Quantitative Studies on Ligand Binding to Recombinant Human Serum Albumin (rHSA) and Synthetic HSA Domains with Defined Glycation Patterns

• Applicant: Professor Dr. Ralf Hoffmann
• Subject Area: Analytical Chemistry ; Biochemistry
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 585683586

The aldehyde group of glucose can react with the amino group of lysine residues in proteins to form a Schiff base, which can rearrange into the relatively stable fructosyllysine residue, often referred to as an Amadori product. This modification affects virtually all proteins, including human serum albumin (HSA), which represents 50% to 55% of the total protein content in plasma. Mature HSA is a transport protein consisting of three domains that bind many endogenous and exogenous substances, such as fatty acids and drugs, with high affinity at different binding sites. Glycation of lysine residues, specifically when nearby the binding site, is known to increase or decrease the binding affinity of these compounds, including fatty acids and drugs. However, fatty acids bound to HSA can also affect the glycation rates of nearby lysine residues. Although these effects have been studied, the glycation status of HSA differed much among studies and was often not characterized in detail, which allows only qualitative comparison of the published data. Most importantly, the HSA was extremely heterogeneously glycated and typically contained advanced glycation end products (AGEs), which were also not characterized further. Most studies have only considered the total glycation degree of HSA, but they lacked precise quantitation of the relevant glycation site. To address this issue, we will use recombinant HSA (rHSA) and rHSA domains that are glycated in continuous-flow reactors using well defined reaction conditions and near to physiological sugar concentrations. This method mostly eliminates byproducts resulting from degradation of glucose or the initially formed Schiff base during incubation periods of up to six weeks. Using mass spectrometry and stable isotope-labeled authentic peptides, we will precisely quantify all glycation sites and study their effect on the binding constants of different fatty acids and anti-diabetic drugs by isothermal titration calorimetry (ITC). Glycation in the presence of fatty acids will demonstrate how bound fatty acids alter the degree of glycation near their binding sites. Finally, to confirm and quantify the influence of the relevant modification sites on binding affinities, we will chemically synthesize rHSA domains with defined fructosyllysine and AGE-modifications at one or two sites of interest. These results will enable us to determine the impact of elevated glycation rates in diabetic patients on the structure and function of HSA, and how this may contribute to diabetes-related complications.

• DFG Programme: Research Grants