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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

Treatment of tunicamycin suggested N-glycosylation within ECM1. (A) Schematic diagram of human ECM1 protein. ECM1 mutations observed in LP patients are indicated by the black inverted triangles. Three putative N-glycosylation sites (Asn354, Asn444 and Asn530) are indicated by sugar chains. Signal peptide is indicated by the black box. (B) HT1080-neo and HT1080-ECM1-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.
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f0010: Treatment of tunicamycin suggested N-glycosylation within ECM1. (A) Schematic diagram of human ECM1 protein. ECM1 mutations observed in LP patients are indicated by the black inverted triangles. Three putative N-glycosylation sites (Asn354, Asn444 and Asn530) are indicated by sugar chains. Signal peptide is indicated by the black box. (B) HT1080-neo and HT1080-ECM1-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.

Mentions: Previous report showed that nonsense mutations in exons 6 and 7 of ECM1 are frequently observed in LP patients [10]. Moreover, three putative N-glycosylation sites in ECM1 exist between exon 7 and the C-terminal domain (Fig. 2A). Thus, N-linked glycan cannot attach to mutated ECM1 observed in LP patients. Furthermore, some studies have reported that N-linked glycan is important for protein secretion [21,22,30–32]. For these reasons, we hypothesized that the aberrant N-glycosylation by gene mutation in ECM1 is one of the causes of LP. To validate this hypothesis, we analyzed N-glycosylation of ECM1.


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)

Treatment of tunicamycin suggested N-glycosylation within ECM1. (A) Schematic diagram of human ECM1 protein. ECM1 mutations observed in LP patients are indicated by the black inverted triangles. Three putative N-glycosylation sites (Asn354, Asn444 and Asn530) are indicated by sugar chains. Signal peptide is indicated by the black box. (B) HT1080-neo and HT1080-ECM1-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.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0010: Treatment of tunicamycin suggested N-glycosylation within ECM1. (A) Schematic diagram of human ECM1 protein. ECM1 mutations observed in LP patients are indicated by the black inverted triangles. Three putative N-glycosylation sites (Asn354, Asn444 and Asn530) are indicated by sugar chains. Signal peptide is indicated by the black box. (B) HT1080-neo and HT1080-ECM1-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.
Mentions: Previous report showed that nonsense mutations in exons 6 and 7 of ECM1 are frequently observed in LP patients [10]. Moreover, three putative N-glycosylation sites in ECM1 exist between exon 7 and the C-terminal domain (Fig. 2A). Thus, N-linked glycan cannot attach to mutated ECM1 observed in LP patients. Furthermore, some studies have reported that N-linked glycan is important for protein secretion [21,22,30–32]. For these reasons, we hypothesized that the aberrant N-glycosylation by gene mutation in ECM1 is one of the causes of LP. To validate this hypothesis, we analyzed N-glycosylation of ECM1.

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