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

Abstract

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.

Keywords: Differential interacting proteins; N-glycosylation; Pichia pastoris; Prochymosin; Secretion.

Fig. 1

The effect of N-glycosylation on molecular weight, autocatalytic cleavage activity, thermal stability of prochymosin and the secretion in P. pastoris GS115. A Mutant site of N-glycosylation in the propeptide of prochymosin, conservated N-glycosylated sequences were indicated by underlines; B molecular weight and de-glycosylation analysis of chy and chy34 by Western-blot; C Determination of the secretion of chy and chy34 by ELISA; D The mRNA levels of chy and chy34 in host cells determined by qRT-PCR; E Analysis of autocatalytic cleavage of the propeptide by confirming the milk-clotting acidity, The control was the supernatant of GS115 culture carried the pPIC9K vector; F Thermostability of chy and chy34 when stored at different temperatures for 8 h prior to enzymatic activity assay. Error bars indicate the standard deviation of tree independent experiments, student’s t-test was used to compare difference between two groups, *P < 0.05

Fig. 2

Bioinformatics analysis of IP-MS data. A Number of differential interacting proteins (DIPs); B Distribution of DIPs and no differentially interacting proteins (ND) in chy and chy34 samples; C GO function classification of DIPs; D KEGG enrichment analysis of DIPs; E COG analysis of DIPs. The terms of p Value < 0.05 were displayed

Fig. 2

Bioinformatics analysis of IP-MS data. A Number of differential interacting proteins (DIPs); B Distribution of DIPs and no differentially interacting proteins (ND) in chy and chy34 samples; C GO function classification of DIPs; D KEGG enrichment analysis of DIPs; E COG analysis of DIPs. The terms of p Value < 0.05 were displayed

Fig. 3

Diagram of glycoprotein processing in ER. The box represents the enzyme in the pathway, red means up-regulated enzyme in chy symple, black indicates ND or undetected proteins, green square represents mannose and red circle represents glucose. The blue arrows indicated ER-associated misfolded protein and degradation pathways

Fig. 4

The effect of five DIPs co-expression and UGGTs knockout on the secretion of prochymosin in P. pastoris. A The effect of five DIPs co-expression on the secretion of chy; B The effect of five DIPs co-expression on the secretion of chy34; C The effect of UGGTs knockout on the secretion of chy and chy34. Data are presented as the mean ± SD from three independent experiments, *P < 0.05