The functional glycoprotein CD9 is variably acylated: localization of the variably acylated region to a membrane-associated peptide containing the binding site for the agonistic monoclonal antibody 50H.19

Jutta G. Seehafer, Shou-Ching Tang, Joseph R. Slupsky, Andrew R.E. Shaw

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Recent studies have shown that [3H]palmitic acid strongly labels both glycosylated forms (gp22 and gp24) of the signal-initiating cell surface glycoprotein CD9. We performed a two-dimensional limited proteolysis analysis with Staphylococcus aureus V8 proteinase in order to localize the palmitylation sites to final peptides on both glycosylated forms of CD9. Analysis of [3H]leucine- and [3H]amino acid mixture-labeled gp22 delineated 4 final peptides of 11, 8, 7 and 4 kDa. gp24 produced a similar pattern with the exception that the 11 kDa peptide was replaced by an N-glycosylated 13 kDa peptide. Since all four final peptides (total molecular mass of 30 32 kDa) could not be accommodated by a parent molecule of 22 24 kDa, it is likely that one of the final peptides coexists in two differently modified states. Palmitic acid labeled the 11kDa 13 kDa final peptides, and the 7 kDa final peptide, with equal intensity, but was not incorporated into the 4 kDa final peptide, demonstrating that fatty acid is ligated in two distinct regions of the molecule. The 8 kDa final peptide was strongly labeled by [3H]palmitic acid, but only weakly by [3H]eucine. We present evidence that this peptide is derived by further acylation of the region defined by the 7 kDa peptide, and that this occurs in only 15% of the molecules. Palmitic acid is turned over faster at these additional sites, indicating that they may be more accessible to membrane transacylases. Proteolysis of CD9 on the intact cell with papain enabled the highly acylated region to be localized to a membrane-associated fragment which contains the binding site for the agonistic monoclonal antibody 50H.19. The co-localization of a functional domain with a region of variable acylation suggests that acylation events may play a role in the transduction of the signal initiated by interaction of the antibody with CD9.

Original languageEnglish (US)
Pages (from-to)399-410
Number of pages12
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume957
Issue number3
DOIs
StatePublished - Dec 2 1988

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Glycoproteins
Binding Sites
Monoclonal Antibodies
Membranes
Peptides
Palmitic Acid
Acylation
Proteolysis
Molecules
Papain
Membrane Glycoproteins
Molecular mass
Leucine
Staphylococcus aureus
Labels
Signal Transduction
Peptide Hydrolases
Fatty Acids
Amino Acids
Antibodies

Keywords

  • Glycoprotein CD9
  • Membrane glycoprotein
  • Protein acylation
  • Signal transduction

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

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title = "The functional glycoprotein CD9 is variably acylated: localization of the variably acylated region to a membrane-associated peptide containing the binding site for the agonistic monoclonal antibody 50H.19",
abstract = "Recent studies have shown that [3H]palmitic acid strongly labels both glycosylated forms (gp22 and gp24) of the signal-initiating cell surface glycoprotein CD9. We performed a two-dimensional limited proteolysis analysis with Staphylococcus aureus V8 proteinase in order to localize the palmitylation sites to final peptides on both glycosylated forms of CD9. Analysis of [3H]leucine- and [3H]amino acid mixture-labeled gp22 delineated 4 final peptides of 11, 8, 7 and 4 kDa. gp24 produced a similar pattern with the exception that the 11 kDa peptide was replaced by an N-glycosylated 13 kDa peptide. Since all four final peptides (total molecular mass of 30 32 kDa) could not be accommodated by a parent molecule of 22 24 kDa, it is likely that one of the final peptides coexists in two differently modified states. Palmitic acid labeled the 11kDa 13 kDa final peptides, and the 7 kDa final peptide, with equal intensity, but was not incorporated into the 4 kDa final peptide, demonstrating that fatty acid is ligated in two distinct regions of the molecule. The 8 kDa final peptide was strongly labeled by [3H]palmitic acid, but only weakly by [3H]eucine. We present evidence that this peptide is derived by further acylation of the region defined by the 7 kDa peptide, and that this occurs in only 15{\%} of the molecules. Palmitic acid is turned over faster at these additional sites, indicating that they may be more accessible to membrane transacylases. Proteolysis of CD9 on the intact cell with papain enabled the highly acylated region to be localized to a membrane-associated fragment which contains the binding site for the agonistic monoclonal antibody 50H.19. The co-localization of a functional domain with a region of variable acylation suggests that acylation events may play a role in the transduction of the signal initiated by interaction of the antibody with CD9.",
keywords = "Glycoprotein CD9, Membrane glycoprotein, Protein acylation, Signal transduction",
author = "Seehafer, {Jutta G.} and Shou-Ching Tang and Slupsky, {Joseph R.} and Shaw, {Andrew R.E.}",
year = "1988",
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language = "English (US)",
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T1 - The functional glycoprotein CD9 is variably acylated

T2 - localization of the variably acylated region to a membrane-associated peptide containing the binding site for the agonistic monoclonal antibody 50H.19

AU - Seehafer, Jutta G.

AU - Tang, Shou-Ching

AU - Slupsky, Joseph R.

AU - Shaw, Andrew R.E.

PY - 1988/12/2

Y1 - 1988/12/2

N2 - Recent studies have shown that [3H]palmitic acid strongly labels both glycosylated forms (gp22 and gp24) of the signal-initiating cell surface glycoprotein CD9. We performed a two-dimensional limited proteolysis analysis with Staphylococcus aureus V8 proteinase in order to localize the palmitylation sites to final peptides on both glycosylated forms of CD9. Analysis of [3H]leucine- and [3H]amino acid mixture-labeled gp22 delineated 4 final peptides of 11, 8, 7 and 4 kDa. gp24 produced a similar pattern with the exception that the 11 kDa peptide was replaced by an N-glycosylated 13 kDa peptide. Since all four final peptides (total molecular mass of 30 32 kDa) could not be accommodated by a parent molecule of 22 24 kDa, it is likely that one of the final peptides coexists in two differently modified states. Palmitic acid labeled the 11kDa 13 kDa final peptides, and the 7 kDa final peptide, with equal intensity, but was not incorporated into the 4 kDa final peptide, demonstrating that fatty acid is ligated in two distinct regions of the molecule. The 8 kDa final peptide was strongly labeled by [3H]palmitic acid, but only weakly by [3H]eucine. We present evidence that this peptide is derived by further acylation of the region defined by the 7 kDa peptide, and that this occurs in only 15% of the molecules. Palmitic acid is turned over faster at these additional sites, indicating that they may be more accessible to membrane transacylases. Proteolysis of CD9 on the intact cell with papain enabled the highly acylated region to be localized to a membrane-associated fragment which contains the binding site for the agonistic monoclonal antibody 50H.19. The co-localization of a functional domain with a region of variable acylation suggests that acylation events may play a role in the transduction of the signal initiated by interaction of the antibody with CD9.

AB - Recent studies have shown that [3H]palmitic acid strongly labels both glycosylated forms (gp22 and gp24) of the signal-initiating cell surface glycoprotein CD9. We performed a two-dimensional limited proteolysis analysis with Staphylococcus aureus V8 proteinase in order to localize the palmitylation sites to final peptides on both glycosylated forms of CD9. Analysis of [3H]leucine- and [3H]amino acid mixture-labeled gp22 delineated 4 final peptides of 11, 8, 7 and 4 kDa. gp24 produced a similar pattern with the exception that the 11 kDa peptide was replaced by an N-glycosylated 13 kDa peptide. Since all four final peptides (total molecular mass of 30 32 kDa) could not be accommodated by a parent molecule of 22 24 kDa, it is likely that one of the final peptides coexists in two differently modified states. Palmitic acid labeled the 11kDa 13 kDa final peptides, and the 7 kDa final peptide, with equal intensity, but was not incorporated into the 4 kDa final peptide, demonstrating that fatty acid is ligated in two distinct regions of the molecule. The 8 kDa final peptide was strongly labeled by [3H]palmitic acid, but only weakly by [3H]eucine. We present evidence that this peptide is derived by further acylation of the region defined by the 7 kDa peptide, and that this occurs in only 15% of the molecules. Palmitic acid is turned over faster at these additional sites, indicating that they may be more accessible to membrane transacylases. Proteolysis of CD9 on the intact cell with papain enabled the highly acylated region to be localized to a membrane-associated fragment which contains the binding site for the agonistic monoclonal antibody 50H.19. The co-localization of a functional domain with a region of variable acylation suggests that acylation events may play a role in the transduction of the signal initiated by interaction of the antibody with CD9.

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KW - Protein acylation

KW - Signal transduction

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