Identification and characterization of calreticulin as a novel plasminogen receptor.

Calreticulin (CRT) was originally identified as a key calcium-binding protein of the endoplasmic reticulum. Subsequently, CRT was shown to possess multiple intracellular functions, including roles in calcium homeostasis and protein folding. Recently, several extracellular functions have been identified for CRT, including roles in cancer cell invasion and phagocytosis of apoptotic and cancer cells by macrophages. In the current report, we uncover a novel function for extracellular CRT and report that CRT functions as a plasminogen-binding receptor that regulates the conversion of plasminogen to plasmin. We show that human recombinant or bovine tissue-derived CRT dramatically stimulated the conversion of plasminogen to plasmin by tissue plasminogen activator or urokinase-type plasminogen activator. Surface plasmon resonance analysis revealed that CRT-bound plasminogen (KD = 1.8 µM) with moderate affinity. Plasminogen binding and activation by CRT were inhibited by ε-aminocaproic acid, suggesting that an internal lysine residue of CRT interacts with plasminogen. We subsequently show that clinically relevant CRT variants (lacking four or eight lysines in carboxyl-terminal region) exhibited decreased plasminogen activation. Furthermore, CRT-deficient fibroblasts generated 90% less plasmin and CRT-depleted MDA MB 231 cells also demonstrated a significant reduction in plasmin generation. Moreover, treatment of fibroblasts with mitoxantrone dramatically stimulated plasmin generation by WT but not CRT-deficient fibroblasts. Our results suggest that CRT is an important cellular plasminogen regulatory protein. Given that CRT can empower cells with plasmin proteolytic activity, this discovery may provide new mechanistic insight into the established role of CRT in cancer.

Electrochemical capacitance spectroscopy based determination of antibodies against SARS-CoV-2 virus spike protein.

In this study, we are reporting a novel electrochemical capacitance spectroscopy (ECS) platform designed for the sensitive and label-free detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus spike protein (anti-rS) in diluted blood serum. The determination of anti-rS is crucial for identification individuals who have been infected by SARS-CoV-2 virus and may have acquired immunity. The rS protein was immobilized on a screen-printed carbon electrode, which was incubated in diluted blood serum containing anti-rS antibodies. Label-free ECS was applied for the determination of interaction between immobilized rS and free-standing anti-rS. Here reported bioanalytical platform demonstrated high sensitivity and specificity in detecting anti-rS, achieving a limit of detection of 4.38 nM. This versatile platform could be further enhanced by applying various electrode materials and adapting this platform to detect antibodies against some other proteins. Our findings have significant implications for the development of affordable, scalable biosensing platforms capable to provide rapid and accurate public health screening and monitoring, particularly in the context of the coronavirus disease 2019 (COVID-19) pandemic.

Antibodies to expanded virus antigen panels show elevated diagnostic sensitivities in multiple sclerosis and optic neuritis.

An antigen panel consisting of Epstein-Barr, measles, mumps, varicella zoster and rubella viruses (EMMRZ) was recently presented, which may aid in the diagnosis of multiple sclerosis (MS). The aim of this study was to validate and extend the EMMRZ panel. Various candidates, such as Cytomegalovirus and John Cunningham virus were analysed in relapsing-remitting MS (RRMS) and optic neuritis (ON) samples by enzyme-linked immunosorbent assay. IgG levels were elevated in RRMS samples and correlations were found between serum and cerebrospinal fluid levels. Cohort-dependent optimized panels were obtained for RRMS and ON, which obtained the highest sensitivity when combined with the status of oligoclonal bands.

Investigation of SARS-CoV-2 nucleocapsid protein interaction with a specific antibody by combined spectroscopic ellipsometry and quartz crystal microbalance with dissipation.

Detailed evaluations of the antigen and antibody interaction rate and strength of the immune complex formed are very important for medical and bioanalytical applications. These data are crucial for the development of sensitive and fast immunosensors suitable for continuous measurements. Therefore, combined spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation (QCM-D) technique (SE/QCM-D) was used for the evaluation: (i)of covalent immobilization of SARS-CoV-2 nucleocapsid protein (SCoV2-N) on QCM-D sensor disc modified by self-assembled monolayer based on 11-mercaptoundecanoic acid and (ii)interaction of immobilized SCoV2-N with specific polyclonal anti-SCoV2-N antibodies followed by immune complex formation process. The results show that the SCoV2-N monolayer is rigid due to the low energy dissipation registered during the QCM-D measurement. In contrast, the anti-SCoV2-N layer produced after interaction with the immobilized SCoV2-N formed a soft and viscous layer. It was determined, that the sparse distribution of SCoV2-N on the surface affected the spatial arrangement of the antibody during the formation of immune complexes. The hinge-mediated flexibility of the antibody Fab fragments allows them to reach the more distantly located SCoV2-N and establish a bivalent binding between proteins in the formed SCoV2-N/anti-SCoV2-N complex. It was noted that the SE/QCM-D method can provide more precise quantitative information about the flexibility and conformational changes of antibody during the formation of the immune complex on the surface over time.

Electrochemical Determination of Interaction between SARS-CoV-2 Spike Protein and Specific Antibodies.

The serologic diagnosis of coronavirus disease 2019 (COVID-19) and the evaluation of vaccination effectiveness are identified by the presence of antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we present the electrochemical-based biosensing technique for the detection of antibodies specific to the SARS-CoV-2 proteins. Recombinant SARS-CoV-2 spike proteins (rSpike) were immobilised on the surface of a gold electrode modified by a self-assembled monolayer (SAM). This modified electrode was used as a sensitive element for the detection of polyclonal mouse antibodies against the rSpike (anti-rSpike). Electrochemical impedance spectroscopy (EIS) was used to observe the formation of immunocomplexes while cyclic voltammetry (CV) was used for additional analysis of the surface modifications. It was revealed that the impedimetric method and the elaborate experimental conditions are appropriate for the further development of electrochemical biosensors for the serological diagnosis of COVID-19 and/or the confirmation of successful vaccination against SARS-CoV-2.

Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases.

Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.

Yeast Secretes High Amounts of Human Calreticulin without Cellular Stress.

The ER chaperone calreticulin (CALR) also has extracellular functions and can exit the mammalian cell in response to various factors, although the mechanism by which this takes place is unknown. The yeast Saccharomyces cerevisiae efficiently secretes human CALR, and the analysis of this process in yeast could help to clarify how it gets out of eukaryotic cells. We have achieved a secretion titer of about 140 mg/L CALR in our S. cerevisiae system. Here, we present a comparative quantitative whole proteome study in CALR-secreting yeast using non-equilibrium pH gradient electrophoresis (NEPHGE)-based two-dimensional gel electrophoresis (2DE) as well as liquid chromatography mass spectrometry in data-independent analysis mode (LC-MSE). A reconstructed carrier ampholyte (CA) composition of NEPHGE-based first-dimension separation for 2DE could be used instead of formerly commercially available gels. Using LC-MSE, we identified 1574 proteins, 20 of which exhibited differential expression. The largest group of differentially expressed proteins were structural ribosomal proteins involved in translation. Interestingly, we did not find any signs of cellular stress which is usually observed in recombinant protein-producing yeast, and we did not identify any secretory pathway proteins that exhibited changes in expression. Taken together, high-level secretion of human recombinant CALR protein in S. cerevisiae does not induce cellular stress and does not burden the cellular secretory machinery. There are only small changes in the cellular proteome of yeast secreting CALR at a high level.

Peptide Antibody Reactivity to Homologous Regions in Glutamate Decarboxylase Isoforms and Coxsackievirus B4 P2C.

Two isoforms of the glutamate decarboxylase (GAD) enzyme exist, GAD65 and GAD67, which are associated with type 1 diabetes (T1D) and stiff-person syndrome (SPS), respectively. Interestingly, it has been reported that T1D patients seldom develop SPS, whereas patients with SPS occasionally develop T1D. In addition, coxsackievirus B4 (CVB4) has previously been proposed to be involved in the onset of T1D through molecular mimicry. On this basis, we aimed to examine antibody cross-reactivity between a specific region of GAD65 and GAD67, which has high sequence homology to the nonstructural P2C protein of CVB4 to determine potential correlations at antibody level. Monoclonal peptide antibodies generated in mice specific for a region with high similarity in all three proteins were screened for reactivity along with human sera in immunoassays. In total, six antibodies were generated. Two of the antibodies reacted to both GAD isoforms. However, none of the antibodies were cross-reactive to CVB, suggesting that antibody cross-reactivity between GAD65 and CVB, and GAD67 and CVB may not contribute to the onset of T1D and SPS, respectively.

Molecularly imprinted polypyrrole based sensor for the detection of SARS-CoV-2 spike glycoprotein.

This study describes the application of a polypyrrole-based sensor for the determination of SARS-CoV-2-S spike glycoprotein. The SARS-CoV-2-S spike glycoprotein is a spike protein of the coronavirus SARS-CoV-2 that recently caused the worldwide spread of COVID-19 disease. This study is dedicated to the development of an electrochemical determination method based on the application of molecularly imprinted polymer technology. The electrochemical sensor was designed by molecular imprinting of polypyrrole (Ppy) with SARS-CoV-2-S spike glycoprotein (MIP-Ppy). The electrochemical sensors with MIP-Ppy and with polypyrrole without imprints (NIP-Ppy) layers were electrochemically deposited on a platinum electrode surface by a sequence of potential pulses. The performance of polymer layers was evaluated by pulsed amperometric detection. According to the obtained results, a sensor based on MIP-Ppy is more sensitive to the SARS-CoV-2-S spike glycoprotein than a sensor based on NIP-Ppy. Also, the results demonstrate that the MIP-Ppy layer is more selectively interacting with SARS-CoV-2-S glycoprotein than with bovine serum albumin. This proves that molecularly imprinted MIP-Ppy-based sensors can be applied for the detection of SARS-CoV-2 virus proteins.

Endoplasmic reticulum stress-induced release and binding of calreticulin from human ovarian cancer cells.

BACKGROUND: Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone, but can appear surface bound on cancers cells, including ovarian cancers (OC). We investigated at what stage of cell viability, CRT appeared associated with surface of human OC cells. CRT on pre-apoptotic tumour cells is thought to initiate their eradication via a process termed immunogenic cell death (ICD). METHODS: We treated OC cells with the chemotherapeutic-doxorubicin (DX) known to induce translocation of CRT to some tumour cell surfaces, with and without the ER stressor-thapsigargin (TG)-and/or an ER stress inhibitor-TUDCA. We monitored translocation/release of CRT in pre-apoptotic cells by flow cytometry, immunoblotting and ELISA. We investigated the difference in binding of FITC-CRT to pre-apoptotic, apoptotic and necrotic cells and the ability of extracellular CRT to generate immature dendritic cells from THP-1 monocytes. RESULTS: Dx-treatment increased endogenously released CRT and extracellular FITC_CRT binding to human pre-apoptotic OC cells. DX and TG also promoted cell death in OC cells which also increased CRT release. These cellular responses were significantly inhibited by TUDCA, suggesting that ER stress is partially responsible for the changes in CRT cellular distribution. Extracellular CRT induces maturation of THP-1 towards a imDC phenotype, an important component of ICD. CONCLUSION: Collectively, these cellular responses suggest that ER stress is partially responsible for the changes in CRT cellular distribution. ER-stress regulates in part the release and binding of CRT to human OC cells where it may play a role in ICD.

Mapping human calreticulin regions important for structural stability.

Calreticulin (CALR) is a highly conserved multifunctional chaperone protein primarily present in the endoplasmic reticulum, where it regulates Ca2+ homeostasis. Recently, CALR has gained special interest for its diverse functions outside the endoplasmic reticulum, including the cell surface and extracellular space. Although high-resolution structures of CALR exist, it has not yet been established how different regions and individual amino acid residues contribute to structural stability of the protein. In the present study, we have identified key residues determining the structural stability of CALR. We used a Saccharomyces cerevisiae expression system to express and purify 50 human CALR mutants, which were analysed for several parameters including secretion titer, melting temperature (Tm), stability and oligomeric state. Our results revealed the importance of a previously identified small patch of conserved surface residues, amino acids 166-187 ("cluster 2") for structural stability of the human CALR protein. Two residues, Tyr172 and Asp187, were critical for maintaining the native structure of the protein. Mutant D187A revealed a severe drop in secretion titer, it was thermally unstable, prone to degradation, and oligomer formation. Tyr172 was critical for thermal stability of CALR and interacted with the third free Cys163 residue. This illustrates an unusual thermal stability of CALR dominated by Asp187, Tyr172 and Cys163, which may interact as part of a conserved structural unit. Besides structural clusters, we found a correlation of some measured parameter values in groups of CALR mutants that cause myeloproliferative neoplasms (MPN) and in mutants that may be associated with sudden unexpected death (SUD).

Epitope Mapping of Monoclonal Antibodies to Calreticulin Reveals That Charged Amino Acids Are Essential for Antibody Binding.

Calreticulin is a chaperone protein, which is associated with myeloproliferative diseases. In this study, we used resin-bound peptides to characterize two monoclonal antibodies (mAbs) directed to calreticulin, mAb FMC 75 and mAb 16, which both have significantly contributed to understanding the biological function of calreticulin. The antigenicity of the resin-bound peptides was determined by modified enzyme-linked immunosorbent assay. Specific binding was determined to an 8-mer epitope located in the N-terminal (amino acids 34-41) and to a 12-mer peptide located in the C-terminal (amino acids 362-373). Using truncated peptides, the epitopes were identified as TSRWIESK and DEEQRLKEEED for mAb FMC 75 and mAb 16, respectively, where, especially the charged amino acids, were found to have a central role for a stable binding. Further studies indicated that the epitope of mAb FMC 75 is assessable in the oligomeric structure of calreticulin, making this epitope a potential therapeutic target.

Structural Analysis of Calreticulin, an Endoplasmic Reticulum-Resident Molecular Chaperone.

Calreticulin (Calr) is an endoplasmic reticulum (ER) chaperone involved in protein quality control, Ca2+ regulation and other cellular processes. The structure of Calr is unusual, reflecting different functions of the protein: a proline-rich ß-hairpin arm and an acidic C-terminal tail protrude from a globular core, composed of a ß-sheet sandwich and an α-helix. The arm and tail interact in the presence of Ca2+ and cover the upper ß-sheet, where a carbohydrate-binding site gives the chaperone glycoprotein affinity. At the edge of the carbohydrate-binding site is a conserved, strained disulphide bridge, formed between C106 and C137 of human Calr, which lies in a polypeptide-binding site. The lower ß-sheet has several conserved residues, comprised of a characteristic triad, D166-H170-D187, Tyr172 and the free C163. In addition to its role in the ER, Calr translocates to the cell surface upon stress and functions as an immune surveillance marker. In some myeloproliferative neoplasms, the acidic Ca2+-binding C-terminal tail is transformed into a polybasic sequence.

Evaluation of kinetics and thermodynamics of interaction between immobilized SARS-CoV-2 nucleoprotein and specific antibodies by total internal reflection ellipsometry.

During the pandemic, different methods for SARS-CoV-2 detection and COVID-19 diagnostics were developed, including antibody and antigen tests. For a better understanding of the interaction mechanism between SARS-CoV-2 virus proteins and specific antibodies, total internal reflection ellipsometry based evaluation of the interaction between SARS-CoV-2 nucleoprotein (SCoV2-rN) and anti-SCoV2-rN antibodies was performed. Results show that the appropriate mathematical model, which takes into account the formation of an intermediate complex, can be applied for the evaluation of SCoV2-rN/anti-SCoV2-rN complex formation kinetics. The calculated steric factor indicated that SCoV2-rN/anti-SCoV2-rN complex formation has very strict steric requirements. Estimated Gibbs free energy (ΔGAssoc) for SCoV-rN and anti-SCoV-rN binding was determined as -34 kJ/mol. The reported findings are useful for the design of new analytical systems for the determination of anti-SCoV2-rN antibodies and for the development of new anti-SARS-CoV-2 medications.

Antibodies to Epstein-Barr virus and neurotropic viruses in multiple sclerosis and optic neuritis.

Antibody indices to Measles, Mumps, Varicella Zoster (MRZ) are of diagnostic value in multiple sclerosis (MS). Here, we have investigated, if this panel could be extended to increase diagnostic value. Samples from relapsing-remitting (RR) MS and optic neuritis (ON) patients were tested for reactivity to antigens from Epstein-Barr, Varicella Zoster, Measles, Mumps and Rubella (EMMRZ) viruses. Increased IgG levels in serum and cerebrospinal fluid (CSF) were found in RRMS patients, along with a significant correlation between serum and CSF. The sensitivity of the EMMRZ panel was increased approximately 40% compared to the MRZ panel, suggesting that the EMMRZ panel may be useful in MS and ON diagnostics.

Comparison of immunological adjuvants.

In this study, several innate immunological adjuvants and related compounds were compared with respect to complement activation in serum and induction of cytokine release in whole blood samples using immunoassays. As found, simple lipids had no effect on the complement system or on cytokine release, whereas lipopolysaccharides induced prominent release of pro-inflammatory cytokines (IL1ß, TNF and IFNγ) without affecting the complement system, except for one, which activated the lectin pathway (LP). Moreover, saponin induced IL1ß and MCP1 release and did not affect the complement system. The polysaccharide inulin exhausted the alternative pathway (AP) completely without affecting the LP and the classical pathway (CP), whereas zymosan exhausted the AP and had a major effect on the LP and CP as well. Peptidoglycans mainly affected the LP. Inulin, agarose and cellulose induced IL1ß and MCP1 release, while dextran had no effect on cytokine secretion. Zymosan mainly induced IL1ß release. The inorganic compound aluminium hydroxide, Al(OH)3 , activated the complement system very efficiently (all three pathways) but only induced MCP1 release. Other compounds tested had minor/individual effects. Collectively, well-known adjuvants, such as aluminum hydroxide, activated the complement system and/or induced pro-inflammatory cytokine release. Since complement activation generates anaphylactic peptides, a simple definition of an (innate) immunological adjuvant can be inferred: it activates the (innate) immune system by complement activation and/or release of cytokines so as to attract cells of the adaptive immune system.

Mapping the Ca(2+) induced structural change in calreticulin.

UNLABELLED: Calreticulin is a highly conserved multifunctional protein implicated in many different biological systems and has therefore been the subject of intensive research. It is primarily present in the endoplasmatic reticulum where its main functions are to regulate Ca(2+) homeostasis, act as a chaperone and stabilize the MHC class I peptide-loading complex. Although several high-resolution structures of calreticulin exist, these only cover three-quarters of the entire protein leaving the extended structures unsolved. Additionally, the structure of calreticulin is influenced by the presence of Ca(2+). The conformational changes induced by Ca(2+) have not been determined yet as they are hard to study with traditional approaches. Here, we investigated the Ca(2+)-induced conformational changes with a combination of chemical cross-linking, mass spectrometry, bioinformatics analysis and modelling in Rosetta. Using a bifunctional linker, we found a large Ca(2+)-induced change to the cross-linking pattern in calreticulin. Our results are consistent with a high flexibility in the P-loop, a stabilization of the acidic C-terminal and a relatively close interaction of the P-loop and the acidic C-terminal. BIOLOGICAL SIGNIFICANCE: The function of calreticulin, an endoplasmatic reticulin chaperone, is affected by fluctuations in Ca(2+)concentration, but the structural mechanism is unknown. The present work suggests that Ca(2+)-dependent regulation is caused by different conformations of a long proline-rich loop that changes the accessibility to the peptide/lectin-binding site. Our results indicate that the binding of Ca(2+) to calreticulin may thus not only just be a question of Ca(2+) storage but is likely to have an impact on the chaperone activity.

Survey of molecular chaperone requirement for the biosynthesis of hamster polyomavirus VP1 protein in Saccharomyces cerevisiae.

A number of viruses utilize molecular chaperones during various stages of their life cycle. It has been shown that members of the heat-shock protein 70 (Hsp70) chaperone family assist polyomavirus capsids during infection. However, the molecular chaperones that assist the formation of recombinant capsid viral protein 1 (VP1)-derived virus-like particles (VLPs) in yeast remain unclear. A panel of yeast strains with single chaperone gene deletions were used to evaluate the chaperones required for biosynthesis of recombinant hamster polyomavirus capsid protein VP1. The impact of deletion or mild overexpression of chaperone genes was determined in live cells by flow cytometry using enhanced green fluorescent protein (EGFP) fused with VP1. Targeted genetic analysis demonstrated that VP1-EGFP fusion protein levels were significantly higher in yeast strains in which the SSZ1 or ZUO1 genes encoding ribosome-associated complex components were deleted. The results confirmed the participation of cytosolic Hsp70 chaperones and suggested the potential involvement of the Ydj1 and Caj1 co-chaperones and the endoplasmic reticulum chaperones in the biosynthesis of VP1 VLPs in yeast. Likewise, the markedly reduced levels of VP1-EGFP in Δhsc82 and Δhsp82 yeast strains indicated that both Hsp70 and Hsp90 chaperones might assist VP1 VLPs during protein biosynthesis.

High-level secretion of native recombinant human calreticulin in yeast.

BACKGROUND: Calreticulin (CRT) resides in the endoplasmic reticulum (ER) and functions to chaperone proteins, ensuring proper folding, and intracellular Ca(2+) homeostasis. Emerging evidence shows that CRT is a multifunctional protein with significant roles in physiological and pathological processes with presence both inside and outside of the ER, including the cell surface and extracellular space. These recent findings suggest the possible use of this ER chaperone in development of new therapeutic pharmaceuticals. Our study was focused on human CRT production in two yeast species, Saccharomyces cerevisiae and Pichia pastoris. RESULTS: Expression of a full-length human CRT precursor including its native signal sequence resulted in high-level secretion of mature recombinant protein into the culture medium by both S. cerevisiae and P. pastoris. To ensure the structural and functional quality of the yeast-derived CRTs, we compared yeast-secreted human recombinant CRT with native CRT isolated from human placenta. In ESI-MS (electrospray ionization mass spectrometry), both native and recombinant full-length CRT showed an identical molecular weight (mass) of 46,466 Da and were monomeric by non-denaturing PAGE. Moreover, limited trypsin digestion yielded identical fragment patterns of calcium-binding recombinant and native CRT suggesting that the yeast-derived CRT was correctly folded. Furthermore, both native and recombinant CRT induced cellular proliferation (MTS assay) and migration of human dermal fibroblasts (in vitro wound healing assay) with the same specific activities (peak responses at 1-10 ng/ml) indicating that the functional integrity of yeast-derived CRT was completely preserved. Simple one-step purification of CRT from shake-flask cultures resulted in highly pure recombinant CRT protein with yields reaching 75 % of total secreted protein and with production levels of 60 and 200 mg/l from S. cerevisiae and P. pastoris, respectively. Finally, cultivation of P. pastoris in a bioreactor yielded CRT secretion titer to exceed 1.5 g/l of culture medium. CONCLUSIONS: Yeasts are able to correctly process and secrete large amounts of mature recombinant human CRT equally and fully biologically active as native human CRT. This allows efficient production of high-quality CRT protein in grams per liter scale.

Heat shock at higher cell densities improves measles hemagglutinin translocation and human GRP78/BiP secretion in Saccharomyces cerevisiae.

The yield of heterologous proteins is often limited by several bottlenecks in the secretory pathway of yeast Saccharomyces cerevisiae. It was shown earlier that synthesis of measles virus hemagglutinin (MeH) is inefficient mostly due to a bottleneck in the translocation of viral protein precursors into the endoplasmic reticulum (ER) of yeast cells. Here we report that heat shock with subsequent induction of MeH expression at 37°C improved translocation of MeH precursors when applied at higher cell densities. The amount of MeH glycoprotein increased by about 3-fold after heat shock in the late-log phases of both glucose and ethanol growth. The same temperature conditions increased both secretion titer and yield of another heterologous protein human GRP78/BiP by about 50%. Furthermore, heat shock at the late-log glucose growth phase also improved endogenous invertase yield by approximately 2.7-fold. In contrast, a transfer of yeast culture to lower temperature at diauxic shift followed by protein expression at 20°C almost totally inhibited translocation of MeH precursors. The difference in amounts of MeH glycoprotein under expression at 37°C and 20°C was about 80-fold, while amounts of unglycosylated MeH polypeptides were similar under both conditions. Comparative proteomic analysis revealed that besides over-expressed ER-resident chaperone Kar2, an increased expression of several cytosolic proteins (such as Hsp104, Hsp90 and eEF1A) may contribute to improved translocation of MeH.