Brain Research, 559 (1991) 118-129 © 199l Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939116952P

118

BRES 16952

A carbohydrate epitope defined by monoclonal antibody VCI.1 is found on N-CAM and other cell adhesion molecules Janice R. Naegele and Colin J. Barnstable Department of Ophthalmology and Visual Sciences, Yale University School of Medicine, New Haven, CT 06510 (U.S.A.)

(Accepted 23 April 1991) Key words: Monoclonal antibody; Neural cell adhesion molecule; Adhesion molecule; HNK-1

VCI.1 is a monoclonal antibody that stains the surfaces of neuronal subsets in the brain. Previous work showed that VCI.1 recognizes 3 polypeptide bands with molecular weights of 95-105 kDa, 140 kDa and 170 kDa and two high molecular weight proteoglyeans with weights of approximately 680 and 650-700 kDa. The heterogeneity and molecular weight range of these bands suggested that VCI.1 might recognize a carbohydrate moiety associated with a family of cell surface molecules. It had been previously demonstrated that a separate monoclonal antibody, HNK-1 also recognized a cell surface associated epitope characterized as a sulfate- and glueuronic acid-containing N-linked carbohydrate. This epitope has been shown to be present on members of the N-CAM adhesion molecule family. In this report, we demonstrate that VCI.1 recognizes an N-linked carbohydrate group that is attached to myelin-associated glycoprotein and N-CAM. Immunoeytochemical and biochemical comparisons of VCI.1 and HNK-1 staining in rat and cat brain indicate that these two antibodies probably recognize overlapping, or identical carbohydrate epitopes. INTRODUCTION Many interactions that occur between neurons and their environment may be modulated by carbohydrate groups on the cell surface. Studies of carbohydrate moieties associated with cell surface molecules have led to the discovery of neuronal subsets in the brain that express unique surface glycoconjugates 3"12"26"45-48'57'68'69. Although these studies suggested that cell surface glycoconjugates may be expressed by cells with distinct functional properties, the exact link between molecular and functional properties is not yet clear. Cell adhesion molecules (CAMs) are one of the major classes of glycoconjugates in the nervous system which participate in intercellular recognition during development. N - C A M and N-cadherin, two of the most abundant CAMs, are expressed on the perikarya, dendrites and axons of most neurons 14'22"65. Spatial and temporal regulation of N - C A M contributes to the histogenesis of the embryonic CNS 7"9'16'20"23"40'54'62. There are suggestions that N - C A M may also facilitate later stages of cellcell recognition such as synapse formation and stabilization; however, the mechanism is not yet known 12'53'55. The polypeptides and genes of many CAMs, including N - C A M , N-cadherin, myelin-associated glycoprotein ( M A G ) , a n d L1 have been isolated. If one considers the

numbers of identified genes for the known adhesion molecules, there are too few to account for the complexity of neuronal circuits. There is evidence, however, that greater diversity can be achieved through transcriptional and post-translational modifications which generate multiple forms of each adhesion molecule. For example, differential splicing gives rise to the 3 major polypeptide chains of N - C A M and a number of other small exons can be spliced in to give tissue-specific and developmentallyregulated forms of the molecule 19"59"67. The other major source of variation in the N - C A M molecule is in its carbohydrate chains. In the embryo, N - C A M contains long chains of a - 2 - 8 linked polysialic acid that accounts for about one-third of the molecular weight 15'29. During development, the amount of polysialic acid decreases and this enhances adhesion between cells 27'53. N - C A M (140/170), M A G and L1 have been shown to express a carbohydrate epitope that is recognized by the HNK-1 monoclonal antibody 37. This epitope has been identified as a glucuronic acid- and sulfate-containing carbohydrate 5'6 on approximately 15-20% of the N - C A M molecules in mouse brain 37. This same carbohydrate epitope is also expressed on a variety of other adhesion or extracellular matrix molecules. These include cytotactin, an extracellular matrix glycoprotein synthesized by glia (also known as tenascin, and related or identical to

Correspondence: C.J. Bamstable, Box 3333, Department of Ophthalmology and Visual Sciences, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06510, U.S.A. Fax: (1)(203) 785-6123.

119 J1) s'36'38 a n d c y t o t a c t i n - b i n d i n g ( C T B ) p r o t e o g l y c a n s, which is synthesized b y n e u r o n s 28, the 680 k D a c h o n d r o i t i n sulfate p r o t e o g l y c a n identified b y m A b Cat-30170, a n d several p e r i p h e r a l n e r v e glycolipids 2'6"37. A l t h o u g h n e a r l y all n e u r o n s express N - C A M to varying degrees, o n l y rare subsets express forms of N - C A M b e a r i n g the H N K - 1 e p i t o p e 11'68. I n o u r p r e v i o u s studies, we d e s c r i b e d a m o n o c l o n a l a n t i b o d y called V C I . 1 which stains the surfaces of rare n e u r o n a l subsets in cat r e t i n a , t h a l a m u s a n d c e r e b r a l cortex 3'70. Similar to H N K - 1 , V C I . 1 reacts with m u l t i p l e n e r v o u s system glycoconjugates with m o l e c u l a r weights similar to N - C A M a n d M A G 3. It was r e c e n t l y discovered that V C I . 1 also recognizes at least two high m o l e c u l a r weight glycoconjugates, i n c l u d i n g a 680 k D a c h o n d r o i t i n sulfate p r o t e o glycan a n d a different 6 5 0 - 7 0 0 k D a p r o t e o g l y c a n with k e r a t i n sulfate chains 7°. B e c a u s e the r e p o r t e d distribution of H N K - 1 s t a i n i n g in the m o u s e CNS strongly res e m b l e d the p a t t e r n of V C I . 1 s t a i n i n g in the cat C N S , we have p e r f o r m e d i m m u n o c y t o c h e m i c a l a n d b i o c h e m i cal c o m p a r i s o n s of these two m a r k e r s in the s a m e tissues. T h e s e studies indicate that the V C I . 1 a n d H N K - 1 d e t e r m i n a n t s are similar or identical. MATERIALS AND METHODS

Immunological reagents. Mouse monoclonal antibodies VCI.1 (IgM) and VC5.1 (IgGx) were generated as described previously3. Mouse monoclonal cell line HNK-1 (IgM) 1 was obtained from American Type Culture Association (Rockville, MD) and propagated in vivo. Antibody 7. lb is specific for p38 (synaptophysin) and was a gift from Dr. R. Jahn. Each of the above antibodies were used as a diluted ascites fluid. Monoclonal antibodies 5D12, 0Bll and CB7.5 were all generated in our laboratory and recognize NCAM (140/170) 5°. The epitope recognized by 5D12 and 01311 appears to be on the cytoplasmic portion of the molecule close to the point at which the 140 kDa and 170 kDa forms differ (E. Book, personal communication). All 3 antibodies also recognize polysialated embryonic forms of N-CAM. All were used as undiluted hybridoma supernatants. Because all 3 gave essentially identical results, they were used interchangeably in the experiments described. A rabbit antiserum against L-MAG, directed against the last 23 amino acids of large myelinassociated glycoprotein, was generated by Drs. D. Colman and J. Seltzer (Columbia University, NY, NY). To produce 35S-labeled antibodies, VCI.1 and HNK-1 producing hybridomas were grown in medium containing 10 gC/ml 3SS-methionine (1,000 Ci/millimol) for 48 h. Culture medium was dialyzed against RPMI 1640 to remove unincorporated label and then used in binding assays. lmmunocytochemistry. Three adult cats and 3 adult rats were used for immunocytochemistry. Animals were deeply anesthetized with an overdose of sodium pentobarbital and perfused through the heart with 0.1 M sodium phosphate buffer (pH 7.4) containing 0.9% saline (PBS), followed by a fixative solution containing 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.5) for 30-60 min. Brains were removed, blocked and cryoprotected in 30% sucrose in sodium phosphate buffer and stored frozen at -80 °C. Blocks were thawed and 50-/zm thick sections were cut on a vibratome. Sections were pre-treated with 0.3% Triton X-100 in PBS for 3-5 h, blocked in 3% normal horse serum in PBS, incubated in primary antibodies, biotinylated secondary antibodies and

ABC-peroxidase (Vector) as described previously47. Specific immunoreactivity was visualized by DAB precipitation or using an intensification mixture of 0.05% nickel chloride and DAB 3o. Western blots. P2" membrane fractions were prepared from whole rat cortex or cat cortex and subjected to electrophoresis as described3. Between 50 and 100 #g was used per lane. Following 2 h of electrophoretic transfer onto nitrocellulose63, blots were washed in blocking buffer (10 mM Tris, pH 8.2, 0.5 M NaCI, 0.5% Tween 20) containing 5% NGS for 1-2 h, then incubated overnight at 4 °C in primary antibody. The primary antibodies were used at dilutions of 1:1000-1:4000 (VCI.1, VC5.1, HNK-1, L-MAG, 7.1b) or undiluted hybridoma supernatant (5D12, 0Bll, CB7.5). After washing in blocking buffer, the filters were incubated in anti-mouse or antirabbit IgG conjugated to alkaline phosphatase (Promega, 1:7500) for 1 h, and then reacted in alkaline phosphatase substrate (1% NTB, 0.5% BCIP in 50 mM Tris, pH 10.0, 3 mM MgCI2). Immune precipitation. Immunobeads conjugated with goat antimouse IgG antibody (Biorad) were washed with 1% BSA in 10 mM Tris-HCl (pH 7.5) and then incubated for 1 h at room temperature with monoclonal antibody, either VCI.1 or anti N-CAM (CB7.5) Unbound and non-specifically bound antibody was removed by multiple washes in 10 mM Tris-HCl, pH 7.5 and 0.5 M NaC1 in the same buffer. P2" fractions from rat cortex were dissolved in 1% NP-40 in 10 mM Tris-HCI, pH 7.5. Insoluble material was removed by centrifugation at 100,000 g for 1 h. Immunobeads with bound monoclonal antibody were then incubated with soluble membrane proteins for 1 h at 4 °C. They were then washed extensively, as described above. The final pellet of beads, antibody, and bound membrane proteins was then dissolved in SDS sample buffer and aliquots were run on 7.5% polyacrylamide gels and analyzed by Western blot. Deglycosylation. Cytosolic or P2" membrane fractions from cat cortex were prepared as described previously3. Samples were diluted to a final protein concentration of 0.5 mg and added to 100 mM phosphate buffer, pH 8.5 containing 25 mM EDTA, 0.5% TX-100, 0.1% SDS and 1.0% mercaptoethanol. Samples were boiled for 3 min, cooled on ice, and 25 units of glycopeptidase F enzyme (Boehringer-Mannheim) was added. Control samples were treated identically except that glycopeptidase F was omitted. Samples were incubated with shaking at 37 °C for 18 h, concentrated in a Speedvac, and loaded onto 7.5% SDS polyacrylamide gels. Competition assays. Rat cortical membranes were run on SDS polyacrylamide gels and transferred to nitrocellulose filters as described above. The regions of the filters containing bands reactive with antibody VCI.1 were cut out and non-specific binding sites blocked by incubation with 10 mM Tris, pH 8.2, 0.5 M NaCI, 0.5% Tween 20 and 5% NGS. Filters were then incubated in mixtures of 3ss-labelled monoclonal antibody and unlabelled antibody of the same or different specificity overnight at 4 °C. After washing, illters were dried and exposed to X-ray film. RESULTS

Patterns o f V C I . 1 immunoreactivity in rat and cat cerebral cortex A l t h o u g h similar s u b p o p u l a t i o n s of n o n - p y r a m i d a l n e u r o n s are labelled b y a n t i b o d y V C I . 1 in the cat a n d rat, side-by side c o m p a r i s o n of labelling in the two species reveals differences in the density of s t a i n e d n e u r o n s a n d in the e x t e n t of labelling in the n e u r o p i l (Fig. 1). I n b o t h species, V C I . 1 shows s t r o n g pericellular labelling a r o u n d n e u r o n a l cell b o d i e s a n d p r o x i m a l d e n d r i t e s (Fig. 1 A , B ) . I n rat c e r e b r a l cortex, there is m o r e extensive labelling of distal d e n d r i t e s (Fig. 1A). I n b o t h cat a n d rat, there are regional v a r i a t i o n s in the e x t e n t of diffuse

120 neuropil labelling. In rat sensorimotor area, shown in Fig. 1A, the diffuse neuropil staining is not evident. In other cortical areas, including primary olfactory and entorhinal cortices, neuropil staining p r e d o m i n a t e s (not shown). In cat, all p r i m a r y cortical areas exhibited neuropil staining to varying degrees. In addition to pericellular and neuropil-immunoreactive structures in gray matter, myelinated fiber tracts and cortical white matter were stained in both rat and cat cerebral cortex (see below). On immunoblots of cortical m e m b r a n e s from rat and cat, differences can also be detected. In both species, V C I . 1 reacted with p r o m i n e n t bands of approximately 170 k D a nd 145 k D a and with a n u m b e r of m i n o r bands (Fig. 2). The major species difference was an intensely stained band at 95-105 k D a that was detected in cat, but not rat. Membrane fractions from rat retina also gave similar results to rat cortical membrane fractions (Fig. 3).

The VCI.1 epitope is an N-linked carbohydrate M e m b r a n e fractions from rat retina or cat brain were

incubated with specific glycosidases prior to Western blotting to remove N-linked carbohydrates. Western blots were subsequently p e r f o r m e d and then p r o b e d with antibody VC1.1 to d e t e r m i n e whether the VC1.1 epitope included an N-linked carbohydrate. In Fig. 3, lanes A and B were loaded with rat retinal m e m b r a n e s and for comparison, lanes C - F were loaded with cat cortical m e m b r a n e s . Similar to the results shown in Fig. 2 for rat brain samples, rat retinal samples incubated in buffer lacking N-glycanase, exhibited V C I . I immunoreactive polypeptides of 145 and 170 k D a (Fig. 3A). In addition, several higher molecular weight polypeptide bands were detected in rat nervous tissue, including bands at approximately 160 and 200 k D a (Fig. 3A). None of these polyp e p t i d e bands were detectable after N-gtycanase treatment, indicating that the enzyme t r e a t m e n t had r e m o v e d the V C I . 1 e p i t o p e (Fig. 3, lane B). In Fig. 3C, cat cortical m e m b r a n e s from cat exhibited the same major V C l . l - i m m u n o r e a c t i v e band corresponding to 95-105 k D a as described in Fig. 2. D u e to the experimental

RAT

CAT

~-'gin

170--

145--

105-95--

~nt

Fig. 1. lmmunofluorescence staining of multipolar neurons in rat (A) and cat (B) visual cortex with antibody VCI.1. Extensive labelling of distal dendrites was observed in rat, but not cat visual cortex stained with VCI.1. VCI.1 immunoreactivity is also present as punctate labelling in the neuropil staining was observed with VCI .l (see Fig. 4). Fifteen-/~mthick cryostat sections. Bar = 1130/~m.

Fig. 2. Comparison of VCl.l-immunoreactive bands on Western blots of rat or cat cortical membranes. Two prominent bands of 170 kDa and 140 kDa are evident in the rat, as well as additional bands between these, in cat, the same bands are stained and a third prominent band can be detected at 95 kDa-105 kDa. Higher molecular weight bands are also evident in both species. The mobility of standard protein markers are indicated on the right.

121 conditions, less protein was loaded per lane and the two additional polypeptide bands at 145 and 170 kDa were only faintly detected. In Fig. 3D, the sample of cat cortical membranes that was treated with N-glycanase lacked the VCl.l-immunoreactive bands. Together, these findings demonstrate that the VCI.1 epitope contains N-linked carbohydrate which is cleaved by enzymatic digestion with peptide-N-glycosidase F (N-glycanase). In Fig. 3E, cat cortical membranes were incubated in control buffer without N-glycanase and then probed with a control antibody, 7.1b. This antibody recognizes a polypeptide epitope on a 38 kDa integral membrane glycoprotein present on synaptic vesicles (p38, synaptophysin o r SVP38) 1°'31. When the N-linked carbohydrate groups on p38 were removed by N-glycanase, the immunoreactive band shifted from 38 kDa to 34 kDa (Fig. 3F) as shown in previous deglycosylation studies 49, thus demonstrating that the effects shown are not due to proteolysis.

Antibodies VCI.1 and HNK-1 recognize similar epitopes The antibody HNK-1 and a similar antibody, 4F4, have been shown to recognize sulfate- and glucuronic acid-containing epitopes on the surfaces of interneurons in the mouse hippocampus and cerebral cortex 68. The reported distribution was similar to that of V C I . 1 3 and prompted us to make further comparisons between HNK-1 and VCI.1. As shown in Fig. 4, sections from rat somatosensory cortex that had been stained with either VCI.1 (Fig. 4A) or HNK-1 (Fig. 4B) had nearly identical patterns of immunoreactive labelling of non-

VC 1.1

VC 1.1

P38

200

116

97

66

42

Front -

4-

--

-I-

--

+

A

B

C

D

E

F

Fig. 3. Deglycosylation experiments demonstrating that the VCI.1 epitope is part of an N-linked carbohydrate. Rat retinal membranes (lanes A, B) or cat cortical membranes (lanes C-F) were treated with buffer alone (-) or with buffer containing N-glycanase (+) as described in Materials and Methods. N-glycanase treatment abolished the binding of mAb VCI.1 to polypeptide bands on the Western blots (lanes B,D). This effect was specific for N-linked carbohydrates and was not due to proteolysis since identical enzymatic treatments of membrane fractions in buffer alone (lane E) or in buffer containing N-glycanase (lane F) and subsequent incubation of the antigen transfers in antibody 7.1b, against a polypeptide epitope on the synaptic vesicle protein synaptophysin (P38), resulted in a shift of the immunoreactive band to a lower molecular weight, as described previously 31.

Fig. 4. Patterns of VCI.1 (A) or HNK-1 (B) immunoperoxidase staining in 50-#m thick vibratome sections from adult rat somatosensory cortex. Both antibodies outline the somata and proximal dendrites but not the axons of non-pyramidal multipolar neurons. VC1.1 and HNK-1 both label structures in the neuropil in somatosensory cortex. Bar = 100/lm.

122 pyramidal cell bodies and dendrites. VCI.1 and HNK-1 antibodies were also found to inhibit the binding of each other. 35S-methionine-labelled VCI.1 and HNK-1 bound to characteristic bands corresponding to 145 and 170 kDa on immunoblots even in the presence of a high concentration of mouse IgM (Fig. 5A,E). Unlabelled VCI.1 or HNK-1 were able to block binding of either radiolabelled antibody (Fig. 5B,C,F,G) whereas antibody CB7.5 (specific for N-CAM) did not (Fig.5D,H). These results suggest that the epitopes recognized by antibodies VCI.1 and HNK-1 are either identical or sufficiently close to cause steric hindrance in a binding assay.

A

E

B

F

C

G

D

H

Fig. 5. Competition experiments revealed that the epitopes recognized by antibodies VCI.1 and HNK-I are either identical or sufficiently close to cause steric hindrance in a binding assay. 3SSmethionine-labeUed VCI.1 and HNK-1 antibodies bound to char-

acteristic bands on immonoblots (A and E). Unlabeled VCI.1 or HNK-1 blocked the binding of either radiolabelled antibodies (B, C, F, G) whereas antibody CB7.5 did not (D and H).

The 145 kDa and 170 kDa VCI.I bands are forms o[ N-CAM The two VCl.l-immunoreactive bands with molecular weights of 145 and 170 kDa had similar mobilities to the polypeptide bands stained by 3 separate monoclonal anti-N-CAM antibodies CB7.5, GC-5D12 and GC-0BII. To determine whether the VCI.1 carbohydrate epitope was expressed on N-CAM polypeptides or separate polypeptides which had similar mobilities, we carried out a series of immunoprecipitations with detergent solubilized rat cortical membranes, as described in Materials and Methods. Antigen recognized by VC1.1 or CB7 was precipitated using a secondary antibody coupled to latex microspheres. Equal volumes of each redissolved precipitate were analyzed on immunoblots using VCI.1 and CB7. Although the intensities of labelled bands varied, Fig. 6 shows that antigen precipitated by VCI.1 reacted with both CB7 (lane A) and VCI.1 (lane D) and antigen precipitated by CB7 reacted with both CB7 (lane B) and VCI.1 (lane E). Control lanes contained starting membrane material that was not reacted with the precipitating antibodies, VCI.1 or CB7 (Fig. 6C, F). No specific bands were observed when other unrelated monoclonal antibodies were used to precipitate proteins (data not shown). Several low molecular weight bands were present in the lanes containing precipitated antigens, but were not seen in control lanes, and probably represent reactivity of the secondary antibodies with the precipitating antibodies that were also run on the gel. The 95-105 kDa VC1.1 band is myelin-associated glycoprotein In addition to the 145 and 170 kDa bands stained by VCI.1 on Western blots, VCI.1 reacted with a prominent 95-105 kDa glycoprotein in cat, but not rat brain (see Fig. 2). In a previous study, McGarry et al 43 demonstrated that HNK-1 reacts primarily with a band of 108 kDa present in crude myelin fractions from human brain and immunoprecipitation experiments demonstrated that this immunoreactive band corresponded to myelin-associated glyeoprotein (MAG). These observations led us to hypothesize that the 95105 k D A band stained by VCI.1 in membrane fractions from cat brain might also correspond to myelin-associated glycoprotein. To test this possibility, nitrocellulose filters from Western blots of rat and cat brain were reacted with VCI.1, HNK-1 and a rabbit polyclonal antibody against large myelin associated glycoprotein (LMAG), as well antibodies against N-CAM (CB7) and an irrelevant control antibody (RP-1). The results for each antibody are shown in Fig. 7. VCl.l-immunoreactive bands on Western blots of adult rat brain (Fig. 7, lane A) were identical to the HNK-l-immunoreactive bands

123 in rat brain (Fig. 7, lane C). Similarly, VCI.1 immunoreactive bands in cat brain (Fig. 7, lane B) were identical to the HNK-1 immunoreactive bands in cat brain (Fig. 7, lane D). L - M A G antisera strongly stained M A G , which appeared as a prominent immunoreactive band with a mobility of approximately 95-110 kDa in rat and cat (Fig. 7, lanes E,F), with slight species differences in the molecular weights. L - M A G antisera also reacted with a series of minor bands, some of which probably represent antigen degradation components. Only the major L - M A G reactive band of 95-105 kDa that was stained in cat (Fig. 7, lane F) was the same as the 95 kDa band recognized by VCI.1 and HNK-1 (Fig. 7, lanes B,D). These findings indicate that the VCI.1/HNK-1 carbohydrate epitope is present on L - M A G in the cat brain. In

the rat however, L - M A G does not appear to express detectable levels of the VCI.1/HNK-1 carbohydrate epitope (Fig. 7, compare lanes A,C,E). Similar to the findings presented in Fig. 6, an antibody against N-CAM (CB7) reacted with two bands at 145 and 170 kDa in membrane fractions from either rat (Fig. 7, lane G) or cat brain (Fig. 7, lane H). These bands corresponded to two of the prominent bands stained by VCI.1 (Fig. 7, lanes A,B) and by HNK-1 (Fig. 7, lanes C,D) in rat and cat brain.

Some of the neuronal cell surface staining is due to N-CAM, but not L-MAG molecules, carrying the VCI.1 epitope Given the multiple glycoproteins expressing the VCI.1 epitope, we were interested in determining which of

CB 7

VCI - 2O0

-116

-

A

B

C

D

E

97

-

66

-

42

-

Front

F

Fig. 6. Immunoprecipitation experiments demonstrating that VCI.1 immunoprecipitates N-CAM and that antibodies against N-CAM immunoprecipitate VCI.1 reactive antigens. VCI.1 or anti-N-CAM antibody (CB7) were coupled to anti-mouse immunobeads, the beads were incubated in detergent solubilized membrane fractions from rat brain to immunoprecipitate their respective antigens and the immunoprecipitates were separated by SDS-PAGE and transferred onto nitrocellulose, as described in Materials and Methods. Antigens precipitated by mAb VCI.1 reacted with both mAb CB7 (lane A) and VCI.1 (lane D). Similarly, antigens precipitated by mAb CB7 were recognized by both CB7 (lane B) and VCI.1 (E). Control lanes containing detergent solubilized membranes (starting material before immunoprecipitations) reacted with CB7 (lane C) and VCI.1 (F). Mobilities of molecular weight standard proteins are shown to the right of the figure.

124 these molecules accounted for the strong pericellular neuronal staining observed in retina and cerebral cortex. lmmunocytochemical studies were carried out in both structures to compare the staining patterns. Comparison of VCI.1 and N-CAM (mAb 5D12) staining in rat retina is shown in Fig. 8. With both VCI.1 and N-CAM antibodies, strong staining was observed in the fiber layers of the retina, the outer and inner plexiform layers (OPL, IPL). Both antibodies also stained the surfaces of neurons in the inner nuclear layer (INL). Nearly all cells in the INL were N-CAM-immunoreactive, whereas VCI.1 labelled only a minor subset of these which probably correspond to subsets of horizontal and amacrine cells. Similar patterns were seen in the ganglion cell layer where only a minor subset of N-CAM reactive cells were stained by VCI.1. These observations indicate that in rat retina, most cell types and their processes exhibit N-CAM immunoreactivity. In contrast, the V C I . I carbohydrate epitope is probably expressed on a subset of

VC 11

HNK-1

L-MAG

N-CAM molecules associated with a minor subset of retinal neurons. Although we have not studied the staining patterns given by M A G antibodies in the retina, in previous reports, MAG was reported to be present along oligodendroglia processes only. In addition, we have shown that in the rat, M A G does not carry the VCI.1/ HNK-1 carbohydrate epitope. Therefore, the V C I . I staining in retina cannot be due to expression of this carbohydrate on MAG. Since the VCI.1 carbohydrate has been shown to be present on several high molecular weight proteoglycans, our findings do not rule out the possibility that some of the pericellular staining in retina is due to the expression of the VCI.1/HNK-1 carbohydrate antigen on retinal proteoglycans and studies are currently underway to test this hypothesis. We further compared cellular staining patterns in sections of cat and rat cerebral cortex which had been incubated in VCI.1, anti-L-MAG or anti-NCAM antibodies. A comparison of cerebral cortex gray matter stained

N- CAM

RP-1

Fig. 7. VCI.1 and HNK-1 give identical patterns of staining on Western blots of rat (lanes A. C, E, G, I) or cat (lanes B, D, F, H) cortical membranes. Each antibody recognizes bands at 140 kDa in rat and cat. Neither VCI.1 or HNK-1 recognizes a band of 95,000-105 k D A in the rat. Lanes E and F are stained for L-MAG, demonstrating that although this adhesion molecule is present in both species, VCI.1 and HNK-1 only recognize it in the eat, Lanes G and H were stained with O B l l , a mouse monoclonal against N-CAM and demonstrate that two of the higher molecular weight bands stained by VCI.1 and HNK-1 in both rat and cat correspond to N-CAM 145,17o. Lane 1 is stained with monoclonal antibody RP-I, specific for opsin and thus demonstrates bands which are stained non-specifically by the secondary antibodies.

125 with each of these markers is shown in Fig. 9. As described above, VCI.1 outlines subsets of neurons in the gray matter (Fig. 9A). Similar to the findings presented for retina, some N-CAM immunoreaetivity is associated with neuropil in cerebral cortex. In contrast to the retina however, only a few neurons exhibit strong surface staining with the anti-NCAM antibody CB7.5 (Fig. 9B) and there was a striking increase in the number of neurons outlined by VCI.1 as compared with CB7.5. Furthermore, pericellular N-CAM immunoreactivity was observed only on medium to small diameter neurons. VCI.1 immunoreactivity was present on neurons with medium and large diameter perikarya. Together, these observations suggest that only a minority of the VCI.1 pericellular staining in cerebral cortex can be due to the VCI.1 epitope on N-CAM. We also made comparisons of gray matter staining with VCI.1 and the anti-L-MAG antiserum. These data indicated that only small oligodendroglia in gray and white matter reacted with the L - M A G antiserum (Fig. 9C). Since this rabbit antiserum was generated against the last 20 amino acids of L-MAG, the antiserum should detect L - M A G regardless of whether the VCI.1 carbohydrate epitope is expressed or not. Since only a few cells in gray matter were stained and these were oligo-

dendroglia, it is unlikely that forms of L - M A G carrying the VCI.1 epitope are expressed by cerebral cortical neurons. DISCUSSION The results presented in this paper indicate that mAb VCI.1 recognizes an epitope that is part of an N-linked carbohydrate with properties similar, or identical, to the HNK-1 epitope. The conclusion that the VCI.1 and HNK-1 epitopes are similar or overlapping is based upon (1) the reactivity of each monoclonal antibody with the same set of polypeptides on Western blots, including N-CAM and MAG; (2) similarities in the immunocytochemical staining patterns in cerebral cortex; and (3) upon blocking experiments in which either antibody inhibited the binding of the other. Although it would be ideal to show that these two epitopes co-localize on the same neurons, double-labelling studies with VCI.1 and HNK-1 were not possible due to the fact that both are mouse IgM's. In closely spaced sections from retina or cerebral cortex, however, each antibody gave nearly identical staining patterns. The HNK-1 epitope has been shown previously to depend upon a sulphated glucuronic acid residue that is

~S

NL PL NL

CL

Fig. 8. Immunocytochemical comparisons of the distribution of the VCI.1 carbohydrate epitope with N-CAM polypeptides in cryostat sections of rat retina. The VCI.1 carbohydrate epitope is expressed on surface molecules associated with horizontal and amacrine cells. A few ganglion cells are also immunoreactive (A). In contrast, most cell types and their processes exhibit N-CAM immunoreactivity (B). These findings suggest that a subset of N-CAM-immunoreactive molecules in retina carry the VCI.1 epitope. Both the VCI.1 epitope and N-CAM polypeptides show complete overlap in their distributions in the fiber layers of the retina (OPL, IPL). A phase-contrast image of rat retina is shown in C. Bar = 50 gin.

126

Fig. 9. Comparisons of patterns of immunoperoxidase staining in vibratome sections from rat cerebral cortex with VCI.1, anti-N-CAM antibodies, and anti-L-MAG antiserum. The resulting comparisons indicate that VCI.1 cell surface staining in cerebral cortex is probably due to the expression of this epitope on several different molecules, including N-CAM but probably not L-MAG. Upper row (A, B, C); sections from rat cerebral cortical gray matter. Lower row (D, E, F) sections from rat cerebral cortical white matter. VCI.1 reacts with subsets of neurons in gray matter (A) and also stains oligodendroglia and myelinated axons in the white matter (D). Antibodies against N-CAM give staining of cell bodies and neuropil in cortical gray matter (B) and axons in the white matter (E). L-MAG antiserum stains oligodendroglia in gray (C) and white (F) matter. Bar = 50/~m. part of an N-linked carbohydrate group attached to N - C A M , M A G , L1 and cytotactin. Based on identification of the VC1.1-reactive polypeptides on Western blots, we have now shown that the VCI.1 epitope is attached to N - C A M and M A G . Because of the extensive similarities between the V C I . 1 and HNK-1 epitopes, we postulate that the VC1.1 epitope is also attached to L1 and cytotactin; however, specific antisera against the polypeptide chains of these molecules would be necessary to determine this. Multiple chondroitin sulfate proteoglycans with HNK-1 antigenic determinants have also been identified in the brain 18'28'7°. It is not yet clear whether these are different from the forms of Cat-301 chondroitin sulfate proteoglycan which bears the HNK-1/VCI.1 epitope 7°. Although it was previously shown that about 15-20% of mouse brain N - C A M molecules express the HNK-1 epitope 37, our results indicate that the HNK-1/VCI.1 epitope is expressed by relatively few neurons. It seems likely that a cell which expresses the enzymatic machin-

ery to synthesize the HNK-1/VCI.1 epitope will attach this carbohydrate to whichever array of cell adhesion molecules it is expressing. For example, in cat white matter many oligodendroglia express M A G . Those cells that also synthesize the VCI.1/HNK-1 epitope will attach it to the M A G polypeptide chain. In gray matter, cells synthesizing the VCI.1/HNK-1 carbohydrate epitope probably attach it to N - C A M , L1, the 680 kDa chondroitin sulphate proteoglycan recognized by Cat-301, and possibly other molecules. Most of the molecules which have previously been identified as carrying the HNK-1 epitope are cell adhesion molecules of the immunoglobulin supergene family which includes N - C A M , L1, M A G and cytotactin. It has not been established whether the Cat-301 proteoglycan bears a homology to these other molecules, but there are a number of lines of evidence showing that this molecule is developmentally regulated in an activity-dependent fashion 21'25"33"6~. A number of different monoclonal antibodies which react to an epitope similar or identical to the HNK-1

127 determinant have been described and, for some of these, there are published descriptions of their staining patterns in adult brain. HNK-1 (anti-Leu-7) was originally generated against a lymphoblastoma and found to cross-react with nervous tissue 1'41"52'56. The antibody NC-1, raised against quail embryo ciliary ganglion 66, was later shown to have identical reactivity with HNK-164. Similarly, mAb 4F4, raised against E15-E17 rat forebrain cell suspensions 5s and a panel of L2 mAbs, raised against a glycoprotein fraction of mouse brain 37 were shown to recognize the HNK-1 determinant 51. Several other antibodies identify subsets of neurons with similar distributions to the VCl.1/HNK-l-positive subsets and these include antibody 6A2, generated against heads from Drosophila melanogaster17"24 and Tor 23, generated against Torpedo electric organ synaptosomes 39'6°. It remains to be shown whether these additional antibodies also recognize the same sulfate and glucuronic acid-containing epitope identified by HNK-1. Although the HNK-1 epitope appears to be expressed in many different tissues and in diverse species (from insects to human), the central nervous system exhibits striking regional variations in the number of cells expressing this epitope. The HNK-1/VCl.l-immunoreactive subset in cerebral cortex consists almost entirely of GABAergic neurons 47'68. Depending on the species and cortical area examined, the proportion of G A B A cells expressing this epitope ranges from approximately 15% to 35% 35'45'47. The HNK-1NCl.l-immunoreactive subset in cortex is also stained by the lectin VVA, which binds to O-linked carbohydrate groups containing N-acetylgalactosamine 44'46. Although detailed morphological studies of the VCl.l-positive cells have not yet been done, studies of the VVA-positive neurons revealed that the two major cell types labelled in layer 4 of cat area 17 were the basket and neurogliaform cell types 46. About 90% of the HNK-1/VCl.l-positive neurons are VVA-positive 35, thus it is likely that most cortical neurons expressing the HNK-1/VCI.1 carbohydrate epitope are types of cortical basket and neurogliaform cells. Our results indicate that the expression of the HNK1/VCI.1 epitope on N-CAM alone can only account for a minority of the pericellular staining in cerebral cortex. Comparisons between L-MAG and VCI.1 immunoreactivity indicate that little, if any, of the HNK-1NCI.1 pericellular staining of neurons in cortex arises as a consequence of MAG carrying the VCI.1/HNK-1 carbohydrate. Thus, we think it likely that a majority of the neuronal staining in cerebral cortex is due to the expression of HNK-1/VCI.1 carbohydrate epitope on Cat-301 surface-associated chondroitin sulfate proteoglycan and on a larger keratin sulfate proteoglycan which also car-

ries this epitope 7°. Biochemical and immunocytochemical experiments indicate that there are two forms of the Cat-301 proteoglycan: one with and the other without the HNK-1NCI.1 carbohydrate 7°. These results suggest that in cerebral cortex, neuronal subsets could express both the VCl.l-positive and the VCl.l-negative forms of this proteoglycan or different neurons could express one form but not the other. In brain regions other than the cerebral cortex, the pericellular staining produced by VCI.1 may be due to the expression of this carbohydrate epitope on N-CAM, MAG or other adhesion molecules such as cytotactin. This is supported by our biochemical findings in retina, where it was demonstrated that VCI.1 reacted with N-CAM(140,170). Our immunocytochemical studies also support this conclusion since it was shown that VCI.1 immunoreactivity was strong on the same retinal cell types (amacrines and horizontal cells) expressing the VCI.1/HNK-1 carbohydrate epitope. Since the Cat-301 proteoglycan is not expressed in retina, the pericellular staining given by VCI.1 in the retina is not due to the expression of the VCI.1/HNK-1 carbohydrate on the Cat-301 proteoglycan. Studies now underway are examining the possibility of whether there are other high molecular species of proteoglycans with the VCI.1/HNK-1 carbohydrate epitope that are expressed in retina or retinal cell lines. In vivo and in vitro studies have demonstrated that the HNK-1 antibody can block axonal growth and modify neuronal migration patterns 4'16"23. The HNK-1 carbohydrate epitope has been shown to act as a ligand in cell-cell adhesion and to promote process outgrowth 3s" 42. It has also been demonstrated that N-CAM function depends upon having the correct glycosylation pattern 32. These studies suggest that it is possible to modify the selectivity of cell-cell interactions by expression of the HNK-1NCI.1 carbohydrate moiety on cell surface molecules. During early embryogenesis, neurons may express the HNK-1 epitope on N-CAM and related adhesion molecules to facilitate migration or axonal outgrowth. Later, during postnatal maturation, the expression of this epitope on extracellular matrix molecules may serve to anchor or stabilize synapses or other types of permanent cell-ceU contacts. Further studies of the mechanisms by which the VCI.1 and other carbohydrate antigens are regulated should help define their role in the developing and mature brain. Furthermore, by defining the interactions between cells expressing the VCI.1/HNK-1 epitope at different stages of development, it will be possible to design experiments to test the more general hypothesis that cell adhesion molecules can play a significant role in the establishment or maintenance of neural circuits.

128 Acknowledgements. We thank Laura Milroy for technial assistance and Robert Brown for photography. This work was supported by National Institutes of Health Grants EY08914, EY05206, EY07119, and EY00785, the Klingenstein Foundation, and Re-

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