Summary
Reductive methylation of botulinum neurotoxin (NT) serotypes A and B at various ratios of protein to reagent modified up to 75° 10 of the lysine residues. Amino acid analysis of the modified proteins (HCl hydrolysed) confirmed selective modifications of lysine. The derivative N,N-dimethyl lysine was more abundant than monomethyl lysine; trimethyl lysine was not detected. Distribution of modified lysine residues among the heavy and light chains (Mr ∼100000 and ∼50000, respectively) of the dichain type A NT (Mr ∼150000) was approximately proportional to the lysine contents of the two subunit chains of the NT. Toxicity (mouse lethality) and serological reactivity (polyclonal antibody) of serotype A NT were not (or insignificantly) damaged following methylation of up to 72 lysine residues. Modification of 3 additional residues caused precipitous loss in toxicity. Toxicity of serotype B NT, unlike type A, appeared more sensitive to lysine modification. The large number of lysine residues that can be methylated without damaging toxicity of type A NT can be exploited to a) radiolabel the dichain protein exclusively in one chain keeping the other chain unlabelled, b) restrict the number of tryptic cleavage sites of the NT, and c) tag the protein with various markers or reactive ligands.
Similar content being viewed by others
References
DasGupta BR, Sugiyama H: Biochem Biophys Res Commun 48:108–112, 1972
Sathyamoorthy V, DasGupta BR: Biochem Biophys Res Commun 127:768–772, 1985
Schmidt JJ, Sathyamoorthy V, DasGupta BR: Arch Biochem Biophys 238:544–548, 1985
Kozaki S, Miyazaki S, Sakaguchi G: Infect Immun 18:761–766, 1977
Kozaki S, Togashi S, Sakaguchi G: Jpn J Med Sci Biol 34:61–68, 1981
Syuto B, Kubo S: J Biol Chem 256:3712–3717, 1981
Sathyamoorthy V, DasGupta BR: J Biol Chem 260:10461–10466, 1985
Bandyopadhyay S, Clark AW, DasGupta BR, Sathyamoorthy V: J Biol Chem 262:2660–2663, 1987
Simpson LL: J Pharmacol Exp Ther 212:16–21, 1980
Simpson LL: Annul Rev Pharmacol Toxicol 26:427–453, 1986
Dolly JO, Black J, Williams RS, Melling J: Nature 307:457–460, 1984
Ackers GK, Smith FR: Ann Rev Biochem 54:597–629, 1985
DasGupta BR, Sathyamoorthy V. Toxicon 22:415–424, 1984
DasGupta BR, Woody M: Toxicon 22:312–315, 1984
Weber K, Osborn M: J Biol Chem 244:4406–4412, 1969
Means GE, Feeney RE: Biochemistry 7:2192–2201, 1968
DasGupta BR, Sugiyama H: Infect Immun 6:587–590, 1972
DasGupta BR, Rasmussen S: Arch Biochem Biophys 232:172–178, 1984
DasGupta BR, Rasmussen S: Biochem Biophys Res Commun 101:1209–1215, 1981
DasGupta BR, Rasmussen S: Toxicon 21:566–569, 1983
Kuehl WM, Adelstein RS: Biochem Biophys Res Commun 37:59–65, 1969
Wellhoner HH: Rev Physiol Biochem Pharmacol 93:1–68, 1982
DasGupta BR, Sugiyama H: In: Bernheimer AW (ed) Perspectives in Toxinology. John Wiley & Sons, New York, 1977, pp 87–119
Schmidt JJ, Sathyamoorthy V, DasGupta BR: Biochem Biophys Res Commun 119:900–904, 1984
Eisel U, Jarausch W, Goretzki K, Henschen A, Engels J, Weller U, Hudel M, Habermann E, Heiner N: EMBO J 5:2495–2502, 1986
Schmitt A, Dreyer F, John C: Naunyn-Schmiedebergs Arch Pharmacol 317:326–330, 1981
Simpson LL: J Pharmacol Exp Ther 229:182–187, 1984
Sellin LC, Thesleff S, DasGupta BR: Acta Physiol Scand 119:127–133, 1983
Kozaki S, Sakaguchi G, Nishimura M, Iwamori M, Nagai Y FEMS Microbiol Lett 21:219–223, 1984
Montecucco C: Trends in Biochemical Sciences 11:314–317, 1986
Poncz L, Dearborn DG: J Biol Chem 258:1844–1850, 1983
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Sathyamoorthy, V., DasGupta, B.R. Reductive methylation of lysine residues of botulinum neurotoxin types A and B. Mol Cell Biochem 83, 65–72 (1988). https://doi.org/10.1007/BF00223199
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00223199