Brain Research, 580 (1992) 233-240 (~ 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993192/$05.00
233
BRES 17729
A novel monoclonal antibody against carbohydrates of L1 cell adhesion molecule causes an influx of calcium in cultured cortical neurons K o u i c h i I t o h a, H i d e k i K a w a m u r a b a n d H i r o a k i A s o u c aChildren's Hospital Research Foundation, Cincinnati, OH 45229-2899 (USA), bTsumura Research Institute for Pharmacology, lbarakd, (Japan) and CDepartment of Physiology, Keio University, School of Medicine, Tokyo (Japan) (Accepted 24 December 1991)
Key words: L1 molecule; Calcium; Neuron; Monoclonal antibody; Fura-2; Carbohydrate; Cell adhesion molecule; Culture
We have studied the function of carbohydrates of the L1 molecule, a member of the immunoglobulin superfamily of adhesion molecules, using a novel monoclonal antibody, mAb-LI(2E12), against L1 molecule. This antibody was specific for the 200 kDa component of mouse L1 molecule and its epitope was N-linked for complex-type oligosaccharides. The mAb-LI(2E12) was found to induce a rise in intracellular Ca2+ concentration ([Ca2+]i) in cultured mouse embryonic cortical neurons. The rise in [Ca2+]i was dependent on the concentrations of mAb-LI(2E12). The rise seemed to be due to an influx of extracellular Ca2+ as EGTA treatment abolished it. Both cadmium and nifedipine blocked the effect of mAb-LI(2EI2), suggesting the Ca2÷ influx was through voltage-operated Ca2+ channels, particularly L-type Caz+ channels. These results provide an important insight for understanding the mechanisms by which oligosaccharides of the L1 molecule influence various functions of neural cells. INTRODUCTION Carbohydrate structures have recently received attention for their role in cell-cell interaction and signal transduction. In particular, carbohydrate groups present on neural cell surface proteins such as N C A M , L1 and myelin associated glycoprotein ( M A G ) have b e e n found to participate in cell-cell adhesion and cellular signaling29. These glycoproteins have been shown to be involved in initiating a variety of biological events such as morphogenesis and cell recognition8'15. However, the mechanisms for the intracellular transduction of any signals which may be caused by the adhesion remain unknown. Recently, m o n o c l o n a l anti-Thy-1 antibody has been seen to increase a voltage-activated calcium current in cultured sensory n e u r o n s obtained from mouse dorsal root ganglia and polyclonal a n t i - N C A M and L1 antibodies increase intracellular Ca 2÷ concentration ([Ca2+]i) in rat PC12 pheochromocytoma cells 27'32. The adhesion mediated by these molecules is, however, i n d e p e n d e n t of extracellular Ca 2÷ 28,38. N e u r o n s have b e e n reported to contain multiple types of Ca 2+ channels 12'37, and the rise in [Ca2+]i could be a trigger for a variety of intracellular p h e n o m e n a such as activation of protein kinases or expression of i m m e d i a t e - e a r l y genes 24'25.
In this study, to examine whether interactions of carbohydrates of the L1 molecule would influence second messengers, especially [Ca2+]i , we made a novel m o n o clonal antibody against oligosaccharide chains of L1 molecule. To test this hypothesis, we measured whether the monoclonal antibody causes changes of Ca 2÷ influx using the calcium-sensitive fluorescent dye, fura-2, in primary mouse brain cultured n e u r o n s and glia cells. MATERIALS AND METHODS
Production of monoclonal antibody Monoclonal antibody against the L1 molecule was prepared by the method as described previously26. Briefly, a mixture of 140 and 200 kDa components of L1 molecule was isolated from the brains of CD-1 mice on monoclonal and L1 antibody immunoaffinity column and was used as an antigen. F1 hybrid rats (Lou x SpragueDawley) were immunized with 50/~g of the antigen on days 0 (first immunization), 14, 21, 35 and 45. The first immunization was performed by subcutaneous injection using complete Freund's adjuvant and immunizations thereafter were by intraperitoneal injections using incomplete Freund's adjuvant. The antibody titer on day 42 after the first immunization was determined by staining cerebellar cell cultures using an indirect immunofluorescence staining method 31. On day 48, the animals were killed for preparation of spleen cells. Fusion of the spleen cells and a myeloma cell line X63-Ag 8.65316 was performed according to the method of de St. Groth and Scheidegger6. The fused cells were first screened by the enzyme linked immunosorbent assay against purified L1 molecule, and then the positive supernatants were examined by the indirect
Correspondence: K. Itoh, Children's Hospital Research Foundation, Division of Basic Science Research, Elland and Bethesda Avenues, Cincinnati, OH 45229-2899, USA. Fax: (1) (513)-559-4317.
234 immunofluorescent staining method on the cell cultures of 4-dayold mouse cerebellum maintained for 5 days in vitro 31. Nine wells had anti-Ll antibody. We report here on one monoclonal antibody (mAb), mAb-Ll(2E12). This mAb was determined to belong to the IgG2a subclass by subclass specific antibodies (Mono Ab-10, rat-EIA kit, Lab. Inc.). The mAb was purified from ascites fluid of pristane-primed nude mice (Charles River Japan Inc., Japan) injected with cells of the L1 hybridoma clone.
Analytical procedures Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) analysis was performed on linear gradient (4-20%) polyacrylamide gels according to the method of Laemmli ~9. In order to elucidate the specificity of mAb-LI(2E12), the proteins fractionated by SDS-PAGE were transferred to nitrocellulose filters (Millipore) and processed for Western blot analysis with horseradish peroxidase-conjugated anti-rat IgG antibodies (Cappel). Digestion of L1 molecule with glycopeptidase F treatment (Boehringer Mannheim) was carried out as described previously9. Protein determinations were carried out according to the method of Bradford 3. Isolation of plasma membranes from embryonic mouse cultured neurons and from neonatal mouse cultured astroglial cells grown in primary cultures was performed according to the method of Itoh 14 and Rathjen and Schachner26. Briefly, the cultured neurons and astrocytes were homogenized in 5 ml of homogenization buffer: phosphate-buffered saline containing CaC12 0.2 mM, MgC12 0.2 mM, spermidin 1.0 mM, NaHCO 3 1.0 mM, soybean trypsin inhibitor 10 mg/ml, eggwhite trypsin inhibitor 10 mg/ml, phenylmethyl sulfonyl fluoride 1.0 mM, iodoacetamide 0.5 mM, leupepsin 0.1 mM and aprotinin 40 U/ml at pH 7.9. The homogenate was centrifuged for 20 min at 1,200 x g, 4°C. After repeating the above procedure 3 times on the pellet, the combined supernatants were centrifuged for 30 min at 30,000 x g at 4°C. This pellet was solubilized in 5 ml of solubilization buffer (Tris 25 mM, NaC1 15 mM, pH 8.2) containing 0.1% deoxycholate and protease inhibitors as described above and centrifuged for 1 h at 100,000 × g at 4°C. The final supernatant was used as a crude membrane fraction.
Cell cultures Pregnant mice (ICR, Charles River Japan Inc., Japan) were sacrificed by cervical dislocation on the 14th day of gestation (El4). The embryos were staged according to the method of Gruneberg 1°. Embryos with visible deformities were discarded. The process of dissection and dissociation was carried out entirely in ice-cold Ca2+/ Mg2+-free-Hank's balanced salt solution (CMF-HBSS) (Gibco). In sterile hood, brains from fetuses (E14) were removed and cleared of meninges and blood vessels under a dissection microscope. The cerebral cortex was dissected from the cerebral hemisphere. Tissue fragments were washed 3 times to remove meningeal fibroblasts and other small debris. The fragments were incubated in CMFHBSS containing 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) (Dojin, Japan) and 0.4 mg/ml dispase II (Boehringer Mannheim) for 20 min at room temperature. After removing the supernatant, the fragments were treated with 0.05% DNase I (Boehringer Mannheim), and they were mechanically dissociated with fire-polished Pasteur pipettes. After standing for 5 min in ice, large settled particles were removed and the single cell suspension centrifuged for 5 min at 4°C at 800 rpm. The cells were washed by centrifugation 3 times with the same medium. Finally, they were suspended in Eagle's minimal essential medium (EMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) plated at a density of 1 × l0 s cells/well where each well held one glass coverslip which had been previously coated overnight with poly-L-lysine (100 /~g/ml), and cultured under an atmosphere of 95% air and 5% CO 2 at 37°C. Primary astroglial cell cultures were obtained from newborn (P1) mouse cerebral cortex as described previously1. Our neuronal cultures and glial cultures were judged as containing approximately 80% neurons and more than 95% glia by the immunocytochemical criteria of expressing the markers, neurofilament protein and gfial fibrillary acidic protein, respectively.
2001~
1161~ 971~ 6 6 D.43m,-
Tiiiiiiiiii!iiiiil
I
2
3
4
Fig. 1. L1 molecule was treated with (lanes 3 and 4) or without (lanes 1 and 2) glycopeptidase F, and then analyzed for their specificity by the protein blot method using polyclonal L1 antibody (lanes 1 and 3) and mAb-LI(2E12) (lanes 2 and 4).
lmmunofluorescence staining with laser scan microscope Cultures were washed 3 times in Dulbecco's phosphate-buffered saline (D-PBS, Gibco) and incubated for 30 min at room temperature firstly with antibody purified from ascites diluted 1:50 in D-PBS, pH 7.2 containing 1% bovine serum albumin (BSA) and 10% heat-inactivated horse serum (HS). Cultures were then washed 3 times and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rat IgG (Cappel), also diluted 1:200 in D-PBS containing 1% BSA and 10% HS. After washing 3 times, cultures were fixed with a 2% periodate-lysine-paraformaldehyde (PLP) solution (McLean and Nakane, 1974) and washed with D-PBS and were mounted in Perma Fluor Aqueous mounting medium (Lipshaw). Cells were examined with Zeiss laser scan microscope equipped with Argon laser source at 488 nm for FITC-staining.
Measurement of [Ca2+]i The method used for measuring [Ca2÷]i was described in detail elsewhere 11'3s. Briefly, [Ca2+]i was measured using the calcium-sensitive fluorescent dye, fura-2 acetoxymethylester (fura-2/AM; Dojin, Japan) 36. The cultured cells were loaded with fura-2/AM 3 mM in CMF-HBSS containing HEPES 10 mM (pH 7.4) and CaCI 2 5 mM without phenol red for 30 min at 37°C. The fura-2-1oaded cells were washed 3 times, and incubated for 60 min with the same buffer to allow hydrolysis of the ester. The cultures were maintained at 30°C, and viewed on a Nikon inverted microscope with a SIT video camera (C-2400; Hamamatsu Photonics, Japan); the camera output was fed into an Argus-100/CA (Hamamatsu Photonics, Japan), which controlled the image acquisition and display. [Ca2+]i was subsequently computed and determined from images at 340 and 380 nm by the ratio method. The fluorescent signal was measured by focusing on the soma. In these experiments, the relative elevation in [Ca2+]i is more important than the absolute value of [Ca2+]i . In our experiments, cells which had a fluorescence signal which was abnormally high (10% greater than the average) prior to antibody
235 TABLE I
Effects of mAb-L1(2E12), mAb-NCAM(H28) and rat normal lgG on [Ca2+]i in neurons and astrocytes Cell type
Agent (l~g/ml)
Max. [Ca2+]i (nM) a
Neuron
mAb-LI(2E12) 0.00 3.25 7.50 15.0 30.0
98.9 103.0 139.6 150.1 194.7
Neuron
Neuron
Astrocyte
mAb-NCAM(H28) 30.0 Rat normal 15.0 30.0
+ + + + +
-~200
8.5 b 5.8 3.9 6.4 3.4
-'~116 ~[ 97
99.5 + 10.4 c
-~43
IgG
2E12 (30/tg/ml)
101.0 + 3.2 c 108.2 + 2.4 c 109.6 + 2.0c
aMax. [Ca2+]i (nM) represents the maximum [CaZ+]i (nM) throughout the measurement (up to 300 s after the addition of the antibodies). bData represent the mean + S.E.M. of at least 28 cells for each condition. CNot statistically different from Max. [Ca2+]i in the absence of antibody.
addition were excluded from these measurements. Changes in [Ca2+]i of cultured cells were examined under the following conditions; application of (1) mAb-LI(2E12), monoclo-
12
34
Fig. 2. Western blot analysis using mAb-LI(2E12) in terms of plasma membrane fraction of embryonic mouse cultured neurons and astrocytes. Lanes 1, 3: astrocytes; lanes 2, 4: neurons. Samples were analyzed by Western blot method using polyclonal NCAM (lanes 1 and 2), and mAb-LI(2E12) (lanes 3 and 4).
Fig. 3. Immunocytochemical staining with mAb-LI(2E12) of primary cultures of mice cortical neurons. Cultures of primary neurons were prepared and immunocytochemistry performed as described in Materials and Methods. Cells were examined by Zeiss laser scan microscope equipped with Argon laser source 488 nm for FITC-staining. Arrow indicates astrocyte. Bar -- 25 gin.
236
Q
,i (nM)
!":, 400
0
0
200
,0
0 Fig. 4, Pseudo color images of fura-2 fluorescence intensity ratio imaging (340 nm/380 nm imaging) in primary cultured neurons treated with mAb-LI(2E12), a: normal resting levels; b: 180 s after addition of 30/zg/ml mAb-LI(2E12); c: resting levels in Ca2+-free medium plus 2 mM EGTA; d: 180 s after addition of 30/~g/ml mAb-LI(2E12) in Ca2+-free medium plus 2 mM EGTA.
nal anti-NCAM antibody (mAb-NCAM(H28) or non-immune rat IgG at several concentrations in CMF-HBSS containing HEPES and CaCI2, (2) mAb-Ll(2E12) at 30 pg/ml in CMF-HBSS containing HEPES in the presence of ethyleneglycol bis (b-aminoethylether)-N,N,N',N'-tetraacetic (EGTA) 2 raM, (3) mAb-Ll(2E12) at 30/~g/ml followed by application of CdC12 100/tM, nifedipine 1 pM, O,L-2-amino-5-phosphonovalericacid (AVP) 100/~M, phencyclidine (PCP) 1/~M, MgCl2 2 mM, phorbol-12-myristate-13-acetate (PMA) 100 nM, 1-(5-iso-quinolinesulfonyl)-2-methylpiperazine (H7) 100/~M, or tetrodotoxin (TI'X) 10/tM, (4) Ll-mAb-Ll(2E12) performed complex. For these applications, mAb-Ll(2E12), mAbNCAM(H28), EGTA, CdC12, APV, PCP, MgCI2, I-/7, TTX and L1 were dissolved in the CMF-HBSS containing HEPES and CaC12, and nifedipine and PMA were dissolved in dimethylsulfoxide (DMSO). APV was purchased from Cambridge Research Biochemicals. H7 and poly-L-lysine were from Seikagaku Kougyou, Japan. mAb-NCAM(H28) was purchased from Funakoshi Chemical Co., Japan. All other reagents were from Sigma Chemical Co. RESULTS
Specificity of mAb-L1 (2E12) According to the method of Western blot analysis, the specificity of m A b - L I ( 2 E 1 2 ) was determined using a
plasma membrane fraction from embryonic cultured neurons. Polyclonal L1 antibody reacted with all 3 previously identified L1 molecule components 26, whereas mAb-LI(2E12) reacted with only the 200 kDa component (Fig. 1, lines 1 and 2). The molecular weight of L1 molecule was reduced by approximately 30% by treatment overnight with glycopeptidase F which is known to cleave N-linked oligosaccharides from glycoproteins (Fig. 1, lines 1 and 3). Polyclonal L1 antibody still detected the digested proteins but m A b - L I ( 2 E 1 2 ) no longer reacted with the 200 kDa component (Fig. 1, lines 3 and 4). mAb-LI(2E12) immunoreacted with the 200 kDa of L1 molecule derived from cultured neurons but not with membrane preparations from astrocytes (Fig. 2, lines 3 and 4). Furthermore, from our immunocytochemical studies, we showed that the L1 molecule was recognized by m A b - L l ( 2 E 1 2 ) in primary cultured neurons, but not in astrocytes (Fig. 3). In contrast, N C A M was detected on both neurons and astrocytes by polyclonal N C A M antibody (Fig. 2, lines 1 and 2).
237 m A b - L 1 ( 2 E 1 2 ) causes a rise in [Ca2+]i in neurons [Ca2+]i increases rapidly and dramatically in cultured neurons treated with 30/~g/ml of m A b - L I ( 2 E 1 2 ) but not after treatment with normal rat IgG (Figs. 4 and 5). The [Ca2÷]i was elevated as early as 10 s after m A b - L I ( 2 E 1 2 ) addition. A peak of [Ca2+]i (approximately 200% increase) was seen at 180 s after the addition and persisted as long as 600 s. Longer times were not examined. Of the 25 cells present in the field, 17 cells were responsive to m A b - L I ( 2 E 1 2 ) (Fig. 4, a and b). In combined experiments, the percentage of responsive cells was approximately 70% when treated with this mAb. In control experiments, m A b - L I ( 2 E 1 2 ) had no such [Ca2+]i increasing effect on astrocytes (Table I). L I - m A b - L I ( 2 E 1 2 ) complexes did not cause [Ca2+]i increases in neurons. The [Ca2÷]i-increasing effect of m A b - L I ( 2 E 1 2 ) was dependent on the concentration of the antibody used (Fig. 6 and Table I). In contrast, m A b - N C A M ( H 2 8 ) failed to elicit a change in [Ca2+]i in cultured neurons (Table I). Furthermore, the subsequent addition of m A b L1(2E12) to neurons after treatment with m A b N C A M ( H 2 8 ) produced an increase in [Ca2+]i, and the magnitude and time course pattern of this increase were identical to those observed in the treatment with m A b Ll(2E12) alone (data not shown). m A b - L l ( 2 E 1 2 ) causes an influx o f extracellular Ca 2+ through L-type voltage-operated Ca 2+ channels ( V O C C s ) When neurons were cultured in Ca2+-free medium supplemented with E G T A 2 mM, the increase in [Ca2+]i by m A b - L I ( 2 E 1 2 ) (30/~g/ml) was not observed (Figs. 4
220
•
2E12 30~g/ml
a)
i)
IgG 30-g/ml
T
~
it)
180 i
,
~
[] mAb-LI(2EI2) ~i 180' • nonimmune lgG 160
~
-
/I
~
140-
y
/ -
,oo 80 1 0
•
,
•
,
10 IgG
•
20
concentration
30
(~g/mi)
Fig. 6. Dose-response relationship between mAb-Ll(2E12) concentrations and [Ca2+]i in neurons. Values represent the mean + S.E.M. of at least 28 neurons for each condition. [Ca2+]i represents the maximum concentrations throughout the measurement (up to 300 s after the addition of the antibody).
and 7). The addition of cadmium 100 ~M or nifedipine 1 ~M, inhibitors of L-type VOCCs, also completely abolished the increase of [Ca2+]i by m A b - L I ( 2 E 1 2 ) (Fig. 7A). On the other hand, the increase in [Ca2+]i by m A b Ll(2E12) was not antagonized by treatment of T-FX, a sodium channel blocker. N-Methyl-D-aspartate (NMDA)operated Ca 2÷ channel blockers such as A P V (100/~M), PCP (1/~M) and MgCI2 (2 mM) 5'22, had no significant effects on the increase of [Ca z÷] by mAb-LI(2E12) (Fig.
TABLE II ~j)
200 ')
200
"r
")
")
")
*)
160
Effects of PMA and H7 on the rise in [Ca]i of neurons induced by rnAb-L1(2E12) Agent
Max. [Ca2+]i (nM) a
mAb-L1(2E12) ~g/rnl) 0.0 15.0
98.9 + 8.5b 150.1 + 6.4
14o.
~
PMA (100 nM, 18 h) plus 2E12 (15/tg/ml)
116.9 + 4.1c'a
120 '. 100 j.
H7 (100/~M, 18 h) plus 2E12 (15 gg/ml)
109.8 + 8.7c'a
[Ca2+]i
80 -60
0
60
120 TIME
in
180
240
300
(sec)
Fig. 5. Changes [Ca2+]i after treatment of neurons with mAbLl(2E12). Either mAb-Ll(2E12) or normal rat IgG was added to the cultures at the 0 time point (arrow). a) Statistically significant, P < 0.01; mAb-LI(2E12) vs. normal rat IgG.
maximum[Ca2+]i
aMax. (nM) represents the (nM) throughout the measurement (up to 300 s after the addition of the antibodies). bData represent the mean + S.E.M. of at least 28 cells for each condition. cStatistically significant, P < 0.01 as compared with [Ca2+]i in the presence of antibody. dNot statistically different from [Ca2+]i in the absence of antibody.
Max.
Max.
238
B
A 220
•
220'
2E12 30~0/m|
b)
*Ca f r l e (EGTA)
A
200
b)
200'
*Cd 0.1rnM
180
180'
160-
160'
b)
b)
b)
T
T~II
I
b)
b)
b)
0 0 m
3
140-
U
120-
140"
V
2
"; :
"
1~I
120 • I
~
80 -60
•
~f ~
----o--
.APV0.1mM
f
---O---
~PCP I~M
¢
,Mg 1ram
100' •
.60
b)
i
0
-
i
60
-
i
120 TIME
-
w
180
•
|
240
•
i
300
(sec)
i
0
•
i
60
•
i
i
i
i
120
180
240
300
TIME
(see)
Fig. 7. Effect of EGTA and L-type voltage-operated Ca2+ channels (VOCCs)-antagonists (A) and N-methyl-o-aspartate (NMDA)-antagonists (B) on the rise in [Ca2÷]i of neurons induced by mAb-LI(2E12), mAb-LI(2E12) was added to the medium at the 0 time point (arrow) after treatment with EGTA or each of L-type VOCCs- or NMDA-antagonists. a)Statistically significant, P < 0.01; mAb-LI(2E12) alone vs. EGTA or L-type VOCCs-antagonistsin the presence of antibody, b)Not statistically different from [Ca2+]iin the presence of antibody alone.
7B). These combined results indicate that the mAbLl(2E12)-induced Ca 2+ influx into neurons seems to be through the VOCCs, particularly L-type VOCCs.
Protein kinase C (PKC) activity influences the rise in [Ca2+]i in neurons exposed to mAb-L1(2E12) The exposure of neurons to PMA (100 nM) or H7 (100/~M) for long (18 h) periods had no significant effects on the basal levels of [Ca2+]i (data not shown). However, the magnitude of [Ca2÷]i elevation by mAb-L1 was significantly reduced after the long-term exposure of neurons to PMA (Table II). This suggests a relationship between mAb-Ll(2E12)-induced Ca 2÷ influx and PKC activity in our culture system. DISCUSSION We studied a potential functional mechanism of carbohydrates of the LI molecule in the cell-cell interactions of neuronal cells using a monoclonal antibody against oligosaccharides of L1 molecule because a monoclonal antibody is often a useful tool both for identifying its specific epitope and also for triggering the signal transduction in which its epitope is involved. We selected the mAb-LI(2E12) which specifically reacted with only the 200 kDa component of L1 molecule• This mAb lost the reactivity with the 200 kDa component after treatment with glycopeptidase F which is reported to cleave asparagine-linked glycans34, demonstrating that the epitope recognized by this mAb is a carbohydrate part of L1 molecule (Fig. 1). Endoglycosidase H mainly releases mannose- and hybrid-type
N-linked oligosaccharides, and endoglycosidase F additionally cleaves certain complex-type oligosaccharides 17. Treatment with either endoglycosidase H or F did not alter the reactivity of mAb-LI(2E12) at all (data not shown). Therefore, the carbohydrate epitope appeared to consist of a complex-type glycan which is only susceptible to glycopeptidase F. The L2/HNK-1 epitope, being expressed on several adhesion molecules, was also found to carry a complex-type glycan4. In addition, the adhesion molecules L1, NCAM and MAG were reported to be L2/HNK-1 epitope-positive TM. The epitope recognized by mAb-LI(2E12), however, seems to be distinct from the L2/HNK-1 epitope because mAb-LI(2E12) did not react with NCAM and MAG. We determined the influence of the carbohydrate epitopes of L1 molecule on Ca 2+ influx in cultured neurons using antibodies to mimic ligand binding 3°. Treatment of cultured neurons with mAb-LI(2E12) caused an increase in [caa*]i . The magnitude of the increase was dependent on the concentrations of the mAb (Fig. 6 and Table I). When the Ca 2+ in the medium (extracellular Ca 2*) was eliminated by the addition of EGTA, the increase in [Ca2*]i by mAb-LI(2E12) did not occur (Fig. 7A). The increase in [Ca2+]i, therefore, is due to an influx of extracellular Ca 2+ into the neurons rather than release from intracellular stores. In general, two types of Ca 2+ channels, voltage-operated Ca 2+ channels (VOCCs) and receptor-operated Ca 2+ channels (ROCCs), have been shown to mediate Ca 2+ influx. The VOCCs can be distinguished pharmacologically: L-type VOCCs are affected by both dihydropyridines (DHP) and ~o-conotoxin; N-type VOCCs are
239 effectively inhibited by to-conotoxin but not by the DHP. The T-type V O C C s are relatively resistant to inhibition by both the D H P and to-conotoxin 37. Our results demonstrated that nifedipine and Cd 2+, two L-type V O C C s antagonists, were effective in abolishing increases in [Ca2+]i after m A b - L I ( 2 E 1 2 ) treatment (Fig. 7A). We concluded that the influx of Ca 2+ in neurons exposed to m A b - L l ( 2 E 1 2 ) was particularly mediated by L-type VOCCs, because L-type V O C C s were modulated by D H P antagonists but T- and N-type V O C C s were not, and L- and N-type V O C C s were potently blocked by Cd 2+, whereas T-type V O C C s were much less sensitive. However, it will have to be determined whether T- and N-type V O C C s blockers influence the increase in [Ca2+]i by mAb-Ll(2E12). We then examined the involvement of NMDA-linked R O C C s in the increase in [Ca2+]i by m A b - L I ( 2 E 1 2 ) (Fig. 7B). A P V and PCP at the concentrations sufficient to block completely the NMDA-linked R O C C s 5 failed to inhibit the effect of mAb-L1(2E12), suggesting that the NMDA-linked R O C C s are not associated with the Ca 2÷ influx induced by m A b - L l ( 2 E 1 2 ) in our neuronal culture system. The increase in [Ca2+]i was seen quickly (10 s) after the addition of the antibody, reached a plateau at 180 s, and persisted throughout the experiment (Figs. 5 and 7). This is similar to the [Ca2÷]i increase induced by the addition of anti-galactocerebroside (GalC) antibody to oligodendrocytes 7. These authors suggested that V O C C s in oligodendrocytes are not responsible for the Ca 2+ influx caused by anti-GalC antibody in contrast to our results with neurons. Since the interactions of PKC with Ca2÷ and neural adhesion molecules have been demonstrated in several neural types 2'13'14, it is of interest to determine if P K C
influences the elevated [Ca2+]i in neurons, m A b Ll(2E12)-induced increase in [Ca2+]i was lowered by approximately 70% in neurons previously exposed to P M A (PKC-deficient cells) for 18 h 21. This inhibition was also seen when cells were treated with H7, a PKC inhibitor. These results suggest that PKC activation at least partially influences the increase in [Ca2+]i by m A b Ll(2E12). However, further studies are required to confirm the relationship between P K C and the increase in [Ca2+]i by mAb-LI(2E12). Our results may suggest an important clue for clarifying one mechanism by which carbohydrate chains of L1 molecule may mediate in neuronal functions. L1 molecule-dependent adhesion of neurons to neurons may influence neuronal morphological changes by modulating VOCCs, because [Ca2+]i may control neurite elongation in part by regulating the stability of actin filaments 2°. Moreover, these changes may contribute to intracellular molecular events leading to adhesion-linked gene activation, because Ca 2+ is a major second messenger regulating immediate-early gene expression in excitable cells 33. Taken together these observations provide evidence that the expression of a particular carbohydrate epitope may have a neuronal functional significance. Further studies on the structure and function of the carbohydrate epitope of L1 molecule and gene regulations by these glycans of L1 molecule are necessary to obtain a more complete understanding of the role of carbohydrates in cell-cell interaction.
REFERENCES
(1980) 1-21. 7 Dyer, C.A. and Benjamins, J.A., Glycolipids and transmembrane signaling: antibodies to galactocerebroside cause an influx of calcium in oligodendrocytes, J. Cell Biol., 111 (1990) 625633. 8 Edelman, G.M., Morphoregulatory molecules, Biochemistry, 27 (1988) 3533-3543. 9 Fahrig, T., Schmitz, B., Weber, D., Kucherer-Ehret, A., Faissner, A. and Schachner, M., Two monoclonal antibodies recognizing carbohydrate epitopes on neural adhesion molecules interfere with cell interactions, Eur. J. Neurosci., 2 (1990) 153161. 10 Gruneberg, A., The development of some external features in mouse embryos, J. Hered., 34 (1943) 89-92. 11 Grynkiewicz, G., Poenie, M. and Tsien, R.Y., A new generation of Ca indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440-3450. 12 Hess, P., Calcium channels in vertebrate cells, Annu. Rev. Neurosci., 13 (1990) 337-356. 13 Itoh, K., Asou, H., Ikarashi, Y. and Maruyama, Y., Morphological changes and neural cell adhesion molecule expression in
1 Asou H. and Brunngraber, E.G., Absence of ganglioside GM x in astroglial cells from newborn rat brain, Neurochem. Int., 6 (1984) 81-89. 2 Bixby, J.L. and Jhabvala, P., Extracellular matrix molecules and cell adhesion molecules induce neurites through different mechanisms, J. Cell Biol., 111 (1990) 2725-2732. 3 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 4 Chou, D.K.H., Ilyas, A.A., Evans, J.E., Costello, C., Quarles, R.H. and Jungalwala, EB., Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNK-1 antibody and some IgM paraproteins in neuropathy, J. Biol. Chem., 261 (1986) 383-388. 5 Cotman, C.W. and Iversen, L.L., Excitatory amino acids in the brain focus on NMDA receptors, Trends Neurosci., 10 (1987) 263-265. 6 de St. Groth, S.E and Scheidegger, D., Production of monoclonal antibodies: strategy and tactics, J. lmmunol. Methods, 35
Acknowledgements. We would like to thank Dr. Richard Akeson for his review of the manuscript. H.A. is grateful to Dr. M. Schachner for the opportunity to do the related experiments in her laboratory. This work is supported in part by Deutsche Forschungsgemeinschaft (SFB 317) and Tsumura and Co.
240 mouse cerebrum primary cultures following long-term exposure to phorbol ester, Neurosci. Res., 6 (1989) 350-357. 14 Itoh, K., Neural adhesion molecule L1 associated with protein kinase C, J. Med. Soc. Toho, Jpn., 37 (1990) 465-479. 15 Jessell, T.M., Adhesion molecules and the hierarchy of neural development, Neuron, 1 (1988) 3-13. 16 Kearney, J.F., Radbruch, A., Liesegang, B. and Rajewski, K., A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines, J. lmmunol., 123 (1979) 1548-1550. 17 Kobata, S., Use of endo- and exoglycosidases for structural studies of glycoconjugates, Anal. Biochem., 100 (1979) 1-14. 18 Kruse, J., Mailhammer, R., Wernecke, H., Faissner, A., Sommer, I., Goridis, C. and Schachner, M., Neural cell adhesion molecules and myeline-associated glycoprotein share a common carbohydrate moiety recognized by monoclonal antibodies L2 and HNK-1, Nature, 311 (1984) 153-155. 19 Laemmli, U.K., Cleavage of structural protein during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. 20 Lankford, K,L. and Letourneau, P.C., Evidence that calcium may control neurite outgrowth by regulating the stability of actin filaments, J. Cell Biol., 109 (1989) 1229-1243. 21 Manev, H., Costa, E., Wroblewski, J.T. and Guidotti, A., Abusive stimulation of excitatory amino acid receptors: a strategy to limit neurotoxicity, FASEB J., 4 (1990) 2789-2797. 22 Mayer, M.L. and Westbrook, G.L., Permeating and block of N-methyl-o-aspartic acid receptor channel by divalent cations in mouse central neurones, J. Physiol., 394 (1987) 501-527. 23 McLean, I.W. and Nakane, P.K., Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy, J. Histochem. Cytochem., 22 (1974) 1077-1083. 24 Morgan, J.I. and Curran, T., Calcium as a modulator of the immediate-early gene cascade in neurons, Cell Calcium, 9 (1988) 303-311. 25 Nishizuka, Y., Studies and perspectives of protein kinase C, Science, 233 (1986) 305-312. 26 Rathjen, F.G. and Schachner, M., Immunocytological and biochemical characterization of a new neural cell surface component (L1 antigen) which is involved in cell adhesion, EMBO J., 3 (1984) 1-10. 27 Saleh, M., Lang, R.J. and Bartlett, P.E, Thy-l-mediated regula-
tion of a low-threshold transient calcium current in cultured sensory neurons, Proc. Natl. Acad. Sci. USA, 85 (1988) 4543-4547. 28 Salzer, J.L. and Colman, D.R., Mechanisms of cell adhesion in the nervous system: role of the immunoglobulin gene superfamily, Dev. Neurosci., 11 (1989) 377-390. 29 Schachner, M., Antonicek, H., Fahrig, T., Faissner, A., Fischer, G., Kiinemund, V., Martini, R., Mayer, A., Persohn, E., Pollerberg, E., Probstmeier, R., Sadoul, R., Seilfeimer, B. and Thor, G., Families of neural cell adhesion molecules. In G.M. Edelman, B.A. Cunningham, and J.-P. Thiery, (Eds.), Morphoregulatory Molecules, John Wiley and Sons, New York, 1989, pp. 443-468. 30 Schlessinger, J. Lax, I., Yarden, Y., Kanety, H. and Libermann, T., Monoclonal antibodies against the membrane receptor for epidermal growth factor: a versatile tool for structural and mechanistic studies, In P. Cuatrecasas and M.F. Greaves (Eds.), Receptors and Recognition. Antibodies and Receptors: Probes for Receptor, Structure and Function, Chapman and Hall, London, 1984, pp. 279-303. 31 Schnitzer, J. and Schachner, M., Expression of Thy-1, H-2 and NS-4 cell surface antigen and tetanus toxin receptors in early postnatal and adult mouse cerebellum, J. Neuroimmunol., 1 (1981) 429-456. 32 Schuch, U., Lohse, M.J. and Schachner, M., Neural cell adhesion molecules influence second messenger systems, Neuron, 3 (1989) 13-20. 33 Sheng, M. and Greenberg, M.E., The regulation and function of c-fos and other immediate early genes in the nervous system, Neuron, 4 (1990) 477-485. 34 Tarentino, A.L., Gomez, C.M. and Plummer Jr., T.H., Deglycosylation of asparagine-linked glycans by peptide: N-glycosidase F, Biochemistry, 24 (1985) 4665-4671. 35 Thayer, S.A., Murphy, S.N. and Miller, R.J., Widespread distribution of dihydropyridine-sensitive calcium channels in the central nervous system, Mol. Pharmacol., 30 (1987) 505-509. 36 Tsien, R.Y., Fluorescent probes of cell signaling, Annu. Rev. Neurosci., 12 (1989) 227-253. 37 Tsien, R.W. and Tsien, R.Y., Calcium channels, stores, and oscillations, Annu. Rev. Cell Biol., 6 (1990) 715-760. 38 Yoshihara, Y., Oka, S., Ikeda, J. and Mori, K., Immunoglobulin superfamily, molecules in the nervous system, Neurosci. Res., 10 (1991) 83-105.
233
BRES 17729
A novel monoclonal antibody against carbohydrates of L1 cell adhesion molecule causes an influx of calcium in cultured cortical neurons K o u i c h i I t o h a, H i d e k i K a w a m u r a b a n d H i r o a k i A s o u c aChildren's Hospital Research Foundation, Cincinnati, OH 45229-2899 (USA), bTsumura Research Institute for Pharmacology, lbarakd, (Japan) and CDepartment of Physiology, Keio University, School of Medicine, Tokyo (Japan) (Accepted 24 December 1991)
Key words: L1 molecule; Calcium; Neuron; Monoclonal antibody; Fura-2; Carbohydrate; Cell adhesion molecule; Culture
We have studied the function of carbohydrates of the L1 molecule, a member of the immunoglobulin superfamily of adhesion molecules, using a novel monoclonal antibody, mAb-LI(2E12), against L1 molecule. This antibody was specific for the 200 kDa component of mouse L1 molecule and its epitope was N-linked for complex-type oligosaccharides. The mAb-LI(2E12) was found to induce a rise in intracellular Ca2+ concentration ([Ca2+]i) in cultured mouse embryonic cortical neurons. The rise in [Ca2+]i was dependent on the concentrations of mAb-LI(2E12). The rise seemed to be due to an influx of extracellular Ca2+ as EGTA treatment abolished it. Both cadmium and nifedipine blocked the effect of mAb-LI(2EI2), suggesting the Ca2÷ influx was through voltage-operated Ca2+ channels, particularly L-type Caz+ channels. These results provide an important insight for understanding the mechanisms by which oligosaccharides of the L1 molecule influence various functions of neural cells. INTRODUCTION Carbohydrate structures have recently received attention for their role in cell-cell interaction and signal transduction. In particular, carbohydrate groups present on neural cell surface proteins such as N C A M , L1 and myelin associated glycoprotein ( M A G ) have b e e n found to participate in cell-cell adhesion and cellular signaling29. These glycoproteins have been shown to be involved in initiating a variety of biological events such as morphogenesis and cell recognition8'15. However, the mechanisms for the intracellular transduction of any signals which may be caused by the adhesion remain unknown. Recently, m o n o c l o n a l anti-Thy-1 antibody has been seen to increase a voltage-activated calcium current in cultured sensory n e u r o n s obtained from mouse dorsal root ganglia and polyclonal a n t i - N C A M and L1 antibodies increase intracellular Ca 2÷ concentration ([Ca2+]i) in rat PC12 pheochromocytoma cells 27'32. The adhesion mediated by these molecules is, however, i n d e p e n d e n t of extracellular Ca 2÷ 28,38. N e u r o n s have b e e n reported to contain multiple types of Ca 2+ channels 12'37, and the rise in [Ca2+]i could be a trigger for a variety of intracellular p h e n o m e n a such as activation of protein kinases or expression of i m m e d i a t e - e a r l y genes 24'25.
In this study, to examine whether interactions of carbohydrates of the L1 molecule would influence second messengers, especially [Ca2+]i , we made a novel m o n o clonal antibody against oligosaccharide chains of L1 molecule. To test this hypothesis, we measured whether the monoclonal antibody causes changes of Ca 2÷ influx using the calcium-sensitive fluorescent dye, fura-2, in primary mouse brain cultured n e u r o n s and glia cells. MATERIALS AND METHODS
Production of monoclonal antibody Monoclonal antibody against the L1 molecule was prepared by the method as described previously26. Briefly, a mixture of 140 and 200 kDa components of L1 molecule was isolated from the brains of CD-1 mice on monoclonal and L1 antibody immunoaffinity column and was used as an antigen. F1 hybrid rats (Lou x SpragueDawley) were immunized with 50/~g of the antigen on days 0 (first immunization), 14, 21, 35 and 45. The first immunization was performed by subcutaneous injection using complete Freund's adjuvant and immunizations thereafter were by intraperitoneal injections using incomplete Freund's adjuvant. The antibody titer on day 42 after the first immunization was determined by staining cerebellar cell cultures using an indirect immunofluorescence staining method 31. On day 48, the animals were killed for preparation of spleen cells. Fusion of the spleen cells and a myeloma cell line X63-Ag 8.65316 was performed according to the method of de St. Groth and Scheidegger6. The fused cells were first screened by the enzyme linked immunosorbent assay against purified L1 molecule, and then the positive supernatants were examined by the indirect
Correspondence: K. Itoh, Children's Hospital Research Foundation, Division of Basic Science Research, Elland and Bethesda Avenues, Cincinnati, OH 45229-2899, USA. Fax: (1) (513)-559-4317.
234 immunofluorescent staining method on the cell cultures of 4-dayold mouse cerebellum maintained for 5 days in vitro 31. Nine wells had anti-Ll antibody. We report here on one monoclonal antibody (mAb), mAb-Ll(2E12). This mAb was determined to belong to the IgG2a subclass by subclass specific antibodies (Mono Ab-10, rat-EIA kit, Lab. Inc.). The mAb was purified from ascites fluid of pristane-primed nude mice (Charles River Japan Inc., Japan) injected with cells of the L1 hybridoma clone.
Analytical procedures Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) analysis was performed on linear gradient (4-20%) polyacrylamide gels according to the method of Laemmli ~9. In order to elucidate the specificity of mAb-LI(2E12), the proteins fractionated by SDS-PAGE were transferred to nitrocellulose filters (Millipore) and processed for Western blot analysis with horseradish peroxidase-conjugated anti-rat IgG antibodies (Cappel). Digestion of L1 molecule with glycopeptidase F treatment (Boehringer Mannheim) was carried out as described previously9. Protein determinations were carried out according to the method of Bradford 3. Isolation of plasma membranes from embryonic mouse cultured neurons and from neonatal mouse cultured astroglial cells grown in primary cultures was performed according to the method of Itoh 14 and Rathjen and Schachner26. Briefly, the cultured neurons and astrocytes were homogenized in 5 ml of homogenization buffer: phosphate-buffered saline containing CaC12 0.2 mM, MgC12 0.2 mM, spermidin 1.0 mM, NaHCO 3 1.0 mM, soybean trypsin inhibitor 10 mg/ml, eggwhite trypsin inhibitor 10 mg/ml, phenylmethyl sulfonyl fluoride 1.0 mM, iodoacetamide 0.5 mM, leupepsin 0.1 mM and aprotinin 40 U/ml at pH 7.9. The homogenate was centrifuged for 20 min at 1,200 x g, 4°C. After repeating the above procedure 3 times on the pellet, the combined supernatants were centrifuged for 30 min at 30,000 x g at 4°C. This pellet was solubilized in 5 ml of solubilization buffer (Tris 25 mM, NaC1 15 mM, pH 8.2) containing 0.1% deoxycholate and protease inhibitors as described above and centrifuged for 1 h at 100,000 × g at 4°C. The final supernatant was used as a crude membrane fraction.
Cell cultures Pregnant mice (ICR, Charles River Japan Inc., Japan) were sacrificed by cervical dislocation on the 14th day of gestation (El4). The embryos were staged according to the method of Gruneberg 1°. Embryos with visible deformities were discarded. The process of dissection and dissociation was carried out entirely in ice-cold Ca2+/ Mg2+-free-Hank's balanced salt solution (CMF-HBSS) (Gibco). In sterile hood, brains from fetuses (E14) were removed and cleared of meninges and blood vessels under a dissection microscope. The cerebral cortex was dissected from the cerebral hemisphere. Tissue fragments were washed 3 times to remove meningeal fibroblasts and other small debris. The fragments were incubated in CMFHBSS containing 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) (Dojin, Japan) and 0.4 mg/ml dispase II (Boehringer Mannheim) for 20 min at room temperature. After removing the supernatant, the fragments were treated with 0.05% DNase I (Boehringer Mannheim), and they were mechanically dissociated with fire-polished Pasteur pipettes. After standing for 5 min in ice, large settled particles were removed and the single cell suspension centrifuged for 5 min at 4°C at 800 rpm. The cells were washed by centrifugation 3 times with the same medium. Finally, they were suspended in Eagle's minimal essential medium (EMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) plated at a density of 1 × l0 s cells/well where each well held one glass coverslip which had been previously coated overnight with poly-L-lysine (100 /~g/ml), and cultured under an atmosphere of 95% air and 5% CO 2 at 37°C. Primary astroglial cell cultures were obtained from newborn (P1) mouse cerebral cortex as described previously1. Our neuronal cultures and glial cultures were judged as containing approximately 80% neurons and more than 95% glia by the immunocytochemical criteria of expressing the markers, neurofilament protein and gfial fibrillary acidic protein, respectively.
2001~
1161~ 971~ 6 6 D.43m,-
Tiiiiiiiiii!iiiiil
I
2
3
4
Fig. 1. L1 molecule was treated with (lanes 3 and 4) or without (lanes 1 and 2) glycopeptidase F, and then analyzed for their specificity by the protein blot method using polyclonal L1 antibody (lanes 1 and 3) and mAb-LI(2E12) (lanes 2 and 4).
lmmunofluorescence staining with laser scan microscope Cultures were washed 3 times in Dulbecco's phosphate-buffered saline (D-PBS, Gibco) and incubated for 30 min at room temperature firstly with antibody purified from ascites diluted 1:50 in D-PBS, pH 7.2 containing 1% bovine serum albumin (BSA) and 10% heat-inactivated horse serum (HS). Cultures were then washed 3 times and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rat IgG (Cappel), also diluted 1:200 in D-PBS containing 1% BSA and 10% HS. After washing 3 times, cultures were fixed with a 2% periodate-lysine-paraformaldehyde (PLP) solution (McLean and Nakane, 1974) and washed with D-PBS and were mounted in Perma Fluor Aqueous mounting medium (Lipshaw). Cells were examined with Zeiss laser scan microscope equipped with Argon laser source at 488 nm for FITC-staining.
Measurement of [Ca2+]i The method used for measuring [Ca2÷]i was described in detail elsewhere 11'3s. Briefly, [Ca2+]i was measured using the calcium-sensitive fluorescent dye, fura-2 acetoxymethylester (fura-2/AM; Dojin, Japan) 36. The cultured cells were loaded with fura-2/AM 3 mM in CMF-HBSS containing HEPES 10 mM (pH 7.4) and CaCI 2 5 mM without phenol red for 30 min at 37°C. The fura-2-1oaded cells were washed 3 times, and incubated for 60 min with the same buffer to allow hydrolysis of the ester. The cultures were maintained at 30°C, and viewed on a Nikon inverted microscope with a SIT video camera (C-2400; Hamamatsu Photonics, Japan); the camera output was fed into an Argus-100/CA (Hamamatsu Photonics, Japan), which controlled the image acquisition and display. [Ca2+]i was subsequently computed and determined from images at 340 and 380 nm by the ratio method. The fluorescent signal was measured by focusing on the soma. In these experiments, the relative elevation in [Ca2+]i is more important than the absolute value of [Ca2+]i . In our experiments, cells which had a fluorescence signal which was abnormally high (10% greater than the average) prior to antibody
235 TABLE I
Effects of mAb-L1(2E12), mAb-NCAM(H28) and rat normal lgG on [Ca2+]i in neurons and astrocytes Cell type
Agent (l~g/ml)
Max. [Ca2+]i (nM) a
Neuron
mAb-LI(2E12) 0.00 3.25 7.50 15.0 30.0
98.9 103.0 139.6 150.1 194.7
Neuron
Neuron
Astrocyte
mAb-NCAM(H28) 30.0 Rat normal 15.0 30.0
+ + + + +
-~200
8.5 b 5.8 3.9 6.4 3.4
-'~116 ~[ 97
99.5 + 10.4 c
-~43
IgG
2E12 (30/tg/ml)
101.0 + 3.2 c 108.2 + 2.4 c 109.6 + 2.0c
aMax. [Ca2+]i (nM) represents the maximum [CaZ+]i (nM) throughout the measurement (up to 300 s after the addition of the antibodies). bData represent the mean + S.E.M. of at least 28 cells for each condition. CNot statistically different from Max. [Ca2+]i in the absence of antibody.
addition were excluded from these measurements. Changes in [Ca2+]i of cultured cells were examined under the following conditions; application of (1) mAb-LI(2E12), monoclo-
12
34
Fig. 2. Western blot analysis using mAb-LI(2E12) in terms of plasma membrane fraction of embryonic mouse cultured neurons and astrocytes. Lanes 1, 3: astrocytes; lanes 2, 4: neurons. Samples were analyzed by Western blot method using polyclonal NCAM (lanes 1 and 2), and mAb-LI(2E12) (lanes 3 and 4).
Fig. 3. Immunocytochemical staining with mAb-LI(2E12) of primary cultures of mice cortical neurons. Cultures of primary neurons were prepared and immunocytochemistry performed as described in Materials and Methods. Cells were examined by Zeiss laser scan microscope equipped with Argon laser source 488 nm for FITC-staining. Arrow indicates astrocyte. Bar -- 25 gin.
236
Q
,i (nM)
!":, 400
0
0
200
,0
0 Fig. 4, Pseudo color images of fura-2 fluorescence intensity ratio imaging (340 nm/380 nm imaging) in primary cultured neurons treated with mAb-LI(2E12), a: normal resting levels; b: 180 s after addition of 30/zg/ml mAb-LI(2E12); c: resting levels in Ca2+-free medium plus 2 mM EGTA; d: 180 s after addition of 30/~g/ml mAb-LI(2E12) in Ca2+-free medium plus 2 mM EGTA.
nal anti-NCAM antibody (mAb-NCAM(H28) or non-immune rat IgG at several concentrations in CMF-HBSS containing HEPES and CaCI2, (2) mAb-Ll(2E12) at 30 pg/ml in CMF-HBSS containing HEPES in the presence of ethyleneglycol bis (b-aminoethylether)-N,N,N',N'-tetraacetic (EGTA) 2 raM, (3) mAb-Ll(2E12) at 30/~g/ml followed by application of CdC12 100/tM, nifedipine 1 pM, O,L-2-amino-5-phosphonovalericacid (AVP) 100/~M, phencyclidine (PCP) 1/~M, MgCl2 2 mM, phorbol-12-myristate-13-acetate (PMA) 100 nM, 1-(5-iso-quinolinesulfonyl)-2-methylpiperazine (H7) 100/~M, or tetrodotoxin (TI'X) 10/tM, (4) Ll-mAb-Ll(2E12) performed complex. For these applications, mAb-Ll(2E12), mAbNCAM(H28), EGTA, CdC12, APV, PCP, MgCI2, I-/7, TTX and L1 were dissolved in the CMF-HBSS containing HEPES and CaC12, and nifedipine and PMA were dissolved in dimethylsulfoxide (DMSO). APV was purchased from Cambridge Research Biochemicals. H7 and poly-L-lysine were from Seikagaku Kougyou, Japan. mAb-NCAM(H28) was purchased from Funakoshi Chemical Co., Japan. All other reagents were from Sigma Chemical Co. RESULTS
Specificity of mAb-L1 (2E12) According to the method of Western blot analysis, the specificity of m A b - L I ( 2 E 1 2 ) was determined using a
plasma membrane fraction from embryonic cultured neurons. Polyclonal L1 antibody reacted with all 3 previously identified L1 molecule components 26, whereas mAb-LI(2E12) reacted with only the 200 kDa component (Fig. 1, lines 1 and 2). The molecular weight of L1 molecule was reduced by approximately 30% by treatment overnight with glycopeptidase F which is known to cleave N-linked oligosaccharides from glycoproteins (Fig. 1, lines 1 and 3). Polyclonal L1 antibody still detected the digested proteins but m A b - L I ( 2 E 1 2 ) no longer reacted with the 200 kDa component (Fig. 1, lines 3 and 4). mAb-LI(2E12) immunoreacted with the 200 kDa of L1 molecule derived from cultured neurons but not with membrane preparations from astrocytes (Fig. 2, lines 3 and 4). Furthermore, from our immunocytochemical studies, we showed that the L1 molecule was recognized by m A b - L l ( 2 E 1 2 ) in primary cultured neurons, but not in astrocytes (Fig. 3). In contrast, N C A M was detected on both neurons and astrocytes by polyclonal N C A M antibody (Fig. 2, lines 1 and 2).
237 m A b - L 1 ( 2 E 1 2 ) causes a rise in [Ca2+]i in neurons [Ca2+]i increases rapidly and dramatically in cultured neurons treated with 30/~g/ml of m A b - L I ( 2 E 1 2 ) but not after treatment with normal rat IgG (Figs. 4 and 5). The [Ca2÷]i was elevated as early as 10 s after m A b - L I ( 2 E 1 2 ) addition. A peak of [Ca2+]i (approximately 200% increase) was seen at 180 s after the addition and persisted as long as 600 s. Longer times were not examined. Of the 25 cells present in the field, 17 cells were responsive to m A b - L I ( 2 E 1 2 ) (Fig. 4, a and b). In combined experiments, the percentage of responsive cells was approximately 70% when treated with this mAb. In control experiments, m A b - L I ( 2 E 1 2 ) had no such [Ca2+]i increasing effect on astrocytes (Table I). L I - m A b - L I ( 2 E 1 2 ) complexes did not cause [Ca2+]i increases in neurons. The [Ca2÷]i-increasing effect of m A b - L I ( 2 E 1 2 ) was dependent on the concentration of the antibody used (Fig. 6 and Table I). In contrast, m A b - N C A M ( H 2 8 ) failed to elicit a change in [Ca2+]i in cultured neurons (Table I). Furthermore, the subsequent addition of m A b L1(2E12) to neurons after treatment with m A b N C A M ( H 2 8 ) produced an increase in [Ca2+]i, and the magnitude and time course pattern of this increase were identical to those observed in the treatment with m A b Ll(2E12) alone (data not shown). m A b - L l ( 2 E 1 2 ) causes an influx o f extracellular Ca 2+ through L-type voltage-operated Ca 2+ channels ( V O C C s ) When neurons were cultured in Ca2+-free medium supplemented with E G T A 2 mM, the increase in [Ca2+]i by m A b - L I ( 2 E 1 2 ) (30/~g/ml) was not observed (Figs. 4
220
•
2E12 30~g/ml
a)
i)
IgG 30-g/ml
T
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180 i
,
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[] mAb-LI(2EI2) ~i 180' • nonimmune lgG 160
~
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/I
~
140-
y
/ -
,oo 80 1 0
•
,
•
,
10 IgG
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20
concentration
30
(~g/mi)
Fig. 6. Dose-response relationship between mAb-Ll(2E12) concentrations and [Ca2+]i in neurons. Values represent the mean + S.E.M. of at least 28 neurons for each condition. [Ca2+]i represents the maximum concentrations throughout the measurement (up to 300 s after the addition of the antibody).
and 7). The addition of cadmium 100 ~M or nifedipine 1 ~M, inhibitors of L-type VOCCs, also completely abolished the increase of [Ca2+]i by m A b - L I ( 2 E 1 2 ) (Fig. 7A). On the other hand, the increase in [Ca2+]i by m A b Ll(2E12) was not antagonized by treatment of T-FX, a sodium channel blocker. N-Methyl-D-aspartate (NMDA)operated Ca 2÷ channel blockers such as A P V (100/~M), PCP (1/~M) and MgCI2 (2 mM) 5'22, had no significant effects on the increase of [Ca z÷] by mAb-LI(2E12) (Fig.
TABLE II ~j)
200 ')
200
"r
")
")
")
*)
160
Effects of PMA and H7 on the rise in [Ca]i of neurons induced by rnAb-L1(2E12) Agent
Max. [Ca2+]i (nM) a
mAb-L1(2E12) ~g/rnl) 0.0 15.0
98.9 + 8.5b 150.1 + 6.4
14o.
~
PMA (100 nM, 18 h) plus 2E12 (15/tg/ml)
116.9 + 4.1c'a
120 '. 100 j.
H7 (100/~M, 18 h) plus 2E12 (15 gg/ml)
109.8 + 8.7c'a
[Ca2+]i
80 -60
0
60
120 TIME
in
180
240
300
(sec)
Fig. 5. Changes [Ca2+]i after treatment of neurons with mAbLl(2E12). Either mAb-Ll(2E12) or normal rat IgG was added to the cultures at the 0 time point (arrow). a) Statistically significant, P < 0.01; mAb-LI(2E12) vs. normal rat IgG.
maximum[Ca2+]i
aMax. (nM) represents the (nM) throughout the measurement (up to 300 s after the addition of the antibodies). bData represent the mean + S.E.M. of at least 28 cells for each condition. cStatistically significant, P < 0.01 as compared with [Ca2+]i in the presence of antibody. dNot statistically different from [Ca2+]i in the absence of antibody.
Max.
Max.
238
B
A 220
•
220'
2E12 30~0/m|
b)
*Ca f r l e (EGTA)
A
200
b)
200'
*Cd 0.1rnM
180
180'
160-
160'
b)
b)
b)
T
T~II
I
b)
b)
b)
0 0 m
3
140-
U
120-
140"
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120 • I
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----o--
.APV0.1mM
f
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100' •
.60
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i
0
-
i
60
-
i
120 TIME
-
w
180
•
|
240
•
i
300
(sec)
i
0
•
i
60
•
i
i
i
i
120
180
240
300
TIME
(see)
Fig. 7. Effect of EGTA and L-type voltage-operated Ca2+ channels (VOCCs)-antagonists (A) and N-methyl-o-aspartate (NMDA)-antagonists (B) on the rise in [Ca2÷]i of neurons induced by mAb-LI(2E12), mAb-LI(2E12) was added to the medium at the 0 time point (arrow) after treatment with EGTA or each of L-type VOCCs- or NMDA-antagonists. a)Statistically significant, P < 0.01; mAb-LI(2E12) alone vs. EGTA or L-type VOCCs-antagonistsin the presence of antibody, b)Not statistically different from [Ca2+]iin the presence of antibody alone.
7B). These combined results indicate that the mAbLl(2E12)-induced Ca 2+ influx into neurons seems to be through the VOCCs, particularly L-type VOCCs.
Protein kinase C (PKC) activity influences the rise in [Ca2+]i in neurons exposed to mAb-L1(2E12) The exposure of neurons to PMA (100 nM) or H7 (100/~M) for long (18 h) periods had no significant effects on the basal levels of [Ca2+]i (data not shown). However, the magnitude of [Ca2÷]i elevation by mAb-L1 was significantly reduced after the long-term exposure of neurons to PMA (Table II). This suggests a relationship between mAb-Ll(2E12)-induced Ca 2÷ influx and PKC activity in our culture system. DISCUSSION We studied a potential functional mechanism of carbohydrates of the LI molecule in the cell-cell interactions of neuronal cells using a monoclonal antibody against oligosaccharides of L1 molecule because a monoclonal antibody is often a useful tool both for identifying its specific epitope and also for triggering the signal transduction in which its epitope is involved. We selected the mAb-LI(2E12) which specifically reacted with only the 200 kDa component of L1 molecule• This mAb lost the reactivity with the 200 kDa component after treatment with glycopeptidase F which is reported to cleave asparagine-linked glycans34, demonstrating that the epitope recognized by this mAb is a carbohydrate part of L1 molecule (Fig. 1). Endoglycosidase H mainly releases mannose- and hybrid-type
N-linked oligosaccharides, and endoglycosidase F additionally cleaves certain complex-type oligosaccharides 17. Treatment with either endoglycosidase H or F did not alter the reactivity of mAb-LI(2E12) at all (data not shown). Therefore, the carbohydrate epitope appeared to consist of a complex-type glycan which is only susceptible to glycopeptidase F. The L2/HNK-1 epitope, being expressed on several adhesion molecules, was also found to carry a complex-type glycan4. In addition, the adhesion molecules L1, NCAM and MAG were reported to be L2/HNK-1 epitope-positive TM. The epitope recognized by mAb-LI(2E12), however, seems to be distinct from the L2/HNK-1 epitope because mAb-LI(2E12) did not react with NCAM and MAG. We determined the influence of the carbohydrate epitopes of L1 molecule on Ca 2+ influx in cultured neurons using antibodies to mimic ligand binding 3°. Treatment of cultured neurons with mAb-LI(2E12) caused an increase in [caa*]i . The magnitude of the increase was dependent on the concentrations of the mAb (Fig. 6 and Table I). When the Ca 2+ in the medium (extracellular Ca 2*) was eliminated by the addition of EGTA, the increase in [Ca2*]i by mAb-LI(2E12) did not occur (Fig. 7A). The increase in [Ca2+]i, therefore, is due to an influx of extracellular Ca 2+ into the neurons rather than release from intracellular stores. In general, two types of Ca 2+ channels, voltage-operated Ca 2+ channels (VOCCs) and receptor-operated Ca 2+ channels (ROCCs), have been shown to mediate Ca 2+ influx. The VOCCs can be distinguished pharmacologically: L-type VOCCs are affected by both dihydropyridines (DHP) and ~o-conotoxin; N-type VOCCs are
239 effectively inhibited by to-conotoxin but not by the DHP. The T-type V O C C s are relatively resistant to inhibition by both the D H P and to-conotoxin 37. Our results demonstrated that nifedipine and Cd 2+, two L-type V O C C s antagonists, were effective in abolishing increases in [Ca2+]i after m A b - L I ( 2 E 1 2 ) treatment (Fig. 7A). We concluded that the influx of Ca 2+ in neurons exposed to m A b - L l ( 2 E 1 2 ) was particularly mediated by L-type VOCCs, because L-type V O C C s were modulated by D H P antagonists but T- and N-type V O C C s were not, and L- and N-type V O C C s were potently blocked by Cd 2+, whereas T-type V O C C s were much less sensitive. However, it will have to be determined whether T- and N-type V O C C s blockers influence the increase in [Ca2+]i by mAb-Ll(2E12). We then examined the involvement of NMDA-linked R O C C s in the increase in [Ca2+]i by m A b - L I ( 2 E 1 2 ) (Fig. 7B). A P V and PCP at the concentrations sufficient to block completely the NMDA-linked R O C C s 5 failed to inhibit the effect of mAb-L1(2E12), suggesting that the NMDA-linked R O C C s are not associated with the Ca 2÷ influx induced by m A b - L l ( 2 E 1 2 ) in our neuronal culture system. The increase in [Ca2+]i was seen quickly (10 s) after the addition of the antibody, reached a plateau at 180 s, and persisted throughout the experiment (Figs. 5 and 7). This is similar to the [Ca2÷]i increase induced by the addition of anti-galactocerebroside (GalC) antibody to oligodendrocytes 7. These authors suggested that V O C C s in oligodendrocytes are not responsible for the Ca 2+ influx caused by anti-GalC antibody in contrast to our results with neurons. Since the interactions of PKC with Ca2÷ and neural adhesion molecules have been demonstrated in several neural types 2'13'14, it is of interest to determine if P K C
influences the elevated [Ca2+]i in neurons, m A b Ll(2E12)-induced increase in [Ca2+]i was lowered by approximately 70% in neurons previously exposed to P M A (PKC-deficient cells) for 18 h 21. This inhibition was also seen when cells were treated with H7, a PKC inhibitor. These results suggest that PKC activation at least partially influences the increase in [Ca2+]i by m A b Ll(2E12). However, further studies are required to confirm the relationship between P K C and the increase in [Ca2+]i by mAb-LI(2E12). Our results may suggest an important clue for clarifying one mechanism by which carbohydrate chains of L1 molecule may mediate in neuronal functions. L1 molecule-dependent adhesion of neurons to neurons may influence neuronal morphological changes by modulating VOCCs, because [Ca2+]i may control neurite elongation in part by regulating the stability of actin filaments 2°. Moreover, these changes may contribute to intracellular molecular events leading to adhesion-linked gene activation, because Ca 2+ is a major second messenger regulating immediate-early gene expression in excitable cells 33. Taken together these observations provide evidence that the expression of a particular carbohydrate epitope may have a neuronal functional significance. Further studies on the structure and function of the carbohydrate epitope of L1 molecule and gene regulations by these glycans of L1 molecule are necessary to obtain a more complete understanding of the role of carbohydrates in cell-cell interaction.
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