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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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Residue N1036 is critical in determining the functional  differences between SERCA2 isoforms. (a) Differential migration patterns between SERCA2bN1036A (lane 1) vs. GFP-SERCA2bN1036A (lane 2) on a Western blot probed with the  same N1 anti-SERCA2 Ab from Fig. 2 b. Fractions from control  oocytes (H2O replacing mRNA) were run on lane 3. (b) Confocal  images of GFP fluorescence intensity at high resolution (60× objective; 52 μm × 36 μm) and at low resolution (10× objective;  745 μm × 530 μm) in a GFP-SERCA2bN1036A oocyte matched  for GFP fluorescence intensity with the oocytes shown in Fig. 3 a.  (c) Spatiotemporal stack (left) of Ca2+ wave activity from the  GFP-SERCA2bN1036A overexpressing oocyte in b. Confocal  image of Ca2+ wave activity at the indicated time (right). Imaging  parameters were similar to those described in Fig. 3 b.
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Figure 7: Residue N1036 is critical in determining the functional differences between SERCA2 isoforms. (a) Differential migration patterns between SERCA2bN1036A (lane 1) vs. GFP-SERCA2bN1036A (lane 2) on a Western blot probed with the same N1 anti-SERCA2 Ab from Fig. 2 b. Fractions from control oocytes (H2O replacing mRNA) were run on lane 3. (b) Confocal images of GFP fluorescence intensity at high resolution (60× objective; 52 μm × 36 μm) and at low resolution (10× objective; 745 μm × 530 μm) in a GFP-SERCA2bN1036A oocyte matched for GFP fluorescence intensity with the oocytes shown in Fig. 3 a. (c) Spatiotemporal stack (left) of Ca2+ wave activity from the GFP-SERCA2bN1036A overexpressing oocyte in b. Confocal image of Ca2+ wave activity at the indicated time (right). Imaging parameters were similar to those described in Fig. 3 b.

Mentions: To compare the properties of SERCA2bN1036A with those of the wild-type SERCA2 isoforms on Ca2+ wave activity accurately, we tagged SERCA2bN1036A with GFP at the NH2 terminus under conditions of equivalent levels of expression. A Western blot probed with an anti-SERCA2 antibody demonstrated that SERCA2bN1036A migrated differentially from GFP-SERCA2bN1036A protein as expected (Fig. 7 a). GFP fluorescence imaging at high magnification (60× objective) of a GFP-SERCA2bN1036A-overexpressing oocyte (Fig. 7 b) demonstrated that the fusion protein was targeted to the same ER compartment as shown for both wild-type (Fig. 1 b) and GFP-tagged SERCA2 isoforms (Fig. 2 c). GFP fluorescence intensity of equal magnitude to that in the oocytes shown in Fig. 3 a was measured at low magnification (10× objective) for a GFP-SERCA2bN1036A oocyte shown in Fig. 7 b, allowing us to compare the Ca2+ wave properties of this oocyte directly (Fig. 7 c) with those of the GFP-SERCA2a and GFP-SERCA2b oocytes shown in Fig. 3 b. We carried out detailed analyses of oocytes that were matched in GFP flluorescence intensity in the study (Table I). Remarkably, Ca2+ wave activity normalized for GFP fluorescence revealed that the single amino acid mutation in SERCA2b (N1036A) yielded a Ca2+ ATPase with uptake properties indistinguishable from those of SERCA2a. Furthermore, there was no inhibitory effect of ΔC when it was coexpressed with either SERCA2a or SERCA2bN1036A (Figs. 4 b and 6 c). Together, these data indicate that the functional differences between SERCA2a and SERCA2b on Ca2+ wave activity reported here and elsewhere (Lytton et al., 1992; Verboomen et al., 1992; Verboomen et al., 1994) can be attributed to the presence of the luminal glycosylated residue on SERCA2b that is absent in SERCA2a.


Differential modulation of SERCA2 isoforms by calreticulin.

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

Residue N1036 is critical in determining the functional  differences between SERCA2 isoforms. (a) Differential migration patterns between SERCA2bN1036A (lane 1) vs. GFP-SERCA2bN1036A (lane 2) on a Western blot probed with the  same N1 anti-SERCA2 Ab from Fig. 2 b. Fractions from control  oocytes (H2O replacing mRNA) were run on lane 3. (b) Confocal  images of GFP fluorescence intensity at high resolution (60× objective; 52 μm × 36 μm) and at low resolution (10× objective;  745 μm × 530 μm) in a GFP-SERCA2bN1036A oocyte matched  for GFP fluorescence intensity with the oocytes shown in Fig. 3 a.  (c) Spatiotemporal stack (left) of Ca2+ wave activity from the  GFP-SERCA2bN1036A overexpressing oocyte in b. Confocal  image of Ca2+ wave activity at the indicated time (right). Imaging  parameters were similar to those described in Fig. 3 b.
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Figure 7: Residue N1036 is critical in determining the functional differences between SERCA2 isoforms. (a) Differential migration patterns between SERCA2bN1036A (lane 1) vs. GFP-SERCA2bN1036A (lane 2) on a Western blot probed with the same N1 anti-SERCA2 Ab from Fig. 2 b. Fractions from control oocytes (H2O replacing mRNA) were run on lane 3. (b) Confocal images of GFP fluorescence intensity at high resolution (60× objective; 52 μm × 36 μm) and at low resolution (10× objective; 745 μm × 530 μm) in a GFP-SERCA2bN1036A oocyte matched for GFP fluorescence intensity with the oocytes shown in Fig. 3 a. (c) Spatiotemporal stack (left) of Ca2+ wave activity from the GFP-SERCA2bN1036A overexpressing oocyte in b. Confocal image of Ca2+ wave activity at the indicated time (right). Imaging parameters were similar to those described in Fig. 3 b.
Mentions: To compare the properties of SERCA2bN1036A with those of the wild-type SERCA2 isoforms on Ca2+ wave activity accurately, we tagged SERCA2bN1036A with GFP at the NH2 terminus under conditions of equivalent levels of expression. A Western blot probed with an anti-SERCA2 antibody demonstrated that SERCA2bN1036A migrated differentially from GFP-SERCA2bN1036A protein as expected (Fig. 7 a). GFP fluorescence imaging at high magnification (60× objective) of a GFP-SERCA2bN1036A-overexpressing oocyte (Fig. 7 b) demonstrated that the fusion protein was targeted to the same ER compartment as shown for both wild-type (Fig. 1 b) and GFP-tagged SERCA2 isoforms (Fig. 2 c). GFP fluorescence intensity of equal magnitude to that in the oocytes shown in Fig. 3 a was measured at low magnification (10× objective) for a GFP-SERCA2bN1036A oocyte shown in Fig. 7 b, allowing us to compare the Ca2+ wave properties of this oocyte directly (Fig. 7 c) with those of the GFP-SERCA2a and GFP-SERCA2b oocytes shown in Fig. 3 b. We carried out detailed analyses of oocytes that were matched in GFP flluorescence intensity in the study (Table I). Remarkably, Ca2+ wave activity normalized for GFP fluorescence revealed that the single amino acid mutation in SERCA2b (N1036A) yielded a Ca2+ ATPase with uptake properties indistinguishable from those of SERCA2a. Furthermore, there was no inhibitory effect of ΔC when it was coexpressed with either SERCA2a or SERCA2bN1036A (Figs. 4 b and 6 c). Together, these data indicate that the functional differences between SERCA2a and SERCA2b on Ca2+ wave activity reported here and elsewhere (Lytton et al., 1992; Verboomen et al., 1992; Verboomen et al., 1994) can be attributed to the presence of the luminal glycosylated residue on SERCA2b that is absent in SERCA2a.

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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