Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored membrane proteins in intact cells: specific amino acid requirements adjacent to the site of cleavage and GPI attachment

Mutational studies were previously carried out at the omega site intact cells (Micanovic, R., L. Gerber, J. Berger, K. Kodukula, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA. 87:157-161; Micanovic R., K. Kodukula, L. Gerber, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA: 87:7939-7943) and at the omega + 1 and omega + 2 sites in a cell- free system (Gerber, L., K. Kodukula, and S. Udenfriend. 1992. J. Biol. Chem. 267:12168-12173) of nascent proteins destined to be processed to a glycosylphosphatidyl-inositol (GPI)-anchored form. We have now mutated the omega + 1 and omega + 2 sites in placental alkaline phosphatase (PLAP) cDNA and transfected the wild-type and mutant cDNAs into COS 7 cells. Only glycine at the omega + 2 site yielded enzymatically active GPI membrane-anchored PLAP in amounts comparable to the wild type (alanine). Serine was less active and threonine and valine yielded very low but significant activity. By contrast the omega + 1 site was promiscuous, with only proline being inactive. These and the previous studies indicate that the omega and omega + 2 sites of a nascent protein are key determinants for recognition by COOH-terminal signal transamidase. Comparisons have been made to specific requirements for substitution at the -1, -3 sites of amino terminal signal peptides for recognition by NH2-terminal signal peptidase and the mechanisms of NH2 and COOH-terminal signaling are compared.

[1]  P. Goodfellow,et al.  The human Thy-1 gene: structure and chromosomal location. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[2]  G. Vonheijne.  The signal peptide. , 1990 .

[3]  Ikezawa Hiroh,et al.  Purification and properties of phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis , 1980 .

[4]  H. Riezman,et al.  Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane , 1991, Molecular and cellular biology.

[5]  B. Cullen.  Use of eukaryotic expression technology in the functional analysis of cloned genes. , 1987, Methods in enzymology.

[6]  M. Ferguson,et al.  Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. , 1988, Annual review of biochemistry.

[7]  K. Kodukula,et al.  Structural requirements of a nascent protein for processing to a PI-G anchored form: studies in intact cells and cell-free systems. , 1991, Cell biology international reports.

[8]  K. Kodukula,et al.  Biosynthesis of phosphatidylinositol glycan-anchored membrane proteins. Design of a simple protein substrate to characterize the enzyme that cleaves the COOH-terminal signal peptide. , 1991, The Journal of biological chemistry.

[9]  M. Takeya,et al.  LOCALIZATION OF ALKALINE PHOSPHATASE ISOZYMES IN HUMAN CANCER CELL LINES IN VITRO , 1979 .

[10]  B. Dobberstein,et al.  A tripartite structure of the signals that determine protein insertion into the endoplasmic reticulum membrane , 1989, The Journal of cell biology.

[11]  J. Novotný,et al.  Trypanosoma congolense: structure and molecular organization of the surface glycoproteins of two early bloodstream variants. , 1987, Biochemistry.

[12]  Y. Misumi,et al.  Membrane-anchoring domain of rat liver 5'-nucleotidase: identification of the COOH-terminal serine-523 covalently attached with a glycolipid. , 1990, Biochemistry.

[13]  M. G. Low.  The glycosyl-phosphatidylinositol anchor of membrane proteins. , 1989, Biochimica et biophysica acta.

[14]  G. Blobel.  Control of intracellular protein traffic. , 1983, Methods in enzymology.

[15]  I. Caras,et al.  Fusion of sequence elements from non-anchored proteins to generate a fully functional signal for glycophosphatidylinositol membrane anchor attachment , 1991, The Journal of cell biology.

[16]  N. Takami,et al.  Chemical characterization of the membrane-anchoring domain of human placental alkaline phosphatase. , 1988, The Journal of biological chemistry.

[17]  W. McMaster,et al.  Molecular cloning of the major surface antigen of leishmania , 1988, The Journal of experimental medicine.

[18]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[19]  K. Kodukula,et al.  Selectivity of the cleavage/attachment site of phosphatidylinositol-glycan-anchored membrane proteins determined by site-specific mutagenesis at Asp-484 of placental alkaline phosphatase. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[20]  S. Udenfriend,et al.  Site-directed antibodies for probing the structure and biogenesis of phosphatidylinositol glycan-linked membrane proteins: application to placental alkaline phosphatase. , 1988, Analytical biochemistry.

[21]  G. Blobel.  [52] Control of intracellular protein traffic , 1983 .

[22]  R. Mccomb,et al.  Study of optimum buffer conditions for measuring alkaline phosphatase activity in human serum. , 1972, Clinical chemistry.

[23]  S. Udenfriend,et al.  Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[24]  S Udenfriend,et al.  Phosphatidylinositol glycan (PI-G) anchored membrane proteins. Amino acid requirements adjacent to the site of cleavage and PI-G attachment in the COOH-terminal signal peptide. , 1992, The Journal of biological chemistry.

[25]  G von Heijne,et al.  Patterns of amino acids near signal-sequence cleavage sites. , 1983, European journal of biochemistry.

[26]  G. Cross,et al.  Glycolipid anchoring of plasma membrane proteins. , 1990, Annual review of cell biology.

[27]  K. Kodukula,et al.  Selectivity at the cleavage/attachment site of phosphatidylinositol-glycan anchored membrane proteins is enzymatically determined. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[28]  W. Kohr,et al.  Glycophospholipid membrane anchor attachment. Molecular analysis of the cleavage/attachment site. , 1991, The Journal of biological chemistry.

[29]  S. Williams,et al.  Analysis of the signal for attachment of a glycophospholipid membrane anchor , 1989, The Journal of cell biology.

[30]  G. von Heijne.  The signal peptide. , 1990, The Journal of membrane biology.

[31]  S. Udenfriend,et al.  Processing at the carboxyl terminus of nascent placental alkaline phosphatase in a cell-free system: evidence for specific cleavage of a signal peptide. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Y. Gluzman.  SV40-transformed simian cells support the replication of early SV40 mutants , 1981, Cell.

[33]  M. Ferguson,et al.  Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease. , 1990, The Journal of biological chemistry.

[34]  Gunnar von Heijne,et al.  Patterns of Amino Acids near Signal‐Sequence Cleavage Sites , 1983 .