Cloning of murine glycosyl phosphatidylinositol anchor attachment protein, GPAA1

Y. Hiroi1, R. Chen2, H. Sawa3, T. Hosoda1, S. Kudoh1, Y. Kobayashi3, H. Aburatani1, K. Nagashima3, R. Nagai1, Y. Yazaki1, M. E. Medof2, and I. Komuro1

1 Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo 113-8655; and 3 Department of Pathology, University of Hokkaido School of Medicine, Sapporo, Japan; and 2 Institute of Pathology, Case Western Reserve University, Cleveland, Ohio


    ABSTRACT
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INTRODUCTION
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Glycosyl phosphatidylinositols (GPIs) are used to anchor many proteins to the cell surface membrane and are utilized in all eukaryotic cells. GPI anchoring units are attached to proteins via a transamidase reaction mediated by a GPI transamidase complex. We isolated one of the components of this complex, mGPAA1 (murine GPI anchor attachment), by the signal sequence trap method. mGPAA1 cDNA is about 2 kb in length and encodes a putative 621 amino acid protein. The mGPAA1 gene has 12 small exons and 11 small introns. mGPAA1 mRNA is ubiquitously expressed in mammalian cells, and in situ hybridization analysis revealed that it is abundant in the choroid plexus, skeletal muscle, osteoblasts of rib, and occipital bone in mouse embryos. Its expression levels and transamidation efficiency decreased with differentiation of embryonic stem cells. The 3T3 cell lines expressing antisense mGPAA1 failed to express GPI-anchored proteins on the cell surface membrane.

GPAA1; GAA1; glycosyl phosphatidylinositol; anchor; transamidase


    INTRODUCTION
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INTRODUCTION
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TO OBTAIN NEW MOLECULES that play important roles in the development of heart, we tried the signal sequence trap method using developing embryonic stem (ES) cells (32). We isolated some novel clones, and one of them was a homologue of yeast glycosyl phosphatidylinositol (GPI) anchor attachment (GAA1) (8).

GPI attachment is a general mechanism for anchoring proteins to the cell surface membrane. This anchoring system is used by all mammalian cell types (31, 34, 36) as well as lower eukaryotes including yeast (9) and protozoa (7, 19, 22, 33). GPI anchors share a common core structure consisting of an ethanolamine phosphate, three mannose residues, and a nonacetylated glucosamine linked to phosphatidylinositol. The mechanism of mammalian GPI biosynthesis has been studied using mutant cells that are defective in different steps of the biosynthetic pathway. Many of the affected genes underlying the defects have been isolated, including PIG (phosphatidylinositol glycan)-A (24), PIG-B (30), PIG-C (12), PIG-F (11), PIG-H (13), PIG-L (25), and GPI1 (35).

Anchor incorporation into proteins occurs in a concerted reaction in which preassembled GPI donors are substituted for specific COOH-terminal signal sequences in nascent polypeptides after NH2-terminal processing of these polypeptides in the endoplasmic reticulum (7, 34). In contrast to the above mutants, all of which are defective in GPI assembly, in class K mutant cells the complete GPI donor is formed, but a defect in the transfer of this donor to proproteins is responsible for its inability to display GPI-anchored proteins (5, 24).

Several of the affected genes in yeast mutants that fail to express GPI-anchored proteins have been identified (3, 17, 18). Among these mutants, the two affected genes, GAA1 and GPI8, have been isolated and characterized. GAA1 and GPI8 are yeast mutants that can synthesize the complete GPI precursor but fail to express GPI-anchored proteins on the cell surface (2, 8). Their phenotypes are similar to that of the mammalian class K mutant for which a defect in the activity of the transamidase has been documented (4, 24). The affected gene in mutant K cells, the PIG-K gene, recently has been shown to be hGPI8 (human GPI8), a homologue of yGPI8 (yeast GPI8) (37).

In this study, we isolated murine GPAA1 cDNA and the murine GPAA1 gene, examined its expression, and characterized GPAA1 protein function.


    METHODS
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INTRODUCTION
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The signal sequence trap method and murine cDNA library screening. The signal sequence method was performed as described (10, 32). We used poly(A)+ RNA from embryoid bodies (EB) that had been differentiated for six days. We screened ~1.0 × 106 plaques of a murine heart cDNA library in Uni-ZAP XR Vector (Stratagene) with a 32P-labeled cDNA fragment of clone 82D3 that appeared to be a new gene. Hybridization was performed at 42°C for 12 h in 2× sodium chloride-sodium citrate (SSC), 50% deionized formamide, 10% dextran sulfate, 1% SDS, and 20 mg/ml heat-denatured salmon sperm DNA. The filters were washed twice with 2× SSC/0.1% SDS, once with 1× SSC/0.1% SDS, and twice with 0.1× SSC/0.1% SDS at 65°C. Plasmids were excised from the phage vectors in vivo by the ExAssist helper phage (Stratagene). Nucleotide sequences of clones were determined on both strands using the fluorescent dideoxy terminator sequencing kit (Perkin-Elmer).

Murine genomic library screening. Approximately 1.0 × 106 plaques of a murine 129SV genomic library in lambda FIXII Vector (Clontech) were screened with 32P-labeled mGPAA1 (murine GPI anchor attachment) cDNA. Phage DNA was purified from the isolated clone, digested with EcoR I, and subcloned into the EcoR I site of pBluescript II SK(+). This plasmid was digested with several restriction enzymes to produce a restriction enzyme map. About 8.9 kb of nucleotide sequence was determined on both strands.

Northern blot analysis. Total RNA was extracted from murine fetal, neonatal, and adult tissues with RNAzol B (Biotecx Laboratories). Twenty micrograms of total RNA were separated on 1.0% formaldehyde-agarose gel and transferred to Hybond-N filters (Amersham). The filters were hybridized with 32P-labeled mGPAA1 cDNA at 42°C for 12 h in 5× sodium chloride-sodium phosphate-EDTA, 50% formamide, 5× Denhardt's solution, 4% dextran sulfate, 0.5% SDS, and 20 mg/ml heat-denatured salmon sperm DNA. The filters were washed twice with 2× SSC/0.1% SDS, once with 1× SSC/0.1% SDS, and twice with 0.1× SSC/0.1% SDS at 42°C.

Decay accelerating factor expression and miniPLAP in vitro translation assay of the development of ES cells. Trypsinized ES cells were cultured in bacterial dishes in medium without leukemia inhibitory factor (LIF), feeding every other day. Cells (2.5 × 105) were harvested at 3, 6, and 9 days. Cells were stained for surface decay-accelerating factor (DAF) expression using rat anti-murine DAF monoclonal antibody (MAb) 2C6 [generously provided by Dr. Paul Morgan (Cardiff)] and FITC-labeled goat anti-rat immunoglobulin (Pharmingen). Stained cells were analyzed in a FACScan (Becton Dickinson) flow cytometer (5). Cells were stained without and with prior incubation with recombinant Bacillus thuringensis phosphatidyl inositol-specific phospholipase C (PI-PLC) (5). Rough microsomes and the miniPLAP in vitro translation assay were performed as described before (4).

In situ hybridization analysis. The in vivo expression of mGPAA1 mRNA was examined by in situ hybridization (ISH) as described previously (27, 29). For this purpose a 1,014-bp Sma I fragment of mGPAA1 cDNA was subcloned into the Sma I site of pBluescript II SK(+). Plasmids with the insert in both directions were prepared, and sense and antisense RNAs were generated by T7 RNA polymerase. After death, whole fetal mice were immediately fixed with 4% paraformaldehyde in 100 mM phosphate buffer at 4°C overnight. Consecutive sections of fetal mice were used for analysis. Tissue sections on silane-coated glass slides were fixed for 15 min at room temperature with 4% paraformaldehyde in 100 mM phosphate buffer and treated for 30 min at 37°C with 10 mg/ml proteinase K [in 10 mM Tris·HCl (pH 8.0), 1 mM EDTA]. The sections were then incubated for 10 min at room temperature in 4% formaldehyde in 100 mM phosphate buffer, 0.2 M HCl, and 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). After these treatments, the sections were washed with phosphate buffer (pH 7.4) and gradually dehydrated in ethanol. Hybridization was carried out for 20 h at 50°C using 10 ng/ml tRNA, 0.05% heparin, 0.1% BSA, and 1% SDS. Subsequently, the sections were washed, and the ISH signals were detected by staining for overnight at room temperature with the alkaline phosphatase substrate solution of nitro blue tetrazolium chloride (Boehringer Mannheim). Each slide was examined by three experienced pathologists.

Isolation and analysis of 3T3 cell lines expressing antisense mGPAA1. We subcloned the 5'-terminal 550-bp fragment of mGPAA1 cDNA into pMAM2-BSD vector (Kaken Seiyaku, Japan) in an inverted direction: this fragment contains a dexamethazone-inducible promoter and a blasticidin-resistant gene (15). We transfected 3T3 cells with this construct and selected them with 8 µg/ml of blasticidin. Total RNA was extracted from isolated clones cultured in the medium containing 1 µg/ml dexamethasone. Expression of 550-base antisense RNA was detected by Northern blot analysis with 32P-labeled mGPAA1 cDNA. Parental 3T3 cell lines and selected clones were stained with anti-Ly6 or CD24 monoclonal antibody/FITC-conjugated anti-mouse immunoglobulin. They were analyzed on the FACScan flow cytometer.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Isolation of murine GPAA1 cDNA. We obtained 36 positive plasmids from the EB library using the signal sequence method. The deduced amino acid sequence of one clone, 82D3, showed no homologies to any known sequences except yeast Gaa1 protein (yGaa1p) (10). When we screened a murine heart cDNA library with the 82D3 cDNA fragment, we obtained mGPAA1 cDNA and determined its nucleotide sequence (Fig. 1). mGPAA1 cDNA was ~2 kb in length, a size almost the same as that of the mRNA transcript detected in Northern blot analysis. The sequence around the predicted translation initiation site (CGCCATGGG) was consistent with the Kozak (16) consensus sequence. The overall sequence showed a long open reading frame encoding 621 amino acids. In the 3'-terminal untranslated region, there was a polyadenylation signal (AATAAA) 26 bp upstream of the poly(A) tract. Computer analysis showed that the deduced protein had a 47-amino acid signal sequence at its NH2 terminus (26), one cAMP- and cGMP-dependent protein kinase phosphorylation site, seven protein kinase C (PKC) phosphorylation sites, two putative N-glycosylation sites, and eight putative transmembrane domains. The overall deduced amino acid sequence was 91% identical to that of hGpaa1p (human Gpaa1 protein) (10) and 25% identical and 57% homologous to that of yGaa1p (8).


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Fig. 1.   DNA and amino acid sequences of mGPAA1. The nucleotide sequence of mGPAA1 cDNA is shown with nucleotide positions indicated on the right. The deduced amino acid sequence of mGPAA1 is shown in single-letter code with the amino acid numbers on the left. The putative 8-transmembrane domains are underlined. Two N-linked glycosylation sites are bold. A phosphorylation site by a cAMP- and cGMP-dependent protein kinase is bold, and protein kinase C phosphorylation sites are indicated with asterisks. Four leucins in leucin zipper pattern are indicated by italics. A polyadenylation signal is doubly underlined. The European Molecular Biology Laboratory (EMBL), GenBank, and DNA Data Bank of Japan (DDBJ) accession number is AB006970.

Genomic structure of mGPAA1 gene. We recovered only a single positive clone from a murine genomic library. This clone contained about 16 kb DNA, and it was confirmed by Southern blot analysis using 32P-labeled mGPAA1 cDNA. Restriction enzyme mapping showed that this clone had a 3.6-kb BamH I fragment. This BamH I fragment was the same length as a single positive band obtained by BamH I digestion of murine genomic DNA Southern blot analysis (data not shown). To confirm that the BamH I fragment contains mGPAA1 genomic DNA and to determine the exact exon and intron locations, we determined nucleotide sequence of an 8.9-kb fragment encompassing the BamH I fragment. The analysis revealed that the mGPAA1 gene has 12 small exons and 11 small introns and that the BamH I fragment contains all of the exons and introns (Fig. 2). A putative translation initiation site is present in the exon 1 and a termination site in exon 12. All exon-intron boundary sequences except the intron 8 sequence are in accordance with the GT-AG rule (Table 1).


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Fig. 2.   mGPAA1 gene restriction map and exon-intron structure. mGPAA1 gene consists of 12 small exons and 11 small introns. A putative transcription initiation site is present in exon 1 and the termination signal in exon 12. All exons are included in the 3.6-kb BamH I fragment. Approximately 8.9-kb genomic DNA sequence containing this BamH I fragment was determined. The EMBL, GenBank, and DDBJ accession number is AB006971.


                              
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Table 1.   Exon-intron boundaries of mGPAA1 gene

Expression of mGPAA1 mRNA. At first we performed Northern blot analyses of murine tissues in fetal, neonatal, and adult stages to examine the expression of mGPAA1 mRNA. Strong expression of mGPAA1 was detected in murine fetal (Fig. 3A) and neonatal (Fig. 3B) tissues. mGPAA1 mRNA was detected as a single band, and its size was about 2 kb. This size was almost the same as that of the mGPAA1 cDNA that we isolated. In the adult, mGPAA1 also was expressed ubiquitously, but its expression levels differed among tissues. In general, they were weaker than in fetal and neonatal tissues.


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Fig. 3.   Northern blot analysis of mGPAA1 mRNA in murine tissues. mGPAA1 mRNA is ubiquitously expressed in murine fetal (A), neonatal (B), and adult (C) tissues. Twenty micrograms of total RNA were hybridized with 32P-labeled mGPAA1 cDNA probe. Expression levels were higher in fetal and neonatal tissues than in adult tissues. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

To examine the expression of mGPAA1 mRNA in different cell types, we performed in ISH analysis of murine embryos. mGPAA1 was expressed ubiquitously, and we found mGPAA1 mRNA expression at high levels in the choroidea of the fourth ventricle (Fig. 4, A-C), skeletal muscle cells (Fig. 4, D-F), osteoblasts of ribs (Fig. 4, G-I), and occipital bone (Fig. 4, J-L).


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Fig. 4.   In situ hybridizations of mGPAA1. Strong signals were detected in choroidae of the fourth ventricle (A-C), skeletal muscle (D-F), rib (G-I), and occipital bone (J-L). Hematoxylin-eosin staining sections are shown (left). Antisense and sense sections are also shown (middle and right, respectively). Arrows indicate strong signal of mGPAA1.

DAF expression and miniPLAP in vitro translation assay of the development of ES cells. We previously had shown that mGPAA1 mRNA expression decreased with the development of ES cells (10). We compared surface expression levels of the DAF that is a widely expressed GPI-anchored protein. We confirmed that DAF on the cells was GPI-anchored by showing that it could be released by PI-PLC and that levels of the protein declined during the differentiation (Fig. 5, A and B). DAF protein decreased with mGPAA1 mRNA levels.


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Fig. 5.   Decay-accelerating factor (DAF) expression on embryonic stem (ES) cells. After removal of leukemia inhibitory factor, ES cells were harvested at 3, 6, and 9 days, at which time beating was observed. Histograms showing surface DAF levels on the cells are shown (A) and mean DAF fluorescence levels are given diagrammatically (B). The open lines (A) show the staining with nonrelevant isotype-matched control monoclonal antibody. Surface DAF levels progressively declined with the appearance of beating.

We used the miniPLAP in vitro translation assay to examine the transamidation reaction directly. In this assay, COOH-terminal processing of 27.0-kDa prominiPLAP yields 24.7-kDa GPI-anchored mature miniPLAP. We found that in accordance with Northern blot data of mGPAA1 mRNA levels, the efficiency of the transamidation reaction went down with the appearance of beating ES cells (Fig. 6, A and B).


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Fig. 6.   MiniPLAP in vitro translation assays. The miniPLAP mRNA was incubated for 90 min with rough microsomes prepared from 3-, 6-, and 9-day different ES cells, and after immunoprecipitation with anti-PLAP antibody (Sigma Chemical, St. Louis, MO) and protein A-Sepharose (Pharmacia, Piscataway, NJ), the products were analyzed on 15% SDS-PAGE gels. The positions of the 28.2-kDa preprominiPLAP translation product, 27.0-kDa NH2-terminal processed proprotein, and 24.7-kDa GPI-anchored mature miniPLAP products are shown (A). The percentage of prominiPLAP converted to the mature GPI-anchored end product declined with differentiation (B).

Analysis of function of cell lines expressing antisense mGPAA1. We prepared 3T3 cell lines expressing antisense mGPAA1 controlled by a dexamethasone-inducible promoter to clarify mGPAA1 function. We selected 12 clones with blasticidin, and we detected that 6 of them overexpressed 550-bp antisense mGPAA1 by Northern blot analysis. Ly6 and CD24 are known to be GPI-anchored proteins and to exist in 3T3 cells. We could detect both Ly6 and CD24 with monoclonal antibodies on the surface of parental 3T3 cells (Fig. 7A). Neither Ly6 nor CD24 could be detected on any of the six cell lines expressing antisense mGPAA1 (Fig. 7B). In conjunction with the above miniPLAP data, these results indicated that mGpaa1p participates in GPI-anchoring acting at the GPI transfer step like yGaa1p.


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Fig. 7.   Expression of GPI-anchored Ly6 on the cell surface of 3T3 cell lines. The ordinate gives the cell number and the abscissa the fluorescent intensity of cells stained with antibodies. The results of staining with a nonrelevant antibody control are shown by the line without fill and the results with Ly6 antibody by the filled area. Overlap of the filled and unfilled areas means no expression of Ly6. Wild-type 3T3 cells expressed Ly6 (B), and cell line expressing antisense mGPAA1 failed to express Ly6 on the cell surface membrane (A). Analysis of CD24 on the cell surface expression gave the same result.


    DISCUSSION
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INTRODUCTION
METHODS
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DISCUSSION
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Several results suggest that the final step in GPI-anchored protein synthesis is accomplished by a transamidase (1, 20, 21); a transamidation mechanism for GPI attachment to protein has recently been confirmed in trypanosomes (28), but the mammalian cellular machinery of this GPI transamidase reaction is not yet defined. It is difficult to directly measure the activity of the transamidase in crude extracts because the transamidase requires two substrates, a proprotein and the GPI anchor donor (34). Recently, two components of the GPI transfer machinery have been identified based on the analysis of the two yeast mutants gaa1 (8) and gpi8 (2), both of which synthesize the complete GPI anchor precursor but fail to attach it to proteins. Overexpression of yGAA1 restored the ability of gaa1 mutant cells to anchor GPIs to proproteins (8).

In this study, we have isolated mGPAA1 cDNA and genomic DNA by the signal sequence trap method. mGpaa1p has an NH2-terminal signal sequence like that of yGaa1p that consists of 47 amino acids and functions as an NH2-terminal signal sequence in this method. The nucleotide sequences of mGPAA1 and human GPAA1 showed no significant homology with any known genes, but the deduced amino acid sequences showed 25% identity and 57% homology with that of yGaa1p. mGPAA1 cDNA has a long open reading frame with short 5'- and 3'-untranslated regions. A putative translation initiation site is fully consistent with Kozak (16) consensus, and a polyadenylation signal and a poly(A) tract exist at the 3' terminus. The size of the cDNA is almost the same as that of the mRNA detected on Northern blot analysis (Fig. 3). We also determined the murine gene structure and compared its sequence with the cDNA sequence. Our data indicated that we isolated a full-length mGPAA1 cDNA.

There are several consensus motifs in the deduced proteins of mGPAA1 and hGPAA1. All of them were conserved in both Gpaa1p proteins. They have two putative N-glycosylation sites and seven PKC phosphorylation sites, but yGaa1p has only three PKC phosphorylation sites. Unlike yGaa1p, they have one cAMP- and cGMP-dependent protein kinase phosphorylation site. These putative phosphorylation sites may be important for the regulation of Gpaa1p function, and in higher eukaryotes, a cAMP- and cGMP-dependent protein kinase phosphorylation site might play an important role. Leucin zipper motif is also conserved, and this motif is known to take part in protein interaction. It may be important for the formation of transamidase complexes.

Southern blot analyses of murine genomic DNA suggested that the mGPAA1 gene is a single copy gene in the haploid murine genome. It also exists as a single gene in yeast (8). The mGPAA1 gene has 12 small exons and 11 small introns that are contained in a 3.6-kb BamH I fragment. The mGPAA1 gene is small compared with other PIG genes that participate in GPI anchor synthesis.

mGPAA1 mRNA is expressed ubiquitously in the fetus, neonate, and adult (Fig. 3, A-C). mGPAA1 resembles housekeeping genes, and yGAA1 is necessary for yeast survival. In mammalian cells, GPI anchor-deficient cell lines survive, but PIG-A-deficient mice cannot be produced from PIG-A-deficient ES cells (6, 14). These results suggest that GPIs or GPI-anchored proteins are necessary to form particular organs or tissues in the early embryonic stage. In our experiments, mGPAA1 mRNA expression levels were higher at the embryonic stage than at the adult stage, and in the in vitro differentiation system of ES cells, mGPAA1 mRNA levels decreased in the course of development (10). Moreover, GPI-anchored DAF expression and the transamidation efficiency directly measured by miniPLAP assay decrease with the differentiation of ES cells (Figs. 5 and 6). These results also support the above proposition.

ISH analysis elucidated the high expression of mGPAA1 mRNA at the cellular level. It revealed that mGPAA1 mRNA is expressed abundantly in choroidea of the fourth ventricle of brain. The reason for the high expression in muscle cells and osteoblasts is unknown.

Recently, another protein, Gpi8p, which is essential for GPI transfer, has been characterized, and the yeast gpi8 mutant cannot be rescued by overexpression of yGAA1 (2). Expression of yGPI8 rescues the yeast gpi8 mutant, and expression of hGPI8 rescues class K cells (37). These results indicate that at least two gene products participate in the transamidation reaction both in mammalian cells and in yeast. Gpi8p has homology to a family of plant endopeptidases, one of which has transamidase activity (37). Gpi8p may be a catalytic component of transamidase machinery complex, and Gpaa1p possibly could function to hold COOH-terminal signal sequences of nascent proteins.

Previously, we showed that transient overexpression of antisense hGPAA1 significantly reduced the production of a reporter GPI-anchored protein in human K562 cells. So, we selected 3T3 cell lines expressing antisense mGPAA1 under the dexamethasone-inducible promoter. GPI-anchored Ly6 and CD24 expressions on the cell surface were completely inhibited by antisense mGPAA1 (Fig. 7). These results suggested that mGpaa1p has a function in the GPI anchor attachment step similar to yeast Gaa1p.

In summary, we have isolated mGPAA1 cDNA and the mGPAA1 gene. Gpaa1p protein presumably plays an important role with Gpi8p in the transfer step of GPI anchor attachment to protein. Their precise functions need further investigation.


    ACKNOWLEDGEMENTS

We thank Dr. Honjo for pcDL-SRa-Tac (3'), Dr. Uchiyama for anti-Tac antibody, and Dr. Morgan for DAF MAb 2C6. We also thank Fumiko Harima and Maki Kitagawa for excellent technical assistance.


    FOOTNOTES

The sequences reported in this paper have been deposited in the European Molecular Biology Laboratory, GenBank, and DNA Data Bank of Japan nucleotide sequence databases with the following accession numbers: AB006970 and AB006971.

Address for reprint requests and other correspondence: I. Komuro, Dept. of Cardiovascular Medicine, Univ. of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan (E-mail: komuro-tky{at}umin.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 8 September 1998; accepted in final form 29 January 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 279(1):C205-C212
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society




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