Fig. 5: The C19F variant in MGP results in impaired processing of the signal peptide and intrecallular accumulation of the mutated protein. | Nature Communications

Fig. 5: The C19F variant in MGP results in impaired processing of the signal peptide and intrecallular accumulation of the mutated protein.

From: Specific heterozygous variants in MGP lead to endoplasmic reticulum stress and cause spondyloepiphyseal dysplasia

Fig. 5

Impaired processing, intracellular accumulation and poor secretion of C19F MGP in ATDC5 (a–f) and HEK-293 (g–o) cells. a Scheme showing the pMgp-FLAG-IRES-GFP or pC19FMgp-FLAG-IRES-GFP plasmids expressing the FLAG-tagged WT or C19F MGP. The constructs also express internal ribosome entry site (IRES)-driven GFP. b Fluorescence images showing GFP expression indicate comparable transfection efficiencies by both the plasmids in ATDC5 cells. Cell extracts or culture media from the transfected ATDC5 cells were enriched by immunoprecipitation (IP) using anti-FLAG magnetic beads and subjected to immunoblotting using an anti-FLAG antibody. C19F MGP expressing cells show~17 kDa and ~15kDa bands in the extracts (c), but not in the culture medium (d). e Plasmid vectors pMgp-GFP and pC19FMgp-GFP expressing the WT or C19F MGP fused in frame to GFP. f Fluorescence images showing higher retention of C19F MGP-GFP in the transfected ATDC5 cells. g Scheme showing the FLAG-tagged WT (pMgp-FLAG) or mutated (pC19FMgp-FLAG) MGP expressing plasmids. h Co-transfection of HEK-293 cells with GFP-expressing pMaxGFP and pMgp-FLAG or pC19FMgp-FLAG plasmids shows comparable transfection efficiency. i qRT-PCR showing comparable levels of WT and mutant Mgp RNA in the transfected HEK-293 cells (n = 3 independent biological samples/group). Graph shows mean±SD. Anti-FLAG immunoblots of the cell extracts (j) and culture media (k) from HEK-293 cells transfected with the pMgp-FLAG or pC19FMgp-FLAG constructs. Culture media were enriched for the tagged protein by IP as above. C19F MGP expressing cells show the ~17 kDa and ~15 kDa bands and higher amount of tagged proteins in the extract, but not in the medium. Molecular weights were shown in kilodalton (kDa). Densitometric analysis shows a consistent increase of C19F MGP protein in the cell lysate compared to that of the WT MGP. Graph shows mean of three independent experiments. l. Immunofluorescence imaging using the anti-FLAG antibody shows accumulated C19F MGP inside the transfected HEK-293 cells. The nuclei were stained by DAPI (blue). m WT/C19F MGP sequence. Peptides examined (n) are shown in color. n Representative mass spectrometry analyses of cell extracts or culture media from the transfected HEK-293 cells show more abundant tagged proteins in the extracts expressing C19F MGP, while their amount is very low in the culture medium. Analyses of cells expressing WT MGP result in an opposite pattern. o A “no enzyme” or semi-trypsin search shows that the C19F MGP samples contain several MGP peptides carrying the mutated F19 residue, often with some upstream amino acids. Source data are provided as a Source Data file.

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