Site-1 protease deficiency causes human skeletal dysplasia due to defective inter-organelle protein trafficking
Yuji Kondo, … , Patrick M. Gaffney, Lijun Xia
Yuji Kondo, … , Patrick M. Gaffney, Lijun Xia
Published July 26, 2018
Citation Information: JCI Insight. 2018;3(14):e121596. https://doi.org/10.1172/jci.insight.121596.
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Research Article Cell biology Genetics

Site-1 protease deficiency causes human skeletal dysplasia due to defective inter-organelle protein trafficking

Abstract

Site-1 protease (S1P), encoded by MBTPS1, is a serine protease in the Golgi. S1P regulates lipogenesis, endoplasmic reticulum (ER) function, and lysosome biogenesis in mice and in cultured cells. However, how S1P differentially regulates these diverse functions in humans has been unclear. In addition, no human disease with S1P deficiency has been identified. Here, we report a pediatric patient with an amorphic and a severely hypomorphic mutation in MBTPS1. The unique combination of these mutations results in a frequency of functional MBTPS1 transcripts of approximately 1%, a finding that is associated with skeletal dysplasia and elevated blood lysosomal enzymes. We found that the residually expressed S1P is sufficient for lipid homeostasis but not for ER and lysosomal functions, especially in chondrocytes. The defective S1P function specifically impairs activation of the ER stress transducer BBF2H7, leading to ER retention of collagen in chondrocytes. S1P deficiency also causes abnormal secretion of lysosomal enzymes due to partial impairment of mannose-6-phosphate–dependent delivery to lysosomes. Collectively, these abnormalities lead to apoptosis of chondrocytes and lysosomal enzyme–mediated degradation of the bone matrix. Correction of an MBTPS1 variant or reduction of ER stress mitigated collagen-trafficking defects. These results define a new congenital human skeletal disorder and, more importantly, reveal that S1P is particularly required for skeletal development in humans. Our findings may also lead to new therapies for other genetic skeletal diseases, as ER dysfunction is common in these disorders.

Authors

Yuji Kondo, Jianxin Fu, Hua Wang, Christopher Hoover, J. Michael McDaniel, Richard Steet, Debabrata Patra, Jianhua Song, Laura Pollard, Sara Cathey, Tadayuki Yago, Graham Wiley, Susan Macwana, Joel Guthridge, Samuel McGee, Shibo Li, Courtney Griffin, Koichi Furukawa, Judith A. James, Changgeng Ruan, Rodger P. McEver, Klaas J. Wierenga, Patrick M. Gaffney, Lijun Xia

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Figure 3

S1P is important but is not the sole enzyme that activates GPT.

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S1P is important but is not the sole enzyme that activates GPT. (A) Supe...
(A) Supernatants from WT and mutant Saos2 cells were immunoprecipitated with anti-cathepsin D antibody (anti-CtsD) or normal goat IgG or precipitated with trichloroacetic acid (TCA)/acetone (input). M6P moieties were detected with anti-M6P. WT Saos2 cells were treated with NH4Cl to mimic ML-II/III cells (positive control). p, cathepsin D precursor. Asterisks indicate nonspecific bands. As expected, M6P moiety was detected on secreted cathepsin D from WT cells induced by NH4Cl treatment but not on secreted cathepsin D from untreated GPT-KO and S1P-KO cells. (B) Western blotting of M6P modification of lysates. Anti-GAPDH was used as loading control. Unlike GPT-KO cells, M6P bands were detected in lysates from S1P-KO cells. (C) Established clones were transfected with myc3-tagged GPT reporter (top). Cleavage of the reporter substrate in S1P-KO cells was considerably impaired but a small amount of cleaved reporter substrate was detected, supporting an alternative S1P-independent GPT-activating mechanism (bottom). (D) GPT activity in membrane fractions of WT cells, mutant HEK293T cells, or WT or S1P-KO Saos2 cells. Compared with WT cells, GPT-KO cells showed a substantial reduction in GPT activity, which was rescued by WT cells but not by the cleavage resistant GPT-R925A construct, which has missense mutation in the cleavage site by S1P to endow the resistance to the cleavage. Residual GPT activity was observed in S1P-KO cells, suggesting an alternative S1P-independent GPT-activating mechanism. (E) β-Hexosaminidase activity in lysates and culture supernatants from WT and mutant Saos2 cells. S2P-KO Saos2 cells do not show excessive secretion of β-hexosaminidase. Mean ± SEM for 4 independent experiments are shown. *P < 0.05, 1-way ANOVA followed by Student’s t test. (F) Serum lysosomal enzyme activities. Nor, normal individual; Fa, father; Mo, mother; Pa, patient. S2P patient does not have lysosomal abnormality. Mean ± SEM for 3 independent experiments are shown. *P < 0.05, 1-way ANOVA followed by Student’s t test. (G) Models of lysosomal enzyme trafficking in WT and mutant cells.