Study On The Mechanism Of Protein Retention In Endoplasmic Reticulum Of Saccharomyces Cerevisiae And Its Application In Protein Engineering

Protein endoplasmic reticulum?ER?retention is an important physiological phenomenon in eukaryotic cells.Characteristic ER structural and functional proteins could interact with specific receptor proteins on the ER membrane via a carboxy-terminal ER retention sequence?ERS?,leading to their retention in the ER to affect post-translational modification,folding and secretion of proteins.It is currently known that proteins that are incorrectly folded and secreted could cause a range of diseases such as Parkinson's disease,Alzheimer's disease and heart diseases.However,due to the lack of systematic studies of ER retention proteins and their retention mechanisms,their importance is often underestimated.To solve this problem,we use Saccharomyces cerevisiae as the research model to explore the protein retention mechanisms in the ER of eukaryotic cells.Preliminary studies showed that ERS was the C-terminal specific sequence of ER retention proteins in eukaryotic cells,which was identified as the conserved HDEL and KDEL sequences in yeast and human cells,respectively.In our studies here,a new method was developed in S.cerevisiae,which combined yeast surface display,fluorophore-conjugated antibody labeling and flow cytometry analysis to comprehensively quantify the ER retention effect of various ERS in a fast manner.After the systematic analysis of the HDEL-type ERS in S.cerevisiae,we found that the properties of amino acids at-4,-5 and-6 positions were important for maintaining the retention effect of ERS(X_-6X_-5H_-4DEL),especially the His at-4 position and the aromaticity of the amio acid at-6 position.In addition,Erd2 was identified as the major retention receptor protein to interact with the HDEL-type ERS in S.cerevisiae.Through the protein structure simulation guided biochemical analysis,we further identified that the amino acids at-4 and-6 positions of X_-6X_-5H_-4DEL-type ERS formed an ionic bond and aromatic ring-mediated?-?stacking force with the Phe54,Trp56,and the aromatic network formed by aromatic amino acids of Erd2 located in the ER lumen,which mainly determined the strength of the XXHDEL-type ERS.Interestingly,comparing the Erd2 with human KDELR1,a Erd2 homologous protein in human cells,indicated that this characteristic aromatic residue-mediated ER retention mechniasm might lead to the different ERS in yeast or human cells.Moreover,based on our studies,a new ERS,WEHDEL,which presented more than two-fold strength of the best known ERS,FEHDEL,was engineered,which was later applied in the subsequent protein engineering experiments.In our studies of the ERS-mediated protein ER retention in yeast cells,we found that the C-terminal sequences of some ER resident proteins did not belong to the HDEL-type ERS.For example,YKLDIQ?the C-terminal sequence of Ero1?,a non-HDEL type sequence,exhibited ER retention ability,which was significantly enhanced by the overexpression of Erd2.It is possible that non-HDEL-type ERS along with uncharacterized new protein ER retention mechanisms might exist in eukaryotic cells.To evaluate this speculation,we established an ERS library with a 2.7×10⁸size to cover 6-amino acid length,which was further analyzed to characterize the non-HDEL-type ERS in S.cerevisiae.The ERS library was sorted by multi-rounds to enrich the cells bearing plasmids with strong ERS using flow cytometry.Enriched cells from each round were then subjected to plasmid DNA extration and high-throughput sequencing analysis using Next-generation Sequencing?NGS?technology to decipher the abundance of characteristic ERS.Interestingly,our preliminary results clearly indicated that non-HDEL-type ERS existed in S.cerevisiae,which might be associated to new retention mechanisms.The studies on the ERS could not only help us further understand the protein ER retention in eukaryotic cells,but also be applied to facilitate the protein engineering.The yeast endoplasmic reticulum retention screening?YESS?system was a high-throughput platform technology that integrated yeast surface display and protein ER retention for protease engineering.Using our engineered strong ERS,WEHDEL,we could further increase the retention time and concentration of the target proteases and substrates in yeast ER,thus amplifying weak protease hydrolysis reaction signals to enable the engineering of low-activity proteases,such as engineering human MMP7 protease to efficiently cleave human Ig G.Other than protease engineering,ERS were also applied to develop an optimized Fab yeast cell surface display?YSD?technology based on the YESS system,which presented advantages over the current sc Fv YSD method.In this approach,the VH-CH1 and VL-CL regions of Fab were constructed into the bidirectional promoter vector of the YESS system,leading to the simultaneous induction and expression of VH-CH1 and VL-CL domains,which were subsequenctly assembled into Fab fragments followed by being displayed on the yeast cell surface.Through this Fab YSD system,the Fab fragments of four Adalimumab variants were displayed on yeast cell surface,which were identified to have higher cell surface display efficiency and TNF-?binding ability than those of sc Fv fragments.Additionally,our library screening experiments showed that Adalimumab variants with high activity could be effectively sorted out from 10⁵:1 mixed cells at?1 p M concentration of TNF-?using this Fab YSD system.Furthermore,achoring the WEHDEL to the C-terminus of the VH-VL regions of Adalimumab and Infliximab variants or co-expressing molecular chaperones such as Pdi1 or Kar2 could facilitate the Fab fragment assembly from the VH-CH1 and VL-CL domains in the yeast ER,thus enhancing the functional activity of the surface displayed Fab fragments.Our results showed that using WEHDEL and Pdi1 significantly increased the functional activity of surface displayed Infliximab variant against TNF-?antigen for up to 2.5-fold and 3.6-fold,respectively.In summary,our comprehensive studies on the protein ER retention in S.cerevisiae not only promoted the further understanding of the protein retention mechanisms in ekaryotic cells,but also effectively expanded the development of new technologies in protease and antibody engineering.