshift mutation in the prominin-1 gene, which results in a truncated protein that is no longer transported to the cell surface
(12). In keeping with the preferential association of prominin-1 with
plasma membrane protrusions (1, 2, 7), prominin-1 is concentrated in
the membrane evaginations at the base of the outer segment of rod
photoreceptor cells, which are precursor structures in the biogenesis
of photoreceptor disks (12). Given the ability of prominin to interact
with cholesterol within a specific membrane microdomain (3), it has
therefore been proposed that this pentaspan membrane protein has a role in establishing and/or maintaining certain plasma membrane protrusions (8).
The existence of several open reading frames
(ORFs)2 in the
Caenorhabditis elegans genome encoding predicted proteins
structurally related to prominin-1 (9) raised the possibility that
prominin-1 may be the first characterized members of a novel family of
polytopic membrane proteins occurring throughout the animal kingdom.
The existence of a prominin paralogue in humans could potentially explain why the homozygous carriers of the frameshift mutation in the
prominin-1 gene resulting in retinal degeneration (12) lack
other obvious pathological signs.
In the present study, we report the identification, molecular cloning,
cell biological characterization, tissue distribution, and genomic
organization of novel mouse, rat, and human cDNAs encoding a
membrane glycoprotein structurally related to prominin-1 and referred
to as prominin-2. Furthermore, sequence analyses of mammalian
prominin-2 as well as proteins predicted from several expressed
sequence tag (EST) clones derived from various other vertebrate and
invertebrate organisms, such as chicken, fish, fly, and worm, provide a
first description of the prominin family.
Data Base Searches and Computer Analyses--
Nucleotide and
protein sequence data bases were searched at the National Center for
Biotechnology Information, Berkeley Drosophila Genome
Project data base, and Celera Discovery System by using the BLAST
network services (13). ScanProsite searches in Swiss-Prot and TREMBL
protein data bases (including weekly releases of Swiss-Prot) were
performed at the ExPASy Molecular Biology www server (14) of the Swiss
Institute of Bioinformatics (www.expasy.ch/). The exonic organization
of human and mouse prominin-2, prominin-1, and
Drosophila melanogaster prominin-like genes were determined by comparing a given cDNA with the corresponding genomic sequence using BLAST. Pairwise protein sequence comparisons were performed using
the ALIGN program with a BLOSUM 50 matrix (15) and multiple sequence
alignments using the ClustalW program (16). Molecular phylogenetic
analysis was done using PHYLIP software (17) at the infobiogen server
(www.infobiogen.fr). Reliability of the branching was analyzed by the
bootstrap method with 200 replications (18).
EST Clones and DNA Sequencing--
The EST clones, Life Tech
mouse embryonic clone mp20c10 (GenBankTM accession number
AA396526) and human IMAGE clone qu80c08 (GenBankTM
accession number AI285647), were obtained from Resource Center/Primary Data of the German Human Genome Project at the Max-Planck-Institute for
Molecular Genetics; the human IMAGE clone qk36c08
(GenBankTM accession number AI92608), Danio
rerio clones fb75c01 (GenBankTM accession number
AI545699) and fm10h05 (GenBankTM accession number
BG307062), D. melanogaster clones LD23965 (GenBankTM accession number AA820383), LD22538
(GenBankTM accession number AA817301), and LD16666
(GenBankTM accession number AA441662) were purchased from
Research Genetics, Inc. (Huntsville, AL); the human clone au65a10
(GenBankTM accession number AI929095) and chicken clone
pgf1n.pk006.j1 (GenBankTM accession number BI065967) were
from Genome System Inc. (St. Louis, MO) and University of
Delaware Chicken EST data base of Delaware Biotechnology Institute
(Newark, DE), respectively. All EST clones were completely sequenced on
both strands by primer walking along the cDNA, using either the
AutoreadTM sequencing kit and an ALFexpressTM
sequencer (Amersham Biosciences) or the ABI PRISMTM Dye
Terminator Cycle Sequencing kit and an Applied Biosystems model 377 DNA
sequencer (Applied Biosystems, Foster City, CA).
Cloning of the Murine, Human, and Rat Prominin-2 cDNA
Sequences--
Cloning of the murine, human, and rat prominin-2
cDNA sequences is described in detail in the Supplemental Material.
Plasmid Construction--
The eukaryotic expression plasmid
pCMV-prominin-2, containing the entire coding sequence of mouse
prominin-2, was obtained by subcloning the
HindIII-XhoI cDNA fragment released from the pCR-Blunt II TOPO-mouse prominin-2 plasmid into pCMV-Tag5c (Stratagene) digested with the same restriction enzymes. To obtain the pCMV-mouse prominin-2-myc plasmid, an XhoI site, destroying the
wild-type amber stop codon of the mouse prominin-2 cDNA and
allowing the fusion in-frame with the Myc tag cDNA of
pCMV-prominin-2, was introduced by PCR using m3 (see Supplemental
Material) and the primer 5'-cggtgggttctcgagcttcagagag-3'. The PCR
product was digested with AccI and XhoI, and the
resulting AccI-XhoI cDNA fragment containing
the mutated 3' region of the mouse prominin-2 cDNA was used to
replace the corresponding wild-type cDNA fragment in the
pCMV-prominin-2 plasmid. The pEGFP-N1-prominin-2 plasmid, containing
the entire coding sequence of rat prominin-2 fused in-frame to the N
terminus of green fluorescent protein (GFP), was constructed by
selective PCR amplification of the corresponding cDNA
(GenBankTM accession number AF508942) using the
oligonucleotides 5'-tgcttcccagaattccgaaaccatgacgcgca-3' and
5'-tcaccttgcccggtggatcccatagcttcagg-3' as 5' and 3' primers, respectively. The 5' and 3' primers created an EcoRI and
BamHI restriction site, respectively. In addition, the
nucleotide sequence flanking the initial start codon in the 5' primer
(see underlined letters above) was converted to a Kozak consensus
translation initiation site to further increase the translation
efficiency. The resulting PCR fragment was digested with
EcoRI and BamHI and cloned into the corresponding
sites of pEGFP-N1vector (Clontech).
The bacterial expression plasmid pGEX-E3, containing the mouse
prominin-2 cDNA from nt 1891 to 2193 (residues
Ile582-Thr682) fused in-frame to glutathione
S-transferase, was constructed by selective PCR
amplification of the corresponding cDNA using the oligonucleotides
5'-atacccacagaattcagcaggagc-3' and 5'-gacactgaattcgagctaggtcacaagg-3' as 5' and 3' primers, respectively. Both oligonucleotides created an
EcoRI restriction site, and the 3' primer introduced in
addition an amber stop codon. The resulting PCR fragment was digested
with EcoRI and cloned into the corresponding site of pGEX-2T
vector (Amersham Biosciences).
In all cases, the PCR DNA fragments were verified by sequencing.
Antiserum against Recombinant Prominin-2--
The recombinant
glutathione S-transferase-prominin-2 fusion protein,
containing a fragment of the second extracellular loop of prominin-2
(residues Ile582-Thr682), was expressed in
BL21 Escherichia coli, purified on glutathione-Sepharose 4B
beads (Amersham Biosciences) as described previously (2), and used as
antigen to generate the rabbit antiserum Cell Culture and Transfection--
CHO cells were cultured as
described previously (7) and either double-transfected with the
eukaryotic expression plasmid pRc/CMV-prominin containing the mouse
prominin-1 cDNA (1) and the pEGFP-N1-prominin-2 plasmid, or
transfected with the pCMV-mouse-prominin-2 plasmid or the pCMV-mouse
prominin-2-myc plasmid alone, using LipofectAMINE (Invitrogen)
according to the supplier's instructions. After 24 h, the medium
was changed, and the cells were incubated for an additional 17 h
in the presence of 5 mM sodium butyrate. Transiently
transfected cells were then either used for immunofluorescence or
solubilized in ice-cold solubilization buffer (1% Triton X-100, 0.1%
SDS, 150 mM NaCl, 5 mM EGTA, 50 mM
Tris-HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 10 µg/ml aprotinin), and the detergent extract
obtained after centrifugation (10 min, 10,000 × g) was
used for deglycosylation, SDS-PAGE, and immunoblotting.
Endoglycosidase Digestions and Immunoblotting--
CHO cell
detergent extracts corresponding to one-fifth of an 80% confluent
60-mm dish, membranes from adult mouse kidney (50 µg of protein)
(11), or detergent extracts (100 µg protein) corresponding to
one-eighth of an adult mouse eye prepared as described (12) were
incubated overnight at 37 °C in the absence or presence of either 1 unit of peptide:N-glycosidase F (PNGase F) or 10 milliunits
of endo- Immunofluorescence and Confocal Microscopy--
Cell surface
immunofluorescence analysis was carried out as described previously
(2). Prominin-1 and/or prominin-2-GFP transfected CHO cells grown on
glass coverslips were washed with Ca/Mg-PBS (PBS containing 1 mM CaCl2 and 0.5 mM
MgCl2), first at room temperature and then on ice, and
surface-labeled for 30 min at 4 °C by the addition of the rat mAb
13A4 (10 µg/ml) against prominin-1 (1) or mouse mAb anti-GFP (clones
7.1 and 13.1; 1/500) (Roche Molecular Biochemicals) diluted in
immunofluorescence buffer (Ca/Mg-PBS containing 0.2% gelatin). Unbound
antibodies were removed by five washes with ice-cold immunofluorescence
buffer. Fixative, 3% paraformaldehyde in PBS, was added to the cells
on ice, and the coverslips were placed at room temperature for 30 min.
The fixative was removed by three washes with immunofluorescence buffer, and the residual formaldehyde was quenched for 30 min with 0.1 M glycine in PBS. The cells were then incubated for 30 min
at room temperature with Cy3-conjugated goat anti-rat IgG (H + L) or
rhodamine Red-X conjugated goat anti-mouse IgG (H + L) (Jackson
ImmunoResearch). Coverslips were rinsed sequentially with
immunofluorescence buffer, PBS, and distilled water, and mounted in
Mowiol 4.88 (Calbiochem).
In experiments with fixed, permeabilized cells, prominin-2,
prominin-2-myc, or prominin-2-GFP transfected CHO cells grown on glass
coverslips were washed with PBS and fixed with 3% paraformaldehyde in
PBS for 30 min at room temperature. Coverslips were then rinsed with,
and incubated for 10 min in, PBS containing 50 mM ammonium chloride. In the case of prominin-2-GFP transfected cells, the fixed
cells were permeabilized and blocked with 0.2% saponin, 0.2% gelatin
in PBS (blocking solution) for 30 min at room temperature. Cells were
then incubated sequentially for 30 min at room temperature with mouse
mAb anti-GFP (1/500) and rhodamine Red-X conjugated goat
anti-mouse IgG (H + L), both in blocking solution. Coverslips were
rinsed and mounted as described above. In the case of prominin-2 or
prominin-2-myc transfected cells, the fixed cells were incubated sequentially with 0.2% gelatin in PBS for 30 min, PNGase F buffer (0.1% Triton X-100, 0.05% SDS, 25 mM EDTA, 50 mM sodium phosphate, pH 7.2, and 1%
The cells were observed with a Leica TCS SP2 confocal laser scanning
microscope using a ×100/1.40-0.7 objective. The confocal settings
were such that the photomultipliers were within their linear range.
With prominin-1 and prominin-2-GFP double-transfected cells, the GFP
and Cy3 fluorophore were excited sequentially to minimize potential of
cross-collection of signal. The images shown were prepared from the
confocal data files using Adobe Photoshop software.
mRNA Expression Analysis--
Northern blot analyses were
performed using either mouse Northern blot membrane
(Clontech, catalogue number 7762-1) or human Northern blot membranes (Clontech, catalogue
numbers 7780-1 and 7782-1). Blots 7782-1 and 7780-1/7762-1 contain ~1
and 2 µg of poly(A)+ RNA per lane, respectively.
A BstEII-digested PCR fragment corresponding to nt
1880-2634 of the murine prominin-2 cDNA (GenBankTM
accession number AF269062) and an EcoRV-BstEII
fragment (nt 1852-2617) derived from the 5.0-kb human prominin-2
cDNA (GenBankTM accession number AF245303) were used to
generate the murine and human prominin-2-specific cDNA probes,
respectively. For the selective PCR amplification of the murine probe,
the sense m6 (5'-atacccacagaattcagcaggagc-3') and antisense m7
(5'-gtcatgaggagaaagtgc-3') primers were used together with the murine
EST clone mp20c10 as template (see Supplemental Material). The PCR
product was digested with BstEII, and the resulting 755-bp
fragment was purified on an agarose gel. The human prominin-1 probe has
been described previously (7).
Probes were labeled with [
Northern dot blot analysis was performed using Human Multiple Tissue
Expression (MTETM) Array membrane
(Clontech, catalogue number 7775-1). The amount of
poly(A)+ RNA in each dot of the array was normalized by the
manufacturer to yield similar hybridization signal for eight
housekeeping genes. Therefore, the amounts of mRNA in each dot vary
from 50 to 956 ng, and the array allows for comparative analysis of
gene expression in various tissues. The blot was hybridized with the
human prominin-2-radiolabeled probe for 6 h at 65 °C and washed
according to the Clontech protocol (manual
PT3307-1). The blot was exposed for 2 days at Identification and Molecular Cloning of Prominin-2--
To
identify potential vertebrate paralogues of prominin-1, data bases were
searched with human and mouse prominin-1 sequences, and EST clones
showing homology were sequenced and used to obtain full-length
cDNAs by PCR amplification. This eventually resulted in the
identification and molecular cloning of human, mouse, and rat
prominin-2, as described in detail in the Supplemental Material.
Prominin-2 Is Structurally Related to Prominin-1--
The ORF of
human and rat prominin-2 cDNAs predicts an 834-amino acid protein
with a molecular weight of 91,900 and 92,800, respectively, whereas
that of mouse prominin-2 cDNA predicts an 835-amino acid protein
with a molecular weight of 93,200 (Fig. 1). For all three prominin-2 sequences,
hydropathy analysis indicates six hydrophobic segments (data not
shown). The first N-terminal hydrophobic segment most likely
corresponds to a signal peptide. A potential signal peptidase cleavage
site is found after serine 21 (Fig. 1, triangle). The other
five hydrophobic segments constitute putative transmembrane domains
(Fig. 1, solid lines). Strikingly, the similarity of the
hydropathy profiles of prominin-2 and prominin-1 proteins suggests the
same membrane topology. Consequently, human and rodent prominin-2, like
prominin-1, are predicted to be pentaspan membrane proteins with two
large extracellular loops, each containing more than 250 residues. Like
prominin-1, prominin-2 contains a cysteine-rich region of as yet
unknown function, which is located at the transition of the first
transmembrane segment to the first small cytoplasmic loop (residues
128-136) (Fig. 1, asterisks). In all three prominin-2
sequences, the up to nine potential N-glycosylation sites
are found in the two extracellular loops (data not shown), with six
conserved in position, two in the first and four in the second loop
(Fig. 1,
The predicted primary structures of mouse and rat prominin-2 are highly
related to each other, with an overall amino acid identity and
similarity of 88 and 93%, respectively. Human and rodent prominin-2
show
To corroborate further that prominin-2 has the same membrane topology
as prominin-1 and, specifically, that its C-terminal domain is located
in the cytoplasm, prominin-2 was expressed in CHO cells as a fusion
protein, with GFP tagged to its C-terminal domain. By using an antibody
against GFP as topological probe in immunofluorescence experiments on
either intact (Fig. 2, A and
B) or saponin-permeabilized (Fig. 2, C and
D) prominin-2-GFP-transfected CHO cells, we could show that
the GFP tag of the fusion protein was indeed located inside the cells
(Fig. 2, B versus D), consistent with
a cytoplasmic location of the C-terminal domain of prominin-2. Similar
results were obtained with C-terminally Myc-tagged prominin-2 (data not
shown), further supporting a prominin-1-like membrane topology of
prominin-2.
Prominin-2 Is a Plasma Membrane Glycoprotein--
To analyze
further the expression of prominin-2, a rabbit antiserum, referred to
as
In CHO cells transiently transfected with the full-length mouse
prominin-2 cDNA, immunoblotting with
Surprisingly, upon immunoblotting of mouse kidney membranes using the
Co-localization of Prominin-2 with Prominin-1 in Plasma Membrane
Protrusions--
Because prominin-1 is specifically localized in
microvilli and related plasma membrane protrusions in various cell
types, it was of interest to investigate if prominin-2 is also
associated with plasma membrane protrusions. To this end, we
co-expressed prominin-1 and prominin-2-GFP in CHO cells and examined
their potential co-localization in plasma membrane protrusions by
confocal laser scanning microscopy. The pattern of green fluorescence
of the prominin-2-GFP fusion protein (Fig.
5, top panel) largely coincided with that of prominin-1 immunofluorescence (Fig. 5, bottom panel), indicating the specific association of
prominin-2-GFP with plasma membrane protrusions.
Tissue Distribution of Murine and Human Prominin-2
mRNAs--
We examined the expression of prominin-2 mRNA in
different mouse tissues by Northern blot analysis. A 4.4-kb-long
transcript was found in kidney and testis (Fig.
6A, arrow), whereas
a shorter transcript of 1.35 kb was detected in heart and skeletal
muscle (Fig. 6A, asterisk). The length of the
long transcript corresponded to what would be expected for a prominin-2
mRNA as deduced from the cDNA sequences (4307 nt). The nature
of the 1.35-kb transcript, which is too short to encode full-length
prominin-2, was not investigated further, also because no such
transcript was detected in human tissues (see below).
As to human prominin-2, we first examined the relative abundance of its
mRNA in 76 different human tissues and tumor cell lines using a
multiple tissue expression array (Fig.
7A). Human prominin-2 showed
strong expression in the adult kidney (A7). Prominin-2
mRNAs were also detected in all tissues of the digestive tract
(columns 5 and 6, A-C), prostate
(E8), trachea (H7), salivary gland
(E9), thyroid gland (D9), mammary gland
(F9), and placenta (B8). No expression was
evident in any of the eight tumor cell lines (column 10),
although two EST clones encoding part of human prominin-2 that derived
from a colon adenocarcinoma have been deposited in the
GenBankTM data base (accession numbers BE867546 and
AI792608), and other EST clones (accession numbers AW292801 and
BE762637) indicate that prominin-2 can be expressed in tumors of the
lung and nervous system.
To characterize further human prominin-2 transcripts, Northern blot
analyses were performed on poly(A)+ RNA. Consistent with
the two cDNAs that were amplified by PCR (Supplemental Material,
Fig. 1B), two main transcripts of
Although the cDNA probes used for the detection of human and mouse
prominin-2 encompassed equivalent regions (i.e. the
C-terminal third of the molecule), no 1.35-kb mRNA was detected in
human skeletal muscle or heart (Fig. 7B), as opposed to the
mouse (Fig. 6A). As for both mouse and human, the long
transcripts (Figs. 6A and 7B) showed the highest
expression in the kidney, and the nature of the short transcript in
mouse heart and skeletal muscle is questionable. Moreover, no such
signal was observed in mouse when an N-terminal probe was used (data
not shown).
Expression of Prominin-1, but Not Prominin-2, in the
Eye--
Patients carrying a mutation in the prominin-1
gene resulting in a truncated protein that is no longer transported to
the cell surface are afflicted with retinal degeneration but do not show obvious pathological signs in other tissues expressing prominin-1 (12). This may be so because, given that prominin-2 is expressed in
most of the tissues known to express prominin-1 (Fig. 7) (5, 7),
prominin-2 may perhaps be able to functionally substitute for the lack
of prominin-1. It was therefore of particular importance to investigate
whether or not prominin-2 occurs in the retina. Prominin-2 was not
detectable in immunoblots of the adult mouse eye (Fig.
8, lanes 3 and
4, arrowhead and arrow), in contrast to prominin-1 which was readily detectable (Fig. 8, lanes 5 and 6), confirming previous observations (12). As a positive
control to detect prominin-2 immunoreactivity in immunoblots, kidney
membranes were analyzed in parallel (Fig. 8, lanes 1 and
2, arrowhead and arrow), which yielded
the same results as in Fig. 4. We conclude that prominin-2, in contrast
to prominin-1, is not expressed in the retina.
Occurrence of Prominins throughout the Animal Kingdom--
Data
base searches and the sequencing of several EST clones displaying
similarities to mammalian prominin-1 and prominin-2 (see Supplemental
Material, Table I) indicate that related molecules exist throughout the
animal kingdom. Prominin-like proteins are found in other vertebrates
such as chicken (GenBankTM accession number AF406812) and
fish (D. rerio, GenBankTM accession numbers
AF160970 and AF373869). With regard to invertebrates, prominin-like
proteins are found in fly (D. melanogaster, GenBankTM accession numbers AF127935 and AF197345) and worm
(1, 9). Additional prominin-like proteins are also present in the data
base as EST clones originating from various other species (Ascaris suum, GenBankTM accession numbers
AW165790 and BG733718; Strongylocentrotus purpuratus,
accession number AF122176). The evolutionary relationship between these
proteins is displayed in Fig.
9A. The phylogenetic tree
clearly indicates the segregation of two orthologous groups in mammals,
prominin-1 and prominin-2 (Fig. 9A, pink boxes).
The orthologue versus paralogue relationship of the chicken
and fish prominin-like proteins with regard to the prominin-1 or
prominin-2 group is more ambiguous. As can be seen from the greater
consensus among vertebrate sequences (see Fig. 9B,
below), invertebrate prominins are more distantly related (Fig.
9A).
A Prominin Signature--
Multiple amino acid sequence alignment
of mammalian prominin-1 and -2 as well as the prominin-like proteins
from other species indicated that very few residues are conserved in
all sequences. Several of such conserved residues cluster in a region
including the end of the second extracellular loop and the fifth
transmembrane domain (Fig. 9B). In this region, four
completely conserved residues (Cys, Pro, Cys, and Trp) followed by a
stretch of hydrophobic residues are found. Furthermore, a consensus
core sequence
CXPX(12,13)CX(5)(P/S)X(4)WX(2)hX(4)hhXh can be noticed, in which X stands for any residue; the
number of X is indicated in parentheses; residues in
parentheses indicate alternatives for a given position, and h stands
for any hydrophobic residue (Fig. 9B). Interestingly,
ScanProsite searches in Swiss-Prot and TREMBL protein data bases using
this consensus core sequence identified no other protein. Because all
the membrane proteins structurally related to prominin-1 and prominin-2
share this consensus core sequence, we propose to refer to it as the
prominin signature.
The Prominin Genes--
Running a BLAST search with the 5.0-kb
human prominin-2 cDNA sequence as query identified the clone
RP11-468G5 located to chromosome 2 (GenBankTM accession
number AC009238) as containing the corresponding gene. Similarly, the
celera clone GA_X5J8B7W49H2 (Celera Discovery System), located to mouse
chromosome 2, was identified as containing the murine
prominin-2 gene. Human and mouse prominin-2 genes
contain 24 exons distributed over 16,853 and 14,492 bp, respectively
(Fig. 10). All of the human
prominin-2 and all except one of the mouse prominin-2 gene exon/intron boundaries conform to the GT-AG
rule (see Supplemental Material, Table II). Introns are conserved in position and phase. In both prominin-2 genes, exon 1 contains the translation-initiation codon and exon 23 the stop codon,
with the 3'-untranslated region being almost entirely encoded by the exon 24. The two, 5.0 and 4.2 kb, human prominin-2 cDNAs result from alternative splicing due to the presence of an internal acceptor site within exon 24 (data not shown).
The exon/intron organization of prominin-2 gene is
strikingly similar to that of the prominin-1 gene (Fig. 10),
which was partially determined by Maw et al. (12) and
completed in the present study (see Supplemental Material, Table III).
The human prominin-1 gene is composed of 27 exons
distributed over more than 75 kb on chromosome 4 (locus p16.2-p12). We
also determined the structure of the murine prominin-1 gene
(see Supplemental Material, Table III), which is located to chromosome
5 (20). As in the case of prominin-2, the structure of the mouse
prominin-1 gene turns out to be identical to that of its
human orthologue. The exon/intron boundaries of prominin-1 and
prominin-2 can be largely superimposed (Fig. 10). Introns, although
larger in mouse and human prominin-1 than
prominin-2, are remarkably concordant in position and phase.
In comparison to the prominin-2 genes, an additional
symmetric exon (human exon 3 and mouse exon 4) encoding the 9-amino
acid stretch PETVILGLK and PEIIVLALK is alternatively spliced into the
N-terminal extracellular domain of human and murine prominin-1,
respectively (see Refs. 10 and 21; GenBankTM accession
number AK027420). Prominin-1 variants containing these additional 9 amino acid residues have been previously referred to as B1
isoforms (8). Two other additional exons coding for part of the
cytoplasmic C-terminal tail of prominin-1 are also inserted in the
prominin-1 genes (Fig. 10). Remarkably, the
prominin-2 genes are over four times more compact than the
prominin-1 genes (
The genomic organization of mammalian prominins also shows significant
similarity with the invertebrate prominin-like proteins such as the one
encoded by the C. elegans F08B12.1 gene (22); of the 19-exon
boundaries of the F08B12.1 gene, 16 are concordant in
position with the mammalian prominin genes (Fig. 10).
Similarly, the genomic organization of D. melanogaster prominin-like protein (see FlyBase annotation
CG7740) also exhibits several conserved exon/intron boundaries with
worm and mammalian prominins (Fig. 10).
We have identified and characterized prominin-2, a novel polytopic
membrane protein. Despite the low level of amino acid identity, this
protein displays structural features characteristic of prominin-1, i.e. five transmembrane domains and two large extracellular
glycosylated loops. The membrane topology of prominin-2 is supported by
analyses of N-glycosylation and epitope accessibility.
Likewise, the genomic organization of prominin-1 and prominin-2 is
nearly identical, indicating that prominin-2 is indeed a paralogue of
prominin-1. This is also supported by phylogenetic analysis.
Identification of several prominin-like proteins in various species and
the analysis of their evolutionary relationship strongly suggest that
the prominin family arose through an early gene duplication event.
Interestingly, whereas the prominins are conserved through metazoan
evolution, they appear to be absent in yeast, suggesting that their
role is related to some aspect of multicellularity.
The presence of prominin-2 in plasma membrane protrusions of
transfected CHO cells, as shown by its co-localization with prominin-1, raises the possibility that prominin-2 may exert a similar function as
prominin-1, which is thought to be involved in the organization of
plasma membrane microdomains (8). In light of our observation that the
tissue distribution of prominin-2 largely overlaps with that of
prominin-1 (Fig. 7) (5, 7), this in turn would suggest that in tissues
expressing both prominins, the loss of prominin-1 may be compensated by
prominin-2 and vice versa. Two tissues deserve special comment in this
regard, the retina and the esophagus. A mutation in the human
prominin-1 gene resulting in the loss of cell surface
prominin-1 causes retinal degeneration but no obvious pathological
signs in other tissues (12). If this were due to prominin-2
compensating for the loss of prominin-1 in these tissues, one would
expect that prominin-2 should not be expressed in the retina, which
would provide a possible explanation of the pathology observed upon the
loss of cell surface prominin-1 in this tissue. Indeed, in contrast to
prominin-1, prominin-2 was not detected in the eye (Fig. 8).
The opposite situation is the case for the esophagus, which expresses
prominin-2 but not prominin-1. The epithelium of the esophagus is known
to lack microvilli on the apical surface. Preliminary data using
Madin-Darby canine kidney cells transiently transfected with
prominin-2-GFP suggest that whereas prominin-1 is restricted to
microvilli of the apical surface of these epithelial cells (2),
prominin-2 is found not only on the apical but also the lateral plasma
membrane, which also forms
protrusions.3 It therefore
appears that in terms of both tissue distribution and subcellular
localization, prominin-1 and prominin-2 are largely overlapping but not identical.
Both prominin-1 and prominin-2 are subject to alternative splicing. In
fact, both prominin genes contain numerous short exons, which raises the possibility of a greater modular structure of these
proteins than suggested by their domain organization derived from their
pentaspan topology. Indeed, several human prominin-2 clones amplified
in the present study revealed differential splicing, with variable
consequences with respect to the structure of the protein. Two clones
lacked exon 6, resulting in an in-frame deletion of 30 amino acids in
the first large extracellular loop. Eight clones lacked the entire
fifth exon, and two clones were missing part of exon 7 due to use of an
alternative internal splice donor site (nucleotide 1039). The latter
two deletions result in a frameshift with premature termination of the
prominin-2 ORF. This would lead to the translation of short splice
variants of 209 and 312 amino acid residues, respectively, with only
two transmembrane domains. Given that
similar observations on alternative splicing have been made with
prominin-1 (8),4,5 it appears
that a wide variety of related polypeptides can be generated from the
two prominin genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
E3 (alias SA7564)
(EUROGENTEC Bel S.A. Seraing, Belgium).
-N-acetylglucosaminidase H (endo H) according to
the manufacturer's instructions (Roche Molecular Biochemicals).
Proteins were then analyzed by SDS-PAGE and transferred to
poly(vinylidene difluoride) membranes (Millipore Corp. Bedford, MA;
pore size 0.45 µm) using a semi-dry transfer cell system (Cti, Idstein, Germany). After the transfer, membranes were incubated overnight at 4 °C in blocking buffer (PBS containing 5% low fat milk powder and 0.3% Tween 20). Prominin-2 was then detected using
E3 antiserum (1/3000) followed by horseradish peroxidase-conjugated secondary antibody, both diluted in blocking buffer. Antigen-antibody complexes were visualized using enhanced chemiluminescence (ECL system,
Amersham Biosciences).
-mercaptoethanol)
containing 1 unit of PNGase F for 3 h, and 0.2% gelatin in PBS
for 30 min, all at room temperature. Cells were then double-labeled for
30 min at room temperature with
E3 antiserum (1/500) and mouse mAb
anti-c-Myc antibody (clone 9E10; 1/100) (Sigma) followed by
Cy3-conjugated goat anti-rabbit IgG (H + L) and Cy2-conjugated goat
anti-mouse IgG (H + L) (Jackson ImmunoResearch), all diluted in PBS
containing 0.2% gelatin. Coverslips were rinsed and mounted as
described above.
-32P]dCTP by the
random-prime method using the Rediprime kit (Amersham Biosciences). In
all cases, blots were prehybridized at 68 °C for 30 min and
incubated with the appropriate radiolabeled probe at 68 °C for
1 h in the ExpresshybTM hybridization solution
(Clontech). After hybridization, blots were washed
at room temperature with solution 1 (2× SSC, 0.05% SDS), followed by
solution 2 (0.1× SSC, 0.1% SDS) at 50 °C according to the
Clontech protocol (manual PT1190-1). Blots were
analyzed by autoradiography at
80 °C using Kodak X-Omat AR x-ray
film and an intensifying screen (3 and 7 days of exposure). The blots were then stripped and, before a new hybridization, re-exposed for 3 days to ascertain the complete removal of the radioactive probe.
80 °C. For
quantification, non-saturated exposures were scanned with an ARCUS II
scanner and quantified using MacBas software (Raytest Isotopenmessgeräte GmbH, Pforzheim, Germany).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
). The two large
extracellular loops also contain six cysteine residues conserved in
position between prominin-2 and prominin-1 that are likely to form
disulfide bridges (Fig. 1, #).
View larger version (126K):
[in a new window]
Fig. 1.
Comparison of the amino acid sequence of
rodent and human prominin-2 with human prominin-1. The mouse
(m), rat (r), and human (h) prominin-2
(PROM-2) sequences determined in the present study were
aligned with human prominin-1 (PROM-1). Shaded
boxes, amino acid residues that are identical between at least
three of the sequences; triangle, predicted cleavage site of
the signal peptide of prominin-2; solid lines, predicted
membrane-spanning segments; asterisks, cysteine-rich region;
, conserved potential
N-glycosylation sites in prominin-2; #, conserved
cysteine residues in the extracellular domains of prominin-2 and
prominin-1.
75% overall amino acid identity. Remarkably, the C-terminal
domain, constituted in all three molecules by the last 34 residues, is
perfectly conserved. (The lack of conservation of the C-terminal domain
in the rat prominin-2 sequence reported by Zhang et al.
(19), i.e. after our deposition of the first prominin-2
sequences in the data base in 2000 (8), is due to a sequence error; see
Supplemental Material). Besides the C-terminal tail, the best conserved
regions of human and rodent prominin-2 are the first (residues
106-127) and fifth (residues 782-801) transmembrane domains, with 91 and 95% amino acid identity, respectively. Human, mouse, and rat
prominin-2 show only 26, 29, and 30% amino acid identity,
respectively, to their prominin-1 paralogues.
View larger version (57K):
[in a new window]
Fig. 2.
Intracellular localization of the C-terminal
domain of prominin-2. CHO cells were transiently transfected with
the expression vector containing the rat prominin-2-GFP fusion protein
cDNA. Intact cells at 4 °C (A and B) or
paraformaldehyde-fixed, saponin-permeabilized cells (C and
D) were incubated with anti-GFP antibodies (anti-GFP)
followed by rhodamine Red-X conjugated goat anti-mouse antibody
and double fluorescence analysis using confocal microscopy. GFP
fluorescence of prominin-2-GFP fusion protein is shown in A
and C. Single optical xy plane sections near the
bottom of the cell are shown. Bar in B, 5.4 µm;
bar in D, 6.2 µm.
E3, was raised against a portion of the second large
extracellular loop of murine prominin-2 (amino acids 582-682).
Although this antiserum, upon cell surface immunofluorescence of
transfected CHO cells, did not recognize the native form of prominin-2
(data not shown), it did reveal, upon treatment of fixed cells with
PNGase F, the presence of prominin-2 at the plasma membrane (Fig.
3A). The authenticity of this
immunostaining was confirmed by its dependence on the presence of
prominin-2 cDNA upon transfection of CHO cells (Fig. 3,
A versus B) and by the essentially
identical pattern observed with the
E3 antiserum and anti-Myc
antibodies upon immunostaining of CHO cells transfected with Myc-tagged
prominin-2 (Fig. 3, C and D).
View larger version (83K):
[in a new window]
Fig. 3.
E3 antiserum recognizes the
authentic mouse prominin-2. CHO cells were transiently transfected
with the expression vector containing the mouse prominin-2 cDNA
(A), mouse prominin-2-myc cDNA (C and
D), or, as a control, vector DNA alone (B).
Paraformaldehyde-fixed, PNGase F-treated cells were double-labeled with
E3 antiserum (A-C) and anti-c-Myc antibody
(D) followed by appropriate Cy3- and Cy2-conjugated
secondary antibodies and double immunofluorescence analysis using
confocal microscopy. Single optical xy plane sections near
the bottom of the cell are shown. Bar in A, 11.5 µm; bar in B, 14 µm; bar in
D, 7.8 µm.
E3 antiserum revealed two
bands with apparent molecular mass of
112 and
95 kDa (Fig. 4, top and bottom
panels, lane 3, arrowheads and
asterisks, respectively). No immunoreactivity was detected
when CHO cells were transfected with the expression vector alone (Fig.
4, top and bottom panels, lane 5). The
two-band pattern of prominin-2 was reminiscent of that of prominin-1
ectopically expressed in CHO cells (1, 12) and Madin-Darby canine
kidney cells (2, 3). The 95-kDa form of prominin-2 was sensitive to
digestion with endo H (Fig. 4, bottom panel, lane
3, asterisk, and lane 4, arrow)
and therefore represented the high mannose form localized in the
endoplasmic reticulum and/or an early Golgi compartment. The 112-kDa
form of prominin-2 was resistant to the endo H (Fig. 4, bottom
panel, lanes 3 and 4, arrowhead),
indicating that it had passed through the Golgi apparatus. PNGase F
treatment converted both the 112- and 95-kDa forms of recombinant
prominin-2 found in transfected CHO cells into an
88-kDa product
(Fig. 4, top panel, lane 4, arrow),
which is in agreement with the predicted molecular weight (92,100) of
unglycosylated prominin-2 after cleavage of its signal peptide. PNGase
F digestion also resulted in a
2-fold increase in prominin-2
immunoreactivity detected after immunoblotting, suggesting that complex
N-glycans masked a significant proportion of the epitopes
recognized by the
E3 antiserum. This is consistent with our
observation that upon immunofluorescence analysis of prominin-2-transfected CHO cells using
E3 antiserum, plasma membrane staining for prominin-2 was only observed if the fixed cells were treated with PNGase F prior to addition of the antibody (see above, Fig. 3). Taken together, the immunofluorescence data (Fig. 3) and the
immunoblotting data (Fig. 4, lanes 3-6) obtained with prominin-2-transfected CHO cells show that the
E3 antibody is specific for prominin-2, at least in these cells.
View larger version (47K):
[in a new window]
Fig. 4.
Expression of prominin-2 in CHO cells and
murine kidney. CHO cells were transiently transfected with either
the expression vector containing the mouse prominin-2 cDNA
(PROM-2) or, as a control, vector DNA alone
(MOCK). Lysates from the CHO cells and, for comparison, from
adult mouse kidney membranes (KIDNEY) were incubated in the
absence ( ) or presence of 1 unit of PNGase F
(F, top panel) or 10 milliunits of endo H
(H, bottom panel) and analyzed by immunoblotting
with
E3 antiserum. Arrowheads, PNGase F-sensitive, endo
H-resistant form of prominin-2; asterisks, PNGase F- and
endo H-sensitive form; arrows, product after
N-deglycosylation; diamonds, PNGase F- and endo
H-insensitive, immunoreactive bands of unknown identity. The position
of prestained apparent molecular mass markers (in kDa) is indicated on
the right.
E3 antiserum, the pattern of immunoreactive bands was more complex.
First, we observed four immunoreactive bands that showed no change in
electrophoretic mobility upon PNGase F and endo H digestions (Fig. 4,
top and bottom panels, lanes 1 and 2,
diamonds) and presumably were unglycosylated proteins. The identity of these bands and their possible relationship to prominin-2 has not been investigated further in the present study. Second, we
observed a 112-kDa band that showed the same endo H resistance (Fig. 4,
bottom panel, lanes 1 and 2,
arrowhead) and PNGase F sensitivity (Fig. 4, top
panel, lane 1, arrowhead and lane
2, arrow) as authentic prominin-2 expressed in CHO
cells. This establishes that prominin-2 is endogenously expressed in
the kidney in vivo. Furthermore, given that all potential
N-glycosylation sites of prominin-2 are found in the two
large loops, our results also indicate that these loops are indeed
located extracellularly and, hence, that prominin-2 displays the same
membrane topology as prominin-1.
View larger version (26K):
[in a new window]
Fig. 5.
Co-localization of prominin-1 and prominin-2
in plasma membrane protrusions. CHO cells were transiently
double-transfected with the expression vectors containing either the
rat prominin-2-GFP fusion protein cDNA or mouse prominin-1
cDNA, cell surface-labeled with rat mAb 13A4 against prominin-1
followed by Cy3-conjugated anti-rat antibody, and examined for the
fluorescent signal of prominin-2-GFP fusion protein (top)
and immunostaining of prominin-1 (bottom). Single optical
xy plane section at the apex of the cell is shown.
Arrowheads indicate co-localization of prominin-2 and
prominin-1. Bar, 8 µm.
View larger version (59K):
[in a new window]
Fig. 6.
Northern blot analysis of mouse prominin-2
expression. A and B, mouse multiple tissue
Northern blot membrane was probed with mouse prominin-2 cDNA
(A) or actin cDNA (B). Arrow,
transcripts in kidney and testis; asterisk, transcripts in
heart and skeletal muscle.
View larger version (54K):
[in a new window]
Fig. 7.
Tissue distribution of human
prominin-2 mRNA. A, human multiple tissue expression
array membrane was probed with human prominin-2 cDNA. The various
dots represent the following: A1, whole brain;
B1, cerebral cortex; C1, frontal lobe;
D1, parietal lobe; E1, occipital lobe;
F1, temporal lobe; G1, paracentral gyrus of
cerebral cortex; H1, pons; A2, cerebellum left;
B2, cerebellum right; C2, corpus callosum;
D2, amygdala; E2, caudate nucleus; F2,
hippocampus; G2, medulla oblongata; H2, putamen;
A3, substantia nigra; B3, nucleus accumbens;
C3, thalamus; D3, pituitary gland; E3,
spinal cord; A4, heart; B4, aorta; C4,
atrium left; D4, atrium right; E4, ventricle
left; F4, ventricle right; G4, interventricular
septum; H4, apex of the heart; A5, esophagus;
B5, stomach; C5, duodenum; D5,
jejunum; E5, ileum; F5, ileocecum; G5,
appendix; H5, colon ascending; A6, colon
transverse; B6, colon descending; C6, rectum;
A7, kidney; B7, skeletal muscle; C7,
spleen; D7, thymus; E7, peripheral blood
leukocytes; F7, lymph node; G7, bone marrow;
H7, trachea; A8, lung; B8, placenta;
C8, bladder; D8, uterus; E8, prostate;
F8, testis; G8, ovary; A9, liver;
B9, pancreas; C9, adrenal gland; D9,
thyroid gland; E9, salivary gland; F9, mammary
gland; A10, promyelocytic leukemia HL-60; B10,
HeLa S3; C10, chronic myelogenous leukemia K-562;
D10, lymphoblastic leukemia MOLT-4; E10,
Burkitt's lymphoma, Raji; F10, Burkitt's lymphoma, Daudi;
G10, colorectal adenocarcinoma, SW480; H10, lung
carcinoma, A549; A11, fetal brain; B11, fetal
heart; C11, fetal kidney; D11, fetal liver;
E11, fetal spleen; F11, fetal thymus;
G11, fetal lung; A12, yeast total RNA;
B12, yeast tRNA; C12, E. coli rRNA; D12, E. coli
DNA; E12, poly(rA); F12, human C0t-1
DNA; G12, human DNA 100 ng; H12, human DNA 500 ng. B and C, human multiple tissue Northern blot
membrane was probed with human prominin-2 cDNA (B) or
actin cDNA (C). D-F, human
digestive tract Northern blot membrane was probed with human prominin-2
cDNA (D), human prominin-1 cDNA (E), or
actin cDNA (F). B and D,
solid arrows, major transcripts; open arrows,
arrowhead and asterisks, minor transcripts.
G, the data obtained from Northern blot analysis of
prominin-1 (E) and prominin-2 (D, solid
arrow; upper (5.0 kb) and lower (4.2 kb)) transcripts were
quantified by densitometric scanning, normalized with respect to actin
mRNA (F) in each tissue sample, and expressed as ratio
of mRNA signal.
5.0 and
4.2 kb (Fig.
7, B and D, solid arrows) were
detected in kidney and placenta (Fig. 7B) and at variable
levels along the digestive tract (Fig. 7D), whereas in the
liver, a 1.5-kb transcript (too short to encode full-length prominin-2)
was detected (Fig. 7, B and D,
asterisk). The results of the dot blot analysis (Fig. 7A) were therefore confirmed. With longer time of exposure,
a
3.2-kb transcript appeared in kidney, placenta, colon, and
esophagus (Fig. 7, B and D, open
arrow), a
6.0-kb transcript in heart and skeletal muscle (Fig.
7B, arrowhead), and a
5-kb transcript in the
lung (data not shown). With respect to the expression of prominin-2 in
the human digestive tract, the amount of the two main transcripts (5.0-4.2 kb, Fig. 7D, solid arrows) was compared
with prominin-1 (Fig. 7E), normalized to actin mRNA
levels (Fig. 7F), and plotted (Fig. 7G). The
distribution of prominin-2 transcripts all along the digestive tract
roughly paralleled that of prominin-1 transcript, with the esophagus
being a notable exception; here the high expression of prominin-2
transcripts (Fig. 7D) was in contrast to the lack of
prominin-1 transcript (Fig. 7E).
View larger version (38K):
[in a new window]
Fig. 8.
Prominin-2 is not expressed in the adult
mouse eye. Lysates from adult mouse eyes (EYE) and, for
comparison, adult mouse kidney membranes (KIDNEY) were
incubated in the absence ( ) or presence of 1 unit of PNGase F
(F) and analyzed by immunoblotting with either
E3
antiserum against prominin-2 (left panel) or mAb 13A4
against prominin-1 (right panel). Left panel,
arrowhead, prominin-2; arrow,
N-deglycosylated form of prominin-2; diamonds,
PNGase F-insensitive immunoreactive bands of unknown identity.
Right panel, bracket, PNGase F-sensitive, endo
H-resistant form of prominin-1 in the eye (12); asterisk,
PNGase F- and endo H-sensitive form of prominin-1 in the eye (12);
open arrow, N-deglycosylated form of prominin-1.
The position of apparent molecular mass markers (in kDa) is indicated.
Note that although the three unidentified bands present in the eye are
at least as strongly labeled as in the kidney (diamonds),
prominin-2 (arrowhead and arrow) is detected in
the kidney but not the eye, in contrast to prominin-1. No prominin-2
immunoreactivity was detected in the eye upon longer exposure of the
immunoblot (not shown).
View larger version (52K):
[in a new window]
Fig. 9.
Sequence comparison of members of the
prominin family. A, reduced unrooted phylogenetic tree of
the prominin family. The alignment in B was used to infer
the tree by the maximum parsimony method. Bootstrap values above 50%
are indicated at the nodes. Branching with bootstrap values below 50%
were collapsed (dot). Mammalian prominin-1 and prominin-2
relatives appear in pink boxes. B, sequence
alignment of prominins. The region encompassing the end of the second
large extracellular loop and the fifth transmembrane domain (red
line) of the different prominin family members were aligned using
ClustalW. Sequences are displayed according to their ranking in the
alignment. Number indicates the amino acid residues in the
respective complete protein sequences. Residues identical in more than
53% of the sequences are boxed. Blue and
red color indicate hydrophilic and hydrophobic residues
(over a window of seven residues), respectively, the intensity of the
color denoting the degree of hydrophilicity and hydrophobicity. In the
consensus sequence, regular uppercase letters indicate
strict (100%) consensus residues, italic uppercase letters
preferred (>53%) residues, and h preferred hydrophobic
residues. Abbreviations and data base accession numbers are as
following Homo sapiens prominin-2 (Hs prom-2,
AF245303), Mus musculus prominin-2 (Mm prom-2,
AF269062), Rattus norvegicus prominin-2 (Rn prom,
AF508942), M. musculus prominin-1 (Mm prom-1,
AF026269), R. norvegicus prominin-1 (Rn prom-1,
AF386758), H. sapiens prominin-1 (Hs prom-1,
AF027208), D. rerio prominin-like 2 (Dr proml2,
AF373869), D. rerio EST clone fo25d03 (Dr
fo25d03, BI884488), Gallus gallus prominin-like
(Gg proml, AF406812), C. elegans M28.8 and M28.9
(Ce M28.8 and Ce M28.9, Z49911), C. elegans F08B12.1 (Ce F08B12.1, Z68104), A. suum EST clone MBAsBWA207 (As MBAsBWA207, AW165790),
and D. melanogaster prominin-like (Dm proml,
AF127935).
View larger version (15K):
[in a new window]
Fig. 10.
Genomic organization of human
prominin-2 and its comparison with other
prominins. The top of the figure shows part of the
genomic clone RP11-468G5 encompassing the human prominin-2 exons, which
appear as boxes. The corresponding human (Hs)
prominin-2 (prom-2) 5.0-kb cDNA is shown below at a
larger scale. Coding region appears in gray, and 5'- and
3'-untranslated region are in white. Vertical lines indicate
the exon boundaries and hatched zones the predicted
transmembrane domains. Bottom of figure, mouse
(Mm) prominin-2 (prom-2) and prominin-1
(prom-1) cDNAs, human (Hs) prominin-1
(prom-1) cDNA, C. elegans (Ce)
F08B12.1 predicted gene product, and D. melanogaster
prominin-like cDNA (Dm proml). The chromosome carrying a
given gene is indicated in parentheses.
16 versus >75 kb).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank D. Buck for the human prominin-1 cDNA; M. Maw for drawing our attention to clone NH0468G05, which contains the human prominin-2 gene; R. Jelinek for valuable technical assistance; G. Wiebe for the excellent sequencing service; and K. Opherk for the prominin-2-GFP construct. Part of the sequencing was performed at the Sequencing Facility of the Zentrum für Molekulare Biologie of Heidelberg University. We are also grateful to Celera Discovery System and Celera Genomics for the use of their data bases. D. C. and M. F. are indebted to Prof. G. Ehninger for financial support.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains additional text, Fig. 1, and Tables
I-III.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF508942, AF269062, AF128113, AF245303, AF245304, AF160970, AF373869, AF127935, AF197345, and AF406812.
¶ Supported by Deutsche Forschungsgemeinschaft Grants Hu 275/7-1 and Hu 275/8-1, the German-Israeli Foundation for Scientific Research and Development, and the Fonds der Chemischen Industrie. To whom correspondence may be addressed. Tel.: 49-351-210-1500; Fax: 49-351-210-1600; E-mail: HUTTNER@mpi-cbg.de.
To whom correspondence may be addressed. Tel.:
49-351-210-1488; Fax: 49-351-210-1489; E-mail:
CORBEIL@mpi-cbg.de.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M210640200
1 M. Florek, D. Freund, G. Ehninger, W. B. Huttner, and D. Corbeil, manuscript in preparation.
3 M. Florek and D. Corbeil, unpublished observations.
4 C. A. Fargeas, A. Joester, A. Hellwig, W. B. Huttner, and D. Corbeil, manuscript in preparation.
5 A. Joester, C. Corbeil, C. A. Fargeas, A. Hellwig, and W. B. Huttner, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ORF, open reading
frames;
CHO, Chinese hamster ovary;
PNGase F, peptide:N-glycosidase;
endo H, endo--N-acetylglucosaminidase H;
nt, nucleotide;
GFP, green fluorescent protein;
PBS, phosphate-buffered saline;
mAb, monoclonal antibody.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Weigmann, A.,
Corbeil, D.,
Hellwig, A.,
and Huttner, W. B.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12425-12430 |
2. |
Corbeil, D.,
Röper, K.,
Hannah, M. J.,
Hellwig, A.,
and Huttner, W. B.
(1999)
J. Cell Sci.
112,
1023-1033 |
3. | Röper, K., Corbeil, D., and Huttner, W. B. (2000) Nat. Cell Biol. 2, 582-592[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Yin, A. H.,
Miraglia, S.,
Zanjani, E. D.,
Almeida-Porada, G.,
Ogawa, M.,
Leary, A. G.,
Olweus, J.,
Kearney, J.,
and Buck, D. W.
(1997)
Blood
90,
5002-5012 |
5. |
Miraglia, S.,
Godfrey, W.,
Yin, A. H.,
Atkins, K.,
Warnke, R.,
Holden, J. T.,
Bray, R. A.,
Waller, E. K.,
and Buck, D. W.
(1997)
Blood
90,
5013-5021 |
6. | Bhathia, M. (2001) Leukemia (Baltimore) 15, 1685-1688 |
7. |
Corbeil, D.,
Röper, K.,
Hellwig, A.,
Tavian, M.,
Miraglia, S.,
Watt, S. M.,
Simmons, P. J.,
Peault, B.,
Buck, D. W.,
and Huttner, W. B.
(2000)
J. Biol. Chem.
275,
5512-5520 |
8. | Corbeil, D., Röper, K., Fargeas, C. A., Joester, A., and Huttner, W. B. (2001) Traffic 2, 82-91[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Corbeil, D.,
Röper, K.,
Weigmann, A.,
and Huttner, W. B.
(1998)
Blood
91,
2625-2626 |
10. |
Miraglia, S.,
Godfrey, W.,
and Buck, D.
(1998)
Blood
91,
4390-4391 |
11. | Corbeil, D., Fargeas, C. A., and Huttner, W. B. (2001) Biochem. Biophys. Res. Commun. 285, 939-944[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Maw, M. A.,
Corbeil, D.,
Koch, J.,
Hellwig, A.,
Wilson-Wheeler, J. S. C.,
Bridges, R. J.,
Kumaramanickavel, G.,
John, S.,
Nancarrow, D.,
Röper, K.,
Weigmann, A.,
Huttner, W. B.,
and Denton, M. J.
(2000)
Hum. Mol. Genet.
9,
27-34 |
13. |
Altschul, S. F.,
Madden, T. L.,
Schaffer, A. A.,
Zhang, J.,
Zhang, Z.,
Miller, W.,
and Lipman, D. J.
(1997)
Nucleic Acids Res.
25,
3389-3402 |
14. | Appel, R. D., Bairoch, A., and Hochstrasser, D. F. (1994) Trends Biochem. Sci. 19, 258-260[CrossRef][Medline] [Order article via Infotrieve] |
15. | Myers, E. W., and Miller, W. (1988) Comp. Appl. Biosci. 4, 11-17[Medline] [Order article via Infotrieve] |
16. | Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680[Abstract] |
17. | Felsenstein, J. (1989) Cladistics 5, 164-166 |
18. | Felsenstein, J. (1985) Evolution 39, 783-791 |
19. |
Zhang, Q.,
Haleem, R.,
Cai, X.,
and Wang, Z.
(2002)
Endocrinology
143,
4788-4796 |
20. | Hurle, B., Lane, K., Kenney, J., Tarantino, L. M., Bucan, M., Brownstein, B. H., and Ornitz, D. M. (2001) Genomics 77, 189-199[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Yu, Y.,
Flint, A.,
Dvorin, E. L.,
and Bischoff, J.
(2002)
J. Biol. Chem.
277,
20711-20716 |
22. | Wilson, R., Ainscough, R., Anderson, K., Baynes, C., Berks, M., Bonfield, J., Burton, J., Connell, M., Copsey, T., and Cooper, J. (1994) Nature 368, 32-38[CrossRef][Medline] [Order article via Infotrieve] |