Glycosylation Contributes to Thermostability and Proteolytic Resistance of rFIP-nha (<i>Nectria haematococca</i>)
Yusi Liu,
Tamara Hoppenbrouwers,
Yulu Wang,
Yingying Xie,
Xue Wei,
Haowen Zhang,
Guoming Du,
Khandader Md Sharif Uddin Imam,
Harry Wichers,
Zhen Li,
Shanna Bastiaan-Net
Affiliations
Yusi Liu
Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, China
Tamara Hoppenbrouwers
Wageningen Food and Biobased Research, Wageningen University and Research, 6708 WG Wageningen, The Netherlands
Yulu Wang
Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, China
Yingying Xie
Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, China
Xue Wei
Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, China
Haowen Zhang
Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, China
Guoming Du
Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, China
Khandader Md Sharif Uddin Imam
Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, China
Harry Wichers
Wageningen Food and Biobased Research, Wageningen University and Research, 6708 WG Wageningen, The Netherlands
Zhen Li
Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, China
Shanna Bastiaan-Net
Wageningen Food and Biobased Research, Wageningen University and Research, 6708 WG Wageningen, The Netherlands
Glycosylation is an important post-translational modification of proteins, contributing to protein function, stability and subcellular localization. Fungal immunomodulatory proteins (FIPs) are a group of small proteins with notable immunomodulatory activity, some of which are glycoproteins. In this study, the impact of glycosylation on the bioactivity and biochemical characteristics of FIP-nha (from Nectria haematococca) is described. Three rFIP-nha glycan mutants (N5A, N39A, N5+39A) were constructed and expressed in Pichia pastoris to study the functionality of the specific N-glycosylation on amino acid N5 and N39. Their protein characteristics, structure, stability and activity were tested. WT and mutants all formed tetramers, with no obvious difference in crystal structures. Their melting temperatures were 82.2 °C (WT), 81.4 °C (N5A), 80.7 °C (N39A) and 80.1 °C (N5+39A), indicating that glycosylation improves thermostability of rFIP-nha. Digestion assays showed that glycosylation on either site improved pepsin resistance, while 39N-glycosylation was important for trypsin resistance. Based on the 3D structure and analysis of enzyme cleavage sites, we conclude that glycosylation might interfere with hydrolysis via increasing steric hindrance. WT and mutants exerted similar bioactivity on tumor cell metabolism and red blood cells hemagglutination. Taken together, these findings indicate that glycosylation of FIP-nha impacts its thermostability and digestion resistance.
