Journal of Cell Science, Supplement 17,109-117 (1993) Printed in Great Britain © The Company o f Biologists Limited 1993
109
Ankyrin-binding activity of nervous system cell adhesion molecules expressed in adult brain Jonathan Q. Davis and Vann Bennett Howard Hughes Medical Institute and Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA
SUMMARY
A family of ankyrin-binding glycoproteins have been identified in adult rat brain that include alternatively spliced products of the same pre-mRNA. A composite sequence of ankyrin-binding glycoprotein (ABGP) shares 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides and ankyrin associate as pure proteins in a 1:1 molar stoichiometry at a site located in the predicted cytoplasmic domain. ABGP polypep tides are expressed late in postnatal development to approximately the same levels as ankyrin, and comprise a significant fraction of brain membrane proteins. Immunofluorescence studies have shown that ABGP polypeptides are co-localized with ankyrinB. Major dif ferences in developmental expression have been
reported for neurofascin in embryos compared with the late postnatal expression of ABGP, suggesting that ABGP and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. Predicted cytoplasmic domains of rat ABGP and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, including LI, Nr-CAM and Ng-CAM of vertebrates, and neuroglian of Drosophila. A hypothesis to be evaluated is that ankyrinbinding activity is shared by all of these proteins.
INTRODUCTION
cated in macromolecular recognition in a variety of pro teins (reviewed by Michaely and Bennett, 1992). Initial characterization of membrane-binding sites for ankyrin in mammalian brain revealed a class of integral membrane proteins capable of high affinity association with the membrane-binding domain of ankyrinB, and these pro teins are present in amounts comparable to ankyrin (Davis and Bennett, 1986). The sites detetected in these binding assays are likely to be distinct from membrane proteins cur rently known to associate with ankyrin, such as the volt age-dependent sodium channel (Srinivasan et al., 1988; Kordeli et al., 1990; Srinivasan et al., 1992) and the Na/K ATPase (Nelson and Veshnock, 1987; Koob et al., 1987; Morrow et al., 1989), based on their abundance and resis tance to high pH.
This article will focus on recent biochemical evidence for a direct association between a family of membrane-span ning cell adhesion molecules and ankyrins in the adult ner vous system of mammals. Ankyrins are a family of struc tural proteins strategically located on the cytoplasmic surface of the plasma membrane with recognition sites for both membrane-spanning integral membrane proteins and spectrin (reviewed by Bennett, 1990; 1992). Ankyrins are ancient components of the nervous system that are expressed in Caenorhabditis elegans (Otsuka et al., 1991), and comprise 0.5-1% of the membrane protein in adult ver tebrate brain (Bennett, 1979; Davis and Bennett, 1984). Multiple isoforms of ankyrin are expressed in brain, with diversity due to distinct genes as well as to alternative splic ing of mRNAs: 220 kDa ankyrinB, which is generally dis tributed in neurons and glial cells of adult brain; 440 kDa ankyrinB, an alternatively spliced form highly expressed in neonatal development and located in neuronal processes; 215 kDa ankyrinR, which is confined to cell bodies and den drites of a subset of neurons, and ankyrinnode, localized at axonal initial segments and nodes of Ranvier. A common feature of the ankyrin family is a membrane-binding domain comprising 24 repeats of a 33-residue motif, impli-
Key words: cell adhesion molecule, ankyrin, spectrin, membranecytoskeletal interaction
IDENTIFICATION OF ANKYRIN-BINDING GLYCOPROTEINS (ABGPs) IN ADULT BRAIN A family of membrane glycoproteins was identified in adult brain and found to associate with 33-residue ankyrin repeats, using the following strategy (Davis et al., 1993). The membrane-binding domain of ankyrinB (residues 190 947, comprising repeats 5-24 plus a portion of the spectrin-
110
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Fig. 1. Identification in adult rat brain of a 186 kDa membrane 2 3 4 5 6 2 3 4 5 6 2 3 4 5 6 glycoprotein with ankyrin-binding activity. Triton-X-100-solubilized m embrane proteins from 10 g o f kDa adult rat brain were applied at 4°C to an ankyrins (residues 190-947)260. affinity column, and the column 225 washed with ten column volumes of 21 5 ' 186 —1 8 6 0.1 M KC1 dissolved in column buffer (0.5 % Triton X-100, 0.01 % phosphatidylcholine, 10 mM Hepes, 2 mM dithiothreitol, 1 mM sodium azide, pH 7.4). The adsorbed proteins were eluted with 1 M NaBr 72in column buffer and applied to a wheat germ agglutinin-agarose affinity column, which was washed with ten volumes o f loading buffer, and adsorbed proteins eluted with 0.2 M A?-acetyl glucosamine. Peak fractions were analyzed by SDSelectrophoresis, and polypeptides detected either by silver stain (A) or by blot-binding with 10 nM 125I-labeled ankyrinB (residues 190-947) following electrophoretic transfer of polypeptides to nitrocellulose paper (B). A control blot was performed with a 20-fold excess o f unlabeled ankyrin (C). Lane 1, Coomassie Blue-stained gel o f ankyrine (residues 190-947) expressed in E. coli (Davis et al., 1991); lane 2, total rat brain membranes; lane 3, Triton X-100 extract of brain membranes; lane 4, Triton X-100 extract follow ing passage over the ankyrin-affinity column; lane 5, polypeptides eluted from the ankyrin-affinity column; lane 6, polypeptides eluted from the wheat germ affinity column. (From Davis et a l, 1993.)
binding dom ain) w as expressed in bacteria (Davis et al., 1991) and used to prepare an affinity adsorbent. Proteins from detergent extracts o f brain m em branes were adsorbed to the ankyrin-affinity colum n, resulting in selection o f at least ten polypeptides (Fig. 1, lane 5). G lycoproteins in this group o f ankyrin-binding polypeptides w ere isolated using a w heat germ agglutinin-affinity colum n (Fig. 1, lane 6). Finally, a 186 kD a polypeptide capable o f direct associa tion w ith ankyrin was identified in the eluate from the w heat germ agglutinin-affinity colum n by blot-binding w ith 125Ilabeled ankyrin (Fig. 1, right panels). D irect evidence that the 186 kD a polypeptide is glycosylated w as provided by its reduction to a m olecular m ass o f 180 kD a follow ing digestion w ith endoglycosidase H and to 165 kD a by diges tion with endoglycosidase F (D avis et al., 1993). A ffinity-purified antibody against the 186 kD a polypep tide also cross-reacted with tw o sequence-related polypep tides o f m olecular m asses 155 and 140 kD a (Fig. 2). The 155 and 140 kD a polypeptides were isolated and found to have identical N -term inal sequences to the 186 kD a polypeptide, except for a deletion o f six residues (Fig. 2). These polypeptides w ere alternatively spliced products o f the same pre-m RN A based on alternate sequences observed in cD N A clones (see below ), and exhibited distinct patterns o f expression in regions o f the brain. W ithin the forebrain, 186 kD a and 155 kD a A BG P exhibited a striking segrega tion betw een w hite m atter and grey m atter, with 186 kD a A B G P present alm ost exclusively in grey m atter and the 155 kD a polypeptide restricted to w hite matter. The pres ence o f 155 kD a ABGP-1 in w hite m atter, and its location in spinal cord and peripheral nerve, suggested that this polypeptide is a com ponent o f m yelinated axon tracts. A ssociation o f ankyrin and A B G P polypeptides was
m easured in quantitative assays using native proteins im m o bilized through their carbohydrate residues by adsorption to Concanavalin A -coated beads (Fig. 3). T he ankyrin dom ain (residues 190-947) bound to purified A B G P 186 w ith a K d o f 65 nM and w ith a stoichiom etry close to 1:1. A ssociation o f ankyrin dom ain w as also m easured w ith iso lated A B G P155/140 that as separated from A BG P186 (Fig. 3). A 270 kD a polypeptide identified as the IP3 receptor (data not show n) w as also present in this preparation (Fig. 2). H ow ever, the 270 kD a polypeptide did n ot interfere with the assay, since it was not adsorbed to the beads under these experim ental conditions. T he affinity o f A BG P 155/140 for ankyrin was reduced 10-fold com pared to A B G P186, with a K d o f 600 nM , although the stoichiom etry was approxi m ately 1:1. T he values for affinity and 1:1 m olar stoi chiom etry are consistent w ith a selective, site-specific in ter action betw een ankyrin and A B G P186, 155 and 140. The differences in affinity betw een A B G P186 and 155/140, com bined with different regional distributions, suggests that these polypeptides perform related but distinct functions.
PRIMARY STRUCTURE AND DOMAIN ORGANIZATION OF ANKYRIN-BINDING GLYCOPROTEINS A com posite sequence o f A B G P encoding 1347 residues was deduced from analysis o f m ultiple overlapping clones, including clones w ith internal deletions, presum ably due to alternative splicing o f pre-m R N A (Fig. 4). T he predicted sequence of the ankyrin-binding glycoproteins contains five types o f dom ains: (1) six Ig dom ains o f the C2 type (residues 25-611); (2) four fibronectin type 3 dom ains
Ankyrin-binding proteins
C. blue 1 2
3
4
B
Immunoblot antrABGP Ig
C. blue 1 2
5
1 2
3 4
3
111
Im m unoblot antrABG P Ig 1 2 3
5
kDa
kDa
186
186 — 155—
155.
k—
— 186 — 155
140 '
Ld -
>*•»*
N-Terminal Sequences ABGP186
I-E-I-P-M-D- P-S-I-Q-N-E L-T-Q-P - - -
ABGP155
I-E-I-P-M-D
L-T-Q-P-P-T-I
ABGP140
- E-I-P-M-D - - - - - -
L-T-Q-P-P-T-I
186-155 -140
Fig. 2. The 186 kDa ankyrin-binding glycoprotein is a mem ber o f a family of sequence-related polypeptides with distinct regional expression. Immunoblots of various regions of rat brain were prepared using affinity-purified Ig against the 186 kDa ankyrin-binding glycoprotein. Coomassie Blue-stained gels are also shown. (A) lane 1, forebrain; lane 2, cerebellum; lane 3, brain stem; lane 4, spinal cord; lane 5, sciatic nerve. In a separate experiment (B), bovine forebrain was dissected into white matter and grey matter and analyzed with the same antibody: lane 1, total forebrain; lane 2, grey matter; lane 3, white matter. (C) The 186 kDa (1) and cross-reacting 155 and 140 kDa polypeptides (2) were isolated from detergent extracts o f cerebellar membranes, and their Nterminal amino acid sequences determined. The 155 and 140 kDa polypeptides were isolated by adsorption to an ankyrin-agarose affinity column, followed by a wheat germ agglutinin-agarose affinity column to remove the 186 kDa polypeptide, and finally by adsorption to a Concanavalin Aagarose affinity column and fractionation on a Mono-S cation exchange column. (From Davis et al., 1993.)
(residues 626-1029); (3) a 173-residue dom ain with a high percentage o f proline and threonine (residues 1030-1203); (4) a hydrophobic stretch o f 23 am ino acids (residues 1216 1238) representing a putative m em brane-spanning segm ent; (5) a putative cytoplasm ic dom ain o f 109 am ino acids (residues 1239-1347). T he Ig dom ains are in the C2 cate gory, based on spacing o f conserved cysteines 48-55 am ino acids apart and distinctive residues near the C -term inal cys
teines (W illiam s and Barclay, 1988). The fibronectin type 3 repeats have characteristic conserved tryptophans spaced about 50 am ino acids from tyrosines (Patthy, 1990), and the third repeat contains a RG D motif. The 173 residues from 1030-1203 have an unusual am ino acid com position with a high proportion o f proline (9% ) and threonine (27% ), as w ell as alanine (9% ) and valine (8%). D om ains with a sim ilar com position are present in other m em brane glyco-
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J. Q. Davis and V. Bennett
Fig. 3. M easurement of association of 125I-labeled ankyrins (brain ankyrin) (residues 190-947) with ankyrin-binding glycoproteins: 186 kDa polypeptide (A) and 155/140 kDa polypeptides (B). Ankyrin-binding glycoproteins adsorbed to the surface of Concanavalin A-coated beads were incubated for 2 hours on ice with increasing concentrations of 125I-labeled ankyrin. Samples were layered onto 200 JJ.1 o f 10% glycerol dissolved in assay buffer and beads with adsorbed proteins pelleted by centrifugation at 5,000 g for 15 minutes. The tubes were frozen and tips Brain Ankyrin, nM Brain Ankyrin, nM containing the beads cut off and assayed for 125I in a gamma counter. Values for nonspecific binding were determined using a 50-fold excess o f unlabeled ankyrin over radiolabeled ankyrin (10 nM), and were subtracted. Data points are the mean o f duplicate determinations, and are expressed as moles of ankyrin bound per mole o f ankyrin-binding glycoprotein (assuming a monomer), and by the method of Scatchard (insert). (From Davis et al., 1993.)
proteins including the LD L receptor (Y am am oto et al., 1984), platelet glycoprotein lb (Lopez et al., 1987), and NC A M (W alsh et al., 1989), and are sites o f O -glycosylation. F our candidate sites for alternative splicing of pre-m R N A can be deduced based on alternative sequences am ong the cD N A clones: one encoding six residues at the N term inus (residues 31-36), one encoding 15 residues betw een Ig and fibronectin type 3 dom ains (residues 611-625), and tw o sites in the proline/threonine dom ain, residues 1030-1035 and 1036-1203 (Fig. 8). The alternate six residues at the N ter m inus correspond to the additional am ino acids present in the 186 kD a polypeptide and are m issing from the 155 and 140 kD a form s (Fig. 4). T he 15 residues betw een Ig and fibronectin dom ains include two prolines and m ay be con figured as an unstructured loop that provides som e flexi bility betw een the dom ains. T he potential for m ultiple alter native sites raises the question o f w hat com bination of deletions/insertions are actually expressed as polypeptides. It also follow s that the com posite sequence does n o t nec essarily correspond to that o f any o f the m ajor polypep tides. Ig and fibronectin type 3 dom ains are independently folded, and m olecules with m ultiple dom ains o f this would be expected to have the configuration of a relatively rigid rod (H all and R utishauser, 1987; Becker et al., 1989; Staunton et al., 1990). The 186 kD a ankyrin-binding gly coprotein, visualized by electron m icroscopy follow ing rotary shadow ing w ith platinum and carbon, also has an elongated shape, 40-60 nm in length (Fig. 5). Som e im ages w ere tw ice this length, and could be due to head-head hom ophilic interactions. Rosettes o f 3-5 m olecules w ere also observed, as has been found for N -C A M , and inter preted as association o f these proteins through their hydrophobic dom ains (B ecker et al., 1989). The fact that the ankyrin-binding glycoprotein has the predicted config uration, in addition to correlation o f the protein-derived
sequence and deduced sequence provides com pelling ev i dence that the cD N A clones actually encode this protein. The ankyrin-binding site o f 186 kD a A B G P is located in the predicted cytoplasm ic dom ain, based on tw o observa tions. C leavage o f the C-term inal 21 kD a, w hich includes the cytoplasm ic dom ain, results in loss o f binding of ankyrine (Fig. 6).
ABGPS ARE MEMBERS OF THE Ig SUPERFAMILY OF CELL ADHESION MOLECULES T he am ino acid sequence o f A B G P is closely related to p re viously identified nervous system cell adhesion m olecules o f the Ig super fam ily (Fig. 7). These proteins contain six Ig type C2 dom ains and four to five fibronectin type 3 dom ains, and highly conserved cytoplasm ic dom ains. T he highest degree o f sim ilarity, w ith 71% sequence identity, is w ith chicken neurofascin, w ith low er degrees o f identity w ith m ouse L I (36% ), chicken N r-C A M (47% ) and chicken N g-C A M (30% ) (Table 1). D rosophila neuroglian, a cell adhesion m olecule with sim ilarity to L I (B ieber et al., 1989), also shares extensive hom ology w ith the vertebrate proteins in the cytoplasm ic domain. T he p redicted cytoplasm ic dom ains are m ost conserved
Table 1. Comparison of amino acid sequence similarity of ABGP with vertebrate nervous system cell adhesion molecules Percentage identity to ABGP Domains Proteins Chicken neurofascin Chicken Nr-CAM M ouse LI Chicken Ng-CAM
Overall 71 47 36 30
Ig
FNIII
PAT
Cytoplasm ic
80 53 38 36
71 45 38 29
19
86 73 50 34
— — -
Ankyrin-binding proteins M AROOAPPWV H V A L I L F L L S LGGAIEIPMD IPSIONFtLTOP PTITKQSVKD
50
HIVDPRDNIL IECEAKGNPA PSFHWTRNSR FFNIAKDPRV SMRRRSGTLV
100
IDFRSGGRPE e y e g e y q C f a r n k f g t a l s n RIRLQVSKSP LWPKENLDPV
150
WQEGAPLTL o C n p p p g l p s PVIFWMSSSM EPITQDKRVS QGHNGDLYFS
200
NVMLQDMQTD y s C n a r f h f t HTIQQKNPFT LKVLTTRGVA ERTPSFMYPQ
250
GTSSSQMVLR g m d l l l e C ia SGVPTPDIAW YKKGGDLPSD KAKFENFNKA
300
LRITHVSEED s g e y f C l a s n KMGSIRHTIS VRVKAAPYWL DEPKNLILAP
350
GEDGRLVCRA NGNPKPTVQW LVNGDPLQSA PPNPNREVAG DTIIFRDTQI
400
ss ra vyqCet
SNEHGYLLAN AFVSVLDVPP RMLSPRNQLI RVILYHRTRL
450
dC p f f g s p i p
TLRWFKNGQG SNLDGGNYHV YQHGSLEIKM IRKEDQGIYT
500
CVATNILGKA e n q v r l e v k d PTRIYRMPED QVAKRGTTVQ LECRVKHDPS
550
LKLTVSWLKD DEPLYIGNRM KKEDDSLTIF GVAERDQGSY TCMASTELDQ
600
DLAKAYLTVL Ia d o a t p t n r l AALPKGRPDR pr d l e Jl t d l a e r s v r l t W ip
650
GDDNNSPITD YWQFEEDQF QPGVWHDHSK FPGSVNSAVL h l s p y v n Y q f
700
RVIAVNEVGS SHPSLPSERY RTSGAPPESN PSDVKGEGTR k n n m e i t W tp
750
MHATSAFGPN LRYIVKWRRR ETRETWN^VT VWGSRYWGQ t p v y v p Y e i r
800
VQAENDFGKG PEPETVIGYS GEDYPRAAPT EVKIRVLfiST a i s l q W n r v y
850
PDTVQGQLRE YRAYYWRESS LLKNLWVSQK RQQASFPGDR PRGWGRLFP
900
ysnY k l e m w
VNGRGDGPRS ETKEFTTPEG VPSAPRRFRV RQPNLETINL
950
ewdhpehpng
ILIGYTLRYV PFfiGTKLGKQ MVENFSPfiQT KFSVQRADPV 1000
srY r f sl sar
TQVGSGEAAT EESPTPPNEA TPTAAPPTLP PTTVGTTGLV 1050
SSTDATALAA TSEATTVPII PTWPTTVAT TIATTTTTTA
AATTTTTTES
1100
PPTTTTGTKI HETAPDEQSI WfiVTVLPNSK WANITWKHNF R PGTDFWEY 1150 IDSJJHTKKTV PVKAQAQPIQ LTDLFPGMTY TLRVYSRDNE GISSTVITFM 1200 TS1 AYTNHQT DTATO GWFTG L M C A IA L L V L I L L I V C F I K R SRGGKYPVRE 1250 KKDVPLGPED PKEEDGSFDY ¿DEDNKPLQG SQTSLDG1IK QQESDDSLVD 1300 YGEGGEGQFN EDGSFIGQYT VRKDKEETEG NESSEATSPV NAIYSLA
1347
betw een ankyrin-binding glycoproteins, neurofascin, L I, N r-C A M and N g-C A M , w ith som e regions th a t are id en tical am ong all five o f these proteins (Table 1, Fig. 8). In addition to sequence sim ilarity, cytoplasm ic dom ains o f chicken neurofascin (V olkm er et al., 1992) ra t and hum an L I (M iura et al., 1991; K obayashi et al., 1991; R eid and
113
Fig. 4. Primary structure o f ankyrin-binding glycoproteins deduced from analysis o f cDNAs isolated from rat brain. An initial cDNA clone was isolated from a lambda g tl 1 expression library prepared from rat brain using poly(A) and random hexamers as primers (Clonetech), using affinity-purified polyclonal antibody against the 186 kDa ankyrin-binding glycoprotein. cDNAs encoding the complete polypeptide were isolated using the first clone and subsequent clones as probes. A stop codon was present in three independent clones. Portions of sequence corresponding to nucleotides 1130-1142, 2870-2914, 4131-4145 and 4146-4652 were deleted in some of the clones and represent candidates for alternative splicing o f pre-mRNA. Hydrophobic portions of the sequence representing a predicted signal peptide (residues 1-24) and membrane-spanning region (residues 1216-1238) are underlined and in italics. Alternate sequences are boxed: residues 31-36; 611-625 and 1001-1203. Conserved cysteines o f the six Ig-like repeats, and tryptophans and tyrosines of the four fibronectin type 3 repeats are enlarged in bold. Potential /V-glycosylation sites in the predicted extracellular domain and phosphorylation sites in the cytoplasmic domain are bold and underlined, and a RGD m otif (residues 914-916) is in bold. Portions of the deduced sequence confirmed by analysis of N-terminal sequences of polypeptides isolated by Mono Q chromatography of V8 proteolytic digests are underlined. (From Davis et al., 1993.)
H em perly, 1992) an d chicken N r-C A M (G rum et et al., 1991; K ayyem et al., 1992) also share alternative splicing involving a tetrapeptide, RSLE. T he cytoplasm ic dom ain o f rat A B G P con tain s the ankyrin-binding site (Fig. 6; M ichaely and B ennett, 1993), suggesting the possibility that other m em bers o f this group also interact w ith either the sam e ankyrin or w ith a related m em ber o f the ankyrin fam ily. A 440 kD a alternatively spliced form o f an k y rin s is expressed prior to birth (K unim oto et al., 1991), and it also is possible that o th er form s o f ankyrin are present in early developm ent. N eurofascin w as initially characterized as a m em brane protein, hypothesized to play a role in extension o f neurites and stabilization o f bundles o f axons during devel opm ent o f the nervous system (R athjen et al., 1987; V olk m er et al., 1992). A significant discovery from this study is that A BG Ps, w hich are closely related to neurofascin, are m ost prom inent in adult brain, with a 10-20-fold increased expression follow ing the m ajor phases o f neu ronal m igration (see below ; Fig. 9). It is not certain at this
Fig. 5. Visualization of the 186 kDa ankyrin-binding •V-\ glycoprotein by electron vS \'V microscopy. The 186 kDa :".y ' v :r l: ankyrin-binding glycoprotein (300 |J.g/ml) in a buffer containing 0.1% Triton X-100, 0.01% phosphatidylcholine, 10 mM Hepes, pH 7.4, was diluted twenty-fold with 30% glycerol, 0.1 M ammonium formate, pH J 7.0, and immediately sprayed onto freshly clcavcd mica. The samples were then rotary shadowed at an angle of 6 degrees with platinum followed by carbon, and the replicas visualized by electron microscopy. (A) Single molecules (arrowheads); (B) head-head pairs; (C) rosettes of 3-5 molecules. Bar, 100 nm. (From Davis et al., 1993.)
: >.> -
B
C
114
J. Q. Davis and V. Bennett C. blue 1
1251-Labeled Ankyrin Blot *
1 2
ABGP/NEUROFASCIN
+ unlabeled protein 1 2
1 2
kD a I t/N gC A fi/N rC A M /N E U R O G LI AN
186 «165
Fig. 7. Schematic model for the domain organization of ankyrinbinding glycoproteins, neurofascin and related nervous system cell adhesion molecules. PM, plasma membrane; Ig, type C2 domains; FNIII, fibronectin type 3 domains (from Davis et al., 1993).
N-Terminal Sequence ABGPl86 I-E-I-P-M-D-P-S-I-Q-N-E-L-T-Q-PV8 165 I-E-I-P-M-D-P-S-I-Q-N-E-L-T-Q-PFig. 6. Localization of the ankyrin-binding site to the C-terminal 21 kDa of the ABGP186 polypeptide. ABGP186 (0.1 mg/ml in a buffer containing 50 mM NaCl. 10 mM Hepes, pH 7.4, 0.5 % Triton X-100, 0.01% phosphatidylcholine. 0.5 mM dithiothreitol, 1 mM sodium azide) was digested with Staphylococcal V8 protease (20 pg/m l for 60 minutes at 24°C), and the products resolved by chromatography on a Mono-Q anion exchange column. A 165 kDa polypeptide was resolved, which retained the N terminus based on amino acid sequence, and was missing the Cterminal 21 kDa. Binding o f l25I-labeled ankyrinB (residues 190 947) to 186 kD a and 165 kDa polypeptides was determined following electrophoretic transfer of polypeptides to nitrocellulose paper. (A) Coomassie Blue-stained polypeptides; (B,C) autoradiograms o f nitrocellulose paper incubated with 36 nM 125Ilabeled ankyrine (residues 190-947), either alone or with a 75-fold excess unlabeled ankyrin to displace nonspecific binding. (From Davis et al., 1993.)
tim e w hether the A B G P polypeptides are encoded by the identical gene to chicken neurofascin. T he sim ilarity in am ino acid sequence and conservation o f sites o f altern a
ABGP composite (1239-1347) Neurofascin (1160-1272) Nr-CAM (1155-1268) LI (1147-1260) Ng-CAM (1153-1266) Neuroglian (1155-1239) Consensus ABGP composite (1239-1347) Neurofascin (1160-1272) Nr-CAM (1155-1268) LI (1147-1260) Ng-CAM (1153-1266) Neuroglian (1155-1239) Consensus
tive splicing strongly support the interpretation that A B G Ps and neurofascin genes share a com m on ancestor. H ow ever, the m ajor differences in developm ental expression and divergence in som e areas o f the sequence (Fig. 9) suggest that A B G Ps and neurofascin represent products o f gene duplication events that have subsequently evolved in p ar allel w ith distinct roles in developm ent. Im portant issues for the future w ill be to determ ine w hether m ultiple copies o f the A BG P and neurofascin genes indeed exist, or if the sam e gene plays roles both in fetal developm ent and in adult brain.
ANKYRIN AND ABGPs ARE CO-LOCALIZED AND CO-EXPRESSED DURING DEVELOPMENT T he question o f w hether the association o f ankyrin and ankyrin-binding glycoproteins actually occurs in vivo will require experim ents beyond the scope o f this initial, b io chem ically oriented work. H ow ever, circum stantial evi dence consistent w ith interaction of these proteins in brain tissue is that A B G P polypeptides are expressed at approx im ately the sam e levels as ankyrin, co-expressed w ith the adult form o f ankyrinB late in postnatal developm ent, and are co-localized w ith ankyrinB by im m unofluorescence. A nkyrin-binding glycoproteins are expressed in the fo re brain at relatively low levels until after birth, w hen the level
L -Q i
TS :D j T i k -
1290 1215
L-P 1rs D j TIKLKK' TP ;d *t v k GS i?s N j D I K A SGSC GS A : S'G : SPGRG \GR< #PG ”
1203 1212 1206
. . .K-
60
A IY S LA DDSLVDYGEGfl QFNEDGSFIG' GC i TVRKDKEETEGNESSEATSP DDSLVDYGEC :QFNEDGSFIG' 3C i TVKKDKEETEGNESSEATSPV NAIYSLA DDSLVDYGEGf. ■q f n e d g s f i g c £ SGKKEKEPAEGNESSEAPSP A-1NSFV 1SDDSLADYGGS j QFNEDGSFIG' 3C t SGKKEKEAAGGNDSSGATSP I MlP W A L E EDSLAGYGGS : d * QtiFNEDGSFIG' GC i RG-----PGAGPGSSGPASP( ,G PPLDQ TDSMAEYGDG IG *LGLGMNEDGSFIGC ..K .K E ...GN.SS.A.SP L U . ..L.
1211
1347 1272 1268 1260 1266 1239 119
Fig. 8. Aligned sequences of putative cytoplasmic domains of rat ankyrin-binding glycoproteins, and related neural adhesion molecules. (From Davis et al., 1993.)
Ankyrin-binding proteins
Fig. 9. Ankyrin-binding glycoproteins and the adult form of ankyrinB are expressed late in post-natal development o f rat brain. Samples of forebrain and cerebellum obtained from rats of various ages were analysed by immunoblots using 125I-labeled Protein A and affinity-purified Ig against the 186 kDa ankyrin-binding glycoprotein and ankyrins, following resolution of polypeptides by SDS-electrophoresis and transfer to nitrocellulose paper. Amounts of immunoreactive 186, 155 and 140 kDa polypeptides were compared by densitometry o f autoradiograms, and these values were then normalized with respect to amount o f protein applied to the gel. Protein was estimated by elution of dye from Coomassie blue-stained gels with 25% pyridine followed by measurement of absorbance at 550 nm. The ratios of im munoreactivity/ protein are expressed as a percentage of the adult values. (From Davis et al., 1993.)
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after day 20, alm ost tw o w eeks after A B G P186 and A G BP155. Events know n to occur in the cerebellum late in postnatal developm ent follow ing the m ajor phases of neuronal m itosis and m igration w hich coincide with expression o f ankyrin-binding glycoproteins include synaptogenesis follow ed by ensheathing o f P urkinje cells by glial cells (A ltm an, 1972). Im m unofluorescence studies in frozen sections o f brain and peripheral nerve indicate that ankyrin and the ankyrinbinding polypeptides are both present in low am ounts in central tracts o f m yelinated axons, highly expressed in unm yelinated axons, and present at this level o f resolution in both neurons and glial cells. A nkyrin-binding glycopro tein im m unoreactivity is highly concentrated at nodes of R anvier in m yelinated axons o f peripheral nerve (Fig. 10), and this is also the site o f localization o f a form o f ankyrin (Kordeli and Bennett, 1991; Kordeli et al., 1990). T he stain ing at nodes o f R anvier is probably due to the 155 kDa polypeptide w hich is the only form detectable by im m unoblots in peripheral nerve (Fig. 2). Punctate staining could also be detected at higher m agnification in m yelinated regions o f the forebrain and cerebellum , w hich may repre sent nodes o f R anvier sm aller and m ore difficult to resolve by light m icroscopy than those in peripheral nerve (not shown). Since the 155 kD a form is the m ajor polypeptide detectable in dissected w hite m atter from forebrain (Fig. 2), the 155 kD a polypeptide is probably a com ponent o f nodes o f R anvier in the central nervous system as w ell as periph eral nerve.
CONCLUSION
Fig. 10. (a) Ankyrin-binding glycoproteins are highly concentrated at the node of Ranvier in peripheral nerve. Frozen sections (10 |im ) of rat sciatic nerve were examined by immunofluorescence using affinity-purified Ig against the ankyrinbinding glycoproteins, (b) Sciatic nerve with a DIC image showing a node of Ranvier (arrowhead) below the same section viewed by immunofluorescence. Bar, 20 |im. (From Davis et al., 1993.)
o f expression increases over 20-fold betw een day 10 and day 50 (Fig. 9, left panel). T he 220 kD a adult form of ankyrine exhibited a sim ilar tim e course, w ith expression accelerating after day 10 (Fig. 9). Sim ilar profiles w ere observed for forebrain and cerebellum (Fig. 10, right panel), although 186 kD a A BG P is expressed in the cerebellum (less than 25% o f adult values) before day 10. A BG P140 is selectively expressed in the cerebellum , and appears only
A ssociations of A BG Ps w ith ankyrin, o f ankyrin w ith spec trin (D avis and Bennett, 1984), and o f spectrin w ith actin (B ennett et al., 1982; G lenney et al., 1982) provide an exam ple o f a series o f protein-protein interactions extend ing from the extracellular space to the cytoplasm that have been defined in term s o f affinity and stoichiom etry with pure com ponents. T hese proteins are abundant in brain, with spectrin representing 3%, ankyrins 1% and A BG Ps 0.3-0.5% o f the total m em brane protein. This system o f pro teins thus represents a m ajor class o f m em brane-cytoskeletal linkages in the nervous system. Interactions am ong these proteins have the potential to be utilized in diverse contexts in cells, since A B G Ps and ankyrins are each capable o f m ultiple types o f interaction. A BG Ps include several polypeptides derived from alternative splicing o f prem RNA , and these form s exhibit differences in affinity for ankyrin, regional distribution and tim e o f expression during developm ent (Fig. 9). In addition to alternative splicing, another level o f diversity is provided by the subgroup o f cell adhesion m olecules with cytoplasm ic dom ains related to A BG P and neurofascin w hich are candidates for associ ation with ankyrins (Fig. 9). Interactions, in addition to hom ophilic associations, have been observed fo r other cell adhesion m olecules containing Ig and fibronectin type 3 repeats and may also be available to A BG Ps. Exam ples include binding to soluble extracellular m atrix m olecules (Reyes et al., 1990; K uhn et al., 1991; Rathjen et al., 1991), lateral association w ith other cell adhesion m olecules to
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J. Q. Davis and V. Bennett
form complexes capable of cell-cell interactions (Kadmon et al., 1990) and heterophilic interactions with integral membrane proteins on adjacent cells (Marlin and Springer, 1987). It is clear that a simple interaction between ankyrins and ABGPs and perhaps other cell adhesion molecules with conserved cytoplasmic domains can, in principle, be uti lized to establish a variety of membrane-cytoskeletal con nections in the nervous system. The node of Ranvier is one example of a specialized membrane domain containing isforms of ankyrin and ABGP polypeptides (Fig. 7) (Kordeli et al., 1990; Kordeli and Bennett, 1991). The high expression of rat ABGP in postnatal and adult brain strongly suggests that the function(s) of this protein includes roles in addition to activities attributed to cell adhesion molecules during prenatal development. Possibil ities include a role in developing and or maintaining spe cialized membrane domains such as unmyelinated axons and the node of Ranvier discussed above. Another conse quence of associations between these proteins may be struc tural support for the adult brain, which is dependent on cell cell contacts and lacks a collagen-based basement membrane utilized by cells in most tissues. Coupling between the elongated extracellular domain of ABGP polypeptides and the cytoskeleton would provide a mechan ical buffer, allowing distribution of shear stresses and defor mations throughout the tissue. Another possible role of ABGPs could be related to cell signaling, and result in sta bilizing neurons and glial cells that maintain appropriate cellular contacts. A prominent role of ABGPs in adult mammalian brain has potential clinical implications, since defects in these proteins may be compatible with survival but with impaired neurological or cognitive development. It is of interest in this regard that abnormal splicing of human LI premRNA, a related cell adhesion molecule, results in hydrocephalus and mental retardation (Rosenthal et al., 1992). Another potential clinical implication is that ABGPs may play a role as receptors for neurotropic viruses, as occurs for Rhinoviruses in lymphocytes (Staunton et al., 1990). Brenda Sampson is gratefully acknowledged for help in prepar ing the manuscript.
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