Drorit Neumann

In vitro proliferative responses and antibody titers specific to human acetylcholine receptor synthetic peptides in patients with myasthenia gravis and relation to HLA class II genes.

To investigate which parts of the acetylcholine receptor are involved in the initiation and development of myasthenia gravis (MG), peptides representing different sequences of the human acetylcholine receptor alpha-subunit were synthesized. These peptides were tested for their ability to stimulate T cells of myasthenic patients and healthy control patients in proliferation assays and to bind to sera antibodies. Three of eight peptides discriminated significantly between the two groups in the proliferation assay, as well as in their ability to bind to serum antibodies. HLA-DR3 and DR5 were associated with proliferative responses to specific AChR peptides in the group of myasthenics. Acetylcholine receptor epitopes that might play a specific role in myasthenia gravis thus were demonstrated.

Regulation of mRNA levels for microtubule proteins during nerve regeneration

The molecular regulation of tubulin synthesis was investigated in the regenerating goldfish retina. Previous in vivo studies pointed to an increase in tubulin synthesis in the retina during regeneration of the injured goldfish optic nerve. Using labeled cDNA probes, we showed that this increase occurs as a result of enhanced tubulin mRNA levels. Analysis of labeled in vivo products revealed enhanced β2‐tubulin synthesis accompanied by an increase in the level of the low‐M r microtubule‐associated proteins identified as TAU factors. The results are discussed with respect to the possible involvement of these proteins in the process of nerve regeneration.

Intracellular Retention and Degradation of the Epidermal Growth Factor Receptor, Two Distinct Processes Mediated by Benzoquinone Ansamycins

Epidermal growth factor (EGF) stimulates the growth of various types of cells via its cell surface tyrosine kinase receptor. The EGF receptor (EGF-R) has an oncogenic potential when overexpressed in a wide range of tumor cells. Geldanamycin (GA) and herbimycin (HA), specific inhibitors of the cytosolic chaperone HSP 90 and its endoplasmic reticulum homologue GRP 94, were shown to accelerate degradation of the EGF-R and of its homologue p185c- erbB-2 . Here we compared the effects of GA and HA on intracellular degradation and maturation of EGF-R. By using an inhibitor of proteasomal degradation, we learned that GA, but not HA, blocks processing of newly synthesized EGF-R. The effects of GA and HA on receptor degradation are mediated by the cytosolic portion of EGF-R and could be conferred to the erythropoietin receptor (EPO-R), by employing the respective chimera. Neither HA nor GA affected stability of newly synthesized EGF-R lacking the cytosolic domain (Ex EGF-R), but GA caused intracellular retention of this mutant. Taken together, our results imply that GA has two distinct targets of action on the EGF-R, one for promoting its degradation and another for mediating its intracellular retention. Apparently, degradation of the EGF-R mediated by GA or HA requires the presence of the EGF-R cytosolic domain, whereas intracellular retention in the presence of GA is coupled to the extracellular domain of the EGF-R.

Localization of a highly immunogenic region on the acetylcholine receptor α-subunit

Abstract Antibodies to synthetic peptides were employed in order to map domains on the α-subunit of the acetylcholine receptor to which several monoclonal antibodies are directed. Five peptides corresponding to residues 1–20, 126–143, 169–181, 330–340 and 351–368 of the receptor α-subunit were synthesized and antibodies against them were elicited. The anti-peptide antibodies were employed along with the monoclonal antibodies to identify fragments of S. aureus V8 protease digested-α-subunit in immunoblotting experiments. Our results demonstrate that a highly immunogenic region of the α-subunit is located on a carboxy-terminal 14 kDa portion of the α-subunit. This region also seems to undergo antigenic changes during muscle development. A monoclonal antibody directed against the cholinergic binding site of the acetylcholine receptor reacted with an 18 kDa segment of the α-subunit which bound α-bungarotoxin as well as antibodies directed against peptide 169–181.

T-cell antigenic sites involved in myasthenia gravis: correlations with antibody titre and disease severity.

We have evaluated the ability of eight synthetic peptides corresponding to selected regions of the alpha-subunit from human (H) or Torpedo (T) acetylcholine receptor (AChR) to stimulate proliferative responses of peripheral blood lymphocytes (PBL) and thymic cells from patients with Myasthenia Gravis (MG) in comparison to healthy controls. Using PBL, two of the peptides were most reactive: in the 40 myasthenic patients tested, peptide 169-181 (H) induced significant proliferative responses in 10 patients and peptide 351-368 (H) in five, while there was no response in any of the 34 healthy controls tested. Interestingly, clear associations between proliferation to peptides and clinical data were observed. Indeed, among responding patients, all presented thymic hyperplasia and most showed a high anti-AChR Ab titre and/or a severe form of the disease. In addition, responses to AChR cytoplasmic sequences were observed only in severely affected patients. Correlation with HLA-DR haplotype, sought in a subgroup of patients, indicated that response to 169-181 (H) is associated with HLA-DR5 in the patients presenting a high anti-AChR antibody titre. Using thymic lymphocytes, few responses were obtained with the human peptides, suggesting that the frequency of autoreactive cells is lower than in the blood. Similar to PBL, responses to peptides were observed only with lymphocytes isolated from hyperplastic thymuses. The correlations observed between responses to peptides and clinical parameters underline the pathophysiological relevance of our data and indicate that pathogenic and nonpathogenic T-cell antigenic sites involved in the anti-AChR response could be identified by this approach.

Acetylcholine receptor gene expression in experimental autoimmune myasthenia gravis.

Acetylcholine receptor (AChR) gene expression was analyzed in experimental autoimmune myasthenia gravis (EAMG) in rabbits, rats and mice. An increase in AChR transcripts was demonstrated to be exclusively associated with myasthénie symptoms and with a severe loss in membrane AChR. An increase of α‐, β‐, ϵ‐, and δ‐subunit specific mRNAs (5.2‐, 1.6‐, 3.2‐ and 3.7‐fold, respectively), which code for the adult type of AChR (α2βϵδ) was observed in EAMG in rats. The γ‐subunit transcript was not detectable in myasthenie or healthy rats. It appears that the regulatory control of AChR gene expression in EAMG is different from that observed upon denervation.

Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

Three peptides corresponding to residues 354‐367, 364‐374, 373‐387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP‐dependent protein kinase, the tyrosine‐specific protein kinase and the calcium/phospholipid‐dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP‐dependent protein kinase it was shown that only peptides 354‐367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP‐dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non‐phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP‐dependent protein kinase. Antibodies to peptide 354‐367 were found to react preferentially with non‐phosphorylated receptor whereas the two other anti‐peptide antibodies bound equally to phosphorylated and non‐phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms.

Anti-acetylcholine receptor response achieved by immunization with a synthetic peptide from the receptor sequence.

A synthetic peptide corresponding to the first twenty amino acids of the N-terminal region from the alpha-subunit of the Torpedo acetylcholine receptor cross reacts with antibodies to the receptor. A conjugate of this peptide to bovine serum albumin elicits in rabbits an immune response towards the synthetic peptide as well as towards the acetylcholine receptor. Blotting experiments demonstrate that the antipeptide antibodies react exclusively with the alpha-subunit of the acetylcholine receptor. Antibodies against synthetic peptides from various regions of the receptor sequence may provide useful reagents for structural and developmental analysis of the acetylcholine receptor as well as for the regulation of experimental autoimmune myasthenia gravis.

Increased levels of acetylcholine receptor α‐subunit mRNA in experimental autoimmune myasthenia gravis

To gain insight into the regulatory mechanisms underlying the blockade and loss of acetylcholine receptor (AChR) in myasthenia, we have followed AChR α‐subunit mRNA levels in leg muscles of myasthenic and normal rabbits and rats. Northern blots of RNA preparations from normal and myasthenic animals were hybridized with a mouse AChR α‐subunit cDNA probe. Our experiments indicate a specific increase (4–7‐fold) in the levels of α‐subunit mRNA in animals with experimental autoimmune myasthenia gravis (EAMG), in comparison with control animals. Actin mRNA levels were essentially unchanged. Our results thus suggest that EAMG is accompanied by an increased level of AChR gene transcription.

Mapping of the cAMP-dependent phosphorylation sites on the acetylcholine receptor.

We have synthesized a tetradecapeptide corresponding to residues 354‐367 of the delta‐subunit of Torpedo acetylcholine receptor. This peptide contains the sequence Arg‐Arg‐Ser‐Ser which has been proposed as the site for phosphorylation of the acetylcholine receptor (AChR) by an endogenous cAMP‐dependent protein kinase. We have shown that the synthetic peptide can be phosphorylated by the catalytic subunit of bovine heart cAMP‐dependent protein kinase. Antibodies elicited against peptide 354‐367 were shown to cross‐react with native AChR and to bind specifically to the delta‐ and gamma‐subunit as detected by immunoblotting. Furthermore, antipeptide antibodies were shown to inhibit specifically the cAMP‐dependent phosphorylation of both the delta‐ and gamma‐subunits. This suggests that the phosphorylation sites in the delta‐ and gamma‐subunits are highly cross‐reactive, and is in agreement with the demonstration that an endogenous cAMP‐dependent kinase phosphorylates these two subunits, probably on homologous sequences. Tryptic digestion of the delta‐subunit isolated from phosphorylated AChR yields a single 25‐kd phosphorylated fragment. Immunoblotting experiments allowed us to map peptide 354‐367 within this phosphorylated fragment.