Novel immunotoxin: a fusion protein consisting of gelonin and an acetylcholine receptor fragment as a potential immunotherapeutic agent for the treatment of Myasthenia gravis.
In continuation of our attempts for antigen-specific suppression of the immune system [I.L. Urbatsch, R.K.M. Sterz, K. Peper, W.E. Trommer, Eur. J. Immunol. 23(1993) 776-779] a novel fusion protein composed of amino acids 4-181 of the extracellular domain of the alpha-subunit of the human muscle acetylcholine receptor and the plant toxin gelonin was expressed in Escherichia coli. The fusion protein formed inclusion bodies but could be solubilized in the presence of guanidinium hydrochloride. After a simple two step purification and refolding procedure, it exhibited a native structure at least in the main immunogenic region as shown by antibodies recognizing a conformational epitope. Half maximal inhibition of translation was achieved at 46 ng/ml as compared to 4.6 ng/ml for native and 2.4 for recombinant gelonin. Its use as therapeutic agent for the treatment of Myasthenia gravis was investigated in an animal model. Female Lewis rats were immunized with complete acetylcholine receptor from the electric ray Torpedo californica and developed thereafter experimental autoimmune M. gravis. Quantitative assessment of the disease was achieved by repetitive stimulation of the Nervus tibialis. Rats showed no symptoms of M. gravis, neither visually nor electrophysiologically after treatment with the fusion protein as determined one and seven weeks after the second application. This approach may also be useful for the therapy of further autoimmune diseases by substituting other autoantigens for the AchR fragment in the fusion protein.
Hossann
Martin
M
Department of Chemistry, TU Kaiserslautern, P.O. Box 3049, D-67653 Kaiserslautern, Germany.
Li
Zhuoyu
Z
Shi
Yawei
Y
Kreilinger
Ulrike
U
Büttner
Jörn
J
Vogel
Pia D
PD
Yuan
Jingming
J
Wise
John G
JG
Trommer
Wolfgang E
WE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005260:Female; D005813:Genes, Synthetic; D006801:Humans; D000922:Immunotoxins / Q000627:therapeutic use*; D023421:Models, Animal; D008969:Molecular Sequence Data; D009157:Myasthenia Gravis / Q000188:drug therapy* / Q000276:immunology*; D010446:Peptide Fragments; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000627:therapeutic use; D051381:Rats; D011917:Rats, Inbred Lew; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000627:therapeutic use; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use*; D015183:Restriction Mapping; D054789:Ribosome Inactivating Proteins, Type 1; D014101:Torpedo
D000922:Immunotoxins; D010446:Peptide Fragments; D010940:Plant Proteins; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C025674:GEL protein, Gelonium multiflorum