ABSTRACT
Miraculin is a glycoprotein with the ability to make sour substances taste sweet. The safety of miraculin has been evaluated using an approach proposed by the Food and Agriculture Organization of the United Nations and the World Health Organization for assessing the safety of novel proteins. Miraculin was shown to be fully and rapidly digested by pepsin in an in vitro digestibility assay. The proteomic analysis of miraculin's pepsin digests further corroborated that it is highly unlikely that any of the protein will remain intact within the gastrointestinal tract for potential absorption. The potential allergenicity and toxigenicity of miraculin, investigated using in silico bioinformatic analyses, demonstrated that miraculin does not represent a risk of allergy or toxicity to humans with low potential for cross-reactivity with other allergens. The results of a sensory study, characterizing the taste receptor activity of miraculin, showed that the taste-modifying effect of miraculin at the concentration intended for product development has a rapid onset and disappearance with no desensitizing impact on the receptor. Overall, the results of this study demonstrate that the use of miraculin to impact the sensory qualities of orally administered products with a bitter/sour taste profile is not associated with any safety concerns.
Subject(s)
Glycoproteins/toxicity , Sweetening Agents/toxicity , Allergens/chemistry , Allergens/isolation & purification , Allergens/toxicity , Computer Simulation , Fruit/chemistry , Glycoproteins/chemistry , Glycoproteins/isolation & purification , Humans , Pepsin A/chemistry , Proteolysis , Sweetening Agents/chemistry , Sweetening Agents/isolation & purification , Synsepalum/chemistry , Taste/drug effectsABSTRACT
The agouti-related protein (AgRP) plays a central role in energy balance by reducing signaling through the hypothalamic melanocortin receptors (McRs) 3 and 4, in turn stimulating feeding and decreasing energy expenditure. Mature AgRP(83-132), produced by endoproteolytic processing, contains a central region that folds as an inhibitor cystine knot (ICK) stabilized by a network of disulfide bonds; this domain alone carries the molecular features for high affinity McR binding and inverse agonism. Outside of the ICK domain are two polypeptide segments, an N-terminal extension and a C-terminal loop, both completely conserved but of unknown function. Here we examine the physiological roles of these non-ICK segments by developing a panel of modified AgRPs that were administered to rats through intracerebroventricular (ICV) injection. Analysis of food consumption demonstrates that basic (positively charged) residues are essential for potent short- and long-term AgRP stimulated feeding. Moreover, we demonstrate an approximate linear relationship between protein charge density and 24 h food intake. Next, we developed artificial AgRP(83-132) analogues with increased positive charge and found that these species were substantially more potent than wild type. A single dose of one protein, designated AgRP-4K, results in enhanced feeding for well over a week and weight gain that is nearly double that of AgRP(83-132). These studies suggest new strategies for the development of potent orexigenic species and may serve as leads for the development of therapeutics for treating wasting conditions such as cachexia.
Subject(s)
Agouti-Related Protein/chemistry , Agouti-Related Protein/pharmacology , Feeding Behavior/drug effects , Agouti-Related Protein/administration & dosage , Agouti-Related Protein/metabolism , Amino Acid Sequence , Animals , Cachexia/drug therapy , Drinking/drug effects , HEK293 Cells , Humans , Male , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Rats , Sequence AlignmentABSTRACT
The melanocortin-4 receptor (MC4R) plays a critical role in the control of energy balance. Of its two pro-opiomelanocortin (POMC)-derived ligands, alpha- and beta-MSH, the majority of attention has focused on alpha-MSH, partly reflecting the absence of beta-MSH in rodents. We screened the POMC gene in 538 patients with severe, early-onset obesity and identified five unrelated probands who were heterozygous for a rare missense variant in the region encoding beta-MSH, Tyr221Cys. This frequency was significantly increased (p < 0.001) compared to the general UK Caucasian population and the variant cosegregated with obesity/overweight in affected family members. Compared to wild-type beta-MSH, the variant peptide was impaired in its ability to bind to and activate signaling from the MC4R. Obese children carrying the Tyr221Cys variant were hyperphagic and showed increased linear growth, both of which are features of MC4R deficiency. These studies support a role for beta-MSH in the control of human energy homeostasis.
