ABSTRACT
Transglutaminase 2 (TG2) catalyzes transamidation or deamidation of its substrates and is ordinarily maintained in a catalytically inactive state in the intestine and other organs. Aberrant TG2 activity is thought to play a role in celiac disease, suggesting that a better understanding of TG2 regulation could help to elucidate the mechanistic basis of this malady. Structural and biochemical analysis has led to the hypothesis that extracellular TG2 activation involves reduction of an allosteric disulfide bond by thioredoxin-1 (TRX), but cellular and in vivo evidence for this proposal is lacking. To test the physiological relevance of this hypothesis, we first showed that macrophages exposed to pro-inflammatory stimuli released TRX in sufficient quantities to activate their extracellular pools of TG2. By using the C35S mutant of TRX, which formed a metastable mixed disulfide bond with TG2, we demonstrated that these proteins specifically recognized each other in the extracellular matrix of fibroblasts. When injected into mice and visualized with antibodies, we observed the C35S TRX mutant bound to endogenous TG2 as its principal protein partner in the small intestine. Control experiments showed no labeling of TG2 knock-out mice. Intravenous administration of recombinant TRX in wild-type mice, but not TG2 knock-out mice, led to a rapid rise in intestinal transglutaminase activity in a manner that could be inhibited by small molecules targeting TG2 or TRX. Our findings support the potential pathophysiological relevance of TRX in celiac disease and establish the Cys370-Cys371 disulfide bond of TG2 as one of clearest examples of an allosteric disulfide bond in mammals.
Subject(s)
Celiac Disease/metabolism , GTP-Binding Proteins/metabolism , Intestine, Small/metabolism , Thioredoxins/metabolism , Transglutaminases/metabolism , Animals , Celiac Disease/genetics , Celiac Disease/pathology , Cell Line , GTP-Binding Proteins/genetics , Humans , Intestine, Small/pathology , Mice , Mice, Knockout , Protein Glutamine gamma Glutamyltransferase 2 , Thioredoxins/genetics , Transglutaminases/geneticsABSTRACT
The controlled attachment of synthetic groups to proteins is important for a number of fields, including therapeutics, where antibody-drug conjugates are an emerging area of biologic medicines. We have previously reported a site-specific protein modification method using a transamination reaction that chemoselectively oxidizes the N-terminal amine of a polypeptide chain to a ketone or an aldehyde group. The newly introduced carbonyl can be used for conjugation to a synthetic group in one location through the formation of an oxime or a hydrazone linkage. To expand the scope of this reaction, we have used a combinatorial peptide library screening platform as a method to explore new transamination reagents while simultaneously identifying their optimal N-terminal sequences. N-Methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport's salt, RS) was identified as a highly effective transamination reagent when paired with glutamate-terminal peptides and proteins. This finding establishes RS as a transamination reagent that is particularly well suited for antibody modification. Using a known therapeutic antibody, herceptin, it was demonstrated that RS can be used to modify the heavy chains of the wild-type antibody or to modify both the heavy and the light chains after N-terminal sequence mutation to add additional glutamate residues.
Subject(s)
Aldehydes/chemistry , Proteins/chemistry , Pyridinium Compounds/chemistry , Amination , Amino Acid Sequence , Glutamic Acid/chemistry , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Indicators and Reagents , Models, Molecular , Peptide Library , Peptides/chemistry , Receptor, ErbB-2/immunologyABSTRACT
Notwithstanding an extensive literature on assembly line polyketide synthases such as the 6-deoxyerythronolide B synthase (DEBS), a complete naturally occurring synthase has never been reconstituted in vitro from purified protein components. Here, we describe the fully reconstituted DEBS and quantitatively characterize some of the properties of the assembled system that have never been explored previously. The maximum turnover rate of the complete hexamodular system is 1.1 min(-1), comparable to the turnover rate of a truncated trimodular derivative (2.5 min(-1)) but slower than that of a bimodular derivative (21 min(-1)). In the presence of similar concentrations of methylmalonyl- and ethylmalonyl-CoA substrates, DEBS synthesizes multiple regiospecifically modified analogues, one of which we have analyzed in detail. Our studies lay the foundation for biochemically interrogating and rationally engineering polyketide assembly lines in an unprecedented manner.
