Mutants of β2-glycoprotein I: Their features and potent applications

Lianhua Shen, Nuriza Ulul Azmi, Xian Wen Tan, Shinsuke Yasuda, Arum Tri Wahyuningsih, Junko Inagaki, Kazuko Kobayashi, Eiji Ando, Takanori Sasaki, Eiji Matsuura

Research output: Contribution to journalReview article

Abstract

β2-Glycoprotein I (β2GPI) is a highly-glycosylated plasma protein composed of five homologous domains which regulates coagulation, fibrinolysis, and/or angiogenesis by interacting to negatively charged hydrophobic molecules and/or with plasminogen and its metabolites. The present study focused on structural and functional characterization of β2GPI's domain I (DI) and V (DV). Through N-terminal amino acid sequencing, a novel plasmin-cleaved site at K 287 –C 288 was identified in DV. We further modified the intact DV by altering two amino acids at specific proteolytic cleavage sites to generate three stable DV mutants: DV(PP), (PE), and (AA). Results of both SDS-PAGE and MALDI-TOF-MS showed that all three DV mutants were more stable than the intact DV, and DV(PE) was predominantly resistant to proteolysis. Competitive ELISA assessed affinities of intact β2GPI and those mutants to cardiolipin. In culture system, all DV and DI mutants potently inhibited HUVEC's proliferation by 18–30% as compared to control. Only DI and nicked β2GPI showed significant inhibition in HUVEC's tube formation. Moreover, DV(PE)-coated affinity columns demonstrated its binding property towards anionic lipids and could substantially isolate anionic DOPS from zwitterionic DOPC as a purification model. In summary, the proteolytic resistant and unhindered phospholipid (PL) binding properties of DV(PE) have made it an appealing element for subsequent prospective studies. Future in-depth characterization and optimized applications of cleavage-resistant DV(PE) would complement its full capacity as a novel clinical modality in the field of vascular imaging and/or lipidomics studies.

Original languageEnglish
JournalBest Practice and Research: Clinical Rheumatology
DOIs
Publication statusPublished - Jan 1 2019

Fingerprint

Glycoproteins
Cardiolipins
Plasminogen
Fibrinolysin
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Protein Sequence Analysis
Fibrinolysis
Proteolysis
Blood Vessels
Blood Proteins
Polyacrylamide Gel Electrophoresis
Phospholipids
Enzyme-Linked Immunosorbent Assay
Prospective Studies
Lipids
Amino Acids
1,2-oleoylphosphatidylcholine

Keywords

  • Amino acid substitutions
  • Domain I and domain V of β2GPI
  • Lipidomics
  • Nicked β2GPI
  • Proteolytic resistance

ASJC Scopus subject areas

  • Rheumatology

Cite this

Mutants of β2-glycoprotein I : Their features and potent applications. / Shen, Lianhua; Azmi, Nuriza Ulul; Tan, Xian Wen; Yasuda, Shinsuke; Wahyuningsih, Arum Tri; Inagaki, Junko; Kobayashi, Kazuko; Ando, Eiji; Sasaki, Takanori; Matsuura, Eiji.

In: Best Practice and Research: Clinical Rheumatology, 01.01.2019.

Research output: Contribution to journalReview article

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abstract = "β2-Glycoprotein I (β2GPI) is a highly-glycosylated plasma protein composed of five homologous domains which regulates coagulation, fibrinolysis, and/or angiogenesis by interacting to negatively charged hydrophobic molecules and/or with plasminogen and its metabolites. The present study focused on structural and functional characterization of β2GPI's domain I (DI) and V (DV). Through N-terminal amino acid sequencing, a novel plasmin-cleaved site at K 287 –C 288 was identified in DV. We further modified the intact DV by altering two amino acids at specific proteolytic cleavage sites to generate three stable DV mutants: DV(PP), (PE), and (AA). Results of both SDS-PAGE and MALDI-TOF-MS showed that all three DV mutants were more stable than the intact DV, and DV(PE) was predominantly resistant to proteolysis. Competitive ELISA assessed affinities of intact β2GPI and those mutants to cardiolipin. In culture system, all DV and DI mutants potently inhibited HUVEC's proliferation by 18–30{\%} as compared to control. Only DI and nicked β2GPI showed significant inhibition in HUVEC's tube formation. Moreover, DV(PE)-coated affinity columns demonstrated its binding property towards anionic lipids and could substantially isolate anionic DOPS from zwitterionic DOPC as a purification model. In summary, the proteolytic resistant and unhindered phospholipid (PL) binding properties of DV(PE) have made it an appealing element for subsequent prospective studies. Future in-depth characterization and optimized applications of cleavage-resistant DV(PE) would complement its full capacity as a novel clinical modality in the field of vascular imaging and/or lipidomics studies.",
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T2 - Their features and potent applications

AU - Shen, Lianhua

AU - Azmi, Nuriza Ulul

AU - Tan, Xian Wen

AU - Yasuda, Shinsuke

AU - Wahyuningsih, Arum Tri

AU - Inagaki, Junko

AU - Kobayashi, Kazuko

AU - Ando, Eiji

AU - Sasaki, Takanori

AU - Matsuura, Eiji

PY - 2019/1/1

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N2 - β2-Glycoprotein I (β2GPI) is a highly-glycosylated plasma protein composed of five homologous domains which regulates coagulation, fibrinolysis, and/or angiogenesis by interacting to negatively charged hydrophobic molecules and/or with plasminogen and its metabolites. The present study focused on structural and functional characterization of β2GPI's domain I (DI) and V (DV). Through N-terminal amino acid sequencing, a novel plasmin-cleaved site at K 287 –C 288 was identified in DV. We further modified the intact DV by altering two amino acids at specific proteolytic cleavage sites to generate three stable DV mutants: DV(PP), (PE), and (AA). Results of both SDS-PAGE and MALDI-TOF-MS showed that all three DV mutants were more stable than the intact DV, and DV(PE) was predominantly resistant to proteolysis. Competitive ELISA assessed affinities of intact β2GPI and those mutants to cardiolipin. In culture system, all DV and DI mutants potently inhibited HUVEC's proliferation by 18–30% as compared to control. Only DI and nicked β2GPI showed significant inhibition in HUVEC's tube formation. Moreover, DV(PE)-coated affinity columns demonstrated its binding property towards anionic lipids and could substantially isolate anionic DOPS from zwitterionic DOPC as a purification model. In summary, the proteolytic resistant and unhindered phospholipid (PL) binding properties of DV(PE) have made it an appealing element for subsequent prospective studies. Future in-depth characterization and optimized applications of cleavage-resistant DV(PE) would complement its full capacity as a novel clinical modality in the field of vascular imaging and/or lipidomics studies.

AB - β2-Glycoprotein I (β2GPI) is a highly-glycosylated plasma protein composed of five homologous domains which regulates coagulation, fibrinolysis, and/or angiogenesis by interacting to negatively charged hydrophobic molecules and/or with plasminogen and its metabolites. The present study focused on structural and functional characterization of β2GPI's domain I (DI) and V (DV). Through N-terminal amino acid sequencing, a novel plasmin-cleaved site at K 287 –C 288 was identified in DV. We further modified the intact DV by altering two amino acids at specific proteolytic cleavage sites to generate three stable DV mutants: DV(PP), (PE), and (AA). Results of both SDS-PAGE and MALDI-TOF-MS showed that all three DV mutants were more stable than the intact DV, and DV(PE) was predominantly resistant to proteolysis. Competitive ELISA assessed affinities of intact β2GPI and those mutants to cardiolipin. In culture system, all DV and DI mutants potently inhibited HUVEC's proliferation by 18–30% as compared to control. Only DI and nicked β2GPI showed significant inhibition in HUVEC's tube formation. Moreover, DV(PE)-coated affinity columns demonstrated its binding property towards anionic lipids and could substantially isolate anionic DOPS from zwitterionic DOPC as a purification model. In summary, the proteolytic resistant and unhindered phospholipid (PL) binding properties of DV(PE) have made it an appealing element for subsequent prospective studies. Future in-depth characterization and optimized applications of cleavage-resistant DV(PE) would complement its full capacity as a novel clinical modality in the field of vascular imaging and/or lipidomics studies.

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KW - Nicked β2GPI

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