Pigs express multiple forms of decay-accelerating factor (CD55), all of which contain only three short consensus repeats

We report the cloning of cDNAs encoding multiple isoforms of the pig analogue of human decay-accelerating factor (DAF; CD55). Screening of a pig muscle cDNA library using a human DAF probe identified a single clone that encoded a DAF-like molecule comprising three short consensus repeats (SCR) homologous with the amino-terminal three SCR in human DAF, a serine/threonine-rich (ST) region, and sequence compatible with a transmembrane domain and cytoplasmic tail. Northern blot and RT-PCR analysis showed that pig DAF was expressed in a wide range of tissues. Additional isoforms of DAF were sought using RT-PCR and 3'-rapid amplification of cDNA ends followed by sequencing. Isoforms containing a GPI anchor and with differing lengths of ST region were identified; no isoform containing a fourth SCR was found. Cloning of the GPI-anchored isoform from granulocytes confirmed that it was identical with the original transmembrane isoform through the three SCR and first portion of ST and was derived from a frame shift caused by splicing out 176 bp of sequence. A panel of mAbs was generated and used to analyze the distribution and anchoring of pig DAF in circulating cells. Pig DAF was expressed on all circulating cells and was transmembrane anchored on erythrocytes, but completely or partially GPI anchored on granulocytes and mononuclear cells. The transmembrane isoform of pig DAF was expressed on Chinese hamster ovary cells and was shown to affect regulatory activity for the classical pathway of human complement, but was only marginally active against pig serum.     

GPI锚固型Met-RANTES真核表达载体的构建和表达

目的：在仓鼠卵巢细胞（CHO细胞）中高效表达糖基磷脂酰肌醇（GPI） 锚定修饰的Met-RANTES融合蛋白,以研制新型免疫抑制分子GPI锚固型 Met-RANTES。方法：构建真核表达载体PEF/GPI-Met-RANTES,利用电转化法转染CHO-DHFRˉ细胞,氨甲喋呤（MTX）筛选抗性克隆。用流式细胞仪、细胞免疫荧光和免疫金标记电镜检测细胞膜上 GPI 锚固型Met-RANTES融合蛋白的表达情况。结果：构建了阅读框完整的 GPI 锚固型Met-RANTES 嵌合分子,经测序是正确的,并在CHO-DHFRˉ细胞膜上稳定表达。结论：在CHO 细胞表面高效表达GPI修饰的Met-RANTES融合蛋白,GPI 锚固型Met-RANTES分子有可能作为新型的免疫抑制剂用于器官移植中,抑制移植排斥反应。     

Molecular cloning and characterization of novel splicing variants of human decay-accelerating factor

Decay-accelerating factor (DAF) is one of the complement regulatory proteins. Two isoforms of DAF have been identified in humans. In this study, we isolated novel cDNAs encoding five isoforms of DAF from the human lung, which were generated by insertion of new exonic sequences. RT-PCR revealed that all isoforms were expressed in almost all tissues tested, although the expression patterns and levels differed among the tissues. Transfection of isoform vDAF1, 2, and 3 cDNAs into CHO cells showed that these molecules are soluble forms secreted after glycosylation. Isoform vDAF4 and vDAF5 cDNAs included a part of and the entire intron 7 sequence, respectively, and the transfection of vDAF4 cDNA produced a large, glycosylated, membrane-bound form. These results suggest that more than seven isoforms of human DAF are involved in the regulation of complement activation under physiological conditions through their specific structures and localization.     

The glycan processing and site occupancy of recombinant Thy-1 is markedly affected by the presence of a glycosylphosphatidylinositol anchor

Thy-1 is a cell surface glycoprotein containing three N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The effect of the anchor on its N-linked glyco-sylation was investigated by comparing the glycosylation of soluble recombinant Thy-1 (sThy-1) with that of recombinant GPI anchored Thy-1, both expressed in Chinese hamster ovary cells. The sThy-1 was produced in a variety of isoforms including some which lacked carbohydrate on all three sequons whereas the GPI anchored form appeared fully glycosylated like native Thy-1. This was surprising as the attachment of N-linked sugars occurs cotranslationally and it was not expected that the presence of a C-terminal GPI anchor signal sequence would affect sequon occupancy. Furthermore sThy-1 lacking glycosylation could be produced with the inhibitor tunicamycin but in contrast cell surface expression of unglycosylated GPI anchored Thy-1 could not be obtained. The GPI anchored form appeared less processed with almost 4-fold more oligo-mannose oligosaccharides than in sThy-1 and also with less sialylated and core fucosylated biantennary glycans. Possible mechanisms whereby the anchor or the anchor signal sequence, control site occupancy and maturation are discussed.     

Epitope analysis and primary structures of variable regions of anti-human FcepsilonRI monoclonal antibodies, and expression of the chimeric antibodies fused with human constant regions

The structural analysis of monoclonal antibodies (mAbs) against the alpha subunit of the high affinity IgE receptor (FcepsilonRIalpha) is an alternative approach to obtaining information for the design of inhibitors that will block complementary interaction between IgE and FcepsilonRIalpha and to analyzing the various biological effects induced by anti-FcepsilonRIalpha autoantibodies in chronic urticaria. In this study, epitopes for mouse anti-human FcepsilonRIalpha mAbs and primary structures of variable regions of the mAbs were analyzed. Three mAbs inhibitory for IgE-binding reacted to the deletion mutants of FcepsilonRIalpha containing the whole second immunoglobulin-like domain as well as IgE did. On the other hand, two uninhibitory mAbs reacted to those containing the whole first immunoglobulin-like domain. The cDNAs for variable regions of the five mAbs were cloned and sequenced. Two inhibitory mouse/human chimeric antibodies were expressed in COS7 cells and bound to Chinese hamster ovary transfectant cells expressing FcepsilonRI (CHO/alphabetagamma), and these inhibited the binding of IgE to CHO/alphabetagamma cells.     

Effect of glycoinositolphospholipid anchor lipid groups on functional properties of decay-accelerating factor protein in cells.

The inositol ring in the glycoinositolphospholipid (GPI) anchor of human decay-accelerating factor (DAF) is unmodified in nucleated cells, whereas it is fatty acid acylated in erythrocytes (Ehu). To assess the effect of this and of the glycerol sn-2-associated acyl substituent on the abilities of DAF to cell membrane incorporate and function, 1) endogenous (physiologically anchored) DAF proteins bearing three- and two-"footed" GPI anchors were purified from Ehu and HeLa cells and 2) synthetic DAF variants bearing alternative one- "footed" anchors (retaining either the sn-1 glycerol- or inositol-associated lipid) were prepared by alkaline hydroxylamine treatment and phosphatidylinositol-specific phospholipase D digestion of Ehu DAF, respectively. The different DAF species were added to antibody-sensitized sheep erythrocytes (EshA) and their abilities to insert into the plasma membranes of the cells and control subsequent complement activation on their surfaces were compared. DAF proteins bearing all four GPI anchor structures adhered to the Esh hemolytic intermediates and inhibited expression of C3 convertase (C4b2a) activity. However, mixing of DAF-treated EshA with untreated EshAC142 and stripping of cell-associated DAF proteins with vesicles showed that only the physiologically anchored proteins remained stably associated with the lipid bilayer and functioned intrinsically. Both three- and two-"footed" Ehu and HeLa DAF proteins exhibited comparable ability to incorporate and function in the intermediates as well as to accumulate to levels 1000-fold higher/cell in Schistosoma mansoni schistosomula. These findings indicate that 1) an intact inositolphospholipid-containing GPI anchor is necessary for stable membrane integration and intrinsic function, 2) endogenous GPI anchors (with either unsubstituted and acylated inositol) incorporate and function with comparable efficiency, and 3) the transfer of either endogenous DAF form can account for the previously described circumvented uptake of human C3b by blood stage schistosomula.     

The HeLa cell receptor for enterovirus 70 is decay-accelerating factor (CD55).

Enterovirus 70 (EV70) is a recently emerged human pathogen belonging to the family Picornaviridae. The ability of EV70 to infect a wide variety of nonprimate cell lines in vitro is unique among human enteroviruses. The importance of virus receptors as determinants of viral host range and tropism led us to study the host cell receptor for this unusual picornavirus. We produced a monoclonal antibody (MAb), EVR1, which bound to the surface of HeLa cells and protected them against infection by EV70 but not by poliovirus or by coxsackievirus B3. This antibody also inhibited the binding of [35S]EV70 to HeLa cells. MAb EVR1 did not bind to monkey kidney (LLC-MK2) cells, nor did it protect these cells against virus infection. In Western immunoassays and in immunoprecipitations, MAb EVR1 identified a HeLa cell glycoprotein of approximately 75 kDa that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Decay-accelerating factor (DAF, CD55) is a 70- to 75-kDa GPI-anchored membrane protein that is involved in the regulation of complement and has also been shown to function as a receptor for several enteroviruses. MAb EVR1 bound to Chinese hamster ovary (CHO) cells constitutively expressing human DAF. Anti-DAF MAbs inhibited EV70 binding to HeLa cells and protected them against EV70 infection. Transient expression of human DAF in murine NIH 3T3 cells resulted in binding of labelled EV70 and stably, transformed NIH 3T3 cells expressing DAF were able to support virus replication. These data indicate that the HeLa cell receptor for EV70 is DAF.     

Endocytosis of glycophospholipid-anchored and transmembrane forms of CD4 by different endocytic pathways.

A glycophosphatidylinositol (GPI)-anchored form of the CD4 receptor was constructed by fusing the extracellular domain of CD4 to the COOH-terminus of decay accelerating factor (DAF), containing a signal for GPI-anchor attachment. The internalization of GPI-linked CD4 (CD4DAF) was compared to that of transmembrane CD4 using both [125I]gp120 and anti-CD4 antibodies. We show that transmembrane CD4 is rapidly endocytosed in transfected CHO cells, while CD4DAF is internalized at a rate approximately 3-fold slower. Immunoelectron microscopy suggests that whereas transmembrane CD4 is endocytosed via clathrin-coated vesicles, CD4DAF enters cells by an alternative pathway involving non-coated microinvaginations of the plasma membrane. Following internalization CD4DAF recycles through a primaquine-insensitive compartment, whereas the recycling of transmembrane CD4 is inhibited by primaquine, suggesting that the two receptors may recycle from distinct populations of early endosomes. Colocalization of both CD4DAF and CD4 with an antibody against a lysosomal membrane protein suggests that the two endocytic pathways may converge.     

超抗原SEA的GPI锚定修饰和在CHO-dhfr~-细胞膜上的表达

将GPI锚定修饰的免疫分子直接转移到肿瘤细胞膜上,是研究治疗性肿瘤疫苗的一种有潜力的新策略。通过将从衰变加速因子(DAF)来源的GPI修饰性信号序列与SEA嵌合,获得了GPI修饰的SEA分子,构建的真核表达载体pCIGPI-SEA,用脂质体方法转染到CHOdhfr-细胞中,并用氨甲喋呤(MTX)进行筛选,细胞免疫荧光分析证实,SEA能够在细胞膜上表达。上述GPI锚定修饰的SEA,可用于进一步研究治疗性肿瘤疫苗。     

Release of GPI-anchored membrane proteins by a cell-associated GPI-specific phospholipase D.

Although many glycosylphosphatidylinositol (GPI)-anchored proteins have been observed as soluble forms, the mechanisms by which they are released from the cell surface have not been demonstrated. We show here that a cell-associated GPI-specific phospholipase D (GPI-PLD) releases the GPI-anchored, complement regulatory protein decay-accelerating factor (DAF) from HeLa cells, as well as the basic fibroblast growth factor-binding heparan sulfate proteoglycan from bone marrow stromal cells. DAF found in the HeLa cell culture supernatants contained both [3H]ethanolamine and [3H]inositol, but not [3H]palmitic acid, whereas the soluble heparan sulfate proteoglycan present in bone marrow stromal cell culture supernatants contained [3H]ethanolamine. 125I-labeled GPI-DAF incorporated into the plasma membranes of these two cell types was released in a soluble form lacking the fatty acid GPI-anchor component. GPI-PLD activity was detected in lysates of both HeLa and bone marrow stromal cells. Treatment of HeLa cells with 1,10-phenanthroline, an inhibitor of GPI-PLD, reduced the release of [3H]ethanolamine-DAF by 70%. The hydrolysis of these GPI-anchored molecules is likely to be mediated by an endogenous GPI-PLD because [3H]ethanolamine DAF is constitutively released from HeLa cells maintained in serum-free medium. Furthermore, using PCR, a GPI-PLD mRNA has been identified in cDNA libraries prepared from both cell types. These studies are the first demonstration of the physiologically relevant release of GPI-anchored proteins from cells by a GPI-PLD.     

PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG_2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.     

Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil podoplanin

Monoclonal antibodies (mAbs) against not only human, mouse, and rat but also rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. PDPN is used as a lymphatic endothelial cell marker in pathological diagnoses. However, mAbs against Tasmanian devil PDPN (tasPDPN), which are useful for immunohistochemical analysis, remain to be developed. Herein, mice were immunized with tasPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/tasPDPN) cells, and hybridomas producing mAbs against tasPDPN were screened using flow cytometry. One of the mAbs, PMab-233 (IgG₁, kappa), specifically detected CHO/tasPDPN cells by flow cytometry and recognized tasPDPN protein by western blotting. Furthermore, PMab-233 strongly detected CHO/tasPDPN cells by immunohistochemistry. These findings suggest that PMab-233 may be useful as a lymphatic endothelial cell marker of the Tasmanian devil.     

Ligation of human CD46 with purified complement C3b or F(ab')(2) of monoclonal antibodies enhances isoform-specific interferon gamma-dependent nitric oxide production in macrophages

CD46, a complement regulatory protein widely expressed on human cells, serves as an entry receptor for measles virus (MV). We have previously shown that the expression of human CD46 in mouse macrophages restricts MV replication in these cells and enhances the production of nitric oxide (NO) in the presence of gamma interferon (IFN-gamma). In this study, we show that crosslinking human CD46 expressed on the mouse macrophage-like cell line RAW264.7 with purified C3b multimer but not monomer enhances NO production. The enhanced production of NO in response to IFN-gamma was observed again with C3b multimer but not monomer. The augmentation of NO production is human CD46-dependent with a CYT1>CYT2 profile. Thus, the reported MV-mediated NO production, irrespective of whether it is IFN-gamma-dependent or -independent, should be largely attributable to CD46 signaling but not to MV replication. Similar CYT1-dependent augmentation of NO production was reproducible with two CD46 ligating reagents, CD46-specific monoclonal antibodies (mAb) or their F(ab')(2) and MV hemagglutinin (H) and fusion (F) glycoproteins. Co-cultivation of mouse macrophages bearing human CD46 with Chinese hamster ovary (CHO) cells expressing MV H and F enhanced IFN-gamma-induced NO production. Yet, the NO levels induced by F(ab')(2) against CD46 or MV H/F on CHO cells were much lower than those induced by CD46-crosslinking mAb with Fc or MV infection. Removing the cytoplasmic tails of CD46 abrogated the augmentation of NO production triggered by all three stimulators. Thus, the CD46 CYT1 and CYT2 isoforms functionally diverge to elicit innate immune responses, which can be modulated by purified C3b multimer or anti-CD46 mAbs.     

A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells

Glycosyl-phosphatidylinositol- (GPI) anchored proteins contain a large extracellular protein domain that is linked to the membrane via a glycosylated form of phosphatidylinositol. We recently reported the polarized apical distribution of all endogenous GPI-anchored proteins in the MDCK cell line (Lisanti, M. P., M. Sargiacomo, L. Graeve, A. R. Saltiel, and E. Rodriguez-Boulan. 1988. Proc. Natl. Acad. Sci. USA. 85:9557-9561). To study the role of this mechanism of membrane anchoring in targeting to the apical cell surface, we use here decay-accelerating factor (DAF) as a model GPI-anchored protein. Endogenous DAF was localized on the apical surface of two human intestinal cell lines (Caco-2 and SK-CO15). Recombinant DAF, expressed in MDCK cells, also assumed a polarized apical distribution. Transfer of the 37-amino acid DAF signal for GPI attachment to the ectodomain of herpes simplex glycoprotein D (a basolateral antigen) and to human growth hormone (a regulated secretory protein) by recombinant DNA methods resulted in delivery of the fusion proteins to the apical surface of transfected MDCK cells. These results are consistent with the notion that the GPI anchoring mechanism may convey apical targeting information.     

A quantitative method for comparison of expression of alternatively spliced genes using different primer pairs

In this paper we describe a novel two-step method for comparison of expression of alternatively spliced genes by quantitative PCR (QPCR) applying different primer pairs. As a model system we used rat decay accelerating factor (DAF; CD55) mRNA, which comprises three different isoforms: soluble (sDAF), transmembrane (tmDAF) and glycosyl-phosphatidylinositol (GPI) anchored (gpiDAF) forms. The first step was to prepare solid phase specific for each mRNA isoform and purify the three DAF-forms from total RNA. We then assessed amplification efficiency of primer pairs designed to recognise each of the isoforms using equimolar amounts of the three purified DAF mRNAs. The final step in our assay was to compare expression of the three DAF-isoforms in testis by QPCR taking into account the efficiency of their amplification to enable quantification. The RNA capture/QPCR method we described here can be used for quantifying the expression ratios of alternatively spliced mRNAs from a single gene or for direct comparison of expression of different genes.     

Comparison of the N-linked glycans from soluble and GPI-anchored CD59 expressed in CHO cells

The N-linked glycosylation of recombinant human CD59, expressed in Chinese hamster ovary (CHO) cells with and without a membrane anchor, was compared to examine the effect of the anchor on glycan processing. N-Linked glycans were released with peptide-N-glycosidase F (PNGase F) within gel from SDS-PAGE-isolated soluble and glycosylphosphatidylinositol (GPI)-anchored human CD59 expressed in CHO cells. The anchored form contained core-fucosylated neutral and sialylated bi-, tri-, and tetraantennary glycans with up to four N-acetyllactosamine extensions. Exoglycosidase digestions and analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry were used to define the relative amounts of the bi-, tri-, and tetraantennary glycans and to investigate the distribution of N-acetyllactosamine extensions between their antennae. Biantennary structures accounted for about 60% of the glycans, 30% of the triantennary structures, and about 10% of the tetraantennary structures. For tri- and tetraantennary glycans, those with extended antennae were found to be more abundant than those without extensions. The soluble form of CD59, expressed in CHO cells without the GPI anchor signal sequence, consisted almost entirely (97%) of biantennary glycans, of which 81% were unmodified, 17% contained one N-acetyllactosamine extension, and 2% contained two extensions. No compounds with longer extensions were found. A MALDI spectrum of the intact glycoprotein showed a distribution of glycans that matched those released with PNGase F. In addition, the protein was substituted with several small glycans, such as HexNAc, HexNAc-->Fuc, and HexNAc-->HexNAc, probably as the result of degradation of the mature N-linked glycans. The results show that the presence of the anchor increases the extent of glycan processing, possibly as the result of longer exposure to the glycosyltransferases or to a closer proximity of the protein to these enzymes.     

Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells

A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol-phospholipase C (PI-PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI-PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re-expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44-day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins. (c) 1993 John Wiley & Sons, Inc.