Construction of synthetic signals for glycosyl-phosphatidylinositol anchor attachment. Analysis of amino acid sequence requirements for anchoring.
Many membrane proteins are anchored to the cell surface through covalent attachment to a glycosyl-phosphatidylinositol (GPI) structure. The GPI anchor is added to proteins in the endoplasmic reticulum following recognition of a signal in the COOH terminus of the protein. We show that the GPI anchoring signal can be completely recreated by the synthetic polymer Ser3-Thr8-Leu14, but not Thr11-Leu14, inserted at the COOH terminus of a protein. This is consistent with previous reports that a small amino acid such as Ser, Gly, or Ala, but not Thr, is required at the GPI attachment site. Analysis of synthetic amino acid sequences established a basic three-part signal for GPI anchoring: a cleavage/attachment domain that requires small amino acids at the first (GPI anchor attachment) and third positions but with little specificity at the middle position, a spacer domain of approximately 8-12 amino acids, and a hydrophobic domain of at least 11 amino acids. The ability to design a totally synthetic GPI anchoring signal will allow precise probing of the fine structure of this signal.
Coyne
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Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
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Lublin
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000138:chemical synthesis / Q000378:metabolism*
D000596:Amino Acids; D015703:Antigens, CD; C497857:CD46 protein, human; D017261:Glycosylphosphatidylinositols; C497859:Mcp protein, mouse; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D021382:Protein Sorting Signals