Molecular recognition of acetylcholine receptor. Recognition by α-neurotoxins and by immune and autoimmune responses and manipulation of the responses

Pergamon

Advances in Neuroimmunology Vol. 4. pp. 403-432, 1994 Copyright 0 Elsevier Science Ltd

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Molecular recognition of acetylcholine receptor. Recognition by wneurotoxins and by immune and autoimmune responses and manipulation of the responses

M. Zouhair Atassi

Department of Biochemistry, Baylor College of Medicine, Houston. TX 77030, USA

Keywords-Acetylcholine receptor. autoimmune recognition epitopes, autodeterminants, myasthenia gravis, a-neurotoxins, synthetic peptides.

Introduction

The nicotinic acetylcholine receptor (AChR) plays a central role in post-synaptic neuromuscular transmission by mediating ion flux across the cell membrane in response to binding of acetylcholine (Karlin, 1980; Changeux et al., 1984). The receptor is a pentamer composed of four subunits (+P$). This regulatory activity is inhibited by binding to an a-neurotoxin (Lee, 1979) or by some anti-receptor antibodies. Indeed the human disease myasthenia gravis (MG) is caused by an autoimmune response to AChR (Appel et al., 1975; Tzartos et al., 1982). Functional studies have focussed mostly on the cx-subunit because it has been shown to be responsible for binding acetylcholine (Sobel et al., 1977; Moore and Raftery, 1979; Tzartos and Changeux, 1983; McCormick and Atassi, 1984) and a-neurotoxins (Lee, 1979). Snake venom postsynaptic neurotoxins form a large fami- ly of related proteins of which two sub- groups, the long and short neurotoxins, are major constituents. Both long and short neurotoxins are known to bind specifically to the a-chain of AChR in a competitive

manner with cholinergic ligands (Meunier et al., 1974; Maelicke et al., 1977; Haggerty and Froehner, 1981), but display differences in their association and dissociation kinetics.

The primary structures of the four AChR subunits of Torpedo culifornicu (t) (Noda et al., 1982, 1983a,b; Claudio et al., 1983) and the cw-subunits of human (h), bovine (Noda et al., 1983~) and mouse (m) (Boulter et al., 1986) have been deduced from the respective cDNA sequences. From the primary structure of each AChR-subunit, it was possible to identify transmembrane hydrophobic regions and the extracellular part of the chain (Noda et al., 1983b; Guy, 1983; Finer-Moore and Stroud, 1984). Immunological and toxin-binding studies to intertransmembrane synthetic peptides confirmed (Atassi et al., 1988b) the model postulating five transmembrane regions (Guy, 1983; Finer-Moore and Stroud, 1984).

Previously, this laboratory introduced (Kazim and Atassi, 1980) a comprehensive synthetic approach that was specifically designed to localize the full profile of the continuous regions of antibody and T-cell recog-

403

404 Advances in Neuroimmunology

nition (as well as other recognition regions) on a protein molecule. This approach con- sists of the examination of the activities of consecutive synthetic overlapping peptides, of uniform size and overlaps, that encompass the entire protein chain (Kazim and Atassi, 1980). This strategy has been applied to the a-chain of tAChR to localize the continuous regions recognized by anti-AChR antibodies (Mulac-Jericevic et cd., 1987) and T cells (Yokoi et al., 1987). It has also been applied to map the regions of hAChR and lAChR recognized by autoimmune T cells and antibodies (Oshima et al., 1990; Ashizawa et al., 1992; Atassi et al., 1992). By employing a similar strategy we were able to map the full profile of the binding regions for long cx-neurotoxins (a-bungarotoxin (BTX) and cobratoxin) on tAChR (Mulac-Jericevic and Atassi, 1986, 1987a,b) and hAChR (Mulac- Jericevic et al., 1988). More recently, we have mapped the binding regions for short neurotoxins (erabutoxin and cobrotoxin) on the a-subunits of tAChR and hAChR (Ruan et al., 1991). Conversely, the binding sites for AChR on BTX were mapped by synthetic peptides representing each of toxin loops (Atassi et al., 1988a; McDaniel et al., 1987). Identification of the binding sites on the toxin for AChR and on the receptor for the toxin has provided a molecular explanation for the observed differences between the two toxin groups in their actions on AChR (Ruan

125

et al., 1991). Furthermore, by studying the interaction between peptides representing the binding regions of the two proteins, it was possible to derive a three-dimensional description of the neurotoxin-binding cavity on the receptor (Ruan et al., 1990).

The work in this laboratory which has progressed on two fronts, biochemical and immunological, has served to map and understand the function of the molecular features of recognition of AChR and the employment of these recognition features for the manipulation of the autoimmune antibody and T-cell responses in myasthenia gravis (MG). The following describes the various aspects of the studies.

Interaction of acetylcholine receptor with acetylcholine and a-neurotoxins

Localization and synthesis of the acetylcholine-binding site

On the basis of sequence analysis and structural topology of the a-subunit, it has been proposed that the invariant cysteine residues 128 and 142 form a disulfide bridge, the integrity of which is essential for the binding of acetylcholine to the receptor (Karlin, 1980; Noda et al., 1982, 1983b, 1983~; Devillers- Thiery et al., 1983). We have undertaken studies to localize, by peptide synthesis, the acetylcholine-binding site in both human and Torpedo receptors (McCormick and Atassi,

130 Lys-Ser -Tyr-Cys-Glu-Ile-Ile-Val~Thr

I S Ais

I 135 Ghe S Prb

145 I 140 Ile-Gly-Leu-Lys-Met-Thr-Cys-Asn-Gln-Gln-Asp c

Ph’e

Fig, I. Structure of the synthetic peptide corresponding to the region 01125-147 of tAChR. This region has been shown to bind acetylcholine (McCormick and Atassi, 1984). cobratoxin. and a-bungarotoxin (McCormick and Atassi, 1984; Mulac-Jericevic and Atassi, 1986), anti-AChR antibodies (Lennon et al., 19X5) and, when used as an immunogen (Lennon et ul., 1X35), to induce helper T-cell responses, delayed hypersensitivity, antibodies to native AChR, and experimental autoimmune myasthenia gravis. (From Atassi et al.. 1987.)

Molecular recognition of AChR 405

1984). A peptide (Fig. 1) containing this loop (Lennon et al., 1985; Atassi et al., 1992). region (residues 1~125-147) was synthesized. It has been noted (McCormick and Atassi, Solid-phase radiometric binding assays dem- 1984), however, that the results do not pre- onstrated a high binding of 1251-labeled IX- elude the possibility that additional residues, bungarotoxin to the synthetic peptide. It was residing outside the region (~125-147, are further shown that the free peptide bound involved in the binding of acetylcholine to well to [3H] acetylcholine. Pretreatment of AChR. peptide ~125-147 with 2-mercaptoethanol destroyed its binding activity, clearly showing that the integrity of the disulfide bond was essential for binding. Unlabeled acetylcholine also inhibited the binding of labeled acetylcholine to the synthetic peptide. The region (-w125-147, therefore, contains essential elements of the acetylcholine-binding site of tAChR. It is not surprising, therefore, that immune responses to this peptide are involved in the pathogenesis of EAMG

Mapping of the acetylcholine receptor- binding sites on a-bungarotoxin

The amino acid sequences of numerous snake venom toxins have been determined (Dufton and Hider, 1983; Endo and Tamiya, 1987). These sequences tend to fall under three classifications: short neurotoxins, long neurotoxins, and cytotoxins (for recent review, see Atassi, 1991). Pharmacologically

Fig. 2. Diagrammatic representation of the location of the various synthetic peptides (see Fig. 3) within the three-dimensional structure of BTX. In each box, the region that was synthesized is highlighted in heavy lines within BTX and is also shown by itself to the left for clarity. The disulfide bond in the loops is shown by a dotted line. (From Atassi et cd.. 1988a.)


active peptides, with effects ranging from those of the neurotoxins (i.e. muscle pa- ralysis and respiratory failure) to those of the cytotoxins (i.e. hemolysis, cytolysis, cardiotoxic effects, and muscle depolariza- tion), can be designed and synthesized based on the structure of short neurotoxins, long neurotoxins, and cytotoxins (Atassi, 1991). Therefore, we have adopted a synthetic approach to dissect the activities of these toxins. The approach has, thus far, been applied to localize the distinct AChR-binding regions on BTX. The general type of approach is shown for the BTX molecule in Fig. 2,

Peptide

and the covalent structures of the synthetic peptides are shown in Fig. 3. The entire toxin molecule is essentially subdivided into unique, potentially active regions, and the peptides are designed to mimic as closely as possible the native regional structure.

Accordingly the following battery of peptides (shown in Figs 2 and 3) were constructed (Atassi et al., 1988a):

Loop 1 peptide (LI): BTX residues 3-16 with an artificial disulfide between two terminal cysteines.

NH,-terminal extension of the loop 1 peptide

Structure

316 LI C-H-T-T-A-T-I-P-S-S-A-V-T-C-(G)

3 iPl16 LI/N-tail I-V-C-H-T-T-A-T-I-P-S-S-A-V-T-C-(G)

126 30 40 I LII C-K-M-W-A-D-A-F-T-S-S-R-G-S-V-V-E-C-G

48[_,59 LIII C-P-S-K-K-P-Y-E-E-V-T-C-(G)

45 159 LIII/Ext A-A-T-C-P-S-K-K-P-Y-E-E-V-T-C-(G)

601165 74 LIV/C-tail C-S-T-D-K-C-N-H-P-P-K-R-Q-R-G

66 74 C-Tail N-H-P-P-K-R-Q-P-G

Fig. 3. Covalent structures of the BTX synthetic peptides. When applicable, the monomeric cyclic structures were prepared and purified. The disulfide-bond in peptides LI and LI/N-tail is an artificial bridge because in BTX, Cys-3 is linked to Cys-23 and Cys-116 is linked to Cys-44. Similarly. the disulfide bond in peptide LII is artificial, because arginine and lysine occupy positions 25 and 42, respectively. The artificial disulfide bond at this position effectively dispenses with the interior part of loop LII (which would not be expected to participate in the binding of BTX to AChR) while also maintaining the relative distances of the two antiparallel sides of the loop (McDaniel et al., 1987). From examination of the three-dimensional structure of BTX, these artificial disulfide bonds will not be expected to introduce any appreciable distortion in the potential region acquisition of native like conformation. The residues underlined in peptide LII were either replaced or chemically modified in synthetic analogues or both (McDaniel et al., 1987). The glycine residues in parentheses were not part of the BTX sequence, but the peptides were synthesized on a Gly-resin for convenience. (From Atassi et al., 1988a.)


Peptide Number

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Sequence Position

al-16

~~12-27

(r23-38

a34-49

a45-60

a56-71

ff67-82

ff78-93

a89-104

a100-115

~111-126

(~122-138

a134-150

o146-162

~~158-174

0170-186

a182-198

cr194-210

~262-276

Species

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Human Torpedo

Structure

SEHETRLVAKLFKDYS ---------N-LEN-N

FKDYSSVVRPVEDHRQ LEN- NK-I----H-TH

EDHRQVVEVTVGLQLI -H- THF_DI__--___

GLQLIQLINVDEVNQI ______--S_______

EVNQIVTTNVRLKQQW w-w_- _K_____R___

LKQQWVDYNLKWNPDD _R_-- I-VR-R---A-

WNPDDYGGVKKIHIPS ---A----I---RL--

IHIPSEKIWRPDLVLY -RL-- DDV_L______

DLVLYNNADGDFAIVK -------------m-H

FAIVKFTKVLLQYTGH __ --HM--L--D---K

QYTGHITWTPPAIFKS D-- _K-M----_____

AIFKSYGEIIVTWFPFD -w-w-_------_____

HFPFDEQNGSMKLGTWT ___-- Q- _ _ -T _ - _ _ 1 _ _

LGTWTYDGSVVAINPES --I-----TK-S-S---

INPESDQPDLSNFMESG _S_-- -R----T-----

FMESGEWVIKESRGWKH _-------M-DY-----

RGWKHSVTYSGGPDTPY __-- -W-Y-T-------

PDTPYLDITYHFVMQRL ______------I---I

ELIPSTSSAVPLIGK __---__-----~--

Fig. 4. Covalent structures of the synthetic overlapping peptides representing the extracellular part of each of the o-chains of human and Torpedocalifornica AChRs. The upper sequences of each pair of peptides give the full primary structures of the human AChR peptides and, under these, only the residues that are different in the corresponding Torpedo peptides are given. Segments in bold type represent the five-residue overlaps between consecutive peptides.


(LIiN-tail): constructed as in LI but further extended with the hydrophobic potentially interactive NH,-terminal residues 1 and 2.

Initially discovered active-binding peptide (LII): comprised residues 26-41, with cysteine substitutions at both ends of the peptide providing an artificial disulfide linkage between these two residues; threo- nine and alanine substitutions at residues 29 and 33, respectively, to eliminate the disulfide of bungarotoxin loop 5 and, thereby, avoid the formation of disulfide- linked polymers (McDaniel et al., 1987).

Loop peptide corresponding to loop 3 (LIII): residues 48-59 clasped at naturally occur- ring cysteines 48 and 59 of BTX on the two terminals of the peptide.

Loop 3 extended toward the NH,-terminal by three residues (LIIIlExt): Ala-45, Ala-46 and Thr-47 added at the N-terminal of loop 3 (i.e. residues 45-59).

An extension of the COOH-terminated tail (LIVIC-tail): residues 60-74 which includ- ed the fourth loop of BTX between Cys-60 and Cys-65.

A COOH-terminal linear peptide (C-tail): residues 6674.

In all experiments, the peptides were purified and, when appropriate, the monomeric cyclic structures were prepared.

The ability of these peptides to bind tAChR was studied (Atassi et al., 1988a) by radiometric adsorbent titrations. Three regions, represented by peptides l-16, 2641,

44 c (J)

” c ,_ 6 - (b) “- x .Tl b 4-

2

n m

Pept ide

Fig. 5. Summary of the binding profiles of (a) BTX and (b) cobratoxin to the synthetic overlapping peptides of tAChR. The bars represent the maximum binding values to 25 ~1 of a 1: 1 (v/v) suspension of each peptide adsorbent. (From Mulac-Jericevic and Atassi. 1987a,b.)


and 45-59, were able to bind 1251-labeled tAChR and, conversely, ‘ZsI-labeled peptides were bound by tAChR. In these regions, residues IIe-1, Val-2, Trp-28, Lys-26, and/or Lys-38, and one or all of the three residues Ala-45, Ala-46, and Thr-47, are essential contact residues in the binding of BTX to receptor. Other synthetic regions of BTX showed little or no tAChR-binding activity. The specificity of tAChR binding to peptides 1-16, 26-41, and 45-49 was confirmed by inhibition with unlabeled BTX.

Other parts of the BTX molecule make little or no contribution to its binding to AChR. The region within peptide LII makes a higher contribution to the binding activity of BTX than do the regions within the peptides LI/N-tail and LIII/Ext. The radioiodination of peptide LI/N-tail (most

likely at His-4) and LIIUExt (most likely at Tyr-54), appear to have some adverse effects on the binding of the respective peptide to tAChR. Thus, peptide LUN- tail and LIII/Ext exhibited lower affinity than peptide LII when the binding of the labeled peptides to tAChR absorbent was inhibited by unlabeled BTX (IC,, values: LII, 8.4 X lesM; LUN-tail, 8.2 X 1t7M; LIIUExt, 4.4 X 1e7M). By contrast, the three peptides had comparable affinities

(I%” values: LII, 1.5 X 1e7M; LUN-tail, 4.2 x 1@7~; LIIUExt. 5.1 x 10-7~) when binding of 1*51-labeled tAChR to peptide absorbents was inhibited by unlabeled BTX, giving almost superimposable inhibition curves.

It was concluded (Atassi et al., 1988a) that BTX has three main AChR-binding regions (loop I with NH,-terminal extension, loop

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Fig. 6. Summary of the binding profiles of (a) BTX and (b) cobratoxin to the synthetic overlapping peptides of hAChR a-subunit. The bars represent the binding values to 200 ~1 of a 1:l suspension (v/v) of each peptide adsorbent. Titrations were carried out in 0.01 M phosphate buffer (pH 7.2), 0.15 M NaCl (PBS) containing 0.1% bovine serum albumin (BSA). The reaction volume was 250 ~1. The amounts of label added were: BTX, 3.25 x lo5 cpm; Cobratoxin, 2.8 x lo5 cpm. Identical volumes (i.e. 200 ~1 of a 1:l suspension) of adsorbents of T. californica AChR were used as positive controls. Under these conditions, the amounts of label bound to T. californica AChR were: BTX, 243,580 cpm; Cobratoxin, 145,590 cpm. Note that the adsorbents of unrelated proteins and peptides did not bind either of these toxins. The peptide numbers refer to the sequences shown in Fig. 4. (From Mulac-Jericevic et al., 1988.)

410

AChR species

1. californica

2. sapiens

1. californica

H. sapiens -

T_ californica

fi. sapiens

1. californica

5. sapiens

1. californica

l. sapiens

Advances in Neuroimmunology

1. californica binding region 1

1 16 SEHETRLVANLLEDYN

SEHETRLVAKLFKDVS

1. californica binding region II

23 49 EHHTHFVDITVGLQLIQLISVDEVNDI

EDHRQVVEVTVGLQLIQLINVDEVDOI

1. californica binding region III

100 115 FAIVHHTKLLLDVTGK

FAIVKFTKVLLQYTGH

1. californica binding region IV

122 139 143 150 AIFKSYCEIIVTHFPFDQQNCTnKLGTWT

AIFKSYCEIIVTHFPFDEQNCSMKLGTUT

1. californica binding region V

182 198 206 RGWKHWVYYTCCPDTPV LDITYHFI

RGWKHSVTYSCCPDTPY LDITYHFV

Fig. 7. Sequence comparisons of the toxin-binding regions of the AChR cu-subunit of T. califorrzica with the corresponding regions of the a-chain of human (H. sapiens). The amino acid replacements in human, relative to the T. californica toxin-binding regions 1, 3 and 5, explain the reason these regions in human AChR have low binding to w-neurotoxin. Since region 4 in the human AChR retains the ability to bind toxin despite replacements at positions 139 (Gln + Glu) and 143 (Thr + Ser), the toxin-binding region may be further narrowed down and would reside within residues ~1122-138. The strongest toxin-binding region in T. californica AChR resides within residues (-u182-I98 (Mulac-Jericevic and Atassi, 1986, 1987a,b). This region in human AChR is unable to bind toxin because of the replacements at positions 187, 189 and 191. The low-binding activities present to the right of this region (residues ot192-205) and region a3241 (Mulac-Jericevic and Atassi, 1986, 1987a,b)are retained. (From Mulac-Jericevic’er al. 1988.)

II, and loop III extended toward the NH,- terminal by residues 45547).

The a-neurotoxin binding regions on human and Torpedo A ChR

A comprehensive synthetic peptide strategy we had originated (Kazim and Atassi, 1980),

was applied to tAChR and hAChR (Fig. 4) and enabled us to map the full profile of the continuous binding regions for long (Y- neurotoxins on the extracellular part (residues al-210) of the a-chains of tAChR (Fig. 5), (Mulac-Jericevic and Atassi, 1987a,b) and hAChR (Fig. 6) (Mulac-Jericevic et al., 1988; Ruan et al., 1990). In tAChR, the


binding regions reside within (but may not thetic overlapping peptides spanning the include all of) residues tcul-10, ta32-49, entire extracellular part of the respective to1100-115, tor122-138 and tc1182-198 (Fig. 7). a-chains. On tAChR (Fig. S), five Cot- In human AChR, long neurotoxins bind binding regions were found to reside within to regions ha32-49, halOG115, ha122-138 peptides tell-16, &x23-38ltcx34-49 overlap, and ha194210 (Fig. 6). In recent work t(ulOO-115, t&122-138 and t&94-210. The (Ruan et al., 1991), the continuous re- Eb-binding regions were localized within gions for short-neurotoxin binding on the peptides ta23-38/t(w34--49/to145-60 overlap, a-chain of tAChR and hAChR were local- t(ulOO-115 and tc1122-138. The main bind- ized by reaction of ‘251-labeled cobrotoxin ing activity for both toxins resided within (Cot) and erabutoxin b (Eb) with syn- region t(u122-138. Therefore, the binding of

BINDING OF COT AND EB TO TORPEDO ACHR P?,PTIDES

6 -- COT

1 2 3 4 5 6 7 6 9 10 11 12 13 14 15 1617 18

Peptide Number

Fig. 8. Summary of the profiles of (a) cobrotoxin (Cot) and (b) erabutoxin (Eb) binding to the synthetic overlapping peptides of the extracellular part of the a-chain of tAChR. The peptide numbers refer to the sequences given in Fig. 4. The binding values of ““I-labeled Cot and Eb to tAChR were 55140 f 1350 and 68550 + 1520 cpm, respectively. Binding to unrelated proteins (BSA, horse myoglobin) and peptides ((sperm-whale myoglobin synthetic peptides l-17, 2541 and 121-137) (Bixler and Atassi, 1983)) (negative controls) was 650 + 220 cpm. (From Ruan et al., 1991.)


long a-neurotoxins (c-w-bungarotoxin (BTX) strong and medium binding activities to Eb, and cobratoxin (Cbt)) involved the same but low activity to Cot, whereas peptide regions on tAChR as well as an additional hal-16 exhibited low binding to Cot and region within the residues al82-198 (com- no binding to Eb (Fig. 9). Comparison pare Figs 5 and 8). Thus region ~182-198, with previous studies indicated that, for which is the strongest binding region for hAchR, the binding regions of short and long neurotoxins on tAChR, was not a long neurotoxins were essentially the same. binding region for short neurotoxins (Ruan The finding that the region within resi- et al., 1991). On hAChR, peptide ho122-138 dues (~122-138 of both human and Torpedo possessed the highest activity with both short AChR possessed the highest binding activity neurotoxins, and lower activity was found in with short neurotoxins indicated that this the overlap ha23-38/ha34-49/ha45-60 and region constitutes a universal binding site for in peptide ho1194-210 (Fig. 9). In addition, long and short neurotoxins on AChR from peptides halo&115 and hor56-71 showed various species (Ruan et al., 1991).

BINDING OF COT AND EB TO HUMAN ACHR PEPTIDEB

6 COT

4 --

‘0 C 12

: EB m 10 -- c .- x : a --

I

1 2 3 4 5 6 7 a 9 10 1112 13 14 15 1617 la

Peptide Number

Fig. 9. Summary of the binding profiles of Cot and Eb to the synthetic overlapping peptides of the extracellular part of the a-chain of human AChR. The peptide numbers refer to the sequences given in Fig. 4. (From Ruan et al., 1991.)


The a-neurotoxin binding cavity of human AChR

We have recently described an approach for studying the details of protein-protein recognition (Ruan et al., 1990). Each of the active peptides of one protein is allowed to interact with each of the active peptides of the other protein. Based on the relative binding affinities of peptide-peptide interactions, the disposition of two protein molecules in a complex can be described if the three-dimensional (3-D) structure of one of the two molecules is known. The peptides of the binding site of one protein (whose 3-D

structure is not known) are docked onto the appropriate regions of the other whose 3-D structure is known, by computer graphics and energy minimization thus allowing a 3-D model to be constructed of the unknown binding-site cavity. The validity of this approach was first established with peptides corresponding to regions on the B-chain of human hemoglobin involved in binding to the a-chain (Ruan et al., 1990).

As mentioned above, the regions on hAChR and tAChR which bind BTX have been localized. Also, the binding regions for tAChR on BTX were mapped by synthetic peptides representing each of the BTX loops

A. SYNTHETIC PEPTIDES OF ACHR (r-CHAIN WHICH ARE INVOLVED IN BINDING TO BGT

Peptide 34-49

Peptide loo-115

Peptide 122-138

Peptide 194-210

Z!L-Q-L-I-Q-L-I-N-v-D-E-V-D-Q-~TG)

100 115 F-A-I-V-K-E-T-K-V-L-L-Q-Y-T-G-H(G)

122 138 A-I-F-K-S-Y-C-E-I-I-V-T-H-F-P-F-D(G)

194 210 P-D-T-P-Y-L-D-I-T-Y-H-F-V-M-Q-R-L(G)

B. SYNTHETIC LOOP PEPTIDES OF BGT WHICH ARE INVOLVED IN BINDING TO ACHR

LlN I-V-;-H-T-T-A-T-I-P-S-S-A-V-T-:!(G) I

S------ m-___s

L2 C-:zM-W-A-D-A-F-T-S-S-R-G-K-V-V-k-G(G) I

S S

45 59 L3E A-A-T-C-P-S-K-K-P-Y-E-E-V-T-C-(G)

I I S S

Fig. 10. Covalent structures of the synthetic peptides used in this work. (A) Peptides of the hAChR 01 chain that are involved in binding to cx-neurotoxins (Mulac-Jericevic et al., 1988). (B) Peptides of the BTX molecule that are involved in binding to AChR (McDaniel et cd., 1987; Atassi et al., 1988). Note that the disulfide bonds in LIN and L2 are artificial (for details, see Atassi et al., 1988). The binding areas on the AChR peptides have been assigned (Mulac-Jericevic et al., 1988) to residues 3241, 100-110, 125-136, and 198-208. Note that the glycine residues in parenthese are not part of the BTX or the AChR sequences, but the peptides were synthesized on a Gly-resin for covenience. (From Ruan et al., 1990.)


(McDaniel et al., 1987; Atassi et al., 1988a). In recent work (Ruan et al., 1990), peptides representing the active regions of one molecule were allowed to bind to each of the active-region peptides of the other molecule. Thus, the interaction of three BTX synthetic loop peptides with four synthetic peptides representing the toxin-binding regions on hAChR (Fig. 10) permitted the determination of the region-region interactions between BTX and the human receptor. Based on the known 3-D structure of BTX (Love and Stroud, 1986), the active peptides

125’

of hAChR were then assembled to their appropriate toxin-contact regions by computer model building and energy minimization. This allowed the three-dimensional construction of the toxin-binding cavity on hAChR (Fig. 11). The cavity appears to be conical, 30.5A in depth, involving several receptor regions that make contact with the BTX loop regions. One AChR region (within residues al25136) involved in the binding to BTX also resides in a known ACh binding site (McCormick and Atassi, 1984). This demonstrates in three

Fig. Il. A stereo drawing of a 3-D construction of the toxin-binding cavity in AChR. with the BTX molecule (backbone only) bound in the cavity (Upper) and without the BTX molecule (Lower). The somewhat conical cavity has the following dimensions: residues 10&136, 21.32A; residues 13632, 35.OA; residues 32-198, 16.06A; and residues 198-100, 22.13A. The depth of the cavity is 30.48A. (From Ruan et al., 1990.)


dimensions a critical site involved in both ACh activation and BTX blocking. Thus, studying the interaction between peptides representing the binding regions of two protein molecules may provide an approach in molecular recognition by which the binding site on one protein can be described if the 3-D structure of the other protein is known (Ruan et al., 1990).

Mapping of the antibody and T-cell recognition sites in tAChR

The synthetic overlapping peptides which spanned the entire extracellular part (resi-

dues (-wl-210) of the a-chain of tAChR were used to systematically screen for the profiles of the continuous regions that are recognized by antibodies against free, or membrane-sequestered AChR (Fig. 12).

The antigenic sites of tAChR are similarly recognized by rabbit and mouse antibodies (Mulac-Jericevic et al., 1987). This is consistent with the evidence (Atassi, 1975, 1978,198O; Sakata and Atassi, 1980a,b; Kazim and Atassi, 1982; Yoshioka and Atassi, 1986; see Atassi, 1984 for review) that the regions of protein antigenic sites are not dependent on the immunized species, but rather are determined by their

I

I_ IL .I1

1 2 3 4 5 6 7 8 9 l0 11 12 13 I4 1516 17 18 PEPTIDE

Fig. 22. Summary of profiles of binding of the anti-tAChR antibodies to synthetic peptides. (ax) Mouse antisera to isolated AChR: antiserum 3, antiserum 45, and antiserum 41, respectively. (d and e) Mouse antisera to membrane-bound AChR; antiserum 55 and antiserum 53, respectively. (f) Rabbit antiserum to isolated AChR. Peptide numbers refer to the sequences given in Fig. 4. (From Mulac-Jericevic et al., 1987.)


conformational locations (Kazim and Atassi, 1978; Atassi and Kazim, 1978). The im- munodominance of a given site (i.e. the amounts of anti-site antibodies) varied with the antiserum (Mulac-Jericevic et al., 1987). This is consistent with the antibody response to other protein sites (Atassi, 1975, 1978, 1984; Yoshioka and Atassi, 1986). In the recognition of a multi-determinant complex protein antigen, the responses to each site are under separate Ir-gene control (Okuda

et al., 1979). It has been postulated (Tzartos and Lindstrom, 1980; Tzartos et al., 1983) that AChR contains a ‘main immunogenic region’ (MIR) to which an overwhelming fraction of anti-AchR antibodies are directed. However, no single MIR was found (Mulac-Jericevic et al., 1987), but rather a fluctuating dominance of several sites.

The synthetic overlapping peptide strategy has also been employed to localize the T-cell recognition sites of the a-chain of tAChR in

3 C57BL16

2 -

l-

peptides

SWR

?? m I I, I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 16

Fig. 13. Schematic presentation of the in vitro responses of tAChR-primed T cells to the synthetic tAChR peptides. The diagram shows the net counts per minute at the optimum challenge dose of each peptide. Peptide numbers refer to the sequences given in Fig. 4. (From Yokoi et al., 1987.)


four mouse strains (Yokoi et al., 1987) representing different haplotypes (C57BL/6 (H- 2h), C3H/He (H-2k), SWR (H-24) and SJL (H-2s)) (Fig. 13). Two of the strains (H-2s and H-2s) were high responders to tAChR, one strain (H-2b) was a moderate responder. A broad localization of the major continuous T-cell recognition sites by the four strains is given in Fig. 14. The boundary frame shifts observed for the T sites from strain to strain are similar to boundary shifts of T sites on the other aforementioned proteins.

The responses to the T sites were subject to genetic control operating at the antigenic site level (Yokoi et al., 1987) (i.e. each site is under separate genetic control). It is significant to note that AChR-primed T cells of C3H/He (H-2k) mice gave significant responses to at least three different peptides even though they exhibited virtual- ly no in vitro response to intact AChR. The lack of responsiveness of a given strain may be due to Ir-gene restriction. On the other hand, regulating inter-site cellular influences

(help or suppression) play an important role in the expression of the response to a protein (Atassi et al., 1981). It would appear, therefore, that in the H-2k haplotype, the cellular responses of some site(s) have the effect of turning off the responses to other sites. These site-directed cell-cell interactions are now being studied.

These findings permitted comparison, at the submolecular level, of the sites of recognition on tAChR by antibodies (Mulac- Jericevic et al., 1987) and by T-cells (Yokoi et al., 1987) (Fig. 14). Three of the T sites on the a-chain coincide with the antigenic (antibody binding) sites. There are, however, at least two sites (and possibly three) which are exclusively T-cell specific and are not recognized by B cells. These sites reside within regions ~~113-124, ~148%160 and possibly a14-25 (which shifts in H-2s and H-2q). This is similar to the aforementioned findings with several other proteins, each of which contains sites that are recognized by both B and T cells as well as sites that

AChR (00

Antigenic Sites

out bred mouse

T Sites

1 2’0

2536=3 6gS log4 ‘20l&8 1722 1888

C57BLI 6(H-2’) “kzIzP ‘U&60 18z6

C3HIHe (H-$1 142_5 69&O

SJL (H-23 192 6

“Z ‘48 160

I

SWR (H-f) 6&3 69 80 ‘ago

Fig. 14. Schematic diagram showing the full profile of the regions of the tAChR a-subunit that carry the continuous antigenic (i.e. antibody recognition) sites and the sites of T cell recognition (T sites) in four mouse strains. The T cell recognition sites are compared with the sites of antibody (B cell) recognition in the outbred mouse (which were also similarly recognized by other host species) (Mulac-Jericevic et al., 1987). The T site notations are as follows: filled bars, regions stimulating high T cell responses (Acpm >20,000); stippled bars, regions stimulating intermediate responses (Acpm, lO,OOO-20,000); open bars, regions stimulating low responses (Acpm, 500~~10,000). It is not implied that the entire regions shown comprise the T sites, rather that the sites reside within these regions. Indeed, the T sites are localized within intentionally larger regions than their expected size. (From Yokoi et al., 1987.)


are recognized exclusively either by T cells or by B cells. The functions of the T sites that are specific for T cells may be concerned with intersite regulatory influences (Atassi et al., 1981) mentioned above.

Mapping of the B6 T-cell recognition sites in EAMG-susceptible mice with T-lymphocyte lines and clones

Immunization of C57BL6 (B6) mice with tAChR stimulates both cell-mediated and humoral anti-tAChR and anti-mAChR immune responses, which cause neuromuscular dysfunction closely resembling human MG, called experimental myasthenia gravis (EAMG). The pivotal cells in the initial development of the anti-AChR response and its propagation are AChR-specific helper proliferative T cells (Thp) (Hohlfeld et al., 1986). In the preceding section, we described the mapping of the regions on tAChR that are recognized by LNC of B6 mice. In order to define the epitopes of tAChR identified by AChR-specific T cells, we generated T-cell lines and T-cell hybridoma clones from tAChR-immunized B6 mice and tested their reactivity to the synthetic uniform-sized overlapping peptides representing the entire extracellular portion of the a-chain of the tAChR (Pachner et al., 1989). We found that the reactivity of the T- cell clones and the parent lines was directed against the peptides tollll-126, t(u146-162 and t(u182-198, with the immunodominant response being against t(u146-162 (Pachner et al., 1989). This data is consistent with a highly limited recognition of AChR determi- nants in murine EAMG by tAChR-specific T cells (Pachner et al., 1989).

The localization of a specific region of AChR that is recognized immunodominant- ly by antigen-specific T cells represented an advance toward the modification of the autoimmune process in an antigen-specific manner. This was in fact achieved and those studies are described below.

MHC control of EAMG: Comparison of the responses of B6 and bm12 mouse strains

MG has been associated with certain HLA antigens (Safwenberg et al., 1978; Bell et al., 1988). As already mentioned, immunization of C57BL6(B6) mice with tAChR pro- duces an autoimmune disease mimicking MG called experimental autoimmune myasthenia gravis (EAMG). EAMG susceptibil- ity has been mapped to the I-A subregion of the mouse major histocompatibility complex (MHC) (Christadoss, 1989; Christadoss et al., 1990, 1992). The development of EAMG is primarily influenced by the class- II genes (Christadoss, 1989; Christadoss et al., 1990, 1992). Class II-restricted AChR- reactive T-helper (Th) cells are activated in MG patients and appear to contribute to the postsynaptic pathology (Oshima et al., 1990; Hohlfeld et al., 1984; Yokoi et al., 1987; Pachner et al., 1989).

A gene conversion event between I-EBb and I-Al3 in the bm12 strain (Widera and Flavell, 1984; Dinaro et al., 1984), which altered three amino acids in the C-terminal half of the first domain of I-ABb (Ile- 67 + Phe; Arg-70 -+ Gln; Thr-71 + Lys) (McIntire and Seidman, 1984), resulted in resistance (of bm12) to EAMG development (Christadoss, 1989) and lower cellular and humoral immune responses to tAChR. Very recently, we studied the effect of the gene conversion at I-Al3 positions 67, 70, 71 on the T-cell responses to epitopes of tAChR (Y- subunit in B6 and bm12 mice (Shenoy et al., 1993). The mice were primed with tAChR, and the profiles of T-lymphocyte proliferation were determined (Shenoy et al., 1993) with synthetic overlapping peptides encompassing the entire extracellular portion of the tAChR a-subunit (Fig. 15). The proliferative responses of tAChR-primed bm12 lymphocytes were markedly reduced to two (ta146-162 and toL182-198) of the three tAChR peptides (tcylll-126, to1146-162, and t(u182-198) that are immunodominant

Molecular recognition of AChR

Peptide recognition profiles of B6 and bm12 AChR-specific T cells

2 16

-cJ c 14 -

.E 12

s z 10 .- tz6

6

419

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1'8 AChR

Challenge antigen (peptide or AChR)

Fig. 15. Proliferative responses to the synthetic tAChR-a chain peptides of LNC from B6 mice and bm12 mice. Unstimulated cells gave the following cpm: B6, 6208; bm12, 6872. The diagram shows the stimulation index at the optimum challenge dose of each peptide. Peptide numbers refer to the sequences shown in Fig. 4. Results were expressed as stimulation index (stimulation index = mean cpm incorporated by stimulated cells/mean cpm incorporated by unstimulated cells). (Plotted from data in Shenoy et al., 1993.)

in B6 mice (Yokoi et al., 1987; Pachner et al., 1989). Thus, the I-APb residues encompassing the region 67-71 determine the immunogenicity of two of the AChR a-subunit T-cell epitopes in t&146-162 and t(u182-198. In determinant selection, the Ia molecule is believed to specifically bind some, but not all, peptide antigens during presentation, thereby selecting the deter- minants that are to be presented by the antigen presenting cells (Rosenthal, 1978; Benacerraf, 1978). Therefore, the reduced lymphocyte proliferative response seen in bm12 to peptides to1146-162 and t(u182-198 may be due to inefficient Aabm12 binding and/or presentation of these epitopes to bm12 T cells. Alternatively, the low response seen in bm12 to these peptides may be due to the reduced frequency, or absence, of T cells responding to these peptides, due to clonal elimination or anergy. The bm12

mutation also leads to lower expression of Apbml2: Aab, by directly decreasing the efficiency of ol:p heterodimer formation and/or surface membrane expression (Ronchese et al., 1978), and this might contribute to the suppressed response to the dominant epitopes.

Manipulation of the autoimmune response

Suppression of EAMG by epitope-specific neonatal tolerance to an AChR peptide

Because bm12 resistance to EAMG correlates with reduced proliferative response to the otherwise immunodominant epitopes within peptides tcJ4&162 and ta182-198 on the tAChR-a subunit, the data impli- cated the importance of these epitopes in EAMG pathogenesis (Shenoy et al., 1993). We had previously achieved epitope-specific


neonatal tolerance with synthetic peptides (Young and Atassi, 1983). This capability has been exploited in the animal model of experimental autoimmune encephalomyelitis (Clayton et al., 1989). To test the involvement of tAChR a-chain epitope(s) within peptide t(u146-162 in EAMG pathogenesis, B6 mice were neonatally tolerized with soluble peptide to1146-162 and subsequent-

ly immunized with tAChR in complete Freund’s adjuvant (Shenoy et al., 1993). Neonatal tolerance to tAChR or to peptide to1146162 reduced the incidence of clinical MG (Table 1) and suppressed serum anti- AChR antibodies and the T-cell response to peptide to1146162 (Table 2) (Shenoy et al., 1993). This indicates the involvement of T-cell epitope(s) within region to1146162 in

Table I. Effect of neonatal tolerance to tAChR and tAChR o-chain peptides in the development of clinical EAMG

Neonatal injection

Muscle weakness (grade)

0 1 2 3 Total (%) P”

- 4 2 4 0 6/10 (60) rAChR 8 0 1 0 l/9 (11) 0.04h ~~~146-162 10 2 0 0 2112 (16) 0.04h t(u182--198 4 2 2 1 519 (55)

UFisher exact probability test was used to determine the statistical significance of the data obtained.

hThe difference between the incidence of muscle weakness in non-tolerized and tAChR- or to1146162-tolerized mice is statistically significant at p = 0.04. The difference between the incidence of muscle weakness in non-tolerized and totl82-198-tolerized mice did not attain statistical significance. (From Shenoy et al., 1993.)

Table 2. Effect of neonatal tolerance to tAChR and tAChR o-chain peptides on serum autoantibody to mAChR and lymphocyte proliferative response

Neonatal injection

Antibody to mAChRa (Xl@‘“M)

[-iHI Thymidine uptakeb (ACPM f SEM)

rAChR t(u146-162

6.89 f 1.85 tAChR 1.68 ?Z 1.85 a146-162 (Torpedo) 3.14 t 1.31 a182-198 (human) 4.87 + 1.73

18.4 XL 4.7 2.8 ?c 0.5 c-u146-162 (Torpedo) 15.4 + 10.3 1.3 * 0.5

aExpressed as bungarotoxin binding sites precipitated per liter of serum. hValues represent mean [3H] thymidine uptake of triplicate cultures (x10-3 2 SEM). [XH]

Thymidine incorporation is expressed as cpm, with background (cells without antigen) values subtracted (Acpm). (From Shenoy et al., 1993.)


EAMG pathogenesis. Neonatal tolerance to peptide tcx146162 could have caused specific clonal deletion, and/or clonal anergy, and/or recruited suppressor cells to prevent clinical EAMG. Presumably, epitope(s) within t(u146-162, in the context of the I-Apb region 67-71, stimulate(s) specific T-helper cells which interact with specific B cells to produce pathogenic antibodies which cause the end plate lesion in patients with MG.

Epitope-specific suppression of antibody response in EAMG by a monomethoxypolyethylene glycol (mPEG) conjugate of a synthetic peptide

The majority of the autoantibodies in EAMG is directed against the main extracellular part of the c-w-subunit of AChR. As described in the preceding sections, the mapping of the complete antibody recognition profile of the entire extracellular part

of the tAChR a-chain demonstrated that the peptide ta125-148 contains a major antigenic site (Mulac-Jericevic et al., 1987), is a potent region for induction of EAMG (Lennon et al., 1985) and is involved in the binding of ACh to AChR (McCormick and Atassi, 1984). We have shown in recent studies (Atassi et al., 1992) that a peptide &x125-14%mPEG conjugate suppresses the development of the tAChR-induced EAMG in B6 mice by electrophysiological criteria.

Previous studies have shown that conjuga- tion of mPEG, or PVA with protein antigens causes a loss of most of the antigenicity of the antigens (Abuchowski et al., 1977; Lee and Sehon, 1977; Davis et al., 1980; Savoca et al., 1984; Sehon and Lang, 1986), and prior injection of animals with antigen-mPEG conjugates leads to the development of tolerance to subsequent immunization with the native antigen (Lee and Sehon, 1977; Savoca et al., 1984). In more recent studies,

Fig. 16. An example of electrophysiological findings from a normal mouse and an EAMG-positive mouse. (A) EAMG response of a normal mouse to a train of 3 Hz repetitive stimulation. (B) A typical decremental response (-33%) in an EAMG-positive mouse. (C) The decremental amplitude in (B) was substantially restored towards normal (-13%) 3 min after an i.p. injection of 250 pg of edrophonium chloride. (From Atassi et al., 1992.)


we described the synthesis of mPEG-peptide conjugates (Atassi and Manshouri, 1991). We have also shown that treatment with an mPEG-peptide conjugate prior to immunization with the intact protein resulted in suppression of the antibody response specific for the respective region without effect on the antibody responses to other antigenic sites of the protein (Atassi et al., 1992). Injection of mice with mPEG-&x125-148 and subsequent immunization with whole tAChR suppressed the development of EAMG by electrophysiological criteria (Figs 16 and 17). In anti-tAChR sera from these animals, the antibody response against unconjugated to1125-148 was decreased, while the antibody responses against whole tAChR and other epitopes were not altered (Fig. 18) (Atassi et al., 1992). There were no de-

Group 1 Group 2 n-25 n=27

-6O- .

-5o- ??

-4o-

. -3o- . .

.

tectable changes in T-cell proliferation responses to t&125-148 or to whole tAChR in these animals. Prior injections with a ‘nonsense’ peptide-mPEG conjugate had no effect on responses to the subsequent immunization with whole tAChR. The results indicated that the mPEG-&x125-148 conjugate has epitope-specific tolerogenicity for antibody responses in EMAG and that the tAChR a-subunit region t(r125-148 plays an important pathophysiological role in EAMG (Atassi et al., 1992). The epitope- directed tolerogenic conjugates may be useful for future immunotherapies of human MG. The strategy of specific suppression of the antibody response to a predetermined epitope by using a synthetic mPEG-peptide conjugate may prove useful in manipulation and suppression of unwanted immune re-

Group 3 Group 4 n-16 n=24

0

Fig. 17. Effects of prior administration of mPEG-tAChRtcul25148 on the development of electrophysiological EAMG (see Fig. 16). Note that in group 1 mice, both the mean amplitude change and the proportion of mice showing >lO% decrement were smaller than in group 2 or 3 @ < 0.05) but greater than in group 4 0, < O.OS), suggesting that the mPEG-AChRtol25-148 conjugate suppresses development of electrophysiological EAMG although the suppression is incomplete. Prior to immunization with tAChR, the mice received: (Group 1) mPEG-tAChRa(l25-148), (Group 2) mPEG-nonsense peptide conjugate, (Group 3) unaltered free peptide tAChRo(125-148). (Group 4) none. (From Atassi et al., 1992.)


10 55

t B

Fig. 18. Effects of preadministration of mPEG-tAChRoll25-148 on the antibody response to immunization with tAChR. The anti-rAChR antisera from the three groups of mice (groups l-3) were studied for antibody binding to tAChRa125-148 (A), whole rAChR (B), tAChR&-60 (C), and tAChRa182-198 (D). In A. group 1 mice showed significant suppression of the antibody population that binds to tAChRn125-148 (mean net cpm f standard deviation = 1414 +- 1801) compared with the mice in group 2 (3334 k 2318, p < 0.05) and group 3 (3626 f 2214, p < 0.05; by the f test). Antibodies against whole receptor (B) (JJ > 0.5), tAChRa45-60 (C) (p > O.l-OS), and tAChRu182-198 (D) (p > O.lWJ.5) showed no significant suppression in group 1 compared with the control groups. The groups of mice are described in Fig. 17. (From Atassi er al., 1992.)

sponses such as autoimmunity and allergy (Atassi et al., 1992).

Autoimmune recognition of human AChR in myasthenia gravis

Mapping of the sites of autoimmune T-cell recognition

To determine, at the epitope level, the role of T-cell recognition of hAChR in MG, it was necessary to obtain human MG T cells in quantities that are sufficient to map in vitro the T-cell recognition profile of the entire extracellular part of the a-chain

of hAChR. Pure hAChR was difficult to obtain in amounts needed for passage in vitro of autoreactive T cells from MG patients in order to prepare tAChR-specific autoimmune T-cell lines. Others have used tAChR for passage in vitro of human PBL from MG patients (Hohlfeld et al., 1988; Harcourt et al., 1988; Melms et al., 1988). But in spite of the extensive homology between Torpedo and human AChR (about 80% ; Noda et al., 1982, 1983b), this method will select for, and cause expansion of, only the human T cells that are cross-reactive with tAChR. In recent work (Oshima et al., 1990), we took advantage of a technique we


had introduced (Yoshioka et al., 1983) which employs passage in vitro with a selected peptide to obtain peptide-specific T cell lines and clones. We used, for passage of PBL from MG patients, a panel of 18 uniform sized overlapping synthetic peptides which encompass the entire extracellular part of the hAChR c-w-chain (Mulac-Jericevic et al., 1988). The response of these hAChR- specific T-cell lines to each of the individual peptides enabled the mapping of the autoimmune T-cell recognition sites on the hAChR a-chain (Oshima et al., 1990). It was found that the profiles of peptides recognized by the T cells (Fig. 19) were different among the five human MG T-cell lines, consistent

Recognition profiles of Human AChR-specific T-Cell lines from MG patients

with genetic control operating at the auto- determinant level. However, other regulatory influences may play important roles in the triggering of the autoimmune responses. The results suggest that the pathogenesis of this autoimmune disease is variable at the cellular-molecular level (Oshima et al., 1990).

The autoantibody-binding regions on the a-chain of human AChR in MG

Myasthenia gravis (MG) is a disabling autoimmune disease in which autoantibodies are produced against hAChR and inhibit its regulatory activity (Appel et al., 1975; Berman

LBA Y.dl. . l.SS EC”

I

PD Y.dl. . n*eo CCY I

jrl

lid II,.!

Fig. 19. Autoimmune response recognition profile by the T-cell lines prepared from five MG patients by passage in vitro with an equimolar mixture of the 18 synthetic peptides of human AChR shown in Fig. 4. (From Oshima et al., 1990.)


and Patrick, 1980; Engel, 1984). Sera of MG patients contain autoantibodies which compete with cholingeric agents for binding to a-subunit (Lindstrom et al., 1976; Falpius et al., 1980. In recent work (Ashizawa et al., 1992), we have employed the aforementioned set of synthetic overlapping peptides encompassing the entire extracellular part (residues hal-210) of the a-chain of hAChR and a 19th peptide (residues a262-276), corresponding to an extracellular connec- tion between two transmembrane regions, for the measurement of the binding of autoantibodies in sera from MG patients. Autoantibodies were found to recognize only a limited number of the synthetic peptides (Fig. 20). The regions recog-

MG

nized resided predominantly within peptides ha1&30, halll-145 and ha175-198 and, less frequently, ha45-77. Differences in the recognition profile from patient to patient indicated that the autoantibody responses were under genetic control at the autodeter- minant level. However, by using a mixture of the appropriate peptides, it was possible to determine autoantibodies in all 15 MG sera and to distinguish between these, normal human sera and other neurological or autoimmune diseases (Fig. 21). The mapping of the continuous autoantigenic regions on the a-chain of hAChR has permitted a comparison of the regions recognized by autoantibodies (Ashizawa et al., 1992) and autoimmune T-cells (Oshima et al., 1990)

Control

m

0 .I t ??

Peptide Number

Fig. 20. Summary of the binding profiles of the autoantibodies in MG plasma and normal human plasma to the synthetic peptides of hAChR a-chain (Fig. 4). (From Ashizawa et al., 1992.)


10

n-15 n-15 n-15 .

.

.

t

; . .

( Fig. 21. Binding of antibodies in plasma samples from 15 MG patients, 15 individuals suf- fering from other neurological or autoimmune diseases (Control 1) and 15 normal individuals (Control 2) to a mixture of the peptides hu12-27, halll-126, ha122-138 and h&32-198. The plasma samples were diluted (1:200, v/v) with PBS containing 0.2% casein. The MG plasma samples showed significant binding (mean net cpm 5278 + 1398, p < 0.001) compared to the other diseases (Control 1, mean net cpm 956 k 652) and the normal plasma samples (Control 2, mean net cpm 518 k 564). (From Ashizawa et al., 1992.)

0

Control 1 Control 2

Comparison of Autoantibody and Autoimmune T-Cell recognition of Human AChR in MG patients

DONOR S8 Antibody

DONOR RL

Antibody !J! -- - - -

1 Cella - - - -

DONOR LBA Antibody

T Cells II_

I I 1 1 I I I 1 I I I I 20 40 60 80 100 120 140 160 180 200

Residue Number of AChR Alpha Chain

Fig. 22. Comparison of autoimmune antibody and T-cell peptide recognition profiles of the extracellular part of human AChR a-chain in three MG patients. The antibody-binding profiles were determined by Ashizawa et al. (1992). The T-cell recognition profiles for T-cell lines originating from the same three donors were obtained from Oshima et ul. 1990). The bars indicate the locations of the stimulating peptides in the sequence. Solid bars represent a strong response while open bars denote an intermediate response. (From Ashizawa et al., 1992.)


from the same donor (Fig. 22). It also provided a peptide-based direct antibody binding method for diagnosis of MG (Ashizawa et al., 1992).

Presently, one of the tests for MG relies on a RIA for anti-hAChR autoantibodies in human sera. Only traces of hAChR in crude preparations are obtainable from muscle ex- tracts and therefore directly-labeled hAChR cannot be used for RIA. The muscle extract is allowed to bind t251-labeled BTX. The hAChR-BTX complex is used for assay of the autoantibodies (Lindstrom et al., 1976). It has been shown, however, that sera of MG patients contain autoantibodies which compete with BTX for the binding sites of AChR (Falpius et al., 1980), particularly when these sera are used in excess (Vincent and Newson-Davis, 1985). Therefore, this method often yields very low or false negatives. A major toxin binding region was found (Mulac-Jericevic and Atassi, 1987a,b; Mulac-Jericevic et al., 1988) to reside within the peptide h&22-138 and two minor regions occur within peptides hcx34-49 and ha194210. The finding that peptide ha122-138 also contains an autoantigenic region in most MG sera would explain the false negatives obtained with the method that relies on the precipitation by the autoantisera of the i2sI-labeled BTX-AChR complex. Determination of the autoantibodies by a peptide-cocktail based assay was shown to reduce ambiguous diagnosis (Ashizawa et al., 1992). Furthermore, the region a122-138 carries contact residues of ACh (McCormick and Atassi, 1984). The finding that autoantibodies also bind to this region provides a molecular explanation for the dysfunction of AChR in MG (Ashizawa et al., 1992).

synthetic strategy, previously introduced by this laboratory. This enabled the synthetic localization on AChR of both species of the profiles of the regions recognized by long and short a-neurotoxins and the 3-D construction of the cavity, on hAChR, which binds the long neurotoxin, BTX. Structural description of the toxin-binding cavity as well as the acetylcholine-binding site is crucial for the understanding of the molecular ion flux across the membrane and its regulation by acetylcholine binding and inhibition by a-neurotoxin. The comprehensive synthetic strategy was also used to map the antibody and T-cell recognition regions in immune responses to tAChR, as well as the profile of regions on tAChR or hAChR recognized by autoantibodies and by autoimmune T cells in EAMG-susceptible mice and in MG patients. These investigations have been extremely valuable in understanding the molecular and cellular events of immune and autoimmune responses. The studies also enabled the identification of some regulatory and/or pathogenic epitopes which were successfully employed for manipulation and prevention of the disease in mice. This has opened up the exciting capability that appropriate synthetic epitopes may be used in the future for control and manipulation of unwanted immune responses (such as autoimmunity and allergy).

Acknowledgements

The work reviewed in this article was sup- ported by a grant (number NS26280) from the National Institutes of Health. The author wishes to thank the Welch Foundation for the award of the Robert A. Welch Chair in Chemistry.

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Molecular recognition of acetylcholine receptor. Recognition by α-neurotoxins and by immune and autoimmune responses and manipulation of the responses