Isolation and Characterization of a cDNA Encoding a Xenopus Immunoglobulin Binding Protein, BiP (Grp78)

Comp. Biochem. Physiol. Vol. 116B, No. 2, pp. 227–234, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0305-0491/97/$17.00 SSDI 0305-0491(96)00219-2

Isolation and Characterization of a cDNA Encoding a Xenopus Immunoglobulin Binding Protein, BiP (Grp78) Dragana Miskovic,1 Luisa Salter-Cid,2 Nicholas Ohan,1 Martin Flajnik, 2 and John J. Heikkila 1 1

Department of Biology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada, and 2 Department of Microbiology and Immunology, PO Box 016960 (R-138), University of Miami, Miami, FL 33101, U.S.A. ABSTRACT. We have isolated a full-length cDNA clone encoding a Xenopus laevis immunoglobulin binding protein (BiP; also called glucose-regulated protein or grp78). The BiP cDNA sequence includes an open reading frame of 1,965 bp encoding a 655 amino acid protein with an N-terminal hydrophobic leader sequence and a C-terminal KDEL tetrapeptide which has been found in other lumenal proteins of the endoplasmic reticulum. The 3′untranslated region contains a polyadenylation and an adenylation control element (ACE) as well as a putative mRNA instability sequence. The Xenopus BiP amino acid sequence displayed high identity with BiP from other vertebrates including chicken (91.3%), rat (90.7%), and human (89.9%). Northern hybridization analysis demonstrated that BiP mRNA was present constitutively in the Xenopus A6 kidney epithelial cell line and that BiP mRNA levels could be enhanced by treatment of the cells with galactose-free media, 2-deoxyglucose, 2-deoxygalactose, glucosamine, tunicamycin, heat shock, dithiothreitol, and the calcium ionophore, A23187. Finally, while BiP mRNA was detected in all of the adult tissues examined, the relative level of BiP mRNA differed dramatically between organs. For example, relatively high levels of BiP mRNA were detected in liver with moderate levels in testis, ovary and heart and reduced levels in eye and muscle tissue. Copyright  1997 Elsevier Science Inc. comp biochem physiol 116B;2:227–234, 1997. KEY WORDS. Xenopus, development, heat shock protein, immunoglobulin binding protein, mRNA, BiP, glucose-regulated protein, grp78, gene expression, chaperone

INTRODUCTION The heat shock protein (hsp) 70 family consists of related molecular chaperones which are localized to various cellular compartments including the cytosol, endoplasmic reticulum (ER), and mitochondria [reviewed in (20,24)]. The resident ER member of this family, immunoglobulin binding protein (BiP; also known as glucose-regulated protein 78 or grp78), comprises approximately 5% of the lumenal protein content [reviewed in (9,10,17,20,24)]. One function of BiP is to assist in the folding and assembly of newly synthesized proteins. BiP recognizes the hydrophobic sequences exposed on unfolded or unassembled proteins and prevents intra- or intermolecular aggregation. These new proteins are maintained until folding or the formation of oligomeric proteins (in an ATP-dependent manner). Several studies have shown that BiP can undergo posttranslational modifications such as ADP-ribosylation and phosphorylation. These modifications of BiP are associated with an inactive dimer while the active form is an unmodified monomer. Address reprint requests to: John J. Heikkila, Department of Biology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada. Received 28 April 1996; accepted 1 August 1996.

The expression of BiP genes in eukaryotic cells is enhanced under conditions of glucose starvation, inhibitors of glycosylation such as tunicamycin, sulfhydryl reducing reagents, and the calcium ionophore, A23187 (9,10,17,24). Mammalian BiP gene promoters have been characterized and contain a relatively complex set of cis-acting regulatory elements for both basal expression and stress induction (9,10,17,24). Mammalian BiP gene promoters contain two stress-responsive elements including a 36 bp grp core element, a number of CCAAT or CCAAT-like motifs needed to interact with upstream elements and a cAMP response element which is important for both basal and stress-induced expression. While BiP gene expression in mammalian cells has been the focus of intensive research, less is known regarding the basal and stress-induced expression of this gene in vertebrate poikilotherms. In previous studies, we and others have examined the expression of the cytosolic members of the hsp70 family, in the frog, Xenopus laevis (1–3,6,11,12,14, 15,26). We have now turned our attention to the resident ER member of this family, BIP. In earlier studies, we have characterized BiP synthesis in Xenopus embryos and in a Xenopus kidney epithelial cell line, A6 (40,41). While we

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were able to detect the presence of BiP mRNA, a complete characterization of BiP mRNA accumulation could not be carried out due to the use of a weakly hybridizing heterologous BiP genomic clone. In other studies, we found that microinjection of promoter deletion mutants of a rat BiP(grp78)/chloramphenicol acetyl transferase fusion gene into Xenopus embryos required the presence of similar promoter sequences for constitutive and tunicamycin-inducible expression as found in mammalian cells (38,42). This finding indicated that the regulatory elements associated with the Xenopus BiP promoter and mammalian BiP promoters may be conserved. However, this possibility cannot be verified since a Xenopus or any other amphibian BiP gene has not yet been isolated. In order to gain more information about BiP gene expression in amphibians, we have isolated and sequenced the first full length cDNA clone encoding Xenopus BiP. The predicted amino acid sequence was compared to mammalian and avian counterparts, as well as to members of the Xenopus hsp70 family. BiP mRNA levels were examined in various adult tissues and in Xenopus A6 cells treated with various agents that have been shown to induce BiP gene expression. MATERIALS AND METHODS Xenopus cDNA Library and Screening A cDNA library was prepared from RNA isolated from liver, spleen and thymus of adult Xenopus laevis, using the Uni-Zap system (Stratagene, La Jolla, CA) (7). The library was screened with a rat BiP cDNA clone [(31); kindly provided by M. J. Gething, University of Texas] as previously described (27,28) except that the hybridization was carried out under low stringency conditions (40% formamide, 63 SSC, 0.2% sodium dodecyl sulfate (SDS), 53 Denhardt’s solution and 200 mg/ml of denatured salmon sperm DNA). The filters were washed in 23 SSC and 0.2% SDS at 55°C. Subcloning and DNA Sequence Analysis BiP cDNA, originally cloned in pBluescript plasmid DNA, was digested with EcoRI, KpnI and PstI restriction endonucleases, which generated four fragments (0.7 kb, 0.5 kb, 0.35 kb and 0.65 kb). The fragments were isolated using DEAEcellulose membranes (29) and subcloned into the plasmid vector, pUC19 (Fig. 1). DNA sequence analysis was performed by the dideoxy chain termination method (30), using Sequenase version 2.0 T7 DNA polymerase and M13 universal and reverse primers (Amersham, Cleveland, OH, U.S.A.) and by automated DNA sequencing (Mobix, McMaster University, Hamilton, Ontario, Canada). The internal primers were custom synthesized (DNA synthesis facility at University of Guelph, Ontario, Canada, and at Mobix, McMaster University, Hamilton, Ontario, Canada). DNA sequence analysis was performed on an Apple Macin-

FIG. 1. The DNA sequencing strategy of the Xenopus BiP

cDNA clone. The boxes A, B, B1, B2 and C represent the subclones used for DNA sequencing. The shaded regions represent the isolated Xenopus BiP cDNA clone, and open boxes refer to the vector sequence. Arrows indicate the directions of the different sequencing reactions. The translational start (ATG) and stop (TAG) codons are labeled. E, EcoRI; P, PstI; K, KpnI.

tosh computer using the DNA strider 1.2 program. Comparison of the different sequences was carried out using the Genbank blastx program. The Genbank accession number for the Xenopus BiP cDNA nucleotide sequence is U55069. Maintenance of Xenopus A6 Cells The Xenopus laevis A6 kidney epithelial cell line was obtained from the American Type Culture Collection (Rockville, MD, U.S.A.). Cells were grown at 22°C in a medium consisting of 55% (v/v) Leibovitz L-15 medium, 10% (v/v) fetal bovine serum (both from Canadian Life Technologies, Inc., Burlington, Ontario) and 35% (v/v) sterile distilled water. Penicillin and streptomycin (both from Flow Laboratories, McLean, VA, U.S.A.) were added to final concentrations of 100 IU/ml and 100 mg/ml, respectively. To obtain glucose-starved cells, A6 cell media was replaced with galactose-free L-15 medium and maintained for 24, 48 and 72 hr at 22°C. A6 cells were also treated with various agents including calcium ionophore A23187 (7 µM), tunicamycin (1 µg/ml), and dithiothreitol (DTT; 0.1 mM) for a period of 24 hr at 22°C or heat shocked at 33°C for 1 hr. In the preparation of stock solutions, A23187 and tunicamycin were dissolved in ethanol and methanol, respectively, while the other agents used in this study were dissolved in sterile water. A6 cells grown in galactose-free media were also treated with either glucosamine (10 mM), 2-deoxyglucose (10 mM) or 2-deoxygalactose (10 mM) for 24 h at 22°C hr. RNA Isolation Total RNA was isolated from A6 cells or adult tissue using the GIT/CsCl centrifugation method (5), with some modifications as described by Ohan and Heikkila (25). Briefly, A6 cells were homogenized in 10 ml of 4 M guanidine isothiocyanate, and layered on top of 3.3 ml of 5.7 M cesium chloride solution. The samples were centrifuged in an SW41 Ti rotor (Beckman, Mississauga, Ontario), at 30K rpm

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for 23 hr. The RNA pellets were recovered and purified by two precipitations in ethanol on dry ice. Northern Hybridization Analysis Fifteen µg of total RNA was used for electrophoresis on 1.2% formaldehyde agarose gels (29), and then transferred to nylon membrane (ICN, Mississauga, Ontario). The Northern blots were UV cross-linked with a GS-Gene linker (Bio-Rad, Hercules, CA, U.S.A.). The hybridization reactions were performed as described previously (14), using nick translated 32P-labeled BiP cDNA, Xenopus cytoskeleton actin cDNA (pXLcA1) (19) or Xenopus hsp70 genomic probe (pXL16P) (3). The blots were washed under high stringency conditions and exposed to Kodak XAR-5 film at –70°C. In some of the experiments appropriately exposed autoradiograms (within the linearity range of the film) were scanned using an Apple Macintosh OneScanner, and the data were analyzed using NIH Image Version 1.55 software. For mRNA analysis, autoradiograms were scanned densitometrically over the area of interest, and the control was subtracted as a background value, allowing comparison of the signals in each sample lane. RESULTS A rat BiP cDNA was used to screen a Xenopus cDNA library. DNA isolated from one hybridizing plaque showed DNA sequence similarity at the 5′ and 3′ ends with BiP genes from other organisms, and was selected for further analysis. The restriction map and sequencing strategy utilized for this BiP cDNA clone is shown in Fig. 1. The DNA sequence of the putative BiP cDNA clone is displayed in Fig. 2. The open reading frame is 1965 bp in length and encodes a protein of 655 amino acids with a predicted molecular weight of 72,192. Examination of the 3′ untranslated region (UTR) revealed the presence of the consensus sequence for polyadenylation, AATAAA, as well as the adenylation control element (ACE; also called the cytoplasmic polyadenylation element or CPE), TTTTTAT, 275 and 160 nucleotides, respectively, downstream from the stop codon. We also detected the consensus element, TATTTA, which is thought to confer instability to a number of mammalian messages (4,31,39). A survey of the GenBank protein database revealed that the highest degree of identity of the Xenopus BiP amino acid sequence occurred with chicken, rat and human BiP proteins. A detailed comparison of the entire predicted amino acid sequence of Xenopus BiP with chicken (34), rat (21) and human BiP (36) is shown in Fig. 3. The Xenopus BiP amino acid sequence exhibited 91.3% identity with chicken, 90.7% with rat and 89.9% with human BiP. Also, we have compared the Xenopus BiP amino acid sequence with C. elegans (13) and yeast BiP (23) and found that there was 74 and 61.8% identity, respectively (data not shown).

FIG. 2. Nucleotide and amino acid sequence of Xenopus BiP

cDNA clone. The encoded amino acids are listed below the cDNA sequence. The stop codon is labeled with an asterisk. The polyadenylation consensus sequence (AATAAA), adenylation control element (TTTTTAT) and mRNA instability element (TATTTA) are underlined. The C-terminal KDEL sequence is shown in bold.

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FIG. 4. A comparison of the amino acid sequence of Xeno-

pus BiP with other members of the hsp70 family. Xenopus BiP (XBiP), hsc70.1 (Xhsc70.1) and hsp70B (Xhsp70B) were aligned and the amino acid identities are shown as dashes. Asterisks indicate amino acid deletions. FIG. 3. A comparison of the amino acid sequence of Xeno-

pus BiP with mammalian and avian BiP. Xenopus (XBiP), chicken (CBiP), rat (RBiP) and human (HBiP) BiP were aligned and the amino acid identities are shown as dashes. Asterisks represent amino acid deletions.

Most of the differences between Xenopus and the other three vertebrates occur in the amino and carboxyl terminal regions of the proteins. Compared to chicken, Xenopus BiP has 51 amino acid substitutions, 4 deletions and 6 additions. Furthermore, all 4 BiP proteins have a hydrophobic N-terminal leader sequence and share the carboxyl terminal ERretention sequence, KDEL, which is found in other lumenal ER proteins. Also, we have made a detailed comparison of the Xenopus BiP amino acid sequence with the amino acid sequences of the two cytosolic members of the Xenopus hsp70 family, namely hsp70 (3) and hsc70.1 (1) (Fig. 4). The identity of BiP with hsp70 and hsc70.1 is only 57 and 55.2%, respectively. Therefore, Xenopus BiP has greater amino acid sequence identity with BiP from other vertebrates than with other members of the Xenopus hsp70 family. This latter result was expected given previous studies in mammals and is detailed in the discussion. BiP gene expression was examined in a Xenopus kidney epithelial cell line, A6, and adult tissues by northern hybridization analysis. After hybridization of the RNA blots with labeled BiP cDNA, the blots were washed under stringent conditions to minimize any potential cross-reactivity with hsp70 or hsc70 transcripts. As shown in Fig. 5, BiP

FIG. 5. Effect of glucose starvation on the relative level of BiP mRNA in A6 cells. A6 cell media was replaced with galactose free L-15 medium and maintained for 24, 48 and 72hr at 22°C. Total RNA was isolated and subjected to Northern hybridization analysis using a 32P-labeled Xenopus BiP cDNA. The same RNA blot was stripped of labeled BiP probe and reprobed with Xenopus cytoskeletal actin cDNA. C, control; Gal2, galactose-free.

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fold). In separate experiments, we have also observed an increase in the relative level of BiP mRNA in A6 cells treated with 3% DMSO and 0.25% β-mercaptoethanol (data not shown). The RNA blots were also hybridized against a Xenopus hsp70 genomic clone (Fig. 6). Only the heat-shocked A6 RNA sample demonstrated hybridizable hsp70 mRNA, which was smaller (2.4 kb) than BiP mRNA (2.7 kb). While BiP mRNA was detected in each of the tissues studied, we observed a marked difference in the relative levels of BiP mRNA after comparing equivalent amounts of total RNA isolated from the different tissues (Fig. 7). For example, the highest relative level of BiP mRNA was detected in liver followed by testis, ovary and heart with reduced levels in eyes and muscle. DISCUSSION FIG. 6. Effect of various agents on the relative level of BiP

mRNA in A6 cells. A6 cells were either maintained in L-15 media (C) or treated with either-tunicamycin (T), A23187 (A), heat shock (HS), galactose-free medium (Gal2), glucosamine (Glc), 2-deoxyglucose (Dgl), 2-deoxygalactose (Dga) or dithiothreitol (DTT) as outlined in Materials and Methods. Total RNA was isolated and subjected to Northern hybridization analysis using a 32P-labeled Xenopus BiP cDNA (upper and lower panels). The RNA blot in the upper panel was stripped and rehybridized against the Xenopus hsp70 genomic clone (middle panel).

mRNA accumulation (2.7 kb) was detected constitutively in Xenopus A6 cells. Also, this figure demonstrates the effect of glucose starvation on the level of BiP mRNA. A6 cells in galactose-free media displayed an increase in the relative level of BiP mRNA after 48 to 72 hr of exposure. These RNA blots were subsequently reprobed with a Xenopus cytoskeletal actin cDNA and found to contain relatively constant levels of actin mRNA. These latter results together with uniform levels of ethidium bromide staining RNA in formaldehyde-agarose gels indicates that the changes in the relative levels of BiP mRNA were not the result of unequal RNA loading. Also, we have examined the effect of various agents, shown to enhance BiP gene expression in mammalian cells, on the relative level of BiP mRNA in Xenopus A6 cells. The optimal concentrations of the various agents employed in this study were based on preliminary studies in this system (40). As displayed in (Fig. 6), the largest increase in the accumulation of BiP mRNA relative to control, as determined by densitometric analysis, occurred in A6 cells treated with 2-deoxyglucose (4.2-fold) and dithiothreitol (5.6-fold) followed by 2-deoxygalactose (3.3-fold), and the calcium ionophore, A23187 (3.4). Moderate increases in the relative level of BiP mRNA accumulation compared to control were found with cells treated with tunicamycin (2.1-fold), heat shock (2.2-fold) and glucosamine (2.0-

In the present study we have isolated and sequenced a full length cDNA for the Xenopus laevis immunoglobulin binding protein, BiP. The nucleotide sequence contains an open reading frame that encodes a 70, 192-Da protein, which has an identity of approximately 90% with chicken, rat and human BiP. The conservation of the amino acid sequence is maintained along the length of the protein except for the amino and carboxyl terminal regions. Previous studies have shown that BiP from a particular species has a higher identity with BiP from other organisms than with members of the hsp70 family from the same species (10). Similarly, the identity between Xenopus BiP and Xenopus hsp70 and hsc70.1 was lower at 57 and 55.2%, respectively. Thus, Xenopus BiP has greater identity with BiP from other vertebrates than with the cytosolic members of the Xenopus hsp70 family. For example, hamster and rat BiP share 98% sequence identity while hamster BiP and hsp70 have only 62% sequence identity. It has been suggested that BiP genes shared a common ancestor, which diverged from other hsp70 genes at the time of the first appearance of eukaryotes (23). The N-terminal regions of Xenopus and chicken BiP are quite different having only 43% identity in the first 30 amino acids. Nevertheless, the N-terminal amino acid se-

FIG. 7. Relative levels of BiP mRNA in different adult Xeno-

pus tissues. Total RNA was isolated from selected adult tissues and subjected to Northern hybridization analysis employing the labeled Xenopus BiP cDNA probe. L, liver; M, muscle; H, heart; E, eyes; T, testis; O, ovary.

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quence of Xenopus BiP is still typical for a secretory leader sequence having a positively charged second amino acid (lysine) and hydrophobic amino acids thereafter as well as nonpolar amino acids at positions 12 and 14 (Figs 2 and 3) (21,34). In comparison with chicken and hamster BiP, it is likely that cleavage of the signal peptide of Xenopus BiP occurs after the valine in position 14 (21,34). All members of the hsp70 family have ATPase activity and bind ATP in the N-terminal portion of the protein, following the leader sequence. The amino acid sequence identities between the ATP-binding domains of Xenopus BiP, hsc70 and hsp70 as well as with mammalian and avian BiP are quite high suggesting that Xenopus BiP has ATPase activity. Gaut and Hendershot (8) have shown that 3 specific amino acids in the ATP-binding domain of BiP were involved in the ATPrelease of proteins. Examination of our amino acid sequence data revealed the corresponding residues, namely, Thr-37, Glu-199 and Thr-227 in Xenopus BiP (Fig. 3). Xenopus, chicken, rat, and human BiP share the carboxyl terminal sequence, KDEL, which has been reported in other soluble resident ER proteins such as glucose-regulated protein 94 and protein disulfide isomerase (10,21). It has been suggested that ER proteins containing this signal are retrieved from post-ER compartments via interaction with a specific receptor (10). As mentioned previously, the carboxyl portion of Xenopus BiP is significantly different from chicken BiP. For example, in a comparison of Xenopus and chicken BiP, the identity in the the carboxyl end (amino acids 603–655) is 73%. The difference between Xenopus BiP and hsp70 and hsc70.1 is even greater in this region. The C-terminal portion of the hsp70 family of proteins appears to be involved in peptide-binding activity (9,10). Thus, the differences between BiP and hsp70 in this are to be expected, because the two Xenopus hsp70 family members are localized to different subcellular compartments and probably interact with different sets of unfolded or malfolded proteins. In the 3′ UTR of BiP mRNA, we have detected the polyadenylation consensus sequence, AAUAAA, and the adenylation control element (ACE), UUUUUAU. In Xenopus immature oocytes, mRNAs require the presence of a poly(A) tail to be translated (33,37). After oocyte maturation, the germinal vesicle breaks down and releases deadenylation factors which deadenylates all mRNAs without the ACE sequence while those mRNAs possessing the ACE sequence are polyadenylated with a cytoplasmic poly(A) polymerase (33,37). Thus, it is likely that Xenopus BiP mRNA is not efficiently translated during oogenesis until after oocyte maturation. Finally, a mRNA instability element, UAUUUA, was also found in the 3′ UTR of BiP mRNA. This sequence element has been shown to be involved in mRNA instability in interferon, c-fos and c-myc mRNAs (4,31,39). This sequence is also present in Xenopus hsp30C mRNAs, which are unstable at control temperatures (14,16). At this time it is not known whether this instability sequence in Xenopus BiP mRNA is functional.

An examination of BiP mRNA levels in Xenopus A6 kidney epithelial cells revealed that this message was present constitutively and that its levels were enhanced by a variety of agents or treatments that have been shown to induce BiP gene expression in mammalian cells (9,17,24). For example, exposure of A6 cells to galactose-free media, tunicamycin, 2-deoxyglucose, 2-deoxygalactose, glucosamine, dithiothreitol, the calcium ionophore A23187, or heat shock resulted in an increase in the relative level of BiP mRNA. In a previous study we did not observe any detectable enhancement of BiP mRNA levels in A6 cells in response to glucosamine or A23187 (40). However, in this earlier study we utilized a heterologous BiP genomic probe that did not hybridize very strongly to Xenopus BiP mRNA. The treatments employed in the present study, which enhanced the relative level of BiP mRNA, may ultimately lead to an increase in the level of unfolded or denatured protein (9,10). It has been suggested that prolonged association of unfolded protein with BiP results in a drop in the level of active BiP, which then initiates a feed-back regulatory signal ultimately activating BiP gene transcription (9,10). Also, it is possible that BiP gene expression may be regulated at the level of mRNA stability given our finding of an mRNA instability sequence in the 3′UTR. Heat shock has been shown to stabilize hsp mRNAs including Xenopus hsp30 mRNA (25). It has been suggested that inhibition of protein synthesis inactivates the mechanism(s) responsible for mRNA degradation (22,25,32). Thus, it is possible that heat shockinduced inhibition of protein synthesis may result in the stabilization of BiP mRNA. In support of this possibility, recent studies have shown that energy restriction may result in the destabilization of BiP mRNA in mouse liver (35). Finally, we have examined the relative levels of BiP mRNA in selected tissues of adult Xenopus. While constitutive levels of BiP mRNA were detected in all tissues examined, the relative levels differed dramatically between certain tissues. For example, liver displayed a very high level of BiP mRNA while muscle tissue had relatively low levels. These findings suggest that the expression level of the BiP gene is adapted to the cellular requirements of a particular tissue. It is possible that liver cells contain a relatively high level of protein destined for the ER, which require proper folding and assembly by BiP. This idea is feasible given the number of proteins continuously secreted by the liver such as albumin, transferrin, and lipoproteins (18). This research was supported by a Natural Sciences and Engineering Research Council of Canada grant to J.J.H. and a National Institutes of Health grant (A127877) to M.F.F.

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