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N-Glycosylation of extracellular matrix protein 1 (ECM1) regulates its secretion, which is unrelated to lipoid proteinosis.

Uematsu S, Goto Y, Suzuki T, Sasazawa Y, Dohmae N, Simizu S - FEBS Open Bio (2014)

Bottom Line: However, an effective therapeutic approach of LP is not established.Here, we showed that ECM1 gene mutation observed in LP patients significantly suppresses its secretion.These results indicate that the defect of N-glycosylation in ECM1 is not involved in the aberration of secretion of LP-derived mutated ECM1.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.

ABSTRACT
Extracellular matrix protein 1 (ECM1) is expressed in a wide variety of tissues and plays important roles in extracellular matrix formation. Additionally, ECM1 gene mutations cause lipoid proteinosis (LP), a rare skin condition of genetic origin. However, an effective therapeutic approach of LP is not established. Here, we showed that ECM1 gene mutation observed in LP patients significantly suppresses its secretion. As ECM1 has three putative N-glycosylation sites and most of mutated ECM1 observed in LP patients are defective in these N-glycosylation sites, we investigated the correlation between LP and N-glycosylation of ECM1. We identified that the Asn(354) and Asn(444) residues in ECM1 were N-glycosylated by mass spectrometry analysis. In addition, an N-linked glycan at Asn(354) negatively regulated secretion of ECM1, contrary to LP patient-derived mutants. These results indicate that the defect of N-glycosylation in ECM1 is not involved in the aberration of secretion of LP-derived mutated ECM1.

No MeSH data available.


Related in: MedlinePlus

Negative regulation of ECM1 secretion by N-glycosylation at Asn354. (A) HT1080-neo, HT1080-ECM1-MH, HT1080-ECM1-N354Q-MH, HT1080-ECM1-N444Q-MH, and HT1080-ECM1-2NQ-MH cells were lysed, and aliquots of the cell lysates were subjected to SDS–PAGE. The proteins were detected by immunoblotting with anti-c-myc or anti-α-tubulin antibodies. (B) HT1080-neo, HT1080-ECM1-MH, and HT1080-ECM1-2NQ-MH cells were treated with tunicamycin (TM) at various concentrations (0, 0.1, 1, and 10 μg/mL) for 24 h. The cells were lysed, and aliquots of the cell lysates were subjected to SDS–PAGE. The proteins were detected by immunoblotting with anti-c-myc or anti-α-tubulin antibodies. (C) HT1080-neo, HT1080-ECM1-MH, HT1080-ECM1-N354Q-MH, HT1080-ECM1-N444Q-MH, and HT1080-ECM1-2NQ-MH cells were cultured in serum-free media for 24 h. Subsequent conditioned media and cell lysates were collected. Conditioned media were incubated with Ni-NTA agarose for 2 h at 4 °C. The bound proteins were eluted with 300 mM imidazole. Obtained samples were subjected to SDS–PAGE. The proteins were detected by immunoblotting with anti-c-myc or anti-α-tubulin antibodies.
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f0020: Negative regulation of ECM1 secretion by N-glycosylation at Asn354. (A) HT1080-neo, HT1080-ECM1-MH, HT1080-ECM1-N354Q-MH, HT1080-ECM1-N444Q-MH, and HT1080-ECM1-2NQ-MH cells were lysed, and aliquots of the cell lysates were subjected to SDS–PAGE. The proteins were detected by immunoblotting with anti-c-myc or anti-α-tubulin antibodies. (B) HT1080-neo, HT1080-ECM1-MH, and HT1080-ECM1-2NQ-MH cells were treated with tunicamycin (TM) at various concentrations (0, 0.1, 1, and 10 μg/mL) for 24 h. The cells were lysed, and aliquots of the cell lysates were subjected to SDS–PAGE. The proteins were detected by immunoblotting with anti-c-myc or anti-α-tubulin antibodies. (C) HT1080-neo, HT1080-ECM1-MH, HT1080-ECM1-N354Q-MH, HT1080-ECM1-N444Q-MH, and HT1080-ECM1-2NQ-MH cells were cultured in serum-free media for 24 h. Subsequent conditioned media and cell lysates were collected. Conditioned media were incubated with Ni-NTA agarose for 2 h at 4 °C. The bound proteins were eluted with 300 mM imidazole. Obtained samples were subjected to SDS–PAGE. The proteins were detected by immunoblotting with anti-c-myc or anti-α-tubulin antibodies.

Mentions: To further confirm the N-glycosylation of ECM1, we prepared Asn-to-Gln single and double mutants corresponding to N-glycosylated Asn residues and established cell lines overexpressing mutant forms of ECM1 (N354Q, N444Q, and 2NQ) in HT1080 cell lines (Fig. 4A). Western blotting of these stable cell lines revealed that the molecular size of the single mutant was lower than that of wild-type ECM1 and that the molecular size of the double mutant (2NQ) was lower than that of the single mutant (N354Q and N444Q) (Fig. 4A). Subsequently, we treated HT1080-ECM1-MH and HT1080-ECM1-2NQ-MH cells with tunicamycin. Treatment with tunicamycin led to a size reduction of wild-type ECM1 but not 2NQ (Fig. 4B). Moreover, the molecular size between wild-type ECM1 treated with tunicamycin and 2NQ was the same. These results indicate that ECM1 is N-glycosylated at the Asn354 and Asn444 residues.


N-Glycosylation of extracellular matrix protein 1 (ECM1) regulates its secretion, which is unrelated to lipoid proteinosis.

Uematsu S, Goto Y, Suzuki T, Sasazawa Y, Dohmae N, Simizu S - FEBS Open Bio (2014)

Negative regulation of ECM1 secretion by N-glycosylation at Asn354. (A) HT1080-neo, HT1080-ECM1-MH, HT1080-ECM1-N354Q-MH, HT1080-ECM1-N444Q-MH, and HT1080-ECM1-2NQ-MH cells were lysed, and aliquots of the cell lysates were subjected to SDS–PAGE. The proteins were detected by immunoblotting with anti-c-myc or anti-α-tubulin antibodies. (B) HT1080-neo, HT1080-ECM1-MH, and HT1080-ECM1-2NQ-MH cells were treated with tunicamycin (TM) at various concentrations (0, 0.1, 1, and 10 μg/mL) for 24 h. The cells were lysed, and aliquots of the cell lysates were subjected to SDS–PAGE. The proteins were detected by immunoblotting with anti-c-myc or anti-α-tubulin antibodies. (C) HT1080-neo, HT1080-ECM1-MH, HT1080-ECM1-N354Q-MH, HT1080-ECM1-N444Q-MH, and HT1080-ECM1-2NQ-MH cells were cultured in serum-free media for 24 h. Subsequent conditioned media and cell lysates were collected. Conditioned media were incubated with Ni-NTA agarose for 2 h at 4 °C. The bound proteins were eluted with 300 mM imidazole. Obtained samples were subjected to SDS–PAGE. The proteins were detected by immunoblotting with anti-c-myc or anti-α-tubulin antibodies.
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f0020: Negative regulation of ECM1 secretion by N-glycosylation at Asn354. (A) HT1080-neo, HT1080-ECM1-MH, HT1080-ECM1-N354Q-MH, HT1080-ECM1-N444Q-MH, and HT1080-ECM1-2NQ-MH cells were lysed, and aliquots of the cell lysates were subjected to SDS–PAGE. The proteins were detected by immunoblotting with anti-c-myc or anti-α-tubulin antibodies. (B) HT1080-neo, HT1080-ECM1-MH, and HT1080-ECM1-2NQ-MH cells were treated with tunicamycin (TM) at various concentrations (0, 0.1, 1, and 10 μg/mL) for 24 h. The cells were lysed, and aliquots of the cell lysates were subjected to SDS–PAGE. The proteins were detected by immunoblotting with anti-c-myc or anti-α-tubulin antibodies. (C) HT1080-neo, HT1080-ECM1-MH, HT1080-ECM1-N354Q-MH, HT1080-ECM1-N444Q-MH, and HT1080-ECM1-2NQ-MH cells were cultured in serum-free media for 24 h. Subsequent conditioned media and cell lysates were collected. Conditioned media were incubated with Ni-NTA agarose for 2 h at 4 °C. The bound proteins were eluted with 300 mM imidazole. Obtained samples were subjected to SDS–PAGE. The proteins were detected by immunoblotting with anti-c-myc or anti-α-tubulin antibodies.
Mentions: To further confirm the N-glycosylation of ECM1, we prepared Asn-to-Gln single and double mutants corresponding to N-glycosylated Asn residues and established cell lines overexpressing mutant forms of ECM1 (N354Q, N444Q, and 2NQ) in HT1080 cell lines (Fig. 4A). Western blotting of these stable cell lines revealed that the molecular size of the single mutant was lower than that of wild-type ECM1 and that the molecular size of the double mutant (2NQ) was lower than that of the single mutant (N354Q and N444Q) (Fig. 4A). Subsequently, we treated HT1080-ECM1-MH and HT1080-ECM1-2NQ-MH cells with tunicamycin. Treatment with tunicamycin led to a size reduction of wild-type ECM1 but not 2NQ (Fig. 4B). Moreover, the molecular size between wild-type ECM1 treated with tunicamycin and 2NQ was the same. These results indicate that ECM1 is N-glycosylated at the Asn354 and Asn444 residues.

Bottom Line: However, an effective therapeutic approach of LP is not established.Here, we showed that ECM1 gene mutation observed in LP patients significantly suppresses its secretion.These results indicate that the defect of N-glycosylation in ECM1 is not involved in the aberration of secretion of LP-derived mutated ECM1.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.

ABSTRACT
Extracellular matrix protein 1 (ECM1) is expressed in a wide variety of tissues and plays important roles in extracellular matrix formation. Additionally, ECM1 gene mutations cause lipoid proteinosis (LP), a rare skin condition of genetic origin. However, an effective therapeutic approach of LP is not established. Here, we showed that ECM1 gene mutation observed in LP patients significantly suppresses its secretion. As ECM1 has three putative N-glycosylation sites and most of mutated ECM1 observed in LP patients are defective in these N-glycosylation sites, we investigated the correlation between LP and N-glycosylation of ECM1. We identified that the Asn(354) and Asn(444) residues in ECM1 were N-glycosylated by mass spectrometry analysis. In addition, an N-linked glycan at Asn(354) negatively regulated secretion of ECM1, contrary to LP patient-derived mutants. These results indicate that the defect of N-glycosylation in ECM1 is not involved in the aberration of secretion of LP-derived mutated ECM1.

No MeSH data available.


Related in: MedlinePlus