Identification of a single amino acid substitution in the diphtheria toxin A chain of CRM 228 responsible for the loss of enzymatic activity

Abstract

CRM 228 (T. Uchida, A. M. Pappenheimer, and R. Greany, J. Biol. Chem. 248:3838-3844, 1973), a mutant form of diphtheria toxin which completely lacks ADP-ribosyltransferase activity, contains five amino acid substitutions. The two amino acid changes that fall within the A chain of the toxin (G79D and E162K) were separately analyzed by substituting a variety of other amino acids at these sites. The substitution at position 79 (G79D) singularly appears to account for the loss of enzymatic activity found in CRM 228.Diphtheria toxin (DT; Mr, 58,342) is secreted by pathogenic strains of Corynebacterium diphtheriae. It is composed of an A and a B subunit; the A subunit (DTA) has ADP-ribosyltransferase (ADPRT) activity, catalyzing the transfer of ADP-ribose from NAD+ to a posttranslationally modified histidine residue, diphthamide, found in elongation factor 2 (12). This results in the inhibition of protein synthesis and ultimately in the death of the intoxicated cell. The toxin B subunit (DTB) is responsible for binding to cell surface receptors and for mediating the translocation of the A subunit through the endosomal membrane into the cytosol.Many of the initially identified structure-function relationships of the toxin were revealed by analysis of mutants produced by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis (29). Toxins with altered function but with cross-reactivity against polyclonal anti-DT antibodies were generated. These cross-reacting materials (CRMs) generally fall into one of two groups: (a) those which are deficient in binding and/or translocation function (CRMs 30, 45, 228 [30,31], 107, 102, 103 [11,20], 9 [13], and 1001 [7]) and (b) those with altered enzymatic activity (CRM 197, 228, and 176 [24,29,30,31] E162K) and three within DTB (S197G, P378S, and G431S) (18). It has 15 to 20% of the binding activity of native DT and has no ADPRT activity (30, 31). It was not known whether the lack of ADPRT activity can be attributed to just one or to both of the DTA substitutions. In an attempt to clarify this, the two affected DTA chain sites from CRM 228 (positions 79 and 162) were independently mutagenized; the native amino acid at each of these sites was substituted with a variety of other amino acids, and the relative enzymatic activities of the mutant proteins were compared.Mutagenesis at position 79 or 162 was carried out with a PCR-based mutagenesis system as previously described (14). Random nucleotide substitutions at positions 235 to 237 (encoding amino acid 79) or at positions 484 to 486 (encoding amino acid 162) enabled a variety of amino acids to be substituted at these sites (Fig. 1). PCR products were cloned into the Bluescript KS+ vector in a region downstream of the T7 RNA polymerase promoter, and Escherichia coli XL1 Blue was transformed with the recombinant library. The cloned region of the toxin gene encodes amino acids 1 to 193 corresponding to the enzymatically active form of DTA (32), without DTB, so the construct is in compliance with the restrictions for cloning toxin genes (8).P...

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