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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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ΔC inhibition of repetitive Ca2+ waves and reversal of  the ΔC effect by glucosidase inhibitors. (a) The percent of oocytes exhibiting repetitive Ca2+ oscillations is significantly reduced in oocytes coexpressing ΔC with SERCA2b (black bars),  but not in oocytes coexpressing ΔC with SERCA2a (gray bars;  **P < 0.01, Chi-squared test). Of those oocytes that did display  repetitive Ca2+ oscillations, the interwave period (middle histogram) and decay time (right histogram) were significantly increased  in oocytes coexpressing ΔC with SERCA2b when compared with  control oocytes overexpressing SERCA2b alone (*P < 0.005). No  significant differences were found between oocytes coexpressing  ΔC with SERCA2a as compared with control oocytes overexpressing SERCA2a alone in either interwave period or in decay  time of individual waves. Note that there is a change in scale values for the ordinate in histograms of wave period and decay time  with respect to Fig. 3 c. The larger scale in this figure reflects the  longer period and longer decay time of Ca2+ waves in SERCA2b +  ΔC-overexpressing oocytes. (b) Western blot analysis demonstrates overexpression of the ΔC mutant of CRT in fractions from  oocytes coexpressing this calreticulin mutant with SERCA2a  (lane 1) and SERCA2b (lane 2). Oocyte extracts from control oocytes (H2O replacing mRNA) were run on lane 3. The membrane  was probed with a primary anti-CRT KDEL Ab that recognizes  the last six amino acids at the COOH terminus of rabbit CRT  (gift of Michalak). (c) Glucosidase inhibition antagonizes the effects of ΔC overexpression on oscillatory Ca2+ waves. Period between waves in SERCA2b + ΔC-overexpressing oocytes is significantly decreased (n = 13) in oocytes injected with 1 mM final DNJ  (Toronto Research Chemicals, North York, Ontario, Canada)  when compared with uninjected SERCA2b + ΔC control oocytes  (n = 18). **Indicates statistical significance at P < 0.025. In the  same groups of oocytes, decay time of individual Ca2+ waves is also  reduced, although the differences are not statistically significant.
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Figure 5: ΔC inhibition of repetitive Ca2+ waves and reversal of the ΔC effect by glucosidase inhibitors. (a) The percent of oocytes exhibiting repetitive Ca2+ oscillations is significantly reduced in oocytes coexpressing ΔC with SERCA2b (black bars), but not in oocytes coexpressing ΔC with SERCA2a (gray bars; **P < 0.01, Chi-squared test). Of those oocytes that did display repetitive Ca2+ oscillations, the interwave period (middle histogram) and decay time (right histogram) were significantly increased in oocytes coexpressing ΔC with SERCA2b when compared with control oocytes overexpressing SERCA2b alone (*P < 0.005). No significant differences were found between oocytes coexpressing ΔC with SERCA2a as compared with control oocytes overexpressing SERCA2a alone in either interwave period or in decay time of individual waves. Note that there is a change in scale values for the ordinate in histograms of wave period and decay time with respect to Fig. 3 c. The larger scale in this figure reflects the longer period and longer decay time of Ca2+ waves in SERCA2b + ΔC-overexpressing oocytes. (b) Western blot analysis demonstrates overexpression of the ΔC mutant of CRT in fractions from oocytes coexpressing this calreticulin mutant with SERCA2a (lane 1) and SERCA2b (lane 2). Oocyte extracts from control oocytes (H2O replacing mRNA) were run on lane 3. The membrane was probed with a primary anti-CRT KDEL Ab that recognizes the last six amino acids at the COOH terminus of rabbit CRT (gift of Michalak). (c) Glucosidase inhibition antagonizes the effects of ΔC overexpression on oscillatory Ca2+ waves. Period between waves in SERCA2b + ΔC-overexpressing oocytes is significantly decreased (n = 13) in oocytes injected with 1 mM final DNJ (Toronto Research Chemicals, North York, Ontario, Canada) when compared with uninjected SERCA2b + ΔC control oocytes (n = 18). **Indicates statistical significance at P < 0.025. In the same groups of oocytes, decay time of individual Ca2+ waves is also reduced, although the differences are not statistically significant.

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)

ΔC inhibition of repetitive Ca2+ waves and reversal of  the ΔC effect by glucosidase inhibitors. (a) The percent of oocytes exhibiting repetitive Ca2+ oscillations is significantly reduced in oocytes coexpressing ΔC with SERCA2b (black bars),  but not in oocytes coexpressing ΔC with SERCA2a (gray bars;  **P < 0.01, Chi-squared test). Of those oocytes that did display  repetitive Ca2+ oscillations, the interwave period (middle histogram) and decay time (right histogram) were significantly increased  in oocytes coexpressing ΔC with SERCA2b when compared with  control oocytes overexpressing SERCA2b alone (*P < 0.005). No  significant differences were found between oocytes coexpressing  ΔC with SERCA2a as compared with control oocytes overexpressing SERCA2a alone in either interwave period or in decay  time of individual waves. Note that there is a change in scale values for the ordinate in histograms of wave period and decay time  with respect to Fig. 3 c. The larger scale in this figure reflects the  longer period and longer decay time of Ca2+ waves in SERCA2b +  ΔC-overexpressing oocytes. (b) Western blot analysis demonstrates overexpression of the ΔC mutant of CRT in fractions from  oocytes coexpressing this calreticulin mutant with SERCA2a  (lane 1) and SERCA2b (lane 2). Oocyte extracts from control oocytes (H2O replacing mRNA) were run on lane 3. The membrane  was probed with a primary anti-CRT KDEL Ab that recognizes  the last six amino acids at the COOH terminus of rabbit CRT  (gift of Michalak). (c) Glucosidase inhibition antagonizes the effects of ΔC overexpression on oscillatory Ca2+ waves. Period between waves in SERCA2b + ΔC-overexpressing oocytes is significantly decreased (n = 13) in oocytes injected with 1 mM final DNJ  (Toronto Research Chemicals, North York, Ontario, Canada)  when compared with uninjected SERCA2b + ΔC control oocytes  (n = 18). **Indicates statistical significance at P < 0.025. In the  same groups of oocytes, decay time of individual Ca2+ waves is also  reduced, although the differences are not statistically significant.
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Figure 5: ΔC inhibition of repetitive Ca2+ waves and reversal of the ΔC effect by glucosidase inhibitors. (a) The percent of oocytes exhibiting repetitive Ca2+ oscillations is significantly reduced in oocytes coexpressing ΔC with SERCA2b (black bars), but not in oocytes coexpressing ΔC with SERCA2a (gray bars; **P < 0.01, Chi-squared test). Of those oocytes that did display repetitive Ca2+ oscillations, the interwave period (middle histogram) and decay time (right histogram) were significantly increased in oocytes coexpressing ΔC with SERCA2b when compared with control oocytes overexpressing SERCA2b alone (*P < 0.005). No significant differences were found between oocytes coexpressing ΔC with SERCA2a as compared with control oocytes overexpressing SERCA2a alone in either interwave period or in decay time of individual waves. Note that there is a change in scale values for the ordinate in histograms of wave period and decay time with respect to Fig. 3 c. The larger scale in this figure reflects the longer period and longer decay time of Ca2+ waves in SERCA2b + ΔC-overexpressing oocytes. (b) Western blot analysis demonstrates overexpression of the ΔC mutant of CRT in fractions from oocytes coexpressing this calreticulin mutant with SERCA2a (lane 1) and SERCA2b (lane 2). Oocyte extracts from control oocytes (H2O replacing mRNA) were run on lane 3. The membrane was probed with a primary anti-CRT KDEL Ab that recognizes the last six amino acids at the COOH terminus of rabbit CRT (gift of Michalak). (c) Glucosidase inhibition antagonizes the effects of ΔC overexpression on oscillatory Ca2+ waves. Period between waves in SERCA2b + ΔC-overexpressing oocytes is significantly decreased (n = 13) in oocytes injected with 1 mM final DNJ (Toronto Research Chemicals, North York, Ontario, Canada) when compared with uninjected SERCA2b + ΔC control oocytes (n = 18). **Indicates statistical significance at P < 0.025. In the same groups of oocytes, decay time of individual Ca2+ waves is also reduced, although the differences are not statistically significant.
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
Related in: MedlinePlus