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Sequence and expression of the monkey homologue of the ER-Golgi Intermediate Compartment lectin, ERGIC-531
Biochimica et Biophysica Acta 1447 (1999) 334^340 www.elsevier.com/locate/bba
Short sequence-paper
Sequence and expression of the monkey homologue of the ER-Golgi Intermediate Compartment lectin, ERGIC-531 Sonia Sarnataro, Maria Gabriella Caporaso, Stefano Bonatti, Paolo Remondelli * Dipartimento di Biochimica e Biotecnologie Mediche, Universita© degli Studi di Napoli `Federico II', via S. Pansini 5, 80131, Naples, Italy Received 22 June 1999; received in revised form 2 September 1999; accepted 7 September 1999
Abstract We obtained the cDNA sequence of the monkey homologue of the intermediate compartment protein ERGIC-53 by both cDNA library screening and RT-PCR amplification. The final sequence of 2422 nts of the monkey ERGIC-53 cDNA is 96.2% identical to the human ERGIC-53 cDNA and 87% and 67% identical to the rat and amphibian cDNA, respectively. The translated CV1 ERGIC-53 protein is 96.47% identical to the human ERGIC-53, 87% identical to the rat p58 and 66.98% to the Xenopus laevis protein. Southern blot analysis of multiple genomic DNAs shows the presence of sequences similar to ERGIC-53 in different species. ERGIC-53 is expressed as a major transcript of about 5.5 kb in either monkey CV1 or in human CaCo2. A shorter transcript of 2.3 kb was detected in both cell lines and in mRNAs derived from human pancreas and placenta. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: ERGIC-53; cDNA cloning; mRNA expression
Animal lectins are a family of proteins having as common feature the ability to recognise speci¢c carbohydrates (i.e. galactose, mannose) [1^5]. In mammals lectins participate in a number of di¡erent functions such as the endocytosis of glycoproteins, regulation of cell adhesion, cell migration, sorting and distribution of microsomal enzymes [6^9]. The human endoplasmic reticulum-Golgi intermediate compartment protein, ERGIC-53 is an intracellular lectin whose binding to mannose residues requires calcium (c-type lectin) and involves speci¢c amino acids present at the carbohydrate recognition domain, CRD [10^13]. ERGIC-53 is able to recycle * Corresponding author. fax. +39-81-7463150; E-mail: [email protected] 1 GenBank submission: bankit276506, Accession number: AF160877
between the ER and the vesicular tubular clusters, VTCs [14^16]. Its cytoplasmic domain contains a double phenylalanine signal (AKKFF) which is required for ER export via the interaction with Sec23 of the COPII-coat anterograde vesicles [17^19]. In addition a di-lysine signal (AKKFF) is required for the interaction with the COPI coat in the VTCs resulting in the incorporation into retrograde COPI vesicles [20^23]. Because for these properties, it has been proposed that ERGIC-53 may act as a chaperone for the transport of glycoproteins between the ER and Golgi [24,25]. Most recently, it has been shown that the inherited disease combined de¢ciency of coagulation factors V and VIII, which results in an autosomal recessive bleeding disorder, could be attributed to mutations in the ERGIC-53 gene [24]. This observation indicated that this lectin could play an important role
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in the selective export of a speci¢c subset of proteins [24,25]. Mammalian cDNAs encoding the human ERGIC53 [23,26] and its homologue in rat (p58) [27^30] were isolated and characterised. A partial cDNA sequence of the amphibian homologue of ERGIC-53 from Xenopus cells was also reported [30]. We screened a 5P stretched Vgt11 cDNA library obtained from CV1 cells (Clontech) to obtain the homologous of the monkey sequence for the intermediate compartment protein ERGIC-53. The library was screened with a K-32 P-labelled cDNA probe (441 bp) corresponding to the region of the ERGIC-53 cDNA spanning from nt 134 and nt 575. This region encodes the calcium dependent carbohydrate recognition domain (CRD) and was obtained by digesting with SacII and HindIII the pECE-myc p53 vector expressing a myc tagged 53 [13]. The DNA probe was isolated and labelled with [K-32 P]dCTP, 3000 mCi/mmol (Amersham) by the random priming method (Promega), puri¢ed by nick columns (Pharmacia) and denatured by boiling for 10 min. The cDNA library from CV1 cells in Vgt11 phage (Clontech) was plated at a density of 105 plaques per 150 mm petri dish. A total of approximately 1.5^ 2.0U106 plaques were analysed and after overnight growth were lifted onto Hybond-C nitrocellulose membranes (Amersham). Blotted DNA was denatured with denaturing solution containing 1.5 M NaCl, 0.5 M NaOH, neutralised and baked in a vacuum oven at 80³C for 2 h. Filters were pre-hybridised at 42³C for 3^4 h in hybridisation bu¡er containing 50% formamide, 5USSPE, 5UDenhart's solution, 0.1% SDS, 100 Wg/ml denatured salmon sperm DNA and hybridised at the same temperature for 16 h in the presence of the probe. Membranes were washed once at room temperature. (2USSC, 0.5% SDS) and twice at 55³C (1USSC, 0.1% SDS). Filters were dried and exposed to X-ray ¢lm (Kodak) at 380³C with an intensifying screen. After three rounds of screening, six independent positive clones were isolated. Phage clones were subsequently plaque puri¢ed and V DNA was isolated (Quiagen). We used PCR analysis to assess the length of the inserts. V DNAs were ampli¢ed with V arms speci¢c primers: forward, 5P-GGTGGCGA-
335
Fig. 1. RT-PCR ampli¢cation of CV1 and CaCo2 mRNAs. RT-PCR ampli¢cation from total CV1 (lane 1) or CaCo2 (lane 2) RNAs. The arrow indicates ERGIC-53 speci¢c ampli¢cation products. Non-speci¢c fragments are indicated by a bracket. Molecular weight size standards are indicated on the left.
CGACTCCTGGAGCC-3P and reverse, 5P-GACACCAGACCAACTGGTAATG-3P. The length of the PCR products was compared to those obtained by restriction analysis of EcoRI digested V DNAs. DNA fragments were excised from the gel, puri¢ed and subcloned in pBluescript SK( ) (Stratagene). PCR products were subcloned into the TA cloning vector pCR 2.1 (Invitrogen). Plasmid clones were sequenced on both strands by automated DNA sequencer (Applied Biosystems) by using as primers T3, T7 and synthesised internal primers. The sequence alignment of six independent cDNA clones revealed a 100% identity to each other thus indicating that the isolated clones represented di¡erent regions of the same transcript. Sequence homology analysis revealed that the larger CV1 cDNA clone corresponded to the human ERGIC-53 cDNA region spanning from nt 311 to nt +970 therefore lacking most of the 3P region. Therefore, we repeated the CV1 cDNA library screening with a 979 nt cDNA probe corresponding to the human ERGIC-53 3P region spanning from nt 575 to nt 1554, obtained by digesting with HindIII, with the vector pECE-myc p53 [13]. After three rounds of screening a single clone hybridising with the 3P ERGIC-53 probe was plaque puri¢ed, V DNA was extracted and the insert subcloned and subjected
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to sequence analysis. This revealed high homology with the human ERGIC-53 cDNA region enclosed between nt 1350 and nt 2480. This cDNA clone thus contained part of the coding region and most of the 3P untranslated region. We next used reverse transcription PCR (RTPCR) to obtain the complete CV1 ERGIC-53 sequence. A ERGIC-53 transcript was previously detected in the human CaCo2 cell line [23]. We therefore used CaCo2 cells as source for positive control. Total mRNA was isolated from CV1 cells cultured in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 50 Wg/ml streptomycin and 2 mM Lglutamine. CaCo2 cells were cultured in the same medium supplemented with 20% FBS. RNA extraction was performed for both cell lines with the acidic phenol method [31]. 1 Wg of total mRNA extracted from CV1 cells was mixed with random primers and used in the reverse transcriptase reaction to obtain the template cDNA. The following oligomers were synthesised: primer 1, 5P-CCAAGATGGCGGGATCCA-3P corresponding to bp 35 to bp +13 of the monkey cDNA sequence; primer 2, 5P-GGCAGCTGCTTCTTGCGT-3P corresponding to bp 1522 to bp 1540 of the monkey cDNA sequence. Both primers were also chosen for their ability to recognise also the human ERGIC-53 sequence. A 50 Wl PCR reaction contained approximately 10 ng of the monkey cDNA as template, 15 WM of primer 1 and 2, 200 nM of each dNTP, 4 mM MgCl2 and 2.5 units of Pfu DNA polymerase. The reactions were performed with the thermal cycler (Promega). The cycle was of 120 s at 95³C followed by 30 cycles, each consisting of 60 s at 95³C, 60 s at 58³C, 120 min at 72³C. We ampli¢ed similar PCR reaction products from both CV1 and CaCo2 RNAs (Fig. 1). The size of both fragments was the one expected for a cDNA encoding the complete ORF of both the monkey and the human ERGIC-53. The fragments were therefore gel puri¢ed, subcloned in the TA cloning
337
vector pCR2.1 (Invitrogen) and sequenced. Sequence comparison of the PCR products ampli¢ed from CV1 and CaCo2 RNAs showed an identity of 97.37%. Sequences of the shorter ampli¢cation products did not reveal similarities with ERGIC-53 or related cDNA sequences present in databases. Sequences obtained by the cDNA library screening were extended by RT-PCR analysis to obtain the ¢nal sequence of 2422 nts of the monkey ERGIC53 cDNA (Fig. 2). Translation of all six reading frames revealed that one of them contained the ATG start codon and the complete ORF of the monkey CV1 ERGIC-53 (Fig. 2). The 2480 nt sequence of the monkey ERGIC-53 cDNA was aligned to those of the human ERGIC-53, rat p58 and the partial Xenopus cDNA. Sequence analysis showed that the CV1 cDNA was 93.27% identical to the human ERGIC-53 cDNA. Lower similarity was revealed when it was compared to the rat p58 (76.3%) or the amphibian cDNA (55.49%). Similar results were obtained from the alignment of the predicted monkey (CV1) protein sequence with those of its relatives in other species. This analysis revealed that the monkey protein is 96.47% identical to the human ERGIC-53, 87% identical to the rat 58 and 66.98% identical to the Xenopus laevis protein. Both the human and monkey proteins have the same number (510) of amino acids. No di¡erences were found in the cytosolic tail sequence, indicating that the CV1 ERGIC-53 has the same recycling properties as its human homologue. Immuno£uorescence microscopy showed the typical localisation of the ERGIC-53 in the intermediate compartment of CV1 cells (data not shown). We next tested the presence of genes similar to ERGIC-53 by Southern analysis of genomic DNAs obtained from di¡erent species (Fig. 3). CV1 ERGIC probe corresponding to the region spanning from nt 311 to nt +970 was prepared as above. Filter (Clontech) was pre-hybridised for 3^4 h at 42³C and hybridised 16 h at 42³C in hybridisation bu¡er containing 5USSPE, 50% formaldehyde, 5UDenhart, 0.5%
6
Fig. 2. Nucleotide sequence of the monkey ERGIC-53 cDNA and deduced amino acid sequence of the protein. The complete coding region along with portion of the 5P- and 3P- non coding sequences is shown together with the predicted amino acid sequence in standard single letter code. The stop codon is indicated by an asterisk.
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SDS, 10% dextran sulphate, 100 Wg/ml salmon sperm DNA and 1.5U106 cpm/ml of DNA probe. Thus, we revealed the presence of sequences similar to ERGIC-53 in all the species analysed (Fig. 3: lanes 1^9). We found diversities in the intensity of the hybridisation signals which suggest di¡erences among the species. Hybridisation patterns obtained from most of the genomes (Fig. 3: lanes 1^3, 5^7, 9) suggest that genes similar to ERGIC-53 could be present in more than one copy in those species. Conversely, mouse and chicken DNAs revealed the presence of an individual signal (Fig. 3: lanes 4 and 8). By this analysis, we also detected the presence of a signal in genomic DNA derived from Saccharomyces cerevisiae (Fig. 3: lane 9). Computer search for sequence homology of the complete yeast S. cerevisiae database (MIPS Yeast Genome Database) failed to detect similarities with expressed sequences. However, weak homology (61%) was revealed with sequences present in yeast chromosome X. A length of about 5.5 kb was reported for the ERGIC-53 transcript by the analysis of mRNA extracted from human CaCo2 cells [23]. We therefore compared the expression of ERGIC-53 in CV1 and CaCo2 cells by the use of Northern blots (Fig. 4a). Cells were lysed in sodium acetate 50 mM, pH 5.2, SDS 1%, EDTA 5 mM and poly-A mRNA was
Fig. 3. Southern blot analysis of genomic DNA obtained from di¡erent species as indicated on top. Membrane was from Clontech laboratories. Each lane contained 5 Wg of EcoRI digested genomic DNA. Molecular weight size standards are reported on the left.
Fig. 4. Expression of ERGIC-53 RNA transcripts. (a) Analysis of the expression of ERGIC-53 in monkey CV1 cells: lanes 1 and 2; and human CaCo2 cells : lanes 2 and 3. Lane 1: 10 Wg total RNA from CV1 cells; lane 2: 1 Wg polyA RNA from CV1 cells ; lane 3: 10 Wg total RNA from CaCo2 cells ; lane 4: 1 Wg polyA RNA from CaCo2 cells. (b) ERGIC-53 mRNA expression in di¡erent human tissues as indicated on the top. Arrows indicate speci¢c ERGIC transcripts. Position of molecular weight size markers is indicated on the left. Membrane was from Clontech laboratories.
extracted by the use of oligo dT-cellulose (Clontech). 10 Wg of total or 1 Wg of poly-A RNA was fractionated on a formaldehyde-1% agarose gel and blotted on Hybond N nylon strips (Amersham). Comparative analysis of mRNA extracted from human CaCo2 cells and monkey CV1 cells revealed the presence of a 5.5 kb transcript in both cell lines (Fig. 4a: lanes 2 and 4). However, we detected also a smaller transcript with a similar size, 2.3 kb, in both species (Fig. 4a: lanes 2 and 4). Neither of the 5.5 or 2.3 kb transcripts were detectable in total RNA extracted from these cell lines (Fig. 4a: lanes 1 and 4)
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Similar results were obtained by analysing ERGIC-53 mRNA expression in di¡erent human tissues (Fig. 4b). Membrane (Clontech laboratories) was analysed with K-32 P-labelled CV1 ERGIC cDNA probe spanning from nt 311 to nt +970 prepared as above. Hybridisation was carried out at 42³C in 5USSPE, 50% formaldehyde, 5UDenhart, 0.5% SDS, 10% dextran sulphate, 100 Wg/ml salmon sperm DNA. 1USSPE was NaCl 180 mM, sodium phosphate 10 mM and EDTA 1 mM. Washes were carried out at low stringency conditions. We thus showed that the ERGIC-53 5.5 kb transcript was expressed in all the tissues analysed (Fig. 4b: lanes 1^8). Quantitative analysis of the expression levels revealed that the 5.5 kb transcript was expressed at similar levels in all the tissues analysed (data not shown). Conversely, a 2.3 kb transcript was detected only in mRNAs obtained from placenta and pancreas tissues (Fig. 4b: lane 3 and 8). The 2.3 kb mRNA was also detected with similar intensities when we hybridised the same ¢lters with ERGIC53 probes containing di¡erent portions of the coding region (data not shown). Therefore, the presence of the minor transcript of 2.3 kb could be due to a di¡erent RNA processing of the same transcript rather than to the expression of a related gene. Our data suggest the presence of genes related to ERGIC-53 in most of the species. Isolation and characterisation of ERGIC-53 related genes could provide additional information about the function of these proteins. Moreover, ERGIC-53 is ubiquitous and constitutively expressed and this is consistent with its supposed housekeeping function during the protein tra¤cking between the ER and Golgi. Acknowledgements We are indebted to Dr H.P. Hauri, Department of Pharmacology, University of Basel, Basel, Switzerland, for providing the plasmid pECE-myc p53. We thank Mr. B. Mugnoz for his technical assistance. This work was supported in part by C.N.R. (P.F. Biotecnologie), European Community (T.M.R. Programme) and M.U.R.S.T. (Prinn 1998).
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References [1] M. Caron, D. Bladier, R. Joubert, Soluble galactoside-binding vertebrate lectins: a protein family with common properties, Int. J. Biochem. 22 (1990) 1379^1385. [2] S.H. Barondes, D.N.W. Cooper, M.A. Gitt, H. Le¥er, Galectins, structure and functions of a large family of animal lectins, J. Biol. Chem. 269 (1994) 20807^20810. [3] W.I. Weis, K. Drickamer, W.A. Hendrickson, Structure of a C-type mannose-binding protein complexed with an oligosaccharide, Nature 360 (1992) 127^134. [4] S.T. Iobst, M.R. Wormand, W.I. Weis, R.A. Dwek, K. Drickamer, Binding of sugar ligand to Ca2 -dependent animal lectins. Analysis of mannose binding by site-directed mutagenesis and NMR, J. Biol. Chem. 269 (1994) 15505^ 15511. [5] A.R. Kolatkar, W. Weis, Structural basis of galactose recognition by C-type animal lectins, J. Biol. Chem. 271 (1996) 6679^6685. [6] H.J. Gabius, Non-carbohydrate binding partners/domain of animal lectins, Int. J. Biochem. 26 (1994) 469^477. [7] H.J. Gabius, Detection and functions of mammalian lectinswith emphasis on membrane lectins, Biochim. Biophys. Acta 1071 (1991) 1^18. [8] H.J. Gabius, Animal lectins, Eur. J. Biochem. 243 (1997) 543^576. [9] S.H. Barondes, Lectins : their multiple endogenous cellular functions, Annu. Rev. Biochem. 50 (1981) 207^231. [10] K. Fiedler, K. Simons, A putative novel class of animal lectins in the secretory pathway homologueous to leguminous lectins, Cell 77 (1994) 625^626. [11] K. Drickamer, Two distinct classes of carbohydrate-recognition domains in animal lectins, J. Biol. Chem. 263 (1998) 9557^9560. [12] K. Drickamer, M.E. Taylor, Biology of animal lectins, Annu. Rev. Cell Biol. 9 (1993) 237^264. [13] C. Itin, A.C. Roche, M. Monsigny, H.P. Hauri, ERGIC-53 is a functional mannose-selective and calcium dependent human homologue of leguminous lectins, Mol. Biol. Cell 7 (1996) 483^493. [14] A. Schweizer, F. Peter, P.N. Van, H.D. Soling, H.P. Hauri, A luminal calcium-binding protein with a KDEL endoplasmic reticulum retention motif in the ER-Golgi intermediate compartment, Eur. J. Cell. Biol. 60 (1993) 366^370. [15] J. Klumperman, A. Schweizer, H. Clausen, B.L. Tang, W. Hong, V. Oorschot, H.P. Hauri, The recycling pathway of protein ERGIC-53 and dynamics of the ER-Golgi intermediate compartment, J. Cell. Sci. 111 (1998) 3411^3425. [16] F. Kappeler, D.R.Ch. Klopfenstein, M. Foguet, J.P. Paccaud, H.P. Hauri, The recycling of ERGIC-53 in the secretory pathway, J. Biol. Chem. 272 (1997) 31801^31808. [17] C. Itin, R. Schindler, H.P. Hauri, Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway, J. Cell Biol. 131 (1995) 57^67.
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[18] T.C. Hobman, B. Zhao, H. Chan, M.G. Farquar, Immunoisolation and characterization of a subdomain of the endoplasmic reticulum that concentrates proteins involved in COPII vesicle biogenesis, Mol. Biol. Cell 9 (1998) 1265^1278. [19] C. Itin, M. Foguet, F. Kappeler, J. Klumperman, H.P. Hauri, Recycling of the endoplasmic reticulum/Golgi intermediate compartment protein ERGIC-53 in the secretory pathway, Biochem. Soc. Trans. 23 (1995) 541^544. [20] E.J. Tisdale, H. Plutner, J. Matterson, W. Balch, p53/p58 binds COPI and is required for selective transport through the secretory pathway, J. Cell Biol. 137 (1997) 581^593. [21] F. Kappeler, C. Itin, R. Schindler, H.P. Hauri, A dual role for COOH-terminal lysine residues in pre-Golgi retention and endocytosis of ERGIC-53, J. Biol. Chem. 269 (1994) 6279^6281. [22] B.L. Tang, S.H. Low, H.P. Hauri, W. Hong, Segregation of ERGIC-53 and the mammalian KDEL receptor upon exit from the 15³C compartment, Eur. J. Cell Biol. 68 (1995) 398^410. [23] R. Schindler, C. Itin, M. Zerial, F. Lottspeich, H.P. Hauri, ERGIC-53, a membrane protein of the ER-Golgi intermediate compartment, carries an ER retention motif, Eur. J. Cell Biol. 61 (1993) 1^9. [24] W.C. Nichols, U. Seligsohn, A. Zivelin, V.H. Terry, C.E. Hertel, M.A. Wheatley, M.J. Moussalli, H.P. Hauri, N. Ciavarella, R.J. Kaufman, D. Ginsburg, Mutation in the ERGolgi intermediate compartment protein ERGIC-53 cause combined de¢ciency of coagulation factors V and VIII, Cell 93 (1998) 61^70.
[25] F. Wallenweider, F. Kappeler, C. Itin, H.P. Hauri, Mistargeting of the lectin ERGIC-53 to the endoplasmic reticulum of Hela cells impairs the secretion of a lysosomal enzyme, J. Cell. Biol. 142 (1998) 377^389. [26] A. Schweizer, A.M. Fransen, T. Bachi, L. Ginsel, H.P. Hauri, Identi¢cation, by monoclonal antibody, of a 53kDa protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus, J. Cell. Biol. 107 (1988) 1643^1653. [27] J. Saraste, G.E. Palade, M.G. Farquar, Antibodies to rat pancreas subfractions: identi¢cation of a 58-kDa cis-Golgi protein, J. Cell Biol. 105 (1987) 2021^2029. [28] U. Lahtinen, U. Hellman, C. Wernstedt, J. Saraste, R.F. Petterson, Molecular cloning and expression of a 58-kDa cis-Golgi and intermediate compartment protein, J. Biol. Chem. 271 (1996) 4031^4037. [29] E. Plutner, H.W. Davidson, J. Saraste, W. Balch, Morphological analysis of protein transport from the ER to Golgi membranes in digitonin-permeabilized cells: role of the p58 containing compartment, J. Cell. Biol. 119 (1992) 1097^1116. [30] U. Lahtinen, B. Dahllof, J. Saraste, Characterisation of a 58 kDa cis-Golgi protein in pancreatic exocrine cells, J. Cell Sci. 103 (1992) 321^333. [31] J. Sambrook, J. Fritch, T. Maniatis, Molecular cloning. A laboratory manual, 2nd edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
BBAEXP 91318 19-10-99
Short sequence-paper
Sequence and expression of the monkey homologue of the ER-Golgi Intermediate Compartment lectin, ERGIC-531 Sonia Sarnataro, Maria Gabriella Caporaso, Stefano Bonatti, Paolo Remondelli * Dipartimento di Biochimica e Biotecnologie Mediche, Universita© degli Studi di Napoli `Federico II', via S. Pansini 5, 80131, Naples, Italy Received 22 June 1999; received in revised form 2 September 1999; accepted 7 September 1999
Abstract We obtained the cDNA sequence of the monkey homologue of the intermediate compartment protein ERGIC-53 by both cDNA library screening and RT-PCR amplification. The final sequence of 2422 nts of the monkey ERGIC-53 cDNA is 96.2% identical to the human ERGIC-53 cDNA and 87% and 67% identical to the rat and amphibian cDNA, respectively. The translated CV1 ERGIC-53 protein is 96.47% identical to the human ERGIC-53, 87% identical to the rat p58 and 66.98% to the Xenopus laevis protein. Southern blot analysis of multiple genomic DNAs shows the presence of sequences similar to ERGIC-53 in different species. ERGIC-53 is expressed as a major transcript of about 5.5 kb in either monkey CV1 or in human CaCo2. A shorter transcript of 2.3 kb was detected in both cell lines and in mRNAs derived from human pancreas and placenta. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: ERGIC-53; cDNA cloning; mRNA expression
Animal lectins are a family of proteins having as common feature the ability to recognise speci¢c carbohydrates (i.e. galactose, mannose) [1^5]. In mammals lectins participate in a number of di¡erent functions such as the endocytosis of glycoproteins, regulation of cell adhesion, cell migration, sorting and distribution of microsomal enzymes [6^9]. The human endoplasmic reticulum-Golgi intermediate compartment protein, ERGIC-53 is an intracellular lectin whose binding to mannose residues requires calcium (c-type lectin) and involves speci¢c amino acids present at the carbohydrate recognition domain, CRD [10^13]. ERGIC-53 is able to recycle * Corresponding author. fax. +39-81-7463150; E-mail: [email protected] 1 GenBank submission: bankit276506, Accession number: AF160877
between the ER and the vesicular tubular clusters, VTCs [14^16]. Its cytoplasmic domain contains a double phenylalanine signal (AKKFF) which is required for ER export via the interaction with Sec23 of the COPII-coat anterograde vesicles [17^19]. In addition a di-lysine signal (AKKFF) is required for the interaction with the COPI coat in the VTCs resulting in the incorporation into retrograde COPI vesicles [20^23]. Because for these properties, it has been proposed that ERGIC-53 may act as a chaperone for the transport of glycoproteins between the ER and Golgi [24,25]. Most recently, it has been shown that the inherited disease combined de¢ciency of coagulation factors V and VIII, which results in an autosomal recessive bleeding disorder, could be attributed to mutations in the ERGIC-53 gene [24]. This observation indicated that this lectin could play an important role
0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 1 7 7 - 3
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in the selective export of a speci¢c subset of proteins [24,25]. Mammalian cDNAs encoding the human ERGIC53 [23,26] and its homologue in rat (p58) [27^30] were isolated and characterised. A partial cDNA sequence of the amphibian homologue of ERGIC-53 from Xenopus cells was also reported [30]. We screened a 5P stretched Vgt11 cDNA library obtained from CV1 cells (Clontech) to obtain the homologous of the monkey sequence for the intermediate compartment protein ERGIC-53. The library was screened with a K-32 P-labelled cDNA probe (441 bp) corresponding to the region of the ERGIC-53 cDNA spanning from nt 134 and nt 575. This region encodes the calcium dependent carbohydrate recognition domain (CRD) and was obtained by digesting with SacII and HindIII the pECE-myc p53 vector expressing a myc tagged 53 [13]. The DNA probe was isolated and labelled with [K-32 P]dCTP, 3000 mCi/mmol (Amersham) by the random priming method (Promega), puri¢ed by nick columns (Pharmacia) and denatured by boiling for 10 min. The cDNA library from CV1 cells in Vgt11 phage (Clontech) was plated at a density of 105 plaques per 150 mm petri dish. A total of approximately 1.5^ 2.0U106 plaques were analysed and after overnight growth were lifted onto Hybond-C nitrocellulose membranes (Amersham). Blotted DNA was denatured with denaturing solution containing 1.5 M NaCl, 0.5 M NaOH, neutralised and baked in a vacuum oven at 80³C for 2 h. Filters were pre-hybridised at 42³C for 3^4 h in hybridisation bu¡er containing 50% formamide, 5USSPE, 5UDenhart's solution, 0.1% SDS, 100 Wg/ml denatured salmon sperm DNA and hybridised at the same temperature for 16 h in the presence of the probe. Membranes were washed once at room temperature. (2USSC, 0.5% SDS) and twice at 55³C (1USSC, 0.1% SDS). Filters were dried and exposed to X-ray ¢lm (Kodak) at 380³C with an intensifying screen. After three rounds of screening, six independent positive clones were isolated. Phage clones were subsequently plaque puri¢ed and V DNA was isolated (Quiagen). We used PCR analysis to assess the length of the inserts. V DNAs were ampli¢ed with V arms speci¢c primers: forward, 5P-GGTGGCGA-
335
Fig. 1. RT-PCR ampli¢cation of CV1 and CaCo2 mRNAs. RT-PCR ampli¢cation from total CV1 (lane 1) or CaCo2 (lane 2) RNAs. The arrow indicates ERGIC-53 speci¢c ampli¢cation products. Non-speci¢c fragments are indicated by a bracket. Molecular weight size standards are indicated on the left.
CGACTCCTGGAGCC-3P and reverse, 5P-GACACCAGACCAACTGGTAATG-3P. The length of the PCR products was compared to those obtained by restriction analysis of EcoRI digested V DNAs. DNA fragments were excised from the gel, puri¢ed and subcloned in pBluescript SK( ) (Stratagene). PCR products were subcloned into the TA cloning vector pCR 2.1 (Invitrogen). Plasmid clones were sequenced on both strands by automated DNA sequencer (Applied Biosystems) by using as primers T3, T7 and synthesised internal primers. The sequence alignment of six independent cDNA clones revealed a 100% identity to each other thus indicating that the isolated clones represented di¡erent regions of the same transcript. Sequence homology analysis revealed that the larger CV1 cDNA clone corresponded to the human ERGIC-53 cDNA region spanning from nt 311 to nt +970 therefore lacking most of the 3P region. Therefore, we repeated the CV1 cDNA library screening with a 979 nt cDNA probe corresponding to the human ERGIC-53 3P region spanning from nt 575 to nt 1554, obtained by digesting with HindIII, with the vector pECE-myc p53 [13]. After three rounds of screening a single clone hybridising with the 3P ERGIC-53 probe was plaque puri¢ed, V DNA was extracted and the insert subcloned and subjected
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to sequence analysis. This revealed high homology with the human ERGIC-53 cDNA region enclosed between nt 1350 and nt 2480. This cDNA clone thus contained part of the coding region and most of the 3P untranslated region. We next used reverse transcription PCR (RTPCR) to obtain the complete CV1 ERGIC-53 sequence. A ERGIC-53 transcript was previously detected in the human CaCo2 cell line [23]. We therefore used CaCo2 cells as source for positive control. Total mRNA was isolated from CV1 cells cultured in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 50 Wg/ml streptomycin and 2 mM Lglutamine. CaCo2 cells were cultured in the same medium supplemented with 20% FBS. RNA extraction was performed for both cell lines with the acidic phenol method [31]. 1 Wg of total mRNA extracted from CV1 cells was mixed with random primers and used in the reverse transcriptase reaction to obtain the template cDNA. The following oligomers were synthesised: primer 1, 5P-CCAAGATGGCGGGATCCA-3P corresponding to bp 35 to bp +13 of the monkey cDNA sequence; primer 2, 5P-GGCAGCTGCTTCTTGCGT-3P corresponding to bp 1522 to bp 1540 of the monkey cDNA sequence. Both primers were also chosen for their ability to recognise also the human ERGIC-53 sequence. A 50 Wl PCR reaction contained approximately 10 ng of the monkey cDNA as template, 15 WM of primer 1 and 2, 200 nM of each dNTP, 4 mM MgCl2 and 2.5 units of Pfu DNA polymerase. The reactions were performed with the thermal cycler (Promega). The cycle was of 120 s at 95³C followed by 30 cycles, each consisting of 60 s at 95³C, 60 s at 58³C, 120 min at 72³C. We ampli¢ed similar PCR reaction products from both CV1 and CaCo2 RNAs (Fig. 1). The size of both fragments was the one expected for a cDNA encoding the complete ORF of both the monkey and the human ERGIC-53. The fragments were therefore gel puri¢ed, subcloned in the TA cloning
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vector pCR2.1 (Invitrogen) and sequenced. Sequence comparison of the PCR products ampli¢ed from CV1 and CaCo2 RNAs showed an identity of 97.37%. Sequences of the shorter ampli¢cation products did not reveal similarities with ERGIC-53 or related cDNA sequences present in databases. Sequences obtained by the cDNA library screening were extended by RT-PCR analysis to obtain the ¢nal sequence of 2422 nts of the monkey ERGIC53 cDNA (Fig. 2). Translation of all six reading frames revealed that one of them contained the ATG start codon and the complete ORF of the monkey CV1 ERGIC-53 (Fig. 2). The 2480 nt sequence of the monkey ERGIC-53 cDNA was aligned to those of the human ERGIC-53, rat p58 and the partial Xenopus cDNA. Sequence analysis showed that the CV1 cDNA was 93.27% identical to the human ERGIC-53 cDNA. Lower similarity was revealed when it was compared to the rat p58 (76.3%) or the amphibian cDNA (55.49%). Similar results were obtained from the alignment of the predicted monkey (CV1) protein sequence with those of its relatives in other species. This analysis revealed that the monkey protein is 96.47% identical to the human ERGIC-53, 87% identical to the rat 58 and 66.98% identical to the Xenopus laevis protein. Both the human and monkey proteins have the same number (510) of amino acids. No di¡erences were found in the cytosolic tail sequence, indicating that the CV1 ERGIC-53 has the same recycling properties as its human homologue. Immuno£uorescence microscopy showed the typical localisation of the ERGIC-53 in the intermediate compartment of CV1 cells (data not shown). We next tested the presence of genes similar to ERGIC-53 by Southern analysis of genomic DNAs obtained from di¡erent species (Fig. 3). CV1 ERGIC probe corresponding to the region spanning from nt 311 to nt +970 was prepared as above. Filter (Clontech) was pre-hybridised for 3^4 h at 42³C and hybridised 16 h at 42³C in hybridisation bu¡er containing 5USSPE, 50% formaldehyde, 5UDenhart, 0.5%
6
Fig. 2. Nucleotide sequence of the monkey ERGIC-53 cDNA and deduced amino acid sequence of the protein. The complete coding region along with portion of the 5P- and 3P- non coding sequences is shown together with the predicted amino acid sequence in standard single letter code. The stop codon is indicated by an asterisk.
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SDS, 10% dextran sulphate, 100 Wg/ml salmon sperm DNA and 1.5U106 cpm/ml of DNA probe. Thus, we revealed the presence of sequences similar to ERGIC-53 in all the species analysed (Fig. 3: lanes 1^9). We found diversities in the intensity of the hybridisation signals which suggest di¡erences among the species. Hybridisation patterns obtained from most of the genomes (Fig. 3: lanes 1^3, 5^7, 9) suggest that genes similar to ERGIC-53 could be present in more than one copy in those species. Conversely, mouse and chicken DNAs revealed the presence of an individual signal (Fig. 3: lanes 4 and 8). By this analysis, we also detected the presence of a signal in genomic DNA derived from Saccharomyces cerevisiae (Fig. 3: lane 9). Computer search for sequence homology of the complete yeast S. cerevisiae database (MIPS Yeast Genome Database) failed to detect similarities with expressed sequences. However, weak homology (61%) was revealed with sequences present in yeast chromosome X. A length of about 5.5 kb was reported for the ERGIC-53 transcript by the analysis of mRNA extracted from human CaCo2 cells [23]. We therefore compared the expression of ERGIC-53 in CV1 and CaCo2 cells by the use of Northern blots (Fig. 4a). Cells were lysed in sodium acetate 50 mM, pH 5.2, SDS 1%, EDTA 5 mM and poly-A mRNA was
Fig. 3. Southern blot analysis of genomic DNA obtained from di¡erent species as indicated on top. Membrane was from Clontech laboratories. Each lane contained 5 Wg of EcoRI digested genomic DNA. Molecular weight size standards are reported on the left.
Fig. 4. Expression of ERGIC-53 RNA transcripts. (a) Analysis of the expression of ERGIC-53 in monkey CV1 cells: lanes 1 and 2; and human CaCo2 cells : lanes 2 and 3. Lane 1: 10 Wg total RNA from CV1 cells; lane 2: 1 Wg polyA RNA from CV1 cells ; lane 3: 10 Wg total RNA from CaCo2 cells ; lane 4: 1 Wg polyA RNA from CaCo2 cells. (b) ERGIC-53 mRNA expression in di¡erent human tissues as indicated on the top. Arrows indicate speci¢c ERGIC transcripts. Position of molecular weight size markers is indicated on the left. Membrane was from Clontech laboratories.
extracted by the use of oligo dT-cellulose (Clontech). 10 Wg of total or 1 Wg of poly-A RNA was fractionated on a formaldehyde-1% agarose gel and blotted on Hybond N nylon strips (Amersham). Comparative analysis of mRNA extracted from human CaCo2 cells and monkey CV1 cells revealed the presence of a 5.5 kb transcript in both cell lines (Fig. 4a: lanes 2 and 4). However, we detected also a smaller transcript with a similar size, 2.3 kb, in both species (Fig. 4a: lanes 2 and 4). Neither of the 5.5 or 2.3 kb transcripts were detectable in total RNA extracted from these cell lines (Fig. 4a: lanes 1 and 4)
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Similar results were obtained by analysing ERGIC-53 mRNA expression in di¡erent human tissues (Fig. 4b). Membrane (Clontech laboratories) was analysed with K-32 P-labelled CV1 ERGIC cDNA probe spanning from nt 311 to nt +970 prepared as above. Hybridisation was carried out at 42³C in 5USSPE, 50% formaldehyde, 5UDenhart, 0.5% SDS, 10% dextran sulphate, 100 Wg/ml salmon sperm DNA. 1USSPE was NaCl 180 mM, sodium phosphate 10 mM and EDTA 1 mM. Washes were carried out at low stringency conditions. We thus showed that the ERGIC-53 5.5 kb transcript was expressed in all the tissues analysed (Fig. 4b: lanes 1^8). Quantitative analysis of the expression levels revealed that the 5.5 kb transcript was expressed at similar levels in all the tissues analysed (data not shown). Conversely, a 2.3 kb transcript was detected only in mRNAs obtained from placenta and pancreas tissues (Fig. 4b: lane 3 and 8). The 2.3 kb mRNA was also detected with similar intensities when we hybridised the same ¢lters with ERGIC53 probes containing di¡erent portions of the coding region (data not shown). Therefore, the presence of the minor transcript of 2.3 kb could be due to a di¡erent RNA processing of the same transcript rather than to the expression of a related gene. Our data suggest the presence of genes related to ERGIC-53 in most of the species. Isolation and characterisation of ERGIC-53 related genes could provide additional information about the function of these proteins. Moreover, ERGIC-53 is ubiquitous and constitutively expressed and this is consistent with its supposed housekeeping function during the protein tra¤cking between the ER and Golgi. Acknowledgements We are indebted to Dr H.P. Hauri, Department of Pharmacology, University of Basel, Basel, Switzerland, for providing the plasmid pECE-myc p53. We thank Mr. B. Mugnoz for his technical assistance. This work was supported in part by C.N.R. (P.F. Biotecnologie), European Community (T.M.R. Programme) and M.U.R.S.T. (Prinn 1998).
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