EBT, A TRYPTOPHAN CONTAMINANT ASSOCIATED WITH EOSINOPHILIA-MYALGIA-SYNDROME, IS INCORPORATED INTO PROTEINS DURING TRANSLATION AS AN AMINO-ACID ANALOG

The tryptophan dimer 1,1'-ethylidenebis[L-tryptophan] was identified a s a contaminant of tryptophan preparations associated with Eosinophili a-Myalgia Syndrome. In this paper, we describe experiments examining t he hypothesis that 1,1'-ethylidenebis[L-tryptophan] acts as an amino a cid analog replacing L-tryptophan during the synthesis of proteins, We propose further that proteins containing 1,1'-ethylidenebisF tryptoph an] are rejected in an autoimmune process identified clinically as Eos inophilia Myalgia Syndrome, Rabbit reticulocyte lysates containing an estimated 1 1 mu M L-tryptophan were used to assay the ability of 1,1' -ethylidenebis[L-tryptophan] to compete with H-3-L-tryptophan for inco rporation into proteins translated from BMV RNA, 1,1'-Ethylidenebis[L- tryptophan] in concentrations of 40, 80 and 110 mu M reduced lysate H -3-L-tryptophan incorporation to 81%, 76% and 75% of control incorpora tion obtained in the absence of 1,1'-ethylidenebis[L-tryptophan]. In t he presence of 20 mu M L-tryptophan, 110 mu M 1,1'-ethylidenebis[L-try ptophan] reduced H-3-L-tryptophan incorporation to 56% of control inco rporation, In contrast, ethyl-L-tryptophan did not significantly reduc e H-3-L-tryptophan incorporation. In the presence of 110 mu M 1,1'-eth ylidenebisF tryptophan] and 20 mu M L-tryptophan, H-3-L-leucine incorp oration was not significantly reduced compared to incorporation in the absence of 1,1'-ethylidenebis[L-tryptophan], demonstrating that prote ins were translated to full length during elongation, These findings s uggest that 1,1'-ethylidenebis[L-tryptophan], but not ethyl-l-tryptoph an, reduced 3H-L-tryptophan incorporation into proteins by substitutin g for Gtryptophan rather than by causing premature termination or sign ificant slowing of nascent protein chains, In further experiments, the ethylidene bridge of 1,1'-ethylidenebis[L-tryptophan] was labeled dur ing synthesis with C-14-acetaldehyde. C-14-1,1'-ethylidenebis[L-trypto phan] was added to rabbit reticulocyte lysates containing BMV RNA to a concentration of 110 mu M 1,1'-ethylidenebis[L-tryptophan]. On SDS-PA GE gels, bands of C-14-labeled proteins were detected at the 5 band po sitions expected from BMV RNA; i.e., at approximately 100, 97, 35, 20 and 15 kD. We conclude from these experiments that trytophanyl t-RNA s ynthetase can activate and attach 1,1'-ethylidenebis[L-tryptophan] to tryptophanyl-t-RNA and that 1,1'-ethylidenebis[L-tryptophan] can be in corporated into proteins in place of L-tryptophan. To determine whethe r Eosinophilia-Myalgia Syndrome was associated with sensitization to 1 ,1'-ethylidenebis[L-tryptophan], we constructed artificial haptens to mimic proteins containing 1,1'-ethylidenebis[L-tryptophan] residues. H aptens were synthesized by coupling 1,1'-ethylidenebis[L-tryptophan], ethyl-l-tryptophan or L-tryptophan to albumin with glutaraldehyde. Per ipheral blood lymphocytes were isolated from patients stably recovered from Eosinophilia-Myalgia Syndrome for 2 yr or more and used in proli feration assays against lymphocytes from controls to determine if they were immunosensitive to synthesized haptens. Only one out of the five recovered Eosinophilia-Myalgia Syndrome patients demonstrated signifi cant reactivity to the synthesized hapten containing 1,1'-ethylidenebi s[l-tryptophan]. This patient also demonstrated an increased reactivit y to the synthesized hapten containing L-tryptophan. A second recovere d Eosinophilia-Myalgia Syndrome patient demonstrated increased sensiti vity to the synthesized hapten containing ethyl-L-tryptophan. These re sults provide inconclusive evidence that incorporated 1,1'-ethylideneb is[l-tryptophan] provides an immunogenic stimulus. However, the synthe sized haptens may not have sufficiently mimicked the configurations of proteins containing 1,1'-ethylidenebis [L-tryptophan] residues to tes t for sensitization or sensitization could have been lost in recovered patients following corticosteroid treatment and the turnover of affec ted proteins.