Digestibility, Allergenicity and the Safety of Novel Proteins

Abstract

Currently, the in vitro pepsin digestion assay is used as part of the safety assessment for genetically modified (GM) crop plants and the foods derived from them. It has been proposed that allergenic proteins are more resistant to digestion with pepsin than non-allergens (Astwood et al., 1996). However, this association has been found not to be absolute. Pepsin is found extracellularly in the stomach, and it is believed that food proteins must remain sufficiently intact in order to be recognised by the immune system and initiate an immune or allergic response. The aim of the current investigations was to determine whether there was a stronger association between resistance to proteolytic digestion within antigen presenting cells (APC) than with pepsin. Dendritic cells and macrophages both act as APC. Thus, murine bone marrow derived - dendritic cells (BM-DC) were cultured for 14 days in the presence of granulocyte macrophage - colony stimulating factor (GM-CSF), BM derived- macrophages were cultured for 9 days in the presence of macrophage colony stimulating factor (M-CSF) and mesenteric lymph node (MLN), peripheral lymph node (PLN) and spleen cells were characterised with particular interest in the DC population of each tissue. Antigen presenting cells were characterised for their surface marker expression, endocytic activity and intracellular expression of cathepsins. Cathepsins are enzymes found in APC known to be involved in antigen processing. Immature BM-DC were found to be endocytic at the later time points (day 12 and 14) and to express high levels of cathepsins D, B and L and low levels of cathepsins E and S. Mesenteric lymph node - DC, PLN-DC and spleen-DC were also found to express cathepsins. Again cathepsins B, L and D were expressed more highly than cathepsins E and S. Recombinant mouse cathepsin D (a highly expressed cathepsin) was used to develop an assay similar to the pepsin digestion assay to compare the digestibility of known allergens with putative non-allergens. A correlation between resistance to digestion with cathepsin D and allergenicity was found, although as already observed for the pepsin digestion assay, there were some false positive and some false negative results. In summary, the question that asks whether there is a relationship between protein allergenicity and proteolytic susceptibility within APC needs to be investigated further, perhaps by studying the digestibility of known allergens and non-allergens with other cathepsins identified inside APC. It must also be acknowledged that resistance to digestion is not the only criterion used to assess whether a food protein has allergenic potential, and that another factor not yet identified may be important in determining whether a Th2 response and allergic sensitisation or elicitation results.

Date of Award	31 Dec 2011
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ian Kimber (Supervisor)