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Biochirnicael BiophjlsicaAvta, 1076O991) 156-160 © 1991 Hscvier Science Publishers B.V. (Biomedical Division)0167.4838/91/$03.50 ADONIS 01674838910U080A

BBAPRO 30279

BBA

Report

Proteolytic modification of neural cell adhesion molecule (NCAM) by the intracellular proteinase calpain A. Sheppard 1, j. W u 1, U. Rutishauser 2 and G. Lynch t Centerfor theNearobidogv of Learmng and Mern~.."yUnioersityofCuhfornia lroine, CA (U.S.A.) and 2 Department of GeneticsCase Weslern Reseme UniversityClevelan~ OH (U~S.A.) (Received 20 February 1990) (Revi~M manmcfipt received 7 June 1990)

Key words: Calpain; NCAM; Synaptie; Pia~tidty; Cyloskdemn

The neural cell adhesion molecule, NCAM, is concentrated in synaptic regions and thus may contribute to the formation and maintenance of connections between brain cells. We present evidence that the cytoplasmic domain el NCAM can be experimentally modified by the intracellular calclum-dependent proteinas¢, ealpain. This degradation could provide a mechanism for rapidly uncoupling and reorganizing synapfic contacts,

Considerable effort has gone into characterizing the molecules that mediate the connections of neurons and their processes to other cells and the extracellular matrix. The most abundant and intensively studied of these ligands is the neural cell adhesion mole~le, or NCAM [1]. From SDS-PAGE analysis, the NGAM found in adult rat brain has been shown to comprise 180, 140 and 120 kDa forms [2]. This variation in molecular weight represents differences in the cytoplasm.~c domain of each form. lmmuno-eytochcmical work indicates that NCAM-180 is highly concentrated in post-synaptic densities while the 140 form is present in both pre- and post-synaptic membranes [3]. We have found that a small subset of proteins arc lost from synaptic plasma membranes following incubation with calpain (Sheppard et aL, unpublished data), a eal,'ium-dependent proteinase isolated previously from ~ynaptic membranes [4]. These proteins are of relatively large molecular weight and nearly all are glycoconjugate in nature, as evidenced by their migration in SDS-PAGE and by their abil/ty to bind the lectin concanavalin A. The present study demonstrates that the transmembrahe forms of NCAM belong to this subset, and with the use of site-specific monoclonal antibodies, reveals

Cor~espcndenca (present address): A. ghcppaxd, Department o[ Cell Biology and Physiology, Washington University School or Medicine, St. Louis, MO 63110, U.S.A,

that the action of calpain is confined to the intracellular domain of NCAM. Spragne-Dawley rats were used in all experiments. Following decapitation, the brain was rapidly dissected in ice-cold buffer, and all subsequent steps were carried out at 4°C, unless otherwise stated. Fractionation of synaptic plasma membranes (SPMs) was carried oat by centrifugadon over Pcreoll gradients. Telencephatie tissue was homogenized in 10 volumes of 0.32 M sucrose, 5 mM Hepas, 0.1 mM EDTA (pH 7.4) (buffer A) and centrifuged at 1090 × g max for 10 rain. The supernatant was diluted with an equal volume of buffer A and centrifuged at 14600 × g max for 15 rain. The pellet was resuspended with 1 ml of buffer A and layered over a freshly prepared 4 x 2 ml gradient consisting o[ 255, 155, 105 and 35 (v/v) solutions of Percoll (Sigma), each adjusted to pH 7.5 just prior to use. The gradient was than centrifuged in a JA-20 fixed angle rotor (Beckman J2-21 centrifuge) at 45700 x g max for exactly 5 rain (not including acceleration and deceleration time). The interracial fractions designated 3 and 4 (the pellet representing fraction 5) were pooled and stored frozen until required. Prior to incubation with calpaia, the $PMs were [y~d:l by resmpension in 6 raM Tris HCi, 0.05 mM EDTA (pH 8.1) and incubation on ice for 60 mia, The suspension was centrifuged at 11400 × g max for | 0 nun ~ d the pellel was washed twice by resuspension and re-centrifuged in 25 mM Hepes, I00 mM NaCl, 0.05 mM EDTA (pH 7.4). For each mg of SPM material, 1 pg of affinity-purified calpain (isolated from rat erythrocyte cytosol) was

157 added to the incubation buffer, 5 mM Hopes, 1.5 mM CaC|2 (pH 6.8). Control incubation also included a final concentration of 1 mM lenpeptin (Boeha'ingerMannheim) and 200 I~M EGTA. Reaetiom wcle carried out at 37°C and terminated after 5 and 15 rain. Controls were incubated for 15 rain. Following incubation, reaction mixtures were centrifuged at 14600 y g max for 15 rain, the pellets were resuspeaded and protein determinations were carried oat prior to SDS-PAGE and Western blot analysis. Proteolysis of affinity-purlfled NCAM was carried oat under identical conditions. Samples of 30/~g total protdn were subject to SDSPAGE on 6-12~ gradient gels and either stained with Coomassie blue or ~ransferred to nitrocellulose using a BioRad transblot apparatus. The blots were incubated overnight at 4°C with rabbit-anti-mouse NCAM polyelonal antibody (30 v g / m l ) or with either of the site specific monoclonal antibodies (7.5/~g/ml) recognizing epitopes on chick NCAM. Incubation with an appropriate alkaline phosphatase linked secondary antibody (BioRad) was carried out for 1 h at room temperature. Visualization was achieved with the 5-bromo-4-

A

B

TABLE I

dpproximot¢ Io~ of different pdypeptide fvona of NCAM from ~t synapt/e plasma membranesJollowinR;realmcnt withcatpeinfor 15 rain, as dc~ermincdby densitomctri¢ scanning of We..ffemblots

NCAMsubtype 180 lz~0

~, loss 70 30

120

I]

chloro-3-indolyl phosphate/nitro blue tetrazofiuro substrate system (BioRad). The incubation of isolated rat synaptie plasma mere, branes with purified calpain leads to the loss of a small subset of the relatively high molecular weight proteins, nearly all of which are glycoennjugate in nature (Sheppard et al., unpublished data). In the present study, we have used a polyclonal antibody to demonstrate that the transmembrane forms of NCAM belong to this subset

C

F

'i°

A

180-

8

116.......

: . . : !;,i:;:,

C

84Fig. i. ~fect of ¢~pain on lh¢ association nf NCAM with ral syrmpfic pls.sma membranes {SPMs), (a) W~lern blot analysis of SPMs inc-bat~/n the p r c s ~ of I mM k~pcpfin for 15 n~n O.an¢A), ~ d in the al~'nc¢ of hlhibltor [or $ mln (hu~ B) and I$ m (kmc C). The dJffe~mt polypcp~id¢form5 of NCAM aye indicar.cd by zuwows.{b) The col~¢spondin8 dcn,sJtomctricscan of each lone,

158

(Fig. la). Densitometric scanning revealed that treatment with calpain results in a substantial reduction in the amount el' both the 180 and 140 kDa forms of NCAM associated with the synaptic plasma membrane (Fig. lb). Significantly, the amount of the 120 kD form of NCAM appeared to be unchanged (Table I). The calpains found in synaptie membrane preparations are identical to those isolated previously from non-neural tissue [4]. Most of the substrates of calpnin are components of the celhilar cytoskeleton and include the microtubule-associated protein 2 (MAP2) [5], ankyrin [6] and brain spectrin [7]. Since the 180 and 140 kDa forms of NCAM are transmembrane proteins, they have the potential to interact with the cytoskeleton. Indeed, evidence already exi.~ts that the lg0 kDa form is able to bind 'spectdn-like' components [8]. As greater than 95% of the membrane-associated spectrin is degraded in our experiments (Sheppard et al., unpublished data), it is possible that the action of ealpain leads indirectly to shedding of NCAM from the membrane, the differential loss of 180 and 140 kDa forms, reflecting the relative proportions of each linked to the submembraneous cytoskeleton. The 120 kDa form would presumably be unaffected as it is anchored to the membrane by a phosphatidylinositol linkage rather than by a membrane spanning segment [9,10]. Our studies suggest the alternative interpretation that NCAM is itself a substrate of calpain and that proteolyric modification leads to its loss from the membrane. We have tested this possibility by in vitro cleavage of purified NCAM antigen. The chick homologue of NCAM was used because of the availability of site specific moncclonal antibodies with which to localize the site of action of calpain [11]. As the major forms of NCAM polypeptides are highly conserved among vertebrates [12] it is likely that our findings are generally applicable. The form of NCAM from embryonic chick brain, while comprising the same polypeptide structure as that of the adult rat, appears on SDS-PAGE as highly glyeosylated 200-250 kDa and 160 kDa forms 113]. Following incubation with calpain, the chick NCAM exhibited a modified mobility in SDS-PAGE appearing approx. 25 kDa smaller than its original molecular weight (Fig. 2). Western blot analysts with antibodies recognizing epitopes on the extra- and intraoellular domains (antibodies 5e and 4d, respectively) indicated that the site of cleavage was on the cytoplasmic domain (Fig. 3). As shown in Figure 3a, antibody to the extracellular domain still recognized NCAM following exposure to activated ealpaln, although as evident "ore its position on the blot, the native protein has been cleaved by the proteinase, By contrast, an antibody (4d) directed against a cytoplasmic region of NCAM-I80 did not recognize high molecular weight NCAM following e~tposure to ca]pain but did detect a major fragment that migrated at approx. 25 kDa. Taken

A

B

C

180.-.*

"1"16...-,.

84---*Fig, 2, In,halloa of pufilledchickNCAMwithcalpain.SDS-PAG£ analysisof 30 lag totalproleinfollowingincubationin the p~esenceof inhibitorfor 15 mill(lane A), and in the absenceof inhibitorfor 5 rain and 15 rain{lanesB and C, respectively).The petitionof native chick NCAM polypeplid¢formsis arrowed. ChickN C A M was isobled a,sdescribedpreviously ill].

together, these observations suggest that the action of ealpain is confined to the intracellular domain. Assum. ing that this component represents a fragment of about 245 amino acids and that the position of the 4d epitope is located approx. 21 kDa from the carboxy terminus [11], it would appear that the action of oalpain shortens the %,toplasmie portion from its normal 362 amino acids [14] to about 117 amino acids. Previous studies have demonstrated the shedding of a number of neuronal cell surface glycoconjugates including NCAM (reviewed in ref. 15) and it has been hypothesized that limited proteolysis is responsible ior facilitating this release [15]. Our results suggest that ca]pain, via proteolytic modification of the intraeellular domain, could release NCAM from cytoskeletal interaction. The potential consequences of this release, such as increased lateral mobility, uncoupling of signals and shedding from the membrane need to be investigated. Such consequences arc of particular interest in that the long-term potentiation of synaptic responses appear to be accompanied by nitrastructural changes [16,17,18] and it is conceivable that such effects involve modifica-

15g A

i

B

C

D

--180-

.,-58-

180-

.,48.5-

,2e,e_

Fig. 3. Detem~nation or cleavage rite an purified cbirk NCAM by Western blot anfly~is u~;7J~g terminU spe~fic monoclonal P.ntibodles.(a) Amino termini antibody 5e. I~cubatiqrt il3 the pRsence of inhibitor for 15 mm (lan©A), and in the a b ~ of inhibitor for 5 rain and 15 rain (lanes B and C, respectively).The positi~l, of native chick NCAM t~|ypcptide foffos is arrowed. (b) Carboxy terminal anti*0ody4d. Pu~ unu'ealed NCAM {lane A), incubation in the preseoce of inhibitoTfor 15 rain 0ane B), and in the absence of in,ttibltnrfor $ min and 15 ~ (lanes C and D, re~e~ively). The position of the 25 kDa proteo|ytic ftagmerll is arro'~ed.

tions in the linkages between pr¢- ~ d post-synaptic membranes. W e wish to t h a n k Dr. M a r c u s Kessler for m a n y helpful discussions a n d Ms. Jackie Porter a n d Mr. Richard Fair for secretarial assistance. Tl~s work was supported b y G r a n t A F O S R 89-0383.

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3 P©rsolm, E., Pollesbet~, K, and Schachner, M. (1989) £ Comp. Neuro]. 288, 92-10q, 4 Siman, 1~, Baudry, M., and Lyach, G J. (]983) Neurochem. 41, 950-956. 5 Sandoval, I.V. arid Weber, K. (1978) Ear. J. Biochem.91,463--470. 6 Bennett, Y. 0985) Am~. Rev. Bi~luml. 54, 273-304. 7 Siman, R., Baudry, M. and Lynch, O. (1984) ~oc. Nad. Acad. Sci. USA 81, 3572-3576. 8 Po]lerbet~ E.G,, Bun-ldge, K., Krebs, K.E., Goodman, $.R. and S~hachner, M. (1987) Celt Tissue Res. 250, 227-2]6, 9 He. H.-T., Barbet, £, Chaix, J,-C and Gorldis, C. 0986) EMBO J. 5, 24~9-2494. 1O Hcmpcrlcy, JJ,, Edelnum, G.M. and Cmmi~em, B.A. (t986) Prec. Nad. Acsd. Sci. USA 83, 9822-9826.

160 11 Frelinger. A.L and Rufishauser, U. (1986} J. Cell Bio]. 103, 1729-1737. ]2 Hoffmaa, S, Chuong, C. and Edelman, O.M~ (1984) Ih'oc. Natl. Acad_ Sci. USA 8t, 6881-6888. 13 Hoffman, S., Sorkin, B.C., White, P.E, Brackenbury, R., Mailhammer. R~ Rutishauser, U.. Cunningham. B.A. and Edelman, G M . ~1987) J. BioL Chem. 257. 7720-7729.

ld Hempefly. JJ., Murray, B.A., P.,dclman, G.M. and Cunningham, B,A, (1986) Prec. NaL Acad. Sci, USA 83, 3037-~3041. 15 Swe~dner0 r j . (1983) i, Ne~Jrasci, 3. 2540-2517. 16 Lee, K.. Sctloltler. F.. Oliver. M. and Lynch, G. (]950) J. Neuxophysiol. 44, 247-258. 17 Desmmd, N.L and Levy. W.B. (t983) Brain Res. 265, 21-30. 18 Chang. E L F . and Greenough, W.T., Brain Res. 31)9(1984) 35-46.