Glycosylation Contributes to Thermostability and Proteolytic Resistance of rFIP-nha (<i>Nectria haematococca</i>)
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...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-08-01
|
| Series: | Molecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1420-3049/28/17/6386 |
| _version_ | 1827727967050006528 |
|---|---|
| author | 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 |
| author_facet | 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 |
| author_sort | Yusi Liu |
| collection | DOAJ |
| description | 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 <i>Nectria haematococca</i>) is described. Three rFIP-nha glycan mutants (N5A, N39A, N5+39A) were constructed and expressed in <i>Pichia pastoris</i> 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. |
| first_indexed | 2024-03-10T23:16:24Z |
| format | Article |
| id | doaj.art-71a7687635b64df4a0a9d33e1c2e7122 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T23:16:24Z |
| publishDate | 2023-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-71a7687635b64df4a0a9d33e1c2e71222023-11-19T08:35:15ZengMDPI AGMolecules1420-30492023-08-012817638610.3390/molecules28176386Glycosylation Contributes to Thermostability and Proteolytic Resistance of rFIP-nha (<i>Nectria haematococca</i>)Yusi Liu0Tamara Hoppenbrouwers1Yulu Wang2Yingying Xie3Xue Wei4Haowen Zhang5Guoming Du6Khandader Md Sharif Uddin Imam7Harry Wichers8Zhen Li9Shanna Bastiaan-Net10Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, ChinaWageningen Food and Biobased Research, Wageningen University and Research, 6708 WG Wageningen, The NetherlandsLaboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, ChinaLaboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, ChinaLaboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, ChinaLaboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, ChinaLaboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, ChinaLaboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, ChinaWageningen Food and Biobased Research, Wageningen University and Research, 6708 WG Wageningen, The NetherlandsLaboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agriculture Sciences, Beijing 100193, ChinaWageningen Food and Biobased Research, Wageningen University and Research, 6708 WG Wageningen, The NetherlandsGlycosylation 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 <i>Nectria haematococca</i>) is described. Three rFIP-nha glycan mutants (N5A, N39A, N5+39A) were constructed and expressed in <i>Pichia pastoris</i> 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.https://www.mdpi.com/1420-3049/28/17/6386immunomodulatory proteinsglycosylationstructurethermostabilitydigestion resistance |
| spellingShingle | 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 Glycosylation Contributes to Thermostability and Proteolytic Resistance of rFIP-nha (<i>Nectria haematococca</i>) Molecules immunomodulatory proteins glycosylation structure thermostability digestion resistance |
| title | Glycosylation Contributes to Thermostability and Proteolytic Resistance of rFIP-nha (<i>Nectria haematococca</i>) |
| title_full | Glycosylation Contributes to Thermostability and Proteolytic Resistance of rFIP-nha (<i>Nectria haematococca</i>) |
| title_fullStr | Glycosylation Contributes to Thermostability and Proteolytic Resistance of rFIP-nha (<i>Nectria haematococca</i>) |
| title_full_unstemmed | Glycosylation Contributes to Thermostability and Proteolytic Resistance of rFIP-nha (<i>Nectria haematococca</i>) |
| title_short | Glycosylation Contributes to Thermostability and Proteolytic Resistance of rFIP-nha (<i>Nectria haematococca</i>) |
| title_sort | glycosylation contributes to thermostability and proteolytic resistance of rfip nha i nectria haematococca i |
| topic | immunomodulatory proteins glycosylation structure thermostability digestion resistance |
| url | https://www.mdpi.com/1420-3049/28/17/6386 |
| work_keys_str_mv | AT yusiliu glycosylationcontributestothermostabilityandproteolyticresistanceofrfipnhainectriahaematococcai AT tamarahoppenbrouwers glycosylationcontributestothermostabilityandproteolyticresistanceofrfipnhainectriahaematococcai AT yuluwang glycosylationcontributestothermostabilityandproteolyticresistanceofrfipnhainectriahaematococcai AT yingyingxie glycosylationcontributestothermostabilityandproteolyticresistanceofrfipnhainectriahaematococcai AT xuewei glycosylationcontributestothermostabilityandproteolyticresistanceofrfipnhainectriahaematococcai AT haowenzhang glycosylationcontributestothermostabilityandproteolyticresistanceofrfipnhainectriahaematococcai AT guomingdu glycosylationcontributestothermostabilityandproteolyticresistanceofrfipnhainectriahaematococcai AT khandadermdsharifuddinimam glycosylationcontributestothermostabilityandproteolyticresistanceofrfipnhainectriahaematococcai AT harrywichers glycosylationcontributestothermostabilityandproteolyticresistanceofrfipnhainectriahaematococcai AT zhenli glycosylationcontributestothermostabilityandproteolyticresistanceofrfipnhainectriahaematococcai AT shannabastiaannet glycosylationcontributestothermostabilityandproteolyticresistanceofrfipnhainectriahaematococcai |