Demonstration of phosphatidylinositol anchors on Ly-6 molecules by specific phospholipase C digestion and gel electrophoresis in octylglucoside

Hammelburger, J W; Palfree, Roger G. E.; Sirlin, Sonia; Hämmerling, Ulrich

doi:10.1016/s0006-291x(87)80275-9

Cited by 35 publications

(6 citation statements)

References 17 publications

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“…The members of this family are all membrane proteins, attached to the cell membrane by a glycosyl-PtdIns anchor (Hammelburger et al, 1987;Williams et al, 1988;Sawada et al, 1990). This sequence similarity prompted an investigation into whether the glycosyl-PtdIns-anchoring position would be a conserved structural feature.…”

Section: Discussionmentioning

confidence: 99%

Structural requirements for glycosyl‐phosphatidylinositol‐anchor attachment in the cellular receptor for urokinase plasminogen activator

Møller

Ploug

Blasi

1992

European Journal of Biochemistry

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The urokinase-plasminogen-activator receptor (u-PAK) is a glycosyl-phosphatidylinositol(g1y-cosyl-Ptd1ns)-anchored membrane protein. Using site-directed mutagenesis, we have studied features in the u-PAR sequence important for successful glycosyl-PtdIns attachment. Two critical sequence elements were identified. In the sequence Ser282-Gly283-Ala284, simultaneous substitution of all of these residues prevented membrane anchoring. Individual substitution of each of the residues indicated that Gly283 is the more critical residue and the likely attachment site. However, it was unexpectedly found that mutation of this residue gave rise only to a partial impairment of glycosyl-PtdIns attachment. We therefore propose that more than one residue within this sequence can be utilized as glycosyl-PtdIns-attachment site. In the last eight COOH-terminal amino acids encoded in u-PAR cDNA, deletion of this sequence (residues 306 -31 3) completely prevented glycosyl-PtdIns attachment. However, the remaining COOH-terminal region proved still to possess a potential glycosylPtdIns signal activity; it could be converted to a new functional glycosyl-PtdIns signal by substitution of a single positively charged residue (Arg304). Substitution of Arg304 by Leu converted this truntaced u-PAR to a glycosyl-PtdTns-anchored protein. indistinguishable from the wild type. Substitution of Arg304 by a negatively charged residue (Glu) led to a partial aquisition of the glycosyl-PtdInsanchoring ability. These findings show that charged amino acids placed in the COOH-terminus interfere negatively with glycosyl-Ptdlns-anchoring, and, furthermore, that this effect is more pronounced for positively charged than for negatively charged amino acid residues.

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Section: Discussionmentioning

confidence: 99%

Structural requirements for glycosyl‐phosphatidylinositol‐anchor attachment in the cellular receptor for urokinase plasminogen activator

Møller

Ploug

Blasi

1992

European Journal of Biochemistry

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“…The lowest molecular weight species appears to be the result of a proteolytic cleavage at the carboxy terminal end of the protein. The two highest molecular weight forms of TAP (Ly-6A.2) are released from the plasma membrane in an intact and soluble form upon digestion with a phosphatidylinositoi specific phospholipase C (PI-PLC) (Reiser et al 1986b, Hammelburger et al 1987) and alco can be biosynthetically labeled with 'H-palmitic acid (Yeh et al 1988). These results indicate that the TAP (Ly-6A.2) protein is anchored to the cell surface via a phosphatidylinositoi linkage.…”

Section: Biochemical Characterization Of Ly-6 Moleculesmentioning

confidence: 99%

“…The electrophoretic mobility of these bands is not affected by labeling in the presence of tunicamycin, suggesting the presence of little or no N-linked carbohydrate (Palfree & Hammearling 1986). Ly-6E.I is removed from the plasma membrane by PI-PLC (Hammelburger et al 1987). The amino-terminal sequence of the Ly-6E.l protein (LeClair et al 1986) was determined and subsequently found to be highly homologous to the amino acid sequence of the TAP (Ly-6A.2) protein .…”

Section: Biochemical Characterization Of Ly-6 Moleculesmentioning

confidence: 99%

The LY‐6 Locus: A Multigene Family Encoding Phosphatidylinositol‐Anchored Membrane Proteins Concerned with T‐Cell Activation

Rock

Reiser

Bamezai

et al. 1989

Immunological Reviews

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“…Like Ly-6 molecules [4,5], CD59 appears to be anchored in the plasma membrane through a phosphatidylinositolglycan (PI-G) attachment, since it is released by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). The strongest evidence of relatedness to Ly-6 came from the N-terminal sequence data which showed intriguing homology with Ly-6AE and C especially in the first 6 of the 17 amino acids reported.…”

Section: Introductionmentioning

confidence: 99%

The CD59 antigen is a structural homologue of murine Ly‐6 antigens but lacks interferon inducibility

et al. 1990

Self Cite

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A cDNA encoding the human leukocyte antigen CD59 has been isolated from the erythroid cell line K-562 and its identity confirmed through expression in COS cells. Northern blotting reveals three message species of approximately 800, 1400 and 2000 bases in size, which are constitutively expressed in all lymphoid, erythroid, myeloid, and neural cell types tested thus far. Southern blotting of human DNA indicates a pattern consistent with the presence of a single gene, which has been mapped to chromosome 11 by somatic cell hybrids. Also, the finding of a transcriptionally active cross-hybridizing gene in monkey cells suggests conservation of CD59 sequences among primates. Comparison of the CD59 protein sequence with those of the Ly-6E and Ly-6C antigens discloses a similarity in overall structure, including the alignment of abundant cysteine residues, hydrophobic carboxy termini and conservation of amino acids surrounding the proposed phosphatidylinositol-glycan modification site for Ly-6 molecules. Unlike Ly-6, however, CD59 expression does not appear to be inducible with interferons. This, along with its limited homology and different tissue distribution, cast doubt upon the functional equivalence of CD59 and either of the well-characterized mouse Ly-6 proteins.

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Demonstration of phosphatidylinositol anchors on Ly-6 molecules by specific phospholipase C digestion and gel electrophoresis in octylglucoside

Cited by 35 publications

References 17 publications

Structural requirements for glycosyl‐phosphatidylinositol‐anchor attachment in the cellular receptor for urokinase plasminogen activator

Structural requirements for glycosyl‐phosphatidylinositol‐anchor attachment in the cellular receptor for urokinase plasminogen activator

The LY‐6 Locus: A Multigene Family Encoding Phosphatidylinositol‐Anchored Membrane Proteins Concerned with T‐Cell Activation

The CD59 antigen is a structural homologue of murine Ly‐6 antigens but lacks interferon inducibility

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