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J Mol Evol (1995) 40:238-248
JOURNAL OF
MOLECULAR
EVOLUTION
© Springer-Verl;i g New York Inc. 1W5
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 Jong1’3
1 Department of Biochemistry, University o f Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
2 CAOS/CAMM Center, University of Nijmegen, Nijmegen, The Netherlands
3 institute of Taxonomic Zoology, University of Amsterdam, Amsterdam, The Netherlands
Received: 8 July 1994 ! Accepted: 28 September 1994
Abstract.
T he ev er-in creasin g num ber of proteins
identified as belonging to the fam ily of small heat-shock
proteins (shsps) and a-crystallins enables us to reassess
the phylogeny o f this ubiquitous protein family. While
the prokaryotic and fungal representatives are not prop­
erly resolved, m ost o f the plant and animal shsps and
related proteins are clearly grouped in distinct clades,
reflecting a history of repeated gene duplications. The
m em bers of the shsp fam ily are characterized by the
presence o f a conserved hom ologous “ a-crystallin dom ain,” which som etim es is present in duplicate. Predic­
tions are m ade of secondary structure and solvent accessibility o f this dom ain, w hich together with hydropathy
profiles and intron positions support the presence o f two
similar hydrophobic P-sheet-rich motifs, connected by a
hydrophilic a-h elical region. Together with an overview
of the newly characterized m em bers o f the shsp family,
these data help to define this fam ily as being involved as
stable structural proteins and as molecular chaperones
during norm al developm ent and induced under patholog­
ical and stressful conditions.
Key words:
M olecular chaperones — Molecular phy­
logeny — M olecular evolution — Structural domains —
Stincture prediction
Correspondence to: W.W. de Jong
Introduction
The small heat-shock proteins (shsps) are one of the four
most common groups of heat-shock proteins (Lindquist
and Craig 1988; Hendrick and Hartl 1993; Morimolo ct
al. 1994). These low-molecular-weight proteins are evolutionarily related to the vertebrate lens protein cx-crystallin, as was first noted by Ingolia and Craig (1982). The
shsps are a diverse family of proteins ol 15-30 kDa
which tend to form large aggregates. Most species have
multiple shsp genes, although in yeast and chicken only
one gene was detected (Susek and Lindquist 1989; Miron
et al. 1991). In plants, the shsp genes are most numerous
(Vierling 1991). Apart from two multigene families of
cytoplasmic shsps (Raschke et al, 1988), plants have also
nucleus-encoded shsps that localize to chloroplasts
(Chen and Vierling 1991), Recently, a fourth group of
plant shsps was found which are localized in the endomembrane system, most likely in the endoplasmic retic­
ulum (Helm et al. 1993).
a-Crystallin is an abundant eye lens protein in verte­
brates. It is usually found as large aggregates, consisting
of two types of subunits, a A and aB . (For reviews see
Wistow and Piatigorsky 1988; Groenen et al. 1994.) It
has a structural function in the lens, warranting proper
refractive properties and transparency (Tardieu and De­
lay e 1988). aB - and to a lesser extent aA -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.
Functionally, the shsps and a-crystallins share the prop-
239
erty of being molecular chaperones (Horwitz 1992; J a ­
kob et al. 1993), and both are able to convey thermotol­
erance (Landry et al. 1989; Klemenz et al. 1991).
Apart from the shsps and a-crystallins, this family
also includes more disparate members, like certain sui>
face antigens in parasitic eukaryotes and bacteria ( None
et al. 1986; Norland et al. 1988; Verbon et al. 1992). All
members of the family are characterized by the presence
o f a homologous sequence of about 80 residues, which
has been dubbed the “ a-erystallin dom ain” and probably forms a distinct structural and functional unit. This
domain is preceded by an N-terminal region of variable
length, which shows little or no similarity between the
various brandies of the family. A short and variable se ­
quence, but containing a conserved motif, extends C-tcrinitially from the “ tt-crystallin dom ain” (Wistow 1985;
de Jong el al. 1988). Unfortunately, no direct information
about the three-dimensional structure of any member o f
the slisp family is available, nor is there any deeper in­
sight into their structural roles and functional activities.
The a-crystallin/sniall lisp family is not related to any
other protein family, although Lee et al. (1993) noticed
som e m inor sequence sim ilarities between the “ a crystallin dom ain” and two separate regions in the hsp7()
family. This might reflect the sharing of some structural
features, possibly pertaining to the shared chaperone
functioning, rather than an evolutionary relationship.
Recently, a considerable number of sequences of dtverse new members of the slisp family have been reported. A comparison of the sequences and the structural
and functional properties of all members of the family
should contribute to a more detailed view of the diver­
gent evolution of this family. The objective of the present
paper is to give an update of the phytogenies presented
earlier (Plesofsky-V iget al. 1992; de Jong etal. 1993), as
well as to analyze the structural and functional variation
and similarity of the proteins. The latter information
might be important for the future unravelling of the
slru c tu re -fu n c lio n o f especially the conserved “ a crystallin dom ain.”
New Members of the Family
A description of the newly characterized members of the
shsp family further highlights the variety of features prosented by this family, as revealed already earlier (de Jong
et al. 1993). The 16-kDa Escherichia coli IhpA and lbpB
proteins, which are 52.2% identical, were found to be
induced in response to expression of several heterologo us proteins (Allen et al. 1992). They are tightly assod a te d with inclusion bodies formed during the expres­
sion of these foreign proteins. The presence of a high
level o f certain unfolded heterologous proteins may in
fact be responsible for induction of IbpA and IbpB. Also,
induction occurred in wild-type Escherichia after heat
shock. A putative heat-shock promoter is indeed located
upstream from the genes, which are separated by 1 10 bp
on the chromosome.
A Clostridium acetobutylicum h s p !8 was show n to be
induced at the m RN A level by heat shock and the onset
o f solventogenesis, a m etabolical shift in w hich excreted
butyric acicl and residual sugar are converted to acetone
and butanol shortly before entry into the stationary phase
(Sauer and Dtirre 1993). T he shift to solventogenesis
seems to be connected with the heat-shock response, but
the underlying m olecular m echanism s are still largely
unknown. In the m yxobacierium Stigm atella aurantiaca
a 21-kD a protein, S P 21, is sy n th esized during heat
shock, fruiting body formation, and stress induced by
oxygen limitation (H cidelbach et al. 1993a,b). The p ro ­
tein scdimented with the m em brane fraction. As there
were no indications o f a direct interaction o f SP21 with
the membranes, this suggests that SP21 forms aggregates
or is organized in larger com plexes.
In the eukaryotic realm, several new plant sequences
were added to the set, including shsps of Arabiclopsis
ihaliana (Battling et al. 1992), m aize Xea m ays (Jorgensen and Nguyen 1994), w heat Triticum aestiviun
(W eng et al. 1991) and rice Oryza saliva (Tseng et al.
1992; R. Nishi et ah, data base ace, nr. P31673). M ost of
these sequences were determ ined at the cD N A level. T he
gene o f the Pharbitis nil 17.1-kDa slisp was induced by
light treatment and by heat shock, w hereas its 18.8-kDa
gene was induced only by heat shock. Both are encoded
by one open reading frame. In the noncoding region of
both genes several h eat-sh o ck elem en ts w ere found
(Krishna et al. 1992). T he Chenopociium rub m m 18.3kDa slisp belongs to the cytosolic shsp subfam ily (class
I). A structure with “ positive D N A -binding regulatory
properties” was predicted that w ould form, a helix-turnhelix region (Knack et al. 1992).
Maximal accum ulation o f the H elianthus cmnuus shsp
mRNA is detected in dry seeds and during em bryo m id­
maturation stage in the absence o f exogenous stress (Almoguera and Jordano 1992). In seedlings, m R N A accumutation to lower levels is found in response to osmotic
stress and abscisic acid treatm ents. T he protein has a
predicted w eight o f 17.6 kDa. A 22-kD a shsp from the
soybean Glycine m ax was found to be endom em brane
localized. It possesses an am ino-term inal signal peptide
and a carboxyl-term inal sequence characteristic o f an
endoplasm ic reticulum retention signal (H elm et al.
1993). The expression o f m eiotic prophase repeat proteins in Li Hum sp. is developm ental fy induced in absence
of stress. A cD N A sequence for one of these proteins was
determined, and dem onstrated it to be a shsp (Bouchard
1990).
A m ong the invertebrates, an additional Schistosom a
m ansoni egg antigen sequence, p40-2 (Cao et al. 1993),
has 57% am ino acid identity to the previously reported
p40 antigen (Nene et al. 1986), and likewise contains a
duplicate “ a-cry stallin d o m a in .” T hese stage-specific
13
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PG ..V D PTQ V
P G . .VDPTLV
PG ..V D PT L V
P G . *VDPTLV
P G . .YEATAV
A. .. .....V
DG..YEADKY
P G ..ÏE A D K V
D G .. YKAEQV
7X3--FD PN EV
K G ..YDPNEV
D D ..VDVSTV
E E -.V Q L D T I
E E . .V Q PD TI
E E ..V Q P D T I
ED..CDLDAV
E D . . AELPSV
ED . . VDX.TSV
E D . .VDVGAV
ED..VDVGAV
E S ..V N P E Q V
EG ..V N PEQ V
KG. .YNKEQV
E G -.V SA E R Ii
K S . . XDDSQL
K S . .XDSNQIi
E T . .VDPLSV
E T . .LD PLSV
EN ____ IHVR
E N -------MEVS
D H .-X D D SK I
____ U 3T E R I
DYPGVDADHX
GR. . VDQÏÜV
- .VGADEDDX
EN ..ANPDGX
E G . -VDGEMV
D K. .VDKDKV
X*i. .CEJUULL
E K . .CEKDKV
r a j . .CEKEKV
DE . . CDKSQV
EN-LT£KXDGX
S I . „AKVEEV
E H . .AKHEEX
H i . .AKMEEV
E H . .AKHDEV
CTf. .AKVDEV
E H . .AJSVBEY
fflT..AKHDKV
S3. .AKMDQV
» S . .AKMDQV
JEN. .AKKDQV
EH..AKVDQV
SCSLSADGHL
SCSLSADGML
TCSLSSDGKL
ACSLSNEGLL
TSSZ jSSDGVL
TSSLSSDGVL
TSSLSSDGVL
TSSLSLDGVL
TCSLSADGVL
TSALSPEGVL
TSALSPEGVL
SSSLSPEGTL
SSSLSPEGTL
SSSLSPEGTL
SSSLSPEGTL
RSSLSPDGML
SAA QGYR
4
.
.
SSSLSDDGVL
ASTLSSDGVL
VSQLSSDGVL
VSTVSSDGVXj
BSTLSSDGIL
KSHLATBGVL
eselsd k g v l
BSHLSDKGVL
ESHLSDKGVX«
HTELDNHGHL
KSATSiqEGKL
KSXX
ASNLSEDGKXi
¿SHLSEPGRt
VCSLSKNGHL
VCSLFKDGHEt
VCjrJuSKDGH [j
TSSLSSDGXZ.
KCRSETUDCVZj
NCSLTSDGVL
SAQVVGHT.L
SAQVXDQT.L
GANL.VMGLL
GATF.VNGLL
DASF.XiDGVL
LASY.QEGYL
KADY.ANGVL
SASIi-NNGXL
K A TY .D K G Ü
TAAH.DKGVL
PADL.KNGVL
KAEL.KHG7I.
XAEL.KSGVL
KAEL.KSGVL
fARTi.niKivii
BA2L.KSGVL,
KAEM.KNGVL
KXSM.ENGVL
K A SH .B PÏV L
KUCH.QiGVIt
n a f .BBCvr.
KAAM.AHGW
XAGH.EUGVI,
K A SM .aiG V I.
K&AM.ESGVL
TaJ^H-ESGTVL
KASM.ESGVI.
O lSH.HSÖVL
muremurKi«? B í . .AKV23EV K&SH.BjGVli
GHFMHRFRIiP E N . .AKVEQV K A SK .Ö G V L
TPSGPKVQSG
TESGPKIPSG
TPSGPKVPSSf
TLCCPRTRPG
TVNGPRKQAS
TVNGPRRQVS
TVNGPRKQAS
TVSAPHKQSD
TÏTGPH3CVAD
SIQ A AP.A SA
SIQ A TP-A SA
TVEAPHPKLA
TVEAPLPKTA
TVEMLPKAV
TVZAPLPKAV
rVEAPLPKPA
FGCHCFHFVG
TISYPNPPGV
TUCVPKPPAI
TVSIPKPQAV
TU A PPPPSK
TVKAPQPLPV
TXXASKKA-.
T ÏG A S K .A T I
TÏCANK- TAY
TISANK.TAX
SIEAPEDGfPS
QXE2LDKKTN.
q x ea pk k tn .
STEAPKKEAV
SXSAPKKEAI
HXQAPRIALP
HXQAPRU lLP
HXKAPWIALP
QXDAPVAVAI
KLE2LPVKVDQ
KLGAPVNVPD
V LEA PLEK -.
VliEAPLHK. .
YIDIxERVIPE
HXDLXRNEPE
R IT L P K . -K
KL5IPVAERA
TIiTVPKLKPQ
TJTV PK ____
TV SV A V SE..
W T V P K . .HE
KiTLPKHPEV
LiXSTPiTTKVE
FXTIPKTKVE
YXTIPKTKVE
YXTIPKTKXE
LVSVPKEETE
KVTVPKJK-.
SVTVPKVQ-.
SV TV PK V P..
TW VPKAP - .
T V W P K . , .B
TVTVPKV.
TVTVPKV----TVTVPK. . .E
TV TV PK .. .E
TVTVPK. . -E
TVTVPK. - . 2
TVTVPK___ E
TVTVPK___ E
TVTVPK. . . 3
.
.
.
LCAG. , BSE.
V D A G ..H5E.
M D P S ..H S E .
DDSH..W QD.
G . ------ P E .
G.............. P E .
G ..............P E .
V
PE.
V
PE.
QA. . PPPAAA
QASLPSPPAA
TQ S____ N E .
TQ S____ A E .
T Q S - - ..A E .
TQ S____ A E .
I Q S ____ S E .
CWSSQYHG.QETL---- K E.
E D K G ...N E .
EDICS . . . jrjs .
EQAK
SE
VKGSLERQE.
.
.
.
.
150
BAIPVSREEK
SAJFVSKEEK
RAIPVSREEK
SPIFV 5B EEK
RPIPVSREEK
RTÏPXTREEK
ÏÏT IP IT R E E K
R T IPIT R E E K
RSXPITREEK
RSVPISRDEK
K......................
K ......................
IT IP V T F E 5 R
IT IPV T PE A R
ITIPV T FE M L
IT IPV T PE A R
ITXPVTVEAK
STISFQGG&Q
KEVTIEQTGE
RIVQIQQVGP
SIIQ IQ Q V G P
RIVQXQQTGP
S IV D IQ Q ISQ
p t s a p s s _____
P S S A P S S ...
.
P S S A P S S .. .
.
PT SA PSS___
.
QGTQPE1BAD p
PAVTAAPKK. .
PAV5Ü&AIKT.
PAVTAAPKK. .
PAXAGSQRK- .
PAVAGPQQK. .
.
AQLGGPEAAK
AQIGGQEAGK
AQXGGPEAGR
AQIGGPE___
KEEPAKK.. .
GAHHTH____
P • . . AjvjSlSAE
AHLNVKEEJPK
AHLNVKANES
AHLSVKAPAP
QQ.KDKDAHR
sd eta a k
SEQSGAK
SEQSGAK
SEQ5GAK
1
EPKDKTASQ. ...................
EAVEQDNGND K .............. ..
EVKGKENGAP HGKDK.. .
EAGDGKXENG SGEKMETS
QSHQR.........................
G ____ .SPAA R N IPIR A SPK EPGAIQKSES MNKEQ.
R N IPIR A SPK EPEAKQKTKK ............
R N IPIR A SPK EPEANQKSAX NDAK.
QKFQSSABSS SAEEPiEVGDT EPVNKRILYK ............
HSIPXHFVAK
RSIPXNFVAK K ....................................................
* Q6a É« « « 4 A H SIP IQ Q A IV
• QG. . *m++m RSXPIQQAFV EQKTSE................................................
TPXPISMDTA PRDAQELPPD AQTSN&EGDQ KVD
PA ---- . .P E . TPXPXSMDTA PBDAQEDPPD AQTSNAEGEQ QHD
P A ---- - . P E . TPXPISHDEH PEMPRKFHPH PRZAHRHD..............
DN. . . . .X X . TAVPVTVEHT X ..............................................................
n q s l t l n e s . GCVAVRPKSD NQIKAVPASQ ALVAKGVHGL SYV,
YQSLTPNDH. GQXAJKPKSH NQVQNAQKLA Y K G V H G Y SIV ____
• ---- AJ.TH___
.Q S ..
.C H . .
.A K K . . . PRR X E IN ..............................................................
PIA A Q H IA IS ERPALNS.................................................
VKGK___IJHG RRXDXH.......................................................
KPKK.............. ISVDRGNNGH QTINKTAHEI ID A . . . .
KDGK___NHV KKTEVSSQES WOT....................................................................
.A K K .. .H E T XRIATN...................... .................................................................
. . GK___ PTE KHXQHLST6T.................................................................................
PPA K . . . PEP KRXAVTGA............................................................................. . .
QPKR............- ..IQ V A S S G T EQKEM,LKAYP APAEPGLAAP LGWPGFS
...................... K KVTCVEXK.............................................................................. .. *
R KVXDVQJQ....................................................................................
...................... R KVXDVQVQ...................... .. ..........................................................
TVXDVQIQ.. ............................................................................
...................... R KVXDVQVQ....................................................................................
EEEK .............. KDVFQYHVD.................................................................................
.E S K -----PEV K SX D ISG .............. ........................................................................
.E K K . . .P E V K SX D IS .............................................. ..................... .................
.E K K ...P Q V KSXDXSGAN. ............................................................................
KKKlC- . .^PH VKAXDISG...................... * . * .....................................................
E I K K ...P E V X2XDX5G*
EMKK. . .P E V K SX EX SG .....................................................................................
E-i RA- . .A SV KSXEXSG.......................................................................................
E IK K ---- PEV KSXETSS
jsvj!k£. . . PEV K T ID IS G
EVKK. . .P E V KSXEXSG.
E I K K .. . PUV KAXDXSG.
EVKK.___ PDV KSXEXSG
EVKK___ PDV KAXEXSG.
H......................
TOirgn-p..............................
9##
« ^ . . KT.P!. . , ,
.............
............. H
«
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.
.
.
.
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G ly c i n e _ 1 5
G ly c in e _ l6
C h e n o p o d i.am _ 1 8
T rltic u m _ 1 6
Z ea_172
O ry z a _ 1 7 a
O ry za_ _ 1 7 b
G ly c in e _ 2 2 a
Pisum_22
P h a r b i t i s_ 1 9
G ly c in e _ 6 X
P h a x b itis _ J L 7
P is \n n _ 1 7
A x a i3 id o p s x s _ 1 7 t>
Z ea_18a
Z ea_ 1 8 i>
C o n sen su s
DWKETQ-EAH
DWXETP.EAH
DWKETP.EAH
DWKETP.EAH
DWKETP.EAH
DWKETP.ESH
DWKJ5TP, EAH
DHKETP,EGH
DWKETP,EGH
DXKNLP. DAY
DVKEYP.NSY
DVKSYP.NSY
DYKEYP .NSY
BVKEHP.NSY
BVXEHP.NAY
DVKELP-GAY
DVKELP.GAY
VLKADXPG.Ii
VP KADXPG.Ii
VFKADLPG.V
VFXVDLPG.V
VFKADLPG.V
VFKADLPG-V
VEKADVPG.L
v m ld v pg .l
VXMVDVPG.L
LYFXDMPG.V
VFXADMPG.V
VFEXDMPG.L
VFXIDM PG.L
VFMVDHPG.V
A F W D H P C .I
AFWDMPG.Xi
AFW DM PG.L
------- D -PG —
K . K E. .EVKV
K .K E.vEV K V
K .K E . .EVKV
K .K E . „EVKV
K .K E ..E V K V
K .K E . .EVKV
K .K E . .EVKV
K .R E ..E IK V
K .K D .»DXKX
R .T G . .E IK V
K.j&A. .E IK V
K .S G ..D X K V
K . SG . , DIKV
K .S G . .D IK V
K .G D . .E IK V
G .T G ..D X R V
G .T G ..D IK V
----- E — B—KV
QIEDDRV^QI
QXEDDKVXiQX
EVElXaiVIiRX
EVEDGNVLW
EVEDGNVLVX
EVEEGNVLVX
EVEDGNVSRS
EVEENKVLRV
EVEENKVLKV
EVEDPSDLVI
QVEDDNVLW
DVEDDNLLLX
QVDGXJNVLSX
QVEDENVLLX
QVENDNVLW
QVEDEHVLW
QVEDERVLVX
-V -D — VL—X
SG ..ER N V EK
S G . .ERNVEK
SG ..Q RA REK
S G . .ERSREK
SG . * QRSREK
SG .-Q RSK EK
A G ..ERXKEQ
SG ..E R K K E E
S G . . iSKJ^KfiK
SG . . liKKHiSiS
S G - -ERTER2
CG . . £RK3U)S
SG . . JStcKiKEA
SG » .ERKREE
SG . .ERQREH
SG .-E R K R E E
S G . .ERRREE
_G--- ---------- —
EDK.
EDK.
EEK .
EDK.
EDK.
EDK.
EEK*
EKK.
DKK.
. . .NDT
. - .NDT
. . -NDT
. . -NDK
« . .DDK
. . -SDK
. ..T D K
. . . GDH
. . . GDH
WHR
WHR.
WHR.
WHR.
WHR.
WHR.
WHR.
WHR.
WHR.
yqi.
KDEK. -DGVK YIoR.
EK E. . . .GAK YXiR.
EEKE. . .GAK YVR.
* . . GVK YLK
KENE . . .GVK YVR,
R E D .. . .DAK YLR,
YIiR.
R E .. .
E ------- ----- D— —H—
m
VDRSS
VERSS
.VERSS
VERSS
VERSS
.VERSS
VERSS
VERSY
VERSY
HERWT
MSRKV
MERHV
.MERHV
MERRX
MERRM
MERRM
MERRM
---- R —
Fig. 1. Alignment of the homologous C-terminal regions o f the shsps and related proteins. Se­
quences are designated by genus name and, for the small hsps, the numbers as used in the databases.
These numbers can be different from those used in the literature. oA and aB —ctA- and aB-crystallin
sequences, respectively. Schistosoma-Nl, -N2, ~C1, and -C2 = N- and C-tenninal copies, respectively,
of the duplicated a-crystallin domains in the egg antigen p40 and p40-2. Mycobac lep and Mycobac
tub = Mycobacterium leprae and M. tuberculosis. Cl = chloroplast-localized hsps. The positions
corresponding to the introns in the genes for Caenorhabditis elegans hsps (L 2 \ Halocynthia roretzi
HR-29 (3, 4 ), mammalian hsp27 (5, (5), and aA - and aB-crystallins (i, 7) are indicated with arrows
above the alignment. All introns fall between codons (phase 0), apart from # 4 , which is between the
second and third nucleotides in the codon for residue 70 (phase 2). “ Consensus” indicates residues
that are present in 50% or more of the entries at a given position. To the alignment of de Jong et al.
(1993) the following sequences are added: Gallus gallus aA-crystallin (database accession number
P02504, residues 63-173), G. gallus aB-crystallin (Q05713, 66-174), Homo sapiens p20 (Kato et al.
1994, 66—160), Rattus norvegicus p20 (Kato et al. 1994, 66—162), R. norvegicus hsp27 (R.R. Giimont
and M J. Welsh, M86389, 91—206), Nippostrongylus brasiliensis hsp20 (S33416, 57—172), Cae
norhabdiiis elegans hspl2.3 (L I5188, translation of residues 36,675-36,752, 37,383—37,496,
GXPMRRPRLP EN
LgAyMRRkriTfP
GQFMRKFRLP
GKFVRRFRLP
GQFXRRFRLP
GQFMRRFRItP
GKFLHRFRLP
GKFWRQFRLP
GKFWRQFKLP
GRHMRKFELP
ŒKFMBXFVLP
6KLHBKFVLP
GKLMRKFVLP
GKLMHKFVT.P
.
EH.
ED,
DD.
EN.
EN.
QK.
QN.
KN.
ST
£21.
ENEN.
■
GKPKRKFVIiP DN<
GKFHKKFVLP XXI.
--- F - R ----- L P ----
G^HP^FQLP
•AKVEQV
AKVEQV
■AKVDQV
■AKVEEV
AKVDQV
.AKVDQV
TKPEQX
VDLDSV
.VDLDSV
ADTKAV
-AHVEAX
ANTHAX
ANKEKX
ANXEAX
ADU3KX
ADVDKV
ADMDKI
KACM-ENGVL T V TX PK .. .E
TV TV PK .. .E
KAGM-EÏJGVL TV TV PK .. -N
KAGL.ENGVL TV TV PK .. .A
Üa GIi . ENGVL TV TV PK .. .A
KA6L »ENGVL TVTVPK. - .A
KASM.ENGVL TVTVPK. . .E
^
*'• - fciwJuT. TLTLDKLSPG
KAKM.ENGVL TLTLHKLSHD
SAVW.KNGVL AVTVERLFVP
NAVY.QDGVi QVTVEKLPPP
SAVC. QDGVL SVTVQKLPPP
TAVC. QDGVL TVTVENVPPP
S A IS . QDGVXi TVTVMKLPPP
SAVC.HDGVL KVTVQKLPPP
AAVC.RDGVL TVTVEKLPPP
SAVC.RDGVL TVT VEKLPPP
-A -L ----- GVL T ----- PK-------
K&SH>£N6?L
EV K K -. .SDV
EVKK. . .PDV
E A P K .. .PQ V
EVKK. . .P E V
EEKK » - . PEV
EVKK. . .P E V
EPKK---- PDV
KXKG-----PKV
KXKG---- PRH
LPTK T...........
EPK K P...........
EPK K P...........
EPKKP...........
EPKKP...........
EPK K P............
EPKKP............
EPKKP............
K PIEX SG ...........................................
KAXEISG........................................... .
K xnavy
KAXEISG...........................................
KAXEISG........................................... .
KAXEISG...........................................
K S IQ IT G ...........................................
VSXAGEDHQQ GNLNNDGAKQ EL
VSXVEEDDKP SKXVNDELK. . .
KSXEVKXA........................................
KTVEVKVA.........................................
RTIQVKVA.........................................
RTIEVKXG.........................................
KTIQVKVA.................„ .....................
KTIQVQVA.................. ......................
KSXEVKVA.........................................
KTXEVKVA........................... .. . . .
— I —
X-----------------------------------------------------------------------
...........................
37,904—38,044), Acanthocheilonema viteae AV25 (S29691, 62-179), Onchocerca volvulus OV25-1
(S29692, 61-173), O. volvulus OV25-2 (S29693, 49-165), Halocynthia roretzi HR-29 (JX0258,
147—252), Schistosoma mansoni p40-2 (M96866, 128-253 and 254—342), Escherichia coli IbpA
(A45245, 39—137), E. coli IbpB (B45245, 37—142), Clostridium acetobutylicum hspl8 (S25534.
48-151), Stigmatella aurantiaca SP21 (M94510, 57-188), Arabidopsis thaliana hspl7.6 (P29830,
49-155), Zea mays hspl7.2 (X65725, 48-152), Triticum aestivum h.sp26.6 (Q0Q445, 134—236),
Oryza sativa hspl7a (P27777, 46-150), O. sativa hspl7b (P31673, 50-154), Pharbitis nil hspl7
(Q01544, 48—155), P. nil hspl9 (Q01545, 59—167), Chenopodium rubrum hspl8 (Q05832, 58-161),
Helianthus annuus hspl8 (P30693, 48-153), Glycine max hsp22 (P30236, 70-192), Lilium sp.
EMPR6 (A61054,43-146). The Homo sapiens otA-crystallin sequence underwent a minor correction
at positions 114-118 of the alignment according to Takemoto and Emmons (1991) and Caspers et al.
(1994). The Mus hsp27 has been corrected as described by Gaestel et al. (1993) and Merck et al.
(1993b). To avoid undue lengthening of the alignment, two unique sequences are left out (marked by
open arrowheads): a 64-residue insert after position 47 in Neurospora hsp30 (A), and a 15-residue
insert after position 104 in Lilium EMPR6 (B).
to
242
antigens are soluble calcium-binding proteins (Moser et
al. 1992). Expression of the gene for the shsp of the
gastrointestinal nematode Nippostrongylus brasiliensis is
developmentally regulated, but not upregulated by heat
shock or other stress conditions (Tweedie et al. 1993).
Another new nematode shsp sequence was found on
chromosome III of Ccienorhabditis elegans (Wilson et al.
1994). Also two sequences of Onchocerca volvulus and
one of Acanthocheilonema viteae are now available (W.
Hoefle, acc. nrs. S29691-3).
A new shoot of the family is represented by the 29kDa protein HR-29 of the ascidian Halocynthia roretzi
(Takagi et al. 1993). The protein is abundant in body
wall muscle and localizes close to the plasma membrane.
In the physiological condition it forms oligomers, ob­
served by electron microscopy as globular aggregates
with a diameter of 16.6 nm, as is also the approximate
size for a-crystallin and shsps.
Finally, an additional shsp was recently reported in
mammals. Kato et al. (1994) found high levels of a 20kDa protein, p20, in rat soleus muscle, heart, and dia­
phragm. These tissues also contain high levels of aBcrystallin and hsp27. The p20 protein is present at lower
levels in other tissues. It occurs both as high-molecularweight aggregates and in dissociated forms. Upon heat­
ing at 45°C of rat diaphragm in vitro, p20 was redistrib­
uted from the cytoplasm to the insoluble fraction, and
dissociation of the aggregated p20 to the small form was
enhanced. The primary structure of rat p20 and its human
homologue were determined at the amino acid level.
*
Sequence Comparisons and Gene Structure
To ensure that all available shsps and related sequences
were included in the present paper, a thorough database
search was performed. To that end a previous alignment
of 57 sequences of “ a-crystallin domains” (de Jong et
al. 1993) was used to create a profile (Gribskov et al.
1990) with which the NBRF, SwissProt, and EMBL da­
tabases were searched to identify new sequences with
similarities to the shsps. These were included in the ex­
panded alignment. As for the extensive set of available
a-crystallin sequences, only those for chicken aA - and
aB-crystallin (de Jong et a l 1984; Sawada et al. 1992)
were added to the previous alignment. Initial alignments
of the expanded set of 85 entries were made with a
multiple alignment program, followed by manual im­
provements, as described earlier (de Jong et al. 1993).
Because a clear similarity among all members of the
family is restricted to the C-terminal parts of the proteins,
only this region could be aligned satisfactorily, as shown
in Fig. 1. In the expanded shsp family more gaps needed
to be introduced in the alignment of this conserved re­
gion. The region presented in Fig. 1 corresponds with the
putative C-terminal structural domain and extending tail
of the a-crystallins and shsps as proposed by Wistow
(1985). A considerable number of residues is highly con­
served throughout the family (indicated as “ consensus”
on the bottom line in Fig. 1), and the conservative nature
of many other positions is conspicuous. The demarcation
between domain and tail is around position 110.
The positions of the conserved “ a-crystallin do­
main” in the various typical representatives of the shsp
family are schematically indicated in Fig. 2. Both Schis­
tosoma egg antigen sequences contain duplicate C-ter­
minal domains. The C-terminal domain of the Neurospora shsp is interrupted by an insertion of 64 amino acids
(after position 47 in the alignment). This figure also re­
veals the length variation in the family. The length of the
N-terminal regions, preceding the a-crystallin-like do­
mains, varies from only 25 residues in the Caenorhabditis 123 gene that was found in the course of the chro­
mosome III sequencing project (Wilson et al. 1994) to
148 residues in the Halocynthia roretzi 29-kDa bodywall protein. Although no sequence similarity can be
detected between the N-terminal regions of all members
of the family, there are still some minor similarities be­
tween the N-termini of the a-crystallins, higher verte­
brate shsps, and Drosophila shsps, as well as between
plant class I shsps and vertebrate shsps (de Jong et al.
1988). It must be concluded that the rate of evolution has
been higher in the N-termini than in the C-terminal re­
gions .
In plants, yeast, and most invertebrates, the shsp
genes are encoded by intronless genes. Four shsp genes
of Caenorhabditis contain only one intron, coinciding
precisely with the first intron of the a-crystallin genes
(Figs. 1 and 2). The aforementioned Caenorhabditis 123
gene contains two introns, of which the second coincides
approximately with the second intron of the a-crystallin
genes. The two introns of mammalian hsp27 are located
at different positions. The Halocynthia body-wall protein
gene contains three introns, of which the second is lo­
cated three nucleotides downstream from intron # 1 in
Caenorhabditis shsps and in a-crystallins, thus directly
preceding the C-terminal domain. The other two introns
are located at different positions than those in other
genes of the family and are phase 2, rather than phase 0
as are all other ones (Takagi et al. 1993). Although in­
trons # 1 and 3 in Fig. 2 demarcate the boundary between
the putative N- and C-terminal domains, there is little
evidence for the assumption that exon shuffling has
played a role in the evolution of this protein family (Patthy 1994).
Phylogeny
Residues 1-104 of the alignment in Fig. 1, encompassing
the “ a-crystallin domain,” were used to construct a phy­
logenetic tree, using the neighbor-joining program (Saitou and Nei 1987) from the PHYLIP package (Felsenstein 1993). The “ a-crystallin domain” was considered
ft
^
243
Mycobacterium leprae
Neurospora
Pharbitis 17
Schistosoma p40
1
T
Caenorhabditis 17
1
2
T
T
Caenorhabditis 123
Drosophila 27
3
4
T
T
Halocynthia
Homo 27
5
6
T
T
1
7
T
T
Bos aB
100
200 residues
Fig. 2. Location of the homologous domains and intron positions in
representative members of the shsp family, The homologous C-terminal “ a-crystallin domains” are indicated by solid bars. N-terminal
domains and C-terminal extensions are indicated by lines, correspond­
ing in length to the number of residues. Intron positions in the corre­
sponding genes are marked by arrowheads. Whether inlrons are present
in the Schistosoma p40 genes is not known. Intron positions are more
precisely indicated in Fig. 1 and its legends. The position of the inser­
tion in Neurospora hsp30 is indicated by the open triangle, Names of
the sequences are as in Fig. 1.
not to extend beyond residue 104, because after this position a 15-residue insert in the Lilium. sequence breaks
up the alignment. Since the structures of all members of
the family are not known at the DNA level, the amino
acid sequences rather than the nucleotide sequences were
used in tree construction. The resulting tree is shown in
Fig. 3, where bootstrap values of 75% and higher are
indicated. Because of the large number of sequences, the
tree is depicted in three parts, comprising prokaryotes
and lower eukaryotes (Fig. 3a), and the higher plant and
animal subtrees (Fig. 3b and c, respectively). The sequences at the deepest branches of the tree, the prokaryote and fungal proteins, are very divergent and not reliably resolved (Fig. 3a). Only the two Escherichia Ibp
proteins group significantly together. The Mycobacterium antigens do not form a sister group, and neither do
the Neurospora and Saccharomyces shsps. However,
these proteins need not be orthologues; it is likely that
only a small subset of the existing shsp variation has
been discovered yet. The present findings indicate that
reconstruction of the earliest divergent evolution of the
shsps will be problematic.
The angiosperm sequences group together, although
at a low bootstrap value (Fig. 3b). A first divergence
occurs between the chloroplast sequences and the cytoplasmic shsps. The latter divide highly significantly into
the class I and class II sequences (Vierling 1991), which
must have emerged before the divergence of monocots
and dicots. Repeated duplications have occurred, espe­
cially of class I genes. The Lilium meiotic prophase re­
peat protein appears to be most closely related to the
class II proteins. The endomembrane-localized Pisum
and Glycine shsps represent an early offshoot of the class
I proteins, as was also noted by Helm et al. (1993).
A monophyletic origin of the animal sequences is
hardly supported (Fig. 3c). The duplicated N- and C-domains within the two Schistosoma egg antigens are very
divergent. The two antigens are apparently the result of a
more recent gene duplication, long after the emergence
of the first antigen with two “ a-crystallin domains.”
All nematode sequences group together, as do all
Drosophila sequences, albeit poorly supported. It was
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
ditis sequence (Wilson et al. 1994) appears to represent a
different lineage, as it is more closely related to the On­
chocerca and Acanthocheilonema sequences. It was tab­
ulated as most similar to aB-crystallin by Wilson et al.
(1994), but the present phylogenetic analysis reveals no
special relationship. The two Onchocerca shsps appear
to have arisen from a very recent gene duplication.
As was noted before, the Xenopus shsps are not
closely related to other vertebrate shsps, and may in fact
be paralogues of these (de Jong et al. 1988). Interest­
ingly, the body-wall protein of the ascidian Halocynthia
appears to be most closely related to the Xenopus shsps.
The other vertebrate shsps form a clade with the aA - and
aB-crystallins, In this clade, the recently discovered rat
and human p20 proteins form a sister group to the mono­
phyletic aA - and aB-crystallins. They are not closer to
aB-crystallins, as Kato et al. (1994) assumed. Three gene
duplications thus have led to the expression of four paralogous genes in higher vertebrates— hsp27, p20, and
aA - and aB-crystallin. These duplications preceded the
earliest vertebrate radiation, since a A - and a B crystalling were already present before the dogfish
Sqiialus diverged from the other vertebrates. Additional
duplications must have occurred in the hsp27 lineage,
The human hsp27 gene family consists of four members
(Hickey et al. 1986), although only a single sequence has
been reported. Cooper and Uoshima (1994) found multiple hsp27 transcripts in murine osteoblasts, which may
be encoded by separate genes. Also a mouse hsp27
pseudogene has been characterized (Frohli et al. 1993).
Secondary Structure and Surface Residues of the
“a-Crystallin Domain”
Insight into the properties of the shsp family is seriously
hampered by the lack of structural information. Using
advanced prediction methods on the accumulated se-
244
a.
c.
loo r Homo aA
Rattus aA
96
771 Bos aA
99
Gallus aA
Squalus aA
9B r Mesocricetus aB
96 “H r Homo aB
t-Bos aB
89
Gailus
aB
74
Squalus aB
100
Homo 20
- c Rattus 20
99 r H o m o 27
“\ j Cricetulus 27
100
iooIMus 27
— Gallus 25
Drosophila 6c
— Drosophila 22
-Drosophila 23
— Drosophila 26
-Drosopnila 27
Drosophila<6a
Caenorhabditis 123
- Acanthocheìlonema
100
- Animals
------ Escherichia a
--------- Escherichia b
--------- Clostridium
------- Mycobac lep
- Saccharomyces
- Neurospora
Mycobac tub
- Chlam ydom onas
— Stigmatella
99
77
¥’
Plants
0.0
0.1
0.2
0.3
0.4
J____ I____ L
L
0.5
-1 .....I
b.
'P e tu n ia 21 Cl
— Arabidopsis 21 Cl
98 — Glycine 22 Cl
Pisum 21 Cl
Triticum 21 Cl
Chenopodium 23 Cl
88i— Arabidopsis 16
- Arabidopsis 17
Arabidopsis 18
Helianthus
Daucus 177
Daucus 179
Pisum 18
ra-Medicago 17
IP-Medicago 18
81 ’— Lycopersicon 18
- Glycine 11
85 'Glycine 14
r- Glycine 13
15
Glycine 16
Chenopodium 18
82 I— Triticum 16
“(j— Zea 172
Oryza 17a
------Oryza 17b
loo I—
r~“ bGlycine
22a
iy
■— Pisum 22
Lilium 6
-Pharbitis 19
— Glycine 61
89
-Pharbitis 17
Pisum 17
— Arabidopsis 17b
100
Zea 18b
{ j Zea 18a
j
66
100
92
100
96
100
b
á
93
r- <
s®1-Onchocerca 252
-------- Nippostrongyjus
100
1Caenorhabditis 16
Caenorhabditis *17
100
^Caenorhabditis 12
Caenorhabditis 161
99 I— Xenopus 30d
r *— Xenopus 30c
*--------Xençpus 30b
■Halocynthia
----------------Schistosoma N1
-------------- -S c h isto so m a N2
Schistosom a C1
------ Schistosom a
C
93
100
100
100
C2
Fig. 3. A phylogenetic tree based on the “ a-crystallin domains” of the shsp family
(residues 1-104 in Fig. 1), The tree was constructed according to the neighbor-joining
method, using the program NEIGHBOR from the PHYLIP package (Felsenstein 1993)
Input distance matrices were calculated according to Fitch and Margoliash (1967),
excluding gaps from the calculations (program HOMOLOGIES, J.A.M. Leunissen,
unpublished). Correction for multiple substitutions was made according to Jukes and
Cantor (1969). Relative bootstrap values (program SEQBOOT from the PHYLIP
package) of 75% and higher (from 1000 replicates) are indicated. Branch lengths are
proportional to the minimum number of mutations per residue (see scale). The tree is
depicted in three parts: (a) prokaryotes, fungi and green alga, (b) higher plants, (c)
animals. Within plants, Arabidopsis 16 to Oryza. 17b belong to the class I shsps, and
Pharbitis 19 to le a 18a to the class 11 shsps. Rattus 27 is not included in the tree,
being identical to Mus 27 and Cricetulus 27 in the “ a-crystallin domain.”
quences in Fig. 1 might allow us to reveal some major
structural features of this conserved domain.
Secondary structure predictions of the a-crystallin do­
main were done for subsets of the alignment, using the
program PHD (Rost and Sander 1993; Rost et al. 1994)
via the PredictProtein e-mail server at EMBL, Heidel­
berg. Solvent accessibility prediction was according to
Rost and Sander (1994), via the same-e-mail server. The
results are shown in Fig. 4. For all subsets, the secondary
structure predictions start with a short a-helix, followed
by three (3-sheet stretches, in the first half of the homol­
ogous domain. This a(3(3(3 motif is repeated in the second
half of the domain, although in plant shsps there are two
initial a-helices and an a-helix stretch is predicted in­
stead of the second (3-sheet stretch. Experimental*spec­
troscopic data support the prevalence of (3-sheet confor­
mation in the mammalian shsps and a-crystallins (Li and
Spector 1974; Merck et al. 1993a),
The residues of the last (3-sheet stretch of both motifs,
as well as the residues of the first (3-sheet stretch of the
first motif, are predicted to be buried, while the a-helix
between the motifs is predicted to be in an exposed re­
gion. Surface residue predictions of the other parts of the
sequence are more equivocal. The location of introns, the
large insertion in Neurospora hsp30, and the extensive
presence of gaps in the region 43-71 (cf. Fig. 1) also
make it likely that this is a more flexible and exposed
region connecting the two more rigid (3-sheet-rich motifs.
10
i
13
30
20
40
PH D _anim al
A c c T a n im a l
15
YY
|
|
!▼
AAAAA-------BBBBBBBB-------------- -BBBBBB----- BBBBBe b b b e e b e e b b b b b e b e - b - e e e e - e e b b b e b e e e bbbbeb
P H D _ p lan t
A c c _ p la n t
AAA-------- BBBBBBB------------------- BBBBBB-------BBBB------e b e e — e e b - b b e b e b e e — e e e e e e e b e b e b e e - e b b b b e e '—
P H D _ o v e ra ll
Acc o v e r a l l
AAAA-------- BBBBBB--------------------BBBBBB------ BBBBB----e b e e e e b e e b - b b b e b e e e - e * -e e e b e b e b e b e e e -b b b b e b ~ b
50
70
7 |
4
80
90
100
ii
j
i
i
\
Y 1
Y
T |
—bbb
-BBBBBB-----■AAAAA---- BBBBBB' -----BBBBB- e - e e e e e e a e e e e e e b b b e b - e b b - e b - b e e e -e-* ebbbbbbbb eetobbbbeb
I9
PH D _anim al
A c c _ a n im a l
60
6
P H D _ p lan t
A c c _ p la n t
AAAAAA-------------------------- AAAAA— BBBBBB---------- AAAAAAAAA-------- BBBBBe — e e e e e e e e e e e - e - b -----b e e b b e e b b — b - b e e e — e e -e e b e b - b - e e b b b b b b b
P H D _ o v e ra ll
A c c ~ * o v e ra ll
----------------------- AAAA—
e e e e e e e e e e e e e e e e -----
mm * * * * * *
-----BBBBBBB----------------BBBBB-------- BBBBB— e b b —■ b -b e e e -e -e ~ e -b e b e b -e e b b b b b -b
This is also the position of the helix-turn-helix with
DNA-binding properties suggested to be present in Chenopoclium shsp (Knack et al. 1992). The possible pres­
ence of two similar structural motifs in the C-terminal
domain of a-crystallin was first proposed by Wistow
(1985) and appears to be corroborated by the present
data.
The composite hydrophobicity profile (Fig. 5) con­
firms the two-motif structure of the “ a-crystallin do­
main.” Peaks of hydrophobicity concur with the first
p-sheet stretches in both motifs, whereas also the other
P-sheet stretches are located in the more hydrophobic
regions, in agreement with the predicted buried position
of these stretches. The exposed a-helix that connects the
two motifs locates with the strongly hydrophilic region
around position 50. The C-terminal arm, beyond position
110, also has a more hydrophilic character. In a-crystallins, the terminal eight to ten residues indeed form very
flexible extensions, as revealed by two-dimensional
NMR spectroscopy (Carver et al. 1992).
Evolution of Function
It is logical to assume that the conserved C-terminal
domain would be responsible for the common structural
and functional properties of the a-crystallin/shsp family.
(For review of the latter, see Arrigo and Landry 1994.)
Deletion of the last 42 amino acids of this domain in
Drosophila hsp27 indeed abolishes its capacity to protect
cells against heat stress (Mehlen et al. 1993). However,
in a-crystallins and mouse hsp25 this separate domain
with its C-terminal extension has no chaperone activity
in vitro (Merck et al. 1993b). It thus seems likely that
also the N-terminal domain is required for proper func­
tioning, and may moderate more specific functions for
various subgroups of shsps. Considering the multitude of
Fig. 4. Secondary structure prediction
{PHD) and solvent accessibility
prediction (Acc) by the methods of Rost
and Sander (1993, 1994) for the
C-terminal “ a-crystallin domain” of
subsets of the shsp family (animal,
animal sequences; plant, plant
sequences; overall, bacterial and fungal
sequences plus Chlamydomonas hsp22,
Petunia hsp21, Zea hspl8a, Helianthns,
Schistosoma p40 N-terminal domain,
Caenorhabditis hsp!2, Drosophila
hsp27, Xenopus hsp30c, Bos
aB-crystallin, and Halocynthia HR-29).
Amino acid position numbers are-as in
Fig. 1. At predicted a-helix; B,
predicted p-sheet; e, predicted exposed
residue; b, predicted buried residue;
no prediction made. The positions of
introns in the conserved domains are
indicated by arrowheads as in Figs. 1
and 2.
functions among the members of the shsp family, one
might wonder what the primordial function of the com­
mon ancestor was. The fact that also in bacteria, like
Escherichia and Clostridium, shsps are now found to be
induced by heat shock, suggests that these proteins, too,
function in chaperoning other proteins under conditions
of stress. The Escherichia Ibp proteins also appear to be
induced by a high level of unfolded proteins (Allen et al.
1992), which further strengthens the notion that chaper­
oning is the primordial function of the family, rather than
a secondarily developed feature.
The abundant expression of shsps as surface antigens
in mycobacteria and in Schistosoma must then be one of
the various derived functions in the family. For the SP21
protein of the myxobacterium Stigmatella it is speculated
that it might help pack mRNAs necessary for the early
steps of germination or might protect mRNAs of house­
keeping genes during periods of development during
which other mRNAs are degraded (Heidelbach et al.
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),
Developmental regulation is also well documented in
many other members of the family, notably in animal
shsps and aB-crystallins (de Jong et al. 1993; Gernold et
al. 1993; Marin et al. 1993). Expression of the gene for
the shsp of the gastrointestinal nematode Nippostrongylus brasiliensis is develop mentally regulated (Tweedie et
a l 1993), mRNA peak expression is concomitant with
the onset of immune damage in the host, but expression
is regulated independently of stress stimuli, apparently
according to a strict developmental program. In plants,
like for Helianthns (Almoguera and Jordano 1992) and
alfalfa (Gyorgyey et al. 1991), the pronounced expres­
sion of shsp mRNA occurs during embryo midmatura­
tion. Shsp mRNAs are stored for long periods of time in
dry seeds of sorghum (Howarth 1990), wheat (Helm and
246
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0 .5
H
£
H» »
o
p
SL
0.0
t
h
y
- 0 .5
-1 . O
1 OO
SO
150
A lig n m e n t P osition
Fig. 5. Composite hydrophobicity profiles of all “ a-crystallin do­
mains” of the shsp family, using the hydrophobicity scale of Sweet and
Eisenberg (1983), as produced by the program CAMELEON (version
3.0, Oxford Molecular Ltd, Oxford, UK). Amino acid position numbers
are as in Fig. I. Hydrophobicity increases toward the top of the figure;
hydrophilic)ty toward the bottom. Predicted p-sheets (see Fig. 4) are
indicated by bars.
Abernethy 1990), and pea (Vierling and Sun 1987). All
this supports the notion that developmental expression of
shsps is quite universal (see Linquist and Craig 1988).
The shsps are apparently involved in cytomorphological
rearrangements, which may relate to their influence on
actin polymerization (Miron et al. 1991) and intermedi­
ate filament assembly (Nicholl and Quinlan 1994).
As pointed out by Bouchard (1990), we find an addi­
tional non-stress-related function in meiotic cells of
three eukaryotic kingdoms. Lilium meiotic prophase re­
peat protein expression is developmentally induced
(Bouchard 1990). Two of the four Drosophila shsps are
expressed in the egg chamber of the ovary during the
meiotic period of oogenesis (Zimmerman et al. 1983).
The controlling regions for the heat shock and the ovar­
ian induction are different (Hoffman et al. 1987). The
Saccharomyces shsp is induced during spoliation and
meiosis (Kurtz et al. 1986).
Gene duplication is a general mechanism to acquire
more functions. For example, in Pharbitis nil, one of the
shsp genes is induced by heat shock only, the other by
light as well (Krishna et al. 1992). The expression of
smaller hsp27 transcripts in murine osteoblasts, which
may be encoded by separate genes, was facilitated by
estrogen treatment prior to heat shock, whereas expres­
sion of the normal, longer, hsp27 was induced by heat
shock alone (Cooper and Uoshima 1994). Gene duplica­
tion must also be the mechanism which enabled an an­
cestral a-crystallin gene to be recruited for its function as
a lens structural protein. Another structural function may
be found for the HR-29 protein of Halocynthia roretzi,
which is localized in body-wall muscle (Shirakata et al.
1986). It may be a component of myofibrils and may act
as a stabilizing protein like aB-crystallin does in skeletal
muscle (Atomi et al. 1991).
This brief and necessarily fragmentary account makes
it clear that gene duplications and divergent evolution
have produced a broad array of structural and functional
properties in the shsp family, using the common “ a crystallin domain” as an essential building block. The
major challenge for the years to come is to elucidate the
tertiary and quaternary structure of members of this fam­
ily and relate these to their various intriguing functions.
Acknowledgments, Part of this work was carried out under the aus­
pices of the Netherlands Foundation for the Life Sciences (SLW) with
financial aid from the Netherlands Organization for Scientific Research
(NWO), The use of the services and facilities of the Dutch CAOS/
CAMM Center is gratefully acknowledged.
References
Allen SP, Polazzi JO, Gierse JK, Easton AM (1992) Two novel heat
shock genes encoding proteins produced in response to heterolo­
gous protein expression in Escherichia coll J Bacteriol J 74:6938—
6947
Almoguera C, Jordano J (1992) Developmental and environmental con­
current expression of sunflower dry-seed-stored low-molecularweight heat-shock protein and Lea mRNAs. Plant Mol Biol 19:
781-792
Arrigo A-P, Landry J (1994) Expression and function of the lowmolecular-weight heat shock proteins. In: Morimoto RJ, Tissieres
A, Georgopoulos C (eds) The biology of heat shock proteins and
molecular chaperones. Cold Spring Harbor Laboratory Press, New
York, pp 335-373
Atomi Y, Yamada S, Strohman R, Nonomura Y (1991) ccB-Crystallin
in skeletal muscle: purification and localization. J Biochem 110:
812-822
»
247
Bartling D, Bulter H, Liebeton K, Weiler EW (1992) An Arabidopsis
lhaliana cDNA clone encoding a 17.6 kDa das II heat shock pro­
tein. Plant Mol Biol 18:1007-1008
Bhat SP, Nagineni CN (1989) aB subunit of lens-specific protein
a-crystallin is present in other ocular and non-ocular tissues. Biochem Biophys Res Commun 158:319-325
Bouchard RA (1990) Characterization of expressed meiotic prophase
repeat transcript clones of Lilium: meiosis-specific expression, re­
latedness, and affinities to small heat shock protein genes. Genome
33:68-79.
Candido EPM, Jones D, Dixon DK, Graham RW, RussnakRH, Kay RJ
(1989) Structure, organization, and expression of the 16-kDa heat
shock gene family of Coenorhabditis elegans. Genome 31:690-697
Cao M, Chao H, Doughty BL (1993) Cloning of a cDNA encoding an
egg antigen homologue from Schistosoma mansoni Mol Biochem
Parasitol 58:169-172
Carver JA, Aquilina JA, Truscott RJW, Ralston GB (1992) Identifica­
tion by 'H NMR spectroscopy of flexible extensions in bovine lens
a-crystallin. FEBS Lett 311:143-149
Caspers G-J, Penning« J, de Jong WW (1994) A partial cDNA se­
quence corrects the human aA-crystallin primary structure. Exp
Eye Res 59:125-126
Chen Q, Vierling E (1991) Analysis of conserved domains identifies a
unique structural feature of a chloroplast heat shock protein. Mol
Gen Genet 226:425-431
Cooper LF, Uoshima K (1994) Differential estrogenic regulation of
small M, heal shock protein expression in osteoblasts. J Biol Chem
269:7869-7873
de Jong WW, Leunissen JAM, Leenen PJM, Zweers A, Versteeg M
(1988) Dogfish a-crystallin sequences: comparison with small heat
shock proteins and Schistosoma egg antigen. J Biol Chem 263:
5141-5149
de Jong WW, Leunissen JAM, Voorter CEM (1993) Evolution of the
a-crystallin/small heat-shock protein family, Mol Biol Evol 10:
103-126
de Jong WW, Zweers A, Versteeg M, Nuy-Terwindt EC (1984) Pri­
mary structures of the a-crystallin A chains of twenty-eight mam­
malian species, chicken and frog, Eur J Biochem 141:131-140
Felsenstein J (1993) PHYL1P: phylogeny inference package, version
3.5c. Distributed by the author, Department of Genetics, UniversiLy
of Washington, Seattle WA
Fitch WM, Margoliash E (1967) Construction of phylogenetic trees.
Science 155:279-284
Frohli E, Aoyama A, Klemenz R (1993) Cloning of the mouse hsp25
gene and an extremely conserved hsp25 pseudogene. Gene 128:
273-277
Gaestel M, Gotthardt R, Miller T (1993) Structure and organization of
a murine gene encoding small heat-shock protein Hsp25. Gene
128:279-283
Gemold M, Knauf U, Gaestel M, Stahl J, Kloetzel P-M (1993) Devel­
opment and tissue-specific distribution of mouse small heat shock
protein hsp25. Dev Genet 14:103-111
Gribskov M, Luethy R, Eisenberg D (1990) Profile analysis. Methods
Enzymol 193:146-159
Groenen PJTA, Merck KB, de Jong WW, Bloemendal H (1994) Struc­
ture and modifications of the junior chaperone a-crystallin: from
lens transparency to molecular pathology. Eur J Biochem 225:1-19
Gyorgyey J, Gartner A, Nemeth K, Magyar Z, Hirt H, Heberle-Bors E,
Dudits D (1991) Alfalfa heat shock genes are differentially ex­
pressed during somatic embryo genesis. Plant Mol Biol 16:9991007
Heidelbach M, Skladny H, Schairer HU (1993a) Purification and char­
acterization of SP21, a development-specific protein of the myxobacterium Stigmatella aurantiaca. J Bacteriol 175:905-908
Heidelbach M, Skladny H, Schairer HU (1993b) Heat shock and de­
velopment induce synthesis of a low-molecular-weight stressresponsive protein in the myxobacterium Stigmatella aurantiaca. J
Bacteriol 175:7479-7482
Helm KW, Abernethy RH (1990) Heat shock proteins and their
mRNAs in dry and early imbibing embryos of wheat. Plant Physiol
93:1626-1633
Helm KW, LaFayette PR, Nagao RT, Key JL, Vierling E (1993) Lo­
calization of small heat shock proteins to the higher plant endomembrane system. Mol Cell Biol 13:238-247
Hendrick JP, Hartl FU (1993) Molecular chaperone functions of heatshock proteins. Annu Rev Biochem 62:349-384
Hickey E, Brandon SE, Potter R, Stein G, Stein J, Weber LA (1986)
Sequence and organization of genes encoding the human 27 kDa
heat shock protein. Nucleic Acids Res 14:4127-4145
Hoffman EP, Gerring SL, Corces VG (1987) The ovarian, ecdysterone,
and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene react very differently to perturbations of DNA
sequence. Mol Cell Biol 7:973-981
Horwitz J (1992) a-Crystallin can function as a molecular chaperone.
Proc Natl Acad Sci USA 89:10449-10453
Howarth C (1990) Heat shock proteins in Sorghum bicolor and Pennisetum americaniun. II. Stored RNA in sorghum seed and its re­
lationship to heat shock protein synthesis during germination. Plant
Cell Environ 13:57-64
Ingolia TD, Craig EA (1982) Four small heat shock proteins are related
to each other and to mammalian a-crystallin. Proc Natl Acad Sci
USA 79:2360-2364
Jakob U, Gaestel M, Engel K, Buchner J (1993) Small heat shock
proteins are molecular chaperones. J Biol Chem 268:1517-1520
Jorgensen JA, Nguyen HT (1994) Isolation, sequence and expression of
a cDNA encoding a Class I heat shock protein (HSP17.2) in maize.
Plant Sci 97:169-175
Jukes TH, Cantor CR (1969) Evolution of protein molecules, In: Munro
HN (ed) Mammalian protein metabolism. Academic Press, New
York, pp 21-132
Kato K, Goto S, Inaguma Y, Hasegawa K, Morishita R, Asano T
(1994) Purification and characterization of a 20-kDa protein that is
highly homologous to aB crystallin. J Biol Chem 269:1530215309
Kato K, Shinohara H, Kurobe N, Goto S, Inaguma Y, Ohshima K
(1991) Immunoreactive aA-crystallin in rat non-lenticular tissues
detected with a sensitive immunoassay method. Biochim Biophys
Acta 1080:173-180
Klemenz R, FrGhli E, Steiger RH, Schafer R, Aoyama A (1991) aBCrystallin is a small heat shock protein. Proc Natl Acad Sci USA
88:3652-3656
Knack G, Liu Z, Kloppstech K (1992) Low molecular mass heat-shock
proteins of a light-resistant photoautotrophic cell culture, Eur J Cell
Biol 59:166-175
Krishna P, Felsheim RF, Larkin JC, Das A (1992) Structure and lightinduced expression of a small heat-shock protein gene of Pharbitis
nil. Plant Physiol 100:1772-1779
Kurtz S, Rossi J, Petko L, Lindquist S (1986) An ancient developmen­
tal induction: heat-shock proteins induced in sporulation and oo­
genesis. Science 231:1154-1157
Landry J, Chretien P, Lambert H, Hickey E, Weber LA (1989) Heat
shock resistance conferred by expression of the human HSP27 gene
in rodent cells. J Cell Biol 109:7-15
Lee DC, Kim RY, Wistow GJ (1993) An avian aB-crystallin: non-lens
expression and sequence similarities with both small (HSP27) and
large (HSP70) heat shock proteins. J Mol Biol 232:1221-1226
Li L-K, Spector A (1974) Circular dichroism and optical rotatory dis­
persion of the aggregates of purified polypeptides of alphacrystallin. Exp Eye Res 19:49-57
Lindquist S, Craig EA (1988) The heat shock proteins, Annu Rev
Genet 22:631-677
Marin R, Valet JP, Tanguay RM (1993) Iisp23 and hsp26 exhibit
distinct spatial and temporal patterns of constitutive expression in
Drosophila adults. Dev Genet 14:69-77
Mehlen P, Briolay J, Smith L, Diaz Latoud C, Fabre N, Pauli D, Arrigo
AP (1993) Analysis of the resistance to heat and hydrogen peroxide
248
stresses in COS cells transiently expressing wild type or deletion
mutants of the Drosophila 27-kDa heat-shock protein. Eur J Biochem 215:277-284
Merck KB, Groenen PJTA, Voorter OEM, de Haard-Hoekman WA,
Horwitz J, Bloemendal H, de Jong WW (1993a) Structural and
functional similarities of bovine a-crystallin and mouse small heatshock protein: a family of chaperones. J Biol Chem 268:1046-1052
Merck KB, Horwitz J, Kersten M, Overkamp P, Gaestel M, Bloemen­
dal H, de Jong WW (1993b) Comparison of the homologous carboxy-terminal domain and tail of a-crystallin and small heat shock
" protein. Mol Biol Rep 18:209-215
Miron T, Vancompernolle K, Yanderkerckhove J, Wilchek M, Geiger
B (1991) A 25-kD inhibitor of actin polymerization is a low mo­
lecular weight mass heat shock protein. J Cell Biol 114:255-261
Morimoto RI, Tissi&res A, Georgopoulos C (eds) (1994) The biology of
heat shock proteins and molecular chaperones. Cold Spring Harbor
Laboratory Press, New York
Moser D, Doenhoff MJ, Klinkert MQ (1992) A stage-specific calciumbinding protein expressed in eggs of Schistosoma mansonl Mol
Biochem Parasitol 51:229-238
Nene V, Dunne DW, Johnson KS, Taylor DW, Cordingley JS (1986)
Sequence and expression of a major egg antigen from Schistosoma
mansoni: homologies to heat shock proteins and a-crys tall ins. Mol
Biochem Parasitol 21:179-188
Nerland AH, Mustafa AS, Sweetser D, Godal T, Young RA (1988) A
protein antigen of Mycobacterium leprae is related to a family of
small heat shock proteins. J Bacteriol 170:5919-5921
Nicholl ID, Quinlan RA (1994) Chaperone activity of a-crystallin
modulates intermediate filament assembly. EMBO J 13:945-953
Nover L, Scharf K-L, Neumann D (1989) Cytoplasmic heat shock
granules are formed from precursor particles and are associated
with a specific set of mRNAs. Mol Cell Biol 9:1298-1308
Patthy L (1994) Introns and exons. Curr Opin Struct Biol 4:383-392
Plesofsky-Vig N, Vig J, Brambl R (1992) Phylogeny of the a-crystal~
lin-related heat-shock proteins. J Mol Evol 35:537-545
Raschke E, Baumann G, SchÖffl F (1988) Nucleotide sequence analysis
of soybean small heat shock protein genes belonging to two differ­
ent multigene families. J Mol Biol 199:549-557
Rost B, Sander C (1993) Prediction of piotein secondary structure at
better than 70% accuracy. J Mol Biol 232:584-599
Rost B, Sander C (1994) Conservation and prediction of solvent ac­
cessibility in protein families. Proteins (in press)
Rost B, Sander C, Schneider R (1994) PHD—an automatic mail server
for protein secondary structure prediction. Comp Appl Biosci 10:
53-60
Saitou N, Nei M (1987) The neighbor-joining method: a new method
for reconstructing phylogenetic trees. Mol Biol Evol 4:406-425
Sauer U, Dürre P (1993) Sequence and molecular characterization of a
DNA region encoding a small heat shock protein of Clostridium
acetobutylicum. J Bacteriol 175:3394-3400
Sawada K, Agata K, Eguchi G (1992) Crystallin gene expression in the
process of lentodogenesis in cultures of chicken lens epithelial
cells. Exp Eye Res 55:879-887
Shirakata M, Takagi T, Konishi K (1986) Isolation and characterization
of a 29,000 dalton protein from ascidian (Halocynthia roretzi) body
wall muscle. Comp Biochem Physiol 85B:71-76
Susek RE, Lindquist SL (1989) HSP26 of Saccharomyces cerevisiae is
related to the superfamily of small heat shock proteins but is with­
out a demonstrable function. Mol Cell Biol 9:5265-5271
Sweet M, Eisenberg D (1983) Correlation of sequence hydrophobicities
measures similarities in three-dimensional protein structures. J Mol
Biol 171:479-488
Takagi T, Yasunaga H, Nakamura A (1993) Structure of 29-kDa pro­
tein from ascidian (Halocynthia roretzi) body wall muscle. J Bio­
chem 113:321-326
Takemoto L, Emmons T (1991) Truncation of aA-crystallin from the
human lens. Exp Eye Res 53:811-813
Tardieu A, Delaye M (1988) Eye lens proteins and transparency: from
light transmission theory to solution X-ray structural analysis.
Annu Rev Biophys Chem 17:47-70
Tseng TS, Yeh KW, Yeh CH, Chang FC, Chen YM, Lin CY (1992)
Two rice {Oryza sativa) full-length cDNA clones encoding lowmolecular-weight heat-shock proteins. Plant Mol Biol 18:963-965
Tweedie S, Grigg ME, Ingram L, Selkirk ME (1993) The expression of
a small heat shock protein homologue is developmentally regulated
in Nippostrongylus brasiliensis. Mol Biochem Parasitol 61:149154
Verbon A, Hartskeerl RA, Schuitema A, Kolk AHJ, Young DB, Lathigra R (1992) The 14,000-molecular-weight antigen of Mycobacte­
rium tuberculosis is related to the alpha-crystallin family of lowmolecular-weight heat shock proteins. J Bacteriol 174:1352-1359
Vierling E (1991) The roles of heat shock proteins in plants. Annu Rev
Plant Physiol Plant Mol Biol 42:579-620
Vierling E, Sun A (1987) Developmental expression of heat shock
proteins in higher plants. In: Cherry J (ed) Environmental stress in
plants: biochemical and physiological mechanisms associated with
environmental stress tolerance in plants. Springer-Verlag, New
York, pp 343-354
Weng J, Wang Z-F, Nguyen HT (1991) Nucleotide sequence of a
Triticum aestivum cDNA clone which is homologous to the 26 kDa
chloroplast-localized heat shock protein gene of maize. Plant Mol
Biol 17:255-258
Wilson R, Ainscough R, Anderson K, Baynes C, Berks M, Bonfield J,
Burton J, Connel M, Copsey T, Cooper J, Coulson A, Craxton M,
Dear S, Du Z, Durbin R, Favello A, Fraser A, Fulton L, Gardner A,
Green P, Hawkins T, Hillier L, Jier M, Johnston L, Jones M, Ker­
shaw J, Kirsten J, Laisster N, Latrielle P, Lightning J, Lloyd C,
Mortimore B, O’Callaghan M, Parsons J, Percy C, Rifken L, Roopra A, Saunders D, Shownkeen R, Sims M, Smaldon N, Smith A,
Smith M, Sonnhammer E, Staden R, Sulston J, Thierry-Mieg J,
Thomas K, Vaudin M, Vaughan K, Waterston R, Watson A, Weinstock L, Wilkinson-Sproat J, Wohldman P (1994) 2.2 Mb of con­
tiguous nucleotide sequence from chromosome III of C. elegans.
Nature 368:32-38
Wistow G (1985) Domain structure and evolution in a-crystallins and
small heat-shock proteins. FEBS Lett 181:1-6
Wistow GJ, Piatigorsky J (1988) Lens crystallins: the evolution and
expression of proteins for a highly specialized tissue. Annu Rev
Biochem 57:479-504
Zimmerman JL, Petri W, Meselson M (1983) Accumulation of a spe­
cific subset of D. melanogaster heat shock mRNAs in normal de­
velopment without heat shock. Cell 32:1161-1170