(Received for publication, April 9, 1997, and in revised form, May 14, 1997)
From the Department of Biology, Dalhousie University,
Halifax, Nova Scotia B3H 4J1, Canada, the § Department
of Medical Biochemistry, University of Leiden,
Leiden, The Netherlands, and ¶ Bodega Marine Laboratory,
University of California, Davis,
Bodega Bay, California 94923
Molecular chaperones protect cells
during stress by limiting the denaturation/aggregation of proteins and
facilitating their renaturation. In this context, brine shrimp embryos
can endure a wide variety of stressful conditions, including
temperature extremes, prolonged anoxia, and desiccation, thus
encountering shortages of both energy (ATP) and water. How the embryos
survive these stresses is the subject of continuing study, a situation true for other organisms facing similar physiological challenges. To
approach this question we cloned and sequenced a cDNA for p26, a
molecular chaperone specific to oviparous Artemia embryos.
p26 is the first representative of the small heat shock/-crystallin family from crustaceans to be sequenced, and it possesses the conserved
-crystallin domain characteristic of these proteins. The secondary
structure of this domain was predicted to consist predominantly of
-pleated sheet, and it appeared to lack regions of
-helix. Unique
properties of the nonconserved amino terminus, which showed weak
similarity to nucleolins and fibrillarins, are enrichments in both
glycine and arginine. The carboxyl-terminal tail is the longest yet
reported for a small heat shock/
-crystallin protein, and it is
hydrophilic, a common attribute of this region. Site-specific
differences between amino acids from p26 and other small heat
shock/
-crystallin proteins bring into question the functions
proposed for some of these residues. Probing of Southern blots
disclosed a multi-gene family for p26, whereas two size classes of p26
mRNA at 0.7 and 1.9 kilobase pairs were seen on Northern blots, the
larger probably representing nonprocessed transcripts. Examination of
immunofluorescently stained samples with the confocal microscope
revealed that a limited portion of intracellular p26 is found in the
nuclei of encysted embryos and that it resides within discrete
compartments of this organelle. The results in this paper demonstrate
clearly that p26 is a novel member of the small heat
shock/
-crystallin family of proteins. These data, in concert with
its restriction to embryos undergoing oviparous development, suggest
that p26 functions as a molecular chaperone during exposure to stress,
perhaps able to limit protein degradation and thus ensure a ready
supply of functional proteins when growth is reinitiated.
Molecular chaperones assist the folding of proteins, protect them
from denaturation/aggregation, aid in their renaturation, and influence
the final intracellular location of mature proteins (1-11). The
chaperones, many of which are induced by exposure of cells to stress,
are divided into several groups, including HSP100, 90, 70, 60 (chaperonin), and small heat shock/-crystallin proteins. The
functions of chaperones differ, but their activities are interrelated
and often dependent on association into macromolecular complexes,
sometimes consisting of representatives from more than one family. Of
most importance to this work are the small heat shock/
-crystallin
proteins, 15-30 kDa in size but with the ability to oligomerize into
particles of varying monomer number (12-14). Proteins in this group
possess a conserved domain of 90-100 amino acid residues, the
-crystallin signature sequence (11, 15-18). Otherwise, they differ
in sequence, although there are conserved residues within variable
regions that may be essential to their chaperone-like activity (15, 19,
20). The ATP-independent chaperone-like activity of these proteins is
seemingly more directed to preventing protein aggregation early in
denaturation, rather than the active refolding of compromised proteins
(19, 21-25). This is an especially important characteristic of the
-crystallins, major proteins of the lens which limit aggregation of
other eye crystallins and maintain lens transparency (23-25). The
-crystallins, and especially
B-crystallin, are also found in
nonlenticular tissues (26-28), where their synthesis is induced by
stress (24, 27-31). The small heat shock/
-crystallin proteins are
thought to protect cells during stress (27, 32-36), and there is
evidence that some of them not only prevent denaturation of
proteins but assist in their renaturation as well (13, 37, 38).
The synthesis of small heat shock/-crystallin proteins is
developmentally regulated in many organisms (29, 34, 39-41), and in
this context, the brine shrimp Artemia is an interesting experimental model. Upon fertilization, Artemia oocytes
undertake one of two developmental pathways. Either they develop
directly into free-swimming larvae and are released from the female. In the alternative mode, development is arrested and embryos are discharged from the female as encysted gastrulae, termed cysts (42).
The cysts, composed of about 4000 cells and enclosed in a shell
impermeable to most molecules, enter diapause, a condition characterized by an extremely low level of metabolic activity (42-46).
They are very resistant to environmental insults including anoxia,
temperature extremes, organic solvents,
-irradiation, and
desiccation (42, 43, 47). Desiccation is a normal part of this path of
development and may be required to break diapause and reinitiate
development. Resistance to environmental insults is not lost
immediately upon resumption of cyst development. For example, fully
hydrated, post-gastrula cysts survive at least 4 years under anoxic
conditions in a state of quiescence
(48-50).1 These animals are essentially
ametabolic, apparently existing in the absence of a continuous free
energy flow (50-52).
The molecular basis for the remarkable stress resistance of
Artemia cysts is poorly understood. However, we previously
purified a low molecular weight protein, termed p26, to apparent
homogeneity and obtained evidence that it protects embryo cells during
encystment, diapause, and anaerobic quiescence (13, 49, 53). Partial sequence analysis, in concert with other characterization, revealed that p26 is a small heat shock/-crystallin protein with chaperone activity in vitro (13). In this paper we report the
isolation and sequencing of a full-length cDNA clone for p26.
Comparison to other small heat shock/
-crystallin proteins at both
primary and secondary levels disclosed novel aspects of p26 structure. Multiple genes for p26 were observed, as were two size classes of
mRNA transcripts, one in much greater abundance than the other. Examination of immunofluorescently stained samples with the confocal microscope revealed that a portion of the p26 in hydrated
Artemia cysts is found within discrete compartments of their
nuclei. We believe that p26 prevents the aggregation of other proteins
when these embryos experience stresses of various kinds, thus playing an important role in their growth and development.
Encysted embryos of Artemia franciscana (Great Salt Lake) obtained from Sanders Brine Shrimp, Ogden, UT, were hydrated in cold distilled water for at least 3 h. Those cysts that sank to the bottom of the container were collected by suction on a Buchner funnel, rinsed several times with cold distilled water, and used immediately.
Cloning and Sequencing of a p26 cDNAPoly(A)+ mRNA, prepared from encysted
Artemia gastrulae (54), was employed as template for the
synthesis of first strand cDNA using the First-strand cDNA
Synthesis KitTM (Pharmacia Biotech Inc.) as described in the
manufacturer's instructions. PCR2 was
performed against the first strand cDNA using a degenerative primer, based on the p26 peptide sequence of 103-DEYGHVQR-110 and an
oligo(dT) primer, NotI-(dT)18, included in the
cDNA synthesis kit. The PCR protocol involved 30 cycles at
92 °C, 30 s; 43 °C, 30 s; 72 °C, 30 s in a
PTC-100 programmable thermal controller (MJ Research Inc., Watertown,
MA). A DNA fragment of 378 bp, termed p26-3, was cloned into PUC18
using the SureCloneTM Ligation Kit (Pharmacia) and sequenced manually
with the T7 SequencingTM Kit (Pharmacia) to ensure that it encoded a
portion of p26. To obtain the full-length p26 clone, a cDNA library
from encysted Artemia gastrulae in
ZAP II (a gift from
Dr. L. Sastre, Instituto de Investigaciones Biomédicas del
C.S.I.C., Madrid, Spain) was converted to the phagemid, Bluescript
SK
, and screened with 32P-labeled p26-3
according to
Sambrook et al. (55). Several putative p26 clones were
plaque-purified two times, and one of these contained an insert termed
p26-3-6-3 shown by sequencing to encode a full-length cDNA for
p26.
Purification of p26, digestion, and separation of peptides by reversed-phase high performance liquid chromatography were as described by Liang et al. (13). The three amino-terminal peptides sequenced in this study were generated by BrCN treatment of p26 (13). As the intact protein was blocked amino-terminally (13) at least one of the peptides formed should be blocked. This peptide could therefore be identified in high performance liquid chromatograms of the entire BrCN mixture on the basis of its insensitivity toward aminopeptidase M (not shown). Removal of the blocking group if it is an acetyl moiety, together with the first amino acid, is possible with acyl amino acid peptidase. The isolated peptide was lyophilized and dissolved in 10 µl of 0.2 M ammonium carbonate, 1 mM 2-mercaptoethanol, and 0.1 mM EDTA (pH 8.5). Deblocking was accomplished by adding 10 µl of acylamino acid peptidase (Boehringer Mannheim), containing 2 µg of lyophilized enzyme, and incubating the mixture for 16 h at 37 °C. The deblocked peptide was loaded on a reversed-phase column (1 × 15 mm) which had been preconditioned by rinsing with 0.5 ml of 0.08% (v/v) trifluoroacetic acid containing 75% (v/v) acetonitrile followed by 0.5 ml of 0.1% (w/v) trifluoroacetic acid. After sample application the column was rinsed with 0.5 ml of 0.1% (w/v) trifluoroacetic acid. The peptide was eluted with 75 µl of 75% (v/v) acetonitrile containing 0.1% (w/v) trifluoroacetic acid, applied to a Polybrene-impregnated glass fiber disk, and analyzed in a model 475A Applied Biosystems pulse liquid sequencer, connected on line to a Model 120A phenylthiohydantoin-derivative analyzer.
Because it was not possible to prepare a suitable carboxyl-terminal peptide, the final eight amino acids were determined by pool sequencing tryptic peptides obtained from a larger carboxyl-terminal peptide, in turn produced by exposure of p26 to endoproteinase Lys-C (13). The large peptide, beginning at Thr-144, was treated on a Polybrene-impregnated sequence glass fiber disk with 20 µl of 10 mM NaHCO3, followed by 20 µl of acetic anhydride/methanol (1:4, v/v). After 5 min at room temperature the remaining liquid was removed by lyophilization, and the disk was wetted with 20 µl of 0.2 M ammonium carbonate (pH 8.5) containing 0.1 µg of trypsin. The disk was transferred to an Eppendorf tube to suppress evaporation and incubated at 37 °C for 2 h. The remaining water and ammonium carbonate were removed by lyophilization, and the disk was placed in the sequencer for analysis. Because there were four internal arginine residues in the large peptide, and the amino terminus of the initial peptide was blocked by acetylation, the simultaneous sequencing of four peptides was expected.
Prediction of p26 Secondary Structure and Solvent AccessibilityThe secondary structure and solvent accessibility of amino acid residues within p26 were predicted by the methods of Rost and Sander via the Predict Protein E-mail server at the European Molecular Biology Laboratory, Heidelberg, as described by Caspers et al. (16).
Analysis of p26 Gene Structure by Southern BlottingDNA was
prepared from Artemia larvae as described previously except
that ethanol precipitation was avoided (54). The DNA was digested with
BamHI, electrophoresed in 0.8% agarose gels with 28 µg of
DNA in each lane, and blotted to HybondTM-N+ nylon membranes (Amersham
Corp.) in 10 × SSC (1.5 M NaCl, 0.15 M
sodium citrate (pH 7.0)). The blots were incubated for 12 h at
68 °C in prehybridization solution that contained 6 × SSC,
5 × Denhardt's, 0.5% SDS, and 100 µg/ml salmon DNA which had
been sheared and denatured. Hybridization was in the same solution for
12 h at 68 °C with DNA fragments corresponding to the
full-length p26 cDNA (p26-3-6-3), 373 bp from the 5-end (p26-5
),
and 378 bp from the 3
-end (p26-3
). The full-length probe was
recovered from agarose gels after incubation of the plasmid containing
p26-3-6-3 with EcoRI and XhoI followed by
electrophoresis. The probe, p26-5
, was generated by PCR amplification
of p26-3-6-3 using a nondegenerate primer based on nucleotides 22-39
of p26-3-6-3 and a degenerate primer based on the peptide sequence,
119-PEHVKPE-126. p26-3
was produced as described previously in this
paper. The 5
- and 3
-probes had a short sequence overlap, but as
revealed by the hybridization patterns, this did not appear to affect
their specificity. The hybridization probes were labeled with
32P by random priming (T7 Quick PrimeTM Kit, Pharmacia)
following the manufacturer's instructions. After hybridization the
blots were washed with 2 × SSC containing 0.1% SDS at room
temperature for 30 min, followed by 1 × SSC containing 0.1% SDS
and then 0.1 × SSC with 0.1% SDS, both at 68 °C for 15 min,
partly air dried, wrapped in Saran WrapTM and exposed to Kodak X-OMATTM
AR film (Picker Scientific, Dartmouth, Nova Scotia) at
70 °C.
The number of p26 genes in the Artemia genome was determined
by hybridizing the 32P-labeled probes just described to
Southern blots that contained known amounts of both p26 cDNA and
restriction-digested Artemia DNA in parallel lanes. For this
quantitation the size of the Artemia haploid genome was set
at 1.37 × 106 kb (1.45 pg) (56), and the size of the
Bluescript Sk plasmid containing p26-3-6-3, which was linearized by
digestion with XhoI before electrophoresis, was 3.64 kb
(3.7 × 10
6 pg). Labeling intensities for bands in
each sample were compared by scanning blots with a Bio-Rad GS-670
Imaging Densitometer and quantitated by use of Molecular Analyst
software from Bio-Rad.
Poly(A)+ mRNA was obtained from hydrated Artemia cysts, electrophoresed in 1.5% agarose gels, blotted to HybondTM-N+ membranes, baked at 80 °C for 2 h, and hybridized to 32P-labeled, full-length p26 cDNA as described by Langdon et al. (54), except that probes were prepared by random priming. Size determinations were made by comparing migration distances of p26 mRNA with RNA fragments of known size in a 0.24-9.5-kb RNA ladder from Life Technologies (Burlington, Ontario).
Preparation of Artemia NucleiNuclei were prepared from
Artemia for immunofluorescent staining by two methods. In
the first procedure hydrated cysts were crushed gently between two
glass slides, one of which was coated with poly-L-lysine.
The slides were separated and the nuclei were fixed immediately. Nuclei
were also isolated by a modification of the technique of Squires and
Acey (57) communicated to us by J. Vaughn (University of Miami, Miami,
OH). Briefly, 10 g of hydrated Artemia cysts were
ground by hand in a chilled mortar and pestle for 2 min in 35 ml of HPC
buffer (0.5 M hexylene glycol, 0.05 M
Pipes-free acid, 1 mM CaCl2 (pH 7.6)) and then
subjected to one passage in a motorized Dounce homogenizer fitted with
a size A pestle. The homogenate was passed through one layer of Miracloth (Calbiochem), centrifuged at 2,000 × g for
10 min at 4 °C, and the supernatant discarded. The pellet was rinsed
once with HPC buffer and then washed two times using 40 ml of HPC
buffer for each wash. The large, greenish pellet of nuclei obtained
after the second wash was resuspended in 17.5 ml of HPC buffer with a
Pasteur pipette. The suspension was layered on a 25-ml cushion of 75%
(v/v) Percoll in 0.15 M NaCl, 10 mM
MgCl2, 10 mM Tris-HCl (pH 7.6) and centrifuged
at 16,000 × g for 30 min at 4 °C in a Beckman JS-13
swinging bucket rotor. The top clear layer was discarded, and the
second layer was transferred to a fresh tube and brought to a final
volume of 10-15 ml with HPC. The solution was applied to a 25-ml
cushion of 75% (v/v) Percoll that had been centrifuged at 16,000 × g for 30 min at 4 °C in a Beckman JS-13 rotor, and the
centrifugation was repeated. The top layer was discarded and the second
layer of 5 to 6 ml was mixed in a fresh tube with an equal volume of
HPC buffer before centrifugation at 16,000 × g for 30 min at 4 °C. The resulting yellow-brown pellets, containing isolated
nuclei, were resuspended in 1.0 ml of HPC buffer. The isolation was
also done with all solutions at pH 6.5 to ensure that p26 was not
extracted from nuclei during their manipulation under basic conditions
(49, 53). Nuclei were mixed with an equal volume of
4,6-diamidino-2-phenylindole hydrochloride (DAPI) (Molecular Probes,
Eugene, OR) at 0.001 µg/ml in H2O for 5 min at room
temperature, allowed to settle onto poly-L-lysine-coated slides for 5 min at room temperature, and examined by light and immunofluorescent microscopy to determine purity. Nuclei at different stages of purification were solubilized, electrophoresed in
one-dimensional, 12.5% SDS-polyacrylamide gels, and either stained
with Coomassie Blue or transferred to nitrocellulose and immunostained
by the enhanced chemiluminescence procedure (RenaissanceR,
NEN Life Science Products) following manufacturer's instructions. The
primary antibody was raised in rabbits to p26 purified as described
previously (13).
Nuclei
prepared by crushing of hydrated cysts and by centrifugation on Percoll
gradients were fixed in either 4% (w/v) paraformaldehyde at room
temperature for 20 min or in methanol at 20 °C for 5 min. The
fixed specimens were hydrated in PBS for 5 min at room temperature and
exposed for 30 min at room temperature to primary antibody, raised to
p26, and diluted 1:500 in phosphate-buffered saline (PBS) (pH 7.4),
containing 0.5% bovine serum albumin and 0.5% Triton X-100 (PBSAT).
The secondary antibody was fluorescein isothiocyanate-conjugated goat
anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) diluted
1:200 in PBSAT. After a 30-min incubation with secondary antibody the
nuclei were rinsed with PBS, incubated with DAPI as above, rinsed
again, and mounted in Vectashield (Vector Laboratories, Burlingame,
CA). Purified nuclei were also stained with propidium iodide (Sigma)
added directly to mounting medium to achieve a final concentration of
0.1 mg/ml. Slides were examined with either a Leitz Aristoplan
epifluorescence microscope or a Zeiss LSM 410 inverted laser scanning
confocal microscope equipped with an argon-krypton laser.
The cloned cDNA,
p26-3-6-3, isolated by screening an Artemia library
contained a single open reading frame that encoded p26, a polypeptide
of 192 amino acid residues beginning with methionine and ending in
alanine (Fig. 1). The first 21 nucleotides of the cDNA clone were noncoding as were nucleotides 598-709. A
termination codon followed the final alanine residue and the
3-noncoding region contained a typical polyadenylation signal of
AATAAA at nucleotides 666-671 and a poly(A) tail (nucleotides
686-709).
The amino acid sequence deduced from the cDNA clone agreed exactly
with the partial sequence of p26, starting with Arg-44 and ending with
Arg-184, that had been derived previously by Edman digestion (13). To
test the remainder of the deduced amino acid sequence, selected
peptides prepared from purified p26, as described under "Materials
and Methods," were analyzed. Three short, nonoverlapping peptides
obtained by BrCN digestion agreed exactly in sequence with amino acid
residues 3-41 of p26 as deduced from the cDNA clone (Fig.
2a). The extreme amino-terminal peptide
showed, after deblocking, the sequence LNPWYGGFGG (Fig. 2a).
Because the acylamino acid peptidase deletes an amino acid from its
substrate, in addition to an acetyl group, it was necessary to depend
on the cDNA sequence to determine the first residue, which in this
case was alanine. Thus, it was revealed that the initiator methionine
observed in p26-3-6-3 (Fig. 1) was removed from mature p26, and
alanine, originally the penultimate residue, was at the amino terminus.
Additionally, methionine residues 13, 30, 42/43 were not observed in
the peptides because they were converted to homoserine lactone residues
during BrCN cleavage, and these are often not visible during
sequencing. The disappearance of these residues was in agreement with
their position as revealed by sequencing of p26-3-6-3 cDNA (Fig.
1).
The cDNA-derived sequence indicated that the high performance liquid chromatography-based isolation of the two tryptic peptides containing the carboxyl-terminal eight amino acid residues would be difficult because of their length and composition. Additionally, the peculiar amino acid composition and sequence in this part of the polypeptide chain precluded other digestion possibilities. Therefore, the carboxyl-terminal sequence obtained by analysis of p26-3-6-3 was confirmed by pool sequencing a mixture of tryptic peptides beginning at arginines 152, 164, 184, and 187. The amino terminus of the original peptide was blocked by acetylation and thus not available for sequencing. The amino acid residues obtained in each sequencing step were as follows: step 1, ITS; step 2, VETG; step 3, PGR; step 4, IGA; step 5, TA; step 6, PT; step 7, AG; and it was possible to align these residues with those deduced from the cDNA sequence (Fig. 2b). To summarize, sequencing of both the cDNA and selected peptides revealed that mature p26 is a polypeptide of 191 amino acid residues with a calculated molecular mass of 20.7 kDa and a predicted pI of 6.6. Throughout the paper numbering of amino acid residues is based on the sequence deduced from the cDNA and not on the mature protein.
Comparison of p26 to Other Small Heat Shock/p26 is divided into three regions that differ in their
similarity to corresponding segments of other small heat
shock/-crystallin proteins (Figs. 3 and
4). The amino-terminal 59 residues of p26 comprise a
variable domain, with limited similarity to other small heat
shock/
-crystallin proteins. A unique characteristic of p26 within
this family of proteins is that 10 of its first 29 amino acid residues
are glycines (34% glycine content), and these are housed in a peptide
of 22 residues (45% glycine content). By comparison, other members of
the small heat shock/
-crystallin group shown in Fig. 3 have 1-4
glycines in the complementary region, which due to gaps in the sequence
alignment are from 43 to 49 residues in length. Also noteworthy is that
residues 36-51 of p26 contain 7 arginines (7/16 = 44%) and that
6 of these arginines are in a 10-residue stretch. This domain of p26
contained 3 regions of predicted
-sheet with the first two separated
by a predicted
-helix. Although there was no strong homology to
other proteins within the first 59 residues of p26, there was a weak
similarity, based on the enrichments in glycine, arginine, and
phenylalanine, to the fibrillarin and nucleolin families. A superficial
similarity with mucins, due to a high serine/threonine content, was
found in the carboxyl-terminal 35 residues of p26. However, this
resemblance was judged to be meaningless because the mucins are heavily
glycosylated and p26 is not.
When data base search programs like FASTA and BLAST were applied to the
complete sequence of p26, only small heat shock/-crystallin proteins
appeared in the results. Membership depended on residues 60-157 that
form the signature
-crystallin domain of p26, a sequence conserved
in all members of the small heat shock/
-crystallin family. We
previously reported on the degree of sequence identity between p26 and
other small heat shock/
-crystallin proteins (13), and these results
are not repeated. In this work we extend study of the p26
-crystallin domain to include its predicted secondary structure,
revealing that this area is divided into discrete regions of
-sheet,
apparently with no intervening
-helix. When HSP27,
A-crystallin,
and
B-crystallin were analyzed by the methods used for
characterization of p26 secondary structure, regions of
-helix
within
-crystallin domains were apparent, a result expected from
published data (16). Of the
-sheet regions in the
-crystallin
domain of p26, the first, beginning at residue 68, and the final three
appeared to be buried. It was not possible to predict, based on our
current analysis, if the
-sheets beginning at residues 83 and 103 are buried or exposed. Residues 158-192 represented a second variable
region, distinguished by a hydrophilic tail longer than that found in
any other small heat shock/
-crystallin protein.
Southern blots
containing Artemia DNA digested with BamHI and
probed with a 32P-labeled DNA fragment, p26-3-6-3,
corresponding to the entire coding region of p26, yielded 4 bands, two
strong and two weak (Fig. 5, lane 5). When
blots were reprobed with PCR-generated DNA fragments, two of the bands
hybridized to p26-5 whereas the other two reacted with p26-3
,
corresponding to the 5
- and 3
-ends of p26-3-6-3, respectively (Fig.
5, lanes 6 and 7). Comparison of values generated
by scanning of bands B1-B4 in Fig. 5, lane 5, to
those obtained by scanning the bands for increasing amounts of
p26-3-6-3 in Fig. 5 (lanes 1-4), indicated that there were at least three copies of the p26 gene in Artemia.
Multiple mRNAs for p26 in Artemia Embryos
Northern blots
of poly(A)+ mRNA from cysts were hybridized to
32P-labeled cDNA consisting of the complete p26 coding
region (Fig. 6). Observation of a major band (0.7 kb)
and a minor band (1.9 kb) revealed that there were two size classes of
p26 mRNA in encysted Artemia embryos.
Compartmentalization of p26 in Nuclei of Artemia Embryos
Because pH has been shown to have a marked effect on
the intracellular location of p26 (see Ref. 49), nuclei were isolated under acidic (pH 6.5) and basic (pH 7.6) conditions. Both preparations were electrophoresed in SDS-polyacrylamide gels and either stained with
Coomassie Blue (Fig. 7a) or blotted to
nitrocellulose and stained with antibody to p26 (Fig. 7b).
Comparison of lanes 1 and 4, respectively, with
lanes 2 and 5, in both Fig 7, a and b, showed that a portion of the p26 within encysted
gastrulae cosedimented with nuclei. When nuclei purified on Percoll
gradients were examined in SDS-polyacrylamide gels, p26 was again
observed, demonstrating that the protein was tightly associated with
the nuclei (Fig. 7, a and b, lanes 3 and
6). To determine if p26 was located internally, purified
nuclei prepared under acidic conditions were double-stained with either
DAPI or propidium iodide and antibody to p26 and then examined with a
fluorescence microscope. All nuclei stained by DAPI/propidium iodide
were labeled by antibody to p26 (Fig. 8a,
a). Examination at a higher magnification indicated that
p26 was dispersed throughout the nuclei, with some of the protein
concentrated in brightly stained foci (Fig. 8b,
b
). That this is the arrangement of p26 was verified by
optical sectioning of nuclei with the confocal microscope (Fig.
9). The brightly stained regions generally appeared in
no more than two consecutive sections, indicating they were discrete,
spherical objects about 1 µm in diameter, a value approximating the
greatest en face diameter of stained foci shown in the
figure. The staining patterns of nuclei prepared at a basic pH and by
crushing cysts between glass slides were identical to those just
shown.
Our previous work revealed that encysted Artemia
embryos contain very large quantities of a low molecular weight protein
termed p26 (13, 49, 53, 58). Partial sequencing of purified p26 indicated that it is a small heat shock/-crystallin protein, while
other experiments demonstrated an in vitro chaperone
activity (13). Our objectives in this work were to analyze further the properties of p26 and to explore more fully the relationship of this
protein to other small heat shock/
-crystallin proteins through study
of its molecular structure. Additionally, we hoped to shed light on the
role of p26 during diapause and other types of stress, thus
contributing to our understanding of small heat shock/
-crystallin proteins in other eukaryotic cells.
Determination of the complete p26 sequence confirmed the presence of an
-crystallin domain and showed that it is positioned toward the
carboxyl terminus, as for other small heat shock/
-crystallin proteins (16, 17). The
-crystallin domain has been reported to
consist of two hydrophobic motifs enriched in
-pleated sheets and
separated by an
-helical, hydrophilic region (16, 17). The first
motif is composed of three
-pleated sheet regions, preceded by an
-helix (
), an arrangement repeated in the second half of
the
-crystallin domain. p26 is unusual in that it appears to lack
regions of
-helix in the
-crystallin domain, although it is
predominantly
-sheet as expected. Similar secondary structure and
hydrophobicity profiles among the small heat shock/
-crystallin proteins suggest that
-crystallin domains have the same function. For example, Smulders et al. (59) have shown that
modification of Asp-69 to Ser within the sequence 63-EVRSDRD-69
disrupts chaperone activity in
A-crystallin, perhaps due to a change
in charge distribution. This residue is close to the beginning of the
-crystallin domain; it occurs in several small heat
shock/
-crystallin proteins, and Asp-67 within the sequence
61-SLRDTAD-67 may be the equivalent residue in p26.
Site-directed mutagenesis of Phe-24 and Phe-27 indicates that a
conserved region (22-RLFDQFFG-29) outside the -crystallin domain is
essential for chaperone-like activity of
B-crystallin (19). Crabbe
and Goode (15) also suggest, through comparisons of published data,
that the peptide RLFDQFF is important in oligomerization of small heat
shock/
-crystallin proteins. The equivalent region in p26, a
multimeric protein with chaperone activity, is 21-FGFGGFGGG-29, interestingly in the glycine-rich sequence. There are two
phenylalanines in positions comparable to those in
B-crystallin, but
the surrounding residues show little similarity. Thus, if the
phenylalanines are mechanistically important in chaperone action
and oligomerization, their neighboring residues are less critical for
these functions. In another case, modification of aspartic acid at the
amino terminus, and two juxtaposed lysines at the carboxyl terminus,
reduced the chaperone-like action of
B-crystallin (19).
Corresponding residues are missing from p26, although its
carboxyl-terminal tail contains two arginines in close proximity to one
another and near the end of the protein. Moreover, p26 has an extended,
hydrophilic tail. Only SEC-1, a developmentally regulated small heat
shock protein of 159 amino acids from Caenorhabditis elegans
possesses a carboxyl-terminal extension with a comparable number of
serines and threonines (34). The carboxyl terminus of p26 and other
small heat shock/
-crystallin proteins may "capture" unfolded
proteins and keep complexes of chaperones and denatured proteins in
solution, explaining why its charged/polar characteristics, but not
necessarily its sequence, are conserved.
Hybridization of p26-3-6-3 to Southern blots of
BamHI-digested DNA yields two strongly labeled bands and two
weak bands, each reacting with a probe from either the 5- or the
3
-end of p26-3-6-3, but not with both. These results, in concert with
the relative staining intensities of the various bands, indicate that
p26 is encoded by a multi-gene family. The heavily stained bands (B1 and B3) contain opposite halves of at least 2 copies of similar p26
genes, perhaps in tandem repeat, whereas the lighter bands are opposite
halves of a separate p26 gene. By comparison, the small heat shock
genes of C. elegans, except for SEC1 (34), are duplicated
and arranged in tandem repeats (61, 62); they contain short introns and
their synthesis is induced by stress. Plants exhibit many highly
conserved small heat shock/
-crystallin proteins (14, 37, 39), and
gene families have been reported for human HSP27 (63) and mouse HSP25
(64). At least a single representative of each family contains two
introns, one of 600-700 bp and another of about 120 bp. Because
p26-3-6-3 lacks a BamHI site, there must be introns in the
p26 genes, and if they exceed approximately 1200 nucleotides, then the
weak 1.9-kb band on Northern blots of cyst mRNA probably represents
nonprocessed transcripts. In support of this, the larger mRNA is
only visible when the 0.7-kb message is most
abundant.3 At this time the transcription
rate may be sufficiently rapid that mRNA production exceeds
splicing, or as discussed by Head et al. (27), processing of
mRNA transcripts is compromised during stress. Differential gene
transcription may generate, at least in part, the p26 isoform
heterogeneity seen previously on Western blots (49).
Small heat shock/-crystallin proteins enter nuclei during stress
(28, 65-67), but their function within the organelle is uncertain. For
example, overexpression of human HSP27 in cultured Chinese hamster
cells leaves stress-induced formation of aggregates within nuclei
unaffected but leads to a faster recovery from damage, suggesting
chaperone activity for HSP27 (38). Large amounts of p26 translocate
into nuclei of encysted Artemia embryos during anoxia,
probably due to a reduced intracellular pH, greatly increasing the
amount of this protein in the organelle under conditions of stress (49,
53, 58). However, its intranuclear distribution was unknown, this being
true for most other small heat shock/
-crystallin proteins. Staining
with antibody to p26 is the same for nuclei released directly from
cysts onto microscope slides and for those purified on Percoll
gradients, under either acidic or basic conditions, regardless of the
fixation method. Identical results obtained by several preparative
protocols is a strong indication that the observed pattern reproduces
the distribution of p26 in vivo. p26 may simply aggregate in
the nuclei during stress and not interact with a particular structure
or protein. Of more interest, the arrangement of p26 into foci suggests
its association with specific nuclear compartments, such as protein
assemblies required for DNA replication (68), or elements of the
structural matrix within the nucleus (69). Sequence analysis failed to
reveal a typical nuclear localization signal consisting of either a
short stretch of lysines (PKKKRKV) or a bipartite signal wherein a
spacer region of 10 to 12 amino acid residues separates a pair of basic
clusters (KRPAATKKAGQAKKKK) (70-72). Of note, however, arginine is
enriched in the nonconserved amino terminus of p26, and potential thus exists for an unusual nuclear localization signal in p26, a proposal best examined by site-directed mutagenesis and deletion analysis.
Nuclear localization of p26 is also interesting from another
perspective. As noted previously, p26 resembles nucleolins and fibrillarins, families of proteins that occur in the nucleus, often in
association with the nucleolus. Nucleolin is a 76-kDa protein which,
like p26, shuttles between the nucleus and the cytoplasm, and it may be
involved in the transport of ribosomal subunits. Fibrillarins are
40-kDa proteins essential for rRNA processing, as are other nucleolar
proteins such as NSR1, SSB1, and GAR1, all of which contain the
so-called GAR domain (73-75). This domain varies from 15 to 80 amino
acid residues in length; it is composed mainly of glycine, arginine,
and phenylalanine, and it exhibits, at least in nucleolin (75),
repeated -turns as a major structural motif. In fibrillarin the GAR
domain is in the amino terminus and its arginines are largely
dimethylated; in nucleolin and NSR1 this domain is in the carboxyl
terminus, and it is in the central part of SSB1 and at each end of GAR1 (73-75). Additionally, the GAR domain occurs in nonnucleolar proteins (76-78). By comparison the GAR domain of p26, located in the amino variable region of the protein, is relatively small and its function has yet to be determined.
To conclude, we present, for the first time, the complete sequence of a
small heat shock/-crystallin protein from a crustacean. This protein
exhibits a conserved
-crystallin domain, as well as unusual
characteristics not found in other members of this group. Equally
interesting to the molecular insights of this work are its
physiological implications. As Artemia embryos enter
diapause, deplete their ATP reserves, and undergo desiccation, a
mechanism must exist to protect macromolecules and subcellular
components. The same is true for fully hydrated cysts that survive
anoxia for years. p26 is ideally suited for this role. Encysted embryos contain large amounts of p26 that exhibit chaperone activity in vitro, and a portion of the protein may engage intranuclear
components. We propose that p26 binds to proteins as they denature
under stress and expose hydrophobic residues, thereby preventing their
aggregation and precipitation. As a consequence, after diapause or
quiescence the embryos have a supply of functional proteins available
for immediate resumption of growth.
We thank Lynne Maillet-Frotten for expert technical assistance with the confocal microscopy and Dr. L. Sastre for supplying the Artemia cDNA library.