From the Department of Biotechnology,
§ Venture Laboratory, Kyoto Institute of Technology,
Kyoto 606-8585, Japan
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ABSTRACT |
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In the central nervous system, many cell adhesion
molecules are known to participate in the establishment and remodeling
of the neural circuit. Some of the cell adhesion molecules are known to
be anchored to the membrane by the glycosylphosphatidylinositol (GPI)
inserted to their C termini, and many GPI-anchored proteins are known
to be localized in a Triton-insoluble membrane fraction of low density
or so-called "raft." In this study, we surveyed the GPI-anchored
proteins in the Triton-insoluble low density fraction from 2-week-old
rat brain by solubilization with phosphatidylinositol-specific phospholipase C. By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the
presence of Thy-1, F3/contactin, and T-cadherin was shown. In addition, one of the major proteins, having an apparent molecular mass of 36 kDa
after the peptide N-glycosidase F digestion, was found to
be a novel protein. The result of cDNA cloning showed that the
protein is an immunoglobulin superfamily member with three C2 domains
and has six putative glycosylation sites. Since this protein shows high
sequence similarity to IgLON family members including LAMP, OBCAM,
neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (a
kindred of IgLON). Kilon-specific monoclonal
antibodies were produced, and Western blotting analysis showed that
expression of Kilon is restricted to brain, and Kilon has an apparent
molecular mass of 46 kDa in SDS-polyacrylamide gel electrophoresis in
its expressed form. In brain, the expression of Kilon is already
detected in E16 stage, and its level gradually increases during
development. Kilon immunostaining was observed in the cerebral cortex
and hippocampus, in which the strongly stained puncta were observed on
dendrites and soma of pyramidal neurons.
Cell adhesion molecules
(CAMs)1 play central roles in
the establishment and the remodeling of the central nervous system.
CAMs are classified into Ca2+-dependent and
Ca2+-independent groups. Cadherins are dependent on
Ca2+ ions for binding. The cytoplasmic domain of different
cadherins is highly conserved, and proteins that bind to the
cytoplasmic domain interact with cytoskeletal proteins and signal
transduction pathways to regulate cell adhesion (1). Integrins make up
a large family of heterodimeric proteins that mediate cell to cell and
cell to extracellular matrix-adhesive connections, and the intracellular domain interacts with the actin cytoskeleton through several intermediate proteins such as Much attention has been paid to a membranous subdomain that is
insoluble in the non-ionic detergent such as Triton X-100 and has a low
density (Triton-insoluble low density fraction: TIF, also called
"raft", "DIGs" (detergent-insoluble, glycolipid-enriched complexes), or "DRMs" (detergent-resistant membrane domains), "Caveolae-like domains," etc.), since this domain has so many signal-transducing molecules such as trimeric G proteins, protein tyrosine kinases, cytoskeletal proteins, and calmodulin-binding proteins (8-14). The detergent insolubility of this fraction is attributed to the enrichment of cholesterol and sphingomyelin (15, 16).
Glycosylphosphatidylinositol (GPI)-anchored proteins, which are
extracellular proteins anchored in the lipid bilayer by GPI instead of
membrane-spanning peptides, are localized in this region (8, 9,
17-20). Some CAMs are known to be the GPI-anchored proteins, and
little is known about their signal-transducing pathways. Since
GPI-anchored proteins are enriched in TIF, this region could be a good
clue to elucidate the molecular mechanisms of signal transduction
through the GPI-anchored cell adhesion molecules. At the first step, we
analyzed the GPI-anchored proteins in the TIF from 2-week-old rat brain
and identified F3/contactin, T-cadherin, and Thy-1 as major
GPI-anchored proteins. In addition, a novel GPI-anchored protein was
identified, and the result of cDNA cloning showed that this protein
belongs to the IgLON family, which contains LAMP, OBCAM, neurotrimin,
CEPU-1, GP55, and AvGP50 (21-26). We call this protein Kilon, for this
protein is a kindred of IgLON.
Preparation of TIF from Rat Brain--
TIF was prepared as
described previously with slight modification (13). All procedures were
carried out on ice or at 4 °C, unless otherwise mentioned. Frozen
whole brains from 2-week-old rat were thawed, minced with scissors, and
homogenized with a Teflon glass homogenizer in 1 volume (w/v) of TME
solution (10 mM Tris-HCl, 1 mM
MgCl2, 1 mM EGTA, pH 7.4) containing 2% Triton X-100. Protease inhibitors used were 1 mM
phenylmethylsulfonyl fluoride, 0.1% aprotinin, 0.01 mg/ml leupeptin,
and 0.01 mg/ml pepstatin. An aliquot of 2.4 M sucrose
solution was added to this homogenate, and the final sucrose
concentration was adjusted to be 0.8 M. The sample was then
placed in the centrifuge tubes. TME solution was overlaid, and the
sample was centrifuged for 6 h at 70,000 × g
using a swing rotor (Hitachi SW27-2). A membrane fraction was
concentrated at the interface of TME solution, and the original sample
solution was collected and recovered as a pellet after dilution with 5 volumes of TME solution containing 1% Triton X-100 and centrifugation
for 1 h at 100,000 × g. This fraction was then
homogenized in 5 volumes of TME solution containing 1 M
NaCl. After centrifugation for 1 h at 100,000 × g, the pellet fraction was recovered and suspended in 5 volumes of 50 mM Tris-HCl, 1 mM EDTA, 10%
glycerol, 10 mM 2-mercaptoethanol, and 5% Nonidet-40, pH
9.5, and was kept for 1 h at 0 °C. After centrifugation for 1.5 h at 100,000 × g, the resulting pellet was
recovered and suspended in 10 mM Tris-HCl, 5 mM
EDTA, pH 7.4. TIF, thus obtained, was frozen at Phosphatidylinositol-specific Phospholipase C (PI-PLC)
Treatment--
GPI-anchored proteins were solubilized from TIF with
PI-PLC (Funakoshi, Japan). TIF (20 mg of protein, 1 mg/ml) was
incubated with 0.125 units/ml PI-PLC for 12 h at 37 °C in the
presence of 0.2% Triton X-100 and the protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, each 0.01 mg/ml pepstatin and leupeptin). After the addition of 4 M
NaCl solution to be 0.1 M, the sample was centrifuged for
3 h at 100,000 × g, and the supernatant was recovered. The supernatant was then lyophilized after dialysis against
50 mM ammonium acetate solution. The fraction was then suspended in a solution containing 50 mM Tris-HCl, 50 mM EDTA, pH 8.6, applied to a Extracti-Gel D column
(0.7 × 5 cm) (Pierce). The flow-through fraction was then
recovered as the PI-PLC supernatant.
Peptide N-Glycosidase F (PNGF) Treatment--
To remove the
N-linked sugar chain, PNGF (Boehringer Mannheim) was used.
The PI-PLC supernatant was incubated first for 30 min at 56 °C in
the presence of 0.1% SDS and 2 mM NaN3. PNGF
was then added at a ratio of 50 units/mg protein and incubated for 24 h at 37 °C.
Partial Amino Acid Sequence--
Samples were incubated for 60 min at 60 °C after the addition of Laemmli sample solution. SDS-PAGE
was done using a 12.5-15% gradient polyacrylamide gel, and proteins
were transferred to a sheet of PVDFSQ membrane (Millipore)
using a buffer containing 10 mM CAPS and 10% methanol, pH
11. The membrane was stained with a solution containing 0.1% Ponceau S
in 1% acetic acid and destained with distilled water. For peptide
sequencing, immobilized protein bands were cut, reduced with
dithiothreitol, pyridyletylated with 4-vinylpyridine, and digested with
endoproteinase Lys-C (Wako, Japan) for 12 h at 37 °C in 20 mM Tris-HCl, 10% acetonitrile, pH 9.0. The peptide fragments were separated on a C18 reversed-phase high performance liquid chromatography column. For N-terminal sequence, immobilized proteins were directly applied to the sequencer. N-terminal amino acid
sequence or internal amino acid sequences were then obtained with an
Applied Biosystems 476A protein sequencer.
cDNA Cloning and Recombinant Protein Expression--
From
the N-terminal peptide sequence (PEP-1, VDFPWAAVDNMLVRK), a
sense PCR primer (N-1, gtNgattttccNtgggcNgcNgtNgaYaaYatgYt) was
constructed. From one of the internal sequences (PEP-3,
DYGNYTCVATNK), an antisense PCR primer (N-3,
cttgttKgtNgccacacaKgtatagtt) was constructed (where Y = c + t,
R = a + g, and K = g + t). Using 2-week-old rat brain
cDNA library (Uni-Zap XR, Stratagene), PCR was carried out 35 cycles at 94 °C for 1 min, 60 °C for 1 min, 74 °C for 1.5 min
using 2.5 µM primers per reaction, and a band of 0.79 kb
was specifically amplified. This product was blunted and subcloned into
the SmaI-cutting site of pBluescript KSII+
(Stratagene). The resulting plasmid (pKS+0.79) was
transformed into E. coli JM109 and was amplified in a large
scale. The pKS+0.79 was double-digested with
ApaI and HindIII, and the resulting 0.79-kb
fragment was purified. Uni-Zap XR library (450,000 plaque-forming units) was screened using the 0.79-kb fragment labeled with a thermostable alkaline phosphatase as described by the AlkPhos Direct
system for chemiluminescence protocol (Amersham Pharmacia Biotech), and
several cDNA clones were isolated.
Nucleotide sequencing was performed by the dideoxy chain termination
method on an Applied Biosystems 373A automated DNA sequencer using the
Thermo Sequenase dye terminator cycle sequencing pre-mix kit (Amersham
Pharmacia Biotech).
To express Kilon in Escherichia coli JM 109, the fragment of
this clone was subcloned into the EcoRI site of pGEX-2T
(Amersham Pharmacia Biotech), and the expression of the GST fusion
protein was induced with 1 mM
isopropyl-1-thio-b-D-galactopyranoside at 37 °C. After
3 h incubation, cells were collected, washed, and disrupted with a
sonicator. After centrifugation at 20,000 × g for 30 min, the supernatant was recovered and applied to a
glutathione-Sepharose column. The unbound proteins were washed with a
solution of 10 mM Tris-HCl, 0.15 M NaCl, 0.2 mM EGTA, pH 7.4, and the bound proteins were eluted with
the above solution containing 10 mM glutathione.
Monoclonal Antibody Production--
Proteins eluted from TIF
with PI-PLC were digested with PNGF and used as the antigen. After PNGF
treatment, proteins were passed through an Extracti-Gel column to
remove detergents in the solution. After dialysis against 50 mM ammonium acetate, the sample was lyophilized. The sample
was finally dissolved in PBS and frozen at Immunocytochemistry--
Rats (9 weeks old) were deeply
anesthetized with sodium pentobarbital (70 mg/kg) and transcardially
perfused with 50 ml of heparinized phosphate-buffered saline (PBS)
followed by 200 ml of 4% paraformaldehyde in 0.1 M sodium
phosphate buffer, pH 7.4. The brain was postfixed with the same
fixative solution for 24 h at 4 °C. The brain blocks including
the cerebral cortex and hippocampus were dissected, and coronal
sections were cut using a vibratome (DTK-1000, DSK, Kyoto, Japan) at
the thickness of 25 µm. Free-floating sections were first pretreated
with 0.1% H2O2 in PBS for 20 min and then with
0.5% sodium borohydride in PBS for 10 min. They were preincubated with
5% normal horse serum in PBS containing 0.3% Triton X-100 for at
least 24 h at 4 °C and then incubated with an mAb 89B3
(dilution 1: 3,000) in PBS/Triton X-100 containing 1% normal horse
serum for 48 h at 4 °C. The sections were further incubated
with biotin-conjugated horse anti-mouse IgG (dilution 1:200, Vector,
CA) for 2 h and then incubated with a avidin and peroxidase ABC
solution (dilution 1:200, elite ABC kit, Vector) for 2 h. The
visualization of the peroxidase was performed with 0.02%
3,3'-diaminobenzidine and 0.005% hydrogen peroxide in 0.05 M Tris-HCl buffer, pH 7.4, containing 0.05% sulfate nickel. All specific immunoreactivity was absent when the primary antibody against Kilon was omitted in control experiments.
Others--
Tissue extracts were prepared from 2-week-old rats
as described previously (27). SDS-PAGE, Western blotting using ECL
system (Amersham Pharmacia Biotech), and protein determination were
performed as described (13). Anti-Thy-1 antibodies were obtained from Cedarlane and Sigma.
Solubilization of Proteins from TIF and Its Treatment with
PNGF--
GPI-anchored proteins are solubilized from the membrane
fractions by PI-PLC. For example, using adult chick brains,
F3/F11/contactin and T-cadherin, both of which are GPI-anchored
proteins, were released from the detergent-insoluble membrane fractions
by PI-PLC (28-31). We also used the PI-PLC to solubilize GPI-anchored
proteins from rat brain-derived TIF and recovered the supernatant
(PI-PLC supernatant) after centrifugation (Fig.
1). Three major broad protein bands with
apparent molecular masses of 110-140, 44-65, and 25-27 kDa were
observed after SDS-PAGE (Fig. 1). With Western blotting using
anti-Thy-1 antibodies, the 25-27-kDa band was identified as Thy-1
(data not shown). Since highly glycosylated proteins are known to show
a broad band on SDS-PAGE, we next deglycosylated the PI-PLC supernatant
using peptide N-glycosidase F (PNGF). After PNGF treatment
(PNGF fraction), the apparent molecular masses of major proteins
changed to 110, 76, 37, and 36 kDa (Fig. 1).
Protein Sequencing--
In order to identify these protein
components, N-terminal peptide sequences of the 110-, 76-, 37-, and
36-kDa protein bands were obtained. The 110-kDa protein, of which the
N-terminal peptide sequence was EFT, was estimated to be F3/contactin,
for this sequence coincided with the reported N-terminal sequence of
F3/contactin, the molecular mass of the deglycosylated band matched
well with the calculated molecular mass of F3/contactin (32, 33), and the presence of the protein in this biochemical fraction was shown by
an immunological method (29). The 76-kDa protein, of which the peptide
sequence was SIVVSPILIPENQR, was identified as T-cadherin, for this
sequence was found in the deduced amino acid sequence of human
T-cadherin (34, 35). The presence of T-cadherin in a Triton-insoluble
fraction is also reported (30, 31). The peptide sequence of the 37-kDa
protein band was GDATFPKAMDXVT, and this sequence was found
in the deduced sequences of OBCAM and neurotrimin (23, 36). The
N-terminal sequence of the 36-kDa protein band, VDFPWAAVDNMLVRK
(PEP-1), however, did not correspond to any identified proteins. Two
internal peptide sequences of the 36-kDa band, GDTAVLRCYLEDGASK (PEP-2)
and DYGNYTCVATNK (PEP-3), were further obtained after the high
performance liquid chromatography separation of the protease digests.
The sequence of PEP-2 was not found in the identified proteins. The
sequence of PEP-3, on the other hand, was found in the sequence of
OBCAM fragment 283-294 (36).
cDNA Cloning--
From the N-terminal (PEP-1) and an internal
amino acid sequence (PEP-3), two degenerative primers for PCR (N-1 and
N-3) were designed and produced. A PCR experiment using a rat brain
cDNA library resulted in the amplification of a single 0.79-kb
band. Since the direct sequencing of this band showed no identity with known genes, this product was used as a probe to screen a full-length cDNA from the same cDNA library. After screening 450,000 clones, 20 positive clones were isolated. The clone having the longest insert was selected. The insert was subcloned into pBluescript KSII+, and its complete sequence was determined (Fig.
2A). The insert contained 1827 base pairs, including an open reading frame of 1044 base pairs coding
for 348 amino acids. The nucleotide sequence at the deduced start
methionine matches the consensus initiation (37). Sequences in the
0.79-kb PCR product was found in the insert. The deduced amino acid
sequence contained two of the peptide sequences determined from the
direct sequence analysis of the protein (PEP-1 and PEP-2 in Fig.
2A). PEP-3 sequence, a sequence used for the design of the
PCR primer, was not found in the sequence. We found, however, that one
of the sequence patterns of the degenerative primer (N-3) matches with
the nucleotide sequence from 949 to 975 (a dashed line in
Fig. 2A). As described below, this region has a considerable
homology within LAMP, OBCAM, neurotrimin, and Kilon. Since the sequence
of Pep-3 is observed in the sequences of OBCAM, and OBCAM-specific
monoclonal antibodies react with the 36-kDa protein band (data not
shown), the 36-kDa band in SDS-PAGE was judged to be a mixture of Kilon
and OBCAM.
From the N-terminal sequence of the protein, the original N-terminal
31-amino acid sequence of Kilon is thought to be cleaved as a signal
sequence (double underlined in Fig. 2A) (38). The presence of a hydrophobic core surrounded by two hydrophilic sequences in this region coincides well with the present concept of the signal
peptide (Fig. 2, A and B). This hydropathy plot
also showed the presence of another hydrophobic region at the C
terminus. In case of GPI-anchored proteins, the addition of the GPI
anchor is known to occur after the cleavage of the C-terminal
hydrophobic region (40-42). Although a putative GPI anchor attachment
site was found (Gly-318 in Fig. 2A), there is no datum to
assign the C-terminal amino acid of this protein at present. A homology
search of the coding sequence of this protein with SWISS-PROT data
bases showed that this protein is a member of the immunoglobulin
superfamily having three C2 domains and six putative glycosylated sites
and has high similarities to rat LAMP (56%), rat OBCAM (49%), rat neurotrimin (48%), chick GP55A (51%), and chick CEPU-1 (49%) (Table I) (21, 23-25, 43, 44). Like the IgLON
family members, this protein has three sets of cysteines that are
likely to form intradomain disulfide linkages in each of its
immunoglobulin-like domains (Fig. 2C). A sequence comparison
of these proteins is shown in Fig. 3. The
regions of highest homology to most of these proteins occur in the
conserved sequences surrounding the cysteines involved in the
intradomain disulfide bonding (shown by dots). Since these proteins are recently classified into a subfamily of the immunoglobulin superfamily (IgLONs, immunoglobulin superfamily containing LAMP, OBCAM,
and neurotrimin), the name, Kilon (kindred of
LON) was coined for this novel protein.
Immunological Analysis of Kilon--
For the further
characterization of Kilon, monoclonal antibody production was attempted
using the PNGF fraction as the antigen. One of the mAbs (89B3) reacted
with the 36-kDa protein of the PNGF fraction (Fig.
4A) and also reacted
specifically with the bacterially expressed GST-Kilon fusion protein
(Fig. 4B) but did not react with the bacterially expressed
GST alone (data not shown). This antibody reacted with one broad band
with an apparent molecular mass of 46 kDa in TIF and PI-PLC fractions
(Fig. 4A). Kilon was highly enriched in the TIF, for no
detectable reaction was observed in the homogenate fraction compared
with TIF under this reaction condition.
The tissue distribution of this protein was examined, and high
expression in brain was shown (Fig.
5A). No expression was detected in other tissues such as kidney, liver, lung, skeletal muscle,
spleen, and testis. Although a broad band of about 38 kDa also reacted
weakly in the skeletal muscle extract, nothing is known about the
nature of this protein at present. Within brain, expression of Kilon
was observed in cerebrum, brain stem, and hippocampus, and much less
expression was detected in cerebellum (Fig. 5B). A
developmental change of the expression of this protein in whole brain
showed that this protein is already expressed in E16 stage, and its
level gradually increases during development (Fig. 5C).
Immunocytochemical studies using the mAb (89B3) showed specific
staining in the rat cerebral cortex (Fig.
6, A and B) and hippocampus (Fig. 6, C and D). In the cerebral
cortex, numerous puncta of Kilon immunoreactivity were visible in all
regions and were most densely distributed in large neurons of layer V
(Fig. 6A). These strongly stained neurons were identified as
pyramidal neurons, because of their soma location in layer V, large
soma size, and extension of their apical dendrite to layer I. Both soma
and dendrite of pyramidal neurons were lightly stained, whereas strongly stained puncta were found on pyramidal neurons (Fig. 6B). The punctate staining appeared to be seen more
frequently on dendrites than on soma of the neurons. The pattern of
Kilon immunoreactivity in the hippocampus was basically similar to that in the cerebral cortex (Fig. 6, C and D). The
strongly stained puncta were observed on dendrites and soma of the
pyramidal neurons. The same pattern of Kilon immunoreactivity was also
observed in Purkinje cell dendrites and soma of the adult rat
cerebellum (data not shown).
A novel GPI-anchored protein that belongs to the Ig superfamily
was detected in the Triton-insoluble low density fraction of rat brain,
and its amino acid sequence was deduced from the cDNA cloning. This
protein, Kilon, has three C2 domains and has a homology to the IgLON
family. IgLON family is a recently recognized protein family named from
its member proteins, LAMP, OBCAM, and neurotrimin, and also contains
CEPU-1, GP-55, and AvGP50 (21-26). From the data of cDNA
sequences, all these proteins without the N-terminal and C-terminal
signal sequences have their calculated molecular masses of about 32 kDa. The molecular masses of the expressed form of these proteins
obtained from SDS-PAGE are much larger and are different each other as
follows: LAMP, 68 kDa; OBCAM, 58 and 51 kDa; neurotrimin, 65 kDa; and
CEPU-1, 51 kDa. The expressed form of Kilon showed a molecular mass of
46 kDa, also much larger than the deduced value from the amino acid
sequences. The difference between the molecular mass obtained from
SDS-PAGE and the molecular mass deduced from cDNA sequencing is
attributed to the post-translational modification of the protein. In
this case, the nascent polypeptide chain receives the cleavage of the N-terminal signal sequence, the cleavage of the retention signal, the
addition of GPI at the C-terminal, and the addition of
N-linked carbohydrate chains. The smaller molecular
mass of this protein suggests the lower level of the glycosylation than
other proteins.
Since the 36-kDa protein band obtained after PNGF treatment contains at
least two proteins (Kilon and OBCAM), and these proteins are very
homologous, it is very important to obtain specific antibodies for
immunological studies. Two independent groups reported that OBCAM in
rat brain has a broad band of more than 50 kDa (46, 47). Our antibodies
against OBCAM reacted with the 36-kDa band in the PNGF fraction and a
broad band around 46 and 51 kDa in the rat brain fraction but did not
react with bacterially expressed Kilon. In contrast, 89B3 antibody
recognized a band of 46 kDa in the brain fraction, reacted with
bacterially expressed Kilon, but did not react with bacterially
expressed OBCAM. Since the expressed form of other IgLON family
proteins are much larger than Kilon, we judged that the mAb (89B3)
reacts with a Kilon-specific sequence, although we have not yet
identified the sequence recognized by the antibody.
The expression of Kilon was evident on the neuronal processes in
the cerebral cortex and hippocampus at adult rat brain. The distribution of Kilon on dendrites and soma of the pyramidal neurons is
similar to that of LAMP (48). The role of LAMP as a cell recognition
molecule is fairly well recognized through the works of Levitt and
colleagues (21, 49). They showed that native, immunoaffinity purified
LAMP exhibits homophilic binding. They also showed that
LAMP-transfected cells selectively facilitated neurite outgrowth of
primary limbic neurons, and the administration of anti-LAMP in
vivo resulted in the abnormal growth of the mossy fiber projection
from developing granule neurons. Chick brain-derived GP-55 is known to
block the neurite outgrowth of dorsal ganglion cells (50). The
expression of GP55 reaches highest levels around post-hatched day 5, after which it reaches a plateau or may decrease a little (25). GP55 is
highly expressed in the central nervous system, and no expression was
observed in non-neural tissues. The expression of neurotrimin (65 kDa
in SDS-PAGE) was recognized as early as E15, increasing through early
postnatal ages and declining in the adult (23). CEPU-1 is a 51-kDa
glycoprotein strongly expressed on cerebellar Purkinje cells, and its
expression on the cells coincides with the growth of the dendritic tree
(24). Recently, identification of another GPI-anchored protein, termed Neurin-1, involved in the neuron-glia interaction, was reported. Neurin-1 (68-kDa protein in SDS-PAGE) was found on the surface of the
axon and growth cone, and its N-terminal sequences were reported to be
TPEGVPG. This sequence is not found in other proteins (51), although
the cDNA cloning of this protein is not yet done. The spatial and
temporal expression pattern of Kilon is fairly similar to some of these
proteins, although a precise comparison is not done at present. Further
biochemical and immunological characterization of these proteins will
be useful in elucidating the molecular mechanism of the construction
and remodeling of the nervous system.
Since the GPI anchor proteins do not have the transmembrane or the
intracellular domains, they may mediate their biological responses by
interaction with other membrane receptors that are able to recruit and
activate intracellular signaling molecules (52). In the chick brain
membrane fraction, association of c-Fyn and Gi
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-actinin, vinculin, talin, and
tensin (2, 3). The largest classes of Ca2+-independent CAMs
are members of the Ig superfamily, which contains Thy-1 (Po), MAG,
NCAM, L1, F3/F11/contactin, and others. These molecules have a number
of Ig motifs and fibronectin-III repeats in their extracellular domain.
The number of Ig and fibronectin repeats differ in the various members
of the Ig superfamily, but generally they share a similar organization.
For many of the molecules, multiple isoforms with distinct functional
effects and expression patterns are known. The method of attachment to
the cell membrane and the length of the cytoplasmic tail are important
structural features with significant functional consequences, and these
consequences are under extensive investigation (2-7).
EXPERIMENTAL PROCEDURES
80 °C until use.
80 °C until use. Each
0.04 mg of protein was injected intraperitoneally after mixing with
Freund's complete adjuvant. After four times immunizations with the
adjuvant, one mouse received four successive intraperitoneal injections
(once a day). Two days after the last injection, the spleen was
dissected and used for cell fusion with myeloma cells. The fusion of
cells and screening of the hybridomas were performed as described
previously (27). TIF and the PNGF fraction were used for screening. Two
clones (89B3 and 89E4) were found to be specific for Kilon, and 89B3 was used for immunological studies. During this screening, two other
monoclonal antibodies (87F2 and 84A3) reactive for the 36-kDa band were
obtained. Using these mAbs, Uni-Zap cDNA library was screened, and
one clone was obtained. DNA sequencing of this clone showed that this
clone contains an insert for OBCAM.
RESULTS
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Fig. 1.
Solubilization of GPI-anchored proteins from
TIF and their deglycosylation with PNGF. TIF was prepared from
2-week-old rat brain (TIF) and treated with PI-PLC.
Solubilized proteins were recovered after centrifugation (PI-PLC
supernatant) and then treated with PNGF (PNGF fraction).
Samples were analyzed with SDS-PAGE using 10% acrylamide gels, and the
gels were stained with CBB. H, homogenate (20 µg of
protein); TIF, Triton-insoluble low density fraction (25 µg of protein); PI, PI-PLC supernatant (15 µg of
protein); PNGF, PNGF fraction (15 µg of protein).
Left bars indicate the positions of molecular mass markers.
Calculated molecular masses of the major proteins in the PI-PLC
supernatant are shown on the right. The N-terminal sequences
of the major proteins in the PNGF fraction (arrows) were
analyzed.
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Fig. 2.
Structure of Kilon. A,
nucleotide sequence and alignment of deduced amino acid sequence of
Kilon coding clone. One obtained amino acid sequence in the Lys-C
endopeptidase digest (PEP-2) and the N-terminal sequence (PEP-1) were
found in the deduced sequence (under lined). The nucleotide sequence, estimated to correspond to
N-3, was indicated by a dashed line. The N-terminal signal
sequence was double underlined. A putative GPI anchor
attachment site was indicated by the . Six potential
N-glycosylation sites were designated by
. The nucleotide
sequence data reported in this paper will appear in the
DDBJ/EMBL/GenBank nucleotide sequence data bases with the accession
number AB017139. B, a hydropathy plot of Kilon. Hydropathy
was calculated according to Kyte and Doolittle (39). Positive values on
the y axis indicate hydrophobic regions. C, a
domain model of Kilon. Ig-related domains were drawn as
loops that are closed by disulfide bridges, and six putative
N-linked glycosylation sites were shown as lines
ending with dots.
Homology of Kilon to other members of the immunoglobulin superfamily
molecules
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Fig. 3.
Comparison of the peptide sequences of Kilon
and other IgLON family members. Amino acids identical to four
proteins are indicated by the *. The boxed regions indicate
the C2 domains. Putative N-terminal signal peptide sequences were
underlined. The six cysteine residues common to all proteins
were designated by the . Putative GPI anchor attachment sites were
indicated by the symbol
. This analysis was done using
the ClustalW (1.7) Multiple Sequence Alignment Program (45).
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Fig. 4.
Western blot analysis with a monoclonal
antibody against Kilon (89B3). A, enrichment of Kilon
in TIF. Hybridomas, which produce antibodies reactive with the 36-kDa
protein in PNGF fraction, were screened as described under
"Experimental Procedures." The mAb (89B3) recognizes the 36-kDa
band in the PNGF fraction and a 46-kDa band in TIF and in PI-PLC
supernatant (shown by arrows). Poor staining in the
homogenate shows the enrichment of Kilon in TIF. H,
homogenate; PI, PI-PLC supernatant; PNGF, PNGF
fraction. Equal amounts of protein (3 µg of protein) were
electrophoresed and processed for Western blotting using the mAb
(89B3). Left bars indicate the positions of molecular mass
markers. B, reactivity of the mAb (89B3) with a bacterially
expressed Kilon. The GST-Kilon fusion protein (60 kDa) was induced in
E. coli JM 109 with 1 mM
isopropyl-1-thio-b-D-galactopyranoside for 3 h at
37 °C (upon induction). After disruption of cells, the
GST-Kilon fusion protein was purified with a glutathione-Sepharose
column (purified GST-Kilon). Cell extracts (2.5 µg of
protein) and the purified protein (0.25 µg of protein) were
electrophoresed and processed for Western blotting using the mAb
(89B3). The location of the GST-Kilon fusion protein (60 kDa) is
indicated by the arrow. Left bars indicate the
positions of molecular mass markers.
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Fig. 5.
Expression of Kilon. A,
tissue distribution. Equal amounts of tissue extracts (20 µg of
protein) from a 2-week-old rat were electrophoresed and processed for
Western blotting using the mAb (89B3). The location of Kilon (46 kDa)
is indicated by the arrow. Left bars indicate the
positions of molecular mass markers. B, distribution in
brain. From 2-week-old rat brains, cerebrum, cerebellum, brain stem,
and hippocampus were dissected and treated for Western blotting using
the mAb (89B3). Equal amounts of protein (5 µg of protein) were
loaded. The location of Kilon (46 kDa) is indicated by an
arrow. Left bars indicate the positions of
molecular mass markers. C, developmental change. Whole brain
extracts were prepared from rats of different embryonic (E)
and postnatal (P) ages. Equal amounts of protein (5 µg of
protein) were electrophoresed and processed for Western blotting using
the mAb (89B3). The location of Kilon (46 kDa) is indicated by the
arrow. Left bars indicate the positions of
molecular mass markers.
View larger version (158K):
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Fig. 6.
Immunocytochemical localization of Kilon in
the adult rat brain. A, low magnification photograph of
the rat cerebral cortex (pial surface is at top). Note
strongly stained pyramidal neurons in layer V.
Bar, 10 µm. B, higher magnification of
pyramidal neurons in the fields of layer V shown in
A. Strongly stained puncta of Kilon immunoreactivity are
seen on apical dendrites (arrowheads) and soma
(arrows) of pyramidal neurons. Bar, 2 µm.
C, Low magnification photograph of the CA1 region of the rat
hippocampus. Note strongly stained pyramidal neurons. Bar, 5 µm. D, higher magnification of pyramidal neurons shown in
C. Strongly stained puncta of Kilon immunoreactivity are
seen on dendrites (arrowheads) and soma (arrows)
of pyramidal neurons. Bar, 1 µm. I, layer I;
II, layer II; III, layer III; IV,
layer IV; V, layer V; VI, layer VI;
or, stratum oriens; pyr, stratum pyramidal;
rad, stratum radiatum.
DISCUSSION
protein
in the activation of signaling pathways through Thy-1 and AvGP50 is
shown (53). In the case of F3/contactin, receptor tyrosine phosphatase
and a novel transmembrane receptor (Caspr) were discovered as the
interacting proteins and characterized (54, 55). Future studies on the
characterization of interacting molecules with GPI-anchored proteins
will elucidate the signal transduction pathways through the cell membrane.
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FOOTNOTES |
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* This work was supported in part by Grant-in-aid for Scientific Research (B) 08459016 and by Grant-in-aid for Exploratory Research 08878124 from the Ministry of Education, Science, Sports, and Culture of Japan.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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB017139.
¶ Supported in part by a Sasagawa Scientific Research grant.
To whom correspondence should be addressed. Tel.: 81 75 724 7789; Fax: 81 75 724 7760; E-mail: smaekawa{at}ipc.kit.ac.jp.
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ABBREVIATIONS |
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The abbreviations used are: CAMs, cell adhesion molecules; TIF, Triton-insoluble low density fraction; GPI, glycosylphosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; PNGF, peptide N-glycosidase F; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; PCR, polymerase chain reaction; kb, kilobase pair; CAPS, 3-[cyclohexylamino]-1-propanesulfonic acid; PBS, phosphate-buffered saline.
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REFERENCES |
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