Proteolytic Cleavage of the Ectodomain of the L1 CAM Family
Member Tractin*
Ying-Zhi
Xu,
Yun
Ji,
Birgit
Zipser
,
John
Jellies§,
Kristen M.
Johansen, and
Jørgen
Johansen¶
From the Department of Zoology and Genetics, Iowa State University,
Ames, Iowa 50011, the § Department of Biological Sciences,
Western Michigan University, Kalamazoo, Michigan 49008, and the
Department of Physiology, Michigan State University,
East Lansing, Michigan 48824
Received for publication, October 22, 2002, and in revised form, November 8, 2002
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ABSTRACT |
Tractin is a member of the L1 family of cell
adhesion molecules in leech. Immunoblot analysis suggests that Tractin
is constitutively cleaved in vivo at a proteolytic site
with the sequence RKRRSR. This sequence conforms to the
consensus sequence for cleavage by members of the furin family of
convertases, and this proteolytic site is shared by a majority of other
L1 family members. We provide evidence with furin-specific inhibitor
experiments, by site-specific mutagenesis of Tractin constructs
expressed in S2 cells, as well as by Tractin expression in
furin-deficient LoVo cells that a furin convertase is the likely
protease mediating this processing. Cross-immunoprecipitations with
Tractin domain-specific antibodies suggest that the resulting
NH2- and COOH-terminal cleavage fragments interact with
each other and that this interaction provides a means for the
NH2-terminal fragment to be tethered to the membrane. Furthermore, in S2 cell aggregation assays we show that the
NH2-terminal fragment is necessary for homophilic adhesion
and that cells expressing only the transmembrane COOH-terminal fragment
are non-adhesive. However, tethering of exogeneously provided
Tractin NH2-terminal fragment to S2 cells expressing only
the COOH-terminal fragment can functionally restore the adhesive
properties of Tractin.
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INTRODUCTION |
The L1 family of cell adhesion molecules
(CAMs)1 consists of
transmembrane proteins with 6 Ig-like domains and 4-5 FNIII-like domains in the extracellular domain (1). In addition, they are
characterized by a highly conserved cytoplasmic segment that contains
an ankyrin binding site. The L1 family currently has four members in
vertebrates (L1, NrCAM, CHL1, and neurofascin) as well as three
invertebrate members, neuroglian in arthropods, Tractin in leech, and
LAD-1 in nematodes (1, 2). Although also expressed in other tissues L1
family CAMs are predominantly found in the nervous system where they
have been implicated in a number of different cellular processes such
as neuronal cell migration, myelination, axonal growth, axon
fasciculation, and axon guidance (3). Of particular interest is that
mutations in the human L1 gene on the X chromosome result in
a complex human mental retardation syndrome referred to as MASA (4, 5)
the symptoms of which include hydrocephalus, spastic paraplegia, and corpus callosum agenesis (6). Furthermore, L1 knockout mice have been
generated, which show severe brain abnormalities similar to those
observed in humans (7, 8). However, the actual developmental mechanisms
by which mutations in the L1 gene cause these brain defects are not
well understood. In humans and mice the severity of the phenotypes
observed is highly dependent on the genetic background suggesting the
participation of modifier genes (8). Thus, the neurological phenotypes
of L1 mutations observed in mammals as well as in Drosophila
(9) cannot be accounted for by disruption of L1's adhesive function
alone, but is likely to also involve interactions with extracellular
ligands and intracellular signaling pathways linked to cytoskeletal
elements (3, 10). For example, L1 family CAMs have an array of
different protein domains that in addition to participating in
homophilic interactions also function in heterophilic interactions with
other CAMs, integrins, and extracellular matrix proteins (1, 3).
The diversity in the structure and potential functional repertoire of
neural CAMs is further amplified with the existence of many splice
variants and various post-translational modifications such as
differential glycosylation and proteolytic processing (11-13). There
is a growing body of evidence that secreted forms of both type I and
type II integral membrane proteins including CAMs, growth factor and
cytokine receptors, and receptor ligands are derived from selective
post-translational proteolysis (14). The biological function of the
proteolytic cleavage of transmembrane proteins is still not well
characterized and may vary. In some cases it may be a process for
rapidly down-regulating the protein from the cell surface, in others it
may be to generate a soluble form of the protein that has functional
properties different than those of the membrane-bound form (12). In
some cases the processing may be necessary for biological activity. For
example, in order to generate a functional Notch receptor in
Drosophila the protein is cleaved constitutively by a furin
convertase (15) to form a disulfide-linked heterodimer (16, 17).
Furthermore, loss-of-function mutations in the kuzbanian
gene, which codes for a disintegrin metalloprotease in
Drosophila embryos show that its proteolytic activity is
required for axonal extension (18). Recently, it has been demonstrated
that ectodomain shedding of human and mouse L1 can promote cell
migration by binding of the secreted ectodomain to integrins (19).
In this study we characterized the proteolytic processing of the
invertebrate L1 family member Tractin in the S2 cell line. We have
provided evidence that the ectodomain of Tractin is constitutively cleaved by a furin-like convertase at a dibasic cleavage site conserved
in many L1 family member CAMs. We further demonstrate that the shedded
ectodomain can be tethered to the membrane by binding to the cleaved
transmembrane fragment and that this interaction promotes homophilic
adhesion. We propose that this novel mechanism may be a general feature
for tethering the NH2-terminal domain of L1 family CAMs
generated by cleavage at this site to the membrane.
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MATERIALS AND METHODS |
Experimental Preparations
For the present experiments we used the two hirudinid leech
species Hirudo medicinalis and Haemopis
marmorata. The leeches were either captured in the wild or
purchased from commercial sources. Dissections of nervous tissue were
performed in leech saline solutions with the following composition (in
mM): 110, NaCl; 4, KCl; 2, CaCl2; 10, glucose;
10, HEPES; pH. 7.4.
Antibodies
The previously reported Tractin monoclonal antibodies (mAbs),
Laz6-56, 4G5, 3A11, and 3A12 (20-22) were used in these studies. In
addition a new antibody, 1H4, was made to a glutathione
S-transferase (GST) fusion protein of the PGYG domain of
Tractin in the pGEX vector system (Amersham Biosciences) encompassing
the sequence Thr1184-Pro1662. The correct
orientation and frame of the construct was verified by sequencing the
insert. The fusion protein was expressed in XL1-Blue cells
(Stratagene), harvested, and purified over glutathione-agarose (Sigma)
columns according to standard protocols (Amersham Biosciences). Balb C
mice were injected with 50 µg of the purified fusion proteins at
21-day intervals. After the third boost spleen cells of the mice were
fused with Sp2 myeloma cells and monospecific hybridoma lines
established using standard procedures (23). All procedures for
monoclonal antibody production were performed by the Iowa State
University Hybridoma facility. V5 and Fc antibody were obtained from
commercial sources (Invitrogen and ICN, respectively).
Full-length and Tractin Deletion Constructs
A full-length Tractin construct (M1-V1880) was cloned into the
pMT/V5-HisB vector (Invitrogen) using standard procedures (24). A
series of deletions of this construct were made in the pMT/V5-HisB vector:
Ac with the acidic domain deleted from
Pro1089-Pro1161,
PGYG with the PGYG domain
deleted from Gly1197-Leu1656, and
Ac/PGYG
with both the acidic and the PGYG domains deleted from
Pro1089-Leu1656. These constructs contain an
in-frame V5 tag at the COOH-terminal end. In addition, a series of
NH2-terminal sequence deletion constructs were cloned into
the pMT/BiP/V5-HisB vector (Invitrogen), which contains the
Drosophila BiP secretion signal to ensure the proper processing of the expressed proteins in transfected S2 cells. The CTF
construct contained the sequence of
Ser905-Val1880 after the first cleavage site,
the
Ig/FN1-3 construct contained the sequence of
Pro957-Val1880 after the third FNIII domain,
and the
Ig/FN construct contained the remaining sequence of
Pro1092-Val1880 after the fourth FNIII domain.
Full-length Tractin (M1-V1880) was also cloned into the pcDNA3.1
vector (Invitrogen) for mammalian cell transfections in LoVo (ATCC) and
HEK293T (Genehunter) cells. For expression in COS cells (ATCC) the
NH2-terminal fragment of Tractin from M1-K890 was ligated
in-frame to a human IgG Fc fragment (AF150959) in the pcDNA3.1
vector (Invitrogen) resulting in the NH2-terminal fragment
(NTF)-Fc fusion construct. For control experiments the Fc fragment
alone was also cloned into the pcDNA3.1 vector. For SDS-PAGE
mobility comparisons a GST fusion protein containing the sequences of
the acidic and the PGYG domain
(Glu1093-Thr1660) was made and expressed in
the pGEX vector system (Amersham Biosciences) as described above. In
addition, a full-length Tractin construct where the sequence RKRRSR was
changed to AAAASA was generated by PCR mutagenesis using standard
procedures (24). The fidelity of all constructs was verified by
sequencing at the Iowa State University Sequencing Facility.
Expression of Tractin Constructs in Transfected Cells
Drosophila Schneider 2 (S2) cells were grown in
Shields and Sang M3 insect medium (Sigma) containing 10% fetal bovine
serum and antibiotics. S2 cells were transfected with Tractin cDNA
clones using a calcium phosphate transfection kit (Invitrogen). Stable lines of each Tractin subclone were established by co-transfection with
pCoHYGRO (Invitrogen) to confer hygromycin resistance. The stable lines
were maintained with 300 µg/ml hygromycin (Invitrogen) in the culture
medium. The expression of Tractin subclones was induced with 0.5 mM CuSO4. COS cells and HEK293T cells were
grown in Dulbecco's modified Eagle's medium with 10% fetal bovine
serum. LoVo cells were grown in Ham's F12 medium with 10% fetal
bovine serum. COS, HEK293T, and LoVo cells were plated overnight to
reach 80% confluence and transiently transfected with
Tractin/pcDNA3.1, NTF-Fc/pcDNA3.1, or Fc/pcDNA3.1 using
LipofectAMINE as per the manufacturer's instructions (Invitrogen).
Cells expressing Tractin or modified versions of Tractin were harvested
24-48 h after transfection and pelleted by centrifugation at 2,000 rpm
for 5 min. For some experiments the supernatant was collected for
further analysis by SDS-PAGE and immunoblotting. The pellet was
resuspended in cell lysis buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, pH 7.4) at 37 °C for 10 min
before centrifugation at 12,000 rpm for 10 min. In some cases the
samples were additionally sonicated. The resulting supernatant was
collected and analyzed with SDS-PAGE and immunoblotting. NTF-Fc and Fc
protein for cell aggregation assays in cultured S2 cells was obtained
by harvesting NTF-Fc- or Fc-expressing COS cells 48 h after
transfection. The cells were resuspended and sonicated in S2 cell
culture medium at 5 × 106 cells/ml. The supernatant
was collected after centrifugation at 12,000 rpm for 10 min and applied
to cultured S2 cells expressing various Tractin deletion constructs for
cell aggregation analysis. The levels of NTF-Fc and Fc protein in the
lysate were verified by SDS-PAGE and immunoblotting.
Biochemical Analysis
SDS-PAGE and Immunoblotting--
SDS-PAGE was performed
according to standard procedures (25). Electroblot transfer was
performed as in Towbin et al. (26) with 1× buffer
containing 20% methanol and in most cases including 0.04% SDS. For
these experiments we used the Bio-Rad MiniPROTEAN II system,
electroblotting to 0.2 µm nitrocellulose, and using anti-mouse
horseradish peroxidase-conjugated secondary antibody (Bio-Rad) (1:3000)
for visualization of primary antibody diluted 1:1000 in Blotto. The
signal was developed with DAB (0.1 mg/ml) and
H2O2 (0.03%) and enhanced with 0.008%
NiCl2 or visualized using chemiluminescent detection
methods (ECL kit, Amersham Biosciences). The immunoblots were digitized
using an Arcus II scanner (AGFA).
Immunoprecipitation--
Immunoprecipitations were performed at
4 °C. Dissected Hemopis leech nerve cord were homogenized
in extraction buffer (20 mM Tris-HCl, 200 mM
NaCl, 1 mM MgCl2, 1 mM
CaCl2, 0.2% Nonidet P-40, 0.2% Triton X-100, pH 7.4 containing the protease inhibitors phenylmethylsulfonyl fluoride and
aprotinin from Sigma and the homogenate (20 µl) incubated with the
nonspecific mouse IgG conjugated to protein G beads for 2 h. The
resulting supernatant was then incubated with anti-Tractin antibody
conjugated to protein G beads (10 µl) overnight. For
immunoprecipitations from Tractin construct expressing S2 cell lines
the cells were harvested and sonicated in immunoprecipitation buffer
(20 mM Tris-HCl, 10 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.2% Triton X-100, 0.2% Nonidet P-40,
pH 7.4) containing the protease inhibitors phenylmethylsulfonyl
fluoride and aprotinin. Appropriate amounts of antibody were conjugated to 10 µl of protein G-Sepharose beads (Sigma) for 2 h on ice. In
the case of immunoprecipitations from S2 cell culture medium the cell
medium was incubated with antibody-conjugated protein G-Sepharose beads
overnight. After a brief spin for 20 s at 2000 rpm the supernatant
was discarded and the immunoaffinity matrix resuspended and washed
three times with 400 µl of extraction buffer for 15 min. The final
pellet was resuspended in 20 µl of SDS-PAGE sample buffer and boiled
for 5 min before centrifugation and analysis of the supernatant by
SDS-PAGE and immunoblotting.
For co-immunoprecipitation experiments with Tractin deletion constructs
Laz6-56 antibody was conjugated to protein G-Sepharose beads as
described above. The resulting Laz6-56-conjugated immunobeads were then
incubated for 12 h with 400 µl of culture medium obtained from
S2 cells that had expressed full-length Tractin for 12 h. After a
brief spin for 20 s at 2000 rpm the supernatant was discarded and
the immunobeads resuspended and washed three times with
immunoprecipitation buffer. The immunobeads were then incubated with
200 µl of lysate from
IgFN,
IgFN1-3, and CTF construct
expressing S2 cells for 12 h. The resulting immunoprecipitate was
processed and analyzed by SDS-PAGE and immunoblotted as described above.
Biotinylation Assays--
S2 cells transfected with the
Tractin/pMT-V5 construct were induced with 0.5 M
CuSO4 for 24 h and subsequently washed three times
with ice-cold PBS (pH 8.0). Cells were resuspended at a concentration
of 2.5 × 107 cells/ml in PBS (pH 8.0).
Sulfo-NHS-LS-Biotin (Pierce) was added to cells at 1 mg/ml or mock
treated for 30 min at room temperature. The cells were then washed
three times with ice-cold PBS to remove any remaining biotinylation
reagent. The cells were sonicated in immunoprecipitation buffer as
described above at a concentration of 5 × 107
cells/ml. The extracts were precleared with protein G beads at 4 °C
for 2 h, and the precleared extracts were directly
immunoprecipitated with 1H4 antibody conjugated to protein G-Sepharose
matrix or incubated with streptavidin-agarose beads overnight at
4 °C. The next day the beads were washed three times for 10 min with
immunoprecipitation buffer at 4 °C. Then the beads were boiled in 20 µl of SDS-PAGE sample buffer for subsequent analysis by SDS-PAGE and immunoblotting.
Furin Inhibition--
For furin convertase inhibition studies
decanoyl-RVKR-chloromethyl ketone (cmk) (Bachem, Switzerland) was added
to S2 cells expressing the full-length Tractin construct for a final
concentration of 50 mM. The cells were induced with 0.5 mM CuSO4 and grown for 12 h before
harvesting and analysis as described above.
Cell Adhesion Assays--
Cell aggregation assays were carried
out essentially as described in Hortsch et al. (27).
Briefly, S2 cells expressing Tractin constructs were plated in 6-well
culture dishes at 1.0 × 106 cells/ml, induced with
0.5 mM CuSO4, and grown for 12 h before the cells were aggregated for 2 h at room temperature on a shaking platform at 100 rpm. Digital images of the aggregated cells were obtained on a Zeiss Axiovert inverted microscope using a Paultek digital camera. To investigate the role of the interaction between NTF-Fc and CTF in cell adhesion, CTF and
IgFN construct-expressing S2 cells were plated at 1.0 × 106 cells/ml. For
experimental cultures 100 µl/ml of NTF-Fc lysate obtained from
NTF-Fc-expressing COS cells as described above were added. For control
cell cultures 100 µl/ml of untransfected COS cell lysate were added.
The cells were induced with 0.5 mM CuSO4 and
grown for 12 h, and aggregation assays were carried out as described above. For quantification a digital image of a field in the
middle of each well was obtained, and the number of aggregates containing more than 10 cells were counted. The difference in the
number of aggregates found in experimental and control wells was
compared using a Student's t test. In some control
experiments 100 µl/ml of lysate from Fc-expressing COS cells were
added as described above. That equivalent levels of Fc and NTF-Fc
expression were obtained was verified by immunoblotting and labeling
with Fc antibody.
For homophilic interaction assays Tractin-transfected S2 cells were
labeled with DiI (Molecular Probes, Eugene, OR). The cells were
incubated with 2 µl of 2 mg/ml DiI solution for each milliliter of
culture medium at room temperature for 1 h before the cells were
washed three times with fresh medium to remove excess dye. Untransfected S2 cells were labeled with DiO using Vybrant DiO cell
labeling solution (Molecular Probes). 5 µl of the DiO solution were
added for each milliliter of culture medium at room temperature for
1 h before the cells were washed three times with fresh medium. DiI- and DiO-labeled cells were mixed at 1:1 to a final concentration of 1.0 × 106 cells/ml. The cell mixture was induced
with 0.5 mM CuSO4 and grown for 12 h, and
aggregation assays carried out as described above. Images of the
labeled preparations were obtained on a Zeiss Axioskop equipped with
the appropriate filter sets and a Paultek digital camera.
Immunohistochemistry--
Tractin-expressing S2 cells were
affixed to polylysine-coated slides and fixed for 2 h in 2%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The
slides were incubated 3 h at room temperature with diluted
antibody in PBS containing 0.4% Triton X-100 and 0.005% sodium azide,
washed in PBS with 0.4% Triton X-100, and incubated with fluorescein
isothiocyanate-conjugated goat anti-mouse antibody (ICN, 1:200
dilution). After three washes in PBS the fluorescently labeled
preparations were mounted in glycerol with 5% n-propyl
gallate. A confocal series of images for each of the labeled
preparations were obtained with a Leica confocal TCS NT microscope at
1-µm intervals using the appropriate laser lines and filter sets.
 |
RESULTS |
Fig. 1A shows a diagram
of the domain structure of Tractin. It contains 6 Ig domains, 4 FNIII-like domains, an acidic domain, 12 repeats of a novel
collagen-like proline- and glycine-rich sequence motif, a transmembrane
domain, and an intracellular tail with an ankyrin and a PDZ domain
binding motif (21, 22). The predicted molecular mass of Tractin is 198 kDa; however, antibodies to the NH2-terminal sequence of
Tractin recognized only a 130-kDa glycosylated fragment on standard
immunoblots (Fig. 1B). Furthermore, antibodies to the fourth
FNIII- and acidic domains and the PGYG-repeat domain (mAb 3A11, mAb
1H4) both recognize a doublet of bands of ~165 and 185 kDa whereas an
antibody to the intracellular domain (mAb 3A12) recognized only the
higher 185-kDa band (Fig. 1B). This and observations with
other domain-specific antibodies suggest that Tractin is cleaved at two
sites (Fig. 1A, arrows), one in the third FNIII
domain, and one just proximal to the transmembrane domain (22).
Previously, a full-length version of the Tractin protein has not been
observed on immunoblots of leech central nervous system proteins (21,
22); however, on greatly overloaded gels the same faint band
corresponding to the expected size for the unprocessed protein can be
detected with different domain-specific antibodies (Fig.
1C). The several hundred-fold ratio of cleaved fragments to
full-length protein indicate that Tractin is constitutively post-translationally processed in the leech central nervous system.

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Fig. 1.
Domain-specific Tractin antibodies.
A, diagram of the Tractin protein. The protein sequence is
organized into six Ig domains, four FNIII domains, an acidic domain, a
PGYG repeat-containing domain, which is collagen-like, a transmembrane
domain (TM), and a cytoplasmic domain with an ankyrin
binding and a PDZ binding (SXV) motif. The two putative
proteolytic cleavage sites in the third FNIII domain (cleavage site I)
and between the PGYG-repeat and transmembrane domains are indicated by
arrows. The monoclonal antibodies 4G5, 3A11, 1H4, and 3A12
were made to fusion proteins or peptides of Tractin sequences as
indicated by the black horizontal bars. mAb Laz6-56
(6-56) recognizes the NH2-terminal fragment of
Tractin. The predicted molecular mass of the fragments generated by
proteolytic cleavage at sites I and II is shown on the line
below the diagram. B, immunoblots of Haemopis
nerve cord proteins labeled with Tractin domain-specific antibodies.
Antibody to the NH2-terminal fragment (4G5) recognized a
130-kDa band, antibody to the fourth FNIII and/or acidic domain (3A11)
recognized a doublet of bands, whereas antibody to the cytoplasmic
domain (3A12) only recognized the high band of the doublet.
C, immunoblots of Haemopis nerve cord proteins
after separation by SDS-PAGE and labeled with mAb 1H4 and Laz6-56. On
these immunoblots of intentionally overloaded lanes, full-length
Tractin is recognized by both antibodies as a weak band
(arrow) migrating with a relative molecular mass of 290 kDa.
In B and C the migration of molecular mass
markers in kDa is indicated in gray.
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Expression and Processing of Tractin in the S2 Cell Line--
To
study the processing of Tractin and to determine the ability of Tractin
to mediate homophilic and/or heterophilic interactions we transfected
Drosophila S2 cells with Tractin expression constructs under
the control of a metallothionein promoter. Fig.
2 shows diagrams of the full-length and
the various truncated Tractin constructs with COOH-terminal V5 epitopes
employed in these studies. In addition, we generated an Fc fusion
construct of the NTF from the starting methionine to the first cleavage
site (Fig. 2). Stably transfected S2 cell lines were obtained for the
full-length construct as well as for the deletion constructs.
Interestingly, the cell line expressing full-length Tractin cleaves
Tractin in a pattern identical to that observed in leech nerve cords
(compare Fig. 3 and Fig. 1B).
In immunoblots of cell lysate-containing cell membranes, antibodies to
the COOH-terminal part of Tractin (3A11 and V5) recognize a 180-kDa
band whereas antibodies to the NH2-terminal fragment
(Laz6-56) recognize a 110-kDa band (Fig. 3A). As a
consequence of artificial overexpression in the cell culture the
full-length protein migrating at ~290 kDa is relatively more abundant
under these conditions than in the in vivo situation (Fig.
3A). The 110-kDa NH2-terminal fragment is found
both in the medium and cell lysate (Fig. 3, A and
B). In contrast, the 160-kDa band is only found secreted
into the medium and is not recognized by antibodies to either the
NH2-terminal fragment or the intracellular domain (Fig.
3B). That the molecular masses of these bands do not
precisely correspond to those from leech nerve cords is due to
differences in glycosylation in the S2 cell line and the added V5
tag.

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Fig. 2.
Diagram of Tractin deletion and expression
constructs. The full-length and the COOH-terminal deletion
constructs have an in-frame V5 tag (V5), whereas the NTF
construct was fused to an Fc tag (Fc). The different
domains of Tractin are indicated above the figure:
Ig, immunoglobulin domains; FN, FNIII domains;
Ac, acidic domain; PGYG, PGYG collagen-like
domain; TM, transmembrane domain.
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Fig. 3.
Proteolytic processing of a full-length
Tractin construct in stably transfected S2 cells. A,
immunoblot of S2 cell lysate that includes cell membranes. The Tractin
construct is tagged with the V5 epitope at the COOH-terminal end.
Full-length Tractin is detected as an ~280-kDa band with both
Laz6-56, 3A11, and V5 antibody, the COOH-terminal transmembrane
fragment as a 180-kDa band by 3A11 and V5 antibody, whereas the
NH2-terminal fragment is detected as a 110-kDa band by the
mAb Laz6-56. That the NH2-terminal fragment is found in the
cell lysate suggests that it is tethered to the cell surface.
B, immunoblot of cell medium from S2 cells expressing
full-length Tractin. In the cell medium the secreted middle fragment is
detected as a 160-kDa band by mAb 3A11 whereas the secreted form of the
NH2-terminal fragment is detected as a 110-kDa band by mAb
Laz6-56. No V5-tagged fragments of Tractin are detected in the cell
medium by V5 antibody. The migration of molecular mass markers in kDa
is indicated in gray.
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The migration on SDS-PAGE of the doublet of bands from leech central
nervous system proteins recognized by Tractin COOH-terminal domain-specific antibodies of 165 and 185 kDa (Fig. 1B) are
much higher than the 76 and 98 kDa predicted for these bands based on
their amino acid sequence. This discrepancy can either be due to homo-
and/or heterodimer formation through the formation of SDS-resistant
covalent bonds or to anomalous gel migration (22). In order to
distinguish between these possibilities we compared the migration on
SDS-PAGE of Tractin constructs expressed in the S2 cell line where
different COOH-terminal sequences had been deleted (Fig.
4). From this analysis we found that when
the acidic and the PGYG domains were deleted gel migration of the
peptides were considerably closer to the molecular mass predicted from their amino acid sequence and that the acidic and PGYG domain when
present together accounted for 50-65 kDa of the anomalous gel
migration (Fig. 4, A and B). This observation was
confirmed by a GST fusion protein construct expressed in bacteria
containing only the acidic and PGYG domains, which also migrated with
the molecular mass of a peptide 63 kDa larger than the predicted 79 kDa
(Fig. 4C). Taken together these data suggest that homo
and/or heterodimer formation through SDS-resistant covalent bonds as previously proposed (22) are unlikely.

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Fig. 4.
Relative migration of Tractin deletion
constructs and domains separated by SDS-PAGE. Immunoblots of cell
lysate from S2 cells expressing full-length Tractin, PGYG, Ac,
and Ac/PGYG constructs, respectively, were labeled with V5 antibody
in A and with Laz6-56 antibody in B. The
predicted molecular mass of the full-length Tractin construct is 200 kDa; however, it migrates as an ~290 kDa protein relative to
molecular mass markers on SDS-PAGE (lanes 1 and
5). The majority of this anomalous gel migration is due to
sequences in the COOH-terminal Tractin fragment as the
NH2-terminal fragment migrates close to its predicted size
of 100 kDa (B). Deleting the Ac and PGYG domains
individually does not significantly change the anomalous gel migration
(lanes 2, 3, 6, and 7);
however, when both the acidic and PGYG domains are deleted the
relative migration of the resulting peptides were 50-65 kDa closer to
the migration predicted from their amino acid sequence (lanes
4 and 8). C, the relative migration of a GST
fusion protein containing only the Ac and PGYG domains detected on an
immunoblot with mAb 1H4. The fusion protein migrates as a 142-kDa
protein. This migration is 63 kDa larger than that predicted for a
peptide with a molecular mass of 79 kDa. The migration of molecular
mass markers in kDa is indicated in gray.
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Tractin Is Constitutively Cleaved by a Furin-like Convertase in the
Third FNIII Domain--
Our data from immunoblots with domain-specific
antibodies (Fig. 1) suggest that Tractin is constitutively cleaved in
the ectodomain. Constitutive processing of precursor proteins are often
mediated by furin convertases, which are calcium-dependent
proteases mainly localized in the trans-Golgi network and
are ubiquitously expressed by eukaryotic cells (14). In addition,
Tractin contains a dibasic sequence conforming to the consensus
cleavage site for furin convertases, RXR/KR (14), in the
third FNIII domain. To investigate the possibility of furin
convertase-dependent cleavage of Tractin we applied the furin inhibitor cmk (28-30) to full-length Tractin-expressing S2 cells
for 12 h at a final concentration of 50 mM. As shown
in Fig. 5 (A and B)
incubation with cmk furin inhibitor prevented the generation of the
180- and 110-kDa polypeptides. This observation indicates that these
polypeptides, directly or indirectly, are the result of furin-mediated
proteolytic processing. To test whether the consensus furin site indeed
is a site of cleavage we introduced a RKRRSR to AAAASA mutation into
the full-length Tractin construct and expressed it in S2 cells.
Immunoblot analysis of cell lysate from these cells show that
processing at this site of the mutated Tractin construct was completely
abolished. Thus, these data indicate that tractin is likely to be
cleaved at the RKRRSR sequence in the third FNIII domain. To test the
implication of furin itself in this process we tested the processing of
the full-length Tractin construct in LoVo cells, a cell line
established from a human colon carcinoma that expresses no functional
furin (15, 31). Expression of Tractin in the mammalian HEK293T cell
line served as a control. Fig. 6 shows
the results of transiently expressing Tractin in these cell lines. Both
the 1H4 and Laz6-56 antibodies detect the expected cleavage products on
immunoblots of cell lysate from the furin-containing HEK293T cells,
whereas no cleaved polypeptides are detectable in the LoVo cells that
lack furin. These results support the hypothesis that Tractin
processing at the first cleavage site is furin-dependent.
To further examine whether full-length Tractin is expressed at the cell
surface we treated Tractin-expressing S2 cells with biotin
succinimidyl-ester or mock-treated them without adding biotin. The
cells were lysed and the protein extracts immunoprecipitated by Tractin
1H4 antibody. Subsequently, the 1H4 immunoprecipitates were separated
by SDS-PAGE and labeled with either 1H4 or anti-biotin antibody on
immunoblots. While both full-length Tractin and the COOH-terminal
fragment could be detected with 1H4 antibody only the transmembrane
fragment was labeled with anti-biotin antibody (Fig.
7). This result suggests that the
COOH-terminal cleavage product but not the full-length precursor are
present at the cell surface.

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Fig. 5.
Furin-dependent cleavage of
Tractin expressed in S2 cells. A and B,
expression of full-length Tractin were induced with CuSO4
in stably transfected S2 cells for 12 h in the absence (cmk ) or
presence (cmk+) of the furin convertase inhibitor cmk. The cell lysate
from the cells were separated by SDS-PAGE, immunoblotted, and Tractin
detected with V5 antibody in A and with mAb Laz6-56 in
B. In the presence of furin inhibitor (cmk+) neither
antibody was able to detect any of the Tractin cleavage fragments
obtained without furin inhibitor (cmk ). C, site-directed
mutagenesis of the consensus furin convertase cleavage site RKRRSR in
Tractin. The lysines and the arginines at the cleavage site were
mutated into alanines by PCR. When this construct was expressed in S2
cells no cleavage fragments, only full-length Tractin, were detected by
the antibodies Laz6-56, 3A11, and V5 on immunoblots of cell lysates.
The migration of molecular mass markers in kDa is indicated in
gray.
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Fig. 6.
Tractin processing in LoVo cells. LoVo
cells do not express any functional furin convertase. In LoVo cells
transiently transfected with a full-length Tractin construct no Tractin
cleavage fragment can be detected on immunoblots of cell lysate by
either mAb 1H4 or Laz6-56 (lanes 2 and 4). In
contrast, in control furin-expressing HEK293T cells (293T) transiently
transfected with the same construct the expected Tractin cleavage
fragment is labeled by the respective antibodies (lanes 1 and 3). The migration of molecular mass markers in kDa is
indicated in gray.
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Fig. 7.
Full-length Tractin is not present at the
cell surface in S2 cells. Tractin-expressing S2 cells were treated
with biotin succinimidyl-ester (biotin +) or mock-treated without
adding biotin ( ). The cell lysate from both groups of cells was
immunoprecipitated by 1H4 antibody followed by SDS-PAGE and
immunoblotting. The immunoblots were labeled with the Tractin
antibody 1H4 (lanes 1 and 2) and with antibody to
biotin (lanes 3 and 4). While both full-length
Tractin and the COOH-terminal fragment could be detected with 1H4
antibody only the transmembrane fragment was labeled with anti-biotin
antibody in biotin-treated S2 cells (lane 3). The migration
of molecular mass markers in kDa is indicated in gray.
|
|
Membrane Tethering of the Cleaved NH2-terminal Tractin
Fragment--
The above studies provide evidence that the
NH2-terminal fragment of Tractin is constitutively cleaved
in the trans-Golgi network although it is known from
immunocytochemistry and immunoelectron microscopy that it is localized
to the surface of axons (32, 33). This raises the question of how it is
tethered to the membrane. One possibility is that it is interacting
with integrins through its RGD integrin binding motif just upstream of
the first cleavage site (21). Another is that it binds to the
transmembrane fragment of Tractin after cleavage. We explored the
latter possibility by performing cross-immunoprecipitation experiments
with Tractin domain-specific antibodies of homogenates from S2 cells
stably transfected with a full-length V5-tagged Tractin construct. Fig. 8A shows that on immunoblots
of immunoprecipitations with the Tractin NH2-terminal mAb
Laz6-56 the transmembrane Tractin fragment is detected as a 180-kDa
band by the mAbs, 3A11, 1H4, and V5. Conversely, on immunoblots of
homogenate from these cells immunoprecipitated by V5 antibody to the
COOH-terminal fragment the NH2-terminal fragment is
detected by Laz6-56 antibody as a 110-kDa band (Fig. 8B).
The 1H4 mAb co-immunoprecipitates the 110-kDa NH2-terminal fragment as detected by mAb Laz6-56 (Fig. 8C, lane
1) from the medium of Tractin-transfected S2 cells in addition to
the secreted 160-kDa fragment (Fig. 8C, lane 2).
The 110-kDa band is not immunoprecipitated from non-Tractin-expressing
control S2 cells (Fig. 8C, lane 3). Consistent
with these findings the 110-kDa NH2-terminal-containing fragment, but not the 160-kDa middle fragment of Tractin, is found in
the cell lysate, which contains cell membranes (Fig. 3A).
Thus, the cleavage fragments of Tractin interact with each other, and this interaction may provide a means for the NH2-terminal
fragment to be tethered to the membrane. To test whether this
interaction of the Tractin fragments also occurs in the leech nervous
system we performed cross-immunoprecipitation experiments with the
Laz6-56 and 1H4 mAbs on protein extracts from leech nerve cords. As
indicated by the immunoblots in Fig. 9
(A and B) both the Laz6-56 and 1H4 antibodies can
co-immunoprecipitate the transmembrane as well as the cleaved
NH2-terminal-secreted fragment demonstrating that the two
fragments are likely to interact in vivo.

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Fig. 8.
The NH2-terminal fragment of
Tractin interacts with the COOH-terminal fragment in
co-immunoprecipitation assays. A, immunoblots of cell
lysate from S2 cells stably transfected with a full-length Tractin
construct and immunoprecipitated with the Tractin
NH2-terminal-specific mAb Laz6-56. The transmembrane
Tractin COOH-terminal fragment is detected as a 180-kDa band by the
3A11, 1H4, and V5 mAbs. B, on immunoblots of cell lysate
from Tractin-expressing S2 cells immunoprecipitated by V5 antibody the
NH2-terminal Tractin fragment is detected by Laz6-56
antibody as a 110-kDa band. C, the 1H4 mAb
co-immunoprecipitates the 110-kDa NH2-terminal fragment as
detected by mAb Laz6-56 (lane 1) from the medium of
Tractin-expressing S2 cells (S2/Tractin) in addition to the secreted
middle fragment of 160 kDa (lane 2). The 110-kDa band is not
immunoprecipitated from non-Tractin-expressing control S2 cells
(lane 3).
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Fig. 9.
The NH2-terminal fragment of
Tractin extracted from leech nerve cords interacts with the
COOH-terminal Tractin transmembrane fragment. A and
B, immunoblots of Haemopis nerve cord proteins
immunoprecipitated with mAbs 1H4 and Laz6-56, respectively, and
compared with nerve cord lysate (lysate). The immunoblots
were labeled with mAb Laz6-56 in A and with mAb 1H4 in
B. Both the Laz6-56 and 1H4 antibodies can
co-immunoprecipitate the transmembrane 185 kDa as well as the cleaved
130-kDa NH2-terminal fragment, demonstrating that the two
fragments are likely to interact in vivo. The migration of
molecular mass markers in kDa is indicated in gray.
|
|
To identify the domain of the transmembrane fragment of Tractin
responsible for binding the NH2-terminal fragment we tested the ability of various truncated transmembrane expression constructs to
interact with the NH2-terminal fragment (Fig.
10). Tractin NH2-terminal fragment obtained from the medium of stably full-length
Tractin-expressing S2 cells was bound to Laz6-56-coated protein G beads
and incubated with homogenates of cell lysate from
Ig/FN,
Ig/FN1-3, or CTF transiently transfected S2 cells. Subsequently,
the protein G beads were spun down, washed, and assayed on immunoblots
for the presence of the V5-tagged transmembrane deletion constructs
with V5 antibody. In all three constructs the entire sequence
NH2-terminal to the furin cleavage site located in the
middle of the third FNIII domain was deleted. In addition, the
remaining part of the third FNIII domain was deleted in the
Ig/FN1-3 construct, and the fourth FNIII domain was further deleted
in the
Ig/FN construct. Fig. 10 shows that of the three deletion
constructs only the CTF construct was detected on the immunoblots with
V5 antibody. These data suggest that the interaction between the
NH2-terminal cleavage fragment and the transmembrane
fragment is mediated by sequences located in the third FNIII domain
just distal to the furin cleavage site.

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Fig. 10.
The NH2-terminal fragment of
Tractin interacts specifically with the third FNIII domain of the
COOH-terminal transmembrane fragment. Tractin
NH2-terminal fragment was bound to Laz6-56-coated protein G
beads and incubated with homogenate of cell lysate from Ig/FN,
Ig/FN1-3, or CTF Tractin construct-expressing S2 cells.
Subsequently, the immunobeads were pelleted and assayed on immunoblots
for the presence of the V5-tagged transmembrane deletion constructs
with V5 antibody. Of the three deletion constructs only the CTF
construct was detected on immunoblots with V5 antibody indicating that
the interaction between the NH2-terminal cleavage fragment
and the transmembrane fragment is mediated by sequences located in the
third FNIII domain. The migration of molecular mass markers in kDa is
indicated in gray.
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|
Homophilic Adhesion of Tractin-expressing S2 Cells--
To
determine the ability of Tractin to mediate homophilic and/or
heterophilic interactions we transiently and stably transfected Drosophila S2 cells with Tractin expression constructs.
Among the stably transfected S2 cell lines one expressed a full-length construct with a COOH-terminal V5 epitope and one expressed a truncated
Tractin construct (
Ac/PGYG) encompassing sequence from the first
cleavage site in the third FNIII domain to the COOH-terminal end but
lacking the acidic and the collagen-like domains (Fig. 2). S2 cells are
ideal for adhesion interaction assays since untransfected cells are
non-adhesive (Fig. 11A) and
do not express any known adhesion molecules (27). When S2 cells are
induced with the full-length Tractin construct it leads to cell
aggregation (Fig. 11B). This aggregation is not observed in
cells transfected with the CTF construct (Fig.
12A) but is still present
when the acidic and PGYG domains are deleted (Fig. 11C).
These data suggest that the cell adhesive properties reside within
sequences of the NH2-terminal domain of Tractin. Expression
of Tractin in the transfected cell lines was confirmed by confocal
imaging of Tractin antibody-labeled cells (Fig. 11D) and by
immunoblot analysis (data not shown). To directly test whether the
induced interaction was homophilic we labeled untransfected S2 cells
with DiO (green) and Tractin-transfected S2 cells with DiI (red). Equal
numbers of DiO- and DiI-labeled cells were mixed and grown for 12 h. As shown in Fig. 11E the Tractin-transfected S2 cells
formed pure aggregates of DiI-labeled cells whereas DiO-labeled untransfected S2 cells were scattered and not part of the aggregates. These data suggest that Tractin induces cell adhesion through homophilic interactions mediated by the NH2-terminal
fragments. To further test whether the NH2-terminal
fragment is necessary for cell adhesion we added NTF-Fc constructs to
S2 cells transfected with Tractin CTF. S2 cells induced to express
Tractin CTF are non-adhesive (Fig. 12A); however, addition
of NTF-Fc to the medium results in aggregation (Fig. 12B).
In control experiments where Fc fragment only was applied to the medium
of the CTF-expressing S2 cells no such aggregation were observed (data
not shown). We quantified NTF-Fc induced interaction by counting the
number of aggregates containing more than ten cells in experimental
versus control wells. In thirteen such experiments control
wells had 1.7 ± 1.3 aggregates whereas experimental wells in
which NTF-Fc had been added had 21.6 ± 8.2 aggregates. This
difference is statistically significant on the p < 0.001 level (Student's t test). The induced aggregation is
a consequence of specific interactions of Tractin NTF-Fc with FNIII
domains 3.5-4 since S2 cells transfected with a COOH-terminal construct
where these sequences were deleted do not become adhesive and do not
form aggregates in the presence of the NTF-Fc construct (Fig.
12D). These findings indicate that tethering of exogeneously
provided Tractin NH2-terminal fragment to S2 cells
expressing only the COOH-terminal fragment can functionally restore the
adhesive properties of Tractin.

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Fig. 11.
Aggregation of Tractin-expressing S2
cells. A, non-induced control S2 cells do not adhere.
B, induction of expression of a full-length Tractin
construct in stably transfected S2 cells leads to cell adhesion and the
formation of large cell aggregates (arrows). C,
cell aggregates are still present when S2 cells express the Ac/PGYG
Tractin construct where the acidic and the PGYG domains are deleted.
D, confocal image of an aggregate of Tractin-expressing S2
cells labeled with mAb Laz6-56. The antibody labeling is predominantly
associated with the cell surface. E, homophilic adhesion
(arrow) of Tractin-transfected S2 cells labeled with DiI
(red) in a mixture of untransfected S2 cells labeled with
DiO (green).
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Fig. 12.
The NH2-terminal Tractin
fragment is necessary for cell aggregation through interaction with the
FNIII domains of the transmembrane fragment. A, S2
cells expressing the CTF construct do not adhere. B,
addition of NTF-Fc fusion protein to the medium of CTF
construct-expressing S2 cells induces adhesion and cell aggregation
(arrows). C, S2 cells expressing the Ig/FN
construct do not adhere. D, addition of NTF-Fc fusion
protein to the medium of Ig/FN construct-expressing S2 cells does
not induce cell aggregation.
|
|
 |
DISCUSSION |
In this study we provide evidence that the L1 family CAM Tractin
is constitutively cleaved in a furin-dependent process and that the full-length precursor protein does not reach the cell surface.
This gives rise to an extracellular NH2-terminal fragment and a COOH-terminal transmembrane fragment that form a heterodimer through non-covalent interactions. This processing is similar to that
of the Notch receptor where furin-dependent cleavage in the
trans-Golgi network also determines the functional structure of the molecule (15, 17). The cleavage site of Tractin, which conforms
to the consensus sequence for processing by furin convertases, is found
at the same location in the third FNIII domain in a majority of L1
family CAMs (3) including mammalian L1, NrCAM, chick NgCAM, and
nematode LAD-1, suggesting this cleavage has a conserved function. This
site in mammalian L1 has previously been shown to be sensitive to
trypsin (34, 35) as well as to plasmin (36, 37) in cell culture
studies; however, the enzyme responsible for in vivo L1
cleavage has yet to be determined. Our studies raise the possibility
that cleavage by furin convertases may be a general mechanism for L1
family CAM processing. While all Tractin molecules are cleaved at the
furin site a fraction of the COOH-terminal fragments are additionally
cleaved just proximal to the membrane, generating a secreted middle
fragment. We do not know the identity of the enzyme responsible for
this processing in leech; however, evidence has been presented that
this cleavage process, which also takes place with mammalian L1, is
mediated by a disintegrin metalloproteinase (19).
Interestingly, the studies performed with limited trypsination of
mammalian L1 in cell culture studies indicated that the NH2-terminal fragment generated by this treatment is not
released after cleavage from the cell surface (35). Based on this
observation it was speculated that it remains in a non-covalent
association with its complementary transmembrane cleavage partner (35). In the present study we provide direct experimental evidence for this
hypothesis. We show that the NH2-terminal fragment of
Tractin expressed in S2 cells can be tethered to the membrane by
interaction with sequences in the third FNIII domain of the
transmembrane fragment and that this interaction is necessary for
establishing homophilic cell adhesion. The COOH-terminal transmembrane
fragment alone did not promote cell adhesion when expressed in the S2
cells suggesting that trans interactions between these
fragments do not occur under physiological conditions. Analysis with
truncated constructs demonstrated that sequences just distal to the
furin cleavage site of the third FNIII domain were necessary for
binding of the NH2-terminal domain; however, the sequence
or sequences responsible for binding within the
NH2-terminal domain remains to be determined. Previous
studies have shown that multiple regions of the
NH2-terminal domain of mammalian L1 can interact with the third FNIII domain including several of the Ig domains (35). These
findings suggest that several regions of the NH2-terminal fragment may be involved in the binding to the third FNIII domain. Recent studies have suggested that the third FNIII domain in mammalian L1 also plays an important role in homomultimerization leading to
trimeric L1 and a concomitant recruitment of integrins by means of
cis interactions (37). The L1 trimerization is regulated by
ligand interactions of the extracellular domain. Interestingly, the
multimerization is abolished by proteolytic cleavage within the third
FNIII domain (37). However, since Tractin is constitutively cleaved at
this site multimerization of Tractin by this mechanism is not likely to occur.
An important issue is whether interactions observed in heterologous
cell culture systems also take place in vivo. We found that
both the Laz6-56 and 1H4 antibodies can co-immunoprecipitate the
transmembrane as well as the cleaved NH2-terminal secreted fragment from leech nerve cord extracts strongly indicating that the
two fragments also are likely to interact in the nervous system. Interestingly, the secreted middle fragment generated from proteolysis at the furin site as well as just distal to the transmembrane segment
is also located to the surface of neurons and axons (22). A likely
explanation for this membrane localization is that the middle fragment
may interact heterophilic with other molecules. It is well established
that many L1 family CAMs can interact with various extracellular matrix
components (1, 38) raising the possibility that the secreted middle
fragment of Tractin may function as a substrate adhesion molecule by
incorporation into the extracellular matrix. Such an interaction may be
facilitated by the collagen-like properties of the PGYG domain
(22).
Interaction with its own transmembrane cleavage fragment may not be the
only mechanism for tethering the NH2-terminal fragment of
Tractin to the membrane as this fragment contains an RGD
integrin-binding motif at the beginning of the third FNIII domain (21).
Consequently, the possibility exists that Tractin
NH2-terminal fragments are linked to integrins as well as
to the Tractin COOH-terminal fragment and in this way may provide a
capability of signaling through two different signal transduction
systems. This notion is supported by the findings that the shedded
ectodomain of mammalian L1 interacts with integrins through a RGD motif
located in the sixth Ig domain and that this interaction may play a
distinct functional role in promoting cell migration (19). Thus,
controlled post-translational proteolysis of CAMs may be a general
mechanism for enhancing functional diversity by generating cleaved
ectodomains that can bind to different molecules involved in distinct
signal transduction pathways or extracellular matrix interactions.
 |
ACKNOWLEDGEMENT |
We thank Dr. Paul Kapke at the Iowa State
University Hybridoma Facility for help with maintaining the monoclonal
antibody lines.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant NS 28857 (to J. Jo.), by National Science Foundation Grant 9724064 (to J. Je.), and by a Fung Graduate Fellowship Award (to Y. X.).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.
¶
To whom correspondence should be addressed: Dept. of Zoology
and Genetics, 3156 Molecular Biology Bldg., Iowa State University, Ames, IA 50011. Tel.: 515-294-2358; Fax: 515-294-4858; E-mail: jorgen@iastate.edu.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M210775200
 |
ABBREVIATIONS |
The abbreviations used are:
CAM, cell adhesion
molecule;
mAb, monoclonal antibody;
PBS, phosphate-buffered saline;
GST, glutathione S-transferase;
DAB, diaminobenzidine;
cmk, decanoyl-RVKR-chloromethyl ketone;
NTF, NH2-terminal
domain;
CTF, COOH-terminal domain.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.