Title: Quantifying N-Acetylneuraminic Acid on Glycoproteins and Assessing the Impact on Bioactivity

Program: Biomolecular Sciences PhD

Committee Chair: Owen McDougal

Committee: Owen McDougal, Henry Charlier, Javier Ochoa-Reparaz, Juliette Tinker

Abstract: Milk is nutrient dense and contains many bioactive components that promote health and wellbeing. Dairy glycoproteins are some of the most well-known bioactive components in milk, and it is thought that glycosylation is a contributor to their functionality. a-Lactalbumin, immunoglobulin G (IgG), and lactoferrin serve various functions including lactose synthesis, regulating immune system function, and mineral-binding. The functionality of IgG and lactoferrin have been connected to the presence of glycosylation. Glycomacropeptide is another glycosylated dairy protein, which is generated during the cheesemaking process. GMP functions as an antiviral, antibacterial, and a prebiotic. The portion of the GMP protein structure that is responsible for these activities is not known, but some studies have hypothesized that the bioactivity of GMP is due to glycosylation. GMP is glycosylated with three different types of sugar: N-acetylneuraminic acid (NANA), N-acetylgalactosamine (GalNAc), and galactose (Gal). NANA part of the sialic acid family and is the most well-studied. Many biologically active proteins—found in dairy and otherwise—are sialylated. Proteins are known for nutrition, but market value can be enhanced due to benefits to health and well-being. The degree of glycosylation may add value for products. This is an area of interest for processors that sell products containing glycosylated proteins. Currently there is no standard method for measuring NANA on glycoproteins. Many methods have been trialed with either an acid or enzymatic hydrolysis followed by detection with colorimetric, fluorometric, ultraviolet (UV), or chromatographic/mass spectrometry assays. The first content chapter of this dissertation compares NANA hydrolysis approaches using either acid or enzymatic conditions, and common detection methods (i.e., colorimetric, fluorometric, and chromatographic/mass spectrometry) to measure the NANA content on GMP. It was determined that a weak acid hydrolysis followed by chromatographic/mass spectrometry detection was the most accurate, precise, and protein-specific approach to achieve the goal of quantifying NANA from GMP. In order to select the optimal hydrolysis conditions for maximum NANA yield, a design of experiments (DOE) was performed. The DOE model predicted the use of 1.1 M acetic acid at 67.5°C for 360 min would yield maximum NANA from GMP. This was slightly altered to 60.0°C overnight with 1.1 M acetic acid, which released 6.18% ± 0.12% (w/w) NANA.
To demonstrate the efficacy of this method, chapter three details the validation process to quantify NANA on a completely different protein, bovine fetuin, using the DOE process described for GMP. The best parameters for fetuin hydrolysis were found to be 86.9°C for 287 minutes, which yielded a w/w of 6.41% ± 0.52%.
Finally, chapter four focused on assessing whether the prebiotic activity of GMP was due to the protein or the carbohydrate. GMP with and without NANA was studied by supplementing enhanced Lactococcus lactis with increasing concentrations of GMP and measuring the amount of ?-aminobutyric acid (GABA) produced. Although no trend was observed, some interesting insights emerged from this preliminary investigation. In addition, the connection between NANA content and the bioactivity of GMP was investigated by performing an exploratory in vitro study on a model microbiome system using both GMP with and without NANA. Evidence was obtained that GMP with NANA may have a greater effect on the composition of the microbiome based on the increased number of unique amplicon sequence variants that were measured compared to samples GMP without NANA, but definite conclusions cannot be made in the absence of more extensive investigation.