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. 2020 Nov 5;107(5):989-999.
doi: 10.1016/j.ajhg.2020.09.009. Epub 2020 Oct 13.

Interaction between KDELR2 and HSP47 as a Key Determinant in Osteogenesis Imperfecta Caused by Bi-allelic Variants in KDELR2

Affiliations

Interaction between KDELR2 and HSP47 as a Key Determinant in Osteogenesis Imperfecta Caused by Bi-allelic Variants in KDELR2

Fleur S van Dijk et al. Am J Hum Genet. .

Abstract

Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G. gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI.

Keywords: HSP47; KDELR2; osteogenesis imperfecta; retrograde Golgi-ER transport.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Pedigrees and Radiographs of P1 (A–D), P2-1 (E–H), P3 (I–K), and P4-1 and P4-2 (L–M) (A) Pedigree of P1. (B and C) Radiographs of the lateral spine at the start of bisphosphonate treatment and after 9 years of treatment. Only mild reshaping of the vertebrae is visible, demonstrating a limited effect of the antiresorptive treatment. Improvement of vertebral morphology is less than reported in the literature for individuals affected with OI. (D) Radiograph of pelvis and both femurs after surgical procedures to stabilize fractures. Right femur is supported by a plate to stabilize the femur shaft fracture, and on the left side, screws have been used to treat a femoral neck fracture. Both femurs demonstrate good differentiation between cortical and trabecular bone with a relatively thick cortex contrary to the severely compressed vertebrae. (E) Pedigree of P2-1; asterisk denotes the number of sons/daughters. (F) Severe left convex scoliosis measuring 80 degrees at the lumbar level and 116 degrees at the thoracic level. (G) Deformation of the left shoulder with severe bowing of the left arm. (H) Deformed pelvis with bilateral protrusio acetabulum. (I) Pedigree of P3. (J) Barrel shaped thorax, dorsal scoliosis, and loss of height of the vertebral bodies; osteopenia. (K) Slender and curved diaphysis of the femur and fibula; osteopenia and muscular atrophy. (L) Pedigree of P4-1 and P4-2. (M and N) Babygrams of P4-1 and P4-2 at 24 weeks and 22+1 weeks of pregnancy, respectively, showed normal ossification of the skull and normal shape of the long bones of the upper extremity. The ribs were slender but showed no fractures. The long bones of the lower extremity were malformed because of multiple fractures, and both femora were more severely affected. A radiological diagnosis of OI type 2B/3 was made. E, a clinical diagnosis of OI type 2B/3; asterisk, documented evaluation.
Figure 2
Figure 2
KDELR2 Variants in P1 and P2-1 Lead to Retention of Collagen Type I in Cultured Fibroblasts without Compensating Effects of KDELR1 and KDELR3 (A) Expression of KDELR1, KDELR2, and KDELR3 in cultured fibroblasts was normalized to GAPDH and log2-transformed. For better visualization, corresponding fold changes are displayed on a log2-transformed axis for controls and fibroblasts derived from affected individuals (P1 [p.His150fs] and P2-1 [p.His12Asp]). Individual values and the mean of technical replicates are given. (B) Collagen type I protein (COL1) was detected in cultured fibroblasts by immunofluorescence microscopy after 1 or 3 days in different areas of the same culture. The brightness and contrast of fluorescent images was adjusted for visualization. Representative pictures are shown. Scale bar represents 20 μm. (C) Soluble fraction of COL1 in the supernatant of cultured confluent fibroblasts was detected by immunoblotting after 1 and 3 days. Ponceau S staining was used as loading control.
Figure 3
Figure 3
Mutations in KDELR2 in Fibroblasts Derived from Affected Individuals Result in Reduction of HSP47 and FKBP65 Accompanied by Reduced Collagen Assembly and Quality due to Mislocalization of HSP47 (A) The protein amount of P3H1 and HSP47 in trypsinized fibroblasts was determined by immunoblotting on day 1 or day 3 in controls and fibroblasts derived from P1 (p.His150fs) and P2-1 (p.His12Asp). Detection of β-actin was used as loading control. (B) The amount of FKBP65 in cultured control and fibroblasts derived from P1 (p.His150fs) and P2-1 (p.His12Asp) on day 1 were examined by immunoblotting. GAPDH was used to control equal protein loading. (C) The protein amount of HSP47 in the supernatant of fibroblasts was determined by immunoblotting on day 1 and day 3 in controls and fibroblasts derived from P1 (p.His150fs) and P2-1 (p.His12Asp). Ponceau S staining was used as loading control. (D) Representative immunoblot showing HSP47 amounts in the cell layer and supernatant of cultured control and fibroblasts derived from P3 (p.Pro133Leu). GAPDH amounts and Ponceau S staining served as loading control. (E) Negative staining EM and TEM were used to visualize triple helical collagen and maturing collagen fibrils, whereas immunogold EM was used to detect localization of HSP47 in the supernatant of cultured control and fibroblasts derived from P1 (p.His150fs) and P2-1 (p.His12Asp) after 3 days. Representative pictures are shown as overviews (upper panel) or higher magnification of diluted samples (lower panel). Scale bars represent 250 nm (upper panel), 100 nm (lower panel).
Figure 4
Figure 4
Evolutionary Conservation of KDELR Members of the PQ-Loop Motif Superfamily (A) KDELR2 residues substituted in affected individuals (black triangles) are identical from yeast to man. Transmembrane regions are shown above the amino acid sequences. His12 and Tyr158 (box A) co-operate to coordinate the KDEL ligand of target proteins in a polar binding cavity. A short hydrogen bond between p.Glu127 and p.Tyr158 (box B) lock this in place. The PQ-loop motif for which the superfamily is named is colored gray. (B) The crystal structure of G. gallus KDELR2 (PDB: 6I6H; colored N-term blue to C-term orange) bound to the TAEKDEL peptide (gray). The PQ-motif and His12 side chain are highlighted. On the right is a close-up view of the peptide binding site, showing the main interacting side chains. Hydrogen bond interactions are shown as yellow dashed lines. The water molecule (W1) linking the C terminus of the peptide with His12 is shown as a red sphere.

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