 |
INTRODUCTION |
Proteolytic processing of cell-surface proteins is of prime
importance for regulating the functional properties of these proteins (for reviews, see Refs. 1-5). Cleavage of recognition molecules at the
cell surface has been implicated in neuronal migration, neurite
outgrowth, and synaptic plasticity (6-13). Among the neural adhesion
molecules, L1 has been shown to undergo proteolytic cleavage, which has
been suggested to be involved in several functions of this molecule.
L1 is a member of the immunoglobulin superfamily consisting of
immunoglobulin-like domains and fibronectin type III repeats (for
reviews, see Refs. 14 and 15). In the central nervous system, L1 is
expressed only by post-mitotic neurons and mainly on non-myelinated
axons, whereas in the peripheral nervous system, it is expressed by
neurons as well as by non-myelinating Schwann cells. L1 is also
expressed by non-neural cells, including normal and transformed cells
of hematopoietic and epithelial origin. L1 is involved in neuronal
migration, neurite outgrowth, and myelination (for review, see Ref. 14)
as well as axon guidance, fasciculation, and regeneration (16, 17).
Furthermore, it enhances cell survival (18) and synaptic plasticity
(19). The importance of L1 in nervous system development is underscored
by the abnormal phenotypes of L1 mutations in humans and mice (for
review, see Ref. 20). L1 engages in homophilic and heterophilic cell
interactions (for reviews, see Refs. 14 and 15) Heterophilic binding
partners are the RGD-binding integrins and TAG-1/axonin-1,
F3/F11/contactin, NCAM, CD9, CD24, and phosphacan (Ref. 21 and
references therein). These interactions are likely to depend on the
presentation of the L1 molecule either as a membrane-bound form or as a
proteolytic fragment, which has been described in various forms (Ref.
22 and references therein). The 140- and 80-kDa fragments resulting from cleavage within the third fibronectin type III
(FNIII)1 domain (23) have
been generated in vitro by trypsin (24) or plasmin (21). The
third FNIII domain containing two RGD-independent integrin-binding
sites (21) is involved in homophilic binding (25), multimerization
(21), and L1-dependent neurite outgrowth (26). Cleavage
within this domain by plasmin reduces multimerization and
RGD-independent integrin binding (21). The 180- and 50-kDa fragments
result from membrane-proximal cleavage of the membrane-spanning 200- and 80-kDa L1 forms, respectively, by a metalloprotease, most likely of
the ADAM (a disintegrin and
metalloprotease) family (27, 28). This cleavage step has
been proposed to be required for cell migration (28). Because
specific proteolytic processing of L1 is important for regulation of
neuronal migration and neurite outgrowth, we have searched for the
proteases responsible for cleaving L1 at the two sites and investigated
some of the structural and functional consequences of this proteolytic cleavage.
 |
EXPERIMENTAL PROCEDURES |
Reagents and Antibodies--
GM 6001 and leupeptin were
purchased from Calbiochem (Bad Soden, Germany), and aprotinin and
1,10-phenanthroline were from Sigma (Taufkirchen, Germany). The
calmodulin inhibitor CGS 9343B was a gift from Novartis Consumer Health
(Nyon, Switzerland). CompleteTM EDTA-free protease
inhibitor mixture was obtained from Roche Molecular Biochemicals
(Mannheim, Germany). Polyclonal anti-L1 antibodies were obtained from
rabbits immunized with a protein A-purified L1-Fc fusion protein
consisting of the extracellular domain of mouse L1 and the Fc portion
of human IgG1 (18). Rat monoclonal antibody 555 (29) reacts with an
epitope at the border between the FNIII homologous repeats 2 and 3. Anti-glyceraldehyde-3-phosphate dehydrogenase antibody was purchased
from Chemicon (Hofheim, Germany). All secondary antibodies were
obtained from Dianova (Hamburg, Germany).
DNA Mutagenesis--
Mutagenesis of L1 was performed using the
QuickChange site-directed mutagenesis kit (Stratagene, Amsterdam, The
Netherlands). Mouse L1 cDNA was cloned into pGEM2 (Promega,
Mannheim), and the resulting vector was used as a template in PCR-based
mutagenesis. The PCR primers used for mutagenesis had the following
sequences: 5'-tgg aag ggc agc cag agc aag cac agc
tcg agc cat atc cac aaa agc-3' and 5'-gct ttt gtg
gat atg gct cga gct gtg ctt gct ctg
gct gcc ctt cca-3'. Bases in boldface indicate exchanges in the mutated
L1 cDNA.
Cell Culture--
Mouse neuroblastoma cells (Neuro2a) were
maintained in Dulbecco's modified Eagle's medium containing 10%
fetal calf serum, 1 mM sodium pyruvate, and antibiotics
(100 units/ml penicillin and 100 µg/ml streptomycin). Cerebellar
granule neurons were prepared from 6-8-day-old C57BL/6J mice as
described (30). In brief, cerebella were digested with a trypsin/DNase
solution and mechanically dissociated. The cells were resuspended and
cultured in serum-free basal medium Eagle culture medium
containing 1 mg/ml bovine serum albumin, 2.2 mg/ml NaHCO3,
100 µg/ml transferrin, 10 µg/ml insulin, 4 nM
thyroxine, 30 nM NaSeO3, 0.027 trypsin
inhibitory units/ml aprotinin, 5 IU/ml penicillin, and 5 µg/ml
streptomycin. Cultures of hippocampal neurons were also prepared by a
combination of enzymatic and mechanical dissociation from 1-3-day-old
C57BL/6J mice. During the first 20 h after plating on a laminin
substrate (40 µg/ml), cells were maintained in minimum
essential medium Eagle containing 100 µg/ml transferrin, 2.5 µg/ml
insulin, 5 µg/ml gentamycin, and 10% horse serum (31).
Treatment of Neuro2a Cells with Inhibitors--
Neuro2a cells
(105 to 106 cells/well) were seeded onto a
six-well plate (Nunc, Wiesbaden, Germany) and grown in Dulbecco's
modified Eagle's medium containing 10% fetal calf serum. After
24 h, the medium was replaced with serum-free medium containing
either a protease inhibitor (10 µM GM 6001 or leupeptin)
or a calmodulin inhibitor (10 µM CGS 9343B). The cells
were maintained for an additional 24 h in the absence or presence
of the inhibitors. Cells and cell culture supernatants were collected
separately. Cell culture supernatants were cleared by centrifugation at
100,000 × g for 1 h at 4 °C. After
centrifugation, proteins from the supernatants were concentrated by
acetone precipitation. 1 volume of the sample was incubated with 7 volumes of ice-cold acetone for 30 min and then subjected to
centrifugation at 14,000 × g for 30 min at 4 °C.
Protein pellets were resuspended in sample buffer. Cells were homogenized in phosphate-buffered saline (pH 7.4) and centrifuged at
1000 × g for 10 min at 4 °C. For SDS-PAGE, the
supernatants were diluted with sample buffer.
Cultures of Dissociated Hippocampal and Cerebellar Granule
Neurons--
Hippocampal and cerebellar granule neurons were
maintained in vitro for 6 days and lysed in radioimmune
precipitation assay buffer (150 mM NaCl, 50 mM
Tris-HCl (pH 7.4), and 1 mM EDTA) containing 1% Nonidet
P-40 and CompleteTM protease inhibitor mixture.
Lysates were centrifuged at 1000 × g for 10 min at
4 °C. Proteins in the supernatants were concentrated by
methanol/chloroform precipitation (32).
Microexplant Cultures--
Microexplant cultures from mouse
cerebella were prepared as described by Fischer et al. (33).
Briefly, cerebella were taken from 6-8-day-old C57BL/6J mice and
transferred to ice-cold Hanks' balanced salt solution. The tissue was
freed from meninges, choroid plexus, and blood vessels and forced
through a Nitrex net with a pore width of 300 µm. The small tissue
pieces were washed twice with Hanks' balanced salt solution, followed
by a washing step with culture medium (minimum Eagle's medium
containing 10% horse serum, 10% fetal calf serum, 6 mM
glucose, 200 µM L-glutamine, 50 units/ml
penicillin, 50 µg/ml streptomycin, 10 µg/ml human transferrin, 10 µg/ml insulin, and 10 ng/ml selenium) (34). The tissue pieces were
collected by sedimentation. 30-40 explants were plated onto
poly-L-lysine-coated glass coverslips (15-mm diameter) or
coverslips additionally coated with 2 µg/ml mouse L1
immunoaffinity-purified from adult mouse brain (35) in ~100 µl of
culture medium/coverslip. Just before plating, the coating solution was
removed, and the coverslips were rinsed once with Hanks' balanced salt
solution. 16 h after plating, 1 ml of culture medium lacking fetal
calf serum was added to the explants containing the metalloprotease
inhibitor GM 6001 at 20 µM (20 mM stock
solution dissolved in Me2SO), the serine inhibitor
aprotinin at 1 µM, or the serine and cysteine inhibitor
leupeptin at 10 µM. In the case of GM 6001, the control
explants were incubated with 0.2% Me2SO in parallel. After
an incubation time of 24 h, the explants were fixed with 2%
glutaraldehyde and 2% paraformaldehyde and stained with 1% toluidine
blue and 1% methylene blue in 1% borax (pH 7.4). The effect of the
different protease inhibitors on neurite outgrowth was analyzed and
quantitated by measuring the length of the 10 longest neurites of 10 aggregates in each experiment with an IBAS Image analysis system
(Kontron, Zeiss, Germany).
Transient DNA Transfection--
For transient expression, the
following constructs were used. The cDNA coding for mouse PC1, PC2,
or PC5A was cloned into the pRcCMV vector (Invitrogen, Karlsruhe,
Germany). The cDNA coding for human furin, human PACE4,
or rat PC7 was inserted into pcDNA3 (Invitrogen). Neuro2a cells
were transfected with the different vectors using LipofectAMINE Plus
(Invitrogen) following the manufacturer's instructions. After 24 h, the transfection medium was replaced with serum-free medium, and the
cells were maintained for an additional 24 h at 37 °C. Cells
and cell culture supernatants were collected separately. Cell culture
supernatants were cleared by centrifugation at 100,000 × g for 1 h at 4 °C. After centrifugation, proteins from the supernatants were concentrated by methanol/chloroform precipitation. Cells were homogenized in phosphate-buffered saline (pH
7.4) and further centrifuged at 1000 × g for 10 min at
4 °C. For SDS-PAGE, the supernatants were diluted with sample buffer.
Infection of HEK293 Cells Using a Vaccinia Virus System--
For
expression of wild-type and mutated L1 via vaccinia virus, the
corresponding cDNAs were cloned into the pMJ601 expression vector
(36). HEK293 cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum. For expression of the
different proprotein convertases (37), HEK293 cells were infected with
pMJ601 constructs carrying the cDNA inserts coding for mouse PC1,
PC2, and PC5A; for rat PC7; and for human PACE4 and furin (38). After
infection of cells with 1 plaque-forming units/cell for 2 h in 5 ml of phosphate-buffered saline and 0.01% bovine serum albumin, cells
were maintained for 48 h in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. Cells were then washed twice
with serum-free medium and further incubated for 6 h in the
absence of serum. Cells were harvested by centrifugation at 500 × g for 10 min at 4 °C and resuspended in radioimmune
precipitation assay buffer containing 1% Nonidet P-40 and
CompleteTM protease inhibitor mixture. Cell lysates
were then subjected to sonification for 1 min at an amplitude of 100%
(UP50 sonifier, Hielscher, Stuttgart, Germany) and cleared by
centrifugation at 14,000 × g for 30 min at 4 °C.
Culture supernatants were cleared by centrifugation at 100,000 × g for 1 h at 4 °C. Cleared cell lysates and culture
supernatants were concentrated by acetone precipitation. Protein
pellets were resuspended in radioimmune precipitation assay buffer
containing CompleteTM protease inhibitor mixture.
In Vitro Assay of Proteolytic Processing--
For analysis of
the proteolytic L1 products, Neuro2a cells and mouse brains from adult
wild-type mice were used. The Neuro2a cells and total mouse brain were
homogenized in 1 mM NaHCO3, 0.2 mM
CaCl2, 0.2 mM MgCl2, and 1 mM spermidine (pH 7.9) and centrifuged at 600 × g for 15 min at 4 °C. After recentrifugation of the
supernatant at 25,000 × g for 45 min at 4 °C, the
pellet was resuspended in RPMI 1640 medium (PAA Laboratories,
Cölbe, Germany) and subdivided into 1-ml aliquots. These aliquots
were incubated at 37 °C for different times with or without the
metalloprotease inhibitors GM 6001 (10 µM) and
1,10-phenanthroline (50 µM), the serine and cysteine
protease inhibitor leupeptin (10 µM), and
CompleteTM EDTA-free protease inhibitor mixture (which
inhibits a broad range of serine and cysteine proteases). As a control,
aliquots were incubated at 4 °C. The samples were then centrifuged
at 100,000 × g for 1 h at 4 °C. Supernatants
were concentrated by acetone precipitation. Protein pellets were
resuspended in sample buffer.
Subfractionation and Sucrose Gradient Analysis--
Brains or
different brain regions (hippocampus and cerebellum) from adult
C57BL/6J mice were homogenized in homogenization buffer (0.32 M sucrose, 50 mM Tris-HCl (pH 7.4), 1 mM CaCl2, and 1 mM
MgCl2). Homogenates of the cerebellum and hippocampus were centrifuged at 1000 × g for 15 min at 4 °C, and the
resulting supernatants were subjected to methanol/chloroform
precipitation (32). The total brain homogenate was centrifuged at
17,000 × g for 15 min at 4 °C. The pellet was
resuspended in homogenization buffer, and the supernatant was cleared
by centrifugation at 100,000 × g for 1 h at
4 °C. For sucrose gradient analysis, the brain homogenate,
17,000 × g pellet, and cleared 17,000 × g supernatant were diluted 1:3 with extraction buffer (1 mM CaCl2, 1 mM MgCl2, 50 mM Tris (pH 7.5), and 1% Triton X-100). After 1 h
of incubation at 4 °C, the samples were layered on top of 10 ml of a
continuous 5-30% sucrose gradient made up in 50 mM
Tris-HCl (pH 7.5), 1 mM CaCl2, 1 mM
MgCl2, and 0.1% Triton X-100. The gradient was centrifuged at 35,000 rpm for 16 h at 4 °C in a Beckman SW 41 rotor.
Fractions of 0.5 ml were harvested from the top of the gradient and
subjected to Western blot analysis. Molecular mass standards (Sigma)
consisting of bovine serum albumin (66 kDa), catalase (260 kDa),
apoferritin (480 kDa), and thyroglobulin (670 kDa) were analyzed in parallel.
Western Blot Analysis--
Protein samples were mixed with
sample buffer and boiled for 5 min. Proteins were separated by SDS-PAGE
under reducing conditions on 8% gels. The proteins were transferred to
a nitrocellulose membrane, and the membrane was blocked with 4% nonfat
dry milk powder in Tris-HCl-buffered saline (pH 7.3). The membrane was incubated with primary antibody overnight at 4 °C with shaking, washed with Tris-HCl-buffered saline (pH 7.3), and probed with horseradish peroxidase-conjugated goat anti-rabbit or anti-rat antibody
(1:10,000 in 4% milk powder in Tris-HCl-buffered saline (pH 7.3)) for
1 h. After washing, immunodetection was performed by chemiluminescence.
 |
RESULTS |
L1 Is Proteolytically Cleaved by PC5A in the Third FNIII
Domain--
In mouse brain, the full-length 200-kDa L1 molecule is
cleaved at a site within the third FNIII domain, generating 140- and 80-kDa fragments (Fig. 1A). A
membrane-proximal cleavage generates 180- and 30-kDa fragments from the
full-length 200-kDa L1 molecule and 50- and 30-kDa fragments from the
80-kDa fragment (Fig. 1A). Because the third FNIII domain of
L1 contains a putative proprotein convertase recognition and cleavage
motif (Fig. 1B), we investigated whether the 140-kDa
fragment could be generated by proprotein convertases
(37).

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Fig. 1.
A, schematic representation of the
different L1 forms in mouse brain. The extracellular domain of the
full-length transmembrane 200-kDa form of L1 consists of six
immunoglobulin-like domains and five FNIII domains. Cleavage within the
third FNIII domain generates the 140-kDa fragment and a transmembrane
80-kDa fragment. Membrane-proximal cleavage of the 200-kDa form and the
80-kDa fragment leads to the generation of 180- and 50-kDa fragments,
respectively, and, in addition, to a 30-kDa transmembrane stump.
B, the putative proprotein convertase recognition motif in
the third FNIII domain of L1. The sequences of third FNIII domain of
human, rat, and mouse L1 (Swiss Protein Database accession numbers
P32004, Q05695, and P11627) are shown. The putative proprotein
convertase recognition motif in the L1 sequence, which is conserved in
all sequences, is depicted in boldface. In the mouse
wild-type L1 sequence, this motif precedes the cleavage site identified
by sequencing the N terminus of the 80-kDa L1 form (23), which is
highlighted in gray. The mutations introduced into the
putative proprotein convertase recognition motif in this study are
indicated with arrows. The underlined sequences
in the human protein indicate two RGD-independent integrin-binding
domains (21).
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Mouse neuroblastoma Neuro2a cells express the full-length transmembrane
200-kDa form of L1 and a 180-kDa proteolytic fragment, but not the
140-kDa L1 fragment (data not shown). Therefore, this cell line was
used for transient transfection with the proprotein convertases furin,
PC1, PC2, PACE4, PC5A, and PC7. As reported previously, control
experiments have shown that each transfected proprotein convertase is
expressed in similar amounts as a functional enzyme (39, 40). Cells
were maintained overnight in serum-free culture medium, and cells and
cell culture supernatants were then collected separately. Western blot
analysis using polyclonal antibodies against the extracellular part of
mouse L1 showed the full-length 200-kDa form in the cell pellet upon
mock transfection (Fig. 2A, lane 1), whereas the 180-kDa fragment was present in both
the cell pellet (Fig. 2A, lane 1) and culture
supernatant (Fig. 2B, lane 1). The 140-kDa
fragment was not detectable in the cell pellet or culture supernatant.
Similar results were obtained upon transfection with PC1 (Fig. 2,
A and B, lanes 2), PC2 (lanes
3), and PACE4 and PC7 (data not shown). In contrast, after
transfection with PC5A, high levels of the 140-kDa form and low levels
of the 200- and 180-kDa forms were found in the cell pellet (Fig.
2A, lane 4). Only the 140-kDa fragment was
detectable in the culture supernatant (Fig. 2B, lane
4). Transfection with furin resulted in the appearance of smaller
amounts of the 140-kDa form in comparison with transfection with PC5A,
whereas the 200- and 180-kDa forms were predominant in the pellet (Fig.
2A, lane 5). The supernatant of furin-transfected cells contained mainly the 180-kDa fragment, whereas the 140-kDa fragment was detectable in minor amounts (Fig. 2B,
lane 5). These results indicate that PC5A cleaves L1 very
efficiently to generate the 140-kDa form, whereas furin is not as
efficient as PC5A in generating this fragment. The proprotein
convertases PC1, PC2, PACE4, and PC7 are not capable of cleaving
L1.

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Fig. 2.
Transfection of Neuro2a cells with proprotein
convertases. Neuro2a cells were mock-transfected (control
(ctrl); lane 1) or transfected with a vector
coding for PC1 (lane 2), PC2 (lane 3), PC5A
(lane 4), or furin (lane 5). The transiently
transfected cells were grown overnight in fetal calf serum-free cell
culture medium. Cell lysates (A) and cell culture
supernatants (B) were subjected to Western blot analysis
using polyclonal anti-L1 antibodies. The positions of the individual L1
forms are indicated to the right.
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Mutation of the Proprotein Convertase Recognition Motif Abrogates
Generation of the 140-kDa L1 Fragment by PC5A--
N-terminal
sequencing of the 140-kDa fragment (23) indicated that this fragment is
generated by proteolytic cleavage at Arg845 (Fig.
1B). To verify this site as the one that is cleaved by PC5A,
the L1 sequence 840RKHSKR845, resembling the
proprotein convertase recognition motif,
(R/K)X0,2,4,6(K/R), was mutated to
840SKHSSS845 (Fig. 1B). The
L1-deficient cell line HEK293 was used for expression of mouse
wild-type and mutated L1 using a vaccinia virus system. Western blot
analysis using polyclonal anti-L1 antibodies showed the 200- and
140-kDa forms in the cell pellet after expression of wild-type L1 (Fig.
3A, lane 1).
Significant amounts of the 180- and 140-kDa forms were found in the
supernatants (Fig. 3A, lane 3), indicating the
presence of L1-processing enzymes in HEK293 cells. Coexpression of
wild-type L1 and PC5A resulted in a strong increase in the amount of
the 140-kDa fragment in the cell pellet (Fig. 3A, lane
2). Only the 140-kDa form was found in the supernatant (Fig.
3A, lane 4), underscoring the efficient cleavage
of L1 by PC5A. Coexpression of wild-type L1 with furin, PC1, PC2,
PACE4, or PC7 showed no increase in the amount of the 140-kDa fragment (data not shown), confirming that PC5A is the most efficient
L1-cleaving proprotein convertase. This difference in cleavage
efficiency is not due to different expression levels of each proprotein
convertase, as shown previously in other studies (38, 41, 42).
In contrast to the expression of wild-type L1, no 140-kDa fragment was
found in the cell pellet (Fig. 3B, compare lanes
2 and 1) or in the culture supernatant (compare
lanes 5 and 4) after expression of the mutated L1
molecule. Even after coexpression of mutated L1 and PC5A (Fig.
3B, lanes 3 and 6) or the other
proprotein convertases (data not shown), the 140-kDa form was not
detectable. These results clearly demonstrate that the proprotein
convertase PC5A cleaves L1 at Arg845.

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Fig. 3.
Infection of HEK293 cells with vaccinia virus
coding for wild-type or mutated L1. A,
L1-deficient HEK293 cells were either infected with a vaccinia
virus carrying a DNA coding for the wild-type (WT) L1
protein (lanes 1 and 3) or co-infected with
PC5A-coding virus (lanes 2 and 4). B,
HEK293 were infected with a vaccinia virus carrying DNA coding for
either wild-type L1 (lanes 1 and 4) or L1
carrying mutations (mut) in the putative proprotein
convertase recognition motif (lanes 2 and 5). In
addition, cells were co-infected with mutated L1 and PC5A (lanes
3 and 6). Cell lysates (lanes 1-3) and
culture supernatants (lanes 4-6) were subjected to Western
blot analysis using polyclonal anti-L1 antibodies. The positions of the
individual L1 forms are indicated to the right.
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The 140-kDa L1 Fragment Is Generated in the Hippocampus, but Not in
the Cerebellum--
Previous studies showed that, in cultured
cerebellar neurons, the 140-kDa fragment is not detectable (24).
Because PC5A is highly expressed in the hippocampus, but not or only
weakly in the cerebellum (44, 45), we investigated whether the 140-kDa form is generated in the hippocampus. Homogenates of adult mouse hippocampus and cerebellum or of cultured early postnatal hippocampal and cerebellar neurons were subjected to Western blot analysis using
monoclonal anti-L1 antibody, which recognizes an epitope at the border
between the second and third FNIII domains. The full-length
membrane-spanning 200-kDa L1 form was detectable in the two brain
regions (Fig. 4, lanes 1 and
2) and cultured neurons (lanes 3 and
4). The 140-kDa fragment was detectable only in the hippocampus and cultured hippocampal neurons (Fig. 4, lanes
1 and 3), but not in the cerebellum and cultured
cerebellar neurons (lanes 2 and 4).

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Fig. 4.
Analysis of L1 processing in cerebellar and
hippocampal cells. Homogenates of the hippocampus and cerebellum
(lanes 1 and 2, respectively) or of cultured
hippocampal and cerebellar granule neurons (lanes 3 and
4, respectively) were subjected to Western blot analysis
using monoclonal anti-L1 antibody. The positions of the individual L1
forms are indicated to the right.
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Dimers Consisting of the Transmembrane 200-kDa L1 Form and the
Tightly Associated 140-kDa Fragment Are Released from the Membrane by a
Metalloprotease--
The generation of the 140-kDa fragment was
analyzed in more detail in an in vitro assay using a crude
membrane fraction from adult mouse brain. After incubation of the
membrane fraction at 4 or 37 °C for 2 or 4 h, the membranes
were pelleted. Pellets and supernatants were subjected to Western blot
analysis using polyclonal anti-L1 antibodies. Membrane pellets
contained high amounts of the 200-, 140-, and 80-kDa forms (Fig.
5A, lanes 2, 4, and 6). After incubation at 4 °C, only low
amounts of the 180-, 140-, and 50-kDa fragments were found in the
supernatants (Fig. 5A, lane 1), whereas
incubation at 37 °C yielded significantly higher amounts of these
fragments in the supernatant (lanes 3 and 5).
After incubation in the presence of the metalloprotease inhibitor GM
6001, neither the 180- nor 140-kDa fragment was detectable in the
supernatant (Fig. 5B, compare lanes 1 and
3). The serine and cysteine protease inhibitor leupeptin
(Fig. 5B, compare lanes 1 and 4) and
CompleteTM EDTA-free protease inhibitor mixture (which
inhibits a broad range of serine and cysteine proteases) (compare
lanes 1 and 2) did not influence the release of
the 180- and 140-kDa fragments. Another metalloprotease inhibitor
(1,10-phenanthroline) reduced the shedding of both fragments (Fig.
5B, compare lanes 1 and 5). Together,
these results indicate that a metalloprotease is involved in the
cleavage of the transmembrane 200- and 80-kDa forms, resulting in the
generation and release of the 180- and 50-kDa fragments. Furthermore,
the membrane-proximal cleavage of the transmembrane forms promotes the
release of the 140-kDa fragment. These observations imply that the
140-kDa fragment is tightly associated with the full-length
transmembrane L1 molecule and that cleavage of the 200- and 80-kDa
forms by the metalloprotease results in the release of the soluble
180-, 140-, and 50-kDa fragments.

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Fig. 5.
Proteolysis of L1 as a function of time,
temperature, and different inhibitors. A, a crude
membrane fraction from brain was incubated for 2 h at 4 °C
(control (ctrl); lanes 1 and 2) or for
2 h (lanes 3 and 4) and 4 h
(lanes 5 and 6) at 37 °C. B, in
parallel, the crude membrane fraction was incubated for 2 h at
37 °C in the absence (control; lane 1) or presence of the
metalloprotease inhibitors GM 6001 (lane 3) and
1,10-phenanthroline (lane 5) as well as
CompleteTM EDTA-free protease inhibitor mixture (lane
2) and the serine and cysteine protease inhibitor leupeptin
(lane 4). The samples were then centrifuged, and the
supernatants (A, lanes 1, 3, and
5; and B, lanes 1-5) and pellets
(A, lanes 2, 4, and 6) were
subjected to Western blot analysis using polyclonal anti-L1 antibodies.
The positions of molecular mass markers are indicated to the left. The
positions of the individual L1 forms are indicated to the right.
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To further investigate whether the 200-kDa form complexes with the
140-kDa fragment, sucrose gradient velocity sedimentation analysis was
performed. A detergent extract from the brain homogenate was separated
in a continuous sucrose gradient, and fractions were analyzed by
Western blotting using monoclonal anti-L1 antibody. The 200- and
140-kDa forms were present in different fractions throughout the
gradient. Based on the sedimentation of the molecular mass markers, the
200- and 140-kDa forms in fractions 4-8 correspond to the 200- and
140-kDa monomers, whereas the 200- and 140-kDa forms in fractions
12-14 sedimented at a molecular mass of ~350 kDa, which is the
expected molecular mass of a 200/140-kDa heterodimer (Fig.
6A). Significant amounts of
the 200-kDa form were present in fractions 16-17 (Fig. 6A),
which correspond to a molecular mass of ~500 kDa and may represent a
high molecular mass complex.

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Fig. 6.
Sucrose gradient sedimentation analysis of L1
and L1 fragments in the total brain homogenate and brain membrane and
soluble protein fractions. Detergent lysates of the total brain
homogenate (A), a detergent-solubilized crude membrane
fraction (B), and a soluble fraction (C) were
applied on top of a 5-30% sucrose gradient. After centrifugation,
fractions were collected from the top of the gradient (fraction 1) to
the bottom (fraction 20) and subjected to Western blot analysis using
monoclonal anti-L1 antibody. The individual L1 forms are indicated to
the right. The positions of the molecular mass standard proteins bovine
serum albumin (BSA), catalase, apoferritin, and
thyroglobulin within the gradient are indicated at the bottom.
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Western blot analysis after sucrose gradient analysis of
detergent-solubilized membranes from mouse brain showed a major portion of the 200- and 140-kDa forms in fractions 12-14, corresponding to a
molecular mass of ~350 kDa (Fig. 6B), the expected
molecular of a 200/140-kDa heterodimer. Only a minor portion of the
200- and 140-kDa forms was present in fractions 4-8 (Fig.
6B), where the 200- and 140-kDa monomers are expected to
migrate. The observation that a large portion of the 200- and 140-kDa
forms was detectable in fractions 16-20 indicates that the two forms
are associated in a high molecular mass complex (Fig. 6B).
Western blot analysis after sucrose gradient analysis of a soluble
protein fraction prepared from the mouse brain homogenate revealed that
the soluble 140- and 180-kDa fragments were present only in fractions
4-9 (Fig. 6C), indicating that the soluble fragments
migrate at molecular masses corresponding to the 140- and 180-kDa
monomers. These results suggest that the membrane-associated
200/140-kDa heterodimers are released from the membrane upon cleavage
of the 200-kDa form by a metalloprotease and that the resulting
180/140-kDa heterodimeric complex dissociates after release from the
cell surface.
Cleavage of L1 by a Metalloprotease Is Increased by a Calmodulin
Inhibitor--
As mentioned above, neuroblastoma Neuro2a cells did not
generate the 140-kDa fragment, but released the 180-kDa fragment into the culture supernatant (Fig. 2, A and B). This
finding prompted us to investigate this particular membrane-proximal
proteolytic process leading to the formation of the soluble 180-kDa
fragment. When Neuro2a cells were cultured in the presence of the
metalloprotease inhibitor GM 6001, release of 180-kDa fragment into the
culture supernatant was strikingly inhibited (Fig.
7, compare lanes 1 and
2), whereas leupeptin, an inhibitor of trypsin-like and
cysteine proteases, had no effect (compare lanes 1 and
3). This finding confirmed that the 180-kDa fragment is
generated by a metalloprotease activity.

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Fig. 7.
Effect of protease and calmodulin inhibitors
on the generation and release of the 180-kDa fragment. Neuro2a
cells were maintained in culture in the absence (control
(ctrl); lane 1) or presence of the
metalloprotease inhibitor GM 6001 (lane 2), the serine and
cysteine protease inhibitor leupeptin (lane 3), and the
calmodulin inhibitor CGS 9343B (lane 4). After incubation
for 24 h at 37 °C, cell culture supernatants were subjected to
Western blot analysis using monoclonal anti-L1 antibody. The position
of the individual L1 form is indicated to the right. For normalization,
cell lysates were analyzed by Western blot analysis using
anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
antibody.
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Because it has been reported that processing by a metalloprotease of
several cell-surface receptors, such as the transforming growth
factor-
receptor (46), amyloid precursor protein (46), tyrosine
receptor kinase TrkA (46), and L-selectin (47), is increased by a
calmodulin inhibitor, we investigated whether cleavage of L1 and
release of the 180-kDa fragment are affected by the calmodulin
inhibitor CGS9343B. The production and release of the 180-kDa fragment
were increased in the presence of this inhibitor (Fig. 7, compare
lanes 1 and 4). As an internal control and for normalization, the cell lysates were analyzed by Western blot analysis
using anti-glyceraldehyde-3-phosphate dehydrogenase antibody (Fig. 7,
lanes 1-4)
The Metalloprotease Inhibitor GM 6001 Inhibits
L1-dependent Neurite Outgrowth--
Cerebellar neurons
from L1-deficient mice do not show neurite outgrowth on
substrate-coated L1 (Ref. 48 and references therein), indicating that
L1-induced neurite outgrowth is mainly mediated by homophilic
interaction and signaling via neuronal cell surface-expressed L1. This
finding allowed us to investigate the effect of GM 6001 on L1-induced
neurite outgrowth and to determine whether processing of L1 by a
metalloprotease is required for L1-induced neurite outgrowth.
Microexplants of early postnatal mouse cerebellum were maintained in
the presence or absence of GM 6001 either on poly-L-lysine or on substrate-coated L1. Explants maintained on
poly-L-lysine showed no significant difference in neurite
length in the presence or absence of GM 6001 (Fig.
8, A, B, and
G). Explants maintained on substrate-coated L1 showed a
decrease in neurite length of ~40% in the presence of GM 6001 compared with explants maintained in its absence (Fig. 8,
C-G), suggesting that a metalloprotease activity is
required for L1-mediated neurite outgrowth. Explants were also grown on
poly-L-lysine or substrate-coated L1 in the presence or
absence of the serine and cysteine protease inhibitor leupeptin and the
serine protease inhibitor aprotinin (Fig. 8G). These
protease inhibitors did not influence the L1-mediated neurite outgrowth
or the neurite outgrowth on the control substrate. These results
confirm that shedding of L1 by a metalloprotease is involved in
L1-mediated neurite outgrowth.

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Fig. 8.
Effect of the metalloprotease inhibitor GM
6001 and the serine and cysteine protease inhibitors aprotinin and
leupeptin on neurite outgrowth of cerebellar neurons. Cerebellar
microexplant cultures were plated onto glass coverslips coated with
poly-L-lysine (PLL) (A and
B) or a combination of poly-L-lysine and L1
(C-F). After incubation for 24 h at 37 °C in the
presence (B, D, and F) or absence
(A, C, and E) of the metalloprotease
inhibitor GM 6001, the explants were fixed and stained. The effect of
the metalloprotease inhibitor GM 6001 and the serine and cysteine
protease inhibitors aprotinin and leupeptin on neurite outgrowth from
the explants was quantitated by measuring the 10 longest neurites of 10 aggregates in three independent experiments (G). The
asterisk indicates a statistically significant difference
(p < 0.05). Bars = 50 µm. The
broken lines in E and F indicate the
margins of the longest neurites.
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DISCUSSION |
Full-length L1 is converted to N-terminal 140-kDa and C-terminal
membrane-spanning 80-kDa complementary proteolytic fragments (24) by
proteolytic cleavage in the third FNIII domain (23). This domain is
functionally important because it contributes to homophilic binding
(25) and multimerization of L1, neurite outgrowth, and RGD-independent
integrin binding to the
1-integrin subunit (21). Two
motifs (821QVKGHLR827 and
837GSQRKHSKR845; see Fig. 2A) within
the third FNIII domain are involved in RGD-independent integrin
binding to L1. Substitution of the dibasic RK and KR in the
837GSQRKHSKR845 motif results in reduced
multimerization of L1 molecules and diminished integrin binding (21).
The functional activity of this particular RGD-independent
integrin-binding motif appears to be regulated by proteolytic cleavage
within this sequence motif: cleavage by plasmin within this motif
disrupts multimerization and RGD-independent integrin binding. Plasmin,
the end product of the plasminogen/plasminogen activator cascade,
cleaves L1 within the 837GSQRKHSKR845 motif at
Lys841 and Lys844, leading to the
generation and release of a 140-kDa fragment (21). These cleavage
sites are both different from the cleavage site obtained by N-terminal
sequencing of the 80-kDa form (23), indicating that proteolytic
cleavage takes place mainly at Arg845. The sequence
840RKHSKR845 preceding this cleavage
site resembles the proprotein convertase recognition
motif, (R/K)X0,2,4,6(K/R) (for review,
see Ref. 37). Here, we have provided evidence that the proprotein
convertase PC5A is responsible for the proteolytic processing at
Arg845, leading to the generation of the 140-kDa L1
fragment. Mutation of this putative recognition motif abolishes
cleavage of L1 by PC5A, clearly demonstrating that this sequence is the
recognition motif for PC5A. Furin is relatively inefficient in
processing L1 to yield a 140-kDa fragment, and other proprotein
proteases do not cleave L1. A similar result was obtained for the
processing of integrin pro-
-subunits: PC5A is more active than
furin, whereas PC1, PC2, PACE4, PC7, and PC5B are inactive (38). In
addition, although furin and other proprotein convertases do not
efficiently process members of the transforming growth factor-
superfamily, PC5A appears to be responsible for the in vivo
processing of these proteins (49). The low processing efficiency of
furin could be due to the fact that furin preferentially cleaves
substrates with the recognition motif RX(R/K)R (50), and
the sequence HXKR, which is present in L1, is a poor
substrate (51).
The particular spatial and temporal expression patterns of PC5A in the
brain (45, 52, 53) overlap with those of L1 (54), whereas furin is
ubiquitously expressed in all brain areas and cell types (55). In
contrast to furin, PC5A expression is restricted to neurons (53), as
has been shown for L1 (for review, see Ref. 20). Proteolysis of L1 by
PC5A in the hippocampus might be involved in synaptic plasticity
underlying hippocampus-dependent spatial learning. Indeed,
generation of the 140-kDa fragment is observed in the hippocampus,
which expresses PC5A, whereas in the cerebellum, which does not show
detectable PC5A expression, this fragment is not generated.
Interestingly, both PC5A (56) and L1 are up-regulated after a lesion in
the peripheral nervous system; and thus, the processing of L1 by PC5A
could be relevant in regeneration, which is highly
L1-dependent (see, for instance, Ref. 17). However, our
results do not exclude that other proteases, such as plasmin, cleave L1
in the third FNIII domain, resulting in the generation of different
140-kDa fragments.
A second proteolytic site has been shown to exist in L1, leading to the
formation of a 180-kDa fragment. This site within an unknown cleavage
sequence is localized close to the plasma membrane and is susceptible
to cleavage by a metalloprotease. Recently, it has been suggested that
the metalloprotease ADAM10 cleaves L1 at this site and generates a
180-kDa fragment (57). In the present study, we have shown that the
metalloprotease inhibitor GM 6001 inhibits formation of this 180-kDa
fragment and interferes with neurite outgrowth. Interestingly,
calmodulin inhibitors enhance proteolytic cleavage at this site, as has
been shown for other functionally important cell-surface receptors
regulating cytokine, neurotrophin, and cell recognition (46, 47). Our
observation thus provides further evidence that intracellular signaling
via calcium influences release of recognition molecule fragments from the cell surface. These fragments may then diffuse into the
extracellular matrix to interact with partner molecules and thereby
modulate the cellular environment. The release of the 180-kDa fragment entails the release of the 140-kDa fragment, which remains tightly associated with the membrane by interacting with the full-length L1
molecule. A prerequisite for this concerted release of the complex of
the 180- and 140-kDa fragments appears to be the dimerization of
full-length L1 at the cell surface, as shown in this study.
cis-Dimerization has been reported for other recognition
molecules belonging to the families of cadherins (58); integrins (for
review, see Ref. 59); selectins (60); and immunoglobulins, such as
ICAM-1 (intercellular adhesion
molecule-1) (61), P0 (62), PEACAM-1 (63), JAM-1
(64), nectin (65), tractin (66), CD4 (67), and CEACAM-1 and CEACAM-2
(68). Dimerization thus appears to be important for signal
transduction, as has been shown for E- and C-cadherins as well as for
the neural N-cadherin (Ref. 58 and references therein).
Furthermore, dimerization of selectins has been shown to enhance
adhesive tethers (60). Interestingly, dimerization of CEACAM-1
and CEACAM-2 is regulated by calmodulin and calcium ions (68),
highlighting the importance of inside-out signaling mechanisms. ICAM-1
exists predominantly as a dimer at the cell surface and binds in this
dimeric state with greatly enhanced affinity to the integrin LFA-1
compared with its monomeric form (43).
The shedding of the L1 dimers from the cell surface could have several
consequences. Because the 180/140-kDa dimer dissociates into its
monomers after release from the cell surface, the soluble diffusible
140- and 180-kDa fragments could constitute important ingredients in the extracellular matrix, possibly playing different functional roles. It might thus be conceivable that L1-synthesizing cells build concentration gradients of either soluble or
matrix-embedded adhesion molecules that modulate cell migration and
axon guidance by "conditioning" the cellular environment for L1
homophilic and heterophilic interactions with the surface of adjacent
cells. Another possibility is that the proteolytic processing uncovers binding sites of the residual transmembrane fragments for different ligands. We thus support the idea that proteolytically processed L1 may
subserve at least two functions: modification of the extracellular milieu and of transmembrane signaling via the residual L1 receptor stumps.