Final Report Summary - LUPIN-CHALLENGE (Characterization of Lupin B-Conglutin Seed Proteins with a Focus on Health Benefits and their Role in Allergenicity)
The research proposed endeavours to reveal the health promoting aspects of Lupinus angustifolius (L.), or narrow-leafed Lupin (NLL) grain such as the prevention of diabetes and obesity, as well as help with problems associated with food allergies through multidisciplinary approaches.
The number of adult-onset or Type-2 diabetes is in rapid increment worldwide, where incidence among younger people is quite alarming, mostly due to life style habits reflected in overweight people which increase the risk of contracting diabetes, conditions that has been denominated with a new term “the Diabesity epidemic”. It is estimated that 285 million people, corresponding to 6.4% of the world’s adult population, already suffer from diabetes, and this is expected to grow to 438 million by 2030, with an enormous direct and indirect medical cost to society. Thus, preventing and treatments for “Diabesity” must be a priority, together with the development of approaches to modulate the ways in which the brain controls metabolism (satiety), body weight and composition.
This project proposes to help address this problem by using anti-diabesity properties of diets supplemented with NLL. NLL seeds exhibit high protein content, which are accumulated in the form of seed storage proteins (SSPs). NLL are getting more and more recognition for multiple benefits, since NLL crops are i) highly beneficial for agriculture; ii) a potential human health food, with specific nutritional attribute, associated with the high protein and dietary fibre content, helping reduce blood pressure and the risk of cardiovascular disease, in addition to the satiety control properties, as food enriched with lupin grain significantly influences subsequent energy intake, and also being gluten-free and low in fat and starch, has a very low glycaemic Index; iii) pharmaceutical attributes similar to the hypoglycemic drug metformin, the drug of choice for the treatment of type 2 diabetes.
From more than 450 species of the Lupinus family, only lupine known as ‘‘sweet lupines’’ such as white lupine (L. albus), yellow lupine (L. luteus), and blue lupine (L. angustifolius) are being used in food manufacturing. Sweet lupine seeds seem to be particularly promising as a source of innovative food ingredients due to averaged protein content similar to soybean and an adequate composition of essential amino acids. Foods based on sweet lupine proteins included in flour in bakery, pasta formulations, gluten-free products and other food items are gaining more attention from industry and consumers because a number of health-promoting benefits have been ascribed to lupin seed components, i.e. prevention of cardiovascular disease, reduction of blood glucose and cholesterol levels.
Seed proteins from lupin species, as with the majority of edible legume grains species, can potentially contribute to food allergy in a small percentage of the population, either separately or together with peanut allergy or other legumes. With the rapid introduction of novel foods and new ingredients in traditional foods, the number of allergenic people is also rising. Food allergy is a serious and a growing problem in the western world. The prevalence of food allergies varies by country, which has increased over the last 10 years. At present in the world, approximately 4 - 6% of children under age 18 and 3% of adults experience food allergy. In Europe, food allergies ranges between 2–5% among the population, base in studies assessed for sensitization and symptoms towards foods (data from EuroPrevall). Therefore, the frequency of sensitization and allergic reactions to lupine in the general population is unknown. However, as lupin becomes more prevalent as an alternative protein source for human use, it can be expected that demand for it will increase and more workers will be exposed to lupin. Despite the above numbers, and the fact that food allergy is a growing concern, there is still a lack in accurate clinical trials (diagnosis and therapies), and additional problems in reliability and consistency in diagnostic tests, especially the poor clinical specificity of skin tests and measurement of food-specific IgE, that it further aggravated in poor-result of allergy therapy.
The objectives of this project can be summarized as follow:
1. To obtain individually purified b-conglutin proteins, and loss-of-function mutant lupin plants for each individual b-conglutin genes and various combinations.
2. To identify key b-conglutin proteins that promotes increasing insulin sensitivity and/or reducing appetite in mice genetic model systems.
3. Characterization of the molecular aspects of allergy to Lupin b-conglutin seed storage proteins.
Beta-conglutin genes were synthetized following optimal codon usage for E. coli expression, and cloned into a vector designed for over-expressing fusion proteins in E. coli. Conglutins b1 to b4 and b6 have been purified using affinity chromatographic techniques, after multiple assays in different E. coli strains for avoiding cytotoxicity effects associated with exogenous expressing proteins. After setting-up optimal conditions, these recombinant b-conglutin proteins (rBCs) were produced in large quantities (25-35mg of purified proteins, purity > 95%).
Approaches to obtain lupin seeds with low b-conglutin proteins expression were attempted by generation of RNAi constructs, allowing simultaneous silencing of multiple members of the b-conglutin family. However, technical - methodological constrains with lupin seed transformation (very low efficiency of transformation) made not possible to obtain RNAi plant lines and seeds with reduced b-conglutin proteins content in the time-frame available for this part of the objective 1.
Co-IP assays were performed using a monoclonal antibody against 6xHisTag, individual purified rBCs and r-human insulin (rHI). Results showed how conglutins b1 to b3 and b6 interact with rHI, but particularly strongly b3 and b6. This knowledge constitutes another main achievement of the project, demonstrating the direct in vitro interaction between rHI and specific members of the b-conglutin family. This interaction may drive physiological effects and possible increase of insulin sensitivity.
Furthermore, model mice feeding experiments using purified b-conglutin proteins were performed as part of the objective 2 of the outgoing phase. These experiments where performed by our colleagues, the group of A/Prof S. Andrikopoulos (The University of Melbourne, Department of Medicine), under Ethical approval, and in a collaborative endeavour with Prof Grant Morahan (The University of Western Australia) as described in the project.
The outcomes of this first trial demonstrated that b-conglutins are major allergens for animal (as they are also major allergen in human). These results are consistent with what we also found in human with the experimental assessment using atopic patient’s sera.
In this line of results, and to continue working in health promoting aspects (diabetes improvement) of NLL seed proteins, using alternative experimental approaches, we have demonstrated that conglutin proteins activate insulin signalling pathway, having beneficial effects through genes up-regulation and increasing translation of biomarker protein. This new achievement could have enormous repercussion about the influence of conglutin proteins in the recovery of patients suffering diabetes, as well as in the diabetes prevention and treatment.
Therefore, the study of the molecular aspects of allergy shed interesting results. These experiments showed differential antigenicity of different classes of recombinant (overexpressed and purified) and native (purified from lupin seeds) conglutin proteins, characterized by using several atopic patient sera collected under Ethical approval. We also identified new reactive polypeptides ranging from 15 to 80kDa among these that constitute multimeric forms of b-conglutins. In addition, and using bioinformatics tools, we have identified the potential reactive IgE-binding, B- and T-cell epitopes that may be responsible for the common and differential reactivity showed by the different patient sera. Therefore, experiments showed potential cross-reactivity through individual polypeptides that recognized sera from atopic patients to lupin, peanut and other seeds.
Antigenic stability of recombinant b-conglutins and native seeds b-conglutins extracted from lupin seeds was examined by treatments with enzymes simulating stomach and intestine conditions, as well as food processing conditions, finding that conglutins form native sources are more resistant to these treatments in comparison to recombinant sources. Molecular allergy studies were mainly performed during the return phase of the project in the Estacion Experimental del Zaidin – CSIC, Granada (Spain).
No doubts, outcome of this project constitute the foundation and a significant advance in knowledge of potential medical benefits of seed proteins of Lupinus sp. The project is providing fundamental knowledge that can help with reducing the impact of diabetes and obesity, by identified specific NLL seed storage proteins with potential, lowering blood glucose effects and increase insulin sensitivity and/or reduce weight gain. This in turn could help with the development of scientifically proven food, enriched with specific NLL ingredients (novel, effective and affordable), with positive health and medical benefits on hunger, blood glucose/insulin control and blood pressure, and the regulation of body metabolism. Overall, the project’s molecular aspects concerning food allergy (antigenicity, cross-reactivity and allergen proteins stability), is providing the railroad for the long-term endeavour to help designing alternative lupin-enriched food with positive health benefits. The results will serve to improve clinical trial, develop new allergens diagnostic kits and food allergy vaccines.
The number of adult-onset or Type-2 diabetes is in rapid increment worldwide, where incidence among younger people is quite alarming, mostly due to life style habits reflected in overweight people which increase the risk of contracting diabetes, conditions that has been denominated with a new term “the Diabesity epidemic”. It is estimated that 285 million people, corresponding to 6.4% of the world’s adult population, already suffer from diabetes, and this is expected to grow to 438 million by 2030, with an enormous direct and indirect medical cost to society. Thus, preventing and treatments for “Diabesity” must be a priority, together with the development of approaches to modulate the ways in which the brain controls metabolism (satiety), body weight and composition.
This project proposes to help address this problem by using anti-diabesity properties of diets supplemented with NLL. NLL seeds exhibit high protein content, which are accumulated in the form of seed storage proteins (SSPs). NLL are getting more and more recognition for multiple benefits, since NLL crops are i) highly beneficial for agriculture; ii) a potential human health food, with specific nutritional attribute, associated with the high protein and dietary fibre content, helping reduce blood pressure and the risk of cardiovascular disease, in addition to the satiety control properties, as food enriched with lupin grain significantly influences subsequent energy intake, and also being gluten-free and low in fat and starch, has a very low glycaemic Index; iii) pharmaceutical attributes similar to the hypoglycemic drug metformin, the drug of choice for the treatment of type 2 diabetes.
From more than 450 species of the Lupinus family, only lupine known as ‘‘sweet lupines’’ such as white lupine (L. albus), yellow lupine (L. luteus), and blue lupine (L. angustifolius) are being used in food manufacturing. Sweet lupine seeds seem to be particularly promising as a source of innovative food ingredients due to averaged protein content similar to soybean and an adequate composition of essential amino acids. Foods based on sweet lupine proteins included in flour in bakery, pasta formulations, gluten-free products and other food items are gaining more attention from industry and consumers because a number of health-promoting benefits have been ascribed to lupin seed components, i.e. prevention of cardiovascular disease, reduction of blood glucose and cholesterol levels.
Seed proteins from lupin species, as with the majority of edible legume grains species, can potentially contribute to food allergy in a small percentage of the population, either separately or together with peanut allergy or other legumes. With the rapid introduction of novel foods and new ingredients in traditional foods, the number of allergenic people is also rising. Food allergy is a serious and a growing problem in the western world. The prevalence of food allergies varies by country, which has increased over the last 10 years. At present in the world, approximately 4 - 6% of children under age 18 and 3% of adults experience food allergy. In Europe, food allergies ranges between 2–5% among the population, base in studies assessed for sensitization and symptoms towards foods (data from EuroPrevall). Therefore, the frequency of sensitization and allergic reactions to lupine in the general population is unknown. However, as lupin becomes more prevalent as an alternative protein source for human use, it can be expected that demand for it will increase and more workers will be exposed to lupin. Despite the above numbers, and the fact that food allergy is a growing concern, there is still a lack in accurate clinical trials (diagnosis and therapies), and additional problems in reliability and consistency in diagnostic tests, especially the poor clinical specificity of skin tests and measurement of food-specific IgE, that it further aggravated in poor-result of allergy therapy.
The objectives of this project can be summarized as follow:
1. To obtain individually purified b-conglutin proteins, and loss-of-function mutant lupin plants for each individual b-conglutin genes and various combinations.
2. To identify key b-conglutin proteins that promotes increasing insulin sensitivity and/or reducing appetite in mice genetic model systems.
3. Characterization of the molecular aspects of allergy to Lupin b-conglutin seed storage proteins.
Beta-conglutin genes were synthetized following optimal codon usage for E. coli expression, and cloned into a vector designed for over-expressing fusion proteins in E. coli. Conglutins b1 to b4 and b6 have been purified using affinity chromatographic techniques, after multiple assays in different E. coli strains for avoiding cytotoxicity effects associated with exogenous expressing proteins. After setting-up optimal conditions, these recombinant b-conglutin proteins (rBCs) were produced in large quantities (25-35mg of purified proteins, purity > 95%).
Approaches to obtain lupin seeds with low b-conglutin proteins expression were attempted by generation of RNAi constructs, allowing simultaneous silencing of multiple members of the b-conglutin family. However, technical - methodological constrains with lupin seed transformation (very low efficiency of transformation) made not possible to obtain RNAi plant lines and seeds with reduced b-conglutin proteins content in the time-frame available for this part of the objective 1.
Co-IP assays were performed using a monoclonal antibody against 6xHisTag, individual purified rBCs and r-human insulin (rHI). Results showed how conglutins b1 to b3 and b6 interact with rHI, but particularly strongly b3 and b6. This knowledge constitutes another main achievement of the project, demonstrating the direct in vitro interaction between rHI and specific members of the b-conglutin family. This interaction may drive physiological effects and possible increase of insulin sensitivity.
Furthermore, model mice feeding experiments using purified b-conglutin proteins were performed as part of the objective 2 of the outgoing phase. These experiments where performed by our colleagues, the group of A/Prof S. Andrikopoulos (The University of Melbourne, Department of Medicine), under Ethical approval, and in a collaborative endeavour with Prof Grant Morahan (The University of Western Australia) as described in the project.
The outcomes of this first trial demonstrated that b-conglutins are major allergens for animal (as they are also major allergen in human). These results are consistent with what we also found in human with the experimental assessment using atopic patient’s sera.
In this line of results, and to continue working in health promoting aspects (diabetes improvement) of NLL seed proteins, using alternative experimental approaches, we have demonstrated that conglutin proteins activate insulin signalling pathway, having beneficial effects through genes up-regulation and increasing translation of biomarker protein. This new achievement could have enormous repercussion about the influence of conglutin proteins in the recovery of patients suffering diabetes, as well as in the diabetes prevention and treatment.
Therefore, the study of the molecular aspects of allergy shed interesting results. These experiments showed differential antigenicity of different classes of recombinant (overexpressed and purified) and native (purified from lupin seeds) conglutin proteins, characterized by using several atopic patient sera collected under Ethical approval. We also identified new reactive polypeptides ranging from 15 to 80kDa among these that constitute multimeric forms of b-conglutins. In addition, and using bioinformatics tools, we have identified the potential reactive IgE-binding, B- and T-cell epitopes that may be responsible for the common and differential reactivity showed by the different patient sera. Therefore, experiments showed potential cross-reactivity through individual polypeptides that recognized sera from atopic patients to lupin, peanut and other seeds.
Antigenic stability of recombinant b-conglutins and native seeds b-conglutins extracted from lupin seeds was examined by treatments with enzymes simulating stomach and intestine conditions, as well as food processing conditions, finding that conglutins form native sources are more resistant to these treatments in comparison to recombinant sources. Molecular allergy studies were mainly performed during the return phase of the project in the Estacion Experimental del Zaidin – CSIC, Granada (Spain).
No doubts, outcome of this project constitute the foundation and a significant advance in knowledge of potential medical benefits of seed proteins of Lupinus sp. The project is providing fundamental knowledge that can help with reducing the impact of diabetes and obesity, by identified specific NLL seed storage proteins with potential, lowering blood glucose effects and increase insulin sensitivity and/or reduce weight gain. This in turn could help with the development of scientifically proven food, enriched with specific NLL ingredients (novel, effective and affordable), with positive health and medical benefits on hunger, blood glucose/insulin control and blood pressure, and the regulation of body metabolism. Overall, the project’s molecular aspects concerning food allergy (antigenicity, cross-reactivity and allergen proteins stability), is providing the railroad for the long-term endeavour to help designing alternative lupin-enriched food with positive health benefits. The results will serve to improve clinical trial, develop new allergens diagnostic kits and food allergy vaccines.