Angiotensin-converting enzyme 2 ectodomain shedding cleavage-site identification: Determinants and constraints

Zon Lai, Iresha Hanchapola, David Steer, Alexander Smith

Research output: Contribution to journalArticleResearchpeer-review

Abstract

ADAM17, also known as tumor necrosis factor alpha-converting enzyme, is involved in the ectodomain shedding of many integral membrane proteins. We have previously reported that ADAM17 is able to mediate the cleavage secretion of the ectodomain of human angiotensin-converting enzyme 2 (ACE2), a functional receptor for the severe acute respiratory syndrome coronavirus. In this study, we demonstrate that purified recombinant human ADAM17 is able to cleave a 20-amino acid peptide mimetic corresponding to the extracellular juxtamembrane region of human ACE2 between Arg(708) and Ser(709). A series of peptide analogues were also synthesized, showing that glutamate subtitution at Arg(708) and/or Arg(710) attenuated the cleavage process, while alanine substitution at Arg(708) and/or Ser(709) did not inhibit peptide cleavage by recombinant ADAM17. Analysis of CD spectra showed a minimal difference in the secondary structure of the peptide analogues in the buffer system used for the ADAM17 cleavage assay. The observation of the shedding profiles of ACE2 mutants expressing CHO-K1 and CHO-P cells indicates that the Arg(708) --> Glu(708) mutation and the Arg(708)Arg(710) --> Glu(708)Glu(710) double mutation produced increases in the amount of ACE2 shed when stimulated by phorbol ester PMA. In summary, we have demonstrated that ADAM17 is able to cleave ACE2 peptide sequence analogues between Arg(708) and Ser(709). These findings also indicate that Arg(708) and Arg(710) play a role in site recognition in the regulation of ACE2 ectodomain shedding mediated by ADAM17.
Original languageEnglish
Pages (from-to)5182 - 5194
Number of pages13
JournalBiochemistry
Volume50
Issue number23
DOIs
Publication statusPublished - 2011

Cite this

Lai, Zon ; Hanchapola, Iresha ; Steer, David ; Smith, Alexander. / Angiotensin-converting enzyme 2 ectodomain shedding cleavage-site identification: Determinants and constraints. In: Biochemistry. 2011 ; Vol. 50, No. 23. pp. 5182 - 5194.
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abstract = "ADAM17, also known as tumor necrosis factor alpha-converting enzyme, is involved in the ectodomain shedding of many integral membrane proteins. We have previously reported that ADAM17 is able to mediate the cleavage secretion of the ectodomain of human angiotensin-converting enzyme 2 (ACE2), a functional receptor for the severe acute respiratory syndrome coronavirus. In this study, we demonstrate that purified recombinant human ADAM17 is able to cleave a 20-amino acid peptide mimetic corresponding to the extracellular juxtamembrane region of human ACE2 between Arg(708) and Ser(709). A series of peptide analogues were also synthesized, showing that glutamate subtitution at Arg(708) and/or Arg(710) attenuated the cleavage process, while alanine substitution at Arg(708) and/or Ser(709) did not inhibit peptide cleavage by recombinant ADAM17. Analysis of CD spectra showed a minimal difference in the secondary structure of the peptide analogues in the buffer system used for the ADAM17 cleavage assay. The observation of the shedding profiles of ACE2 mutants expressing CHO-K1 and CHO-P cells indicates that the Arg(708) --> Glu(708) mutation and the Arg(708)Arg(710) --> Glu(708)Glu(710) double mutation produced increases in the amount of ACE2 shed when stimulated by phorbol ester PMA. In summary, we have demonstrated that ADAM17 is able to cleave ACE2 peptide sequence analogues between Arg(708) and Ser(709). These findings also indicate that Arg(708) and Arg(710) play a role in site recognition in the regulation of ACE2 ectodomain shedding mediated by ADAM17.",
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Angiotensin-converting enzyme 2 ectodomain shedding cleavage-site identification: Determinants and constraints. / Lai, Zon; Hanchapola, Iresha; Steer, David; Smith, Alexander.

In: Biochemistry, Vol. 50, No. 23, 2011, p. 5182 - 5194.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Angiotensin-converting enzyme 2 ectodomain shedding cleavage-site identification: Determinants and constraints

AU - Lai, Zon

AU - Hanchapola, Iresha

AU - Steer, David

AU - Smith, Alexander

PY - 2011

Y1 - 2011

N2 - ADAM17, also known as tumor necrosis factor alpha-converting enzyme, is involved in the ectodomain shedding of many integral membrane proteins. We have previously reported that ADAM17 is able to mediate the cleavage secretion of the ectodomain of human angiotensin-converting enzyme 2 (ACE2), a functional receptor for the severe acute respiratory syndrome coronavirus. In this study, we demonstrate that purified recombinant human ADAM17 is able to cleave a 20-amino acid peptide mimetic corresponding to the extracellular juxtamembrane region of human ACE2 between Arg(708) and Ser(709). A series of peptide analogues were also synthesized, showing that glutamate subtitution at Arg(708) and/or Arg(710) attenuated the cleavage process, while alanine substitution at Arg(708) and/or Ser(709) did not inhibit peptide cleavage by recombinant ADAM17. Analysis of CD spectra showed a minimal difference in the secondary structure of the peptide analogues in the buffer system used for the ADAM17 cleavage assay. The observation of the shedding profiles of ACE2 mutants expressing CHO-K1 and CHO-P cells indicates that the Arg(708) --> Glu(708) mutation and the Arg(708)Arg(710) --> Glu(708)Glu(710) double mutation produced increases in the amount of ACE2 shed when stimulated by phorbol ester PMA. In summary, we have demonstrated that ADAM17 is able to cleave ACE2 peptide sequence analogues between Arg(708) and Ser(709). These findings also indicate that Arg(708) and Arg(710) play a role in site recognition in the regulation of ACE2 ectodomain shedding mediated by ADAM17.

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