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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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Site-directed mutagenesis of SERCA2b residue N1036  creates a protein that is no longer responsive to ΔC coexpression,  and that resembles SERCA2a. (a) Amino acid sequence comparison between the COOH terminus of SERCA2a and SERCA2b.  The eleventh transmembrane segment of SERCA2b is shown  (hatched). The consensus N-linked glycosylation motif is underlined, and the mutated residue N1036A is indicated in bold. (b)  Comparison of Ca2+ wave activity in two oocytes overexpressing  SERCA2bN1036A (top) or SERCA2bN1036A + ΔC (bottom).  (c) The left histogram shows percent of oocytes exhibiting repetitive Ca2+ oscillations when SERCA2bN1036A is expressed alone  or with ΔC. Of those oocytes that displayed repetitive Ca2+ oscillations, no significant differences were found in either interwave  period (middle histogram) or in decay time (right histogram) between oocytes coexpressing ΔC with SERCA2bN1036A and control oocytes overexpressing SERCA2bN1036A alone. These results are similar to those observed for SERCA2a and SERCA2a  + ΔC-overexpressing oocytes (see Fig. 4 b). (d) Western blot  analysis demonstrates overexpression of ΔC in fractions from  SERCA2bN1036A + ΔC oocytes (lane 1). No detectable CRT  product was observed in extracts from control oocytes (H2O replacing mRNA) (lane 2). The membrane was probed with the  anti-CRT KDEL primary Ab from Fig. 4 c.
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Figure 6: Site-directed mutagenesis of SERCA2b residue N1036 creates a protein that is no longer responsive to ΔC coexpression, and that resembles SERCA2a. (a) Amino acid sequence comparison between the COOH terminus of SERCA2a and SERCA2b. The eleventh transmembrane segment of SERCA2b is shown (hatched). The consensus N-linked glycosylation motif is underlined, and the mutated residue N1036A is indicated in bold. (b) Comparison of Ca2+ wave activity in two oocytes overexpressing SERCA2bN1036A (top) or SERCA2bN1036A + ΔC (bottom). (c) The left histogram shows percent of oocytes exhibiting repetitive Ca2+ oscillations when SERCA2bN1036A is expressed alone or with ΔC. Of those oocytes that displayed repetitive Ca2+ oscillations, no significant differences were found in either interwave period (middle histogram) or in decay time (right histogram) between oocytes coexpressing ΔC with SERCA2bN1036A and control oocytes overexpressing SERCA2bN1036A alone. These results are similar to those observed for SERCA2a and SERCA2a + ΔC-overexpressing oocytes (see Fig. 4 b). (d) Western blot analysis demonstrates overexpression of ΔC in fractions from SERCA2bN1036A + ΔC oocytes (lane 1). No detectable CRT product was observed in extracts from control oocytes (H2O replacing mRNA) (lane 2). The membrane was probed with the anti-CRT KDEL primary Ab from Fig. 4 c.

Mentions: Unlike SERCA2a, SERCA2b has a potential glycosylated residue (N1036) at the COOH terminus (Fig. 6 a). If the sustained Ca2+ release and inhibition of oscillatory Ca2+ waves is due to an interaction of calreticulin (or ΔC) with this SERCA2b residue, then site-directed mutagenesis to an unreactive alanine (SERCA2bN1036A mutant) should remove this potential site of calreticulin interaction. Consequently, when coexpressed with SERCA2bN1036A, ΔC should no longer have an effect on repetitive Ca2+ wave activity. To test this hypothesis, we overexpressed either SERCA2bN1036A by itself, or coexpressed it with ΔC (SERCA2bN1036A + ΔC oocytes). Overexpression of SERCA2bN1036A alone resulted in high-frequency Ca2+ oscillations in all oocytes tested (n = 23; Fig. 6 b). As predicted, repetitive Ca2+ activity was not inhibited when ΔC was coexpressed with SERCA2bN1036A (n = 22), suggesting that this mutation removed the site of interaction of calreticulin with SERCA2b (Fig. 6, b and c). Detection of the ΔC mutant protein product in SERCA2bN1036A + ΔC-overexpressing oocytes was corroborated by Western blotting and probing with an anti-KDEL Ab to the COOH terminus of calreticulin (Fig. 6 d). Together, these results implicate a role for calreticulin in the modulation of Ca2+ uptake via SERCA2b, but not SERCA2a, and suggest that the residue N1036 in SERCA2b is a putative target of lectin activity of this chaperone.


Differential modulation of SERCA2 isoforms by calreticulin.

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

Site-directed mutagenesis of SERCA2b residue N1036  creates a protein that is no longer responsive to ΔC coexpression,  and that resembles SERCA2a. (a) Amino acid sequence comparison between the COOH terminus of SERCA2a and SERCA2b.  The eleventh transmembrane segment of SERCA2b is shown  (hatched). The consensus N-linked glycosylation motif is underlined, and the mutated residue N1036A is indicated in bold. (b)  Comparison of Ca2+ wave activity in two oocytes overexpressing  SERCA2bN1036A (top) or SERCA2bN1036A + ΔC (bottom).  (c) The left histogram shows percent of oocytes exhibiting repetitive Ca2+ oscillations when SERCA2bN1036A is expressed alone  or with ΔC. Of those oocytes that displayed repetitive Ca2+ oscillations, no significant differences were found in either interwave  period (middle histogram) or in decay time (right histogram) between oocytes coexpressing ΔC with SERCA2bN1036A and control oocytes overexpressing SERCA2bN1036A alone. These results are similar to those observed for SERCA2a and SERCA2a  + ΔC-overexpressing oocytes (see Fig. 4 b). (d) Western blot  analysis demonstrates overexpression of ΔC in fractions from  SERCA2bN1036A + ΔC oocytes (lane 1). No detectable CRT  product was observed in extracts from control oocytes (H2O replacing mRNA) (lane 2). The membrane was probed with the  anti-CRT KDEL primary Ab from Fig. 4 c.
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Figure 6: Site-directed mutagenesis of SERCA2b residue N1036 creates a protein that is no longer responsive to ΔC coexpression, and that resembles SERCA2a. (a) Amino acid sequence comparison between the COOH terminus of SERCA2a and SERCA2b. The eleventh transmembrane segment of SERCA2b is shown (hatched). The consensus N-linked glycosylation motif is underlined, and the mutated residue N1036A is indicated in bold. (b) Comparison of Ca2+ wave activity in two oocytes overexpressing SERCA2bN1036A (top) or SERCA2bN1036A + ΔC (bottom). (c) The left histogram shows percent of oocytes exhibiting repetitive Ca2+ oscillations when SERCA2bN1036A is expressed alone or with ΔC. Of those oocytes that displayed repetitive Ca2+ oscillations, no significant differences were found in either interwave period (middle histogram) or in decay time (right histogram) between oocytes coexpressing ΔC with SERCA2bN1036A and control oocytes overexpressing SERCA2bN1036A alone. These results are similar to those observed for SERCA2a and SERCA2a + ΔC-overexpressing oocytes (see Fig. 4 b). (d) Western blot analysis demonstrates overexpression of ΔC in fractions from SERCA2bN1036A + ΔC oocytes (lane 1). No detectable CRT product was observed in extracts from control oocytes (H2O replacing mRNA) (lane 2). The membrane was probed with the anti-CRT KDEL primary Ab from Fig. 4 c.
Mentions: Unlike SERCA2a, SERCA2b has a potential glycosylated residue (N1036) at the COOH terminus (Fig. 6 a). If the sustained Ca2+ release and inhibition of oscillatory Ca2+ waves is due to an interaction of calreticulin (or ΔC) with this SERCA2b residue, then site-directed mutagenesis to an unreactive alanine (SERCA2bN1036A mutant) should remove this potential site of calreticulin interaction. Consequently, when coexpressed with SERCA2bN1036A, ΔC should no longer have an effect on repetitive Ca2+ wave activity. To test this hypothesis, we overexpressed either SERCA2bN1036A by itself, or coexpressed it with ΔC (SERCA2bN1036A + ΔC oocytes). Overexpression of SERCA2bN1036A alone resulted in high-frequency Ca2+ oscillations in all oocytes tested (n = 23; Fig. 6 b). As predicted, repetitive Ca2+ activity was not inhibited when ΔC was coexpressed with SERCA2bN1036A (n = 22), suggesting that this mutation removed the site of interaction of calreticulin with SERCA2b (Fig. 6, b and c). Detection of the ΔC mutant protein product in SERCA2bN1036A + ΔC-overexpressing oocytes was corroborated by Western blotting and probing with an anti-KDEL Ab to the COOH terminus of calreticulin (Fig. 6 d). Together, these results implicate a role for calreticulin in the modulation of Ca2+ uptake via SERCA2b, but not SERCA2a, and suggest that the residue N1036 in SERCA2b is a putative target of lectin activity of this chaperone.

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