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Keywords:

  • precrystallization screening;
  • green fluorescent protein;
  • GFPuv;
  • secretory pathway;
  • class C GPCR

Abstract

  1. Top of page
  2. Abstract
  3. Outline
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

Applications of the GFP-fusion technique have greatly facilitated evaluations of the amounts and qualities of sample proteins used for structural analyses. In this study, we applied the GFP-based sample evaluation to secreted protein expression by insect cells. We verified that a GFP variant, GFPuv, retains proper folding and monodispersity within all expression spaces in Sf9 cells, such as the cytosol, organelles, and even the extracellular space after secretion, and thus can serve as a proper folding reporter for recombinant proteins. We then applied the GFPuv-based system to the extracellular domains of class C G-protein coupled receptors (GPCRs) and examined their localization, folding, and oligomerization upon insect cell expression. The extracellular domain of metabotropic glutamate receptor 1 (mGluR1) exhibited good secreted expression by Sf9 cells, and the secreted proteins formed dimer with a monodisperse hydrodynamic state favorable for crystallization, consistent with the results from previous successful structural analyses. In contrast, the extracellular domains of sweet/umami taste receptors (T1R) almost completely remained in the cell. Notably, the T1R and mGluR1 subfractions that remained in the cellular space showed polydisperse hydrodynamic states with large aggregated fractions, without forming dimers. These results indicated that the proper folding and oligomerization of the extracellular domains of the class C GPCR are achieved through the secretory pathway.


Outline

  1. Top of page
  2. Abstract
  3. Outline
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

We have applied a GFP-based sample evaluation protocol to secreted protein expression, using insect cells. We verified that GFPuv, retains proper folding within all expression spaces such as the cytosol, organelles, and the extracellular space after secretion, We then evaluated the properties of the secreted expression of the extracellular domains of class C GPCRs using the system, in terms of the localization, folding, and oligomerization of the expressed products.

Introduction

  1. Top of page
  2. Abstract
  3. Outline
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

About one-third of eukaryotic genes encode membrane proteins and secreted proteins,1, 2 which play important roles in various biological activities such as intercellular communication, signal perception and transduction, permeation of various substances through membranes, and cell adhesion. Eukaryotic membrane proteins and secreted proteins are now important targets for structural biology in terms of basic life sciences and drug discovery. Most of these proteins acquire their properly folded states and functional structures through the common secretory pathway, by way of the endoplasmic reticulum (ER) and the Golgi, to the appropriate cellular compartments, such as the plasma membrane.3–6 During the process, misfolded proteins are removed by quality control systems represented by ER-associated degradation.7, 8 Therefore, for the purpose of sample preparation of eukaryotic membrane and secreted proteins for structural analyses, eukaryotic cells are considered to serve as suitable expression hosts, as they are expected to possess similar folding, quality control, and degradation machineries to those in their native host cells. Nevertheless, overexpression of membrane or secreted proteins in heterologous cells often results in low expression levels, improper translocation/trafficking, or misfolding, probably due to the lack of the same systems for proper protein folding and trafficking in the native hosts. Consequently, the protein expression step has been a serious bottleneck in structural studies of membrane and secreted proteins.

One of the attempts to overcome the difficulties encountered in the protein expression steps is a strategy called the “funnel approach”9 or “precrystallization screening.”10 The process consists of the rational selection of target proteins showing good expression, in terms of the amount and proper folding, at an early stage of the analyses, achieved by extensive screening of genes, expression, and sample-preparation conditions.9–12 Applications of green fluorescent protein (GFP)-fusion techniques have greatly facilitated these target selection processes. A common fundamental feature of the GFP-fusion techniques is that the targets are genetically and exclusively “labeled” with GFP and thus can be specifically detected by its fluorescent signal, enabling rapid and efficient biochemical assessments using small quantities of samples without any tedious purification processes. The GFP-fusion strategy has been applied to a variety of screenings, such as for expression levels by whole-cell fluorescence measurement,11, 13–17 localization of expression by confocal microscopic observation,11 molecular weight and integrity assessment by SDS-PAGE analysis,18 and evaluation of hydrodynamic states by fluorescence-detection size-exclusion chromatography (FSEC)10 or improved Native-PAGE.19 Especially, FSEC screening emphasizes the sample monodispersity, a unimodal distribution of the hydrodynamic volume of the sample, which reflects proper folding and is considered as an essential factor for successful crystallization.20, 21 FSEC has indeed been leading to successes in the crystallographic analyses of eukaryotic membrane proteins, as well as prokaryotic ones.22–27 While those applications have generally used sample proteins with GFP tagged to the cytoplasmic termini of the targets, the GFP-fusion strategies have not yet been extensively applied to secreted proteins or the extracellular side of membrane proteins, which are expressed by way of the secretory pathway. GFP reportedly showed insufficient secretion upon eukaryotic expression,28, 29 and thus the applicability of the GFP-based screening strategy to the secretory pathway was unclear.

In this study, we first examined the applicability of GFP as a reporter for sample evaluation to a eukaryotic secreted expression system. We confirmed that GFPuv, a brighter GFP variant, showed good secretion and proper folding upon insect cell expression, and exhibited monodisperse characteristics suitable for the reporter in all expression spaces, including the cytosol, organelles, and the extracellular space.

We then applied the GFPuv-based sample evaluation system to the extracellular domains of the class C G-protein coupled receptor (GPCR) family. GPCRs mediate a wide array of extracellular signals to intracellular responses by activating heterotrimeric G-proteins, and the class C family includes receptors for neurotransmitters (glutamate and γ-aminobutyric acid), calcium ions, sweet and umami taste substances, and pheromones.30, 31 The class C members possess large extracellular domains (ca. 500–600 residues) responsible for ligand binding, namely the Venus Flytrap module homologous to bacterial periplasmic leucine/isoleucine/valine-binding proteins,32 in addition to the following seven helical transmembrane regions commonly observed in GPCRs. The receptors in this family exist as homo- or heterodimers in physiological states. Among the members, the expression of the ligand binding domains of two samples were tested in this study: one is metabotropic glutamate receptor (mGluR), the sole member whose crystal structure has been solved,33–35 and the other is sweet/umami taste receptors, which have not yet been reported about successful sample preparation using eukaryotic expression systems or crystallization. The GFP-based evaluation dissected the expression properties of these samples through the secretory pathway, in terms of localization, folding, and oligomerization.

Results

  1. Top of page
  2. Abstract
  3. Outline
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

Reporter GFP system

We adopted the GFP variant, GFPuv,36 as the fusion reporter for the expression, folding, and hydrodynamic status (monodispersity) of test proteins produced by insect-cell expression systems. GFPuv shows a 16- to 18-fold higher fluorescence signal, as compared to wild type GFP (wtGFP), when expressed in E. coli,36 and thus is expected to be advantageous to detect difficult target proteins with poor expression. GFPuv is also much less prone to aggregation, as compared to wtGFP,37 and is presumed to be folded properly and to be less likely to undergo GFP-mediated aggregation in a wide variety of expression conditions. We chose the A206K mutant of GFPuv, to avoid dimerization through GFP,10, 38 expecting to act as an ideal reporter for the multimerization state of a target protein.

GFPuv showed good expression and a strong fluorescence signal in insect cells, Sf9 (Fig. 1), although we used the original GFPuv gene with its codon usage optimized for E. coli expression.36 The FSEC elution profiles of GFPuv transiently expressed in the cytosol of Sf9 cells [Fig. 1(C)] and High Five cells (data not shown) exhibited a single, sharp peak, indicating a monodisperse property in solution, and thus representing proper folding and the absence of aggregation. The apparent molecular weight estimated from the retention volume of the elution peak was 41.2 KDa, in agreement with that of the monomeric GFPuv (31.6 KDa, calculated from its amino-acid sequence).

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Figure 1. Transient expression of GFPuv in Sf9 cells, using the vectors for cytosolic expression (A, C, E) and secreted expression (B, D, F). (A, B) Western-blotting analyses of the expression products from Sf9 cells transfected with the pCGFP_SF vector (cytosolic expression, panel A) or the pCGFP_SF+AKH vector (secreted expression, panel B) at 120 h after transfection. Lanes marked “C” and “S” show cellular (cytoplasm and organelles) and secreted fractions, respectively. (C, D) FSEC elution profiles of the samples transfected with the pCGFP_SF vector (C) or the pCGFP_SF+AKH vector (D). The same samples used in panels A and B were subjected to the analyses. Cyan and red lines show the profiles of the cellular (cytoplasm and organelles) and secreted fractions, respectively. The inset shows a magnification of the profile of the cellular fraction. (E, F) Confocal microscopic observation of Sf9 cells transfected with the pCGFP_SF vector (E) or the pCGFP_SF+AKH vector (F) at 72 h after transfection. The scale bars represent 5 μm.

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Notably, GFPuv was secreted well upon the addition of the tobacco hornworm adipokinetic hormone secretion signal (AKHss),39 as judged by western blotting [Fig. 1(B)] and FSEC [Fig. 1(D)]. The ratio of the secreted to cell-remained AKHss-GFPuv fractions observed at 120 h after transfection was 6.0, as estimated by the peak areas of the FSEC profiles of the culture media and cell lysate, respectively. The observed secretion level of GFPuv was much higher than that of wtGFP, which exhibited insufficient secretion from insect culture cells with any cell lines or signal sequences tested (in the range of almost no secretion to a ratio of 1.4).28, 29 Confocal-microscopic observations of cells expressing AKHss-GFPuv revealed the fluorescence localization [Fig. 1(F)], which likely indicated the existence of GFPuv in organelles, undergoing processing and trafficking. In contrast, the cytosolic-expressed GFPuv was distributed throughout the entire cytoplasm [Fig. 1(E)]. We found that the elution profiles of both AKHss-GFP samples undergoing processing (the cellular fraction) and after secretion (the extracellular fraction recovered from the culture media) exhibited single, sharp peaks [Fig. 1(D)], similar to the cytosolic-expressed GFPuv [Fig. 1(C)]. These results indicated that GFPuv retains proper folding and monodispersity at all expression spaces in Sf9 cells, such as the cytosol, organelles, and even after secretion.

The recombinant GFPuv production by the system was quantitatively analyzed by the peak areas of the FSEC elution profiles. The time course of GFP expression and the observed expression levels were comparable to those normally observed with Sf9 cells (Fig. 2). In the case of AKHss-GFPuv expression, the GFPuv was accumulated in the extracellular fractions as expected, while a constant amount of GFPuv was observed in the cellular fractions [Fig. 2(B)], implying that amount of the protein undergoing the processing and trafficking is steady during the secreted expression.

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Figure 2. Quantitative analyses of recombinant GFPuv in transiently transfected Sf9 cells, at each time point after transfection. Line graphs represent relative fluorescence intensity measured by in vivo fluorescence detection with a microplate reader, while cyan and red bars describe the amounts of GFPuv in the cellular (cytoplasm and organelles) and secreted fractions of the same culture samples evaluated by FSEC, respectively. (A) Cytosolic expression of GFPuv (B) secreted expression of GFPuv with the AKH signal sequence. The error bars for each data point represent the standard deviation from triplicate measurements.

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In addition, we confirmed that the high fluorescence intensity of GFPuv enables the easy estimation of recombinant GFP-expression levels by insect cells in vivo. Taking advantage of the transparency of insect cells and their subjectability to monolayer culture, we established a protocol to estimate the relative amount of GFP in cultured cells fairly accurately. We were able to measure GFP-derived fluorescence from living cells while keeping the culture intact, using only conventional culture plates and a microplate reader (for details, please see materials and methods). The quantification results using the protocol were in good agreement with those obtained by FSEC (Fig. 2). In our experience, relative fluorescence intensities above 0.2 in our system are sufficient for quantification.

All together, we confirmed that GFPuv serves as a suitable fluorescent reporter for recombinant test proteins expressed in insect cells, for both cytosolic and secreted expression, as well as its successful application in a prokaryotic expression system.10

Application of GFP-based precrystallization evaluation to the extracellular domains of class C GPCRs

We applied the GFPuv-based expression evaluation to the extracellular domains of the class C GPCR family. The two family members, mGluR and sweet/umami taste receptors, were tested for insect cell expression, and the expression products were evaluated in terms of folding and oligomerization by FSEC, and trafficking by confocal microscopy.

Metabotropic glutamate receptor 1

The mGluRs are receptors for L-glutamate, the major excitatory neurotransmitter in the central nervous system in vertebrates. mGluRs are considered to be involved in neuronal activities by activating slow excitatory potentials at postsynaptic cells, as well as by regulating neuronal excitability, synaptic transmission and plasticity.40 The crystal structures of the extracellular ligand-binding region of rat mGluR1 (mGluR1LBD) were successfully solved using a sample prepared by secreted expression from Sf9 cells.33, 34 In this study, we used the same mGluR1LBD construct previously employed for the crystallographic analyses (residues 1–522, including its original signal sequence),33, 34 and fused GFPuv to its C-terminus.

As judged by western blotting [Fig. 3(A)] and FSEC [Fig. 3(B)], the mGluR1LBD-GFPuv fusion protein showed good secreted expression by Sf9 cells, and the ratio of secreted to cell-remaining fractions observed at 120 h transient expression was 13.3. The FSEC elution profile of the secreted mGluR1LBD-GFPuv exhibited a single, sharp peak [Fig. 3(B)], indicating the monodisperse property of the sample, as expected from the previous successful crystallization results. The molecular weight of mGluR1LBD-GFPuv estimated by the FSEC analysis was 215.7 KDa. Considering the calculated molecular weight from the amino acid sequence is 87.6 KDa and that the expressed sample was likely modified with several glycosylations, this result suggested that the secreted mGluR1-GFPuv forms a dimer, as observed by biochemical41 and crystallographic33 analyses. The secreted mGluR1LBD-GFPuv, expressed with the system, exhibited enhanced intrinsic tryptophan fluorescence upon glutamate binding (Supporting Information Fig. S1), as reported previously.42 This result verified that the monodisperse hydrodynamic state of the secreted fraction of mGluR1LBD-GFPuv, observed with the FSEC analysis, actually reflects the proper, functional folding of the protein.

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Figure 3. Expression analyses of mGluR1LBD-GFPuv by Sf9 cells at 120 h after transfection. (A) Western blotting analysis. Lanes marked “C” and “S” show cellular (cytoplasm and organelles) and secreted fractions, respectively. (B) FSEC profiles. Cyan and red lines show the elution profiles of the cellular (cytoplasm and organelles) and secreted fractions, respectively. The closed and open triangles represent the elution volumes corresponding to dimeric and monomeric mGluR1LBD-GFPuv, respectively. The inset shows a magnification of the cellular fraction profile. (C) Typical observations of Sf9 cells expressing mGluR1LBD-GFPuv by confocal-microscopy at 72 h after transfection. The scale bar represents 5 μm.

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The high secretion ratio and the monodiserse profile for the secreted fraction were also observed upon the expression of the ligand binding domain of the group III mGluR subtype 7 (mGluR-III7; Supporting Information Fig. S2), the other group member of mGluR whose crystal structure has been solved using a sample prepared with insect cells.35 The noticeable secreted populations with favorable FSEC profiles lacking aggregated fractions were also observed for the non-class C GPCR samples, the amino-terminal domains (ATD) of the AMPA receptor GluR243 and the NMDA receptor NR2B,44 the other examples whose samples were prepared by insect-cells secreted expression and resulted successful crystallographic analyses (Supporting Information Fig. S2). These results further verified that the observed features were not mGluR-specific properties, but general characteristics of properly folded proteins.

In contrast, the FSEC elution profile of mGluR1LBD-GFPuv from the cellular fraction was poor, indicating a polydisperse property. This profile included a peak at the retention volume corresponding to 94.4 KDa, as well as a peak at the void volume and a broadly distributed high background, both representing the existence of aggregated fractions [Fig. 3(B)]. The mGluR1LBD-GFPuv showed somewhat localized distribution in the cells under confocal microscopic observation [Fig. 3(C)], but the distribution was not as compact as that observed for the AKHss-GFPuv expression. These results indicate that only the properly folded and oligomerized fraction of mGluR1LBD is properly trafficked to the extracellular space.

Sweet/umami taste receptors

The taste receptors are expressed in taste cells in the oral cavity, and are responsible for direct interactions with tastants at the initial step of taste perception.45 Among these receptors, the sweet and umami receptors are referred to as T1R and belong to the class C GPCRs. The T1R family consists of three members, T1R1, T1R2, and T1R3. The T1R1/T1R3 heterodimer is activated by umami tastants, including L-glutamate as a representative,46, 47 while the T1R2/T1R3 heterodimer is activated by various kinds of sweet taste compounds, such as sugars, artificial sweeteners, and sweet proteins.48

We attempted to transiently express the C-terminal GFP-fusion product of the extracellular, putative ligand binding domains of mouse T1Rs (T1RsLBD), which consist of the corresponding regions of mGluRLBD, with their original putative signal sequences. In contrast to the case for mGluR1LBD-GFPuv, almost all of the expression products by Sf9 cells remained in the cellular fraction, and the secreted fractions were hardly observed (Fig. 4). Confocal microscopic observation revealed that the expression products were either distributed within the cytoplasmic space or somewhat localized similar to mGluR1LBD-GFPuv [Fig. 4(E–G,K,L)]. The FSEC elution profiles of the cellular fractions of T1RsLBD-GFPuv showed polydisperse patterns, indicating the existence of large aggregated fractions and a broad peak which is probably corresponding to the monomeric T1RLBD-GFPuv [Fig. 4(B–D,I,J)]. The elution peaks corresponding to the dimeric T1RLBD-GFPuv were hardly observed, even for the coexpression of the physiological heterodimeric pairs, T1R1/T1R3 and T1R2/T1R3. A western-blotting analysis revealed that the bands for the intact T1RsLBD-GFPuv were the major ones, verifying that the aforementioned FSEC profiles and confocal-microscopic observations were derived from the T1RsLBD-GFPuv samples themselves, and not from their degradation products [Fig. 4(A,H)].

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Figure 4. Expression analyses of T1RsLBD-GFPuv by Sf9 cells at 72 h after transfection. (A–G) Expression of each T1RsLBD-GFPuv gene. (H–L) Coexpression of heterodimeric pairs of T1RsLBD-GFPuv genes. (A, H) Western blotting analyses. Lanes marked “C” and “S” show the cellular (cytoplasm and organelles) and secreted fractions, respectively, and the numbers above each lane correspond to the samples as follows: 1, T1R1; 2, T1R2; 3, T1R3; 1/3, coexpression of T1R1 and T1R3; 2/3, coexpression of T1R2 and T1R3. (B–D) and (I, J), FSEC elution profiles. Cyan and red lines show the cellular and secreted fractions, respectively. The open triangle represents the elution volume of the monomeric T1RsLBD-GFPuv. (B) T1R1; (C) T1R2; (D) T1R3; (I) coexpression of T1R1 and T1R3; (J) coexpression of T1R2 and T1R3. (E–G), (K, L) Typical confocal microscopic observations of T1RsLBD-GFPuv expressing Sf9 cells. (E) T1R1; (F) T1R2; (G) T1R3; (K) coexpression of T1R1 and T1R3; (L) coexpression of T1R2 and T1R3. The scale bars represent 5 μm.

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We further attempted to express T1RsLBD-GFPuv by adding the signal sequence of AKH, which had resulted in the successful secretion of GFPuv (Figs. 1 and 2), upstream of the putative signal sequences of T1Rs. However, the AKHss-T1RsLBD-GFPuv still remained in the cellular fractions and was hardly observed in the secreted fractions, similar to T1RsLBD-GFPuv without AKHss (Fig. 5). The FSEC profiles exhibited polydisperse patterns, including aggregated fractions and a small peak corresponding to the monomer [Fig. 5(B–D, I, J)], and lacking obvious peaks corresponding to the dimer. Similar expression results, such as exclusive remaining in the cellular fraction with no obvious secretion, and polydisperse FSEC profiles, were also observed for the AKHss-added T1RsLBD-GFPuv with deletion of the putative signal sequences of T1Rs (data not shown). Interestingly, the confocal microscopic observations of the cells expressing AKHss-T1RLBD-GFPuv showed some different features, as compared to T1RsLBD-GFPuv without AKHss, and localized regions with high fluorescence intensities existed [Fig. 5(E–G,K,L)]. Notably, these features were considered to be derived from both the intact AKHss-T1RsLBD-GFPuv and its degradation products as judged by western blotting analyses, since they included more small molecular weight protein bands, as compared to T1RsLBD-GFPuv [Fig. 5(A,H)].

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Figure 5. Expression analyses of AKHss-T1RsLBD-GFPuv by Sf9 cells at 72 h after transfection. (A–G) Expression of each AKHss-T1RsLBD-GFPuv gene. (H–L) Coexpression of heterodimeric pairs of AKHss-T1RsLBD-GFPuv genes. (A, H) Western blotting analyses. Lanes marked “C” and “S” show the cellular (cytoplasm and organelles) and secreted fractions, respectively, and the numbers above each lane correspond to the samples as follows: (1) T1R1; (2) T1R2; (3) T1R3; 1/3, coexpression of T1R1 and T1R3; 2/3, coexpression of T1R2 and T1R3. (B–D), (I, J) FSEC elution profiles. Cyan and red lines show the cellular and secreted fractions, respectively. The open triangle represents the elution volume of the monomeric AKHss-T1RsLBD-GFPuv. (B) T1R1; (C) T1R2; (D) T1R3; (I) coexpression of T1R1 and T1R3; (J) coexpression of T1R2 and T1R3. (E–G), (K, L) Typical confocal microscopic observations of AKHss-T1RsLBD-GFPuv expessing Sf9 cells. (E) T1R1; (F) T1R2; (G) T1R3; (K) coexpression of T1R1 and T1R3; (L) coexpression of T1R2 and T1R3. The scale bars represent 5 μm.

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These results indicated that expressed T1RsLBD samples, with either their original signal sequences or an additional secretion signal, were excluded from the secretory pathway, resulting in unsuccessful folding and oligomerization.

Discussion

  1. Top of page
  2. Abstract
  3. Outline
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

GFPuv is a good fluorescent reporter for target selection toward structural study

In this study, we demonstrated that GFPuv showed proper expression, folding, and monodispersity by insect cell expression (Fig. 1). GFPuv also reportedly showed highly improved fluorescence, as compared to wtGFP, when expressed in mammalian culture cells, such as Chinese hamster ovary cells.36 Therefore, GFPuv may function as a good fluorescence reporter for universal recombinant expression systems including eukaryotic cells, as well as the E. coli system, in which the protein was originally developed36 and used as a reporter.10

Notably, GFPuv exhibited favorable secreted expression, as well as cytosolic expression, in Sf9 (Fig. 1) and High Five (data not shown) insect cells. GFPuv was also reported to have good secretion efficiency by an S2 insect cell expression system.49 In this study, we further confirmed that GFPuv itself retains proper folding and monodispersity within all expression spaces in insect cells, such as the cytosol, organelles, and extracellular spaces (Fig. 1), and it properly reported the folding and oligomerization states of the target proteins, mGluR1LBD and T1RsLBD (Figs. 3, 4, 5, 3–5), localized within the various expression spaces. Secreted expression by a eukaryotic cell system is a promising production method for eukaryotic extracellular proteins and the extracellular regions of membrane proteins, because the proteins have better chances of being folded correctly, with proper posttranslational processing and modifications. Nevertheless, the GFP-based precrystallization selection has not been extensively applied to secreted expression. This might be partly because of the poor expression of the reporter GFP, as GFP produces fluorescence only by cytosolic expression in E. coli,50 and wtGFP showed insufficient secretion, even by eukaryotic expression.28, 29 Our study indicated that the application of GFPuv enables GFP-based target evaluation and screening for extracellular proteins or membrane proteins with a GFP-tag on their extracellular side, using eukaryotic secreted expression systems. However, it should be noted that the general applicability of GFPuv to secreted expression might need to be further tested, as the secretion of GFPuv was hardly observed in some cases on HEK293 expression (Takagi, personal communication).

In addition, the high fluorescence intensity from GFPuv also facilitates quantitative expression analysis in living Sf9 cells during culture (Fig. 2). It allows real-time monitoring of the production of the GFP-fusion protein for a single cell culture run, as the culture container can be kept closed, and thus is useful for quick screening of expression conditions or high-expression targets. The preparation of a standard curve or the application of internal standards for each measurement, using known amounts of GFP, will probably enable estimation of exact amounts of the samples of interest in the culture.

Secretory process and structure maturation of the extracellular domains of class C GPCRs

The extracellular domains of the class C GPCR members are important targets for structural studies as well as agonist/antagonist-binding studies, because the regions are generally responsible for binding of the major ligands for the receptors. Although some reports have described the overexpression of those regions,51–54 only the mGluRs have been analyzed successfully by crystallography.33–35 So far, conventional analyses of recombinant protein expression have often focused only on the amounts of the expressed products, evaluated by western blotting, for instance. However, to achieve successful sample preparation for structural studies, expression analyses should also address whether the recombinant proteins acquire their functional structures. We used the aforementioned GFP reporter system and examined the expression of the extracellular domains of the two class C GPCR members, mGluR and T1R, from the standpoint of their folding and oligomerization states in connection with their expression locations, such as the cellular space (cytoplasm and organelles) or the extracellular space after secretion.

In the case of mGluR1LBD, the Sf9-expressed products were secreted well, and the sample showed a narrow, monomodal elution peak (Fig. 3), indicating that the secreted mGluR1LBD was properly folded. These results suggested that mGluR1LBD achieves its proper folding and membrane trafficking by using the ubiquitous systems provided in most cells, including Sf9 cells. These results are also consistent with the fact that mGluR1LBD, expressed in insect cells, served as a successful sample for the previous crystallographic analyses.33, 34

In contrast to the situation with mGluR1LBD, T1RsLBD hardly secreted, and this problem was not solved even by the addition of the AKH signal peptide (Fig. 5), which resulted in the successful secretion of GFPuv (Figs. 1 and 2). T1RsLBD was also not secreted by mammalian cell lines, including HEK293 (Yamashita, Ashikawa, and Takagi, unpublished results). Notably, the FSEC profiles of the cellular-expressed T1RsLBD showed severe polydisperse patterns, with large aggregated fractions. These results clearly indicated that T1RsLBD, expressed under the conditions tested in this study, were unable to achieve proper folding and trafficking in Sf9 cells, and are unsuitable as samples for structural analyses even though some expression was observed in the cellular space. Interestingly, the expression of T1RsLBD-GFPuv and AKHss-T1RsLBD-GFPuv showed some differences upon western blotting and confocal microscopic observations: the former displayed less degradation and a broad distribution in the cells, while the latter included more degradation and spot-like localizations with high fluorescence intensities in the cells (Figs. 4 and 5). These results imply that the former failed in translocation to the ER, while the latter barely translocated to the ER, but was excluded from the subsequent secretory pathway by ER quality-control systems, and at least a part of them was subjected to lysosomal degradation, although further experiments are needed to define their exact localizations in the cells.

There are number of examples that cell-surface membrane proteins possess their specific folding-assistant and/or membrane-escort systems, to prevent misfolded, wrongly organized, or accidentally expressed proteins from reaching the cell surface. In the case of other class C GPCR members, for example, the surface expression of GABAB receptor is achieved through the direct interaction of the C-terminal region of the heterodimeric partner, the GB1 and GB2 subunits, resulting in the masking of the ER retention signal in GB1.55 In the transport of V2R pheromone receptors to the cell surface, the major histocompatibility complex M10 family members, together with β2-microglobulin, reportedly function as escort molecules.56 In a similar manner, T1Rs might require their own chaperones and/or membrane escort systems for proper folding and trafficking, that might specifically exist in taste-receptor expressing cells (such as taste cells), but not in other cells such as Sf9 or HEK293 cells.

The results of mGluR1LBD and T1RsLBD expression displayed the correlation between the localization of the expressed products and their folding status: the secreted sample (the secreted fraction of mGluR1LBD) was favorably folded with a monodisperse hydrodynamic state, while the samples excluded from the secretory pathway (the cellular fractions of mGluR1LBD, T1RsLBD, and AKHss-T1R1LBD) were poorly folded (Figs. 3, 4, 5, 3–5). These results imply that the maturation of the functional structures of the class C GPCR extracellular domains is not attained spontaneously, but through the secretory pathway, by receiving posttranslational-modifications and excluding of the misfolded subfractions by the quality-control systems.

It should be noted that the observed results might not be simply extrapolated to the full-length receptors. Nevertheless, the results in this study might reflect some of the structural maturation processes of cell-surface proteins with extracellular domains, including the class C GPCRs, as well as secreted proteins.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Outline
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

Vector construction

To construct the cytosolic expression vector of GFPuv for expression in Sf9 or HighFive cells (pCGFP_SF), the gene encoding the multicloning site followed by the thrombin digestion site, and GFPuv with an hexa-histidine tag at the C-terminus was amplified by a conventional PCR method using the vector pCGFP-BC (provided from Prof. Eric Gouaux)10 as the template, and the fragment was inserted into the pIEx-4 vector (Novagen) between the blunted NcoI site and the DraIII site. pCGFP_SF+AKH, the vector for secreted expression, was constructed by inserting the AKH signal sequence for secretion upstream of the multicloning site in pCGFP_SF.

The mGluR1 cDNA was obtained from mouse (Balb/c) brain mRNA, the T1R1 and T1R2 cDNAs were obtained from mouse (C57BL/6) tongue mRNA, and the T1R3 cDNA was obtained as described previously.57 To construct the expression vector for the GFP-fusion of the ligand binding domain of mGluR1, the mouse mGluR1 gene was used as the starting material, but Tyr435 was mutated to His by QuikChange (Stratagene), to generate the rat-derived mGluR1 sequence. The coding sequence for residues 1–522, the same region used for crystallographic analyses,33, 34 was then subcloned between the NcoI and XhoI sites in the pCGFP_SF vector. To construct the expression vector for the GFP-fusion of the ligand binding domains of mouse T1Rs, the regions corresponding to mGluR1LBD (T1R1, 1-496; T1R2, 1-497; T1R3, 1-502) were subcloned between the NcoI and XhoI sites (for T1R1 and T1R2) or between the NcoI and NotI sites (for T1R3) in pCGFP_SF or pCGFP_SF+AKH.

Transient protein expression in Sf9 cells

Sf9 cells (GIBCO) were transfected with the expression vectors using Insect Genejuice (Novagen), according to the manufacturer's protocol. Briefly, cells from a confluent monolayer culture were plated into each well of a 6-well tissue culture plate (6 Well Clear TC-Treated microplates, Costar) at a density of 1.0–1.6 × 106 cells/well, and then the well was filled with 1 mL Sf900III SFM (GIBCO) containing 10 μL Insect Genejuice and 2 μg plasmid (for T1RLBD coexpression experiments, 1 μg of T1R3LBD plasmid and 1 μg of either the T1R1LBD or T1R2LBD plasmid were used). The transfected cells were then incubated in Sf900III SFM for 48–144 hrs at 27°C, to allow recombinant protein expression. For in vivo fluorescence detection with a microplate reader, one of the wells in the culture plate was filled with the same amount of the Sf900III SFM and used as a control (background).

Confocal laser scanning microscopy

The Sf9 cells transiently expressing the various proteins at 72-h after transfection were imaged with a confocal laser scanning microscope LSM510, using an EC Plan-Neofluar 40×/0.75 objective (Carl Zeiss, Germany). GFPuv was excited with a 488-nm argon laser line (with a setting of 9–10% intensity), and emission was collected through a 505 to 550-nm bandpass filter.

Sample preparation

Transfected Sf9 cells and culture medium were harvested separately. Cells were washed twice with suspension buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 5 mM KCl), and were suspended in 300 μL of the same buffer. The cells were then sonicated on ice, using a Handy Sonic (TOMY SEIKO) at output 5 for 2–3 min. The samples were centrifuged at 50 k rpm with a TLA-55 rotor (Beckman Coulter) for 30 min at 4°C, and the supernatants were used as the “cellular fraction” (including cytoplasmic and organelle fractions) for further analyses. The culture medium was concentrated with a Vivaspin filter (10k MWCO) (VIVASCIENCE) and exchanged to the suspension buffer by two rounds of dilution and concentration (ca. 400-fold). It was then diluted up to 300 μL with the suspension buffer and centrifuged, and the supernatant was used as the “secreted fraction” for further analyses.

Fluorescence-detection size-exclusion chromatography (FSEC)

The FSEC analysis was performed by loading 200 μL samples on a Superose 6 10/300 GL column (GE Healthcare) connected to an ÄKTA explorer instrument (GE Healthcare), using running buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl) at a flow rate of 0.5–1.0 mL/min. The elution of the size exclusion chromatography was detected with a fluorometer, RF-10AXL (Shimadzu) using an excitation wavelength of 395 nm and an emission wavelength of 507 nm.

Western-blotting

SDS-PAGE was performed with SuperSep Ace 10% gels (Wako), using 2 μL [for Fig. 1(A)], 10 μL [for Fig. 1(D)], 15 μL [for Fig. 3(A)], or 30 μL [for Figs. 4(A,H) and 5(A,H)] of the samples, respectively. The protein bands were electrophoretically transferred onto nitrocellulose membranes (Invitrogen), using an iBlot (Invitrogen). The proteins of interest were detected immunologically, using Penta-His Antibody (Qiagen) as the primary antibody and Anti-Mouse IgG, HRP-linked Whole Antibody (GE Healthcare) as the secondary antibody. The chemiluminescent signals were detected using Immobilon Western HRP substrate (Millipore) and ChemidocXRS (Bio-Rad), according to the manufacturer's instructions.

In vivo fluorescence detection with a microplate reader

The fluorescence intensity derived from the GFPuv expressed in cells grown in tissue culture plates was detected with a microplate reader Varioskan Flash (Thermo Fisher Scientific), using an excitation wavelength of 395 nm and an emission wavelength of 507 nm. The typical measurement conditions utilized multipoint measurements (70–80% circular area from the center of the well), with a measurement time of 500 ms and a bandwidth of 12 nm, using bottom reading optics to measure the culture without removing the lid of the culture plate. The measured fluorescence intensity in each well of the culture plates, F, was normalized by the following equation:

  • equation image

where F0 represents the fluorescence intensity of a control well filled with the same amount of culture medium as the other wells, in the same culture plate of the measured sample.

The amount of expressed GFPuv protein was estimated by integrating the areas of the elution peaks from the FSEC analyses, and converting them to the actual amount of the protein, using a standard curve prepared with known amounts of purified GFPuv as follows. The GFPuv expression vector (pCGFP-BC)10 was transformed into BL21(DE3)pLysS competent cells, and the transformants were cultured in 1L LB medium containing 50 μg mL−1 carbenicillin and 34 μg mL−1 chloramphenicol, at 37°C. When the OD600 reached at ∼0.6, IPTG was added to a final concentration of 1 mM, and the protein expression was induced at 18°C overnight. The cells were harvested by centrifugation, resuspended in 50 mL buffer (20 mM Tris-HCl, 500 mM NaCl, and 0.1 mM PMSF), and sonicated on ice using a SONIFIER 250 (BRANSON) at output 4, with 4 rounds of 10 pulses (1 sec on–1 sec off) at 1 min intervals. The sonicated sample was centrifuged for 40 min at 112,000g. The supernatant was filtered and applied to a HisTrap column (GE Healthcare). GFPuv was eluted by 300 mM imidazole, and was then desalted using a PD-10 column (GE Healthcare). GFPuv was further purified using a HiPrep 16/10 Q XL column (GE Healthcare). The purified GFPuv was stored at −80°C, after adding glycerol to 10%. The standard curve of GFPuv was prepared by subjecting 5, 50, 250, and 500 ng of GFPuv protein in 200 μL of the running buffer, to the FSEC analyses as above, and the data were fit to a linear line with R2 of 0.991.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Outline
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

The authors thank Eric Gouaux for providing the pCGFP_BC and pNGFP_BC vectors, and the GluR2-ATD gene; Hiro Furukawa for the NR2B-ATD gene and sharing the data; The FANTOM consortium for mGluR-III7 gene; Toshiro Oda for help on the fluorescence measurement; Junichi Takagi for discussion about mammalian cell expression; Fumie Iwabuki, Naoko Ono, and Junko Nakamura for assistance.

References

  1. Top of page
  2. Abstract
  3. Outline
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and Methods
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Outline
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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