Coenzyme Q10 attenuates rat hepatocarcinogenesis via the reduction of CD59 expression and phospholipase D activity.

The current study aimed to test the profile of serum lipids, phospholipase D (PLD) activity, and CD59 expression pattern in rat hepatocellular carcinoma (HCC) after therapeutic treatment with Coenzyme Q10 (CoQ10). Three rat groups were allocated as normal control, untreated HCC, and treated HCC (HCC + CoQ10). The levels of serum α-fetoprotein (AFP) and tumour necrosis factor (TNF)-α were assessed using enzyme-linked immunosorbent assay (ELISA), while proliferating cell nuclear antigen (PCNA) was detected using immunohistochemistry (IHC). Serum lipids, classical (CH50), and alternative (APH50) pathways of complement activation, the liver cell HMG-CoA reductase (HMGCR), and PLD activities were assayed colorimetrically. The protein expression of CD59, scavenger receptor class B type 1 (SRB1), B cell lymphoma-2 (Bcl2), and cleaved Caspase-3 (Casp-3) were detected using western blotting, while the level of serum CD59 (sCD59) was assessed using dot-blot. CoQ10 reduced the cell proliferation, histological alterations, and the levels of AFP and TNF-α but increased lipids, CH50, and sCD59 in serum. In the liver cell, CoQ10 decreased and increased PLD and HMGCR enzyme activities, respectively. In addition, reduction of liver CD59, Bcl2, and SRB1 vs increased cleaved Casp-3 expressions was observed. Statistical correlation indicated an inverse relationship between CH50 and each of CD59 expression and PLD activity after treatment with CoQ10. In conclusion, CoQ10 could protect against rat HCC through increased lipids and the reduction of CD59 expression and PLD activity. SIGNIFICANCE OF THE STUDY: To our knowledge, this study is the first to describe the attenuating effect of antitumour natural product like Coenzyme Q10 (CoQ10) via the reduction of CD59 expression and phospholipase D (PLD) activity. This illustrates the important role of CD59 and PLD in relation to lipids in cancer prevention.

Expression of the CD59 Glycoprotein Precursor is Upregulated in an Estrogen Receptor-alpha (ER-α)-Negative and a Tamoxifen-Resistant Breast Cancer Cell Line In Vitro.

BACKGROUND Breast cancer is the most prevalent cancer and the leading cause of cancer death among women. Tamoxifen (TAM) therapy is one of the most widely and successfully used endocrine treatments for estrogen receptor α (ERα)-positive breast cancer. However, resistance to TAM has been a major challenge. In addition, the mechanisms underlying endocrine resistance remain unclear. Here, we report that CD59, a phosphatidylinositol-anchored glycoprotein, is a candidate resistant gene for TAM therapies. MATERIAL AND METHODS The breast cancer cell line MCF-7, the MCF-10A cell line, and the TAM-resistant breast cancer cell line TAMR-MCF-7 were cultured. The TAMR-MCF-7 cells were transfected with CD59 siRNA and control siRNA. Then, the CD59 glycoprotein precursor expression was detected by reverse transcription­quantitative polymerase chain reaction and western blot analysis. Cell counting kit-8 and flow cytometry assay were performed to examine cell proliferation, cell apoptosis, and cell cycle. In addition, the expressions of Bax, Bcl2, cleaved-caspase-8, cleaved-caspase-6, cleaved-caspase-3, and cleaved-PARP were analyzed by western blot analysis in the TAMR-MCF-7 cells treated with CD59 siRNA. RESULTS In the present study, we found that the CD59 glycoprotein precursor was aberrantly upregulated in the ERα-negative breast cancer MCF-10A cells but not the MCF-7 cells. Furthermore, the CD59 glycoprotein precursor expression was elevated in the TAM-resistant breast cancer cells. Importantly, RNAi-mediated attenuation of CD59 was sufficient to rescue the resistance to TAM in the TAMR-MCF-7 cells. CONCLUSIONS In summary, our results proposed a candidate biomarker for predicting TAM resistance in ERa-positive breast cancer via targeting CD59, therefore it could be a novel therapeutic option.

Mutations in PIGY: expanding the phenotype of inherited glycosylphosphatidylinositol deficiencies.

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitously expressed in the human body and are important for various functions at the cell surface. Mutations in many GPI biosynthesis genes have been described to date in patients with multi-system disease and together these constitute a subtype of congenital disorders of glycosylation. We used whole exome sequencing in two families to investigate the genetic basis of disease and used RNA and cellular studies to investigate the functional consequences of sequence variants in the PIGY gene. Two families with different phenotypes had homozygous recessive sequence variants in the GPI biosynthesis gene PIGY. Two sisters with c.137T>C (p.Leu46Pro) PIGY variants had multi-system disease including dysmorphism, seizures, severe developmental delay, cataracts and early death. There were significantly reduced levels of GPI-anchored proteins (CD55 and CD59) on the surface of patient-derived skin fibroblasts (∼20-50% compared with controls). In a second, consanguineous family, two siblings had moderate development delay and microcephaly. A homozygous PIGY promoter variant (c.-540G>A) was detected within a 7.7 Mb region of autozygosity. This variant was predicted to disrupt a SP1 consensus binding site and was shown to be associated with reduced gene expression. Mutations in PIGY can occur in coding and non-coding regions of the gene and cause variable phenotypes. This article contributes to understanding of the range of disease phenotypes and disease genes associated with deficiencies of the GPI-anchor biosynthesis pathway and also serves to highlight the potential importance of analysing variants detected in 5'-UTR regions despite their typically low coverage in exome data.

Altered Expression Pattern of CD55 and CD59 on Red Blood Cells in Anemia of Chronic Kidney Disease.

OBJECTIVE: The aim of this study was to investigate the expression pattern of CD55 and CD59 on red blood cells (RBCs) in anemic chronic kidney disease (CKD) patients, and factors that might influence their expression. SUBJECTS AND METHODS: Nighty-one adult anemic CKD patients and 80 healthy controls (HCs) were enrolled. Anemic CKD patients were divided into 3 subgroups based on receiving erythropoietin and renal replacement therapies. Flow cytometric analysis of CD55 and CD59 expression was performed on RBCs from blood samples obtained from CKD patients and HCs. RESULTS: CD59 deficiency was significantly higher among CKD patients than HCs (n = 68, 74.7%, vs. n = 13, 16.3%, respectively; p < 0.001). The median proportions of CD55- and CD59-deficient RBCs in CKD patients were significantly higher compared to HCs (0.34 vs. 0.15, and 4.3 vs. 2.0, p < 0.001 and p < 0.001, respectively). The mean fluorescence intensity (MFI) of CD55 and CD59 expression was significantly lower in CKD patients compared to HCs (1.2 vs. 2.8, and 17.0 vs. 20.3, p < 0.04 and p < 0. 001, respectively). The hemoglobin level was inversely correlated with the proportions of CD55- and CD59-deficient RBCs (r = -0.37, p < 0.001, and r = -0.22, p < 0.02, respectively). The number of CD59-deficient patients was significantly different between the 3 subgroups of CKD patients (p = 0.001), and a significant difference was present in the MFI of CD55 and CD59 expression among the 3 subgroups (p = 0.04 and p = 0.03, respectively). CONCLUSION: The expression pattern of CD55 and CD59 on RBCs is altered in anemic CKD patients, which could play a role in the pathogenesis of anemia in CKD.

Generation of induced pluripotent stem cells as a potential source of hematopoietic stem cells for transplant in PNH patients.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH, however, lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore, other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs), characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming, and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation, the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients.

[Immunomorphological aspects of miscarriage at late gestational ages].

AIM: to reveal the morphological features of placentas and to define a role of the lectin pathway for activation of the complement system in the development of premature birth. MATERIAL AND METHODS: A complex morphological study was conducted to examine 37 placentas, 25 of which were obtained from women with clinical signs of threatened miscarriage and 12 placentas from apparently healthy pregnant women. RESULTS: Placental tissue CD59 expression was ascertained to be significantly less in the women with threatened miscarriage than in those having full-term babies. CONCLUSION: Decreased CD59 expression in threatened miscarriage at late gestational ages leads to additional activation of a maternal immune response and serves as a possible predictor for premature birth.

Mutations in the glycosylphosphatidylinositol gene PIGL cause CHIME syndrome.

CHIME syndrome is characterized by colobomas, heart defects, ichthyosiform dermatosis, mental retardation (intellectual disability), and ear anomalies, including conductive hearing loss. Whole-exome sequencing on five previously reported cases identified PIGL, the de-N-acetylase required for glycosylphosphatidylinositol (GPI) anchor formation, as a strong candidate. Furthermore, cell lines derived from these cases had significantly reduced levels of the two GPI anchor markers, CD59 and a GPI-binding toxin, aerolysin (FLAER), confirming the pathogenicity of the mutations.

AMP-activated protein kinase as a promoting factor, but complement and thrombin as limiting factors for acquisition of cytoprotection: implications for induction of accommodation.

Accommodation has been termed as a condition without graft rejection even in the presence of antidonor antibody. We previously reported an in vitro accommodation model, which demonstrated that preincubation of A/B antigen-expressing endothelial cells with anti-A/B antibody resulted in ERK inactivation followed by resistance to complement-mediated cytotoxicity through the induction of complement regulatory genes. However, under the in vivo condition, the effects of complement and coagulation system cannot be ignored. The purpose of this study is to find effective ways to navigate accommodation by exploring the relevant signal transduction. Preincubation with a low level of complement or thrombin failed to induce resistance to complement-mediated cytotoxicity. AMP-activated protein kinase (AMPK) activators such as resveratrol, AICAR and metformin protected endothelial cells against complement-mediated cytotoxicity through the increase in CD55, CD59, haem oxygenase-1 (HO-1) and ferritin heavy chain (ferritin H) genes, all of which were attenuated by AMPKα knock-down. Resveratrol counteracted the inhibitory effect of pretreated complement and thrombin on acquisition of resistance to complement-mediated cytotoxicity through AMPKα. AMPK regulation in endothelial cells could become the potential strategy to induce accommodation in clinical pro-inflammation and pro-coagulation.

Improvement of dolichol-linked oligosaccharide biosynthesis by the squalene synthase inhibitor zaragozic acid.

The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc(2)Man(5) in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells.

Post-transcriptional CD59 gene silencing by siRNAs induces enhanced human T lymphocyte response to tumor cell lysate-loaded DCs.

CD59 is a complement regulatory protein known to prevent the membrane attack complex (MAC) from assembling. To investigate the role of CD59 molecules in human T cell activation in response to exogenous antigens, gene silencing via small interfering RNAs (siRNAs) was carried out. Subsequent T cell activation in response to both autologous dendritic cells (DCs) loaded with tumor lysate and beads coated with anti-CD3, anti-CD28 and anti-CD59 antibodies was investigated. The findings demonstrated that decreased CD59 expression on T cells significantly enhanced activation and proliferation of CD4(+) T cells and CD8(+) T cells while the expansion of CD4(+) CD25(+) regulatory T cells (Tregs) was not affected, and CD59 mediated inhibition of T cell activation requires the binding of CD59 with its ligand on antigen-presenting cells (APCs). The data support that CD59 down-regulates antigen-specific activation of human T lymphocytes in a ligand-dependent manner.

Upregulating CD59: a new strategy for protection of neurons from complement-mediated degeneration.

An increasing number of studies have shown a critical role for the membrane attack complex, synthesized on activation of the terminal pathway of the complement system, in causing demyelination and neuronal death in neurodegeneration. The aim of this study was to develop a strategy to increase the resistance of neurons to complement damage by modulating the expression of membrane complement regulatory protein CD59, the only inhibitor of the terminal pathway of the complement cascade. We exploited our recent finding that CD59 expression is regulated by the neural-restrictive silencer factor (REST) and designed a novel REST-derived peptide (REST5) containing the nuclear localization domain of the wild-type protein. The effect of REST5 and the mechanism by which it modulates CD59 expression were modelled in neuroblastoma cells transfected with expression constructs, and then confirmed in human neurons differentiated from neural progenitor cells. REST5 increased the expression of CD59 in neurons by fivefold and protected them from complement-mediated lysis spontaneously triggered by neurons. As a source of complement, we used either human serum or conditioned medium from primary human oligodendroglia. This study brings new insight into immunopharmacological research that may serve to inhibit neuronal death triggered by the terminal pathway of complement activation.

Correlation of donor-specific antibodies, complement and its regulators with graft dysfunction in cardiac antibody-mediated rejection.

Antibody-mediated rejection (AMR) is an immunopathologic process in which activation of complement often results in allograft injury. This study correlates C4d and C3d with HLA serology and graft function as diagnostic criteria for AMR. Immunofluorescence staining for C4d and C3d was performed on 1511 biopsies from 330 patients as part of routine diagnostic work-up of rejection. Donor-specific antibodies were detected in 95% of those with C4d+C3d+ biopsies versus 35% in the C4d+C3d- group (p = 0.002). Allograft dysfunction was present in 84% in the C4d+ C3d+ group versus 5% in the C4d+C3d- group (p < 0.0001). Combined C4d and C3d positivity had a sensitivity of 100% and specificity of 99% for the pathologic diagnosis of AMR and a mortality of 37%. Since activation of complement does not always result in allograft dysfunction, we correlated the expression pattern of the complement regulators CD55 and CD59 in patients with and without complement deposition. The proportion of patients with CD55 and/or CD59 staining was highest in C4d+C3d- patients without allograft dysfunction (p = 0.03). We conclude that a panel of C4d and C3d is diagnostically more useful than C4d alone in the evaluation of AMR. CD55 and CD59 may play a protective role in patients with evidence of complement activation.

Mouse CD4+ CD25+ T regulatory cells are protected from autologous complement mediated injury by Crry and CD59.

Self cells depend on surface complement regulators to protect them from autologous complement mediated attack. CD4(+)CD25(+)foxp3(+) T regulatory (Treg) cells are critical in maintaining immune homeostasis, however, which complement regulators are expressed on them and how they are protected from autologous complement attack remains unknown. We report here that mouse Treg cells express virtually no DAF or CR1. Instead, all of them express Crry and approximately half of them express CD59. Both Crry(-/-) and CD59(-/-) Treg cells exhibit greater complement mediated injury than WT Treg cells. These results clarify the status of cell surface complement regulators on mouse Treg cells and indicate that both Crry and CD59 are required to protect Treg cells from autologous complement mediated injury. Additionally, these data also argue that different from previous assumption, at least in mice, CD4(+)CD25(+)foxp3(+) Treg cells are not homogenous and could be further divided into subgroups based on CD59 expression.

Expression levels of CD47, CD35, CD55, and CD59 on red blood cells and signal-regulatory protein-alpha,beta on monocytes from patients with warm autoimmune hemolytic anemia.

BACKGROUND: Animal models have shown that CD47-deficient mice develop severe autoimmune hemolytic anemia (AIHA) because the binding of red blood cell (RBC) CD47 to signal-regulatory protein (SIRP-alpha) on macrophages contributes to the inhibition of phagocytosis. In contrast, complement-inhibitory proteins such as CD35, CD55, and CD59 may protect RBCs against the lysis by complement. STUDY DESIGN AND METHODS: With the use of flow cytometric analyses, the expression of CD47, CD35, CD55, and CD59 on RBCs and of SIRP-alpha,beta on peripheral monocytes of 36 patients with warm AIHA (wAIHA; 23 with active wAIHA, 13 with wAIHA in remission) and 20 healthy subjects was evaluated. RESULTS: The mean fluorescence intensities (MFIs) of the expression of CD47, CD35, CD55, and SIRP-alpha,beta of active wAIHA patients, wAIHA in remission, and healthy subjects were not statistically different. Patients with active wAIHA showed significantly lower CD59 expression on RBCs than healthy individuals (MFI, 512.5 +/- 59.6 vs. 553.7 +/- 36.6; p = 0.009), while the CD59 expression in patients with wAIHA in remission was not significantly different from that of healthy controls (MFI, 538.4 +/- 48.3 vs. 553.7 +/- 36.6; p > 0.05). The expression of CD59 on RBCs of 3 patients who died from the wAIHA was lower than that seen on RBCs of healthy controls (MFI, 433.6 +/- 69.6 vs. 553.74 +/- 36.6; p = 0.0001). CONCLUSIONS: Our data show that the expression of CD47 on RBCs and SIRP-alpha,beta on monocytes of patients with wAIHA is not different from that seen in healthy individuals. In addition, we detected that patients with active wAIHA present low expression of CD59 and normal expression of CD35 and CD55 on their RBCs. Complement-regulatory proteins may play an important role in protecting RBC destruction through the activation of complement.

Paroxysmal nocturnal hemoglobinuria may cause retinal vascular occlusions.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the classic triad of haemolytic anaemia, thrombophilia and cytopenia with the majority of cases occurring in adulthood. PNH constitutes a nonmalignant clonal disease of hematopoietic stem cells harboring somatic mutations in the X-linked phosphatidyl inositol glycan complementation group-A (PIG-A) gene. METHODS: We report for the first time retinal venous vascular occlusion as the primary manifestation of PNH. A patient of untypical age for retinal vascular occlusions presented with a history of 4 weeks of progressive reduction in visual acuity. RESULTS: The screening tests for thrombophilia were not successful. However, elevated LDH was detected, leading to the diagnosis of PNH. CONCLUSIONS: To date, no report shows retinal vascular occlusion as the primary symptom leading to the diagnosis PNH. This article describes, for the first time, that this rare disease needs to be considered in the differential diagnosis of retinal vascular occlusions.

Relating GPI-Anchored Ly6 Proteins uPAR and CD59 to Viral Infection.

The Ly6 (lymphocyte antigen-6)/uPAR (urokinase-type plasminogen activator receptor) superfamily protein is a group of molecules that share limited sequence homology but conserved three-fingered structures. Despite diverse cellular functions, such as in regulating host immunity, cell adhesion, and migration, the physiological roles of these factors in vivo remain poorly characterized. Notably, increasing research has focused on the interplays between Ly6/uPAR proteins and viral pathogens, the results of which have provided new insight into viral entry and virus-host interactions. While LY6E (lymphocyte antigen 6 family member E), one key member of the Ly6E/uPAR-family proteins, has been extensively studied, other members have not been well characterized. Here, we summarize current knowledge of Ly6/uPAR proteins related to viral infection, with a focus on uPAR and CD59. Our goal is to provide an up-to-date view of the Ly6/uPAR-family proteins and associated virus-host interaction and viral pathogenesis.

Upregulated expression of complement inhibitory proteins on bladder cancer cells and anti-MUC1 antibody immune selection.

Membrane complement inhibitors (CD46, CD55 and CD59) are upregulated in some human cancers indicating that they play a role in immune evasion. We investigated complement inhibitor expression in bladder cancer and examined the hypothesis that selective pressure of an antibody response (anti-MUC1) results in the upregulated expression of complement inhibitors on tumor cells. Paired samples of tumor and normal tissue from 22 bladder cancer patients were analyzed for expression of MUC1, CD46, CD55 and CD59, and matched serum samples analyzed for anti-MUC1 IgM and IgG levels. Relationships between anti-MUC1 antibody levels and complement inhibitor expression were investigated. MUC1 mRNA was upregulated in 86% of tumor samples. CD46 was upregulated in 77%, CD55 in 55% and CD59 in 59% of tumors. Low titer anti-MUC1 IgM was detected in normal human sera, but was elevated in 41% of the bladder cancer patients. Anti-MUC1 IgG was virtually absent from normal sera, but present in 32% of the cancer patients. There was a direct relationship between anti-MUC1 antibody titer and expression level of complement inhibitors. Analysis of the correlation of each antibody with the expression of each complement inhibitor by Spearman's rank test revealed a strong correlation between both anti-MUC1 IgM and IgG levels and increased expression of CD46 and CD55, and combined anti-MUC1 IgM/IgG levels correlated with increased expression of all 3 complement inhibitors. In conclusion, the data demonstrate upregulated complement inhibitor expression and the presence of an anti-MUC1 antibody response in bladder cancer patients and support the hypothesis of antibody-mediated immune selection.

Expression of complement regulatory proteins in accommodated xenografts induced by anti-alpha-Gal IgG1 in a rat-to-mouse model.

Anti-graft antibodies are often associated with graft rejection. Under special conditions, grafts continue to function normally even in the presence of anti-graft antibodies and complement. This condition is termed accommodation. We developed a xenograft accommodation model in which baby Lewis rat hearts are transplanted into Rag/GT-deficient mice, and accommodation is induced by repeated i.v. injections of low-dose anti-alpha-Gal IgG(1). The accommodated grafts survived a bolus dose of anti-alpha-Gal IgG(1), while freshly transplanted second grafts were rejected. To study the mechanism of anti-alpha-Gal IgG(1)-mediated accommodation, both real-time PCR and immunohistochemical staining revealed elevated expression of DAF, Crry and CD59 in the accommodated grafts. In vitro exposure of rat endothelial cells to anti-alpha-Gal IgG(1) also induced the up-regulation of DAF, Crry and CD59, as revealed by Western blot analyses, and was associated with an acquired resistance to antibody and complement-mediated lysis in vitro. Collectively, these studies suggest that the up-regulation of complement regulatory proteins may abrogate complement-mediated rejection and permit the development of xenograft accommodation.

Mammalian PIG-X and yeast Pbn1p are the essential components of glycosylphosphatidylinositol-mannosyltransferase I.

Within the endoplasmic reticulum (ER), mannoses and glucoses, donated from dolichol-phosphate-mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using dolichol-phosphate-monosaccharide.

Cloning of a CD59-like gene in rainbow trout. Expression and phylogenetic analysis of two isoforms.

CD59, the major inhibitor of the complement membrane attack complex, is an 18-20 kDa glycoprotein, linked to the membrane via a glycosylphosphatidylinositol (GPI)-anchor. It restricts binding of C9 to the C5b-8 complex, preventing the formation of the complement membrane attack complex C5b-9. In this study we report the cloning of a second CD59-like gene in the rainbow trout, Oncorhynchus mykiss (referred to as CD59-2 and the previously deposited trout CD59-like gene as CD59-1). Trout CD59-2 is 56% identical to CD59-1 at the amino acid level. Both of trout CD59s show the highest identity score (54%) with putative CD59-like molecules from other teleost, and the overall identity with their mammalian orthologs is less than 30%. Trout CD59s are expressed in brain, heart, intestine, kidney, liver and spleen. Particularly, CD59-2 is abundant in trout brain, while CD59-1 seems to be absent in the trout spleen. Moreover, both of trout CD59 genes seems to be present as a single copy in trout genome.