Enthalpic destabilization of glycosylated lysozymes constructed by genetic modification

• Published in: Biochimica et biophysica acta 2000-08, Vol.1481 (1), p.88-96
• Main Authors: Kato, Akio, Nakamura, Soichiro, Ban, Masanori, Azakami, Hiroyuki, Yutani, Katsuhide
• Format: Article
• Language: English
• Subjects: Binding Sites; Calorimetry, Differential Scanning; Differential scanning calorimetry; Enzyme Stability; Glycoproteins - chemistry; Glycosylation; Glycosylation of lysozyme; Hydrogen-Ion Concentration; Mannose - analysis; Muramidase - chemistry; Muramidase - genetics; Mutagenesis, Site-Directed; Protein Denaturation; Protein Folding; Temperature; Thermal stability of proteins; Thermodynamics
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(00)00123-0

To understand the role of polyglycosylation in protein stability, the thermodynamic changes in the denaturation of various polymannosyl lysozyme mutants (R21T, G49N, R21T/G49N) constructed by genetic modification were analyzed using differential scanning calorimetry (DSC). The denaturation temperature and the enthalpy change for unfolding of the lysozymes were reduced with an increase in the length of the polymannose chain and the number of binding sites to a protein, although the polymannosyl lysozymes revealed apparent heat stability in that no aggregation was observed and the enzymatic activity was conserved under conditions in which the wild-type lysozyme coagulated [S. Nakamura et al., J. Biol. Chem. 268 (1993) 12706–12712]. The reversibility of the denaturation of polymannosyl lysozymes was observed in the DSC curves obtained by reheating after heat denaturation, while it was not observed for the wild-type lysozyme. Based on these results, the polymannosyl lysozyme seems to easily refold due to the excellent reversibility of denaturation, despite the decreases in the enthalpic stabilization due to the strain in the protein molecule by the introduction of a polysaccharide chain.