Sulfate- and sialic acid-containing glycolipids inhibit DNA polymerase α activity

Cynthia Marie G. Simbulan, Takao Taki, Keiko Tamiya-koizumi, Motoshi Suzuki, Ericka Savoysky, Mami Shoji, Shonen Yoshida

Research output: Contribution to journalArticle

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Abstract

The effects of various glycolipids on the activity of immunoaffinity-purified calf thymus DNA polymerase α were studied in vitro. Preincubation with sialic acid-containing glycolipids, such as sialosylparagloboside (SPG), GM3, GM1, and GD1a, and sulfatide (cerebroside sulfate ester, CSE) dose-dependently inhibited the activity of DNA polymerase α, while other glycolipids, as well as free sphingosine and ceramide did not. About 50% inhibition was achieved by preincubating the enzyme with 2.5 μM of CSE, 50 μM of SPG or GM3, and 80 μM of GM1. Inhibition was noncompetitive with both the DNA template and the substrate dTTP, as well as with the other dNTPs. Since the inhibition was largely reversed by the addition of 0.05% Nonidet P40, these glycolipids may interact with the hydrophobic region of the enzyme protein. Apparently, the sulfate moiety in CSE and the sialic acid moiety in gangliosides were essential for the inhibition since neither neutral glycolipids (i.e., glucosylceramide, galactosylceramide, lactosylceramide) nor asialo-gangliosides (GA1 and GA2) showed any inhibitory effect. Furthermore, the ceramide backbone was also found to be necessary for maximal inhibition since the inhibition was largely abolished by substituting the lipid backbone with cholesterol. Increasing the number of sialic acid moieties per molecule further enhanced the inhibition, while elongating the sugar chain diminished it. It was clearly shown that the N-acetyl residue of the sialic acid moiety is particularly essential for inhibition by both SPG and GM3 because the loss of this residue or substitution with a glycolyl residue completely negated their inhibitory effect on DNA polymerase α activity.

Original languageEnglish
Pages (from-to)68-74
Number of pages7
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1205
Issue number1
DOIs
Publication statusPublished - 16-03-1994

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Glycolipids
N-Acetylneuraminic Acid
DNA-Directed DNA Polymerase
Sulfates
Esters
Gangliosides
Ceramides
Galactosylceramides
Sulfoglycosphingolipids
Glucosylceramides
Sphingosine
Enzymes
Sugars
Substitution reactions
Cholesterol
Lipids
Molecules
DNA
Substrates
cerebroside sulfate

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

Cite this

Simbulan, Cynthia Marie G. ; Taki, Takao ; Tamiya-koizumi, Keiko ; Suzuki, Motoshi ; Savoysky, Ericka ; Shoji, Mami ; Yoshida, Shonen. / Sulfate- and sialic acid-containing glycolipids inhibit DNA polymerase α activity. In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular. 1994 ; Vol. 1205, No. 1. pp. 68-74.
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Sulfate- and sialic acid-containing glycolipids inhibit DNA polymerase α activity. / Simbulan, Cynthia Marie G.; Taki, Takao; Tamiya-koizumi, Keiko; Suzuki, Motoshi; Savoysky, Ericka; Shoji, Mami; Yoshida, Shonen.

In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, Vol. 1205, No. 1, 16.03.1994, p. 68-74.

Research output: Contribution to journalArticle

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T1 - Sulfate- and sialic acid-containing glycolipids inhibit DNA polymerase α activity

AU - Simbulan, Cynthia Marie G.

AU - Taki, Takao

AU - Tamiya-koizumi, Keiko

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AU - Yoshida, Shonen

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N2 - The effects of various glycolipids on the activity of immunoaffinity-purified calf thymus DNA polymerase α were studied in vitro. Preincubation with sialic acid-containing glycolipids, such as sialosylparagloboside (SPG), GM3, GM1, and GD1a, and sulfatide (cerebroside sulfate ester, CSE) dose-dependently inhibited the activity of DNA polymerase α, while other glycolipids, as well as free sphingosine and ceramide did not. About 50% inhibition was achieved by preincubating the enzyme with 2.5 μM of CSE, 50 μM of SPG or GM3, and 80 μM of GM1. Inhibition was noncompetitive with both the DNA template and the substrate dTTP, as well as with the other dNTPs. Since the inhibition was largely reversed by the addition of 0.05% Nonidet P40, these glycolipids may interact with the hydrophobic region of the enzyme protein. Apparently, the sulfate moiety in CSE and the sialic acid moiety in gangliosides were essential for the inhibition since neither neutral glycolipids (i.e., glucosylceramide, galactosylceramide, lactosylceramide) nor asialo-gangliosides (GA1 and GA2) showed any inhibitory effect. Furthermore, the ceramide backbone was also found to be necessary for maximal inhibition since the inhibition was largely abolished by substituting the lipid backbone with cholesterol. Increasing the number of sialic acid moieties per molecule further enhanced the inhibition, while elongating the sugar chain diminished it. It was clearly shown that the N-acetyl residue of the sialic acid moiety is particularly essential for inhibition by both SPG and GM3 because the loss of this residue or substitution with a glycolyl residue completely negated their inhibitory effect on DNA polymerase α activity.

AB - The effects of various glycolipids on the activity of immunoaffinity-purified calf thymus DNA polymerase α were studied in vitro. Preincubation with sialic acid-containing glycolipids, such as sialosylparagloboside (SPG), GM3, GM1, and GD1a, and sulfatide (cerebroside sulfate ester, CSE) dose-dependently inhibited the activity of DNA polymerase α, while other glycolipids, as well as free sphingosine and ceramide did not. About 50% inhibition was achieved by preincubating the enzyme with 2.5 μM of CSE, 50 μM of SPG or GM3, and 80 μM of GM1. Inhibition was noncompetitive with both the DNA template and the substrate dTTP, as well as with the other dNTPs. Since the inhibition was largely reversed by the addition of 0.05% Nonidet P40, these glycolipids may interact with the hydrophobic region of the enzyme protein. Apparently, the sulfate moiety in CSE and the sialic acid moiety in gangliosides were essential for the inhibition since neither neutral glycolipids (i.e., glucosylceramide, galactosylceramide, lactosylceramide) nor asialo-gangliosides (GA1 and GA2) showed any inhibitory effect. Furthermore, the ceramide backbone was also found to be necessary for maximal inhibition since the inhibition was largely abolished by substituting the lipid backbone with cholesterol. Increasing the number of sialic acid moieties per molecule further enhanced the inhibition, while elongating the sugar chain diminished it. It was clearly shown that the N-acetyl residue of the sialic acid moiety is particularly essential for inhibition by both SPG and GM3 because the loss of this residue or substitution with a glycolyl residue completely negated their inhibitory effect on DNA polymerase α activity.

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