Limits...
Calreticulin controls the rate of assembly of CD1d molecules in the endoplasmic reticulum.

Zhu Y, Zhang W, Veerapen N, Besra G, Cresswell P - J. Biol. Chem. (2010)

Bottom Line: Initial folding of the heavy chain involves its glycan-dependent association with calreticulin (CRT), calnexin (CNX), and the thiol oxidoreductase ERp57, and is followed by assembly with β(2)m to form the heterodimer.Here we show that in CRT-deficient cells CD1d heavy chains convert to β(2)m-associated dimers at an accelerated rate, indicating faster folding of the heavy chain, while the rate of intracellular transport after assembly is unaffected.However, these free heavy chains are rapidly internalized and degraded in lysosomes, indicating that β(2)m association is required for the exceptional resistance of CD1d to lysosomal degradation that is normally observed.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520-8011, USA.

ABSTRACT
CD1d is an MHC class I-like molecule comprised of a transmembrane glycoprotein (heavy chain) associated with β(2)-microglobulin (β(2)m) that presents lipid antigens to NKT cells. Initial folding of the heavy chain involves its glycan-dependent association with calreticulin (CRT), calnexin (CNX), and the thiol oxidoreductase ERp57, and is followed by assembly with β(2)m to form the heterodimer. Here we show that in CRT-deficient cells CD1d heavy chains convert to β(2)m-associated dimers at an accelerated rate, indicating faster folding of the heavy chain, while the rate of intracellular transport after assembly is unaffected. Unlike the situation with MHC class I molecules, antigen presentation by CD1d is not impaired in the absence of CRT. Instead, there are elevated levels of stable and functional CD1d on the surface of CRT-deficient cells. Association of the heavy chains with the ER chaperones Grp94 and Bip is observed in the absence of CRT, and these may replace CRT in mediating CD1d folding and assembly. ER retention of free CD1d heavy chains is impaired in CRT-deficient cells, allowing their escape and subsequent expression on the plasma membrane. However, these free heavy chains are rapidly internalized and degraded in lysosomes, indicating that β(2)m association is required for the exceptional resistance of CD1d to lysosomal degradation that is normally observed.

Show MeSH

Related in: MedlinePlus

Elevated steady state CD1d protein levels in the absence of CRT. A, the expression of CRT, CD1d, GFP and GAPDH was examined by Western blot in K41 cells, K41 transduced with CD1d-IRES-GFP (CD1d-i-GFP), K42 cells, K42 transduced with CD1d-IRES-GFP, and K42 transduced with CD1d-IRES-GFP and CRT. B, the expression of CRT, CD1d, GFP, and GAPDH in K41.CD1d, K42.CD1d and K42.CD1d.CRT cells was assessed by quantitative Western blot. The level of each protein in K42.CD1d or K42.CD1d.CRT cells was calculated as fold change relative to that in K41.CD1d cells. Data shown are the average ± S.E. of three dilutions from two separate experiments. C, initial CD1d protein synthesis rates in K41.CD1d and K42.CD1d cells were assessed by a short metabolic labeling experiment. Cells were labeled with [35S]methosinine/cysteine for 5 min and extracted in 1% digitonin. The extracts were then immunoprecipitated by D5 (anti-CD1d free heavy chain) together with GAPDH antibody, as the internal control, or by a nonrelated control antibody (Ctr). The immunoprecipitates were eluted by boiling in the reducing SDS-sample buffer and analyzed by 10% SDS-PAGE (left panel). Right panel, quantification of band intensities using Bio-Rad GS-525 phosphorimaging: D5 over GAPDH signal. Data shown are the average ± S.E. of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2992262&req=5

Figure 1: Elevated steady state CD1d protein levels in the absence of CRT. A, the expression of CRT, CD1d, GFP and GAPDH was examined by Western blot in K41 cells, K41 transduced with CD1d-IRES-GFP (CD1d-i-GFP), K42 cells, K42 transduced with CD1d-IRES-GFP, and K42 transduced with CD1d-IRES-GFP and CRT. B, the expression of CRT, CD1d, GFP, and GAPDH in K41.CD1d, K42.CD1d and K42.CD1d.CRT cells was assessed by quantitative Western blot. The level of each protein in K42.CD1d or K42.CD1d.CRT cells was calculated as fold change relative to that in K41.CD1d cells. Data shown are the average ± S.E. of three dilutions from two separate experiments. C, initial CD1d protein synthesis rates in K41.CD1d and K42.CD1d cells were assessed by a short metabolic labeling experiment. Cells were labeled with [35S]methosinine/cysteine for 5 min and extracted in 1% digitonin. The extracts were then immunoprecipitated by D5 (anti-CD1d free heavy chain) together with GAPDH antibody, as the internal control, or by a nonrelated control antibody (Ctr). The immunoprecipitates were eluted by boiling in the reducing SDS-sample buffer and analyzed by 10% SDS-PAGE (left panel). Right panel, quantification of band intensities using Bio-Rad GS-525 phosphorimaging: D5 over GAPDH signal. Data shown are the average ± S.E. of three independent experiments.

Mentions: To investigate the role of CRT in the assembly of CD1d molecules, we used the CRT-sufficient MEF cell line K41 and its CRT-deficient counterpart K42. Because MEF cells have no detectable CD1d expression (20), we retrovirally transduced a CD1d-IRES-GFP construct into K41 and K42 cells to generate the stable transfectants K41.CD1d and K42.CD1d. The internal ribosome entry site (IRES) from encephalomyocarditis virus (ECMV) between the CD1d- and GFP-coding regions permitted both genes to be translated from a single bicistronic mRNA. We used flow cytometry to sort stably transduced cell populations that expressed similar levels of GFP, assessed by Western blotting (Fig. 1, A and B) or flow cytometry (Fig. 4A). As expected, this resulted in equal rates of CD1d protein synthesis in both transfected cell lines as determined by a short metabolic labeling experiment (Fig. 1C). However, when steady state protein levels were assessed by Western blot, CD1d levels were found to be substantially higher in the absence of CRT (Fig. 1A, compare lanes 2 and 4, and Fig. 1B). Quantitative Western blotting showed that steady state protein levels of CD1d in CRT-deficient K42.CD1d cells are ∼2-fold higher than in CRT-sufficient K41.CD1d cells. In contrast, levels of GFP and GAPDH, two non-glycoproteins, were indistinguishable in the two cell lines (Fig. 1, A and B). To confirm that the elevated CD1d level was caused by the absence of CRT, we reconstituted CRT expression in K42.CD1d, generating the cell line K42.CD1d.CRT. Introduction of CRT caused a significant decrease in CD1d steady state protein levels (Fig. 1A, compare lanes 4 and 5 and Fig. 1B), whereas levels of GFP and GAPDH remained unaffected. The CD1d level in K42.CD1d.CRT still remained somewhat above that in K41.CD1d, which may be because of the ∼2-fold higher CRT expression in the latter (Fig. 1A, compare lanes 2 and 5 and Fig. 1B).


Calreticulin controls the rate of assembly of CD1d molecules in the endoplasmic reticulum.

Zhu Y, Zhang W, Veerapen N, Besra G, Cresswell P - J. Biol. Chem. (2010)

Elevated steady state CD1d protein levels in the absence of CRT. A, the expression of CRT, CD1d, GFP and GAPDH was examined by Western blot in K41 cells, K41 transduced with CD1d-IRES-GFP (CD1d-i-GFP), K42 cells, K42 transduced with CD1d-IRES-GFP, and K42 transduced with CD1d-IRES-GFP and CRT. B, the expression of CRT, CD1d, GFP, and GAPDH in K41.CD1d, K42.CD1d and K42.CD1d.CRT cells was assessed by quantitative Western blot. The level of each protein in K42.CD1d or K42.CD1d.CRT cells was calculated as fold change relative to that in K41.CD1d cells. Data shown are the average ± S.E. of three dilutions from two separate experiments. C, initial CD1d protein synthesis rates in K41.CD1d and K42.CD1d cells were assessed by a short metabolic labeling experiment. Cells were labeled with [35S]methosinine/cysteine for 5 min and extracted in 1% digitonin. The extracts were then immunoprecipitated by D5 (anti-CD1d free heavy chain) together with GAPDH antibody, as the internal control, or by a nonrelated control antibody (Ctr). The immunoprecipitates were eluted by boiling in the reducing SDS-sample buffer and analyzed by 10% SDS-PAGE (left panel). Right panel, quantification of band intensities using Bio-Rad GS-525 phosphorimaging: D5 over GAPDH signal. Data shown are the average ± S.E. of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992262&req=5

Figure 1: Elevated steady state CD1d protein levels in the absence of CRT. A, the expression of CRT, CD1d, GFP and GAPDH was examined by Western blot in K41 cells, K41 transduced with CD1d-IRES-GFP (CD1d-i-GFP), K42 cells, K42 transduced with CD1d-IRES-GFP, and K42 transduced with CD1d-IRES-GFP and CRT. B, the expression of CRT, CD1d, GFP, and GAPDH in K41.CD1d, K42.CD1d and K42.CD1d.CRT cells was assessed by quantitative Western blot. The level of each protein in K42.CD1d or K42.CD1d.CRT cells was calculated as fold change relative to that in K41.CD1d cells. Data shown are the average ± S.E. of three dilutions from two separate experiments. C, initial CD1d protein synthesis rates in K41.CD1d and K42.CD1d cells were assessed by a short metabolic labeling experiment. Cells were labeled with [35S]methosinine/cysteine for 5 min and extracted in 1% digitonin. The extracts were then immunoprecipitated by D5 (anti-CD1d free heavy chain) together with GAPDH antibody, as the internal control, or by a nonrelated control antibody (Ctr). The immunoprecipitates were eluted by boiling in the reducing SDS-sample buffer and analyzed by 10% SDS-PAGE (left panel). Right panel, quantification of band intensities using Bio-Rad GS-525 phosphorimaging: D5 over GAPDH signal. Data shown are the average ± S.E. of three independent experiments.
Mentions: To investigate the role of CRT in the assembly of CD1d molecules, we used the CRT-sufficient MEF cell line K41 and its CRT-deficient counterpart K42. Because MEF cells have no detectable CD1d expression (20), we retrovirally transduced a CD1d-IRES-GFP construct into K41 and K42 cells to generate the stable transfectants K41.CD1d and K42.CD1d. The internal ribosome entry site (IRES) from encephalomyocarditis virus (ECMV) between the CD1d- and GFP-coding regions permitted both genes to be translated from a single bicistronic mRNA. We used flow cytometry to sort stably transduced cell populations that expressed similar levels of GFP, assessed by Western blotting (Fig. 1, A and B) or flow cytometry (Fig. 4A). As expected, this resulted in equal rates of CD1d protein synthesis in both transfected cell lines as determined by a short metabolic labeling experiment (Fig. 1C). However, when steady state protein levels were assessed by Western blot, CD1d levels were found to be substantially higher in the absence of CRT (Fig. 1A, compare lanes 2 and 4, and Fig. 1B). Quantitative Western blotting showed that steady state protein levels of CD1d in CRT-deficient K42.CD1d cells are ∼2-fold higher than in CRT-sufficient K41.CD1d cells. In contrast, levels of GFP and GAPDH, two non-glycoproteins, were indistinguishable in the two cell lines (Fig. 1, A and B). To confirm that the elevated CD1d level was caused by the absence of CRT, we reconstituted CRT expression in K42.CD1d, generating the cell line K42.CD1d.CRT. Introduction of CRT caused a significant decrease in CD1d steady state protein levels (Fig. 1A, compare lanes 4 and 5 and Fig. 1B), whereas levels of GFP and GAPDH remained unaffected. The CD1d level in K42.CD1d.CRT still remained somewhat above that in K41.CD1d, which may be because of the ∼2-fold higher CRT expression in the latter (Fig. 1A, compare lanes 2 and 5 and Fig. 1B).

Bottom Line: Initial folding of the heavy chain involves its glycan-dependent association with calreticulin (CRT), calnexin (CNX), and the thiol oxidoreductase ERp57, and is followed by assembly with β(2)m to form the heterodimer.Here we show that in CRT-deficient cells CD1d heavy chains convert to β(2)m-associated dimers at an accelerated rate, indicating faster folding of the heavy chain, while the rate of intracellular transport after assembly is unaffected.However, these free heavy chains are rapidly internalized and degraded in lysosomes, indicating that β(2)m association is required for the exceptional resistance of CD1d to lysosomal degradation that is normally observed.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520-8011, USA.

ABSTRACT
CD1d is an MHC class I-like molecule comprised of a transmembrane glycoprotein (heavy chain) associated with β(2)-microglobulin (β(2)m) that presents lipid antigens to NKT cells. Initial folding of the heavy chain involves its glycan-dependent association with calreticulin (CRT), calnexin (CNX), and the thiol oxidoreductase ERp57, and is followed by assembly with β(2)m to form the heterodimer. Here we show that in CRT-deficient cells CD1d heavy chains convert to β(2)m-associated dimers at an accelerated rate, indicating faster folding of the heavy chain, while the rate of intracellular transport after assembly is unaffected. Unlike the situation with MHC class I molecules, antigen presentation by CD1d is not impaired in the absence of CRT. Instead, there are elevated levels of stable and functional CD1d on the surface of CRT-deficient cells. Association of the heavy chains with the ER chaperones Grp94 and Bip is observed in the absence of CRT, and these may replace CRT in mediating CD1d folding and assembly. ER retention of free CD1d heavy chains is impaired in CRT-deficient cells, allowing their escape and subsequent expression on the plasma membrane. However, these free heavy chains are rapidly internalized and degraded in lysosomes, indicating that β(2)m association is required for the exceptional resistance of CD1d to lysosomal degradation that is normally observed.

Show MeSH
Related in: MedlinePlus