Expression cloning of genes for GPI-anchor biosynthesis

Cloning genes for glycosylphosphatydilinositol (GPI)-anchor biosynthesis is important to further understand its mechanisms and regulation. We have been using expression cloning methods in which a cDNA library was transfected into GPI-anchor-deficient mutant cells. The transfectants which restored surface expression of GPI-anchored proteins were isolated and the plasmids were rescued. In this way we previously cloned cDNAs of genes for complementation classes A and F, and named them PIG-A and PIG-F, respectively. In the present study we have cloned the gene for class B, termed PIG-B. In each case we used different methods. For cloning PIG-A cDNA we used a cDNA library made with an Epstein-Barr-virus-based vector and human class A mutant JY5 which expresses EBNA-1 protein. The EBNA-1 protein allows stable replication of oriP-containing plasmids in the episomal form. For cloning PIG-F cDNA we chose a transient expression method and cotransfected a human T-cell cDNA library made with a vector bearing an origin of replication of polyoma virus with a plasmid bearing polyoma virus large T into the class F murine thymona mutant. This cotransfection strategy was unsuccessful for cloning PIG-B due to low transfection efficiency of the class B thymoma mutant SIA-b. Thus, we first established large T-expressing SIA-b cells and then transfected them with cDNA library. PIG-B cDNA restored the surface expression of Thy-1 on SIA-b cells and also synthesis of mature type GPI-anchor precursors in these cells. The cDNA consists of 1929 bp and codes for a putative new protein of 554 amino acid residues

The developmental regulation and biosynthesis of GPI-related structures in Leishmania parasites

Most macromolecules on the surface of Leishmania parasites, including the major surface proteins and a complex lipophosphoglycan (LPG) are anchored to the plasma membrane via GPI glycolipids. Free glycoinositol-phospholipids (GIPLs) which are not linked to protein or phosphoglycan are also abundant in the plasma membrane. From structural and metabolic labeling studies it is proposed that most Leishmania species express three distinct pathways of GPI biosynthesis. Some of these pathways (i.e those involved in the protein and LPG anchor biosynthesis) are down-regulated during the differentiation of the insect (promastigote) stage to the mammalian (amastigote) stage. In contrast, the GIPLs are expressed in high copy number in both developmental stages. Based on analysis of the lipid moieties of the different GPI species it is possible that the pathways of GPI anchor and GIPL biosynthesis are located in different subcellular compartments. The relative flux through the GIPL and LPG biosynthetic pathways has been examined in L. Major promastigotes. These studies showed that while the rate of synthesis of the GIPLs and LPG is similar, LPG is shed more rapidly from the plasma membrane and has a higher turnover. The possible metabolic relationship between the GIPL and LPG biosynthetic pathways is discussed