Analysis of T cells in paroxysmal nocturnal hemoglobinuria provides direct evidence that thymic T-cell production declines with age. (65/1715)

Peripheral blood T cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) comprise a mixture of residual normal and glycosylphosphatidylinositol (GPI)-deficient PNH cells. Using multicolor flow cytometry, we demonstrated significant differences between the proportions of naive and memory cells within these populations. PNH T cells comprise mainly naive cells (CD45RA(+)CD45R0(-)), whereas normal T cells in the same patients were predominantly memory (CD45RA(-)CD45R0(+)) cells. Functional analyses showed that GPI-deficient CD45RA(+) T cells can convert to a CD45R0(+) phenotype. We present data from a PNH patient in remission for 20 years who still had significant numbers of GPI-deficient T cells; these showed a normal distribution of naive and memory components. The predominantly naive phenotype of GPI-deficient T cells seen in PNH patients with active disease likely reflects the phenotype of recent normal thymic emigrants. In patients where hematopoiesis was predominantly derived from the PNH stem cell, absolute numbers of both naive PNH CD4(+) cells and CD8(+) cells show an inverse correlation with patient age, implying this age-related decline in T-cell production is secondary to a decrease in thymic activity rather than a stem cell defect.  (+info)

Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. (66/1715)

In Drosophila, plexin A is a functional receptor for semaphorin-1a. Here we show that the human plexin gene family comprises at least nine members in four subfamilies. Plexin-B1 is a receptor for the transmembrane semaphorin Sema4D (CD100), and plexin-C1 is a receptor for the GPI-anchored semaphorin Sema7A (Sema-K1). Secreted (class 3) semaphorins do not bind directly to plexins, but rather plexins associate with neuropilins, coreceptors for these semaphorins. Plexins are widely expressed: in neurons, the expression of a truncated plexin-A1 protein blocks axon repulsion by Sema3A. The cytoplasmic domain of plexins associates with a tyrosine kinase activity. Plexins may also act as ligands mediating repulsion in epithelial cells in vitro. We conclude that plexins are receptors for multiple (and perhaps all) classes of semaphorins, either alone or in combination with neuropilins, and trigger a novel signal transduction pathway controlling cell repulsion.  (+info)

Glycosylphosphatidylinositol-anchor intermediates associate with triton-insoluble membranes in subcellular compartments that include the endoplasmic reticulum. (67/1715)

Glycosylphosphatidylinositol (GPI)-anchored proteins are resistant to solubilization with Triton X-100 at 4 degrees C, and they can be recovered in Triton-insoluble membranes (TIMs) that float to a characteristic buoyant density. Because the GPI structure itself has been shown to target GPI-anchored proteins to TIMs, we investigated the association of GPI-anchor intermediates with TIMs. GPI-anchor biosynthesis involves a pathway of some 10 steps that take place in the endoplasmic reticulum (ER). These intermediates include glucosaminyl-acylphosphatidylinositol [GlcN-(acyl)PI] and later mannosylated GPIs, denoted H6, H7 and H8, that are present not only in the ER but also in other cell compartments, including the plasma membrane. At least two-thirds of the GlcN-(acyl)PI in HeLa D cells and mannosylated GPIs in K562 cells were found in TIMs. Although previous reports have considered TIMs to be derived primarily from the plasma membrane, we recovered TIMs from subcellular fractions enriched in ER membranes. The ER marker calnexin and GPI-anchored proteins as well as N-acetylglucosaminyl-phosphatidylinositol and mannosylated GPIs were present in ER-TIMs. Interestingly, GlcN-PI and H7 were less enriched in ER-TIM than the other GPIs, suggesting that ER-TIMs might reflect a compartmentalization of the GPI-anchor biosynthetic pathway in the ER.  (+info)

Molecular cloning and characterization of a novel repeat-containing Leishmania major gene, ppg1, that encodes a membrane-associated form of proteophosphoglycan with a putative glycosylphosphatidylinositol anchor. (68/1715)

Leishmania parasites secrete a variety of proteins that are modified by phosphoglycan chains structurally similar to those of the cell surface glycolipid lipophosphoglycan. These proteins are collectively called proteophosphoglycans. We report here the cloning and sequencing of a novel Leishmania major proteophosphoglycan gene, ppg1. It encodes a large polypeptide of approximately 2300 amino acids. The N-terminal domain of approximately 70 kDa exhibits 11 imperfect amino acid repeats that show some homology to promastigote surface glycoproteins of the psa2/gp46 complex. The large central domain apparently consists exclusively of approximately 100 repetitive peptides of the sequence APSASSSSA(P/S)SSSSS(+/-S). Gene fusion experiments demonstrate that these peptide repeats are the targets of phosphoglycosylation in Leishmania and that they form extended filamentous structures reminiscent of mammalian mucins. The C-terminal domain contains a functional glycosylphosphatidylinositol anchor addition signal sequence, which confers cell surface localization to a normally secreted Leishmania acid phosphatase, when fused to its C terminus. Antibody binding studies show that the ppg1 gene product is phosphoglycosylated by phosphoglycan repeats and cap oligosaccharides. In contrast to previously characterized proteophosphoglycans, the ppg1 gene product is predominantly membrane-associated and it is expressed on the promastigote cell surface. Therefore this membrane-bound proteophosphoglycan may be important for direct host-parasite interactions.  (+info)

Carboxypeptidase M, a glycosylphosphatidylinositol-anchored protein, is localized on both the apical and basolateral domains of polarized Madin-Darby canine kidney cells. (69/1715)

Carboxypeptidase M, a glycosylphosphatidylinositol-anchored membrane glycoprotein, is highly expressed in Madin-Darby canine kidney (MDCK) cells, where it was previously shown that the glycosylphosphatidylinositol anchor and N-linked carbohydrate are apical targeting signals. Here, we show that carboxypeptidase M has an unusual, non-polarized distribution, with up to 44% on the basolateral domain of polarized MDCK cells grown on semipermeable inserts. Alkaline phosphatase, as well as five other glycosylphosphatidylinositol-anchored proteins, and transmembrane gamma-glutamyl transpeptidase exhibited the expected apical localization. Basolateral carboxypeptidase M was readily released by exogenous phosphatidylinositol-specific phospholipase C, showing it is glycosylphosphatidylinositol-anchored, whereas apical carboxypeptidase M was more resistant to release. In contrast, the spontaneous release of carboxypeptidase M into the medium was much higher on the apical than the basolateral domain. In pulse-chase studies, newly synthesized carboxypeptidase M arrived in equal amounts within 30 min on both domains, indicating direct sorting. After 4-8 h of chase, the steady-state distribution was attained, possibly due to transcytosis from the basolateral to the apical domain. These data suggest the presence of a unique basolateral targeting signal in carboxypeptidase M that competes with its apical targeting signals, resulting in a non-polarized distribution in MDCK cells.  (+info)

Expression of serum amyloid A3 mRNA by inflammatory macrophages exposed to membrane glycoconjugates from Trypanosoma cruzi. (70/1715)

Differential display reverse transcriptase-polymerase chain reaction (PCR) was used to identify genes expressed by murine macrophages exposed to glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes. Among the different PCR product bands identified in the differential display gel, one showed high homology with the serum amyloid A3 protein (SAA3). Northern blot assays showed augmentation of SAA3 mRNA expression by inflammatory macrophages exposed to live trypomastigotes or parasite glycolipids, as compared to unstimulated macrophages. Our results also showed the expression of SAA3 mRNA, in liver and heart from animals in the acute phase of Chagas disease. It is important that expression of SAA3 mRNA was closely associated with tissue parasitism and presence of inflammatory cells. Together, our findings indicate the possible involvement of SAA3 protein on immunopathology of Chagas disease and establish a new infectious disease model to study the pathophysiological role of this acute-phase protein.  (+info)

Characterization of glycosylphosphatidylinositol-linked molecule CD55/decay-accelerating factor as the receptor for antibody SC-1-induced apoptosis. (71/1715)

The human monoclonal antibody SC-1 induces apoptosis of stomach carcinoma cells and is currently used in a clinical Phase II trial. The antibody binds to a target molecule that is preferentially expressed on diffuse- and intestinal-type stomach cancer cells and shows a very restricted expression on other normal and malignant tissues. In this paper, we show that the SC-1 receptor is a stomach carcinoma-associated isoform of CD55 [membrane-bound decay-accelerating factor (DAF)-B] with a relative molecular mass of approximately 82 kDa. The antigenic site of SC-1 is an N-linked carbohydrate residue. Cross-linking of the DAF receptor increases apoptotic activity. SC-1 binding induces tyrosine phosphorylation of three proteins of approximately 60, 75, and 110 kDa, whereas a serine residue of an approximately 35-kDa protein is dephosphorylated. Expression of caspase-3 (CPP32) and caspase-8 (FLICE) is elevated, and activation of these caspases occurs. These data show that a tumor-specific variant form DAF is involved in apoptosis and can be used for adjuvant therapeutical purposes on gastric carcinoma.  (+info)

Partial purification and characterization of Bacillus thuringiensis Cry1A toxin receptor A from Heliothis virescens and cloning of the corresponding cDNA. (72/1715)

Although extensively studied, the mechanism of action of insecticidal Bacillus thuringiensis Cry toxins remains elusive and requires further elucidation. Toxin receptors in the brush border membrane demand particular attention as they presumably initiate the cascade of events leading to insect mortality after toxin activation. The 170-kDa Cry1Ac toxin-binding aminopeptidase from the tobacco budworm (Heliothis virescens) was partially purified, and its corresponding cDNA was cloned. The cDNA encodes a protein with a putative glycosyl phosphatidylinositol anchor and a polythreonine stretch clustered near the C terminus with predicted O-glycosylation. Partial purification of the 170-kDa aminopeptidase also resulted in isolation of a 130-kDa protein that was immunologically identical to the 170-kDa protein, and the two proteins had identical N termini. These proteins were glycosylated, as suggested by soybean agglutinin lectin blot results. Cry1Ac toxin affinity data for the two proteins indicated that the 130-kDa protein had a higher affinity than the 170-kDa protein. The data suggest that posttranslational modifications can have a significant effect on Cry1A toxin interactions with specific insect midgut proteins.  (+info)