1. Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation
Melissa E. Heard-Lipsmeyer1, Roberta Besio2, Milena Dimori1, Sarah M. Zimmerman1, Larry J. Suva3, Alan J. Tackett4, David H. Eyre5, Roy Morello1
1Department of Physiology & Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA; 2Department of Molecular Medicine, Universita’ di Pavia, Pavia, Italy; 3Department of Veterinary Physiology and Pharmacology, Texas Veterinary Medical Center, Texas A&M University, College Station, Texas, USA; 4Department of Biochemistry & Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA; 5Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, Washington, USA
ABSTRACT
Figure 5. SC65 directly interacts with prolyl 3-hydroxylase 3 (P3H3) and LH1
Figure 7. Loss of Sc65 results in dermal tears, abnormal collagen fibrils and skin fragility
Figure 1. Sc65KO mouse generation and confirmation of bone loss phenotype
Figure 3. Increased electrophoretic mobility and altered cross-linking of type I collagen
Sc65 (Synaptoneal Complex 65) is an endoplasmic reticulum protein that belongs to the Leprecan family which include the prolyl 3-hydroxylases (P3H1, P3H2, P3H3) and cartilage associated protein (CRTAP). We and others have shown that mutations in both CRTAP and LEPRE1 (encoding P3H1) cause recessive forms of Osteogenesis Imperfecta. Sc65 and CRTAP are both non-enzymatic proteins and share high homology suggesting Sc65 may also function in bone homeostasis. Utilizing a gene trap allele, we recently demonstrated that loss of Sc65 results in low bone mass. Here, a new global knockout mouse (Sc65-KO) derived by deleting exons 7 and 8 using homologous recombination is described. Mice null for Sc65 exhibit a similar bone loss phenotype as the previous model with decreased BV/TV and the loss of cortical and trabecular bone. To ascertain Sc65 function in bone, co-immunoprecipitation (co-IP) of Sc65 candidate interactors in mouse fibroblasts followed by mass spectrometry was performed. These experiments identified several fibrillar procollagen α-chains as likely substrates of Sc65 supporting the idea that Sc65 plays a role in collagen modification, similar to other Leprecans. At the biochemical level, mass spectrometry of type I collagen peptides showed severe under-hydroxylation at helical cross-linking sites K87 and K930/933 in collagen α1(I) and α2(I) chains both from bone and skin which are known LH1 preferred substrate residues but with no effect on sites of prolyl 3-hydroxylation. Direct co-IP assays showed Sc65 interaction with lysyl-hydroxylase 1 (LH1, Plod1), prolyl 3-hydroxylase 3 and cyclophilin B. Western blot revealed dramatic reduction of LH1 and P3H3 in primary osteoblasts and skin fibroblasts from Sc65-KO mice. Size exclusion chromatography confirmed that Sc65 and P3H3 form a stable complex in the ER that affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Further testing showed that Sc65-KO mice also have fragile skin with less tensile strength than control mice, consistent with a collagen cross-linking abnormality. Collectively, these results indicate that Sc65 is a novel adapter molecule that stabilizes a unique ER-resident complex that is essential for proper collagen lysyl-hydroxylation. Loss of Sc65 leads to complex instability and defective fibrillar collagen modifications which negatively impacts bone, skin and likely other connective tissues. c d
A) SDS-6%PAGE of type I collagen extracted from skin and decalcified bone shows increased mobility of α-chains and reduced ratio of cross-linked β to γ components in the Sc65KO skin extracts. . B) Densitometric analysis of collagen bands. Values are means ± SD, n = 6; *p<0.01.
A&C) Murine 714 MEFs stably expressing SC65-Flag or an empty vector (EV) control were transiently transfected with HA-tagged Lh1. IP experiments were conducted using a Flag, HA or P3H3 antibody. Inputs and immuno-precipitates were separated by SDS-PAGE, and probed with antibodies against FLAG and HA or P3H3. B & D) Western blot of primary calvarial osteoblast and skin fibroblast lysates from WT and Sc65KO mice. Densitometric quantification of LH1 or P3H3 protein normalized to β-actin are below (*p<0.05; error bars = SD).
A) Strategy for the creation of the Sc65-null allele. B) PCR genotyping of Sc65KO mice (upper panel) and Western blot confirmation of SC65 protein (arrow) loss in multiple Sc65KO tissues (lower panel: Cal = calvaria, Kid = Kidney). C) IHC detection of SC65 in adult femur section from a WT mouse. Scale bars = 100μM (10x) or 20μM (63x). D) MicroCT analysis of long bones from 6 month-old WT and Sc65KO male mice. (*p<0.05).
A) H&E stained sections of WT and Sc65KO skin. B) Sirius red staining of skin. C) Electron micrographs skin biopsy from WT and Sc65KO mice. D) Distribution of collagen fibril diameter in WT and Sc65KO skin as measured from EM images. E) Skin EMs from Sc65KO mice exhibit significantly more empty space among collagen fibrils compared to WT mice (*p = 0.01). F-H) Biomechanical skin loading test to measure tensile strength of WT and Sc65KO skin. (*p<0.01)
Figure 4. SC65 loss impairs collagen triple-helical lysine hydroxylation
INTRODUCTION
Figure 2. Mass-spectrometry identification of Sc65 interactors
Figure 6. Characterization of a new SC65/LH1/P3H3 complex in the ER
2-oxoglutarate
di-oxygenase
domain
P3H1 / LEPRE1
P3H2 / LEPREL1
P3H3 / LEPREL2
TPR-like domain
CRTAP
SC65
Figure 8. Schematic representation of a fibrillar collagen molecule in the ER
The Leprecan Family. This gene family includes three enzymatic members, prolyl 3-hydroxylase 1 (P3H1), P3H2 and P3H3, and two non-enzymatic members, CRTAP and SC65. All members share a tetratricopeptide-like repeat (TRP-like) domain which is known to mediate protein–protein interactions, while the enzymes also contain a 2-oxoglutarate di-oxygenase domain at the C-terminal end.
A) Coomassie blue gel showing separation of immune-precipitates obtained using the indicated experimental conditions. B) 253 proteins with fold change >1.5 were identified as candidate Sc65 interactors.
Selected prolyl 3-hydroxylase enzymes (P3Hs) and lysyl hydroxylase enzymes (LHs) are shown with some known substrate residues and proposed complex arrangements.
CONCLUSIONS
Table 1. Candidate fibrillar collagen interactors of SC65
Crtap forms an ER complex with P3H1 and cyclophilin B (CypB) that is responsible for the Pro986 3-hydroxylation of collagen a1(I), a1(II) and a2(V)
CRTAP or P3H1 mutations in humans results in severe Osteogenesis Imperfecta
Sc65 shares 55% homology with CRTAP
Sc65 mutant mice have a low bone mass phenotype (Gruenwald, Morello et al. JBMR 2013) but the mechanism in which Sc65 regulates bone homeostasis remains unknown
Hypothesis:
Based on its homology to CRTAP, SC65 forms a complex with other collagen-modifying enzymes in the ER to regulate post- translational modifications of collagen molecules
SC65 is an ER-resident protein that forms a complex with LH1 and P3H3 to regulate post-translational hydroxylation of Lys-87 & 930 of type I collagen
Sc65KO mice exhibit low bone mass and fragile skin likely due to the alterations of hydroxylation and glycosylation of these lysine residues and abnormal collagen cross-links
This is the first work that describes a complex of a “prolyl” and “lysyl” hydroxylase.
FUNDING
Peptides from the two helical domain cross-linking sites in collagen α1(I) prepared from decalcified bone (A) and skin (B) were identified by LC-MS. A) Spectra from the two helical sites (Lys-87 and Lys-930) in bone show that Lys-87 is all glcgalHyl plus galHyl in WT and about 40% is lysine plus hydroxylysine in Sc65KO; Lys-930 is all hydroxylysine in WT and all lysine in Sc65KO B) The same helical sites in skin show that Lys-87 is all glcgalHyl in WT and all lysine plus hydroxylysine in mutant; Lys-930 is all hydroxylysine in wild type and all lysine in Sc65KO. (glcgal = glucosyl-galactosyl; gal = galactosyl; * = hydroxyl group).
A) Lysates of 714 MEFs transiently transfected with HA-tagged LH1 were immuno-precipitated with a HA antibody (upper panel) or a P3H3 antibody (lower panel). Inputs and IPs were separated by SDS-PAGE gel and probed with antibodies against HA and P3H3. B) Lysates of 714 MEFs stably expressing SC65-Flag or EV control and transiently transfected with HA-tagged CYPB were used for IP utilizing an HA antibody. C) WB of primary calvarial osteoblast and skin fibroblast lysates from WT andSc65KO mice showing similar content of CYPB protein in Sc65KOand WT samples.
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Fold change indicates enrichment relative to IP from cells transfected with empty vector.