From the Sars International Centre for Marine
Molecular Biology, Bergen High Technology Centre, N-5008 Bergen,
Norway and the § Protein Interaction Laboratory, Department
of Biochemistry and Molecular Biology, University of Southern
Denmark-Odense, DK-5230 Odense M, Denmark
Received for publication, January 17, 2001, and in revised form, February 28, 2001
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ABSTRACT |
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Appendicularia are protochordates that rely on a
complex mucous secretion, the house, to filter food particles from
seawater. A monolayer of cells covering the trunk of the animal, the
oikoplastic epithelium, secretes the house. This epithelium contains a
fixed number of cells arranged in characteristic patterns with distinct sizes and nuclear morphologies. Certain house structures appear to be
spatially related to defined, underlying groups of cells in the
epithelium. We show that the house is composed of at least 20 polypeptides, a number of which are highly glycosylated, with glycosidase treatments resulting in molecular mass shifts
exceeding 100 kDa. Nanoelectrospray tandem mass spectrometric
microsequencing of house polypeptides was used to design
oligonucleotides to screen an adult Oikopleura dioica
cDNA library. This resulted in the isolation of cDNAs coding
for three different proteins, oikosin 1, oikosin 2, and oikosin 3. The
latter two are novel proteins unrelated to any known data base entries.
Oikosin 1 has 13 repeats of a Cys domain, previously identified as a
subunit of repeating sequences in some vertebrate mucins. We also find
one repeat of this Cys domain in human cartilage intermediate layer
protein but find no evidence of this domain in any invertebrate
species, including those for which entire genomes have been sequenced. The three oikosins show distinct and complementary expression patterns
restricted to the oikoplastic epithelium. This easily accessible
epithelium, with differential gene expression patterns in readily
identifiable groups of cells with distinctive nuclear morphologies, is
a highly attractive model system for molecular studies of pattern formation.
Appendicularia, or larvaceans, are pelagic tunicates (Urochordata)
that feed on dissolved organic carbon and microorganisms by filtering
seawater through a transparent mucous structure called the house. The
name "larvaceans" derives from the fact that the adult animal
resembles the larval form more closely than in the other two classes of
Urochordata. In the latter two classes, metamorphosis drastically
redesigns the body plan, whereas in appendicularia, it consists of a
simple switch in tail position (tailshift) from a straight posteriorly
directed orientation to a definitive arrangement, where the tail is
orthogonal to the trunk and retains the notochord as its axial
structure. For this reason, appendicularia are thought to have diverged
earlier from the chordate ancestor than their sister classes, and
recent phylogenetic analyses of 28 and 18 S rRNA genes confirm this
view (1, 2). Appendicularia have three features common to all chordates
at some stage in the life cycle: gill slits, a tubular nerve chord, and
a rod-shaped notochord. The appendicularia and vertebrates form a
robust monophyletic unit, with two Oikopleura species
forming a sister group to all vertebrates (2).
The term appendicularia refers to the appendices of the animal, namely
their houses. Appendicularian houses are sophisticated filtration
devices with a very complex architecture, featuring different
compartments, valves, septa, and two sets of filters (3-5). The house
is secreted as a rudiment by the oikoplastic epithelium, a specialized
single-layered organ that covers the trunk of the animal (see Fig. 1).
The house rudiment is expanded by means of specific movements of the
tail, which is eventually withdrawn inside the house, such that the
entire animal is contained within this structure. Sinusoidal movements
of the tail control the flow of water through the house filters. The
cells of the oikoplastic epithelium stop dividing before secretion of
the first house. Their number is fixed for a given species of the genus Oikopleura, being about 2,000 in Oikopleura
dioica (6-8). Frequent replacement of the house is needed to
maintain sufficient filtration rates (9), and the cells of the
oikoplastic epithelium sustain high rates of house protein synthesis
and secretion from shortly before filter feeding begins until the end
of the life cycle. For O. dioica, a new house is synthesized
every 3-4 h over a life cycle of 5-6 days at 15 °C. During the
life cycle, which is extremely short for a complex metazoan, the animal
grows about 10-fold in size, and there is a concomitant increase in the
size of the oikoplastic cells. Cell growth is paralleled by an increase
in the sizes of the nuclei and in their DNA content (10).
It is not known whether the increase of DNA content represents partial
or total amplification of the genome. During this process, however, the
shape and size of the terminally differentiated cells of the
oikoplastic epithelium becomes highly variable. These vary from round
and relatively small to lobate, oblong shapes attaining a large size.
The complexity of the house structure is mirrored by the diverse
patterns of the oikoplastic cells. Thus far, different fields of cells
have been identified within the epithelium on the basis of nuclear
morphology, size, and pattern formation (6-8, 10, 11). A correlation
between these morphologically identified fields of cells and the
production of specific structures of the house seems apparent for the
two sets of filters, the inlet filter being secreted by the field of
Eisen and the food concentrating filters being secreted by the field of
Fol (see Fig. 1). These functional correlations remain crude when
compared with the complexity of the filters and the patterns of cells
held responsible for their secretion. The structural characteristics of
different parts of houses of the genus Oikopleura have been
studied by electron microscopy (4), but very little information, based
primarily on histochemical staining, is available concerning their
biochemical composition and properties (5).
Thus, O. dioica has a monolayer of accessible epithelium,
with low and constant cell number, that serves as a template for the
synthesis of diverse house structures from clearly defined regions of
cells with distinct nuclear morphologies. This is an attractive model
system for investigating: (i) coordinate regulation of gene expression,
(ii) cell-cell interactions involved in pattern formation, (iii)
gene/genome amplification, and (iv) the role of nuclear architecture in
regulating gene expression. However, for the oikoplastic epithelium of
Oikopleura to become a bona fide model for
studying the questions posed above, it remains to be established that
the structures in the house are synthesized from a diverse array of
gene products with expression patterns restricted to different regions
of cells. Alternatively, relatively few genes could be involved, with
their products undergoing differential post-translational modifications.
In the present study we report partial biochemical characterization of
the house polypeptides of O. dioica and the cloning of
cDNAs encoding four of them. We demonstrate that the genes coding
for these cDNAs are expressed in well defined complementary regions
of the oikoplastic epithelium. One of the cDNAs codes for a very
large polypeptide, showing distant homology to vertebrate gel-forming
mucins and human cartilage intermediate layer protein, and the
corresponding gene is expressed only in the 14 giant cells of the Fol region.
Appendicularia Culture--
Animals were collected from fjords
around Bergen, Norway and were cultured in 6-liter beakers with
constant stirring at 14-15 °C. The duration of the life cycle was
5-6 days. Mature males and females were placed together in 4-liter
volumes of seawater and allowed to spawn. Metamorphosis took place
12-14 h after spawning, at which time the animals expanded their first
house and began filter feeding. The animals then grew continuously and
on day 5 the gonads increased dramatically in size, surpassing the size of the trunk of the animal at spawning. Cultures were diluted 1:6 on
day 1 and 1:1 on day 2, and then animals were transferred to clean sea
water by pipetting on each of the following days. The appendicularia
were maintained on cultured algal strains of Isochrysis
galbana, Chaetoceros calcitrans, and
Synecococcus sp., supplemented with Rhodomonas
sp. and/or Tetraselmis sp. from day 3 onward.
House Protein Solubilization and Deglycosylation--
To
collect house rudiments free from contaminating particles and
microorganisms, adult animals (day 4-5) were forced out of their
houses and transferred to filtered seawater. House rudiments were then
removed as soon as the animals started expanding them. Aliquots of
50-200 house rudiments were stored at Protein Electrophoresis--
For one-dimensional
SDS-polyacrylamide gel electrophoresis (PAGE) samples were
loaded directly, or in the case of large solubilization volumes,
precipitated with 20% trichloroacetic acid, and then redissolved in
loading buffer. Gel and buffer compositions were according to
Shägger and von Jagow (12), except that 16-cm-long gels with a
5-16% polyacrylamide gradient (3% crosslinking) were used to
maximize resolution over a wide molecular mass range. Gels were stained
with minor modifications of the combined alcian blue-periodic acid
oxidation-silver staining procedure of Møller and Poulsen (13) or by
neutral silver staining (14).
Two-dimensional electrophoresis was performed with isoelectrofocusing
on immobilized pH gradients as the first dimension and SDS-PAGE as the
second dimension. Deglycosylated samples were precipitated with
trichloroacetic acid as above and resuspended in 0.5% immobilized pH
gradient buffer, pH 3-10 (APBiotech), 2% CHAPS, 7 M urea,
2 M thiourea, 5 mM tributyl-phosphine, and
0.005% bromphenol blue. Dry immobilized pH gradients gel strips pH
3-10 NL (nonlinear pH gradient; 7 cm long; APBiotech) were rehydrated with resuspended samples overnight, and first dimension electrophoresis was performed with a Multiphor II apparatus (APBiotech). Electrofocused gel strips were then equilibrated in second dimension loading buffer
(50 mM Tris-HCl, pH 6.8, 6 M urea, 2% SDS,
30% glycerol, 0.05% bromphenol blue) and applied on a vertical second
dimension gel with a 5-16% polyacrylamide gradient as described for
one-dimensional electrophoresis, except that the resolving gel was 11 cm long and no stacking gel was used. Two-dimensional gels were stained with the same neutral silver staining procedure as described for one-dimensional gels. Spots (two-dimensional) and bands
(one-dimensional) corresponding to the added glycosidases were
identified by comparison with gels run with enzyme blanks under
identical conditions.
Peptide Sequencing--
Protein bands were excised from
silver-stained one-dimensional polyacrylamide gels and processed as
described (14). After reduction and alkylation, overnight in-gel
digestion was carried out at 37 °C with an excess of sequencing
grade trypsin (Roche Molecular Biochemicals). After digestion, the
supernatant was acidified with formic acid, loaded onto a Poros R2
(Perseptive Biosystems, Framingham, MA) micro-column, and desalted
(15). The peptides were then eluted with 60% methanol, 5% formic acid directly into a nanoelectrospray needle (MDS Protana, Odense, Denmark).
Peptides were sequenced by nanoelectrospray tandem mass spectrometry
performed on a prototype quadrupole time-of-flight mass spectrometer
(AB/MDS Sciex, Toronto, Canada) (16) equipped with a nanoelectrospray
source (MDS Protana).
Library Construction and Screening--
Total RNA from day 5 animals was extracted with the TRIzol Reagent (Life Technologies,
Inc.), and the poly(A)+ fraction was isolated from ~200
µg of total RNA by two rounds of oligo(dT)-cellulose chromatography
with the Poly(A)+ Quik mRNA isolation kit (Life
Technologies, Inc.). This poly(A)+ RNA was the template for
cDNA synthesis using the ZAP Express cDNA synthesis kit, and
the cDNA was cloned and packaged using the ZAP Express cDNA
Gigapack III Gold Cloning Kit (Stratagene) except that cDNA size
selection was performed with SizeSep 400 Spun Columns (APBiotech). A
primary library of 3 × 105 plaque-forming units was
obtained and amplified once. A plating of 3 × 105
plaque-forming units of the amplified library was screened with degenerate oligonucleotides corresponding to the peptide sequences shown in bold type in Fig. 3. The oligonucleotides were
synthesized at Interactiva Biotechnologie (GmbH): G2,
5'-TGGTAYGGIGAYGAYCARGG-3'; G10, 5'-CAYTTYCCITGGTAYAAYAA-3'; H5,
5'-GAYACIGCITAYCAYGGITT-3'; I1, 5'-GARTTYTGYGGIGTIGAYTT-3'; and N6,
5'-TAYGAYAAYGAYCARGCIGC-3', where Y = C or T, R = G or
A, and I = inosine. Oligonucleotide nomenclature is based on the
labeling of the protein bands from which they were derived.
Oligonucleotides were end-labeled with [
Polymerase chain reaction screening was performed with the standard T3
primer as the vector-anchored primer and with either primer A5'
(5'-CAACGCTGAAGTCATCATC-3'), G5' (5'-AAGCCTTGCATGCGTGC-3'), or
H5' (5'-GGATTTGAACCACGACAAC-3') for amplifying sequences upstream of clones A1, G1, and H, respectively. A first set of primer extention reactions (linear amplification) were carried out in a total volume of
50 µl with 5 µl of amplified library as template, 0.2 µM specific primer, 200 µM of each dNTP,
and 1 unit of DyNAzyme II DNA polymerase (Finnzyme) in DyNAzyme
reaction buffer (10 mM Tris-HCl, pH 8.8, at 25 °C, 50 mM KCl, 1.5 mM MgCl2, and 0.1%
Triton X-100). After 35 cycles, 5 µl of these reactions were used as
templates for exponential amplification reactions under the same
conditions as above, but with the inclusion of 1 µM of T3
primer. Amplified fragments were digested with the appropriate
restriction enzymes, gel purified, and cloned in pBluescript II.
Northern Blotting and Whole Mount in Situ
Hybridization--
Total RNA was prepared from day 4-5 animals by
acid guanidinium thiocyanate-phenol-chloroform extraction, and the
poly(A)+ fraction was isolated by two rounds of
oligo(dT)-cellulose chromatography. Aliquots of 500 ng of the
poly(A)+ fraction were electrophoresed through a 2.2 M formaldehyde, 1% agarose gel and transferred to a
positively charged nylon membrane (Hybond-N+, APBiotech) by capillary
blotting. The RNA was fixed to the wet membrane by UV cross-linking
with 150.000 µJ/cm2 at 254 nm with a Hoefer UVC 500 Ultraviolet Cross-linker (APBiotech). Purified DNA fragments were
radioactively labeled by random priming with the T7 Quick Prime kit
(APBiotech), and unincorporated nucleotides were removed by Sephadex
G-50 spun column chromatography (APBiotech). Membranes were
prehybridized and hybridized in 250 mM sodium phosphate buffer, pH 7.2, 7% SDS, 1 mM EDTA, 1% bovine serum
albumin and washed with 20 mM sodium phosphate buffer, pH
7.2, 1% SDS, 1 mM EDTA. Hybridized blots were analyzed
with a FLA 2000 phosphoimager and Image Gauge software (Fuji).
For whole mount in situ hybridization, sense and antisense
RNA probes were synthesized by in vitro transcription of
linearized plasmids containing the appropriate inserts with either T7
or T3 RNA polymerase (Promega) in the presence of digoxigenin-labeled UTP (digoxigenin RNA Labeling Mix; Roche Molecular
Biochemicals). Day 2-3 animals were fixed with 4% paraformaldehyde in
100 mM MOPS, pH 7.5, and 500 mM NaCl for 20 min
and digested with 10 µg/ml proteinase K in the same buffer for 3 min
at 37 °C. Specimens were post-fixed for 5 min with the same fixing
solution as above. Prehybridization (2 h) and hybridization were
carried out in 50% deionized formamide, 5× SSC, 2% blocking reagent
(Roche Molecular Biochemicals), 0.1% Triton X-100, and 9.2 mM citric acid at 60 °C. Probes were diluted at 400 ng/ml in hybridization mixture and denatured at 85 °C for 5 min
before being added to the specimens and incubated overnight. Samples
were washed once with 4× SSC, 50% formamide, 0.1% Triton X-100 for
30 min at 37 °C; once with 2× SSC, 2 mg/ml bovine serum albumin,
0.1% Triton X-100 for 30 min at 37 °C; and twice with 0.5× SSC, 2 mg/ml bovine serum albumin, 0.1% Triton X-100 for 30 min at 65 °C.
Hybridized specimens were blocked with 1% blocking reagent in
phosphate-buffered saline with 0.1% Triton X-100 for 1 h at room
temperature and incubated in the same medium containing 2 µg/ml of
either fluorescein- or rhodamine-conjugated anti-digoxigenin Fab
fragments (Roche Molecular Biochemicals) for 40 min at room
temperature. After washing three times with phosphate-buffered saline
with 0.1% Triton X-100 for 30 min, specimens were mounted in
Vectashield mounting medium (Vector Laboratories) and analyzed with a
Leica TCS laser scanning confocal microscope equipped with a 40× Leica
oil immersion objective (numerical aperture 1.25-0.75). Images
were acquired with Leica PowerScan software. Parallel hybridizations
with sense RNA probes were run as controls.
Partial Characterization of House Proteins--
To avoid
contamination of house protein preparations by algae and bacteria, all
preparations were made using noninflated prehouse rudiments (Fig.
1). Several solubilization procedures
were tested, including sonication, detergent extraction, and guanidine
HCl extraction. Denaturation by heating in the presence of 1% SDS, followed by titration of SDS with CHAPS was the most effective. One-dimensional SDS-PAGE of solubilized and reduced house material yielded several bands within a range of molecular mass from 5 to over
200 kDa (Fig. 2). Among these, a broad,
prominent band at 36 kDa and several sharper bands appeared as clear
bands upon neutral and particularly upon ammoniacal silver staining
(data not shown). Both molecular mass heterogeneity and negative
staining are associated with highly glycosylated proteins, such as
proteoglycans and mucins (13, 17). Following combined periodic acid
oxidation-alcian blue-silver staining, specific for highly glycosylated
proteins, most of these bands were positively stained (Fig. 2).
Different glycosidases were employed to reduce polypeptide
heterogeneity. After digestion with peptide-N-glycosidase F
(PNGase F), which hydrolyzes the linkage between asparagine and
N-acetylglucosamine common to all N-linked
oligosaccharides, a shift to lower molecular masses was observed for
several polypeptides (Fig. 2A). The heterogeneous band at 36 kDa disappeared, probably giving rise to some of the lower molecular
mass species that appeared following this treatment. After simultaneous
digestion with PNGase F and a broad spectrum neuraminidase, all bands
larger than 70 kDa disappeared, but neuraminidase action produced a
heavy background on gels stained with the combined alcian blue-periodic
acid oxidation-silver procedure. When the gel was neutral silver
stained, the pattern was very simple, with only a few peptide bands
detectable. Two dimensional electrophoresis of a sample treated the
same way showed that at least 20 polypeptide species were detected and
that pI heterogeneity was still present in some of them (Fig.
2B). The electrophoretic patterns from repeated independent
preparations were consistent, and no differences were observed when
these preparations where digested in the presence or absence of a broad
spectrum mixture of protease inhibitors. Thus, these deglycosylation
treatments resulted in substantial decreases in molecular mass
profiles, indicating extensive glycosylation of house proteins, and the
large shifts resulting from treatment with neuraminidase alone suggest
that sialic acid moieties were abundant.
In contrast to PNGase F and neuraminidase treatments, addition of
O-glycosidase (endo- cDNA Cloning and Sequencing--
To isolate
cDNA clones encoding house components, bands prepared from
one-dimensional SDS-PAGE separation of PNGase F digested house proteins
were subjected to nanoelectrospray-tandem mass spectrometry.
Neuraminidase treatment was omitted because it added several enzyme
bands/spots that co-migrated with house peptide bands/spots in both
one- and two-dimensional separations. The most abundant polypeptide
bands were cut out of the gel, digested with trypsin, eluted, and
microsequenced. Two bands at 96 kDa were insufficiently separated and
were processed as a single band labeled G in Fig.
3. The peptide sequences obtained by
microsequencing are shown in Fig. 3.
One of the peptide sequences from band A (bold type in Fig.
3) matched perfectly with the partial sequence of a 3372-bp cDNA clone (clone A1) from an Oikopleura cDNA
shotgun sequencing effort.2
To isolate additional clones, degenerate oligonucleotides were designed
from selected peptide sequences obtained from bands G, H, N, and I
(bold type in Fig. 3). These oligonucleotides were radioactively labeled, pooled, and used as probes to screen a cDNA
library. Thirty-six clones were isolated, and partial sequencing revealed that the amino acid sequences deduced from reading frames present in 20 of them matched with microsequenced peptides.
Eighteen of these clones encoded polypeptides matching peptides
derived from band G (G clones), one for a polypeptide matching
peptides from band H (clone H), and one matched all seven peptides
from band A (clone A2), although no probe sequence derived
from band A was used in this screening (see below). The nucleotide
sequence of this clone was identical to clone A1 but had an
additional 1173 bp at the 5' end. The partial sequences of the
remaining 14 clones did not show significant matches to any of the
sequenced peptides. BLAST-X homology searches showed that seven of
these clones encoded polypeptides similar to known mammalian proteins with functions not obviously related to house polypeptides
(E values between 10
On the basis of alignments of partial sequences, restriction pattern
analysis, and matches with the peptides derived from band G, there were
three distinct types of G clones. The longest clones of each type, G1,
G2a, and G2b were 2542, 2552, and 2559 bp long, respectively, and were
completely sequenced. Clones G1 and G2 share an overall identity of
71% at the nucleotide level and code for polypeptides that show an
identity of 70% (Fig. 4A). Clone G2a differs from G2b by only a few residues, a difference probably caused by allelic polymorphism. Out of the 18 G clones, 13 were of the G1 type, 3 of the G2a type, and 2 of the G2b type. This is
consistent with the presence of multiple bands in the gel slice labeled
G. Clone H, 2189 bp, was also completely sequenced. Translations of
clones A1, G1, G2a, G2b, and H showed uninterrupted reading
frames starting from the 5' end, but there were no putative starting
methionine residues and no signal peptides predicted for any of them,
indicating that all four clones were partial cDNAs.
To obtain full-length coding sequences for clones A1, G1,
and H, both primer extension and rescreening of the library with 5'
fragments of each clone were carried out. Full-length clones were
obtained for both A and H. The 6996-bp nucleotide sequence of clone A
contained an open reading frame that was 2301 amino acids long and
started 10 nucleotides after the 5' end of the cDNA insert. Two
different prediction methods indicated the presence of a signal peptide
with a cleavage site between amino acids 14 and 15 (Fig. 4B)
(18, 19). Interestingly, the predicted molecular mass of the
polypeptide coded by clone A is 256 kDa, whereas band A was only 180 kDa prior to and 170 kDa following PNGase F digestion (Fig.
2A). In addition, upon treatment with neuraminidase, the molecular mass of band A decreased by at least 100 kDa, because no
polypeptide larger than 70 kDa was detected after neuraminidase digestion (Fig. 2B). Because glycosidase digestion patterns
on extracellular house rudiments were the same, both in the presence and absence of protease inhibitors, it is unlikely that this difference in molecular mass can be accounted for by proteolytic degradation, suggesting that polypeptide A is post-translationally processed in vivo.
The full-length sequence for clone H was 2396 bp in length with an
initiation codon at position 43. The predicted molecular mass of the
polypeptide translated from this frame was 67 kDa, the same as that
estimated by SDS-PAGE for band H. For clone G1, neither polymerase
chain reaction extension nor the screening of 20 additional clones in
the 5' probing of the library yielded any sequence upstream of the 5'
end of the existing G1 clone. This suggests that secondary structure
prevented reverse transcription through the 5' region of the G1 transcript.
Nomenclature--
Using the Greek Oikos, for house, as
a root, we have assigned the following names to the clones presented in
this study: clone A = oikosin 1, clone G1 = oikosin 2A, clone
G2a = oikosin 2B1, clone G2b = oikosin 2B2, and clone H = oikosin 3. The EMBL data base accession numbers for the nucleotide
sequences are AJ308491, AJ308492, AJ308493, AJ308494, and
AJ308495, respectively.
Sequence Analysis--
Oikosin 1 contained 13 motifs similar to
the Cys subdomains present in three members of the 11p15 human mucin
gene cluster (MUC2, MUC5B, and MUC5AC), in their rodent homologues, and
in other related mammalian mucin cDNAs (HGM-1 and PGM-2A) (20-22). A similar motif is also present in human cartilage intermediate layer
protein (CILP) (23) and two expressed sequence tag clones from
Xenopus laevis. Several blocks of highly conserved residues were evident in the alignment of the 13 motifs of oikosin 1 and the cysteine subdomains found in these proteins (Fig.
5A). Among these, 6 of the 10 invariant cysteine residues and the putative mannosylation consensus
sequence WXX(W/F) in the mucin Cys subdomains were present
in all repeats of oikosin 1. The CILP Cys domain lacks the same 4 cysteine residues as the oikosin 1 repeats and, in addition, lacks the
first and the penultimate cysteines as well. In the CILP Cys domain the
second tryptophan in the putative mannosylation consensus is also
substituted with leucine, a replacement known to reduce the efficiency
of C-mannosylation of RNase 2 from 73% to 6% in HEK293 cells (24).
Despite this, CILP is the closest match for 11 of 13 of the oikosin 1 repeats when they are used as queries in BLAST-P searches and when the
PRODOM data base is searched with a profile obtained from all 13 repeats. CILP also differs from both the mammalian mucins and oikosin 1 in that it contains only one copy of the Cys domain. Fig. 5B
shows schematic representations of some of the proteins whose Cys
(sub)domains are aligned in Fig. 5A. These motifs were named
Cys subdomains because they were first identified in the 11p15 human
mucin cluster where they form a part of larger repeating units (20). No
significant homologies outside the Cys (sub)domains were found among
the proteins in Fig. 5, making the nomenclature "Cys subdomain"
inappropriate for CILP and the oikosin 1 motifs. To our knowledge, Cys
(sub)domain-like motifs had not been previously identified in CILP or
in any known invertebrate proteins.
The function of the Cys (sub)domain is unknown. Mammalian mucins MUC2,
MUC5B, and MUC5AC, are gel-forming mucins secreted by the epithelia of
the respiratory and digestive tracts, and the presence of several
conserved cysteine residues in this motif invokes possible roles in the
packaging of mucins or in interactions with other components of the
mucus (25). Both the mammalian mucins and oikosin 1 are highly
glycosylated extracellular proteins, and the degree of conservation
among their Cys (sub)domain-like repeats suggests that this domain may
have been present in a common ancestor protein prior to the divergence
of urochordates and vertebrates.
Expression Analysis--
Northern blot analysis (Fig.
6) on poly(A)+ RNA from adult
animals using cDNA probes for oikosin 1, oikosin 2A, and oikosin 3 detected single bands for each clone with estimated molecular masses of
7.0, 2.7, and 2.5 kilobases, respectively. These size estimates
are in agreement with the full-length cDNAs obtained for oikosins 1 and 3 and indicate that the oikosin 2A cDNA sequence lacks 100-200
bp at the 5' end of the complete transcript.
The expression patterns of transcripts corresponding to the isolated
cDNA clones were assayed by whole mount in situ
hybridization (Fig. 7). Probes
corresponding to different fragments of the nucleotide sequence of
oikosin 1 demonstrated that expression of the corresponding gene was
restricted to the seven giant cells of the field of Fol (Fig.
7B). The Fol region of the oikoplastic epithelium appears to
produce different components of the food-concentrating filter, and the
seven giant cells are among the largest cells of the epithelium. No
signal from any other part of the animal was detected with any probes
derived from clone A.
When a nucleotide fragment of oikosin 3 was used as probe, the signal
was detected exclusively from a homogeneous group of cells constituting
the anterior part of the Fol region (Fig. 7C). Probes
prepared from oikosin 2A stained several rows of peri-oral cells in the
anterior part of the epithelium, a single row of cells surrounding the
anterior Fol region, as well as the three rows of small nasse cells
adjacent to the giant cells, and the posterior Fol (Fig. 7,
A and A'). In addition, oikosin 2A was expressed
in the chain of pearls and the three central cells of the seven giant
cells in the field of Eisen, a region implicated in the synthesis of
the inlet filters.
Thus, all of the clones show distinct expression patterns restricted to
the oikoplastic epithelium. The patterns are complementary, with no
overlap and together cover all cells of the Fol anlage, where the
food-concentrating filter is produced. The nuclei of cells in all of
these subregions have distinct morphologies that become increasingly
elaborate as the animal ages.
From small pelagic zooplankton to large baleen whales, filter
feeding is an important and recurrent theme in making a living in the
ocean. Occupying a phylogenetic position near the transition from
invertebrates to vertebrates, the protochordate appendicularians are
filter feeders with a circum-global distribution that play a central
role in marine ecosystems. The discarded mucous houses, in which they
live and filter feed, are a major component of marine snow and
represent a significant contribution to global vertical carbon fluxes.
However, despite the interesting phylogenetic position and ecological
importance of appendicularia, they remain poorly studied.
The apparent association of specific house structures with underlying
fields of cells showing diverse and distinctive nuclear morphologies
led us to analyze the complexity of proteins composing the house, to
clone several cDNAs coding for these proteins, and to examine their
spatial expression patterns in the oikoplastic epithelium. The house is
composed of at least 20 polypeptides, a number of which are highly
glycosylated, because treatments with combined glycosidases resulted in
molecular mass shifts exceeding 100 kDa. This study clearly
demonstrated that mRNAs transcribed from genes corresponding to the
cloned cDNAs were expressed in very restricted and complementary
patterns. Oikosin 1 was restricted to the 14 giant cells in the Fol.
The oikosin 2 family was expressed in a more complex pattern, including
several rows of peri-oral cells in the anterior epithelium, a single
row of cells surrounding the anterior Fol region, and three rows of
small nasse cells adjacent to the Fol giant cells and in the posterior
Fol. In addition, oikosin 2 was expressed in the chain of pearls and
the three central cells of the seven giant cells in the field of Eisen.
Oikosin 3 was expressed only from the anterior cells of the Fol region. Both the oikosin 2 family and oikosin 3 are novel proteins showing no
homology with any known proteins in data bases. On the other hand
oikosin 1 showed limited homology with vertebrate mucins and mammalian
CILP.
Oikosin 2 and oikosin 3 are synthesized in regions of the epithelium
that are overlaid by mesh structures in the house rudiment (Fig. 1).
Oikosin 1 is produced in an intermediate zone between the anterior and
posterior mesh zones of the food-concentrating filter. In this respect,
the weak sequence homology with human CILP is intriguing. CILP is found
only in cartilage and is present in an intermediate layer between
collagen mats (26). Homology between oikosin 1 and CILP is restricted
to a cysteine-rich domain that had previously only been identified as a
subdomain in repeating units found in some vertebrate mucins. The high
concentration of CILP in rib cartilage compared with the low levels in
tracheal cartilage has been interpreted to suggest that compressive
load is a factor in controlling the tissue content of this protein. The
fact that CILP is restricted to an interterritorial zone has been taken
to indicate that this protein has a structural rather than regulatory
role, and it is known that the expression of CILP is increased during
the early stages of osteoarthritis (27). One clear difference among the
different proteins containing the cysteine-rich domain is that oikosin
1 and the mucins contain multiple repeats, whereas CILP contains only
one unit. Tblastn of the oikosin 1 sequence against the entire
Drosophila and Caenorhabditis elegans genomes
revealed no homologies. Therefore, thus far, we are only able to trace
the origins of this domain to a common ancestor protein present prior
to the divergence of urochordates and vertebrates. The conservation of
this domain in organisms as distant as humans and appendicularians
suggests that it is sufficiently important to merit further study.
We have shown that the easily accessible oikoplastic epithelium of
O. dioica serves as a unique template for the synthesis of
different house components from defined cellular regions with distinct
nuclear morphologies. Therefore, we now possess markers to study the
coordinate regulation of gene expression in the house building process,
cell-cell interactions implicated in pattern formation, and the role
that nuclear architecture might play in regulating gene expression.
Superficially, this system resembles synthesis of the insect cuticle
from the underlying epidermis or the production of the egg chorion from
follicle cells in Drosophila. These processes have already
been the targets of some experimental investigation, and the
amplification of chorion genes has been partially characterized (28).
However, neither of the latter two systems presents the same compelling
association of specific structures with underlying subregions of cells.
The insect cuticle contains a chitinous assembly zone directly above
the epidermal microvilli and the perimicrovillar space. During
intermoult, the epidermis secretes peptides constitutively, and these
traverse the perimicrovillar space and form lamellae in the assembly
zone (29). Furthermore, some components of the cuticle are produced in
other organs and transported to the cuticle via the hemolymph (30). In
Drosophila, the composition of the egg chorion appears relatively uniform, although there are some dorsal anterior cells over the oocyte that synthesize specialized chorion structures (28).
Therefore, the oikoplastic epithelium of O. dioica offers significant advantages when compared with alternative model systems and
seems exceptionally well suited to probing the fundamental questions
raised above.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. After thawing,
aliquots were centrifuged at 12,000 × g for 10 min at 4 °C to remove excess sea water. Rudiments were then fragmented by
making a few holes with a 27-gauge needle in the bottom of the tube and
centrifuging this into a second collecting tube. House rudiments were
then heated at 100 °C for 5 min in 1% SDS. The denatured rudiments
were diluted 5-fold with 25 mM sodium phosphate buffer, pH
7.2, 12.5 mM EDTA, 0.625%
CHAPS,1 and 1.25%
-mercaptoethanol and heated again at 100 °C for 5 min. After
cooling, protease inhibitors (a leupeptin, pepstatin, E-64, bestatin,
(4-[2-aminoethyl]benzenesulfonylfluoride, and aprotinin
mixture from Sigma diluted 1:100) were added, samples were centrifuged
at 12,000 × g for 10 min at room temperature, and the
supernatant was recovered. Glycosidases were added to a supernatant
obtained from 50 house rudiments as follows: 0.2 unit of
peptide-N-glycosidase F (Roche Molecular Biochemicals), 5-20 milliunits (as indicated) of
2-3,6,8-neuraminidase
(Calbiochem), and 2 milliunits of bovine serum albumin-free
O-glycosidase (Roche Molecular Biochemicals). When larger
aliquots were processed, enzyme amounts were scaled up proportionally.
Incubation was at 37 °C for at least 18 h. Undigested samples
were incubated under the same conditions.
-32P]ATP using
T4 polynuclotide kinase (New England Biolabs) and pooled in
hybridization buffer in relative concentrations such that each possible
sequence of any of the degenerate sequences was present at a
concentration of 31,25 pM. Filters were prehybridized in
6× SSC, 10 mM sodium phosphate, 5× Denhardt's solution,
0.5% SDS, and 100 µg/ml sheared herring sperm DNA and hybridized in 6× SSC, 10 mM sodium phosphate, pH 7.2, 1× Denhardt's
solution, 0.5% SDS, and 100 µg/ml yeast tRNA at 37 °C for
48 h. Washing was at 37 °C in 6× SSC.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Oikoplastic epithelium and house rudiment of
O. dioica. A, trunk of a fixed animal
showing nuclei stained with Hoechst 33258. The mouth is to the
right, with the maturing gonad to the left. The
gonad is also stained because it contains maturing sperm. B,
epithelial spread stained with Hoechst 33258. The spread was obtained
by removing the tail, the gonad, and the internal organs from a fixed
animal and cutting the oikoplastic epithelium longitudinally along the
mid-ventral line. The mouth is at the top, and the posterior
part of the epithelium at the bottom. Different colors
indicate regions of the epithelium that have been previously defined on
the basis of distinct nuclear morphologies and spatial links with house
structures. The field of Fol includes the anterior Fol
(orange), the seven giant cells (yellow), the
Nasse cells (turquoise green), and the posterior Fol
(pink). The field of Eisen includes the chain of pearls and
the seven giant cells (light blue). The field of Martini
(violet), the anterior (dark blue) and posterior
rosettes (red), and the Leuckart cells (green)
are also indicated. C, house rudiment spread. The
house rudiment was isolated from the animal and cut the same way as the
epithelium in B. fcf, food concentrating filter;
if, inlet filter; ib, bioluminescent inclusion
bodies.
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Fig. 2.
Solubilized, denatured, and reduced house
polypeptides were digested with different combinations of glycosidases
and electrophoretically resolved. A, one-dimensional
SDS-PAGE of house polypeptides without (lane 1) and with
PNGase F treatment (lane 2). In each lane, material
corresponding to 50 house rudiments was loaded, and the bands were
revealed with combined periodic acid oxidation-alcian blue-silver
staining. Following PNGase F treatment, there was a significant
decrease in molecular mass of several bands, including the
disappearance of a broad, prominent band at 36 kDa and the appearance
of lower molecular mass bands. The positions of molecular mass
standards and the PNGase F band are indicated. B,
two-dimensional electrophoresis of house polypeptides simultaneously
digested with PNGase F, neuraminidase, and O-glycosidase.
The gel was stained with neutral silver. Spots corresponding to added
enzymes are indicated and were identified by running a parallel
two-dimensional gel with enzymes only. PNGase F spots migrate in a
basic pH range outside the part of the gel shown here. The two
rectangular boxes inside the neuraminidase box mark house
polypeptides with a wide range of pI heterogeneity. Note the
disappearance of several bands with molecular masses over 70 kDa
compared with PNGase F treatment alone in lane 2 of
A. The nonlinear pH gradient is indicated on top
of the two-dimensional gel, and the positions of molecular mass markers
are on the right side.
-acetylgalactosaminidase) to house rudiment preparations had no detectable effect on the electrophoretic pattern. O-Glycosidase hydrolyzes the bond between serine or
threonine and acetylgalactosamine (GalNAc) in the disaccharide core
unit Gal
1-3GalNAc when the latter is not substituted by sialic acid or other oligosaccharides. Therefore, our result is not surprising, because the Gal
1-3GalNAc core unit is only present in a subset of
O-linked oligosaccharides and a significant amount of this linkage unit would be required to see detectable molecular mass shifts.
Most of the glycosaminoglycans found in proteoglycans and the
oligosaccharide chains of mucins are linked to serine and threonine
through different saccharide moieties.
View larger version (48K):
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Fig. 3.
Neutral silver-stained one-dimensional
SDS-PAGE of a 200-house rudiment sample digested with PNGase F alone,
from which polypeptide bands were cut and microsequenced. Peptide
sequences derived from labeled bands are shown. Where two letters are
in parentheses, the order of those residues could not be
established. XX represents two residues that could not be
identified, and the number in parentheses
indicates the sum of their molecular masses. Stretches of peptide
sequences from which oligonucleotide probes were derived are shown in
bold type. The PNGase F band is marked by an
asterisk, and the positions of molecular mass standards are
indicated.
15 and 10
110
on nonredundant NCBI data bases). The remaining seven clones did not
show significant homology to any known protein in the same data bases
(E value
10
11). Because only partial
sequences of the latter clones were obtained, it is possible that
unsequenced portions match some of the sequenced house peptides. These
clones have not yet been further characterized.
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Fig. 4.
Deduced amino acid sequences from
cDNAs coding for house polypeptides. A, amino acid
sequences are presented for clone A, clones G1, G2a, and G2b, and Clone
H. Potential N-glycosylation sites are indicated in
bold type and underlined. Potential
glycosaminoglycan attachment sites are indicated in italic
type and underlined. Potential RGD cell attachment
sequences are indicated in bold letters within an
oval. For sequence comparisons among G clones,
dots indicate identical residues, and dashes
indicate gaps. B, schematic representations of house
proteins. Scaling for Clone A is contracted 3-fold relative to clones G
and H. Vertical wavy lines indicate potential
O-glycosylation sites, tridents indicate
potential N-glycosylation sites, inverted open
triangles indicate potential glycosaminoglycan attachment sites,
and black boxes indicate predicted secretion signal
peptides. For clone A, the cysteine repeat domains are represented by
open ovals, an epidermal growth factor domain is
represented by a stippled rectangle, and a C type lectin
domain is represented by a dotted hexagon. For clone H, the
location of the RGD cell attachment sequence is indicated by
RGD within a hexagon. Potential
O-glycosylation sites were predicted using the NetOGlyc
prediction server at the Center for Biological Sequence
Analysis. Potential N-glycosylation sequences,
glycosaminoglycan attachment sequences, and RGD cell attachment
sequences were predicted using the PredictProtein server at the
University of Columbia. The epidermal growth factor and C-lectin
domains were predicted using the PFAM version 5.5 data base at
Washington University in St. Louis. Signal peptides were predicted
using the SignalP server at the Center for Biological Sequence
Analysis, and the PSORT server at the University of Osaka, in
combination with data obtained from sequences of known extracellular proteins obtained from shotgun sequencing of
an O. dioica cDNA library.
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Fig. 5.
Oikosin 1 contains repeats of a motif shared
by several vertebrate gel-forming mucins and human CILP.
A, clustalX alignment of the 13 repeats of oikosin 1, the
Cys subdomains of MUC5B, MUC5AC, MUC2, mouse Muc5ac, cervical MUC5B, a
Xenopus expressed sequence tag (Xlmuc), and a
similar motif from CILP. §, hydroxyl residues; #, aromatic
residues; , hydrophobic residues (including aromatic ones); Ø,
acidic residues; *, D, E, N, or Q; +, basic residues. Consensus was
assigned to positions where residues with the given property were
present in at least 50% of the sequences. Differential coloring
indicates highly conserved regions within the repeats. B,
schematic representation of some of the proteins with Cys
subdomain-like motifs (black ovals) aligned in A.
The dotted rectangle in oikosin 1 represents the C type
lectin domain, and the checkered rectangle in CILP
represents a type 1 thrombospondin repeat. Hatched domains
in MUC5B, MUC5AC, and MUC2 represent tandem repeats rich in serine,
threonine, and proline of 29, 8, and 23 amino acid residues,
respectively. In MUC2 there is an additional imperfect
serine-threonine-rich repeat between the two Cys domains. In MUC5B
there are five repeats with 11 copies each of the 29-amino acid
irregular repeat (RI, RII, RIII,
RIV, and RV). All except RV are followed by a
conserved nonrepetitive 111-amino acid sequence (in black)
called the R-end, also rich in serine, threonine, and proline. The
total number of Cys subdomains in MUC5AC is not known. Diagonal
slashes indicate variations in the inner cores of the mucins. The
B, C, CK, and D domains of the mucins are homologous to the
corresponding domains of human prepro-von Willebrand factor.
Representations of MUC5B, MUC5AC, and MUC2 are adapted from Desseyn
et al. (25). Schematics of the polypeptides are all drawn to
the same scale.
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Fig. 6.
Northern blot analysis of
poly(A)+ RNA (500 ng/lane) from adult animals probed with
different cDNA clones. Left lane, oikosin 1 containing a long reading frame matching all peptides from band A (Fig.
3); center lane, oikosin 2A, the longest reading frame
matching with peptides from band G (Fig. 3); right lane,
oikosin 3, the longest reading frame matching with peptides from band H
(Fig. 3). The molecular mass of each transcript as estimated by the
migration of mammalian rRNA bands is indicated. kb,
kilobases.
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Fig. 7.
In situ expression patterns of
genes coding for house proteins. A-C, optical sections
obtained by confocal microscopy on whole mount fluorescent in
situ hybridization preparations. The mouth is on the
left, and the posterior part of the trunk is on the
right. In A and A' a fragment of
oikosin 2A was used as a probe showing staining on the perioral region
and selected regions of the fields of Fol (A) and Eisen
(A'). A' is a section through the field of Eisen
showing staining of the three central giant cells and of the chain of
pearls. In B and C fragments of oikosin 1 and
oikosin 3 stain the seven giant cells and the anterior part of the
field of Fol, respectively. D, epithelial spread stained
with Hoechst 33258, where the expression patterns shown in
A-C are digitally recreated in different colors.
Red corresponds to oikosin 2A (A and
A'); green corresponds to oikosin 1 (B); and yellow corresponds to oikosin 3 (C). For
simplicity the nuclei of the cells have been digitally colored instead
of the cytoplasm. The mouth is at the top, and the posterior
part of the epithelium is at the bottom.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Rein Aasland (University of Bergen) for assistance with the Cys domain alignments.
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FOOTNOTES |
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* This work was supported by the Norwegian Research Council and Ministry of Education. Work in the Protein Interaction Laboratory was funded by a grant from the Danish National Research Foundation to the Center for Experimental BioInformatics.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ308491, AJ308492, AJ308493, AJ308494, and AJ308495.
¶ To whom correspondence should be addressed. Tel.: 47-55-58-43-46; Fax: 47-55-58-43-05; E-mail: Eric.Thompson@sars.uib.no.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M100438200
2 H.-C. Seo, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; PNGase F, peptide-N-glycosidase F; GalNAc, N-acetylgalactosamine; bp, base pair(s); CILP, cartilage intermediate layer protein..
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