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.
View: Text | PDF
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

×

Figure 2

S1P deficiency causes a partially defective M6P-dependent Golgi-to-lysosome transport of lysosomal enzymes.

Options: View larger image (or click on image) Download as PowerPoint
S1P deficiency causes a partially defective M6P-dependent Golgi-to-lysos...
(A) Representative immunofluorescence images of fibroblasts from the patient and her parents. ML-II and ML-IIIa cells are positive controls derived from patients with these conditions. Cathepsin B, lysosome enzyme; LAMP1, lysosome marker; TO-PRO, nuclear counterstaining. Scale bar: 10 μm. The number of N indicates the exact number of samples. (B) M6P-dependent lysosomal enzyme β-hexosaminidase activity from Saos2 lysates or supernatants, as indicated. Data represent mean ± SEM; n = 3. *P < 0.05, 1-way ANOVA followed by Student’s t test. (C) Inter-organelle trafficking of cathepsin D. Without M6P modification, cathepsin D precursor is directly secreted into extracellular space. Ionomycin triggers the release of lysosomal contents through inducing the lysosomal exocytosis of matured cathepsin D. (D) Western blotting of precursor (p), intermediate (i), and mature (m) forms of cathepsin D from Saos2 lysates or supernatants, with or without ionomycin treatment. The precursor form of cathepsin D was detected in supernatant from GPT-KO and S1P-KO cells. However, upon ionomycin treatment, the mature form of cathepsin D was detected in supernatant from S1P-KO cells, indicating correct targeting of cathepsin D in lysosome in S1P-KO cells. (E) Sera from the patient (pat) and her mother (mom) were analyzed by Western blotting (top). Lysates from Saos2 cells were used as a control. The same membrane was stained with Ponceau S to confirm equivalent loading of serum proteins (bottom). (F) Immunofluorescence images of WT and mutant Saos2 cells. Insets show high-magnification images of lysosomes. Scale bars: 5 μm; 125 nm (insets). Enlarged lysosomes were found in GPT-KO and S1P-KO cells. Unlike GPT-KO cells in which cathepsin D is absent in lysosomes, cathepsin D was detected in lysosomes in S1P-KO cells. (G) Phase-contrast images of WT and mutant Saos2 cells. Arrowheads indicate inclusion bodies. Scale bar: 10 μm.