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Differential modulation of SERCA2 isoforms by calreticulin.

John LM, Lechleiter JD, Camacho P - J. Cell Biol. (1998)

Bottom Line: We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences.Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding.In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

ABSTRACT
In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca2+ oscillations in a manner consistent with inhibition of Ca2+ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)2 gene display differential Ca2+ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

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Overexpression of  SERCA2a and SERCA2b  reveals functional differences  between isoforms in IP3-induced repetitive Ca2+ wave  activity. (a) Comparison of the  IP3 (∼300 nM final)-induced  Ca2+ response in a SERCA2a  (left) and a SERCA2b (right)- overexpressing oocyte. In the  top two panels the confocal  images are 745 μm × 745 μm  and are imaged at low magnification (10× objective; bar,  ∼100 μm). In the bottom two  panels, two different oocytes  are confocally imaged at  higher magnification (40×  objective; bar, ∼20 μm) and  the confocal images are 240  μm × 180 μm. Individual images of Ca2+ wave activity  were taken at peak activity.  (b) Confocal immunofluorescence of SERCA2a and  SERCA2b. The top panels  show immunofluorescence  obtained with a primary antibody to rat SERCAs generated in rabbit (C-4, gift of J.  Lytton) and a secondary  FITC-conjugated goat anti– rabbit antibody (Jackson ImmunoResearch Laboratories). The bottom panels are  controls. The left panel  shows immunofluorescence  of a control oocyte (not injected with SERCA2 message) revealing endogenous levels of cross-reactivity with the native Xenopus  oocyte SERCA2b protein. The right panel shows control immunofluorescence omitting the primary antibody. Bar, ∼10 μm. (c)  Western blot of SERCA2 protein levels in either control oocytes  injected with H2O (lane 1) or oocytes overexpressing SERCA2b  (lane 2) and SERCA2a (lane 3) mRNAs. SERCA2 products  were detected by probing with the same C-4 primary antibody  used in b. One oocyte equivalent was loaded per lane and run on  an 8% SDS PAGE gel. Molecular size markers (in kD) are indicated on the left (Hi range; Bio-Rad Laboratories).
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Figure 1: Overexpression of SERCA2a and SERCA2b reveals functional differences between isoforms in IP3-induced repetitive Ca2+ wave activity. (a) Comparison of the IP3 (∼300 nM final)-induced Ca2+ response in a SERCA2a (left) and a SERCA2b (right)- overexpressing oocyte. In the top two panels the confocal images are 745 μm × 745 μm and are imaged at low magnification (10× objective; bar, ∼100 μm). In the bottom two panels, two different oocytes are confocally imaged at higher magnification (40× objective; bar, ∼20 μm) and the confocal images are 240 μm × 180 μm. Individual images of Ca2+ wave activity were taken at peak activity. (b) Confocal immunofluorescence of SERCA2a and SERCA2b. The top panels show immunofluorescence obtained with a primary antibody to rat SERCAs generated in rabbit (C-4, gift of J. Lytton) and a secondary FITC-conjugated goat anti– rabbit antibody (Jackson ImmunoResearch Laboratories). The bottom panels are controls. The left panel shows immunofluorescence of a control oocyte (not injected with SERCA2 message) revealing endogenous levels of cross-reactivity with the native Xenopus oocyte SERCA2b protein. The right panel shows control immunofluorescence omitting the primary antibody. Bar, ∼10 μm. (c) Western blot of SERCA2 protein levels in either control oocytes injected with H2O (lane 1) or oocytes overexpressing SERCA2b (lane 2) and SERCA2a (lane 3) mRNAs. SERCA2 products were detected by probing with the same C-4 primary antibody used in b. One oocyte equivalent was loaded per lane and run on an 8% SDS PAGE gel. Molecular size markers (in kD) are indicated on the left (Hi range; Bio-Rad Laboratories).

Mentions: Oocyte extracts used in Western blots were prepared from pools of 10 oocytes as previously described (Camacho and Lechleiter, 1995). The final pellet of each extract was resuspended in 50 μl of 1% SDS per oocyte equivalent, and was stored frozen in aliquots of one oocyte equivalent each. One oocyte equivalent of each fraction was loaded on an SDS gel, stained with Coomassie blue, and scanned on a UMAX Powerlook II scanner. Two invariant adjacent protein bands of ∼40 kD that appear in each extract were used as densitometric standards. The average of all densitometric readings was used to normalize the sample volume to load on SDS PAGE gels. To detect the ΔC mutant, samples were run on a 12% gel, and to detect the SERCA2 and GFP-tagged SERCA2 proteins, the samples were run on 8% gels by SDS-PAGE. To visualize the SERCA antigen, the membranes were probed with the polyclonal rabbit anti-SERCA antibody (C-4 Ab in Fig. 1 c and N1 Ab in Figs. 2 b and 7 a; both antibodies were a gift from J. Lytton, University of Calgary Health Sciences Centre, Department of Biochemistry and Molecular Biology, Calgary, Alberta, Canada). To detect the ΔC mutant of calreticulin (see Figs. 5 b and 6 d), oocyte fractions were probed with a primary rabbit anti-KDEL Ab that recognizes the COOH-terminal six amino acids of calreticulin (gift of M. Michalak, University of Alberta, Department of Biochemistry, Edmonton, Alberta, Canada). Note that this mutant contains the last six amino acids of calreticulin, including the KDEL ER retention signal, and thus it can be detected with this antibody (Camacho and Lechleiter, 1995). Alkaline phosphatase–conjugated secondary antibodies were used in all Western blots (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), and colorimetric detection was accomplished by NBT/ BCIP (NitroBlue Tetrazolium/5-Bromo-4-Chloro-3-Indolyl Phosphate; Promega Corp., Madison, WI).


Differential modulation of SERCA2 isoforms by calreticulin.

John LM, Lechleiter JD, Camacho P - J. Cell Biol. (1998)

Overexpression of  SERCA2a and SERCA2b  reveals functional differences  between isoforms in IP3-induced repetitive Ca2+ wave  activity. (a) Comparison of the  IP3 (∼300 nM final)-induced  Ca2+ response in a SERCA2a  (left) and a SERCA2b (right)- overexpressing oocyte. In the  top two panels the confocal  images are 745 μm × 745 μm  and are imaged at low magnification (10× objective; bar,  ∼100 μm). In the bottom two  panels, two different oocytes  are confocally imaged at  higher magnification (40×  objective; bar, ∼20 μm) and  the confocal images are 240  μm × 180 μm. Individual images of Ca2+ wave activity  were taken at peak activity.  (b) Confocal immunofluorescence of SERCA2a and  SERCA2b. The top panels  show immunofluorescence  obtained with a primary antibody to rat SERCAs generated in rabbit (C-4, gift of J.  Lytton) and a secondary  FITC-conjugated goat anti– rabbit antibody (Jackson ImmunoResearch Laboratories). The bottom panels are  controls. The left panel  shows immunofluorescence  of a control oocyte (not injected with SERCA2 message) revealing endogenous levels of cross-reactivity with the native Xenopus  oocyte SERCA2b protein. The right panel shows control immunofluorescence omitting the primary antibody. Bar, ∼10 μm. (c)  Western blot of SERCA2 protein levels in either control oocytes  injected with H2O (lane 1) or oocytes overexpressing SERCA2b  (lane 2) and SERCA2a (lane 3) mRNAs. SERCA2 products  were detected by probing with the same C-4 primary antibody  used in b. One oocyte equivalent was loaded per lane and run on  an 8% SDS PAGE gel. Molecular size markers (in kD) are indicated on the left (Hi range; Bio-Rad Laboratories).
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Related In: Results  -  Collection

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Figure 1: Overexpression of SERCA2a and SERCA2b reveals functional differences between isoforms in IP3-induced repetitive Ca2+ wave activity. (a) Comparison of the IP3 (∼300 nM final)-induced Ca2+ response in a SERCA2a (left) and a SERCA2b (right)- overexpressing oocyte. In the top two panels the confocal images are 745 μm × 745 μm and are imaged at low magnification (10× objective; bar, ∼100 μm). In the bottom two panels, two different oocytes are confocally imaged at higher magnification (40× objective; bar, ∼20 μm) and the confocal images are 240 μm × 180 μm. Individual images of Ca2+ wave activity were taken at peak activity. (b) Confocal immunofluorescence of SERCA2a and SERCA2b. The top panels show immunofluorescence obtained with a primary antibody to rat SERCAs generated in rabbit (C-4, gift of J. Lytton) and a secondary FITC-conjugated goat anti– rabbit antibody (Jackson ImmunoResearch Laboratories). The bottom panels are controls. The left panel shows immunofluorescence of a control oocyte (not injected with SERCA2 message) revealing endogenous levels of cross-reactivity with the native Xenopus oocyte SERCA2b protein. The right panel shows control immunofluorescence omitting the primary antibody. Bar, ∼10 μm. (c) Western blot of SERCA2 protein levels in either control oocytes injected with H2O (lane 1) or oocytes overexpressing SERCA2b (lane 2) and SERCA2a (lane 3) mRNAs. SERCA2 products were detected by probing with the same C-4 primary antibody used in b. One oocyte equivalent was loaded per lane and run on an 8% SDS PAGE gel. Molecular size markers (in kD) are indicated on the left (Hi range; Bio-Rad Laboratories).
Mentions: Oocyte extracts used in Western blots were prepared from pools of 10 oocytes as previously described (Camacho and Lechleiter, 1995). The final pellet of each extract was resuspended in 50 μl of 1% SDS per oocyte equivalent, and was stored frozen in aliquots of one oocyte equivalent each. One oocyte equivalent of each fraction was loaded on an SDS gel, stained with Coomassie blue, and scanned on a UMAX Powerlook II scanner. Two invariant adjacent protein bands of ∼40 kD that appear in each extract were used as densitometric standards. The average of all densitometric readings was used to normalize the sample volume to load on SDS PAGE gels. To detect the ΔC mutant, samples were run on a 12% gel, and to detect the SERCA2 and GFP-tagged SERCA2 proteins, the samples were run on 8% gels by SDS-PAGE. To visualize the SERCA antigen, the membranes were probed with the polyclonal rabbit anti-SERCA antibody (C-4 Ab in Fig. 1 c and N1 Ab in Figs. 2 b and 7 a; both antibodies were a gift from J. Lytton, University of Calgary Health Sciences Centre, Department of Biochemistry and Molecular Biology, Calgary, Alberta, Canada). To detect the ΔC mutant of calreticulin (see Figs. 5 b and 6 d), oocyte fractions were probed with a primary rabbit anti-KDEL Ab that recognizes the COOH-terminal six amino acids of calreticulin (gift of M. Michalak, University of Alberta, Department of Biochemistry, Edmonton, Alberta, Canada). Note that this mutant contains the last six amino acids of calreticulin, including the KDEL ER retention signal, and thus it can be detected with this antibody (Camacho and Lechleiter, 1995). Alkaline phosphatase–conjugated secondary antibodies were used in all Western blots (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), and colorimetric detection was accomplished by NBT/ BCIP (NitroBlue Tetrazolium/5-Bromo-4-Chloro-3-Indolyl Phosphate; Promega Corp., Madison, WI).

Bottom Line: We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences.Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding.In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

ABSTRACT
In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca2+ oscillations in a manner consistent with inhibition of Ca2+ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)2 gene display differential Ca2+ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

Show MeSH