(Received for publication, March 17, 1997)
From the Department of Medical Chemistry, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan
We have identified and characterized a cDNA encoding a novel Ca2+-binding protein named calumenin from mouse heart by the signal sequence trap method. The deduced amino acid sequence (315 residues) of calumenin contains an amino-terminal signal sequence and six Ca2+-binding (EF-hand) motifs and shows homology with reticulocalbin, Erc-55, and Cab45. These proteins seem to form a new subset of the EF-hand protein family expressed in the lumen of the endoplasmic reticulum (ER) and Golgi apparatus. Purified calumenin had Ca2+-binding ability. The carboxyl-terminal tetrapeptide His-Asp-Glu-Phe was shown to be responsible for retention of calumenin in ER by the retention assay, immunostaining with a confocal laser microscope, and the deglycosylation assay. This is the first report indicating that the Phe residue is included in the ER retention signal. Calumenin is expressed most strongly in heart of adult and 18.5-day embryos. The calumenin gene (Calu) was mapped at the proximal portion of mouse chromosome 7.
The endoplasmic reticulum (ER)1 is involved in synthesis and modification of secretory and membranous proteins as well as resident proteins in the lumen of the ER, Golgi apparatus, or lysosomes (1). The ER is also known as the major Ca2+ storage compartment in eukaryotic cells. By pumping cytosolic Ca2+ into the ER lumen, cells keep their cytosolic concentration of free Ca2+ at extremely low levels so that they can use Ca2+ as intracellular signal (2). Besides, the ER itself needs luminal Ca2+ for its normal functions such as protein folding and protein sorting (3-6). Among many ER-resident proteins, endoplasmin (GRP94) (7, 8), Bip (GRP78) (9, 10), protein-disulfide isomerase (ERp59) (11, 12), and calreticulin (CRP55) (13) are Ca2+-binding proteins that are associated with Ca2+-dependent folding and maturation of secretory proteins in the ER lumen (14, 15). In addition, two Ca2+-binding ER-resident proteins, reticulocalbin (16) and Erc-55 (17), have been isolated recently. They have multiple EF-hand motifs and constitute a new subset of the EF-hand superfamily, together with a homologous protein in the lumen of the Golgi apparatus, Cab45 (18). However, their physiological functions are still unknown.
ER-resident proteins generally carry a retention signal at their carboxyl terminus. This ER retention signal is first identified to be tetrapeptides, Lys-Asp-Glu-Leu (KDEL) in mammalian cells and His-Asp-Glu-Leu (HDEL) in yeast (19, 20). Soluble ER-resident proteins are trapped by binding to the KDEL receptor expressed in the cis-Golgi and retrieved to the ER (21-23). Further studies have demonstrated that HDEL and several variants of the KDEL sequence can also work as the ER retention signal in mammalian cells (24, 25). Comparison of variants of the KDEL sequence suggests that the replacement of Lys and Asp residues with other amino acid residues does not abolish the ER retention activity. However, the carboxyl-terminal two residues are considered to be critical because the third and fourth positions in all of the ER retention signals are Glu/Asp and Leu/Ile, respectively.
During the embryogenesis, the heart begins to beat already in the 8.5-day mouse embryo, whose heart is still a two-chambered tube with one atrium and one ventricle (26). Beating of cardiac myocytes is maintained by the strict regulation of their cytoplasmic Ca2+ concentration. To achieve this regulation, cardiac myocytes develop their specialized ER, called the sarcoplasmic reticulum, as the Ca2+ storage compartment and produce the rhythmical Ca2+ oscillation between the sarcoplasmic reticulum and the cytosol (27). Molecules involved in this Ca2+ oscillation are reported to be present very early in mouse cardiogenesis (28, 29).
Since we are interested in molecules involved in heart embryogenesis, we screened in a signal sequence trap library (30, 31) of mouse embryonic heart to isolate cDNAs encoding amino-terminal hydrophobic signal sequences. We here report cloning and characterization of cDNA encoding calumenin that binds Ca2+ and carries a new ER retention signal, HDEF, at the carboxyl terminus. Calumenin is a novel member of the reticulocalbin family, a new subset of the EF-hand superfamily in the ER. Calumenin is most strongly expressed in the heart of adult and 18.5-day embryos.
Poly(A) RNA from approximately 100 hearts of 9.5-day postcoitus (dpc) mouse embryos was extracted with TRIzol reagent (Life Technologies, Inc.) and OligotexTM-dT30<Super> (Roche). cDNA was synthesized from 1.35 µg of poly(A) RNA using Super Script II (Life Technologies, Inc.). First strand cDNA was synthesized with 25 pmol of URPX3 primer: GAG-ACG-GTA-ATA-CGA-TCG-ACA-GTA-GCT-CGA-GXX-XXX-XXX-X (where X represents one of the following: A, G, C, or T). After alkali lysis of RNA and the poly(A) tailing procedure, second strand cDNA was synthesized with 25 pmol of ESTN primer: CCG-CGA-ATT-CTG-ACT-AAC-TGA-(T)17XX. Then cDNA of 400-800 base pairs were fractionated by agarose gel electrophoresis and subjected to polymerase chain reaction (PCR) using ExTaq (TaKaRa, Japan) and 25 pmol of ESP primer (CCG-CGA-ATT-CTG-ACT-AAC-TGA-TT) and Ad-P1 primer (GAC-GGT-AAT-ACG-ATC-GAC-AGT-AGG) under the following conditions for Thermal-Cycler (TaKaRa, Japan): 94 °C for 5 min and then 94 °C for 45 s, 52 °C for 60 s, and 72 °C for 2 min for 25 cycles and 72 °C for 10 min. After cloning cDNA unidirectionally between the EcoRI and XhoI sites of pSuc2t7F1ori vector, screening procedures were performed as described (31).
To clone full-length cDNA, the 3-rapid amplification of cDNA
ends (RACE) method was performed with LAtaq (TaKaRa). Mouse 13.5-dpc
embryonic heart cDNA library in Uni-ZAP XR vector was used
(Stratagene). C39N1b outer primer (biotinylated)
(GTG-GAG-CTC-CCG-GGA-AAG-GTT-ATC-ATG) and C39N2 inner primer
(CAT-GGA-CCT-GCG-TCA-GTT-TC) were designed as gene-specific primers and
used with standard T7 primer (CGC-GTA-ATA-CGA-CTC-ACT-ATA-GGG-C). Conditions adopted for Perkin-Elmer 9600 were as follows: 95 °C for
2 min and then 95 °C for 30 s, 68 °C for 30 s, and
72 °C for 2 min for 25 cycles. The reaction mixture of first PCR
with C39N1b and T7 primers was subjected to purification with M280
Dynabeads (Dynal) to decrease backgrounds due to nonspecific
amplification by T7 primer. Second PCR was performed with C39N2 and T7
primers using the purified mixture. For sequencing, amplified cDNA
was cloned into pGEM-T vector (Promega).
Sequencing was performed with an automated sequencer (model 373A; Applied Biosystems), and sequence analysis was done with the computer analysis program, GeneWorks (IntelliGenetics, Inc.). Homology search was performed with BLAST and FASTA, using GenBankTM and EMBL as DNA data bases and PRF, PIR, and SwissProt as protein data bases. Motif search and localization analysis were performed on line at Prosite.
Expression and Purification of ProteinsTo express proteins
in mammalian cells, cDNAs were cloned in XbaI site of
pEF-BOS expression vector (32), and their sequences were confirmed
before assays. cDNAs of FLAG-calumenin (FLAG-C39), FLAG-calumenin-HDEF, and FLAG-calumenin-rHDEL were constructed with
PCR. FLAG epitope (8 amino acids; DYKDDDDK) was incorporated 5 amino
acids downstream of the putative signal sequence cleavage site. The
following primers were used for PCR: FLAG-calumenin primer,
AAG-CCT-ACT-AGT-ATG-GAC-CTG-CGT-CAG-TTT-CTT-ATG-TGC-CTG-TCC-CTG-TGC-ACA-GCC-TTT-GCT-TTG-AGC-AAG-CCT-ACA-GAA-GAC-TAC-AAG-GAC-GAC-GAT-GAC-AAG-AAG-AAG-GAC-CGA-GTA-CAC-CAT-GAG-C; calumenin-HDEF primer,
AAG-CCT-ACT-AGT-TCA-GAA-CTC-ATC-ATG-TCG-TAC-TAA-GG; calumenin-
HDEF
primer, AAG-CCT-ACT-AGT-TCA-TCG-TAC-TAA-GGC-CTC-CCC; and
calumenin-rHDEL primer,
AAG-CCT-ACT-AGT-TCA-CAA-CTC-ATC-ATG-TCG-TAC-TAA-GG. Proteins were
purified from transfected cells according to the standard protocol for
immunoprecipitation, using lysis buffer (150 mM NaCl, 1%
Triton X-100, 10 mM Tris-HCl, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin) and
the anti-FLAG affinity gel (Eastman Kodak Co.).
Mouse anti-FLAG M2 antibody (Kodak), rabbit anti-protein-disulfide isomerase antibody (Stress Gen Biotechnologies), fluorescein isothiocyanate-conjugated affinity-purified goat anti-mouse IgG (H + L) (absorbed with human and rabbit serum) (Kirkegaard & Perry Laboratories Inc.), and rhodamine-conjugated affinity-purified goat anti-rabbit IgG (minimal cross-reaction to mouse IgG) (Biosource International) were used.
Other MethodsThe procedures used to stain cells were
essentially as described (18, 33). Stained cells were analyzed with a
confocal scanning laser microscope (LSM410 UV, Carl Zeiss Inc.)
following the protocol for double stain by fluorescein isothiocyanate
and rhodamine. The Slow Fade Antifade kit (Molecular Probes, Inc.) was
used to prevent photobleaching. Retention of calumenin in ER was
assayed as follows. Cells transfected with FLAG-calumenin and
FLAG-calumenin-HDEF were further incubated for 4 h in
serum-free medium. The media were then concentrated using Centricon-30
(Amicon). Cell extracts and concentrated media were analyzed by 12%
polyacrylamide gel electrophoresis with SDS followed by Western
blotting. The reverse transcriptase-PCR method. After first strand
synthesis using random 9-mer, PCR was performed with C39N3 primer
(GGA-AGA-TGG-ACA-AGG-AAG-AGA-CC) and C39C1b primer (biotinylated)
(AGA-GTT-GTT-CCT-CGG-AAG-CTC-G). Transfection of COS-7 cells was
performed using lipofectamine (Life Technologies) according to the
manufacturer's protocol. Protein synthesis was stopped before protein
purification and immunostaining assay by treating cells with 300 µM cycloheximide for 2 h (34). The filter for
Northern blot was prepared as described (35) and hybridized using
Quick-Hyb solution (Stratagene). The 45Ca2+
binding assay was performed as described (36). Membranes of 45Ca2+ binding assay and Northern blotting was
analyzed using an image analyzer (BAS 2000, Fuji Film). Deglycosylation
assay was done as described (18) and analyzed by SDS-polyacrylamide gel
electrophoresis and Western blotting. For Western blotting, ECL Western
blotting detection reagents (Amersham Life Science, Inc.) was used.
Chromosomal mapping of the calumenin gene was done as described (35,
37).
4.4 × 105 yeast transformants from 9.5 dpc embryonic heart
cDNA library were screened, and 386 positive clones were obtained by the signal sequence trap method described previously (30, 31). Among
these, nucleotide sequences of 17 clones were not reported in any
mammalian species, 15 clones were homologous to sequences reported in
mouse or other mammals and the rest were redundant clones. One of the
novel clones (C39) was picked up for further studies because its
mRNA was most strongly expressed in heart (see below). The
full-length cDNA of C39 was isolated from a 13.5 dpc embryonic
heart cDNA library by the 3-RACE method. Nucleotide sequences of
two independent clones were determined to avoid sequence errors and
shown to encode a 315-amino acid protein (Fig.
1A). The translation start site was assigned
at nucleotide positions 36-38 because of the presence of an
NH2-terminal signal sequence and the comparison with other
family members to be described below.
The C39 Protein Is a Ca2+-binding Protein
cDNA sequence showed that the C39 protein does not
have any other hydrophobic stretch long enough to anchor the protein in the membrane (Fig. 1B). The C39 protein carries six
potential Ca2+-binding EF-hand motifs and a putative
N-glycosylation site. Homology search with the BLAST and
FASTA computer program revealed the presence of many
Ca2+-binding proteins homologous to the C39 protein in the
EF-hand region. Among these, reticulocalbin (16) and Erc-55 (17) in the
ER and Cab45 (18) in the Golgi apparatus had homology in size and
sequence to the C39 protein even outside of the EF-hand region (Fig.
2). These proteins appear to form one subfamily of Ca2+-binding proteins.
Each EF hand in the C39 protein has the general feature for high
affinity Ca2+-binding according to Kretsinger's rule (38);
the presence of the helix-loop-helix motif in which 5 oxygen-containing
residues and the central glycine are conserved for coordination of
Ca2+ binding (Fig. 3A). Although
the central glycine of the third motif is replaced by leucine, the same
replacement was seen in other Ca2+-binding proteins (39,
40) and considered to retain the Ca2+-binding activity by
the secondary structure prediction of computer analysis (41).
To test whether six EF hands in the C39 protein really have Ca2+-binding ability, the 45Ca2+ binding assay was performed as described (36). Strong signal was detected in the FLAG-C39 fusion protein but none in the FLAG-bacterial alkali phosphatase fusion protein (Fig. 3B). The positive band was approximately 57 kDa in size, which was equal to the size of the FLAG-C39 fusion protein detected by Amido Black staining, indicating that EF hands in the C39 protein indeed have Ca2+ binding ability.
Subcellular Localization of the C39 ProteinTo determine the
intracellular localization of the C39 protein, COS-7 cells transfected
with FLAG-C39 were doubly stained with an anti-FLAG antibody and an
anti-protein-disulfide isomerase antibody and observed under a confocal
laser microscope. The staining profile of anti-FLAG antibody (Fig.
4B) was almost identical to that of
anti-protein-disulfide isomerase antibody (Fig. 4A), showing diffuse ER staining patterns as reported previously for
protein-disulfide isomerase, a well characterized protein in the ER
(42). The identical staining patterns with both antibodies (Fig.
4C) indicate that the C39 protein is an ER-resident protein.
Since the C39 protein has Ca2+-binding ability and is
localized in the ER lumen, we named the C39 protein calumenin.
The HDEF Sequence Is a Novel ER Retention Signal
To examine
whether the C-terminal tetrapeptide HDEF of calumenin can serve as a
novel intracellular retention signal, we constructed two expression
vectors: FLAG-calumenin, a fusion protein of FLAG epitope and
calumenin, and FLAG-calumenin-HDEF, a fusion protein of the FLAG
epitope and calumenin lacking its C-terminal HDEF sequence. FLAG
epitopes were incorporated 5 amino acids downstream of the putative
signal sequence cleavage site in each construct. COS-7 cells were
transfected by these constructs, and concentrated media and cell
extracts were analyzed by Western blotting. Most of the
FLAG-calumenin-
HDEF protein was secreted into the medium, while
almost all of the FLAG-calumenin protein was retained in the cell (Fig.
5). These results indicate that HDEF is essential to
maintain calumenin within the cell.
We further examined intracellular localization of calumenin by
treatment with deglycosidases. Proteins translocated into the ER are
exposed to core glycosylation. At this moment, glycosylated proteins
are sensitive to the deglycosidase endoglycosidase H, which cleaves
high mannose oligosaccharides on proteins. However, once proteins are
transported into the medial Golgi and modified by Golgi-mannosidase II,
they become resistant to endoglycosidase H. However, Golgi proteins are
still sensitive to N-glycosidase F, a glycosidase whose
activity is not disturbed by the following carbohydrate modifications
in the medial- and trans-Golgi. Calumenin had one putative
N-linked glycosylation site. Affinity-purified FLAG-calumenin protein was incubated with either endoglycosidase H or
N-glycosidase F and subjected to Western blotting with the anti-FLAG M2 antibody. We also performed the assay against
FLAG-calumenin-rHDEL, whose C-terminal tetrapeptide HDEF was replaced
with HDEL, known as the ER retention signal. Both FLAG-calumenin and
FLAG-calumenin-rHDEL showed strong bands at 57 kDa without the
glycosidases (Fig. 6, lanes 1, 3,
5, and 7). Digestion with either endoglycosidase
H or N-glycosidase F shifted the size of the strong bands to
52 kDa (lanes 2, 4, 6, and
8). We consider that 57-kDa bands correspond to the
glycosylated form of calumenin, and 52-kDa bands correspond to the
deglycosylated form. Calumenin is indeed translocated and glycosylated
in the ER and not modified in the Golgi apparatus, because Endo H
completely cleaved oligosaccharides on FLAG-calumenin (lanes
2 and 6) and FLAG-calumenin-rHDEL (lanes 4 and 8). These results indicate that calumenin resides in the
ER and that the C-terminal tetrapeptide HDEF works as the ER retention
signal like HDEL (16, 17).
Expression and Chromosomal Localization of the Calumenin Gene
Northern blotting analysis showed that calumenin was
expressed ubiquitously in all tissues examined. However, the expression of calumenin was strong especially in heart and lung. Compared with
expression in adult heart, expression in 18.5-dpc heart was slightly
stronger (Fig. 7A). The reverse
transcriptase-PCR method showed that calumenin mRNA was already
expressed as early as 8.5 dpc (Fig. 7B).
To determine the chromosomal localization of the calumenin gene (Calu),2 strain distribution patterns of restriction fragment length polymorphisms of the calumenin gene were determined in 24 independent recombinant inbred strains derived from crosses between AKR/J and DBA/2J (AXD) (Table I). Analysis of the distribution pattern revealed the linkage of the calumenin gene with markers located at the proximal region of chromosome 7 (Fig. 8).
|
We
isolated and characterized a novel Ca2+-binding protein
calumenin from mouse embryonic heart. Calumenin is located in the ER
and homologous to previously reported Ca2+-binding proteins
such as reticulocalbin and Erc-55 in the ER (16, 17), and Cab45 (18) in
the Golgi apparatus. Since all of these proteins including calumenin
have six EF hands and Ca2+-binding activity, they
constitute one subset of the EF-hand superfamily. A data base search
also revealed that cDNA (SCF13) cloned as DNA supercoiling
factor of silkworm (43) also belongs to this family. The deduced amino
acid sequence of
SCF13 has an amino-terminal signal sequence, six EF
hands, and the C-terminal HDEF sequence, which we proved to be the ER
retention signal.
Although many ER Ca2+-binding proteins have been reported so far, their functions are largely not yet well understood. Two possible functions of ER Ca2+-binding proteins have been suggested. First, calreticulin, one of the Ca2+-binding proteins in the ER, is reported to regulate the capacity of Ca2+ in the ER (44, 45) as well as to have chaperone function (46, 47). Another luminal Ca2+-binding protein, calsequestrin, is also reported to regulate Ca2+ flow from the ER (48, 49). These reports suggest that Ca2+ fluxes may be regulated somehow by Ca2+-binding proteins located in the luminal side of the ER. Second, Ca2+ in the ER is reported to be necessary for normal functions of the ER such as protein folding and protein sorting (3-6), suggesting the existence of ER-resident molecules whose function is regulated by Ca2+. In fact, Bip, a Ca2+-binding protein in the ER, is reported to function in the Ca2+-dependent manner (50).
Further study is needed to determine whether or not calumenin is involved in either of the two possible functions. Recently, interaction molecules of Erc-55 have been reported: the E6 protein of papilloma virus, which has p53-independent tumorigenic activity (51), and taipoxin, which blocks neuromuscular transmission at the presynaptic site (52). As discussed above, calumenin belongs to the same subset of the EF-hand superfamily as Erc-55. Thus, the search for proteins that interact with calumenin could provide a clue to elucidate biological functions of this subset.
A Novel ER Retention SignalWe report a novel carboxyl-terminal tetrapeptide HDEF of calumenin as a new ER retention signal to prevent proteins from being secreted and to keep them in the ER. We observed that the HDEF sequence had the intracellular retention activity (Fig. 5). The deglycosylation assay (Fig. 6) and the immunostaining profile (Fig. 4) suggest that the HDEF sequence works as the ER retention signal. The C-terminal tetrapeptide KDEL is the first ER retention signal reported in mammalian cells (20). The C-terminal tetrapeptide HDEL, which was originally identified in yeast, was also found to work as the ER retention signal in mammalian cells (16, 17). Other reports show that the first and second positions of the C-terminal tetrapeptide are not strongly conserved to maintain the ER retention activity. In contrast, replacement of the third and fourth residues is strictly restricted. The third position requires an acidic residue such as Glu or Asp, and the fourth position requires a hydrophobic residue containing the aliphatic side chain such as Leu or Ile, which cannot be replaced by either Val or Ala (24, 25). Since Cab45, a soluble protein in the Golgi apparatus has HEEF sequence at the C terminus, this tetraresidue sequence was presumed to serve as the Golgi retention signal. The C-terminal Phe, a hydrophobic residue containing an aromatic side chain, was thought to work somehow differently from Leu (18). However, since our study clearly showed that the HDEF sequence worked as the ER retention signal, the retention signal to the Golgi apparatus may be localized in another portion of Cab45.
Proteins with ER retention signals such as KDEL are retrieved from Golgi. The convincing mechanism of this retrieval is that such proteins interact with the KDEL receptor distributing in the cis-Golgi and are retrieved to the ER (22, 23). We showed that the C-terminal HDEF sequence worked as the ER retention signal, but further investigation is needed to determine whether HDEF interacts with the KDEL receptor or with an unknown receptor.
Chromosomal Mapping of the Calumenin Gene (Calu)The calumenin gene was mapped on the proximal portion of mouse chromosome 7. This region is well conserved between human and mouse, and the syntenic comparison suggests that this region corresponds to human 19q13.2-13.3 (53), at which the gene for progressive familial heart block type I, autosomal dominantly inherited cardiac bundle-branch disorder, is mapped (54). Since the causal gene of this disease has not been determined so far, further investigation is needed to determine whether the calumenin gene is involved in this disease.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U81829.
We thank Dr. T. Nakano very much for the suggestion of constructing a cDNA library for the signal sequence trap method. We also thank N. Tomikawa for technical assistance, and Y. Horiike for secretarial help.