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. 2015 Apr 1;24(7):1918-28.
doi: 10.1093/hmg/ddu608. Epub 2014 Dec 15.

HSP47 and FKBP65 cooperate in the synthesis of type I procollagen

Ivan Duran  1 , Lisette Nevarez  2 , Anna Sarukhanov  1 , Sulin Wu  1 , Katrina Lee  3 , Pavel Krejci  4 , Maryann Weis  5 , David Eyre  5 , Deborah Krakow  6 , Daniel H Cohn  7
Affiliations

HSP47 and FKBP65 cooperate in the synthesis of type I procollagen

Ivan Duran et al. Hum Mol Genet. .

Abstract

Osteogenesis imperfecta (OI) is a genetic disorder that results in low bone mineral density and brittle bones. Most cases result from dominant mutations in the type I procollagen genes, but mutations in a growing number of genes have been identified that produce autosomal recessive forms of the disease. Among these include mutations in the genes SERPINH1 and FKBP10, which encode the type I procollagen chaperones HSP47 and FKBP65, respectively, and predominantly produce a moderately severe form of OI. Little is known about the biochemical consequences of the mutations and how they produce OI. We have identified a new OI mutation in SERPINH1 that results in destabilization and mislocalization of HSP47 and secondarily has similar effects on FKBP65. We found evidence that HSP47 and FKBP65 act cooperatively during posttranslational maturation of type I procollagen and that FKBP65 and HSP47 but fail to properly interact in mutant HSP47 cells. These results thus reveal a common cellular pathway in cases of OI caused by HSP47 and FKBP65 deficiency.

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Figures

Figure 1.
Figure 1.
Clinical findings and collagen studies. (A–I) Radiographic analysis for patient R92-020A (A–D) at age of 4 and sibling R92-020B (E–I) at age 6 months. (J and K) Electrophoretic mobility of type I procollagen (J) and collagen (K) from cultured fibroblasts and media from case R92-020A. The α1 and α2 type I (pro)collagen chains are identified.
Figure 2.
Figure 2.
Recessive missense mutations in SERPINH1 change a highly conserved HSP47 residue. (A) Pedigree of the family showing two of four affected siblings (R92-020A and B) and a phenotypically undescribed miscarriage. (B) Chromatograms showing the sequence of position c. 710 in SERPINH1 (in yellow) annotated as a thymine (T) in the WT allele and cytosine (C) in R92-020A and the carrier parents. (C) Representation of HSP47 protein with the serine-type endopeptidase inhibitor domain in light blue. Protein alignment of HSP47 sequences from several vertebrate species show high conservation of the residues with OI-related mutations. The R92-020A mutation, resulting in the missense change at amino acid 237, is labeled with a black box and arrow. The previously described human OI case with a missense mutation in another highly conserved residue (L87) is shown in blue, and the dachshund (dog) OI change at residue 326L is shown in green.
Figure 3.
Figure 3.
SERPINH1 mutation causes a reduction in HSP47 and FKBP65 protein levels. (A–C) Protein levels for HSP47 and FKBP65 in HSP47M237T/M237T cells show a decrease in both proteins. (D and E) Transcriptional levels of HSP47 and FKBP65 do not change in HSP47M237T/M237T cells as measured by qRT-PCR. (F–H) Protein levels in FKBP10−/− cells show no change for HSP47 and absence of FKBP65. (I and J) Transcriptional levels for FKBP10−/− cells do not show changes in HSP47 and confirm lack of FKBP10 transcript.
Figure 4.
Figure 4.
HSP47M237T M237T and FKBP10−/− cells show protein accumulation in vesicles. Immunofluorescence against HSP47 (red) and FKBP65 (green) of control cells (A, D, and G), HSP47M237T/M237T (B, E and H) and FKBP65 (C, F and I) show co-localization of these proteins and their presence in massive vesicles in HSP47-mutant cells (arrows in B and E) and ER dilatation in FKBP10−/− (arrows in C and I). Arrow, asterisk and hash in B, E and H show variability of HSP47 signal in HSP47M237T M237T cells. Asterisk shows almost absence of HSP47 protein and hash shows a cell with low HSP47 expression. Bars represent 10 µm.
Figure 5.
Figure 5.
HSP47 accumulates in ER-related vesicles in OI-mutant cells. Immunofluorescence of HSP47 (red) and cell compartments (green): PDI for ER (A–C), Golgin97 for Golgi apparatus (D–F) and ERGIC53 for ER–Golgi intermediate compartment (G–I). Control (A, D and G), HSP47M237T M237T (B, E and H) and FKBP10−/− cells (C, F and I). White arrows identify vesicles accumulating HSP47. Bars represent 10 µm.
Figure 6.
Figure 6.
FKBP65 accumulates in ER-related vesicles in OI-mutant cells. Immunofluorescence of FKBP65 (green) and cell compartments (red): PDI for ER (A–C), Golgin97 for Golgi apparatus (D–F) and ERGIC53 for ER–Golgi intermediate compartment (G–I). Control (A, D and G), HSP47M237T/M237T (B, E and H) and FKBP10−/− cells (C, F and I). White arrows identify vesicles accumulating FKBP65. Bars represent 10 µm.
Figure 7.
Figure 7.
In situ localization of the interaction between HSP47 and FKBP65. Interaction of endogenous chaperones was measured by Proximity Ligation Assay (PLA) in Control (A), HSP47M237T/M237T (B) and FKBP10−/− fibroblasts. (C). Significant reduction was observed in HSP47 and FKBP10-mutant cells (* in D). Bars represent 10 µm.
Figure 8.
Figure 8.
Type I procollagen is present in ER-related abnormal vesicles in OI-mutant cells. Immunofluorescence of COL1 (green) and HSP47 and cell compartments (red): HSP47 (A–C), PDI for ER (D–F) Golgin97 for Golgi apparatus (G–I) and ERGIC53 for ER–Golgi intermediate compartment (J–L). Control (A, D, G and J), HSP47M237T/M237T (B, E, H and K) and FKBP10−/− cells (C, F, I and L). White arrows identify vesicles accumulating HSP47 and type I procollagen. Bars represent 10 µm.
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