Cargando…

The introduction of an N-glycosylation site into prochymosin greatly enhances its production and secretion by Pichia pastoris

BACKGROUND: N-glycosylation is one of the most important post-translational modifications. Many studies have shown that N-glycosylation has a significant effect on the secretion level of heterologous glycoproteins in yeast cells. However, there have been few studies reporting a clear and unified exp...

Descripción completa

Detalles Bibliográficos
Autores principales: Wang, Nan, Yang, Caifeng, Peng, Huakang, Guo, Wenfang, Wang, Mengqi, Li, Gangqiang, Liu, Dehu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9429577/
https://www.ncbi.nlm.nih.gov/pubmed/36042512
http://dx.doi.org/10.1186/s12934-022-01904-3
_version_ 1784779498990862336
author Wang, Nan
Yang, Caifeng
Peng, Huakang
Guo, Wenfang
Wang, Mengqi
Li, Gangqiang
Liu, Dehu
author_facet Wang, Nan
Yang, Caifeng
Peng, Huakang
Guo, Wenfang
Wang, Mengqi
Li, Gangqiang
Liu, Dehu
author_sort Wang, Nan
collection PubMed
description BACKGROUND: N-glycosylation is one of the most important post-translational modifications. Many studies have shown that N-glycosylation has a significant effect on the secretion level of heterologous glycoproteins in yeast cells. However, there have been few studies reporting a clear and unified explanation for the intracellular mechanism that N-glycosylation affect the secretion of heterologous glycoproteins so far. Pichia pastoris is an important microbial cell factory producing heterologous protein. It is of great significance to study the effect of N-glycosylation on the secretion level of heterologous protein. Camel chymosin is a glycoprotein with higher application potential in cheese manufacturing industry. We have expressed camel prochymosin in P. pastoris GS115, but the lower secretion level limits its industrial application. This study attempts to increase the secretion level of prochymosin through N-glycosylation, and explore the molecular mechanism of N-glycosylation affecting secretion. RESULTS: Adding an N-glycosylation site at the 34th amino acid of the propeptide of prochymosin significantly increased its secretion in P. pastoris. N-glycosylation improved the thermostability of prochymosin without affecting the enzymatic activity. Immunoprecipitation coupled to mass spectrometry (IP-MS) analysis showed that compared with the wild prochymosin (chy), the number of proteins interacting with N-glycosylated mutant (chy34) decreased, and all differential interacting proteins (DIPs) were down-regulated in chy34-GS115 cell. The DIPs in endoplasmic reticulum were mainly concentrated in the misfolded protein pathway. Among the five DIPs in this pathway, overexpression of BiP significantly increased the secretion of chy. The knockout of the possible misfolded protein recognition elements, UDP-glycose:glycoprotein glucosyltransferase 1 and 2 (UGGT1/2) had no effect on the growth of yeast cells and the secretion of prochymosin. CONCLUSIONS: In conclusion, N-glycosylation increased the secretion of prochymosin in P. pastoris trough the adjustment of intracellular interacted proteins. The results of our study may help to elucidate the molecular mechanism of N-glycosylation affecting secretion and provide a new research method to improve the secretion of heterologous glycoprotein in P. pastoris. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01904-3.
format Online
Article
Text
id pubmed-9429577
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-94295772022-09-01 The introduction of an N-glycosylation site into prochymosin greatly enhances its production and secretion by Pichia pastoris Wang, Nan Yang, Caifeng Peng, Huakang Guo, Wenfang Wang, Mengqi Li, Gangqiang Liu, Dehu Microb Cell Fact Research BACKGROUND: N-glycosylation is one of the most important post-translational modifications. Many studies have shown that N-glycosylation has a significant effect on the secretion level of heterologous glycoproteins in yeast cells. However, there have been few studies reporting a clear and unified explanation for the intracellular mechanism that N-glycosylation affect the secretion of heterologous glycoproteins so far. Pichia pastoris is an important microbial cell factory producing heterologous protein. It is of great significance to study the effect of N-glycosylation on the secretion level of heterologous protein. Camel chymosin is a glycoprotein with higher application potential in cheese manufacturing industry. We have expressed camel prochymosin in P. pastoris GS115, but the lower secretion level limits its industrial application. This study attempts to increase the secretion level of prochymosin through N-glycosylation, and explore the molecular mechanism of N-glycosylation affecting secretion. RESULTS: Adding an N-glycosylation site at the 34th amino acid of the propeptide of prochymosin significantly increased its secretion in P. pastoris. N-glycosylation improved the thermostability of prochymosin without affecting the enzymatic activity. Immunoprecipitation coupled to mass spectrometry (IP-MS) analysis showed that compared with the wild prochymosin (chy), the number of proteins interacting with N-glycosylated mutant (chy34) decreased, and all differential interacting proteins (DIPs) were down-regulated in chy34-GS115 cell. The DIPs in endoplasmic reticulum were mainly concentrated in the misfolded protein pathway. Among the five DIPs in this pathway, overexpression of BiP significantly increased the secretion of chy. The knockout of the possible misfolded protein recognition elements, UDP-glycose:glycoprotein glucosyltransferase 1 and 2 (UGGT1/2) had no effect on the growth of yeast cells and the secretion of prochymosin. CONCLUSIONS: In conclusion, N-glycosylation increased the secretion of prochymosin in P. pastoris trough the adjustment of intracellular interacted proteins. The results of our study may help to elucidate the molecular mechanism of N-glycosylation affecting secretion and provide a new research method to improve the secretion of heterologous glycoprotein in P. pastoris. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01904-3. BioMed Central 2022-08-30 /pmc/articles/PMC9429577/ /pubmed/36042512 http://dx.doi.org/10.1186/s12934-022-01904-3 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Wang, Nan
Yang, Caifeng
Peng, Huakang
Guo, Wenfang
Wang, Mengqi
Li, Gangqiang
Liu, Dehu
The introduction of an N-glycosylation site into prochymosin greatly enhances its production and secretion by Pichia pastoris
title The introduction of an N-glycosylation site into prochymosin greatly enhances its production and secretion by Pichia pastoris
title_full The introduction of an N-glycosylation site into prochymosin greatly enhances its production and secretion by Pichia pastoris
title_fullStr The introduction of an N-glycosylation site into prochymosin greatly enhances its production and secretion by Pichia pastoris
title_full_unstemmed The introduction of an N-glycosylation site into prochymosin greatly enhances its production and secretion by Pichia pastoris
title_short The introduction of an N-glycosylation site into prochymosin greatly enhances its production and secretion by Pichia pastoris
title_sort introduction of an n-glycosylation site into prochymosin greatly enhances its production and secretion by pichia pastoris
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9429577/
https://www.ncbi.nlm.nih.gov/pubmed/36042512
http://dx.doi.org/10.1186/s12934-022-01904-3
work_keys_str_mv AT wangnan theintroductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT yangcaifeng theintroductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT penghuakang theintroductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT guowenfang theintroductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT wangmengqi theintroductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT ligangqiang theintroductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT liudehu theintroductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT wangnan introductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT yangcaifeng introductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT penghuakang introductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT guowenfang introductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT wangmengqi introductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT ligangqiang introductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris
AT liudehu introductionofannglycosylationsiteintoprochymosingreatlyenhancesitsproductionandsecretionbypichiapastoris