In the verteb-rate lens, the b and c crystallins together account for the majority of the soluble proteins the other major family being the a-crystallins, members of the small heat-shock
Trang 1superfamily in vertebrates
Graeme Wistow1, Keith Wyatt1, Larry David2, Chun Gao1, Orval Bateman3, Steven Bernstein4, Stanislav Tomarev1, Lorenzo Segovia5, Christine Slingsby3and Thomas Vihtelic6
1 National Eye Institute, National Institutes of Health, Bethesda, MD, USA
2 Oregon Health Sciences University, Portland, OR, USA
3 Department of Crystallography, Birkbeck College, London, UK
4 Department of Ophthalmology, University of Maryland School of Medicine, Baltimore, MD, USA
5 IBT ⁄ UNAM, Col Chamilpa, Cuernavaca, Morelos, Mexico
6 Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA
Much of the complexity and diversity of life arises
from the multiplication and evolution of gene
famil-ies, increasing the functional repertoire of the
gen-ome By gene duplication, a single protein function
(or set of functions) can be expanded into a broader
set of more specialized functions The c-crystallins
are a gene family with a complex history in
verteb-rate evolution They encode proteins that are highly
abundant components of the eye lens but are also
expressed at lower levels in other parts of the eye,
perhaps with a stress-like role [1–6] Together with
the related b-crystallins, the c-crystallins belong to an
ancient superfamily (known as the bc-crystallin
super-family) with members ranging from the prokaryotic
sporulation protein, Protein S of Myxococcus xanthus [7], to AIM1, a protein implicated in the control of malignancy in melanoma in man [8,9] In the verteb-rate lens, the b and c crystallins together account for the majority of the soluble proteins (the other major family being the a-crystallins, members of the small heat-shock protein superfamily [10,11]) Although it has been suggested that proteins of this superfamily may have roles in maintenance of cellular architec-ture [8], little is known about their function Like the c-crystallins, b-crystallins have also been detected in the retina [12], and both have been identified in Dru-sen bodies, which form with age in retinal pigment epithelium (RPE) [13]
Keywords
crystallin; eye; gene structure; intron loss;
lens
Correspondence
G Wistow, Section on Molecular Structure
and Functional Genomics, National Eye
Institute, Bg 7, Rm 201, National Institutes
of Health, Bethesda, MD 20892-0703, USA
Tel: +1 301 402 3452
Fax: +1 301 496 0078
E-mail: graeme@helix.nih.gov
(Received 21 January 2005, revised 23
February 2005, accepted 8 March 2005)
doi:10.1111/j.1742-4658.2005.04655.x
The b and c crystallins are evolutionarily related families of proteins that make up a large part of the refractive structure of the vertebrate eye lens Each family has a distinctive gene structure that reflects a history of succes-sive gene duplications A survey of c-crystallins expressed in mammal, rep-tile, bird and fish species (particularly in the zebrafish, Danio rerio) has led
to the discovery of cN-crystallin, an evolutionary bridge between the b and
c families In all species examined, cN-crystallins have a hybrid gene struc-ture, half b and half c, and thus appear to be the ‘missing link’ between the b and c crystallin lineages Overall, there are four major classes of c-crystallin: the terrestrial group (including mammalian cA–F); the aquatic group (the fish cM-crystallins); the cS group; and the novel cN group Like the evolutionarily ancient b-crystallins (but unlike the terrestrial cA–F and aquatic cM groups), both the cS and cN crystallins form distinct clades with members in fish, reptiles, birds and mammals In rodents, cN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the c-crystallins, also in the retina Although well conserved throughout vertebrate evolution, cN in primates has apparently undergone major changes and possible loss of functional expression
Abbreviations
EST, expressed sequence tag; RPE, retinal pigment epithelium.
Trang 2b and c crystallins are related in their highly
sym-metrical structures built from four characteristic
bc-motifs (modified Greek keys) arranged as two
similar domains [14] The two families differ in that
c-crystallins are monomers that lack lengthy
N-ter-minal or C-terN-ter-minal extensions, whereas b-crystallins
have long N-terminal arms and form dimers and
higher oligomers
The two families also differ in gene structure
[3,10,15] In b-crystallins, each protein structural motif
is encoded in a separate exon, with other exons
enco-ding the N-terminal arms The same organization is
seen in the AIM1 gene, and as such presumably
repre-sents the ancestral condition [8].The gene structures of
c-crystallins are clearly related to those of b-crystallins
and AIM1, with precisely conserved intron positions
delineating protein domains; however, in the
c-crystal-lins the introns that divide the sequences encoding the
two motifs of each domain are missing Thus a
c-crys-tallin gene has ‘fused’ exons corresponding to each
two-motif domain
The c-crystallins present a particularly interesting
example of the dynamic evolution of a gene family
They play a key role in determining the optical
proper-ties of the lens, a tissue that is subjected to strong
envi-ronmental selective pressures as species move from
water to land, from dark to light, from the ground to
the air, and the requirements for vision change
accord-ingly As it has adapted in different evolutionary
line-ages, the lens has changed its protein composition
[3,16] This has led to considerable variability in the
content and sequence of c-crystallins in different
verte-brates, which are abundant in species with hard lenses
(such as fish and rodents) but at much lower levels or
missing in other terrestrial species This is in contrast
with the b-crystallins which are well conserved and
have clear orthologs in all vertebrate orders (see for
examples [17–20])
In fish and amphibians, there are multiple, divergent
c-crystallin genes that may exhibit only about 50%
identity at the protein level [17,19,21] This is similar
to the level of divergence among the b-crystallins
[18,19,22,23] and suggests a similar antiquity of these
gene families in the vertebrate lens In contrast, birds,
with soft accommodating lenses, lack the embryonically
expressed c-crystallins that in other vertebrates are
major components of the developing lens, and have
replaced them with the taxon-specific ‘enzyme
crystal-lins’, d and e crystallin [16,24–26]
In placental mammals there is a closely linked
clus-ter of six c-crystallin genes (cA–F) which are generally
expressed in the embryo, and these show 77–97%
iden-tity at the protein level, implying a relatively recent
origin for this family in this lineage It has been sug-gested that, as in birds, c-crystallins may have been on their way to extinction in the ancestors of mammals but were perhaps ‘reinvented’ by successive duplication
of a surviving gene as mammals adapted to principally nocturnal, burrowing habits before the extinction of the dinosaurs [3,16] Indeed, as some mammals have now become diurnal, these genes may again be in a process
of change or loss; in humans two of these genes (cE and cF) are pseudo and others (particularly cA) seem to be expressed at lower levels than in other mammals [15,27] Mammals also express cS-crystallin, a divergent outlier
of the family with a short N-terminal arm, which is the major c-crystallin expressed in the secondary fiber cells
of the mature mammalian lens [27–29]
Little is known about the c-crystallins of marsupials
or reptiles, but it is clear that this family has under-gone considerable changes, particularly during mam-malian evolution These changes illustrate the way in which gene families may expand, contract and adapt They may also help us understand the functions of the c-crystallin family in vision and elsewhere as most changes are presumably driven by specific adaptive requirements in the eyes of different vertebrates Here we describe a survey of the evolutionary his-tory of c-crystallins in vertebrates, including a large analysis of crystallin gene expression in zebrafish lens, and the discovery of a new member of the family, cN-crystallin, which seems to be an evolutionary bridge between the b and c families
Results
c-Crystallin and b-crystallin sequences from several verte-brate species were cloned and sequenced in NEIBank genomics projects or predicted from bioinformatics analysis of genome sequences The novel sequences are shown in Fig 1, aligned using the clustalw algorithm, and their relatedness is illustrated in the phylogenetic tree in Fig 2, drawn using the neighbor joining option
in the program mega [30] Some previously described sequences are also included to illustrate the overall distribution of the superfamily members expressed in vertebrate lenses
cDNA Libraries Approximately 1500 clones were sequenced from the un-normalized mouse whole eye ioip libraries and a further 1000 and 1300, respectively, from the two equalized libraries jajbjc and lglh A total of 1000 clones (code designation mw) were sequenced from a cDNA library made from western grey kangaroo
Trang 4kangD frgM1-1 frgM1-2 zfgM1 zfgM2a zfgM2b zfgM2c carpgM1
carpgM2 carpgM3 zfgM3 zfgM4 zfgM5 zfgM6 zfgM7 zfgMX
Aquatic
γM
γ−type gene
γs
γN
βA
βB
β
γ−type gene
β−type gene
β−type gene
βγ−hybrid
Terrestrial γ γ−type gene
γ
mgB mgC mgD mgE mgF mgA 100
100 100
100 91
74
66
98
99 64
92 99
40 56 100
100
100
100 89
32 71 34 57
72 90
69 61 100
100 mbB3
zfbB3 mbB2 zfbB2
zfbA2 mbA2
zfbA4 mbA4 mbA1 zfbA1-1 zfbgx chickgN iggN mgN zfgN2 zfgN1
zfgSc
zfgSd kangS iggS chickgS mgS zfgSb zfgSaL zfgSa
100 100
100
0.1
94 64 99
76
99
45 53 50 94 29 46
Fig 2 Phylogenetic tree of b and c
crystal-lins in vertebrates Calculated from the
align-ment in Fig 1 and drawn using MEGA , by
neighbor-joining with Poisson correction.
Bootstrap values are indicted for each node.
The major clades are identified Cartoons of
the exon structure of the motif-encoding
regions of genes in each clade are shown.
Red boxes show the typical c-crystallin exon
encoding two motifs, and blue boxes show
the single-motif exons of b-crystallin genes.
Fig 1 Protein sequences for representative members of the bc-crystallin superfamily Sequences are derived from the work described here, with some examples of fish and amphibian sequences taken from GenBank Sequence names beginning with ‘m’ are from mouse, ‘ig’ from iguana, ‘kan’ from kangaroo and ‘zf’ from zebrafish, while carp and chick sequences are so labeled Sequences were aligned by CLUSTAL W The positions of N-terminal and C-terminal arms, the four structural motifs (I–IV) and the connecting peptide between N-terminal and C-ter-minal domains are indicated below the alignment Also shown are the approximate positions of the four b-strands of each motif (a–d), by analogy with known structures Yellow highlights show the principal conserved positions of each motif essential for correct folding Note that the long C-terminal arm of zfbB3 has been truncated to fit the page.
Trang 5(Macropus fuliginosus) lens [31] In a small pilot survey
of expressed genes in a reptile lens, 1000 clones were
sequenced from a PCR-derived library (code
designa-tion hm) made from the lenses of a single individual
iguana Almost 4000 clones from an adult zebrafish
lens library (code designation nab) were sequenced
Further details of this and other zebrafish eye libraries
will be described elsewhere
Vertebrate c-crystallins
Among the collections of sequences obtained for
ver-tebrate lens and eye proteins were a large set of clones
for b and c crystallins Even the small set of clones
from iguana lens yielded complete coding sequences
for two members of the c-crystallin family One of
these (GenBank accession number AY788911) was the
iguana ortholog of cS-crystallin, with 178 amino-acid
residues, including initiator methionine Indeed, our
analyses confirm that cS is well conserved and highly
expressed throughout the vertebrates The other iguana
sequence (GenBank accession number AF445457) was
a novel protein similar in size to cS (183 residues) and
with an N-terminal arm of the same length However,
the new protein is clearly distinct from cS; its overall
sequence identity to iguana cS is only 44% and,
relat-ive to cS, the protein has insertions (mainly of
gly-cines), in the c–d loops of motifs I and III and in the
connection between motifs III and IV (Fig 1) This
protein was given the name cN, for c-new
From the combined mouse whole eye data, complete
sequences were obtained for cA–F and cS from
C57Bl⁄ 6 mice In addition, an almost full-length clone
was obtained for a protein very similar to the iguana
lens cN The complete coding sequence of mouse cN
(GenBank accession number AF445456) was deduced
from this clone, mouse genome sequence, from an
apparently full-length expressed sequence tag (EST) in
dbEST (CK795274), and from interspecies
compari-sons Several other ESTs for both mouse and rat cN
eye are also present in dbEST The mouse gene is on
chromosome 5 at about position 23.2 Mbp, cA–F are
on chromosome 1, and cS is on chromosome 16 [2]
Three full-length c-crystallins were obtained from
kangaroo lens One was the ortholog of cS, identical
in length and 72% identical in sequence with that of
mouse (GenBank accession number AY898646) The
other two were more similar to the cA–F crystallins,
with no N-terminal arm Based on their closest
mat-ches in blast searmat-ches, these were designated cB, with
the longer connecting peptide and a length of 175
codons (accession number AY898644), and cD, with
174 codons (accession number AY898645), although a
more systematic nomenclature will probably be needed when all kangaroo c-crystallin sequences are known
In this small sample, no clones for an ortholog of cN were detected
From zebrafish, a total of 16 distinct c-crystallins were identified along with a b-like sequence (with a long N-terminal arm) that had some sequence similar-ity to c-crystallins and several b-crystallins (GenBank accession numbers AY738742–AY738756) Nine of the c-crystallins were generally similar to the cM-crystal-lins previously cloned from carp lenses [32] and were named accordingly (Figs 1 and 2) Two were named zfcM1 and zfcM3, and three sequences related to carp cM2, including one closely related pair, were named zfcM2a, zfcM2b and zfcM2c The other cM-like sequences were named zfcM4–7 All of these sequences lack the N-terminal arm seen in cS, cN and b crystal-lins and are similar in size to the cA–F group of mam-mals As shown in the phylogenetic tree (Fig 2), the cM-crystallins form a distinct clade of ‘aquatic’ c-crys-tallins
An additional zebrafish c-crystallin was found to be generally similar to the cM class in size but is more divergent in sequence As shown in Fig 2, this protein does not group with either the terrestrial vertebrate c-crystallins or the aquatic cM class Provisionally this has been named zfcMX
Mammalian species possess just one gene for cS-crystallin However, the zebrafish lens has four pro-teins of the cS class Although these propro-teins, named zfcSa–d, show clear sequence similarity to known cS-crystallins, they have considerable variability at the N-terminus Most of the clones for zfcSa, the single most abundant species in the zebrafish lens cDNA library, lack an N-terminal arm altogether However, 13% of the sequences for zfcSa (zfcSaL) revealed an alternative splice at the end of the first exon that added four codons to the coding sequence, enough to make an N-arm of the same length as in mammalian cS-crystallins Like zfcSa, the closely related zfcSb also lacks an N-arm and so far there is no evidence of alternative splicing in this gene A third member of the
cS family in zebrafish, zfcSc has an N-arm that is lon-ger than in other species (although it contains three methionines near the N-terminus that could potentially give rise to alternative translation products), and a fourth member, zfcSd, has a short arm of just a single residue
In addition to the cM and cS crystallins, two of the zebrafish c-crystallins, zfcN1 and zfcN2, are members
of the cN family These two proteins have N-terminal arms identical in length with those of mouse and iguana cN and they also exhibit the characteristic
Trang 6insertions in the c–d loops of motifs I and III and in
the link between motifs III and IV As shown in the
phylogenetic tree, these sequences group with other
members of the cN class in a distinct clade that is
essentially separate from both b and c crystallins In
the tree shown, the cN branch is weakly linked to the
b-crystallin family, but overall the cN family is an
intermediate in the wider bc superfamily
The b-crystallins are represented in the phylogenetic
tree by five members cloned from the zebrafish lens
lib-rary (zfbA1-1, zfbA2, zfbA4, zfbB2 and zfbB4) and
their orthologs from mouse As expected, each fish
sequence is closely related to its mammalian ortholog,
in marked contrast with the relationships among cA–F
and cM crystallins The designation of zfbA1-1 reflects
the fact that the lens library also contains cDNAs for
a second, and possibly a third, bA1-like protein which
has not yet been completely characterized One
remaining zebrafish sequence from the lens library is
an outlier of the b-crystallin family Indeed, in simple
blastcomparisons, this sequence is slightly more closely
related to cN-crystallins than to other crystallins,
although it has a long N-terminal arm like a b-crystallin
In the phylogenetic tree it is placed as an early offshoot
of the main b-crystallin lineages For its currently
ambi-guous status, this has been named zfbcX
In current versions of the zebrafish genome, not
all assembled regions have been assigned to specific
chromosomes However, there is evidence of some
clus-tering of crystallin genes zfcM1, zfcM2b, zfcM2c and
zfcM6 are all located between positions 12.79 and
12.86 Mbp on chromosome 2, and zfcM3 and zfcM5
are both close to position 29.24 Mbp on chromosome 8
It has been known for a long time that bird lenses
lack most c-crystallins, although there has been
evi-dence for the presence of cS in its former guise as
bS-crystallin [33–35] Although no cDNAs are yet
available, inspection of the chicken genome using blat
reveals the presence of well-conserved genes for both
cS and cN crystallins The predicted sequences are
pre-sented in Fig 1, and in the phylogenetic tree (Fig 2)
they group in their respective subfamilies Chicken cN
is located at about position 5.7 Mbp on chromosome
2, and cS is at about 9.3 Mbp on chromosome 9 The
expression of these genes in the chicken remains to be
examined in detail
Novel gene structure of cN-crystallins
Genes for cN orthologs are present in mouse, rat,
chicken, and zebrafish, and, as described below,
orthologous genes are also present in the human and
chimp genomes The most striking feature of the crygn
gene in all these genomes, conserved across over
400 Myr of evolution, is its exon⁄ intron structure (Fig 3) The first half of the gene has the typical struc-ture of a c-crystallin gene [3], with a short first exon encoding the start codon and the short N-terminal
‘arm’ similar to that of cS A phase 0 intron separates that exon from a larger exon, exon 2, which encodes the first two structural motifs, and hence the N-ter-minal domain of the protein, just as in the genes for
cS and cA–F However, the second half of the gene has the structure of a b-crystallin gene, with two exons encoding the two motifs of the C-terminal domain The crygn gene is thus a hybrid of b and c crystallin gene structures, apparently an intermediate in the evo-lution of the c-crystallins from the b-crystallins This observation is concordant with the position of the cN family in the protein sequence phylogenetic tree, where
it is an intermediate between the b and c crystallins
cN in primates: a nonfunctional gene?
A search of the human genome for an ortholog of cN located a highly conserved gene sequence on chromo-some 7q36.1, and a very similar gene is present in the chimp genome, located in an unassembled portion of chromosome 6 This gene sequence contains a well-conserved coding sequence for the cN protein, with one notable exception The stop codon in rodents is TAG, but in the human and chimp genes, this codon has a single base change to CAG (glutamine) (Fig 4)
In principle, this could allow the translation of a larger version of cN with an 11-kDa C-terminal extension rich in glycine and proline (not shown) However, no cDNA clones for a human cN transcript have emerged from any of the NEIBank analyses In an attempt to
Fig 3 Correspondence of exon structure and protein structure for cN-crystallin Red boxes show coding sequence, green boxes show untranslated regions Protein structure is indicated by the stylized Greek key folding pattern of the bc motif The four motifs are labe-led I–IV, as are the corresponding exon sequences that encode those motifs.
Trang 7identify human transcripts, PCR was performed on
template made from human lens, retina, and
RPE⁄ choroid libraries No products were obtained
from lens or retina (a negative result in this procedure
is not proof of absence) A product was obtained from
RPE⁄ choroid template using primers located in exons
2 and 3, equivalent to the N-terminal and C-terminal
domains (Fig 4) However, sequencing showed that
the amplified product contained sequences for the
N-terminal domain of human cN with splice-site
skip-ping and use of a cryptic splice junction in intron 2
Such an alternative splice could produce a truncated,
one-domain protein (Fig 4) However, the crystallin
coding sequence does not appear to be part of a
trans-latable ORF in this transcript Interestingly, an EST
apparently corresponding to a similar transcript from
human hippocampus (BM548090) is present in dbEST,
suggesting that the PCR product from RPE may
repre-sent a transcript found at low levels in neural tissue,
but not one that could produce a viable protein
Screening of available Invitrogen full-length
Gene-Trapper-ready cDNA libraries showed that cN
tran-scripts were detectable only in human testis Two
positive clones were obtained from this tissue
(Gen-Bank accession number AF445455) The testis
tran-script included the first exon (the N-terminal arm
region) and exon 2 (the N-terminal domain) For the
C-terminal domain, however, only exon 3 (the third
motif) was included The exon corresponding to the
fourth motif was skipped and instead an unrelated,
cryptic downstream exon was included (Fig 4) This
exon has no similarity to the bc motif sequence and
could not produce a polypeptide capable of completing
the C-terminal domain of the protein
Currently there is no evidence for expression of
canonical cN in primates This leaves open the
ques-tion of whether the gene for cN retains any funcques-tion
in humans At the very least, the human gene has clearly changed its expression and may indeed be head-ing for extinction, joinhead-ing cE and cF [15]
Recombinant mouse cN Recombinant mouse cN was synthesized in a bacterial host (Fig 5A), purified and verified by MS Initial
Fig 4 Variant splice forms of the human CRYGN gene Exons are
shown by boxes and labeled as in Fig 3 For the full-length cDNA
from human testis, red boxes show the coding sequence
corres-ponding to the cN ORF; the blue box shows the coding sequence
of the cryptic exon; green boxes show untranslated regions; red
lines show the testis splice pattern The change of the stop codon
seen in other species to CAG causes an increase in the potential
ORF of the exons encoding motif IV Potential coding sequence
from intron read-through seen in clones from human RPE is shown
in orange, and the alternative splice of this variant is shown by the
orange lines.
A
B
Fig 5 Recombinant mouse cN-crystallin is less stable than cD-crystallin Expression of recombinant mouse cN Lane 1, pET-cN
E coli whole cell lysate (uninduced); lane 2, pET-cN E coli whole cell lysate (induced); lane 3, purified cN Denaturation profiles for recombinant mouse cN and human cD in increasing urea concen-trations.
Trang 8attempts at obtaining a mass measurement of the
cova-lent structure of cN by MS resulted in a spectrum with
a large number of peaks (including the value
calcu-lated from the sequence), probably due to binding of
multiple sodium ions, making deconvolution difficult
However, prewarming the sample at 37C resulted in
an almost single peak spectrum of 21 270 Da,
corres-ponding to cN lacking the initiator methionine
In solution studies, this protein behaved as a
mono-mer, similar to c-crystallins and in contrast with the
multimeric b-crystallins Indeed, c-crystallins in
solu-tion tend to behave as if they were even smaller than
expected for 20-kDa monomers and cN exhibits the
most extreme version of this behavior seen so far An
estimate of the protein oligomeric size was gained from
gel filtration using two different chemical supports On
both columns, cN was eluted with a higher elution
vol-ume than human cD-crystallin On preparative gel
fil-tration on Sephacryl S300, cN was eluted at 103.7 mL
Under the same conditions, human cD-crystallin was
eluted at 98.5 mL On analytical gel filtration on
Supe-rose 12, cN was eluted at 16.05 mL whereas human
cD-crystallin was eluted at 14.96 mL These results
indicate that cN is eluted at a smaller apparent size
than another monomeric c-crystallin As the two
poly-peptide chains have a similar molecular mass, these
data suggest that cN behaves even more anomalously
on gel filtration than other c-crystallins, possibly
through interactions with the column [36,37] To
pro-vide unambiguous epro-vidence of the oligomer size of cN,
light scattering was performed The molecular mass of
the protein at 5 mgÆmL)1 was evaluated by dynamic
light scattering The average over 15 readings gave a
diffusion coefficient (DT) of 974.5· 10)13 m2Æs)1 The
data were of high standard, with baseline values within
the range 1.000 ± 0.001 Sum-of-squares values were
below 5, and the majority were below 2, showing that
the quality of the data was statistically valid This
measured diffusion coefficient gives an estimated
molecular mass of 23.14 kDa The results showed
clearly that in free solution at 5 mgÆmL)1the protein
was monomeric The likely explanation for the
differ-ent molecular sizes in the gel filtration systems is the
propensity for the crystallin molecule to interact with
the column matrix [36,37] However, efforts to
crystal-lize the protein were unsuccessful
Recombinant mouse cN is less soluble than other
c-crystallins Although not rigorously tested, it seemed
that a concentration of 5 mgÆmL)1 was limiting
Unlike other lens c-crystallins, mouse cN, which has a
calculated pI of 6.27, was not soluble when exposed
for significant lengths of time to pH 5, precluding the
use of cation chromatography for purification Samples
of cN were turbid after storage at 4 C or when thawed Cooling-induced precipitation was not fully reversible Thus, although cN exhibits some of the characteristics of the phase-separation-driven phenom-enon known as ‘cold cataract’, its behavior is not con-sistent with a simple liquid-liquid phase transition seen for some other c-crystallins [38]
Typically, c-crystallins also exhibit very high con-formational stability [39] Recombinant mouse cN was subjected to unfolding in urea under equilibrium con-ditions and compared with human cD-crystallin (Fig 5B) The data show that under conditions in which cD is unchanged, as judged by fluorescence, cN completely unfolds, suggesting a much lower conform-ational stability In common with other c-crystallins, the tryptophans of cN are more quenched when buried
in the folded protein than when exposed to the denatu-rant [40]
cN expression in mouse eye Eye-specific expression of cN was confirmed by Nor-thern blotting In mouse multi-tissue NorNor-thern blot analysis, cN was detectable only in eye (Fig 6A) In Northern blot analysis of rat tissues, cN was detec-ted only in retina (Fig 6B) Lens was not included
on these blots Expression of cN protein in lens was examined by 2D gels and MS Figure 7A shows a Ponceau S-stained blot of soluble protein from a newborn mouse The identities of major crystallins were known from earlier work [41] After destaining, the blot was probed with antibody to cN (Fig 7B)
A single immunoreactive spot was observed just below bA2 The immunoreactive spot was not visible
in Ponceau stain, but Coomassie blue staining of a larger 2D electrophoresis gel of soluble protein from newborn mouse lens did detect a protein spot at this position (cN, Fig 7C) This spot was confirmed to
be cN by in-gel digestion and LC⁄ MS ⁄ MS analysis
of tryptic fragments that identified seven distinct cN peptides covering 48% of the protein sequence (data not shown)
The antibody to cN was used in immunofluores-cence studies of mouse eye sections (Fig 8) In the anterior segment of the eye (Fig 8A), cN immunoreac-tivity was seen specifically in the lens nucleus, the pri-mary site of expression for cA–F crystallins, but not in secondary fibers or lens epithelium, where cS-crystallin
is expressed No expression was evident in other tissues
of the anterior chamber In the retina (Fig 8B), expression was seen in the outer plexiform layer (con-taining photoreceptor axons and synapses) and photo-receptor outer segments
Trang 9In phylogenetic analyses, the b-crystallins form a
dis-tinct clade with bA (acidic) and bB (basic) branches
As has previously been observed, and is illustrated in
Fig 2, most b-crystallins have clear orthologs in all
vertebrates so that mammalian and zebrafish bA2
sequences, for example, are close together on the same
branch of the tree In contrast, the cA–F-crystallins
that are expressed in most mammals have no orthologs
in fish and form a distinct branch of their own that
includes c-crystallins of similar size from amphibians
(two of which, from a frog, Rana catesbeiana [42], are
shown), and from marsupials in a ‘terrestrial’ branch
of the family However, even on this branch, different
orders do not appear to have truly orthologous
crys-tallins, i.e the frog sequences are not orthologs of any
gene in mammals
Whereas most of the zebrafish c-crystallins are
sim-ilar in size to the mammalian cA–F group, with no
N-terminal arm, they too form a distinct branch of the overall family This branch includes the cM-crystallins which have previously been identified in carp, so it seems appropriate to name this subfamily the cM-crys-tallins and to number the new zebrafish sequences
A
B
Fig 6 Expression of cN transcripts is eye specific in rodents (A)
Northern blot of multiple mouse tissues with probe for mouse cN.
Br, brain; Ey, eye; He, heart; Lu, lung; Li, liver; Sp, spleen; Ki,
kidney; Pa, pancreas; Sm, skeletal muscle, Th, thymus Staining
pattern for 28S and 18S rRNA is shown below (B) Northern blot of
multiple rat tissues with probe for mouse cN Ret, retina (two
pre-parations); Lu, lung; Ki, kidney; Te, testis; Li, liver; Sp, spleen; Br,
brain; He, heart The staining pattern for 28S rRNA is shown below.
Fig 7 Expression of cN protein in newborn mouse lens (A) Ponc-eau S-stained blot of 2D electrophoresis gel of soluble protein from
a newborn mouse lens showing major crystallin spots (B) Western blot of destained 2D electrophoresis gel blot shown in (A) using antibody to cN A single spot is detected at the same relative posi-tion occupied by cN in part (C) (C) Coomassie blue-stained large 2D electrophoresis gel of soluble protein from a newborn mouse (from [41]) Protein marked with an arrow was confirmed to be cN
by MS.
Trang 10accordingly (however, the previous cM nomenclature
for Rana may not be appropriate in this overall
con-text) As is seen elsewhere in the family tree, several of
the zebrafish sequences (such as cE and cF in
mam-mals) appear to be the result of relatively recent gene
duplications, probably the result of large-scale genome
duplication events in these and other fish [43]
Mammal, bird and fish cS sequences form a third,
more ancient branch In this subfamily, a pair of
zebrafish genes (cSa and cSb) are close in sequence to
those of chicken and mouse, and a second pair (cSc
and cSd) belong to an earlier offshoot This may
indi-cate that there were two cS genes in the common
ancestor of fish, birds and mammals and that only one
of these survived in the terrestrial species while both
survived in fish and indeed underwent a subsequent
duplication in some species A predicted cS from
chicken and one cloned from iguana belong to this
clade and appear to be orthologs of mammalian cS-crystallin
The cN family is newly discovered In terms of structure and solution behavior, cN most closely resembles c-crystallins However, in the phylogenetic tree, the cN sequences do not associate strongly with either the b or c subbranches Genes from mammals, chicken and zebrafish all show a hybrid gene structure with both c-like and b-like exons Overall, the cN fam-ily appears to be an evolutionary intermediate between the wider b and c crystallin families
It seems likely that the b-crystallin⁄ AIM1 group of genes represents the original gene organization state of the superfamily, with genes built up by successive dupli-cation from an ancestral gene that encoded an individ-ual motif (although, as an individindivid-ual bc motif could not
be a stable structure alone, the protein product must have been an obligate dimer), resulting in each motif
A
B
Fig 8 Immunofluorescence localization of cN-crystallin in mouse eye (A) Expression of cN in the anterior segment Left panel shows DAPI staining for nuclei; center panel shows immunofluorescence stain (red) for cN; right panel shows a control with no primary antibody (B) Expression in the posterior segment Left panel shows combined DAPI (blue) staining of cell nuclei and immunofluorescence (red) signal for
cN GCL, Ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer, OS, outer segments Right panel shows control with no primary antibody White arrows show positive stain in OPL and OS layers.