Effect Of Microbial Transglutaminase On The Structure And Function Of Gluten Peptides Related To Celiac Disease

Wheat is the main food crop,but it can also cause a variety of allergic diseases and autoimmune diseases.Celiac disease(CD)is a chronic intestinal inflammatory disease caused by ingestion of gluten protein in wheat,barley or rye,which is mediated by T cells and develops in genetically susceptible individuals.At present,there is no effective treatment for CD patients except a strict life-long gluten free diet(GFD).Therefore,it is worthwhile to develop wheat products with low celiac toxicity.Recent studies showed that transamidation mediated by microbial transglutaminase(mTG)could reduce the immunogenicity of gluten.33mer(LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF),DQ2.5-glia-?1a(LQPFPQPQL-PYPQ),DQ2.5-glia-?2(PFPQPQLPYPQPQ),gliadin and wheat flour were transamidated by mTG and acyl-acceptor molecules,including L-lysine(Lys),glycine ethyl ester(GEE),hydroxylamine(HA),L-lysine methyl ester(LME),6-Aminocaproic acid(6-AA)and n-butylamine(BL).RP-HPLC,LC-ESI-MS,and MALDI-TOF were performed to explore the effect of transamidation on the properties and structure of gluten peptides and LTQ-FT Ultra-MS was used to identify transamidated sites.R5 ELISA and G12 ELISA were used to evaluate the immunoreactivity of modified peptides and proteins.On the basis of T cell epitopes,anti-DQ2.5-glia-?1a and anti-DQ2.5-glia-?3 monoclonal antibodies were applied to evaluate the immunoreactivity of modified proteins.Moreover,gluten-specific T cells derived from CD patients were used to assess the toxicity of transamidated peptides and proteins.Finally,the effect of tissue transglutaminase(tTG)on the structure and CD toxicity of mTG transamidated peptides and proteins were explored.It will provide theoretical and experimental support for the development of wheat products with low CD toxicity.The main results and conclusions of this study are addressed as follows.1.Based on optimized conditions of transamidation,the purity of transamidated product was more than 99% by one-step mTG-catalyzed reaction.The isopeptide bond formed by mTG transamidation had strong stability.2.It was shown that pH had no significant effect on the transamidation catalyzed by mTG,while it did influence on the deamidation catalyzed by mTG.The deamidated activity of mTG was stronger at acidic pH within the effective range of enzyme activity.3.It was found that Lys,GEE and HA reacted to the same glutamine residue of peptides.Moreover,the transamination modification site is the same as the deamidation modification site catalyzed by mTG/tTG.Therefore,the transamination catalyzed by mTG can inhibit the deamidation of glutamine residues,which resulted in affecting the celiac toxicity of gluten and peptides.4.The competitive ELISA technique based on monoclonal antibody(mAb)was used to evaluate the immunoreactivity of transamidated peptides.Regarding R5 ELISA,the binding activity between R5 mAb and the 33 mer peptide modified with Lys,LME,GEE,6-AA and HA was decreased to 2.57%,1.59%,0.98%,1.66% and 5.17% of the control group,respectively;regarding G12 ELISA,the immunoreactivity of the modified peptides was decreased to 26.21%,29.59%,29.96%,30.26% and 50.22% of its initial level,respectively.5.Gluten-specific T cells were used to evaluate the immunogenicity of the transamidaed peptides and gliadin.It was found that the transamidation with Lys was most effective to abrogate the immune stimulatory properties of gluten while the coupling of smaller acyl-acceptormolecule was less(GEE)effective or mostly ineffective(HA).6.Based on T-cell epitopes and ELISA techniques,it was shown that transamidation can reduce the celiac toxicity of gluten of wheat steambread.7.It was found that tTG can catalyze the reverse reaction of mTG transamination,and further catalyze the deamidation.With the prolonged incubation time of tTG,the degree of reverse reaction catalyzed by tTG increased.Gluten protein-specific T cells were used to evaluate the immunogenicity of the mTG transamidated peptide after tTG treatement.It revealed that the treatment of tTG significantly enhanced the immunogenicity of mTG transamidated peptides and proteins,however,the immunogenicity of mTG transamidated gliadin after tTG treatment was much lower than the positive control.In conclusion,mTG-mediated transamidation using small-molecule acyl receptors had the potential to reduce the CD toxicity of gluten peptides and proteins,which provided a new strategy to solve the CD problem from food source.In addition,it is worthwhile to pay attention to that tTG can catalyze the reverse reaction of mTG transamination,which can generate gluten proteins and peptides with CD toxicity.Therefore,it is necessary to consider the effect of tTG,when evaluate CD toxicity of food.The findings of this study are helpful to improve the awareness of researchers in the field of medicine and food to the special diets for CD patients,as well as to promote the prevention and control of CD and the development of gluten-free food.