　Transmembrane proteins play essential roles in cell signaling, transport of membrane-impermeable molecules, cell-cell communication, and cell adhesion. Our recent work demonstrated that reactive oxygen species-generating NADPH oxidase 4 (Nox4), a protein with multiple transmembrane domains, is involved in cell migration by stabilizing vascular endothelial growth factor receptor 2 (VEGFR-2), a single-span transmembrane protein. During this study and with further verification, we developed a simple method to prepare protein samples without aggregating these membrane proteins for SDS-PAGE, immunoblotting, and deglycosylation assay. We found that heating was unnecessary for protein denaturation for SDS-PAGE and deglycosylation assay. Also, the detectable amounts of VEGFR-2 and Nox4 were increased in the sample treated at 4℃ compared with the sample treated at 98℃ . Moreover, the N-glycan of VEGFR-2 was digested by glycosidase at reaction temperature 4℃ 

KAJIKAWA Mizuho

KANAGAWA Tomoka

KAWAI Chikage

KURIBAYASHI Futoshi

MIYANO Kei

OKAMOTO Shuichiro

TOMINAGA Sayuri

YAMAUCHI Akira

MIYANO, Kei

OKAMOTO, Shuichiro

KAJIKAWA, Mizuho

KAWAI, Chikage

KANAGAWA, Tomoka

TOMINAGA, Sayuri

YAMAUCHI, Akira

KURIBAYASHI, Futoshi

Kawasaki Medical School Institutional Repository / 川崎医科大学学術機関リポジトリ

13Kawasaki Medical Journal 47：13－19，2021　doi：10.11482/KMJ-E202147013Corresponding authorKei MiyanoDepartment of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, 701-0192, JapanPhone : 81 86 462 1111Fax : 81 86 464 1199E-mail: kei-miyano@med.kawasaki-m.ac.jpShuichiro OkamotoDepartment of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, 701-0192, JapanPhone : 81 86 462 1111Fax : 81 86 464 1199E-mail: shuokamoto@med.kawasaki-m.ac.jpThe efficient detection of membrane protein with immunoblotting:  lessons from cold-temperature denaturationKei MIYANO１），＊,  Shuichiro OKAMOTO１），＊,  Mizuho KAJIKAWA２）   Chikage KAWAI１）,  Tomoka KANAGAWA３）,  Sayuri TOMINAGA３）   Akira YAMAUCHI１）,  Futoshi KURIBAYASHI１）1) Department of Biochemistry, Kawasaki Medical School2) Laboratory of Microbiology, Showa Pharmaceutical University3) Second Year Medical Student in fiscal year of 2020, Kawasaki Medical School＊ These authors contributed equally to this work.ABSTRACT   Transmembrane proteins play essential roles in cell signaling, transport of membrane-impermeable molecules, cell-cell communication, and cell adhesion. Our recent work demonstrated that reactive oxygen species-generating NADPH oxidase 4 (Nox4), a protein with multiple transmembrane domains, is involved in cell migration by stabilizing vascular endothelial growth factor receptor 2 (VEGFR-2), a single-span transmembrane protein. During this study and with further verification, we developed a simple method to prepare protein samples without aggregating these membrane proteins for SDS-PAGE, immunoblotting, and deglycosylation assay. We found that heating was unnecessary for protein denaturation for SDS-PAGE and deglycosylation assay. Also, the detectable amounts of VEGFR-2 and Nox4 were increased in the sample treated at 4℃ compared with the sample treated at 98℃ . Moreover, the N -glycan of VEGFR-2 was digested by glycosidase at reaction temperature 4℃ . doi：10.11482/KMJ-E202147013　(Accepted on January 18, 2021)Key words：  Membrane protein,  Endoglycosidase,  Immunoblotting,  SDS-PAGE,   VEGFR-2,  NADPH oxidase 4〈Regular Article〉INTRODUCTION   Membrane proteins are generally classified and named into the following three types: “Type I” membrane protein, the N-terminus is located outside the cell; “Type II” membrane protein, the N-terminus is located inside the cell; and “multiple” transmembrane protein. All three types have at least one transmembrane hydrophobic helix. The helix 14 Kawasaki Medical Journaloften makes protein detection by immunoblotting challenging because its hydrophobicity causes protein aggregation.   Recently, we found that reactive oxygen species-generating Nox4 enhanced the directed migration of endothelial cells by maintaining surface VEGFR-2 levels in the endoplasmic reticulum１）. Nox4 is a transmembrane protein predicted to have 6 transmembrane regions２）. VEGFR-2 is a type I membrane protein３）. During our research, it was necessary to prevent protein aggregation to quantify the amount of these membrane proteins by immunoblotting accurately. This paper aims to provide methodologic details and further verification regarding the denaturation of transmembrane protein for SDS-PAGE, immunoblotting, and deglycosylation assay.MATERIALS AND METHODSReagents and materials   All general ultrapure-grade reagents were purchased from Nacalai Tesque (Kyoto, Japan), Wako Pure Chemicals Industries (Tokyo, Japan), or Sigma-Aldrich Japan (Tokyo, Japan) unless otherwise stated. 2 × Laemmli sample buffer (catalog #1610737) was purchased from Bio-Rad (Tokyo, Japan).Cells and cell culture   EA.hy926 cells were purchased from ATCC, and CHO-K1 cells were obtained from RIKEN Cell Bank (Tsukuba, Japan). The EA.hy926 cells were cultured as previously described１）. The CHO-K1 cells were maintained in Nutrient Mixture F-12 [HAM] medium (Sigma-Aldrich) supplemented with  10% (v/v)  FBS (Sigma-Aldrich)  in  a humidified incubator at 37℃ with 5% (v/v) CO2.Plasmids preparation and Plasmid transfection   pcDNA3.1-human Nox4 was a gift from Karl-Heinz Krause (Addgene plasmid #69352; http://n2t.net/addgene:69352; RRID: Addgene_69352). The cDNA for the human p22phox was prepared by reverse transcribing the mRNA from EA.hy926 cells into cDNA and then cloning the cDNA into pcDNA3.1.   The plasmids were transfected into CHO-K1 cells using the ViofectinTM Transfection Reagent (Viogene). The cells were seeded in 6-well plates at 3.5 × 105 cells per well 24 h before transfection. Before transfection, the plates were washed twice with PBS (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, and 1.47 mM KH2PO4, pH 7.4). Then, 1 ml of serum-free F-12 medium was added. A volume of 7.5μl of Viofectin was diluted with 25μl of serum-free F-12 medium. An amount of 1μg of pcDNA3.1-Nox4 and 0.1μg of pcDNA3.1-p22phox were diluted with 25μl of serum-free F-12 medium. The diluted Viofectin and DNA were mixed and incubated at room temperature for 15 min. The mixtures were added to the plates. The plates were incubated for 2 h at 37℃ in a humidified 5% (v/v) CO2 atmosphere. In order to remove the remaining mixtures, the culture medium was replaced with fresh F-12 medium supplemented with 10% (v/v) FBS. Then, the transfected cells were cultured for 48 h.Sample preparation for immunoblotting and glycosidase treatment   The EA.hy926 cells were cultured in 6-well plates in a confluent state for 48 h. The plates of EA.hy926 cells and transfected CHO-K1 cells were washed twice with ice-cold PBS. Next, 50μL of lysis buffer (20 mM Tris-Cl (pH 7.4 at 4℃ ), 150 mM NaCl, 1% (w/v) Triton X-100, 1% (v/v) protease inhibitor cocktail) was added to each well. The cells were scraped with a scraper and were pipetted into a microcentrifuge tube. The cell lysates were mixed by pipetting ten times and vortexing, and then incubated for 1 h at 4℃ . The lysate was centrifuged at 10,000 × g for 20 min at 4℃ . The supernatant of 15Miyano K, et al. : Mild denaturation of membrane proteins10,000 × g for 15 min at 4℃. The precipitates were resuspended to 50μL of UTM:sample buffer (10 M Urea, 2% (w/v) Triton X-100, 20% (v/v) 2-mercaptoethanol in 1 × Laemmli sample buffer) and then lysed by sonication. Lysates were titrated with 2 M Tris until the color of bromophenol blue changed from yellow to blue.Immunoblotting procedure   The schematic illustration for sample preparation was included (Fig. 1A). The 2 × Laemmli sample each microcentrifuge tube was collected, frozen in small aliquots in liquid nitrogen, and store at -80℃ until the immunoblotting experiments.   For TCA (trichloroacetic acid) treatment, after trypsin harvest of CHO-K1 cells or EA.hy926 cells from 6-well plates, the cells were washed three times with ice-cold PBS and resuspended in 50μL of PBS. 5μL of 100% (w/v) TCA was added to the suspension. The mixtures were mixed by pipetting ten times and vortexing, and then incubated for 1 h at 4℃. The mixtures were centrifuged at Fig. 1. The cold denaturation of transmembrane proteins.A, The schematic illustration of the experiment.B and F, The level of VEGFR-2 protein visualized by immunoblotting. After the indicated treatment of cell lysate (EA.hy926 cells), protein levels of VEGFR-2 were estimated by immunoblot analysis with the anti-Nox4 polyclonal antibody.C–E, The level of Nox4 protein visualized by immunoblotting. CHO cells (C and E) were cotransfected with the indicated plasmids: pcDNA3.1-human Nox4 and pcDNA3.1-human p22phox. After the indicated treatment of cell lysate (C and E, CHO cells; D, EA.hy926 cells), protein levels of Nox4 were estimated by immunoblot analysis with the anti-Nox4 polyclonal antibody.16 Kawasaki Medical Journalbuffer (Bio-Rad catalog #1610737), including 2% (v/v) 2-mercaptoethanol, was added to cell lysates or the glycosidase-treated sample (see Glycosidase treatment for sample preparation). The mixtures were incubated at 4℃ for 1 h (cold temperature denaturation). The denatured samples were loaded into a polyacrylamide gel; an ice-cold running buffer was used during the electrophoresis.   PVDF membrane was soaked in methanol for 1 min and equilibrated in the transfer buffer containing 6.06 g of Tris, 28.8 g of glycine, and 200 ml of methanol in 2L of distilled H2O. The polyacrylamide gel was also equilibrated in the transfer buffer and transferred to the PVDF membrane at 50 V for 2 h using Wet type blotting device (CriterionTM blotter, BioRad). During the transfer, the transfer buffer was maintained at 4℃. After the transfer, the PVDF membrane was temporarily stained with the FAST green, which consisted of 0.1% (w/v) FAST green, 30% (v/v) ethanol, and 10% (v/v) acetic acid, and then rinsed with distilled water to remove the background staining. The membrane was incubated in a blocking buffer of 5% (w/v) skim milk and 0.05% (v/v) Tween in TBS (20 mM Tris-Cl (pH 7.4), 150 mM NaCl) at room temperature for 30 min with shaking. Then, the membrane was incubated with a 1:1,000 diluted anti-Nox4 antibody (Gene Tex, NOX4 antibody [N3C3]) or anti-VEGFR-2 (Cell signaling Technology, 55B11 mAb #2479) in antibody dilution solution (Dream Realization & Communication, DRC-W00H250) at 4℃ overnight. Afterward, the membrane was incubated a 1:5,000 diluted secondary antibody conjugated with horseradish peroxidase in 5% (w/v) skim milk in TBS at room temperature for 30 min. The protein bands on the blots were detected using ImmunoStar® Zeta or LD (FUJIFILM Wako pure chemical corporation).Glycosidase treatment   A volume of 20μl of the lysate from EA.hy926 cells was incubated at 25℃ or 4℃ for 1 h with or without 1μl of peptide N-Glycosidase F (PNGase F) (New England BioLabs, P0704S) or 1μl of Endoglycosidase H (Endo H) ((New England BioLabs, P0702S).RESULTSCold-temperature denaturation for immunoblotting   We investigated whether heat denaturation was required to detect VEGFR-2, a type I membrane protein, by immunoblotting (Fig. 1A). VEGFR-2 protein was observed at its expected position of 200 kDa with cold temperature denaturation (4℃ lane), i.e., without heat denaturation at 98℃. Compared to the sample incubated at 4℃ for 1 h, the detectable amount of VEGFR-2 was reduced in the sample treated at 98℃ (Fig. 1B); the amount of protein in the two samples that had been transferred to the PVDF membrane was comparable. A second band of significant intensity had an apparent molecular weight of 75 kDa; it was derived from the degradation of VEGFR-2. This second band also decreased due to heat treatment (Fig. 1B). This 75-kDa degradation product likely consists of a transmembrane domain and cytosolic domain because it is recognized by the antibody against the cytosolic domain of VEGFR-2, and the molecular weight indicates that the product contains transmembrane and cytosolic domains.   For Nox4, which contains multiple transmembrane domains, the heat treatment might have a more adverse effect on protein detection. Because Nox4 forms a complex with p22phox to function as a ROS-producing oxidase４）, we co-expressed Nox4 with p22phox in CHO-K1 cells, which do not express endogenous Nox4 and p22phox. Indeed, the heat-treated Nox4 was not detected at the expected position according to its molecular weight on the gel (Fig. 1C). Instead, the Nox4 antibody recognized the protein at the top of the running gel, suggesting the aggregation of the Nox4 protein. The detectable 17Miyano K, et al. : Mild denaturation of membrane proteinsamount of Nox4  endogenously expressed in EA.hy926 cells was increased in the sample treated at 4℃ compared with the sample treated at 98℃ (Fig. 1D). Thus, it was found that cold denaturation is suitable for both endogenously expressed Nox4 and overexpressed Nox4.   In previous studies, it was shown that multiple transmembrane proteins were readily detected by TCA treatment of cells without heat denaturation５）. Nox4 in the TCA-treated lysate was detected to the same extent as the cold denatured Nox4 (Fig. 1E). For VEGFR-2, the TCA treatment reduced the amount of full-length VEGFR-2 with an increase of the degradation products (Fig. 1F). Thus, given that the TCA treatment procedure is complicated and some proteins are degraded (Fig. 1F), the cold denatured method is simpler and easier to use.Glycosidase reaction at cold temperature   According to the manufacturer’s protocol, the sample must be treated with heat denaturation (Fig. 2A) and be deglycosylated by glycosidase at the reaction temperature of 37℃. We investigated whether the sugar chains of proteins in Triton X-100 extract (cell lysate) could be cleaved by glycosidase without heat denaturation to avoid sample aggregation. In addition, the reaction temperature of 25℃ or 4℃ was also examined. We previously found that VEGFR-2 existed as doublets in EA.hy926 cells１）. The higher-molecular-weight band was Endo H-resistant and PNGase F-sensitive. In contrast, the lower-molecular-weight band was sensitive to Endo H and PNGase F. In other words, VEGFR-2 was expressed as a protein with high-mannose, localized in the endoplasmic reticulum, and a protein with complex N-glycan, localized in the plasma membrane, in EA.hy926 cells１）. The N-glycan of VEGFR-2 could be digested with PNGase F and Endo H at 4℃ without heat denaturation (Fig. 2B). In the heat denatured sample, although VEGFR-2 was deglycosylated at the reaction temperature of 4℃, the detectable Fig. 2. The denaturation of integral membrane glycoprotein at the cold temperature.A, The schematic illustration of the experiment. The lysate was denatured at 4℃for 1 h; the manufacturer’s protein denaturation protocol (indicated by the dotted arrow) was not used.B and C, Examination of reaction temperature for N-glycan cleavage. VEGFR-2 in the cell lysate (from EA.hy926 cells) was digested with PNGase F or EndoH and subjected to SDS-PAGE, followed by immunoblot analysis with the anti-VEGFR-2 polyclonal antibody.18 Kawasaki Medical Journalamount of VEGFR-2 was reduced (Fig. 2C). This may be due to the aggregation of VEGFR-2 by heat denaturation. Furthermore, the reaction temperature of 37℃ made it more difficult to detect VEGFR-2 bands, suggesting VEGFR-2 protein aggregation progressed further during reaction with glycosidase. DISCUSSION   Protein denaturation by heat treatment is required for immunoblotting of proteins and deglycosylation of glycoprotein. It is challenging to detect proteins with multiple transmembrane domains by immunoblotting because the heat treatment induces protein aggregation (Fig. 1). On the other hand, protein denaturation is caused by cooling the protein from room temperature to lower values６，７）. In the present study, we demonstrated that the cold treatment is more efficient than heat treatment or TCA treatment for denaturation of membrane proteins required for immunoblotting and deglycosylation.   We found that the heat treatment of proteins with the multiple transmembrane domains, such as Nox4, caused a more severe problem than type I membrane proteins, such as VEGFR-2. The difference between the two proteins is the size of their respective transmembrane regions. A standard transmembrane helix is composed of about 20 amino acid residues. VEGFR-2 is assumed to have 21 residues for its transmembrane helix３）, whereas Nox4 is assumed to have about 120 residues (because Nox4 is predicted to have 6 transmembrane regions)２）. The hydrophobic helices are completely exposed by heat denaturation, likely causing protein aggregation. Thus, Nox4 might be more highly aggregated because of the length of its hydrophobic region. Consistent with this hypothesis, Nox4-derived aggregates were observed by immunoblotting (Fig. 1C). On the other hand, VEGFR-2 aggregates were not detected by the anti-VEGFR-2 antibody (Fig. 1B); the VEGFR-2 aggregate might not have migrated through the stacking gel.   Previous ly,  i t  was  shown tha t  the  TCA (trichloroacetic acid) treatment of cells was an effective sample preparation method for detecting phagocyte Nox2, which belonged to the Nox family, in the phagocyte by immunoblotting５）. Neutrophils, rich in protease, often cause undesired proteolysis during sample preparation. Therefore, the TCA treatment is used to prevent proteolysis. This procedure not only avoids proteolysis but also prevents Nox2 aggregation and enables detection by immunoblotting. However, this procedure is a little complicated. In addition, the TCA treatment causes degradation of some membrane proteins (Fig. 1F). The method in our study is more straightforward because we only incubate the cell lysate with the Laemmli sample buffer at 4℃.   The intracellular localization of a membrane protein can be examined by the digestion of N-glycan with glycosidase. However, according to the manufacture’s protocol, membrane proteins need to be denatured by heat. This study showed that heat denaturation was dispensable and the optimal reaction temperature was 4℃ because the reaction temperature of 37℃ made it more difficult to detect VEGFR-2 bands (Fig. 2C). Thus, we believe our method is useful in analyzing the localization of membrane proteins.   In summary, we show that protein denaturation by heat treatment is dispensable for immunoblotting of proteins and deglycosylation of glycoprotein. The cold treatment should be first selected as a denaturing condition for membrane proteins because the cold treated sample can be heat treated later.ACKNOWLEDGMENTS   We are grateful to Aya Morihara (Kawasaki Medical School, Japan) for technical assistance and to the Central Research Institute of Kawasaki Medical School for technical support. This study was supported in part by JSPS KAKENHI grant 19Miyano K, et al. : Mild denaturation of membrane proteinsnumbers JP17K08637 (KM), in part by the Wesco Scientific Promotion Foundation (KM), in part by the Ryobi Teien Memory Foundation (KM), and in part by Research Project Grants [nos. R01S-003 (KM)] from Kawasaki Medical School.CONFLICT OF INTEREST   The authors declare that they have no conflicts of interest.REFERENCES１） Miyano K, Okamoto S, Yamauchi A, Kawai C, Kajikawa M, Kiyohara T, Tamura M, Taura M, Kuribayashi F: The NADPH oxidase NOX4 promotes the directed migration of endothelial cells by stabilizing vascular endothelial growth factor receptor 2 protein. J Biol Chem. 2020; 295: 11877-11890. doi: 10.1074/jbc.RA120.014723.２） Lambeth JD, Neish AS: Nox enzymes and new thinking on reactive oxygen: a double-edged sword revisited. Annu Rev Pathol. 2014; 9: 119-145. doi: 10.1146/annurev-pathol-012513-104651.３）Holmes K, Roberts OL, Thomas AM, Cross MJ: Vascular endothelial growth factor receptor-2: structure, function, intracellular signalling and therapeutic inhibition. Cell Signal. 2007; 19: 2003-2012. doi: 10.1016/j.cellsig.2007.05.013.４）von Löhneysen K, Noack D, Jesaitis AJ, Dinauer MC, Knaus UG; Mutational analysis reveals distinct features of the Nox4-p22phox complex. J Biol Chem. 2008; 283: 35273-35282. doi: 10.1074/jbc.M804200200.５）Yu D, Imajoh-Ohmi S, Akagawa K, Kanegasaki S: Suppression of superoxide-generating ability during differentiation of monocytes to dendritic cells. J Biochem. 1996; 119: 23-28. doi: 10.1093/oxfordjournals.jbchem.a021211.６）Sanfelice D, Temussi PA: Cold denaturation as a tool to measure protein stability. Biophys Chem. 2016; 208: 4-8. doi: 10.1016/j.bpc.2015.05.007.７）Privalov PL: Cold denaturation of proteins. Crit Rev Biochem Mol Biol. 1990; 25: 281-305. doi: 10.3109/10409239009090612.

The efficient detection of membrane protein with immunoblotting: lessons from cold-temperature denaturation

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The efficient detection of membrane protein with immunoblotting: lessons from cold-temperature denaturation

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