The expanding small heat-shock protein family, and structure predictions of the conserved

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UvA-DARE (Digital Academic Repository)
The expanding small heat-shock protein family, and structure predictions of the
conserved .. crystallin domain
Caspers, G.J.M.; Leunissen, J.A.M.; de Jong, W.W.
DOI
10.1007/BF00163229
Publication date
1995
Published in
Journal of molecular evolution
Link to publication
Citation for published version (APA):
Caspers, G. J. M., Leunissen, J. A. M., & de Jong, W. W. (1995). The expanding small heat-
shock protein family, and structure predictions of the conserved .. crystallin domain. Journal of
molecular evolution, 40, 238-248. https://doi.org/10.1007/BF00163229
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Download date:09 Mar 2023
J loM Evol )5991( 842-832:04
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The Expanding Small Heat-Shock Protein Family, and Structure Predictions
of the Conserved "a-Crystallin Domain"
Gert-Jan Caspers, 1 Jack A.M. Leunissen, 2 Wilfried W. de Jong 3'a
1 tnemtrapeD of ,yrtsimehcoiB ytisrevinU of ,negemjiN .O.P xoB ,1019 0056 BH ,negemjiN ehT sdnalrehteN
2 MMAC/SOAC ,retneC ytisrevinU of ,negemjiN ,negemjiN ehT sdnalrehteN
3 etutitsnI of cimonoxaT ,ygolooZ ytisrevinU of ,madretsmA ,madretsmA ehT sdnalrehteN
:devieceR 8 July 4991 / :detpeccA 82 rebmetpeS 4991
Abstract. The ever-increasing number of proteins Introduction
identified as belonging to the family of small heat-shock
proteins (shsps) and a-crystallins enables us to reassess The small heat-shock proteins (shsps) are one of the four
the phylogeny of this ubiquitous protein family. While most common groups of heat-shock proteins (Lindquist
the prokaryotic and fungal representatives are not prop- and Craig 1988; Hendrick and Hartl 1993; Morimoto et
erly resolved, most of the plant and animal shsps and al. 1994). These low-molecular-weight proteins are evo-
related proteins are clearly grouped in distinct clades, lutionarily related to the vertebrate lens protein ~-crys-
reflecting a history of repeated gene duplications. The tallin, as was first noted by Ingolia and Craig (1982). The
members of the shsp family are characterized by the shsps are a diverse family of proteins of 15-30 kDa
presence of a conserved homologous "cz-crystallin do- which tend to form large aggregates. Most species have
main," which sometimes is present in duplicate. Predic- multiple shsp genes, although in yeast and chicken only
tions are made of secondary structure and solvent acces- one gene was detected (Susek and Lindquist 1989; Miron
sibility of this domain, which together with hydropathy et al. 1991). In plants, the shsp genes are most numerous
profiles and intron positions support the presence of two (Vierling 1991). Apart from two multigene families of
similar hydrophobic a-sheet-rich motifs, connected by a cytoplasmic shsps (Raschke et al. 1988), plants have also
hydrophilic co-helical region. Together with an overview nucleus-encoded shsps that localize to chloroplasts
of the newly characterized members of the shsp family, (Chen and Vierling 1991). Recently, a fourth group of
these data help to define this family as being involved as plant shsps was found which are localized in the endo-
stable structural proteins and as molecular chaperones membrane system, most likely in the endoplasmic retic-
during normal development and induced under patholog- ulum (Helm et al. 1993).
ical and stressful conditions. cz-Crystallin is an abundant eye lens protein in verte-
brates. It is usually found as large aggregates, consisting
Key words: Molecular chaperones -- Molecular phy- of two types of subunits, aA and coB. (For reviews see
logeny -- Molecular evolution -- Structural domains -- Wistow and Piatigorsky 1988; Groenen et al. 1994.) It
Structure prediction has a structural function in the lens, warranting proper
refractive properties and transparency (Tardieu and De-
laye 1988). ocB- and to a lesser extent ctA-crystallin have
been shown to occur also in various tissues outside the
lens (Bhat and Nagineni 1989; Kato et al. 1991). Both
subunits are encoded by single-copy genes in humans.
ecnednopserroC :ot W.W. de Jong Functionally, the shsps and tx-crystallins share the prop-
932
erty of being molecular chaperones (Horwitz 1992; Ja- upstream from the genes, which are separated by 110 bp
kob et al. 1993), and both are able to convey thermotol- on the chromosome.
erance (Landry et al. 1989; Klemenz et al. 1991). A Clostridium acetobutylicum hspl8 was shown to be
Apart from the shsps and c~-crystallins, this family induced at the mRNA level by heat shock and the onset
also includes more disparate members, like certain sur- of solventogenesis, a metabolical shift in which excreted
face antigens in parasitic eukaryotes and bacteria ( Nene butyric acid and residual sugar are converted to acetone
et al. 1986; Nerland et al. 1988; Verbon et al. 1992). All and butanol shortly before entry into the stationary phase
members of the family are characterized by the presence (Sauer and Darre 1993). The shift to solventogenesis
of a homologous sequence of about 80 residues, which seems to be connected with the heat-shock response, but
has been dubbed the "o~-crystallin domain" and proba- the underlying molecular mechanisms are still largely
bly forms a distinct structural and functional unit. This unknown. In the myxobacterium Stigmatella aurantiaca
domain is preceded by an N-terminal region of variable a 21-kDa protein, SP21, is synthesized during heat
length, which shows little or no similarity between the shock, fruiting body formation, and stress induced by
various branches of the family. A short and variable se- oxygen limitation (Heidelbach et al. 1993a,b). The pro-
quence, but containing a conserved motif, extends C-ter- tein sedimented with the membrane fraction. As there
minally from the "~-crystallin domain" (Wistow 1985; were no indications of a direct interaction of SP21 with
de Jong et al. 1988). Unfortunately, no direct information the membranes, this suggests that SP21 forms aggregates
about the three-dimensional structure of any member of or is organized in larger complexes.
the shsp family is available, nor is there any deeper in- In the eukaryotic realm, several new plant sequences
sight into their structural roles and functional activities. were added to the set, including shsps of Arabidopsis
The c~-crystallin/small hsp family is not related to any thaliana (Bartling et al. 1992), maize Zea mays (Jor-
other protein family, although Lee et al. (1993) noticed gensen and Nguyen 1994), wheat Triticum aestivum
some minor sequence similarities between the "c~- (Weng et al. 1991) and rice Oryza sativa (Tseng et al.
crystallin domain" and two separate regions in the hsp70 1992; R. Nishi et al., data base acc. nr. P31673). Most of
family. This might reflect the sharing of some structural these sequences were determined at the cDNA level. The
features, possibly pertaining to the shared chaperone gene of the Pharbitis nil 17.1-kDa shsp was induced by
functioning, rather than an evolutionary relationship. light treatment and by heat shock, whereas its 18.8-kDa
Recently, a considerable number of sequences of di- gene was induced only by heat shock. Both are encoded
verse new members of the shsp family have been re- by one open reading frame. In the noncoding region of
ported. A comparison of the sequences and the structural both genes several heat-shock elements were found
and functional properties of all members of the family (Krishna et al. 1992). The Chenopodium rubrum 18.3-
should contribute to a more detailed view of the diver- kDa shsp belongs to the cytosolic shsp subfamily (class
gent evolution of this family. The objective of the present I). A structure with "positive DNA-binding regulatory
paper is to give an update of the phylogenies presented properties" was predicted that would form a helix-turn-
earlier (Plesofsky-Vig et al. 1992; de Jong et al. 1993), as helix region (Knack et al. 1992).
well as to analyze the structural and functional variation Maximal accumulation of the Helianthus annuus shsp
and similarity of the proteins. The latter information mRNA is detected in dry seeds and during embryo mid-
might be important for the future unravelling of the maturation stage in the absence of exogenous stress (A1-
structure-function of especially the conserved "c~- moguera and Jordano 1992). In seedlings, mRNA accu-
crystallin domain." mulation to lower levels is found in response to osmotic
stress and abscisic acid treatments. The protein has a
predicted weight of 17.6 kDa. A 22-kDa shsp from the
New Members of the Family
soybean Glycine max was found to be endomembrane
A description of the newly characterized members of the localized. It possesses an amino-terminal signal peptide
shsp family further highlights the variety of features pre- and a carboxyl-terminal sequence characteristic of an
sented by this family, as revealed already earlier (de Jong endoplasmic reticulum retention signal (Helm et al.
et al. 1993). The 16-kDa Escherichia coli IbpA and IbpB 1993). The expression of meiotic prophase repeat pro-
proteins, which are 52.2% identical, were found to be teins in Lilium sp. is developmentally induced in absence
induced in response to expression of several heterolo- of stress. A cDNA sequence for one of these proteins was
gous proteins (Allen et al. 1992). They are tightly asso- determined, and demonstrated it to be a shsp (Bouchard
ciated with inclusion bodies formed during the expres- 1990).
sion of these foreign proteins. The presence of a high Among the invertebrates, an additional Schistosoma
level of certain unfolded heterologous proteins may in mansoni egg antigen sequence, p40-2 (Cao et al. 1993),
fact be responsible for induction of IbpA and IbpB. Also, has 57% amino acid identity to the previously reported
induction occurred in wild-type Escherichia after heat p40 antigen (Nene et al. 1986), and likewise contains a
shock. A putative heat-shock promoter is indeed located duplicate "o~-crystallin domain." These stage-specific
240
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~+o.++oo++++~o.~m~.o+.+ .... ~m+..
~ ~ ~ m ~ " ~ ; ~ : ~ ' ' ~ +~ ~::~
o+o++++++++++o++.+o~+.++ooo+~o+m~+++,~+ . m++m .... m ~ m ~ ~+::::::::::
+ ° + + + ° ° ° + ° + + + + + + + + ~ + + ~ + + + + + + + + + + + + + + + +
+m++m+aa+++++ m
mr::> +oo oo+o +++
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!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!~!!!!!!!!!!!!!!!!!!!!!!!!
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.... ii ...........
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142
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242
antigens are soluble calcium-binding proteins (Moser et (1985). A considerable number of residues is highly con-
al. 1992). Expression of the gene for the shsp of the served throughout the family (indicated as "consensus"
gastrointestinal nematode Nippostrongylus brasiliensis is on the bottom line in Fig. 1), and the conservative nature
developmentally regulated, but not upregulated by heat of many other positions is conspicuous. The demarcation
shock or other stress conditions (Tweedie et al. 1993). between domain and tail is around position 110.
Another new nematode shsp sequence was found on The positions of the conserved "a-crystallin do-
chromosome III of Caenorhabditis elegans (Wilson et al. main" in the various typical representatives of the shsp
1994). Also two sequences of Onchocerca volvuIus dna family are schematically indicated in Fig. 2. Both Schis-
one of Acanthocheilonema viteae era now available (W. tosoma egg antigen sequences contain duplicate C-ter-
Hoefle, acc. nrs. $29691-3). minal domains. The C-terminal domain of the Neuros-
A new shoot of the family is represented by the 29- pora shsp is interrupted by an insertion of 64 amino acids
kDa protein HR-29 of the ascidian Halocynthia roretzi (after position 47 in the alignment). This figure also re-
(Takagi et al. 1993). The protein is abundant in body veals the length variation in the family. The length of the
wall muscle and localizes close to the plasma membrane. N-terminal regions, preceding the (z-crystallin-like do-
In the physiological condition it forms oligomers, ob- mains, varies from only 25 residues in the Caenorhab-
served by electron microscopy as globular aggregates ditis 123 gene that was found in the course of the chro-
with a diameter of 16.6 nm, as is also the approximate mosome III sequencing project (Wilson et al. 1994) to
size for ot-crystallin and shsps. 148 residues in the Halocynthia roretzi 29-kDa body-
Finally, an additional shsp was recently reported in wall protein. Although no sequence similarity can be
mammals. Kato et al. (1994) found high levels of a 20- detected between the N-terminal regions of all members
kDa protein, p20, in rat soleus muscle, heart, and dia- of the family, there are still some minor similarities be-
phragm. These tissues also contain high levels of ~B- tween the N-termini of the o~-crystallins, higher verte-
crystallin and hsp27. The p20 protein is present at lower brate shsps, and Drosophila shsps, as well as between
levels in other tissues. It occurs both as high-molecular- plant class I shsps and vertebrate shsps (de Jong et al.
weight aggregates and in dissociated forms. Upon heat- 1988). It must be concluded that the rate of evolution has
ing at 45°C of rat diaphragm in vitro, p20 was redistrib- been higher in the N-termini than in the C-terminal re-
uted from the cytoplasm to the insoluble fraction, and gions.
dissociation of the aggregated p20 to the small form was In plants, yeast, and most invertebrates, the shsp
enhanced. The primary structure of rat p20 and its human genes are encoded by intronless genes. Four shsp genes
homologue were determined at the amino acid level. of Caenorhabditis contain only one intron, coinciding
precisely with the first intron of the o~-crystallin genes
(Figs. 1 and 2). The aforementioned Caenorhabditis 123
Sequence Comparisons dna Gene Structure
gene contains two introns, of which the second coincides
To ensure that all available shsps and related sequences approximately with the second intron of the c¢-crystallin
were included in the present paper, a thorough database genes. The two introns of mammalian hsp27 are located
search was performed. To that end a previous alignment at different positions. The Halocynthia body-wall protein
of 57 sequences of "c~-crystallin domains" (de Jong et gene contains three introns, of which the second is lo-
al. 1993) was used to create a profile (Gribskov et al. cated three nucleotides downstream from intron # 1 in
1990) with which the NBRF, SwissProt, and EMBL da- Caenorhabditis shsps and in c~-crystallins, thus directly
tabases were searched to identify new sequences with preceding the C-terminal domain. The other two introns
similarities to the shsps. These were included in the ex- are located at different positions than those in other
panded alignment. As for the extensive set of available genes of the family and are phase 2, rather than phase 0
o~-crystallin sequences, only those for chicken c~A- and as are all other ones (Takagi et al. 1993). Although in-
~B-crystallin (de Jong et al. 1984; Sawada et al. 1992) trons # 1 and 3 in Fig. 2 demarcate the boundary between
were added to the previous alignment. Initial alignments the putative N- and C-terminal domains, there is little
of the expanded set of 85 entries were made with a evidence for the assumption that exon shuffling has
multiple alignment program, followed by manual im- played a role in the evolution of this protein family (Pat-
provements, as described earlier (de Jong et al. 1993). thy 1994).
Because a clear similarity among all members of the
family is restricted to the C-terminal parts of the proteins, ynegolyhP
only this region could be aligned satisfactorily, as shown
in Fig. .1 In the expanded shsp family more gaps needed Residues 1-104 of the alignment in Fig. ,1 encompassing
to be introduced in the alignment of this conserved re- the "o~-crystallin domain," were used to construct a phy-
gion. The region presented in Fig. 1 corresponds with the logenetic tree, using the neighbor-joining program (Sai-
putative C-terminal structural domain and extending tail tou and Nei 1987) from the PHYLIP package (Felsen-
of the c~-crystallins and shsps as proposed by Wistow stein 1993). The "~-crystallin domain" was considered
342
must have emerged before the divergence of monocots
Mycobacterium leprae
and dicots. Repeated duplications have occurred, espe-
Neurospora cially of class I genes. The Lilium meiotic prophase re-
peat protein appears to be most closely related to the
Pharbitis 17
class II proteins. The endomembrane-localized Pisum
and Glycine shsps represent an early offshoot of the class
Schistosoma p40
1 I proteins, as was also noted by Helm et al. (1993).
Caenorhabditis 17 l A monophyletic origin of the animal sequences is
1 2
hardly supported (Fig. 3c). The duplicated N- and C-do-
Caenorhabditis 123 l II l .
mains within the two Schistosoma egg antigens are very
Drosophila 27 l divergent. The two antigens are apparently the result of a
3 4 more recent gene duplication, long after the emergence
Halocynthia of the first antigen with two "t~-crystallin domains."
5 6
All nematode sequences group together, as do all
• T
Homo 27
1 7 Drosophila sequences, albeit poorly supported. It was
Bos ~B III III noted earlier that the Caenorhabditis shsp genes dupli-
cated at an early stage, followed by a more recent, further
duplication (Candido et al. 1989). The new Caenorhab-
0 i00 200 residues ditis sequence (Wilson et al. 1994) appears to represent a
Fig. .2 Location of eht suogolomoh sniamod dna nortni snoitisop ni different lineage, as it is more closely related to the On-
evitatneserper srebmem of eht shsp .ylimaf ehT suogolomoh -imret-C chocerca and Acanthocheilonema sequences. It was tab-
lan nillatsyrc-~t" domains" era detacidni by solid .srab lanimret-N ulated as most similar to t~B-crystallin by Wilson et al.
sniamod dna lanimret-C snoisnetxe era detacidni by ,senil -dnopserroc
(1994), but the present phylogenetic analysis reveals no
gni ni htgnel ot eht number of .seudiser Intron snoitisop ni eht -erroc
gnidnops seneg era dekram by .sdaehworra rehtehW snortni era tneserp special relationship. The two Onchocerca shsps appear
ni eht amosotsihcS 04p seneg si ton .nwonk nortnI snoitisop era erom to have arisen from a very recent gene duplication.
ylesicerp detacidni ni .giF l dna sti .sdnegel ehT noitisop of eht -resni As was noted before, the Xenopus shsps are not
noit ni aropsorueN 03psh si detacidni by eht nepo .elgnairt Names of closely related to other vertebrate shsps, and may in fact
eht secneuqes era sa ni .giF .1
be paralogues of these (de Jong et al. 1988). Interest-
ingly, the body-wall protein of the ascidian Halocynthia
not to extend beyond residue 104, because after this po- appears to be most closely related to the Xenopus shsps.
sition a 15-residue insert in the Lilium sequence breaks The other vertebrate shsps form a clade with the etA- and
up the alignment. Since the structures of all members of c~B-crystallins. In this clade, the recently discovered rat
the family are not known at the DNA level, the amino and human p20 proteins form a sister group to the mono-
acid sequences rather than the nucleotide sequences were phyletic c~A- and t~B-crystallins. They are not closer to
used in tree construction. The resulting tree is shown in c~B-crystallins, as Kato et al. (1994) assumed. Three gene
Fig. 3, where bootstrap values of 75% and higher are duplications thus have led to the expression of four par-
indicated. Because of the large number of sequences, the alogous genes in higher vertebrates---hsp27, p20, and
tree is depicted in three parts, comprising prokaryotes -A~o and txB-crystallin. These duplications preceded the
and lower eukaryotes (Fig. 3a), and the higher plant and earliest vertebrate radiation, since ~A- and c~B-
animal subtrees (Fig. 3b and c, respectively). The se- crystallins were already present before the dogfish
quences at the deepest branches of the tree, the prokary- Squalus diverged from the other vertebrates. Additional
ote and fungal proteins, are very divergent and not reli- duplications must have occurred in the hsp27 lineage.
ably resolved (Fig. 3a). Only the two Escherichia Ibp The human hsp27 gene family consists of four members
proteins group significantly together. The Mycobacte- (Hickey et al. 1986), although only a single sequence has
rium antigens do not form a sister group, and neither do been reported. Cooper and Uoshima (1994) found mul-
the Neurospora and Saccharomyces shsps. However, tiple hsp27 transcripts in murine osteoblasts, which may
these proteins need not be orthologues; it is likely that be encoded by separate genes. Also a mouse hsp27
only a small subset of the existing shsp variation has pseudogene has been characterized (Fr6hli et al. 1993).
been discovered yet. The present findings indicate that
reconstruction of the earliest divergent evolution of the
shsps will be problematic. Secondary Structure and Surface Residues of the
The angiosperm sequences group together, although ' '~-Crystallin Domain' '
at a low bootstrap value (Fig. 3b). A first divergence
occurs between the chloroplast sequences and the cyto- Insight into the properties of the shsp family is seriously
plasmic shsps. The latter divide highly significantly into hampered by the lack of structural information. Using
the class I and class II sequences (Vierling 1991), which advanced prediction methods on the accumulated se-
244
a, C.
Animals ool r Homo aA
e9 I--Jr Rattus aA
Escherichia a 99 F'I tv~ Bos aA
Escherichia b I I l Gallus aA
I - - Squalus aA
Clostridium
1~ 89 r Mesocricetus oB
Mycobac lep II 69 --IrHomo aB
Saccharomyces 8911 I"--I LBos aB
F=-I ~ t_. Gallus oB
Neurospora I I -' Squalus oB
Mycobac tub I I loo r Homo 20
~ LRattus 20
Chlamydomonos
l l 99 r'Homo 27
Stigmotella | I ool F---El Cricetulus 27
--I ' " I 10o'Mus 27
Plants
/ ' Gallus 25
II - - - -- Drosophila 6c
41 I - - " -Drosophila 22
0.0 0.1 0.2 0.3 0.4 0.5 L I 4.., Drosophila 25
I I f I I I I I ' Drosophila 26
_ . j Drosophila 27
b. Drosophila 6a
Caenorhabditis 125
68 I--- Petunia 21 IC 100 I Onchocerca Acanthocheilonema 251
--1 ------Arabidopsis 21 CI
99 Onchocerca 252
~6~2£i-----Glycine 22 CI
Nippostrongylus
29 I I i Pisum 21 IC
ool r- Caenorhabditis 16
--1 i Triticum 21 CI
| Chenopodium 25 Ct 39 i ool -LrCaenorhabCodenoirhatbdiitiss 12 17
16 L Caenorhabditis 1 61
~17 99 ~Xenopus 50d
18 -- om I ' I_Xenopus 50c
--4 ' Xenopus 50b
Halocynthia
ool Schistosoma 1N
I Schistosoma N2
7 ooi l Schistosoma CI
18 , ' Schistosoma C2
)n 18
5
5
Fig. .3 A phylogenetic tree based on the "c~-crystallin domains" of the shsp family
m18 (residues 1-104 in Fig. .)1 The tree saw constructed according to the neighbor-joining
method, using the program NEIGHBOR from the PHYLIP package (Felsenstein .)3991
Input distance matrices were calculated according to Fitch and Margnliash ,)7691(
excluding gaps from the calculations (program ,SEIGOLOMOH J.A.M. Leunissen,
:d .)dehsilbupnu Correction for multiple substitutions saw made according to Jukes and
) Cantor (1969). Relative bootstrap values (program SEQBOOT from the PHYLIP
~6
package) of %57 dna higher (from 1000 replicates) are indicated. Branch lengths are
19
61 proportional to the minimum number of mutations per residue (see scale). ehT tree is
17 depicted in three parts: (a) prokaryotes, fungi dna green alga, (b) higher plants, )e(
animals. Within plants, sispodibarA 61 to azyrO b71 belong to the class I shsps, and
psis 1 7b
sitibrahP 91 to aeZ a81 to the class II shsps. Rattus 72 is not included in the tree,
z-~k4 , ~,ZI being identical to suM 72 dna sulutecirC 72 in the "~-crystallin domain."
quences in Fig. 1 might allow us to reveal some major stead of the second ~-sheet stretch. Experimental spec-
structural features of this conserved domain. troscopic data support the prevalence of ~-sheet confor-
Secondary structure predictions of the (x-crystallin do- mation in the mammalian shsps and (x-crystallins (Li and
main were done for subsets of the alignment, using the Spector 1974; Merck et al. 1993a).
program PHD (Rost and Sander 1993; Rost et al. 1994) The residues of the last ~-sheet stretch of both motifs,
via the PredictProtein e-mail server at EMBL, Heidel- as well as the residues of the first p-sheet stretch of the
berg. Solvent accessibility prediction was according to first motif, are predicted to be buried, while the a-helix
Rost and Sander (1994), via the same e-mail server. The between the motifs is predicted to be in an exposed re-
results are shown in Fig. 4. For all subsets, the secondary gion. Surface residue predictions of the other parts of the
structure predictions start with a short (x-helix, followed sequence are more equivocal. The location of introns, the
by three p-sheet stretches, in the first half of the homol- large insertion in Neurospora hsp30, and the extensive
ogous domain. This ~313~c motif is repeated in the second presence of gaps in the region 43-71 (cf. Fig. 1) also
half of the domain, although in plant shsps there are two make it likely that this is a more flexible and exposed
initial (x-helices and an (x-helix stretch is predicted in- region connecting the two more rigid ~-sheet-rich motifs.
542
.giF .4 yradnoceS erutcurts noitciderp
)DHP( dna tnevlos ytilibissecca
noitciderp )ccA( by eht sdohtem of tsoR
dna ,3991( rednaS )4991 for eht
10 20 30 40
31 l 1 I 51 lanimret-C nillatsyrc-~c" domain" of
vv : l I lv stesbus of eht pshs ylimaf ,lamina(
PHD animal AAAAA ....B ~BBBBBB ......... BBBBBB ....B BBBB--- lamina ;secneuqes ,tnalp tnalp
Acc--animal ebbbeebeebbbbbebe-b-eeee-eebbbebeee-bbbbeb--
;secneuqes ,llarevo lairetcab dna lagnuf
PHD_plant AAA. .... BBBBBBB ........... BBBBBB ....B BBB. ... secneuqes plus sanomodymalhC ,22psh
Ace plant ebee--eeb-bbebebee--eeeeeeebebebee-ebbbbee-- ainuteP ,12psh aeZ ,a81psh ,suhtnaileH
PHD overall AAAA ..... BBBBBB ........... BBBBBB ....B BBBB--- amosotsihcS 04p lanimret-N ,niamod
Acc-overall ebeeeebeeb-bbbebeee-e-eeebebebebeee-bbbbeb-b sitidbahroneaC hspl2, alihposorD
,72psh suponeX ,c03psh soB
,nillatsyrc-B~c dna aihtnycolaH .)92-RH
50 60 70 80 90 100
12 71 4 61 I I onimA dica noitisop srebmun era sa ni
iv ,I • ,', I I .giF .1 ,A detciderp a-helix; ,B
PHD animal - - -AAAAA . . . . . . . . . . . . BBB ..... BBBBBB .......... BBBBBB .... BBBBB - detciderp ;teehs-3[ ,e detciderp desopxe
Acc--animal ....e -eee-eeeeeeeeeebbbeb--ebb-eb-beee-e-ebbbbbbbb-eebbbbbeb
;eudiser ,b detciderp deirub ;eudiser ,-
PHDplant AAAAAA ............... AAAAA--BBBBBB ...... AAAAAAAAA ..... BBBBB- on noitciderp .edam ehT snoitisop of
Accplant e--eeeeeeeeeee-e-b---beebbeebb--b-beee--ee-eebeb-b-eebbbbbbb snortni ni eht devresnoc sniamod era
PHD overall ............. AAA ........... BBBBBBB ......... BBBBB ..... BBBBB- detacidni yb sdaehworra sa ni .sgiF 1
Acc--overall eeeeeeeeeeeeeeee ...... e ....e bb--b-beee-e-e-e-bebeb-eebbbbb-b dna .2
This is also the position of the helix-turn-helix with functions among the members of the shsp family, one
DNA-binding properties suggested to be present in Che- might wonder what the primordial function of the com-
nopodium shsp (Knack et al. 1992). The possible pres- mon ancestor was. The fact that also in bacteria, like
ence of two similar structural motifs in the C-terminal Escherichia and Clostridium, shsps are now found to be
domain of c~-crystallin was first proposed by Wistow induced by heat shock, suggests that these proteins, too,
(1985) and appears to be corroborated by the present function in chaperoning other proteins under conditions
data. of stress. The Escherichia Ibp proteins also appear to be
The composite hydrophobicity profile (Fig. 5) con- induced by a high level of unfolded proteins (Allen et al.
firms the two-motif structure of the "a-crystallin do- 1992), which further strengthens the notion that chaper-
main." Peaks of hydrophobicity concur with the first oning is the primordial function of the family, rather than
p-sheet stretches in both motifs, whereas also the other a secondarily developed feature.
p-sheet stretches are located in the more hydrophobic The abundant expression of shsps as surface antigens
regions, in agreement with the predicted buried position in mycobacteria and in Schistosoma must then be one of
of these stretches. The exposed c~-helix that connects the the various derived functions in the family. For the SP21
two motifs locates with the strongly hydrophilic region protein of the myxobacterium Stigmatella it is speculated
around position 50. The C-terminal arm, beyond position that it might help pack mRNAs necessary for the early
110, also has a more hydrophilic character. In ~-crystal- steps of germination or might protect mRNAs of house-
lins, the terminal eight to ten residues indeed form very keeping genes during periods of development during
flexible extensions, as revealed by two-dimensional which other mRNAs are degraded (Heidelbach et al.
NMR spectroscopy (Carver et al. 1992). 1993b). A tight association during heat shock between
shsps and a specific subset of mRNAs has earlier been
reported for tomato cell cultures (Nover et al. 1989).
Evolution of Function Developmental regulation is also well documented in
many other members of the family, notably in animal
It is logicaI to assume that the conserved C-terminal shsps and c~B-crystallins (de Jong et al. 1993; Gernold et
domain would be responsible for the common structural al. 1993; Marin et al. 1993). Expression of the gene for
and functionalp roperties of the c~-crystallin/shsp family. the shsp of the gastrointestinal nematode Nippostrongy-
(For review of the latter, see Arrigo and Landry 1994.) lus brasiliensis is developmentally regulated (Tweedie et
Deletion of the last 42 amino acids of this domain in al. 1993). mRNA peak expression is concomitant with
Drosophila hsp27 indeed abolishes its capacity to protect the onset of immune damage in the host, but expression
cells against heat stress (Mehlen et al. 1993). However, is regulated independently of stress stimuli, apparently
in c~-crystallins and mouse hsp25 this separate domain according to a strict developmental program. In plants,
with its C-terminal extension has no chaperone activity like for Helianthus (Almoguera and Jordano 1992) and
in vitro (Merck et al. 1993b). It thus seems likely that alfalfa (Gy/3rgyey et al. 1991), the pronounced expres-
also the N-terminal domain is required for proper func- sion of shsp mRNA occurs during embryo midmatura-
tioning, and may moderate more specific functions for tion. Shsp mRNAs are stored for long periods of time in
various subgroups of shsps. Considering the multitude of dry seeds of sorghum (Howarth 1990), wheat (Helm and
642
B B 6 B B 3J
1.O i ,,,m
o:
o.
0.5
::--;i, .
. -i~ -'..
.,I=: ., ; -I~--;
a = =-'-:II':. m .... - c l:il,"" = . -" ° " - . ;l=-,~-i:.s-."= "- o._~+ - -
r .~:.~|11 - -s" I =~llr.. ,- . .;' :.~lZl i~i|~l..:: o .:-.~i-!!;-"::- -'!Pi~-~_ "-#. i:m,.,- -I "=- =. -: .... " ; .~-:- . • . . . . . , ..," .-
o
P 0,0 • : .., "~1 F.;g :: 1"1 - .: ; "..~.i|;.-+~.+. i~l~l i !,+- ~ " - ~.,..-.: '4;,+,+ ;~I ,: ,k "-: i 1'-':
It'~lF':31%7:l " ,-'-::--"l,~::,-~h~- '
Y "1: .~,,l'~'#-';-~~!!l! 'Ll'l~|i~:~ll;, ~!' I.-'~- !{',~-l;liil:~l..:" ~ . . . " : "-'" _:.." .... :~ll~".+-'~;+:"'":1 ::." - -'Ili"a.-;~!-"~i L
I:-". ~., '1- - :.~.,:~= : i'+ 4 "-~1" ",~11 d. " "-I$,F "1t~ .... .~;~.,,.'. ~-":~:~ -- .:'5 " . ,.'-~- -l- . : :+!~: 4"" -'%1 J'i~'l'l II"I : :L " 4t~-,;~:"~. +I" :"" +
;l -t , --* I!; -" *It:'; . ..;; .- .::11:; . . ~. i • .~ .~; -. illl~, l =.. i .-~1 Is'' .
:" -' I- " il| .... + 4--:-i .~; ,! ~i!!+: . ,, ;"
-0.5 - . : ." - * ~= :,- - . ; : i~t!,:- " - "" * •
"-,;,';~ . -= ;~;..-'- . ".. -.-.
" ;i~-~|~: -111 ". -.- "-". :i~':.:.+'::i: :
:~;il I ,:~.:
;dial .
"~M.m "
-1 .0 In: | I
SO 100 150
Alignment Positron
Fig. 5. Composite hydrophobicity profiles of all "c~-crystallin do- are as in Fig. .1 Hydrophobicity increases toward the top of the figure;
mains" of the shsp family, using the hydrophobicity scale of Sweet and hydrophilicity toward the bottom. Predicted R-sheets (see Fig. 4) are
Eisenberg (1983), as produced by the program CAMELEON (version indicated by bars.
3.0, Oxford Molecular Ltd, Oxford, UK). Amino acid position numbers
Abernethy 1990), and pea (Vierling and Sun 1987). All as a stabilizing protein like aB-crystallin does in skeletal
this supports the notion that developmental expression of muscle (Atomi et al. 1991).
shsps is quite universal (see Linquist and Craig 1988). This brief and necessarily fragmentary account makes
The shsps are apparently involved in cytomorphological it clear that gene duplications and divergent evolution
rearrangements, which may relate to their influence on have produced a broad array of structural and functional
actin polymerization (Miron et al. 1991) and intermedi- properties in the shsp family, using the common "~-
ate filament assembly (Nicholl and Quinlan 1994). crystallin domain" as an essential building block. The
As pointed out by Bouchard (1990), we find an addi- major challenge for the years to come is to elucidate the
tional non-stress-related function in meiotic cells of tertiary and quaternary structure of members of this fam-
three eukaryotic kingdoms. Lilium meiotic prophase re- ily and relate these to their various intriguing functions.
peat protein expression is developmentally induced
(Bouchard 1990). Two of the four Drosophila shsps are Acknowledgments. Part of this work was carried out under the aus-
expressed in the egg chamber of the ovary during the pices of the Netherlands Foundation for the Life Sciences (SLW) with
meiotic period of oogenesis (Zimmerman et al. 1983). financial aid from the Netherlands Organization for Scientific Research
(NWO). The use of the services and facilities of the Dutch CAOS/
The controlling regions for the heat shock and the ovar-
CAMM Center is gratefully acknowledged.
ian induction are different (Hoffman et al. 1987). The
Saccharomyces shsp is induced during sporulation and
meiosis (Kurtz et al. 1986).
References
Gene duplication is a general mechanism to acquire
more functions. For example, in Pharbitis nil, one of the
Allen SP, Polazzi JO, Gierse JK, Easton AM (1992) Two novel heat
shsp genes is induced by heat shock only, the other by
shock genes encoding proteins produced in response to heterolo-
light as well (Krishna et al. 1992). The expression of gous protein expression in Escherichia coli. J Bacteriol 174:6938-
smaller hsp27 transcripts in murine osteoblasts, which 7496
may be encoded by separate genes, was facilitated by Almoguera C, Jordano J (1992) Developmental and environmental con-
current expression of sunflower dry-seed-stored low-molecular-
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sion of the normal, longer, hsp27 was induced by heat
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tion must also be the mechanism which enabled an an- molecular-weight heat shock proteins. In: Morimoto RJ, Tissi~res
cestral o~-crystallin gene to be recruited for its function as A, Georgopoulos C (eds) The biology of heat shock proteins and
molecular chaperones. Cold Spring Harbor Laboratory Press, New
a lens structural protein. Another structural function may
York, pp 335-373
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