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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

Suppression of lipoid proteinosis-derived ECM1 secretion. (A) Schematic diagram of human ECM1 protein. ECM1 mutations observed in LP patients are indicated by the black inverted triangles. Signal peptide is indicated by the black box. (B) HT1080-neo, HT1080-ECM1-MH, HT1080-ECM1-Q276X-MH, and HT1080-ECM1-W359X-MH cells were cultured in serum-free media for 24 h. Subsequently, 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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f0005: Suppression of lipoid proteinosis-derived ECM1 secretion. (A) Schematic diagram of human ECM1 protein. ECM1 mutations observed in LP patients are indicated by the black inverted triangles. Signal peptide is indicated by the black box. (B) HT1080-neo, HT1080-ECM1-MH, HT1080-ECM1-Q276X-MH, and HT1080-ECM1-W359X-MH cells were cultured in serum-free media for 24 h. Subsequently, 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: Additionally, an ECM1 gene mutation causes lipoid proteinosis (LP), also known as Urbach–Wiethe disease, which is a rare autosomal recessive genodermatosis mainly observed in the Northern Cape province of South Africa [9]. LP is characterized by hoarseness because of infiltration of the vocal cords and thickening of the skin. Moreover, these patients show vascular anomalies caused by excessive deposition of hyaline-like material and consequent disruption/reduplication of basement membrane around blood vessels. It has been reported previously that exons 6 and 7 are the most common sites for ECM1 mutations in LP [10]. Since most of these mutations are nonsense mutations such as Q276X and W359X (Fig. 1A) or frameshift mutations, synthesis of full-length ECM1 is abolished. Although there have been many therapeutic trials in LP, including oral steroids and oral dimethyl sulfoxide (DMSO) [11,12], a definite therapeutic approach is not established.


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)

Suppression of lipoid proteinosis-derived ECM1 secretion. (A) Schematic diagram of human ECM1 protein. ECM1 mutations observed in LP patients are indicated by the black inverted triangles. Signal peptide is indicated by the black box. (B) HT1080-neo, HT1080-ECM1-MH, HT1080-ECM1-Q276X-MH, and HT1080-ECM1-W359X-MH cells were cultured in serum-free media for 24 h. Subsequently, 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.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215116&req=5

f0005: Suppression of lipoid proteinosis-derived ECM1 secretion. (A) Schematic diagram of human ECM1 protein. ECM1 mutations observed in LP patients are indicated by the black inverted triangles. Signal peptide is indicated by the black box. (B) HT1080-neo, HT1080-ECM1-MH, HT1080-ECM1-Q276X-MH, and HT1080-ECM1-W359X-MH cells were cultured in serum-free media for 24 h. Subsequently, 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: Additionally, an ECM1 gene mutation causes lipoid proteinosis (LP), also known as Urbach–Wiethe disease, which is a rare autosomal recessive genodermatosis mainly observed in the Northern Cape province of South Africa [9]. LP is characterized by hoarseness because of infiltration of the vocal cords and thickening of the skin. Moreover, these patients show vascular anomalies caused by excessive deposition of hyaline-like material and consequent disruption/reduplication of basement membrane around blood vessels. It has been reported previously that exons 6 and 7 are the most common sites for ECM1 mutations in LP [10]. Since most of these mutations are nonsense mutations such as Q276X and W359X (Fig. 1A) or frameshift mutations, synthesis of full-length ECM1 is abolished. Although there have been many therapeutic trials in LP, including oral steroids and oral dimethyl sulfoxide (DMSO) [11,12], a definite therapeutic approach is not established.

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