Abstract
Many cell surface proteins are anchored to the membrane via a glycosylphosphatidylinositol (GPI) moiety, which is attached to the C terminus of the proteins. The core of the GPI anchor is conserved in all eukaryotes but is modified by various side chains. We cloned a mouse phosphatidylinositol glycan-class N (Pig-n) gene that encodes a 931amino acid protein expressed in the endoplasmic reticulum, which is homologous to yeast Mcd4p. We disrupted the gene in F9 embryonal carcinoma cells. In the Pig-n knockout cells, the first mannose in the GPI precursors was not modified by phosphoethanolamine. Nevertheless, further biosynthetic steps continued with the addition of the third mannose and the terminal phosphoethanolamine. The surface expression of Thy-1 was only partially affected, indicating that modification of the first mannose by phosphoethanolamine is not essential for attachment of GPI anchors in mammalian cells. An inhibitor of GPI biosynthesis, YW3548/BE49385A, inhibited transfer of phosphoethanolamine to the first mannose in mammalian cells but only slightly affected the surface expression of GPI-anchored proteins. Biosynthesis of GPI in the Pig-n knockout cells was not affected by YW3548/BE49385A, and yeast overexpressing MCD4 was highly resistant to YW3548/BE49385A, suggesting that Pig-n and Mcd4p are targets of this drug.
Highlights
Many cell surface proteins are anchored to the membrane via a glycosylphosphatidylinositol (GPI) moiety, which is attached to the C terminus of the proteins
Biosynthesis of GPI in the phosphatidylinositol glycan-class N (Pig-n) knockout cells was not affected by YW3548/BE49385A, and yeast overexpressing MCD4 was highly resistant to YW3548/ BE49385A, suggesting that Pig-n and Mcd4p are targets of this drug
There was no information on chromosomal location of Pig-n in data bases, but human MCD4 homologue has been mapped to chromosome 18q21.2 [29]
Summary
Cells and Reagents—Mouse embryonal carcinoma F9 cells were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal calf serum on 0.1% gelatin-coated dishes. Using a forward primer designed in a vector and a reverse primer, fragments containing the 5Ј region of mouse MCD4 homologue were amplified and cloned from the cDNA library. To obtain genomic clones of Pig-n, we screened 1 ϫ 106 pfu of genomic FIXII library from a mouse 129/SvJ liver (Stratagene) using a 1.2-kb fragment containing the 5Ј region of Pig-n cDNA as a probe. In Vivo Mannose Labeling and Characterization of Glycolipids—F9 cells [106] grown in a gelatin-coated six-well plate overnight were incubated for 1 h in a medium containing 20 mM Hepes/NaOH (pH 7.4), 100 g/ml glucose, 10% dialyzed fetal calf serum, 10 g/ml tunicamycin, and penicillin/streptomycin and incubated in the same medium containing 40 Ci/ml [3H] mannose for 45 min [10]. Growth was determined visually after 5 days of incubation at 30 °C, with survivorship scored as the greatest cell dilution exhibiting growth
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