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. 2011;6(10):e26454.
doi: 10.1371/journal.pone.0026454. Epub 2011 Oct 18.

Progranulin, a glycoprotein deficient in frontotemporal dementia, is a novel substrate of several protein disulfide isomerase family proteins

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Progranulin, a glycoprotein deficient in frontotemporal dementia, is a novel substrate of several protein disulfide isomerase family proteins

Sandra Almeida et al. PLoS One. 2011.

Abstract

The reduced production or activity of the cysteine-rich glycoprotein progranulin is responsible for about 20% of cases of familial frontotemporal dementia. However, little is known about the molecular mechanisms that govern the level and secretion of progranulin. Here we show that progranulin is expressed in mouse cortical neurons and more prominently in mouse microglia in culture and is abundant in the endoplasmic reticulum (ER) and Golgi. Using chemical crosslinking, immunoprecipitation, and mass spectrometry, we found that progranulin is bound to a network of ER Ca(2+)-binding chaperones including BiP, calreticulin, GRP94, and four members of the protein disulfide isomerase (PDI) family. Loss of ERp57 inhibits progranulin secretion. Thus, progranulin is a novel substrate of several PDI family proteins and modulation of the ER chaperone network may be a therapeutic target for controlling progranulin secretion.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Biochemical identification of PGRN-interacting proteins.
(A) mPGRN-AP or mPGRN-HA fusion protein constructs were stably transfected into HEK293 cells. Cell lysates or culture medium was analyzed on Western blot with anti-mPGRN antibody. This antibody specifically recognizes mPGRN but not the endogenous hPGRN in HEK293 cells. β-actin was used as the loading control. (B) Stably transfected HEK293 cells expressing mPGRN-HA were treated with cross linkers, followed by immunoprecipitation (IP) with HA antibody or control IgG. Immunoisolates were analyzed on Western blot with anti-mPGRN antibody. UT, untransfected HEK293 cells; ST, stably transfected cells.
Figure 2
Figure 2. Biochemical identification of PGRN-interacting proteins.
(A) Description of the experiment in C. mPGRN-HA stably transfected HEK293 cells were treated with the chemical crosslinker DSS. Immunoprecipitation was performed with an anti-HA antibody. The immunoisolates were analyzed by SDS-PAGE, which was then silver stained. Specific bands were cut out and analyzed by mass spectrometry. (B) Image of a gel after silver staining. The identities of bands 1–4 are listed in Table 1.
Figure 3
Figure 3. Co-localization of mPGRN-HA with its interacting proteins in HEK293 cells.
(A, D, G) Subcellular localization of mPGRN in HEK293 cells. (B, Co-immunostaining of cells in (A) with anti-calreticulin (Calr) antibody. (C) the merged image of (A) and (B). (E) Co-immunostaining of cells in (D) with anti-ERp57 antibody. (F) The merged image of (D) and (E). (H) Co-immunostaining of cells in (G) with anti-ERp72 antibody. (I) Merged image of (G) and (H). The mPGRN antibody did not recognize endogenous hPGRN in HEK293 cells. (J) Subcellular localization of endogenous hPGRN in HEK293 cells. (K) Subcellular localization of endogenous ERp72 in HEK293 cells. (L) Merged image of (J) and (K) indicates that endogenous hPGRN also largely co-localizes with ERp72. Note that the size of scales bars in J–K are different from A–I and mPGRN is overexpressed in A–I and shows a stronger signal in the ER. Scale bars: A–I, 10 µm; J–L, 10 µm.
Figure 4
Figure 4. Expression of endogenous PGRN in different brain cell types.
(A–C) mPGRN is expressed in MAP2-positive cultured mouse cortical neurons. (D–F) mPGRN is present in the processes of astrocytes in mixed brain cell cultures. (G–I) mPGRN seems to be localized in LAMP1-positive vesicles. (J–L) mPGRN is highly expressed in Iba1-positive cultured mouse microglia. (M–O) Anti-mPGRN specifically recognizes the endogenous mPGRN protein since the immunostaining signal is absence in Iba1-positive microglia (M) isolated from GRN knockout mice (N). Scale bar: 20 µm for all panels except G–I (3 µm).
Figure 5
Figure 5. Subcellular localization of endogenous mPGRN in cultured primary microglia.
(A, D, G) Immunostaining of endogenous mPGRN in cultured primary microglia. (B) immunostaining of the ER marker calreticulin in the microglia in (A). (C) Merged image of (A), and (B). (E) Immunostaining of the cis-Golgi marker GM130 in the microglia in (D). (F) Merged image of (D) and (E). (H) Immunostaining of the trans-Golgi marker TGN38 in the microglia in (G). (I) Merged image of (G) and (H). Scale bar: 20 µm.
Figure 6
Figure 6. Loss of ERp57 activity caused reduced PGRN secretion.
(A) Western blot analysis of the efficiencies of siRNA knockdown of ERp57 expression in stably transfected HEK293 cells expressing mPGRN. β-tubulin was used as the loading control. (B) qRT-PCR analysis of relative mPGRN mRNA levels after siRNA treatment. (C) mPGRN levels in the culture medium were measured by Western blot with anti-mPGRN antibody. Asterisk indicates a protein band of unknown identity visualized by staining of the Western blot membrane with Ponceau S to indicate equal loading. (D) Quantification of mPGRN levels in C. The values are mean ± SEM. *p<0.02, **p<0.002 vs. scrambled control. This experiment was repeated three times with similar results. (E) A vector-based shRNA construct reduced ERp5 expression level in stably transfected HEK293 cells expressing mPGRN. β-tubulin was used as the loading control. (F) Levels of secreted mPGRN were reduced in the culture medium of HEK293 cells with partial knockdown of ERp5. Asterisk indicates a protein band of unknown identity visualized by staining of the western blot membrane to indicate equal loading. (G) Quantification of the levels of mPGRN in F. Values are mean ± SEM. *p<0.02. This experiment was repeated three times with similar results.

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