MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta

Uschi Lindert, Wayne A Cabral, Surasawadee Ausavarat, Siraprapa Tongkobpetch, Katja Ludin, Aileen M Barnes, Patra Yeetong, Maryann Weis, Birgit Krabichler, Chalurmpon Srichomthong, Elena N Makareeva, Andreas R Janecke, Sergey Leikin, Benno Röthlisberger, Marianne Rohrbach, Ingo Kennerknecht, David R Eyre, Kanya Suphapeetiporn, Cecilia Giunta, Joan C Marini, Vorasuk Shotelersuk, Uschi Lindert, Wayne A Cabral, Surasawadee Ausavarat, Siraprapa Tongkobpetch, Katja Ludin, Aileen M Barnes, Patra Yeetong, Maryann Weis, Birgit Krabichler, Chalurmpon Srichomthong, Elena N Makareeva, Andreas R Janecke, Sergey Leikin, Benno Röthlisberger, Marianne Rohrbach, Ingo Kennerknecht, David R Eyre, Kanya Suphapeetiporn, Cecilia Giunta, Joan C Marini, Vorasuk Shotelersuk

Abstract

Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia. We identified an X-linked recessive form of OI caused by defects in MBTPS2, which encodes site-2 metalloprotease (S2P). MBTPS2 missense mutations in two independent kindreds with moderate/severe OI cause substitutions at highly conserved S2P residues. Mutant S2P has normal stability, but impaired functioning in regulated intramembrane proteolysis (RIP) of OASIS, ATF6 and SREBP transcription factors, consistent with decreased proband secretion of type I collagen. Further, hydroxylation of the collagen lysine residue (K87) critical for crosslinking is reduced in proband bone tissue, consistent with decreased lysyl hydroxylase 1 in proband osteoblasts. Reduced collagen crosslinks presumptively undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. These mutations provide evidence that RIP plays a fundamental role in normal bone development.

Figures

Figure 1. Extended pedigree of Family I…
Figure 1. Extended pedigree of Family I with linkage and segregation analyses.
(a) Extended pedigree of Family I. The pedigree contains 12 affected members (blackened symbols), with seven living and five deceased (diagonally crossed). Clinical assessments and blood samples were obtained from 18 individuals (short horizontal bars above symbols), including six affected. Dotted circles denote obligate carriers. Arrow indicates Proband P1/I. (b) Linkage and segregation analysis. Thai pedigree shows linkage of mutation with X chromosome and cosegregation of the mutation with phenotype. X-chromosomal markers are shown from Xpter to Xcen (full list of markers in Supplementary Table 3). Linked markers in squares are shown in relation to the members' status. The MBTPS2 c.1376A>G (p.N459S) mutation segregated with phenotype in all affected members. The mutation introduces a BsgI cleavage site, resulting in 375 and 305 bp bands. The normal allele generates the undigested 680 bp band. Marker =100 bp marker; −ve, negative control with no DNA added; an arrowhead indicates the 680 bp band. (c) Pedigree of Family IIA. The segregation analysis was done on all family members still alive at the time of sampling, that is all but individuals I/1, I/2 and II/4. The index patient P1/II and his maternal uncle P2/II show very similar clinical findings compatible with the diagnosis osteogenesis imperfecta type III/IV. The mother of P1/II is of normal height, whereas the maternal grandmother is only 155 cm (<3rd centile), but does not show any signs of osteogenesis imperfecta.
Figure 2. Clinical features of probands and…
Figure 2. Clinical features of probands and mutation identification.
(a) Left, Proband 1/I had blue sclerae, pectus carinatum and anteriorly bowed legs at 2 years of age. Centre, radiographs showed fractures of ribs, humeri, femora and tibiae on day 1 of life. Right, undertubulation and minimal cortex of lower long bones at 2 years of age. (b) Left, Proband 1/II at age 20 years presenting with significant rhizomelia of upper and lower extremities. Centre, radiographs at birth showed bowed tibiae and fibulae (left leg shown). Right, radiographs revealed kyphoscoliosis with anterior vertebral wedging, and flat, biconcave vertebral bodies with significant osteoporosis at 13 years of age. (c) MBTPS2 genomic DNA sequence reveals the c.1376A>G mutation (p.N459S) in proband P1/I and his heterozygous mother, but not in the proband's father. (d) Sequence of P1/II genomic DNA shows the MBTPS2 c.1515G>C mutation (p.L505F), which also occurs in heterozygous form in his mother, but is not present in the proband's father. (e) Species comparison of the S2P amino-acid sequence according to ClustalX. Residues shaded in green denote the NPDG motif required for enzymatic active site metal ion coordination.
Figure 3. Expression of MBTPS2 in OI…
Figure 3. Expression of MBTPS2 in OI and IFAP fibroblasts.
(a) Quantification of MBTPS2 transcripts by real-time RT–PCR shows no significant differences. (b) S2P protein levels are normal in OI, IFAP and KFSD fibroblasts, as detected by western blot. No significant differences in MBTPS2 expression were detected between control and proband fibroblasts. Error bars, s.d.
Figure 4. Functional studies of the MBTPS2…
Figure 4. Functional studies of the MBTPS2 variants.
(a) Western blots of OASIS cleavage by S1P and S2P. Control fibroblast and osteoblast lysates show cleavage of OASIS to its mature active form (S1P/S2P cleaved). Cells from OI, IFAP and KFSD probands contain increased partially cleaved OASIS (S1P cleaved) due to impaired cleavage by S2P (TUN, tunicamycin; ALLN, N-[N-(N-acetyl-L-leucyl)-L-leucyl]-L-norleucine). (b) Luciferase assays of MBTPS2 activation of ATF6 reporters upon induction of stress (+). Renilla luciferase was used to normalize the transfection efficiency. WT, N459S, R429H, and L505F indicate that cells were transfected with normal or mutant MBTPS2 constructs. **P, wild-type versus empty vector, N459S, R429H, and L505F<0.001 by ANOVA. (c) Luciferase assays of MBTPS2 activation of SRE reporters on induction of stress by depletion of sterols (+). Renilla luciferase was used to normalize the transfection efficiency. The results were represented as mean±s.d. WT, N459S, R429H and H171F indicate that cells were transfected with normal or mutant MBTPS2 constructs. **P, wild-type versus empty vector, N459S, R429H and H171F<0.001 by ANOVA. Error bars, s.d.
Figure 5. Effects of MBTPS2 mutations on…
Figure 5. Effects of MBTPS2 mutations on type I collagen.
(a) Expression of COL1A1 in normal control (C), OI proband, IFAP and KFSD fibroblasts, and OI and control (C) osteoblasts. (b) Total procollagen secretion by fibroblast lines harbouring MBTPS2 mutations is decreased compared with normal control (C) fibroblasts. (c) Steady-state type I collagen analysis. The electrophoretic migration of collagen alpha chains synthesized by OI proband fibroblasts (FB) and osteoblasts (OB) was equivalent to normal control (C) fibroblast collagen. *P<0.05, **P<0.001 by t-test. Error bars, s.d.
Figure 6. Effect of S2P deficiency on…
Figure 6. Effect of S2P deficiency on osteoblast differentiation.
(a) Transcripts encoding OASIS (CREB3L1), (b) bone-specific alkaline phosphatase (ALPL), (c) matrilin-1 (MATN1) and (d) Co-SMAD (SMAD4) are decreased in differentiating P1/II versus normal control osteoblasts in culture. *P<0.05, **P<0.001 by t-test. Error bars, s.d.
Figure 7. S2P deficiency causes abnormal post-translational…
Figure 7. S2P deficiency causes abnormal post-translational modification of type I collagen.
(a) Mass spectrometric analysis of bacterial collagenase-digested peptides of control and P1/II bone type I collagen. Hydroxylation and consequent glycosylation of α1(I)K87 residues, involved in crosslink formation, is decreased in the proband sample by more than one-half, while hydroxylation of α1(I)K930 residues is normal, compared with normal control bone. K, lysine; K*, hydroxylysine; gal, galactosyl. (b) Immunoblots of lysates from control (C) and P1/II osteoblasts cultured in the absence or presence (+) of tunicamycin, an inhibitor of N-linked glycosylation that induces ER stress and OASIS cleavage. Despite normal levels of CyPB/PPIB, proband cells have significantly decreased LH1/PLOD1 protein, consistent with decreased hydroxylation of collagen lysyl residues (K87) involved in crosslink formation in extracellular matrix. (c) Schematic of RIP-mediated signalling. OASIS, SREBP and ATF6 translocate to the Golgi in response to ER stress or cholesterol depletion, where they are sequentially cleaved by S1P and S2P. The fully processed forms shuttle to the nucleus to activate transcription of genes involved in protein folding, lipid synthesis and extracellular matrix development.

References

    1. Forlino A. & Marini J. C. Osteogenesis imperfecta. Lancet 387, 1657–1671 (2016).
    1. Mendoza-Londono R. et al. Recessive osteogenesis imperfecta caused by missense mutations in SPARC. Am. J. Hum. Genet. 96, 979–985 (2015).
    1. Lal M. & Caplan M. Regulated intramembrane proteolysis: signaling pathways and biological functions. Physiology (Bethesda) 26, 34–44 (2011).
    1. Murakami T. et al. Cleavage of the membrane-bound transcription factor OASIS in response to endoplasmic reticulum stress. J. Neurochem. 96, 1090–1100 (2006).
    1. Ye J. et al. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol. Cell 6, 1355–1364 (2000).
    1. Wang X., Sato R., Brown M. S., Hua X. & Goldstein J. L. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell 77, 53–62 (1994).
    1. Oeffner F. et al. IFAP syndrome is caused by deficiency in MBTPS2, an intramembrane zinc metalloprotease essential for cholesterol homeostasis and ER stress response. Am. J. Hum. Genet. 84, 459–467 (2009).
    1. Naiki M. et al. MBTPS2 mutation causes BRESEK/BRESHECK syndrome. Am. J. Med. Genet. A 158A, 97–102 (2011).
    1. Aten E. et al. Keratosis Follicularis Spinulosa Decalvans is caused by mutations in MBTPS2. Hum. Mutat. 31, 1125–1133 (2010).
    1. Rudner D. Z., Fawcett P. & Losick R. A family of membrane-embedded metalloproteases involved in regulated proteolysis of membrane-associated transcription factors. Proc. Natl Acad. Sci. USA 96, 14765–14770 (1999).
    1. Lindert U. et al. Urinary pyridinoline cross-links as biomarkers of osteogenesis imperfecta. Orphanet. J. Rare. Dis. 10, 104 (2015).
    1. Cabral W. A. et al. Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta. PLoS Genet. 10, e1004465 (2014).
    1. Murakami T. et al. Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation. Nat. Cell. Biol. 11, 1205–1211 (2009).
    1. Chen Q., Lee C. E., Denard B. & Ye J. Sustained induction of collagen synthesis by TGF-beta requires regulated intramembrane proteolysis of CREB3L1. PLoS ONE 9, e108528 (2014).
    1. Vellanki R. N. et al. OASIS/CREB3L1 induces expression of genes involved in extracellular matrix production but not classical endoplasmic reticulum stress response genes in pancreatic beta-cells. Endocrinology 151, 4146–4157 (2010).
    1. Ishikawa Y. et al. Mutation in cyclophilin B that causes hyperelastosis cutis in American Quarter Horse does not affect peptidylprolyl cis-trans isomerase activity but shows altered cyclophilin B-protein interactions and affects collagen folding. J. Biol. Chem. 287, 22253–22265 (2012).
    1. van Dijk F. S. et al. PLS3 mutations in X-linked osteoporosis with fractures. N. Engl. J. Med. 369, 1529–1536 (2013).
    1. Rawson R. B. The site-2 protease. Biochim. Biophys. Acta 1828, 2801–2807 (2013).
    1. Haghighi A. et al. A missense mutation in the MBTPS2 gene underlies the X-linked form of Olmsted Syndrome. J. Invest. Dermatol. 133, 571–573 (2013).
    1. Bornholdt D. et al. Genotype-phenotype correlations emerging from the identification of missense mutations in MBTPS2. Hum. Mutat. 34, 587–594 (2013).
    1. Rawson R. B. Control of lipid metabolism by regulated intramembrane proteolysis of sterol regulatory element binding proteins (SREBPs). Biochem. Soc. Symp. 70, 221–231 (2003).
    1. Schlombs K., Wagner T. & Scheel J. Site-1 protease is required for cartilage development in zebrafish. Proc. Natl Acad. Sci. USA 100, 14024–14029 (2003).
    1. Patra D. et al. Site-1 protease is essential for endochondral bone formation in mice. J. Cell Biol. 179, 687–700 (2007).
    1. Patra D., DeLassus E., Liang G. & Sandell L. J. Cartilage-specific ablation of site-1 protease in mice results in the endoplasmic reticulum entrapment of type IIb procollagen and down-regulation of cholesterol and lipid homeostasis. PLoS ONE 9, e105674 (2014).
    1. Symoens S. et al. Deficiency for the ER-stress transducer OASIS causes severe recessive osteogenesis imperfecta in humans. Orphanet J. Rare Dis. 8, 154 (2013).
    1. Eyre D. R. & Weis M. A. Bone collagen: new clues to its mineralization mechanism from recessive osteogenesis imperfecta. Calcif. Tissue Int. 93, 338–347 (2013).
    1. Kondo S., Saito A., Asada R., Kanemoto S. & Imaizumi K. Physiological unfolded protein response regulated by OASIS family members, transmembrane bZIP transcription factors. IUBMB Life 63, 233–239 (2011).
    1. Pietrzak A. et al. IFAP syndrome with severe cutaneous, neurologic and skeletal manifestations due to a novel MBTPS2 mutation in a Polish patient. Eur. J. Dermatol. 22, 467–472 (2012).
    1. Leoyklang P., Suphapeetiporn K., Wananukul S. & Shotelersuk V. Three novel mutations in the PORCN gene underlying focal dermal hypoplasia. Clin. Genet. 73, 373–379 (2008).
    1. Yeetong P. et al. A newly identified locus for benign adult familial myoclonic epilepsy on chromosome 3q26.32-3q28. Eur. J. Hum. Genet. 21, 225–228 (2013).
    1. Beever C. L., Penaherrera M. S., Langlois S. & Robinson W. R. X chromosome inactivation patterns in Russell-Silver syndrome patients and their mothers. Am. J. Med. Genet. A 123A, 231–235 (2003).
    1. Lindert U. et al. Molecular consequences of the SERPINH1/HSP47 mutation in the Dachshund Natural Model of osteogenesis imperfecta. J. Biol. Chem. 290, 17679–17689 (2015).
    1. Bonadio J., Holbrook K. A., Gelinas R. E., Jacob J. & Byers P. H. Altered triple helical structure of type I procollagen in lethal perinatal osteogenesis imperfecta. J. Biol. Chem. 260, 1734–1742 (1985).
    1. Christiansen H. E. et al. Homozygosity for a missense mutation in SERPINH1, which encodes the collagen chaperone protein HSP47, results in severe recessive osteogenesis imperfecta. Am. J. Hum. Genet. 86, 389–398 (2010).
    1. Cabral W. A. et al. Type I collagen triplet duplication mutation in lethal osteogenesis imperfecta shifts register of alpha chains throughout the helix and disrupts incorporation of mutant helices into fibrils and extracellular matrix. J. Biol. Chem. 278, 10006–10012 (2003).
    1. Bateman J. F. & Golub S. B. Deposition and selective degradation of structurally-abnormal type I collagen in a collagen matrix produced by osteogenesis imperfecta fibroblasts in vitro. Matrix Biol. 14, 251–262 (1994).
    1. Robey P. G. & Termine J. D. Human bone cells in vitro. Calcif. Tissue Int. 37, 453–460 (1985).

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