(Received for publication, July 27, 1995; and in revised form, September 5, 1995)
From the
We have shown that the 105-kDa heat shock protein (HSP105) and
the 42 °C-specific heat shock protein (42 °C-HSP) constitute
high molecular mass heat shock proteins. To elucidate the structure of
these heat shock proteins, we have screened a cDNA library constructed
with poly(A) RNA derived from mouse FM3A cells
preheated at 42 °C for 2 h using an antibody against murine HSP105.
Two full-length cDNA clones were obtained: the pB105-1 insert encoded
an 858-amino acid protein, and the pB105-2 insert encoded an 814-amino
acid protein and lacked 44 amino acids found in pB105-1. The two clones
contained the amino acid sequence found in the 17-kDa polypeptide
fragments from HSP105 and 42 °C-HSP by lysylendopeptidase
digestion. In vitro translation products of the RNA
transcripts from pB105-1 and pB105-2 migrated to the same positions of
HSP105 and 42 °C-HSP, respectively, on SDS-polyacrylamide gel
electrophoresis. Northern blot analysis showed that the transcript was
4 kilobases in murine FM3A cells and was strongly induced by heat
shock and by treatment with arsenite or an amino acid analog. By
reverse transcription-polymerase chain reaction analysis using primers
by which deletion of 132 nucleotides in pB105-2 could be detected, the
polymerase chain reaction product corresponding to pB105-2 was
increased only after heat shock at 42 °C, whereas the product
corresponding to pB105-1 was induced by heat shock at either 42 or 45
°C and also by other stresses. Thus, the cDNA clones pB105-1 and
pB105-2 encode HSP105 and 42 °C-HSP, respectively, and HSP105 and
42 °C-HSP (a short form of HSP105) are suggested to be produced by
alternative splicing. Here, HSP105 and 42 °C-HSP are renamed
HSP105
and HSP105
, respectively. A protein sequence homology
search revealed that HSP105 shares 54, 34, and 25% amino acid identity
with human HSP70RY, the sea urchin egg receptor for sperm, and murine
inducible HSP70, respectively. Furthermore, by Northern blot analysis,
HSP105 mRNA was revealed to be present in most murine tissues and to be
highly expressed in the brain.
Upon exposure to heat shock, living cells from bacteria to
humans synthesize a set of proteins called heat shock proteins (HSPs). ()Since HSPs are also induced by a variety of stresses,
these proteins are also called stress proteins. However, HSPs are
expressed in considerable amounts in nonstressed cells. HSPs are
involved not only in cell protection and repair of cell damage caused
by a variety of stresses, but also in normal cellular functions (for
recent reviews, see (1, 2, 3) ). These
proteins have been classified into several families according to their
apparent molecular mass: high molecular mass HSP, HSP90, HSP70, HSP60,
HSP47, and low molecular mass HSP. The families such as HSP70, HSP60,
and HSP90 have been studied extensively. These proteins are found to
interact with other proteins to mediate protein folding, unfolding,
assembly, and disassembly of proteins as molecular chaperones.
The members of the mammalian high molecular mass HSP family are not well characterized, although yeast HSP104 has been cloned and sequenced (4, 5) . Deletion of the HSP104 gene has little effect on growth at a normal temperature, and the cells die at the same rate as wild-type cells when exposed to a high temperature. However, the mutant cells do not acquire tolerance to heat and other forms of stress (4, 5, 6) . Interestingly, HSP104 exhibits homology to bacterial ClpA/ClpB proteins, which appear to play a role in an ATP-dependent protein degradation process(7) . Recently, ClpA was found to function as a molecular chaperone like DnaK(8) .
The 110-kDa HSP (HSP110) from Chinese hamster ovary cells (9, 10) and 105-kDa HSP from murine cells (11, 12) have been reported to be the mammalian high molecular mass HSP. Chinese hamster HSP110 was found to be localized in the nucleoli of nonstressed and heat-stressed murine cells by indirect immunofluorescence(9) . Since HSP110 is associated with actively transcribed rRNA genes by immunoelectron microscopy, HSP110 was inferred to participate in ribosome assembly(10) . On the other hand, by indirect immunofluorescence, we have shown that HSP105 is localized to the cytoplasm and nuclei under both nonstressed and stressed conditions in murine cells and has never been found in the nucleoli, unlike HSP110(11) . Furthermore, we have found a specific 90-kDa HSP that is synthesized only when mammalian cells are heat-shocked continuously at 42 °C (42 °C-HSP)(13) . An anti-HSP105 serum reacts not only with HSP105, but also with 42 °C-HSP(11, 14) . By amino acid sequence analysis, HSP105 and 42 °C-HSP were found to be similar proteins containing a particular sequence similar to an adenosine-binding domain of HSP70 family proteins and actin(12) .
Herein, we report the cloning of HSP105 and 42 °C-HSP from murine cells and reveal that HSP105 and 42 °C-HSP are highly homologous to human HSP70RY and moderately homologous to the sea urchin egg receptor for sperm and less so to murine inducible HSP70. The N-terminal half of these proteins contained an ATP-binding domain similar to that of HSP70 family members and was more conserved among these proteins. Northern blot analysis of various murine tissues revealed that HSP105 mRNA was present in most murine tissues and was highly expressed in the brain.
Figure 1: Nucleotide sequence and predicted amino acid sequence of pB105-1. The top line shows the nucleotide sequence of the cDNA for HSP105, and the second line shows the predicted amino acid sequence. The shaded amino acids were determined by amino acid sequencing of HSP105 family members. The boxed nucleotides represent the spliced region of pB105-2. The underlined amino acids indicate the predicted ATP-binding domain of HSP70 family members. The nucleotides underlined by broken lines were used as primers for RT-PCR.
Figure 2: Amino acid sequence homology between mouse HSP105 and human HSP70RY, sea urchin egg receptor, and mouse HSP70. Region I of mouse HSP105, which contained an ATP-binding domain, is highly similar in these four proteins, whereas region III is similar in HSP105, HSP70RY, and the sperm receptor, and the identity of these regions is shown in the respective boxes. Region II represents the spliced-out region in pH105-2. The identity between HSP105 and other proteins is shown at the right. Numbers on the top indicate the positions of amino acid residues for each protein.
Murine HSP105 was 54% identical to human HSP70RY (23, 24) and 34% to the sea urchin sperm receptor (25) and only 25% to murine inducible HSP70(26) . In general, HSP70 family members are conserved in the N-terminal ATP-binding domain and less so in the C-terminal putative peptide-binding domain(27, 28, 29) . The N-terminal 500-amino acid sequence of HSP105 that contained an ATP-binding domain was more homologous to those of human HSP70RY (72% identity) and the sea urchin egg receptor (50%) and less to that of murine HSP70 (32%). Another region between positions 612 and 713 of HSP105 was also highly homologous to HSP70RY (amino acids 594-695; 66% identity) and the sea urchin sperm receptor (amino acids 649-750; 49% identity), but the homologous region was not found in murine inducible HSP70.
Figure 3:
Transcription and translation of pB105-1
and pB105-2. A, 20 µg of cell extracts from control FM3A
cells (lane 1) or cells heat shocked at 42 °C for 4 h (lane 2) was subjected to 8% SDS-PAGE and detected
immunologically using anti-HSP105 antibody. B, linearized
pB105-1 (lane 3) and pB105-2 (lane 2) were
transcribed in vitro with T3 RNA polymerase, and the
translation products were translated in rabbit reticulocyte lysate in
the presence of [S]methionine. Aliquots (3
µl) of translation mixture (25 µl) were subjected to 8%
SDS-PAGE and visualized by autoradiography. Lane 1 represents
the translation product without these plasmids. Molecular masses of
marker proteins are shown at the left, and the upper and lower arrows indicate the positions of HSP105 and 42
°C-HSP, respectively.
Figure 4:
Stress inducibility of HSP105 mRNA in FM3A
cells. A, FM3A cells were incubated at 42 °C for 0, 15,
30, 60, 120, and 300 min (lanes 1-6) or were incubated
at 37 °C for 15, 30, and 60 min after heat shock at 42 °C for
60 min (lanes 7-9). B, cells were treated
without (lane 1) or with heat shock at 45 °C for 15 min
and then incubated at 37 °C for 0, 1, 3, and 6 h (lanes
2-5). C, cells were treated with 100 µM sodium arsenite for 0, 1, 3, and 6 h (lanes 1-4). D, cells were treated with 20 mM azetidine-2-carboxylic acids for 0, 3, 6, and 12 h (lanes
1-4). Total RNAs were extracted from these stress-treated
FM3A cells, and 10 µg of RNA was electrophoresed on 1% agarose
containing 6% formaldehyde. After blotting onto nylon membranes, the
RNAs were hybridized with the cDNA insert from pB105-2 (top
panels), human HSP70 DNA (middle panels), and -actin
cDNA (bottom panels). The positions of 28 S and 18 S rRNAs are
indicated at the right. HSC70 represents heat shock cognate
70.
Figure 5:
RT-PCR
analysis of HSP105 and 42 °C-HSP mRNAs. PCR was performed in the
presence of [-
P]dCTP using total RNA
extracted from control cells (C; lanes 1 and 6), cells heated at 42 °C for 0.5, 1, 2, and 5 h (lanes 2-5), cells heat-shocked at 45 °C for 15 min
and then incubated at 37 °C for 0, 1, 3, and 6 h (lanes
7-10), or cells treated with 100 µM sodium
arsenite (Ar) for 6 h (lane 11) or with 20 mM azetidine-2-carboxylic acid (Az) for 12 h (lane
12) as described under ``Experimental Procedures.'' The
products were electrophoresed on a 3.5% polyacrylamide gel, and the gel
was autoradiographed. Molecular sizes in bases are shown at the
left.
Figure 6: Northern blot analysis of HSP105 mRNA from various murine tissues. Total cellular RNAs (20 µg each) from mouse brain, lung, heart, thymus, spleen, liver, stomach, small intestine, large intestine, kidney, uterus, testis, and bone were electrophoresed on a 1% agarose gel; stained with ethidium bromide (bottom panel); and analyzed by Northern blotting using pB105-2 as a probe. The positions of 28 S and 18 S rRNAs are indicated at the right (top panel).
We have previously reported that HSP105 and 42 °C-HSP, which are serologically related to each other, have an ATP-binding domain similar to that of the HSP70 family and actin(11, 12, 13, 14) . Here, we describe the cloning of the cDNAs encoding HSP105 and 42 °C-HSP from mice.
We have screened a cDNA library constructed with mRNA from heat-shocked mouse FM3A cells using an anti-HSP105 antibody and have obtained two full-length cDNA clones. pB105-1 consists of 858 amino acids, and pB105-2 encodes 814 amino acids. Both clones were exactly the same except that pB105-2 lacked 44 amino acids in the region between positions 530 and 573 of pB105-1, and both contained the amino acid sequence found in the 17-kDa polypeptide fragments from HSP105 and 42 °C-HSP digested by lysylendopeptidase(12) . From in vitro translation experiments and RT-PCR analysis, the cDNA clones pB105-1 and pB105-2 were suggested to encode HSP105 and 42 °C-HSP, respectively. HSP105 mRNA was strongly induced by heat shock at 42 or 45 °C and by treatment with arsenite or an amino acid analog, whereas 42 °C-HSP mRNA was only induced by heat shock at 42 °C.
The 42 °C-specific HSP consists of at least two
polypeptides (basic and acidic) with molecular masses of
90,000(31) . Pulse-chase experiments suggested that the
acidic protein originated from the basic protein by post-translational
modification. HSP105 is induced by heat shock either at 42 or 45
°C, whereas 42 °C-HSP is synthesized only when heated
continuously at 42 °C(13, 14) . Since the amino
acid sequences of HSP105 and 42 °C-HSP deduced from the cDNA
sequences were the same except that 42 °C-HSP lacked 44 amino acids
in the region between positions 530 and 573 of HSP105, 42 °C-HSP
and HSP105 may be produced by alternative splicing from the same
transcript. This possibility was also confirmed by the RT-PCR
experiment (Fig. 5). From these findings, we renamed HSP105 and
42 °C-HSP as HSP105
and HSP105
, respectively.
Previously, we reported that heat shock induces an alternative splicing in the 5`-noncoding region of the collagen-specific stress protein, HSP47(32) . Similarly to the observation for HSP105 (Fig. 5), the alternative splicing of HSP47 was not observed by treatment with other stress inducers like arsenite or azetidine. The finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression. To elucidate the mechanism for this heat shock-induced alternative splicing, we are now cloning the genomic DNA encoding HSP105, which will reported in the near future.
Murine HSP105 and HSP105
contained a single
ATP-binding site similar to that of HSP70 family members, as has been
suggested from our protein sequencing data(12) . By comparison
with GenBank
protein sequences, HSP105
was found to
be 54% identical to human HSP70RY, 34% to the sea urchin sperm
receptor, and 25% identical to murine inducible HSP70. The N-terminal
500-amino acid sequence of HSP105
that contained an ATP-binding
domain similar to that of HSP70 family members shared more identity
with those of human HSP70RY (72% identity) and the sea urchin sperm
receptor (50%). Human HSP70RY is a novel HSP70 of 701 amino acids
cloned from a human B lymphocyte cell line(23) . The HSP70RY gene has been mapped to human chromosome 5, whereas
genes encoding major HSP70 are localized at chromosomes 6, 14, and
21(24) . The function of HSP70RY is not clear, but it may have
distinctive functions in antigen processing and presenting of B
lymphocytes. On the other hand, the sea urchin egg receptor for sperm
is a transmembrane glycoprotein of 1184 amino acids with a short
cytoplasmic domain(25) . The extracellular sperm-binding domain
of the receptor shows sequence similarity to HSP70 family members. The
extracellular portion of the receptor binds to the sperm protein,
bidin, and also inhibits fertilization in a species-specific manner. In
addition, the region between amino acids 612 and 713 of HSP105
was
also highly similar to HSP70RY and the sea urchin sperm receptor
between amino acids 594 and 695 and between amino acids 649 and 750,
respectively, but not to murine inducible HSP70. In the sea urchin
sperm receptor, the second homologous region was also localized to the
extracellular sperm-binding domain of the receptor. Thus, since
HSP105
, HSP70RY, and the sperm receptor all contain not only an
ATP-binding domain but also the second homologous region, these
proteins may constitute a distinct protein family.
Recently, the
cDNA of the murine APG protein was isolated and sequenced. ()The APG protein is 838 amino acids and is found only in
germ cells during spermatogenesis in mice. The amino acid sequence of
the APG protein was 57% identical to murine HSP105
and also shared
high identity at the N-terminal 500 amino acids and the second
homologous region. The APG protein was testis-specific and was not
induced by heat shock, although HSP105
was expressed in most
murine tissues and was significantly induced by heat shock. Thus, the
APG protein may be a testis-specific member of the HSP105 family in
mice, as is testis-specific HSP70 (HSP70T) in the HSP70
family(33) .
HSP104 is a high molecular mass heat shock
protein in yeast, and the antibody against yeast HSP104 cross-reacts
with the high molecular mass heat shock protein from human cells and
Chinese hamster cells(5) . Yeast HSP104 is a protein of 908
amino acids, and the deduced amino acid sequence revealed the presence
of two nucleotide-binding sites in HSP104. Deletion of the HSP104 gene in yeast cells has little effect on growth at a
normal temperature, but has an effect on the acquirement of resistance
to heat and other various stresses (4, 5, 6) . The two nucleotide-binding
domains of HSP104 are essential for thermotolerance(5) .
Recently, HSP104 was found to function to resolubilize heat-aggregated
proteins(34) . Furthermore, yeast HSP104 exhibits homology to
bacterial ClpA/ClpB proteins, which appear to play a role in an
ATP-dependent protein degradation process(7) . In addition,
ClpA functions as a molecular chaperone like DnaK(8) . However,
murine HSP105 did not show any identity to yeast HSP104 or
bacterial ClpA/ClpB proteins.
When preparing this report, the cDNA
sequence of Chinese hamster HSP110 was found in the GenBank protein sequence data base and has recently been
reported(35) . The cDNA of Chinese hamster HSP110 encodes 858
amino acids, and the predicted amino acid sequence shared 96% identity
with murine HSP105
. Chinese hamster HSP110 is a high molecular
mass heat shock protein that was first reported by Subjeck et
al.(9) . This heat shock protein is constitutively
expressed at low levels and appears to increase with heat shock. When
visualized by indirect immunofluorescence, HSP110 is present in nuclei
and essentially in nucleoli. Since HSP110 is released from the nucleoli
by RNase treatment, it is probably complexed to the RNA component of
the nucleoli and possibly participates in ribosome
assembly(9, 10) . However, when we carefully examined
the cellular distribution of HSP105 by immunofluorescence using the
anti-HSP105 antibody, we found that HSP105 is mainly localized in the
cytoplasm and nuclei and is never found in the nucleoli in either
nonstressed or stressed cells(11) . It would also be
interesting to clarify the differences between our murine HSP105 and
Chinese hamster HSP110.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D67016 [GenBank]and D67017[GenBank].