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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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GFP-SERCA2 isoforms retain the characteristics of  their respective unmodified proteins and at equivalent levels of  expression exhibit different Ca2+ wave characteristics. (a) Fluorescence images in GFP-SERCA2a (left) and GFP-SERCA2b  (middle) overexpressing oocytes that have been matched for  equivalent levels of GFP fluorescence intensity. GFP fluorescence (745 μm × 530 μm) was excited at 488 nm. Note that under  these imaging conditions, fluorescence levels in control oocytes  (injected with H2O instead of GFP-SERCA2 mRNA) are not detectable. For quantitation of overexpression levels of GFP fusion  proteins, fluorescence values were measured from images obtained at a low magnification (10× objective; bar, ∼100 μm). (b)  Spatio-temporal patterns (left) of Ca2+ release induced by injection of IP3 (∼300 nM final) in oocytes as labeled. In this experiment, Ca2+ Orange (Molecular Probes, Inc.) was used as indicator of Ca2+ wave activity so that GFP fluorescence and Ca2+  wave activity could be observed in the same oocyte. GFP(S65T)  absorption and emission maxima in the visible spectrum occur at  490 and 509 nm, respectively, while for Ca2+ Orange these are  590 nm and 650 nm, respectively. Thus, for the imaging parameters used, Ca2+ Orange fluorescence does not overlap with GFP  fluorescence emission. Each temporal stack contains 400 images  taken at 0.5-s intervals. A single image (530 μm × 745 μm) of  Ca2+ wave activity is shown at the indicated time (right). (c) At  equivalent levels of GFP fluorescence, the Ca2+ wave properties  are different for oocytes overexpressing GFP-SERCA2a (gray  bars, n = 13) and GFP-SERCA2b (black bars, n = 10). Histogram of GFP fluorescence (left) shows fluorescence intensity  measurements in arbitrary units. Histogram of Ca2+ wave period  (middle) and Ca2+ wave decay time (right) measure each of these  parameters from the time course of the average fluorescence intensity of a 5 × 5 pixel area. Note that GFP-SERCA2a-overexpressing oocytes display a higher Ca2+ wave frequency (i.e.,  shorter periods) than the GFP-SERCA2b-overexpressing oocytes. * Indicates a statistically significant difference at P < 0.005.
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Figure 3: GFP-SERCA2 isoforms retain the characteristics of their respective unmodified proteins and at equivalent levels of expression exhibit different Ca2+ wave characteristics. (a) Fluorescence images in GFP-SERCA2a (left) and GFP-SERCA2b (middle) overexpressing oocytes that have been matched for equivalent levels of GFP fluorescence intensity. GFP fluorescence (745 μm × 530 μm) was excited at 488 nm. Note that under these imaging conditions, fluorescence levels in control oocytes (injected with H2O instead of GFP-SERCA2 mRNA) are not detectable. For quantitation of overexpression levels of GFP fusion proteins, fluorescence values were measured from images obtained at a low magnification (10× objective; bar, ∼100 μm). (b) Spatio-temporal patterns (left) of Ca2+ release induced by injection of IP3 (∼300 nM final) in oocytes as labeled. In this experiment, Ca2+ Orange (Molecular Probes, Inc.) was used as indicator of Ca2+ wave activity so that GFP fluorescence and Ca2+ wave activity could be observed in the same oocyte. GFP(S65T) absorption and emission maxima in the visible spectrum occur at 490 and 509 nm, respectively, while for Ca2+ Orange these are 590 nm and 650 nm, respectively. Thus, for the imaging parameters used, Ca2+ Orange fluorescence does not overlap with GFP fluorescence emission. Each temporal stack contains 400 images taken at 0.5-s intervals. A single image (530 μm × 745 μm) of Ca2+ wave activity is shown at the indicated time (right). (c) At equivalent levels of GFP fluorescence, the Ca2+ wave properties are different for oocytes overexpressing GFP-SERCA2a (gray bars, n = 13) and GFP-SERCA2b (black bars, n = 10). Histogram of GFP fluorescence (left) shows fluorescence intensity measurements in arbitrary units. Histogram of Ca2+ wave period (middle) and Ca2+ wave decay time (right) measure each of these parameters from the time course of the average fluorescence intensity of a 5 × 5 pixel area. Note that GFP-SERCA2a-overexpressing oocytes display a higher Ca2+ wave frequency (i.e., shorter periods) than the GFP-SERCA2b-overexpressing oocytes. * Indicates a statistically significant difference at P < 0.005.

Mentions: To compare the modulation of IP3-mediated Ca2+ release by SERCA2a and SERCA2b, we overexpressed each isoform by injecting synthetic mRNAs (50 nl, 2 μg/μl) into Xenopus laevis oocytes (cDNAs encoding rat SERCAs; Gunteski-Hamblin et al., 1988). Confocal imaging of Ca2+ wave activity was performed 5–7 d later as previously described (Camacho and Lechleiter, 1995). In control oocytes (H2O replacing mRNA), IP3 injection (∼300 nM final) initiates a tidal wave of Ca2+ release that envelopes the entire oocyte, and is followed by low-frequency oscillations (Camacho and Lechleiter, 1993; Camacho and Lechleiter, 1995). In contrast, similar injections of IP3 into oocytes overexpressing SERCA2 isoforms result in high-frequency Ca2+ waves (shorter period between waves) without an initial Ca2+ tide. Confocal images of intracellular Ca2+ release for two oocytes overexpressing either SERCA2a or SERCA2b obtained at low magnification are shown in Fig. 1 a (10× objective, top). Ca2+ wave profiles in SERCA2b-overexpressing oocytes (n = 30) are characterized by broad wave widths and sharply delineated wave fronts. In contrast, the wave profiles in SERCA2a-overexpressing oocytes (n = 19) have shorter wave widths, and the leading wave fronts are poorly delineated. These differences in individual Ca2+ wave characteristics are more clearly observed at a higher magnification (40× objective) in oocytes overexpressing either SERCA2a (n = 4) or SERCA2b (n = 4; Fig. 1 a, bottom). Immunofluorescence images with a SERCA-specific antibody shows that both Ca2+ ATPases are targeted to the same yolk-free ER corridors in the oocyte (Fig. 1 b). The immunofluorescence pattern is identical in SERCA2a and SERCA2b-overexpressing oocytes. A control oocyte (H2O replacing mRNA injection) also demonstrates a similar pattern of low-intensity cross-reactivity of the anti-rat SERCA antibody with the endogenous Xenopus Ca2+-ATPases (Fig. 1 b, lower left). Western blot analysis reveals that the SERCA2 isoforms are overexpressed at roughly equivalent levels (Fig. 1 c). Since quantitation of expression levels by Western blotting is not very precise, we incorporated a fluorescent tag into either SERCA2a or SERCA2b so that Ca2+ wave properties could be compared in oocytes expressing equivalent levels of exogenous pumps. To this end we labeled each SERCA2 isoform with the green fluorescent protein (GFP) S65T mutant (Heim et al., 1995). GFP tagging at the NH2 terminus was the preferred strategy since the sequence differences between the two isoforms reside at the COOH terminus (Fig. 2 a). In these fusion constructs and according to accepted topological maps of SERCA proteins (Clarke et al., 1990; Bayle et al., 1995), GFP is expected to face the cytosol. The resulting fusion constructs were expressed in the oocyte by injecting synthetic mRNAs encoding GFP-SERCA2a and GFP-SERCA2b. Extracts from oocytes overexpressing either the wild-type (SERCA2a and SERCA2b) isoforms or the fusion proteins (GFP-SERCA2a and GFP-SERCA2b) were prepared and analyzed by Western blot probing with an antibody that recognizes the SERCA2 antigens. As expected, GFP fusion products migrate differentially with an apparent Mr ∼27 kD larger than for unmodified proteins, and the mobility differences of the two isoforms are retained (Fig. 2 b). Confocal images acquired at high magnification (60× objective) demonstrate a reticular pattern of GFP fluorescence in oocytes overexpressing either GFP-SERCA2a or GFP-SERCA2b, but not in oocytes overexpressing cytosolic GFP (Fig. 2 c). This immunofluorescence pattern of the GFP-tagged pumps is identical to that of the unmodified SERCA2a and SERCA2b shown in Fig. 1 b, indicating that GFP fusion does not interfere with targeting to the ER compartment. Low-magnification images of GFP fluorescence (10× objective) were used to measure overexpression levels of tagged SERCA2a and SERCA2b. Unlike control oocytes (H2O instead of mRNA) that do not emit fluorescence when excited at 488 nm, oocytes overexpressing either GFP-SERCA2a or GFP-SERCA2b do fluoresce (Fig. 3 a). IP3-mediated Ca2+ wave activity was imaged with the fluorescent indicator Ca2+ Orange (Molecular Probes, Inc.) in the two oocytes that had matching GFP fluorescence levels (Fig. 3 b). Comparison of these two oocytes shows that the GFP-SERCA2b–overexpressing oocytes have longer periods between waves, and exhibit wider wave widths than oocytes overexpressing the GFP-SERCA2a fusion product. Data analysis from oocytes that could be matched for GFP fluorescence levels (Fig. 3 c) also shows longer periods between waves and wave widths in GFP-SERCA2b (n = 13) than in GFP-SERCA2a-expressing oocytes (n = 10). These data suggest that the GFP-tagged pumps behaved like the wild-type isoforms, and allowed us to demonstrate conclusively that the functional differences between the two pumps seen in Fig. 1 a cannot be attributed to differences in expression levels. Rather, differences in Ca2+ uptake properties between SERCA2a and SERCA2b reflect the intrinsic differences in their primary structure (amino acid sequence).


Differential modulation of SERCA2 isoforms by calreticulin.

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

GFP-SERCA2 isoforms retain the characteristics of  their respective unmodified proteins and at equivalent levels of  expression exhibit different Ca2+ wave characteristics. (a) Fluorescence images in GFP-SERCA2a (left) and GFP-SERCA2b  (middle) overexpressing oocytes that have been matched for  equivalent levels of GFP fluorescence intensity. GFP fluorescence (745 μm × 530 μm) was excited at 488 nm. Note that under  these imaging conditions, fluorescence levels in control oocytes  (injected with H2O instead of GFP-SERCA2 mRNA) are not detectable. For quantitation of overexpression levels of GFP fusion  proteins, fluorescence values were measured from images obtained at a low magnification (10× objective; bar, ∼100 μm). (b)  Spatio-temporal patterns (left) of Ca2+ release induced by injection of IP3 (∼300 nM final) in oocytes as labeled. In this experiment, Ca2+ Orange (Molecular Probes, Inc.) was used as indicator of Ca2+ wave activity so that GFP fluorescence and Ca2+  wave activity could be observed in the same oocyte. GFP(S65T)  absorption and emission maxima in the visible spectrum occur at  490 and 509 nm, respectively, while for Ca2+ Orange these are  590 nm and 650 nm, respectively. Thus, for the imaging parameters used, Ca2+ Orange fluorescence does not overlap with GFP  fluorescence emission. Each temporal stack contains 400 images  taken at 0.5-s intervals. A single image (530 μm × 745 μm) of  Ca2+ wave activity is shown at the indicated time (right). (c) At  equivalent levels of GFP fluorescence, the Ca2+ wave properties  are different for oocytes overexpressing GFP-SERCA2a (gray  bars, n = 13) and GFP-SERCA2b (black bars, n = 10). Histogram of GFP fluorescence (left) shows fluorescence intensity  measurements in arbitrary units. Histogram of Ca2+ wave period  (middle) and Ca2+ wave decay time (right) measure each of these  parameters from the time course of the average fluorescence intensity of a 5 × 5 pixel area. Note that GFP-SERCA2a-overexpressing oocytes display a higher Ca2+ wave frequency (i.e.,  shorter periods) than the GFP-SERCA2b-overexpressing oocytes. * Indicates a statistically significant difference at P < 0.005.
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Figure 3: GFP-SERCA2 isoforms retain the characteristics of their respective unmodified proteins and at equivalent levels of expression exhibit different Ca2+ wave characteristics. (a) Fluorescence images in GFP-SERCA2a (left) and GFP-SERCA2b (middle) overexpressing oocytes that have been matched for equivalent levels of GFP fluorescence intensity. GFP fluorescence (745 μm × 530 μm) was excited at 488 nm. Note that under these imaging conditions, fluorescence levels in control oocytes (injected with H2O instead of GFP-SERCA2 mRNA) are not detectable. For quantitation of overexpression levels of GFP fusion proteins, fluorescence values were measured from images obtained at a low magnification (10× objective; bar, ∼100 μm). (b) Spatio-temporal patterns (left) of Ca2+ release induced by injection of IP3 (∼300 nM final) in oocytes as labeled. In this experiment, Ca2+ Orange (Molecular Probes, Inc.) was used as indicator of Ca2+ wave activity so that GFP fluorescence and Ca2+ wave activity could be observed in the same oocyte. GFP(S65T) absorption and emission maxima in the visible spectrum occur at 490 and 509 nm, respectively, while for Ca2+ Orange these are 590 nm and 650 nm, respectively. Thus, for the imaging parameters used, Ca2+ Orange fluorescence does not overlap with GFP fluorescence emission. Each temporal stack contains 400 images taken at 0.5-s intervals. A single image (530 μm × 745 μm) of Ca2+ wave activity is shown at the indicated time (right). (c) At equivalent levels of GFP fluorescence, the Ca2+ wave properties are different for oocytes overexpressing GFP-SERCA2a (gray bars, n = 13) and GFP-SERCA2b (black bars, n = 10). Histogram of GFP fluorescence (left) shows fluorescence intensity measurements in arbitrary units. Histogram of Ca2+ wave period (middle) and Ca2+ wave decay time (right) measure each of these parameters from the time course of the average fluorescence intensity of a 5 × 5 pixel area. Note that GFP-SERCA2a-overexpressing oocytes display a higher Ca2+ wave frequency (i.e., shorter periods) than the GFP-SERCA2b-overexpressing oocytes. * Indicates a statistically significant difference at P < 0.005.
Mentions: To compare the modulation of IP3-mediated Ca2+ release by SERCA2a and SERCA2b, we overexpressed each isoform by injecting synthetic mRNAs (50 nl, 2 μg/μl) into Xenopus laevis oocytes (cDNAs encoding rat SERCAs; Gunteski-Hamblin et al., 1988). Confocal imaging of Ca2+ wave activity was performed 5–7 d later as previously described (Camacho and Lechleiter, 1995). In control oocytes (H2O replacing mRNA), IP3 injection (∼300 nM final) initiates a tidal wave of Ca2+ release that envelopes the entire oocyte, and is followed by low-frequency oscillations (Camacho and Lechleiter, 1993; Camacho and Lechleiter, 1995). In contrast, similar injections of IP3 into oocytes overexpressing SERCA2 isoforms result in high-frequency Ca2+ waves (shorter period between waves) without an initial Ca2+ tide. Confocal images of intracellular Ca2+ release for two oocytes overexpressing either SERCA2a or SERCA2b obtained at low magnification are shown in Fig. 1 a (10× objective, top). Ca2+ wave profiles in SERCA2b-overexpressing oocytes (n = 30) are characterized by broad wave widths and sharply delineated wave fronts. In contrast, the wave profiles in SERCA2a-overexpressing oocytes (n = 19) have shorter wave widths, and the leading wave fronts are poorly delineated. These differences in individual Ca2+ wave characteristics are more clearly observed at a higher magnification (40× objective) in oocytes overexpressing either SERCA2a (n = 4) or SERCA2b (n = 4; Fig. 1 a, bottom). Immunofluorescence images with a SERCA-specific antibody shows that both Ca2+ ATPases are targeted to the same yolk-free ER corridors in the oocyte (Fig. 1 b). The immunofluorescence pattern is identical in SERCA2a and SERCA2b-overexpressing oocytes. A control oocyte (H2O replacing mRNA injection) also demonstrates a similar pattern of low-intensity cross-reactivity of the anti-rat SERCA antibody with the endogenous Xenopus Ca2+-ATPases (Fig. 1 b, lower left). Western blot analysis reveals that the SERCA2 isoforms are overexpressed at roughly equivalent levels (Fig. 1 c). Since quantitation of expression levels by Western blotting is not very precise, we incorporated a fluorescent tag into either SERCA2a or SERCA2b so that Ca2+ wave properties could be compared in oocytes expressing equivalent levels of exogenous pumps. To this end we labeled each SERCA2 isoform with the green fluorescent protein (GFP) S65T mutant (Heim et al., 1995). GFP tagging at the NH2 terminus was the preferred strategy since the sequence differences between the two isoforms reside at the COOH terminus (Fig. 2 a). In these fusion constructs and according to accepted topological maps of SERCA proteins (Clarke et al., 1990; Bayle et al., 1995), GFP is expected to face the cytosol. The resulting fusion constructs were expressed in the oocyte by injecting synthetic mRNAs encoding GFP-SERCA2a and GFP-SERCA2b. Extracts from oocytes overexpressing either the wild-type (SERCA2a and SERCA2b) isoforms or the fusion proteins (GFP-SERCA2a and GFP-SERCA2b) were prepared and analyzed by Western blot probing with an antibody that recognizes the SERCA2 antigens. As expected, GFP fusion products migrate differentially with an apparent Mr ∼27 kD larger than for unmodified proteins, and the mobility differences of the two isoforms are retained (Fig. 2 b). Confocal images acquired at high magnification (60× objective) demonstrate a reticular pattern of GFP fluorescence in oocytes overexpressing either GFP-SERCA2a or GFP-SERCA2b, but not in oocytes overexpressing cytosolic GFP (Fig. 2 c). This immunofluorescence pattern of the GFP-tagged pumps is identical to that of the unmodified SERCA2a and SERCA2b shown in Fig. 1 b, indicating that GFP fusion does not interfere with targeting to the ER compartment. Low-magnification images of GFP fluorescence (10× objective) were used to measure overexpression levels of tagged SERCA2a and SERCA2b. Unlike control oocytes (H2O instead of mRNA) that do not emit fluorescence when excited at 488 nm, oocytes overexpressing either GFP-SERCA2a or GFP-SERCA2b do fluoresce (Fig. 3 a). IP3-mediated Ca2+ wave activity was imaged with the fluorescent indicator Ca2+ Orange (Molecular Probes, Inc.) in the two oocytes that had matching GFP fluorescence levels (Fig. 3 b). Comparison of these two oocytes shows that the GFP-SERCA2b–overexpressing oocytes have longer periods between waves, and exhibit wider wave widths than oocytes overexpressing the GFP-SERCA2a fusion product. Data analysis from oocytes that could be matched for GFP fluorescence levels (Fig. 3 c) also shows longer periods between waves and wave widths in GFP-SERCA2b (n = 13) than in GFP-SERCA2a-expressing oocytes (n = 10). These data suggest that the GFP-tagged pumps behaved like the wild-type isoforms, and allowed us to demonstrate conclusively that the functional differences between the two pumps seen in Fig. 1 a cannot be attributed to differences in expression levels. Rather, differences in Ca2+ uptake properties between SERCA2a and SERCA2b reflect the intrinsic differences in their primary structure (amino acid sequence).

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