No glycolipid anchors are added to Thy-1 glycoprotein in Thy-1-negative mutant thymoma cells of four different complementation classes.

Abstract

Recent evidence shows that the mature Thy-1 surface glycoprotein lacks the C-terminal amino acids 113 to 143 predicted from the cDNA sequence and is anchored in the plasma membrane by a complex, phosphatidylinositol-containing glycolipid attached to the alpha-carboxyl group of amino acid 112. Here we studied the biosynthesis of Thy-1 in two previously described and two newly isolated Thy-1-deficient mutant cell lines. Somatic cell hybridization indicated that their mutations affected some processing step rather than the Thy-1 structural gene. The Thy-1 made by mutants of classes C, F, and H bound detergent but, in contrast to wild-type Thy-1, their detergent-binding moieties could not be removed by phospholipase C. In addition, tryptophan, which only occurs in position 124, was incorporated into Thy-1 of these mutants but not of wild-type cells. Last, the Thy-1 of wild-type but not mutant cells could be radiolabeled with [3H]palmitic acid. Together, these findings strongly suggest that mutants of classes C, F, and H accumulate a biosynthetic intermediate of Thy-1 which retains at least part of the hydrophobic C-terminal peptide. The Thy-1 of these mutants remained endoglycosidase H sensitive, suggesting that it accumulated in the rough endoplasmic reticulum or the Cis-Golgi. A different Thy-1 intermediate was found in a class B mutant cell line: the Thy-1 of this mutant was 2 kilodaltons smaller than the Thy-1 of other cell lines, did not bind detergent, and was rapidly secreted via a normal secretory pathway.

Anchoring and degradation of glycolipid-anchored membrane proteins by L929 versus by LM-TK- mouse fibroblasts: implications for anchor biosynthesis.

“…LM-TK-cells have four to five chromosomes fewer than L929. DAP-3 is a variant of this cell line which also lacks adenosine phosphoribosyltransferase activity (30 ., 4~, 4 after chain termination in the RER and involves replacement of the carboxyl-terminal extremity of the primary translation product (2,5,9,19). In the case of AP, the precursor polypeptide has 29 amino acids at the carboxyl terminus which are excised and replaced (38,42).…”

“…Furthermore, scattered observations indicate that when genes or cDNAs encoding GPL-anchored membrane proteins are transfected into LM-TK-cells, little or none of the corresponding proteins are expressed on the cell surface of the transfectants (13,15,28,54,60). Thus, LM-TK-cells may resemble T-lymphoma mutants which transcribe Thy-1 mRNA of normal size and synthesize Thy-1 but express neither Thy-1 nor other lipid-anchored proteins on the cell surface, apparently because they are unable to add such anchors (9,10,16,18,58). The recessive mutant lymphomas fall into six complementation groups, A to C, E, F, and H (26).…”

“…2G) (33). proteins which it synthesizes or transports these proteins to the cell surface (9,17,18). The Thy-l-mutants, therefore, have intracellular Thy-i.…”

Direct evidence of involvement of glycosylphosphatidylinositol‐anchored proteins in the heavy metal‐mediated signal delivery into T lymphocytes

“…Murine thymoma cell line BW5147 of AKR mouse origin and its Thy-1-mutant class A (Thy-l-a) defective in PIG-A [21,22], were donated by Dr. R. Hyman, Salk Institute, through Dr. T. Kinoshita, Osaka Univesrsity. PIG-A-transfected BW5147 Thy-l-a was a kind gift of Drs.…”

Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core. Implications for paroxysmal nocturnal hemoglobinuria.

“…These mutants cannot express GPI-anchored proteins on the cell surface because their incompletely formed GPI-anchor precursors cannot be transferred to proteins (16)(17)(18)(19). In this paper, we characterize a T cell mutant that expressed reduced levels ofGPI-anchored proteins on the cell surface.…”