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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

N-Glycosylation of human ECM1 at Asn354 and Asn444. (A) HT1080-ECM1-MH cells were cultured in serum-free medium for 24 h. Subsequently, conditioned medium was collected and incubated with Ni-NTA agarose for 2 h at 4 °C. The bound proteins were eluted with 300 mM imidazole, subjected to SDS–PAGE, and detected by CBB staining. (B) Purified recombinant ECM1 was treated with PNGaseF and subjected to SDS–PAGE. The sample that was digested with trypsin was analyzed by LC–MS/MS. Asn354 was converted to Asp residue (underlined “D”) by treatment with PNGaseF. (C) Purified ECM1 was treated with (lower) or without (upper) PNGaseF and subjected to SDS–PAGE. The samples that were digested with trypsin were analyzed by MALDI-TOF MS. The peak of the fragment in which Asn444 was converted to Asp residue (underlined “D”) by PNGaseF was observed at m/z 1360.7. (D) Purified ECM1 was treated with (lower) or without (upper) PNGaseF and subjected to SDS–PAGE. The samples that were digested with trypsin were analyzed by LC–MS. The peak of the fragment including Asn530 was observed at 7.8 min both after treatment with or without PNGaseF.
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f0015: N-Glycosylation of human ECM1 at Asn354 and Asn444. (A) HT1080-ECM1-MH cells were cultured in serum-free medium for 24 h. Subsequently, conditioned medium was collected and incubated with Ni-NTA agarose for 2 h at 4 °C. The bound proteins were eluted with 300 mM imidazole, subjected to SDS–PAGE, and detected by CBB staining. (B) Purified recombinant ECM1 was treated with PNGaseF and subjected to SDS–PAGE. The sample that was digested with trypsin was analyzed by LC–MS/MS. Asn354 was converted to Asp residue (underlined “D”) by treatment with PNGaseF. (C) Purified ECM1 was treated with (lower) or without (upper) PNGaseF and subjected to SDS–PAGE. The samples that were digested with trypsin were analyzed by MALDI-TOF MS. The peak of the fragment in which Asn444 was converted to Asp residue (underlined “D”) by PNGaseF was observed at m/z 1360.7. (D) Purified ECM1 was treated with (lower) or without (upper) PNGaseF and subjected to SDS–PAGE. The samples that were digested with trypsin were analyzed by LC–MS. The peak of the fragment including Asn530 was observed at 7.8 min both after treatment with or without PNGaseF.

Mentions: Next, we performed mass spectrometry analysis to determine the N-glycosylation sites in ECM1. Recombinant ECM1 was purified from conditioned medium of HT1080-ECM1-MH cells using Ni-NTA agarose (Fig. 3A). Purified recombinant ECM1 was digested with trypsin and was treated with or without PNGase F. The resulting peptides were analyzed by MALDI-TOF MS or LC–MS. LC–MS was used when the peptide fragment including putative N-glycosylated Asn residue could not be detected by MALDI-TOF MS. Because PNGase F cleaves between the innermost GlcNAc and Asn residues of N-linked glycans, thereby converting Asn to Asp residues, the new peaks of the fragment showing conversion of glycosylated Asn to Asp can be observed by mass spectrometry analysis. With PNGase F treatment, the peptide fragment including Asp that was converted from Asn354 residue was observed at b3 (283.10) to b5 (535.19) by LC–MS/MS (Fig. 3B). Similarly, with PNGase F treatment, the peptide fragment including Asp that was converted from Asn444 residue was observed at m/z 1360.7 by MALDI-TOF MS (Fig. 3C). However, the peak of the fragment including Asn530 was detected by LC–MS, even though the sample was treated with or without PNGase F (Fig. 3D). Moreover, the peak of the fragment including Asp that was converted from Asn530 residue was not observed (Fig. 3D). These results indicate that the Asn354 and Asn444 residues, but not Asn530, are N-glycosylated.


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)

N-Glycosylation of human ECM1 at Asn354 and Asn444. (A) HT1080-ECM1-MH cells were cultured in serum-free medium for 24 h. Subsequently, conditioned medium was collected and incubated with Ni-NTA agarose for 2 h at 4 °C. The bound proteins were eluted with 300 mM imidazole, subjected to SDS–PAGE, and detected by CBB staining. (B) Purified recombinant ECM1 was treated with PNGaseF and subjected to SDS–PAGE. The sample that was digested with trypsin was analyzed by LC–MS/MS. Asn354 was converted to Asp residue (underlined “D”) by treatment with PNGaseF. (C) Purified ECM1 was treated with (lower) or without (upper) PNGaseF and subjected to SDS–PAGE. The samples that were digested with trypsin were analyzed by MALDI-TOF MS. The peak of the fragment in which Asn444 was converted to Asp residue (underlined “D”) by PNGaseF was observed at m/z 1360.7. (D) Purified ECM1 was treated with (lower) or without (upper) PNGaseF and subjected to SDS–PAGE. The samples that were digested with trypsin were analyzed by LC–MS. The peak of the fragment including Asn530 was observed at 7.8 min both after treatment with or without PNGaseF.
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f0015: N-Glycosylation of human ECM1 at Asn354 and Asn444. (A) HT1080-ECM1-MH cells were cultured in serum-free medium for 24 h. Subsequently, conditioned medium was collected and incubated with Ni-NTA agarose for 2 h at 4 °C. The bound proteins were eluted with 300 mM imidazole, subjected to SDS–PAGE, and detected by CBB staining. (B) Purified recombinant ECM1 was treated with PNGaseF and subjected to SDS–PAGE. The sample that was digested with trypsin was analyzed by LC–MS/MS. Asn354 was converted to Asp residue (underlined “D”) by treatment with PNGaseF. (C) Purified ECM1 was treated with (lower) or without (upper) PNGaseF and subjected to SDS–PAGE. The samples that were digested with trypsin were analyzed by MALDI-TOF MS. The peak of the fragment in which Asn444 was converted to Asp residue (underlined “D”) by PNGaseF was observed at m/z 1360.7. (D) Purified ECM1 was treated with (lower) or without (upper) PNGaseF and subjected to SDS–PAGE. The samples that were digested with trypsin were analyzed by LC–MS. The peak of the fragment including Asn530 was observed at 7.8 min both after treatment with or without PNGaseF.
Mentions: Next, we performed mass spectrometry analysis to determine the N-glycosylation sites in ECM1. Recombinant ECM1 was purified from conditioned medium of HT1080-ECM1-MH cells using Ni-NTA agarose (Fig. 3A). Purified recombinant ECM1 was digested with trypsin and was treated with or without PNGase F. The resulting peptides were analyzed by MALDI-TOF MS or LC–MS. LC–MS was used when the peptide fragment including putative N-glycosylated Asn residue could not be detected by MALDI-TOF MS. Because PNGase F cleaves between the innermost GlcNAc and Asn residues of N-linked glycans, thereby converting Asn to Asp residues, the new peaks of the fragment showing conversion of glycosylated Asn to Asp can be observed by mass spectrometry analysis. With PNGase F treatment, the peptide fragment including Asp that was converted from Asn354 residue was observed at b3 (283.10) to b5 (535.19) by LC–MS/MS (Fig. 3B). Similarly, with PNGase F treatment, the peptide fragment including Asp that was converted from Asn444 residue was observed at m/z 1360.7 by MALDI-TOF MS (Fig. 3C). However, the peak of the fragment including Asn530 was detected by LC–MS, even though the sample was treated with or without PNGase F (Fig. 3D). Moreover, the peak of the fragment including Asp that was converted from Asn530 residue was not observed (Fig. 3D). These results indicate that the Asn354 and Asn444 residues, but not Asn530, are N-glycosylated.

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