Molecular Cloning and Characterization of Spiggin
AN ANDROGEN-REGULATED EXTRAORGANISMAL ADHESIVE WITH STRUCTURAL
SIMILARITIES TO Von Willebrand FACTOR-RELATED PROTEINS*
Iwan
Jones
,
Christina
Lindberg
,
Staffan
Jakobsson§,
Anna
Hellqvist§,
Ulf
Hellman¶,
Bertil
Borg§, and
Per-Erik
Olsson
From the
Department of Cell and Molecular Biology,
Unit of Physiology, Umeå University, SE-901 87 Umeå, Sweden, the
§ Department of Zoology, Stockholm University, SE-106 91 Stockholm, Sweden and the ¶ Ludwig Institute for Cancer Research,
Uppsala Biomedical Center, SE-751 24 Uppsala, Sweden
Received for publication, February 6, 2001, and in revised form, February 28, 2001
 |
ABSTRACT |
One of the most definitive examples of a
vertebrate extraorganismal structural protein can be found in
three-spined sticklebacks (Gasterosteus aculeatus). In the
breeding male the kidney hypertrophies and synthesizes an adhesive
protein called "spiggin," which is secreted into the urinary
bladder from where it is employed as a structural thread for nest
building. This paper describes the first molecular characterization of
spiggin and demonstrates that this adhesive is a protein complex
assembled from a potential of three distinct subunits (
,
, and
). These subunits arise by alternative splicing, and
11-ketoandrogens induce their expression in stickleback kidneys.
Analysis of the predicted amino acid sequence of each subunit reveals a
modular organization whose structural elements display a similarity to
the multimerization domains found within von Willebrand Factor-related
proteins. These results implicate that spiggin utilizes a conserved
multimerization mechanism for the formation of a viscous agglutinate
from its constituent subunits in the urinary bladders of male
sticklebacks. This novel extraorganismal structural protein is
therefore ideally suited to its function as an adhesive thread.
 |
INTRODUCTION |
Extrorganismal proteins are common among
invertebrates. Examples range from the fibroin threads forming spider
webs and silkworm cocoons (1, 2) to the collagen-based matrixes of the
byssus threads of Mytilus and the Drosophila sgs
family (3, 4).
The production of extraorganismal structural proteins is not as common
among vertebrates. However, a distinct example occurs in the teleostean
stickleback family (Gasterosteidae) where the secondary
proximal epithelium cells of the male kidney undergo hypertrophy during
the breeding season (5). In male three-spined stickleback
(Gasterosteus aculeatus) this structural reorganization is
due to the synthesis of an adhesive protein called "spiggin," which
is under the regulation of 11-ketoandrogens (6). This agglutinate is
subsequently secreted into the urinary bladder for storage and employed
as a structural and highly elastic adhesive thread to assemble a nest
from plant material in which the female lays her eggs (5, 6).
Previously we have characterized spiggin from the urinary bladder
content of male sticklebacks as being a 203-kDa cysteine-rich glycoprotein (6). No similarities in amino acid composition could be
observed between spiggin and murine kidney androgen-regulated protein, elastin, collagen, fibroin, or vitelline envelope
proteins (7-9). These observations suggest that spiggin is a novel
structural protein.
This paper describes the first molecular characterization of spiggin
and demonstrates that this extraorganismal adhesive is a protein
complex assembled from a potential of three distinct subunits (
,
, and
). These subunits arise by alternative splicing from a
single gene, and their expression is under the regulation of
11-ketoandrogens. Analysis of each subunit's predicted amino acid
sequence reveals that they exhibit a modular organization whose
structural elements display a similarity to the multimerization domains
found within von Willebrand Factor
(vWF)1-related proteins.
 |
EXPERIMENTAL PROCEDURES |
Fish Maintenance--
Adult sticklebacks were routinely housed
in a 200-liter aquarium containing brackish water (0.5%
salinity) at 20 °C under a photoperiod of 16:8 h light:dark. The
water was aerated and filtered. The fish were fed red midge larvae.
Amino Acid Sequencing--
Urinary bladder contents from 10 mature male sticklebacks were pooled. Proteins were resolved by
SDS-PAGE (10) and visualized by Coomassie staining (11). The 203-kDa
spiggin bands were excised from the gel and subjected to in-gel
digestion with porcine trypsin (Promega) or Lys-C protease (Wako
Chemicals GmbH) (12). Individual peptides were isolated following
acidification by microbore reversed phase liquid chromatography on a
Kromasil C18 column operated in the SMART System (Amersham Pharmacia
Biotech). The amino acid sequences of selected peptides were determined
in an Automated Peptide Sequencer (model 494A, Applied Biosystems).
Reverse Transcriptase-Polymerase Chain Reaction--
Total RNA
was extracted from a pooled sample of five mature male kidneys using
Tri ReagentTM (Sigma). cDNA was synthesized from 1 µg of total
RNA using the First Strand cDNA Synthesis Kit (Amersham Pharmacia
Biotech). PCR reactions were performed in "Thermo Buffer" (Promega)
and contained 500 ng of cDNA template, 0.2 mM dNTPs, 1 mM magnesium chloride, 1 unit of Taq DNA
polymerase (Promega), and 25 pmol of spiggin
NH2-terminal (5'-CARACIAARGARATICARAC-3') and spiggin
peptide-4 (5'-TTRTGIGAIATRTARTTYTCYTT-3') oligonucleotides. Optimized reaction conditions (13) were as follows: 95 °C for 1 min,
50 °C for 1 min, and 72 °C for 1 min for 40 successive cycles
using a PTC-200 Thermal Cycler (MJ Research). Amplified products were
ligated into pGEM®-T (Promega), and recombinant plasmids
were isolated using the Wizard® Plus SV
Miniprep System (Promega). Cycle sequencing was performed using the
Thermo Sequenase (version 2.0) Sequencing Kit (Amersham Pharmacia
Biotech). The reactions were resolved on an ABI PrismTM 377 DNA
Sequencer (PerkinElmer Life Sciences), and the data obtained were analyzed using EditView (version 1.0.1) (PerkinElmer Life Sciences).
Slot Blot Analysis--
Total RNA was extracted from mature male
tissues, from three individual fish, using Tri ReagentTM (Sigma).
Aliquots of 5 µg of total RNA were mixed with denaturing solution
(6 × SSC, 7% (v/v) formaldehyde) and transferred onto a nylon
membrane (Amersham Pharmacia Biotech) using a Minifold II Slot Blot
Apparatus (Schleicher and Schuell). Membranes were probed using a
randomly primed [
-32P]dCTP radiolabeled spiggin
cDNA fragment (636 base pairs) that was isolated by reverse
transcriptase-polymerase chain reaction and sequenced as above.
Hybridizations were performed at 65 °C overnight (6 × SSC, 0.1% (w/v) SDS, 100 µg ml
1 tRNA, and
5 × Denhardt's solution). The membranes were washed for 2 × 30 min periods at 42 and 65 °C in 0.1 × SSC, 0.1% (w/v) SDS and exposed to HyperfilmTM-MP film (Amersham Pharmacia Biotech) at
70 °C. The films were visualized using a Curix 60 Film Developer (AGFA).
Northern Blot Analysis--
Total RNA was extracted from mature
kidneys using Tri ReagentTM (Sigma). Northern blots containing 5 µg
of total RNA were performed as described previously (11). Hybridization
to an [
-32P]dCTP spiggin cDNA probe and washing
were performed as described above.
cDNA Library Construction and Screening--
Total RNA was
extracted from a pooled sample of 20 mature male kidneys using Tri
ReagentTM (Sigma). The mRNA fraction was isolated using the poly(A)
Quik® mRNA Purification Kit (Stratagene), and an
unidirectional cDNA library was constructed in Lambda ZAP
Express® (Stratagene). A total of 2 × 105 plaques were screened (11). Hybridization to an
[
-32P]dCTP spiggin cDNA probe and washing were
performed as described above. Positive plaques were purified through
four successive hybridization rounds, and individual clones were
isolated by phagemid excision. These clones were sequenced by Cybergene
AB (Huddinge, Sweden).
Southern Blot Analysis--
Genomic DNA was isolated from a
whole mature stickleback using the GenEluteTM Mammalian Genomic DNA Kit
(Sigma). Southern blots containing 20-µg aliquots of DNA digested to
completion with BamHI, EcoRI, HindIII,
KpnI, NcoI, and XhoI were performed as
described previously (11). Hybridization to an
[
-32P]dCTP spiggin cDNA probe and washing were
performed as described above.
Effects of Steroid Treatments--
Steroid treatments were
performed using adult females, as their kidneys do not undergo
hypertrophy under natural conditions (6). Nonbreeding females housed at
9 °C and a photoperiod of 8:16 h light:dark were anesthetized with
0.1% (v/v) 2-phenoxyethanol (Sigma) and implanted intraperitoneally
with Silclear silicone tubing (10-mm length, 0.6-mm inner diameter,
1.2-mm outer diameter) containing steroids dissolved in cocoa butter.
The steroids were 11-ketoandrostenedione (11-KA) (0.04, 0.2, 1, 5, and
25 µg µl
1) and cortisol,
5-
-dihydrotestosterone, testosterone, estradiol, and progesterone
(25 µg µl
1 only). Tubes containing cocoa
butter were used as controls. Following implantation the fish were
maintained in a 50-liter aquarium containing brackish water
(0.5% salinity) at 17 °C for 16 days under 16:8 h light:dark prior
to the kidneys being excised and stored at
70 °C. In a time scale
experiment post-breeding females were implanted with 15-mm-long tubes
containing 25 µg µl
1 11-KA and housed in
a 50-liter aquarium containing brackish water (0.5% salinity)
at 20 °C 16:8 h light:dark. Nontreated females served as controls.
Following 1, 3, 5, and 10 h or 1, 3, 5, and 10 days, kidneys were
excised and stored at
70 °C. Total RNA was extracted from each
sample using Tri ReagentTM (Sigma). Aliquots of 5 µg of total RNA
were subjected to slot blot analysis as described above. Relative
spiggin transcript levels were determined using a calibrated GS-250
Molecular Phosphoimager coupled to the Molecular Analyst (version 1.41)
package (Bio-Rad). All determinations were performed on three fish, and
the results were expressed as the mean (±S.D.). The mean value of the
maximal signal intensities was arbitrarily set as 100%.
SDS-PAGE and Immunoblotting--
Kidneys and bladder contents
from three mature males were pooled and homogenized in
phosphate-buffered saline (pH 6.5). The soluble protein fractions were
recovered by centrifugation (13,000 × g for 1 min at
4 °C). The concentration of each preparation was determined using
the Bradford assay (14). Aliquots of 1 µg of protein were resolved by
SDS-PAGE (10) and transferred onto a nitrocellulose membrane (Amersham
Pharmacia Biotech) using a Trans-Blot Semi Dry Electrophoretic Transfer
Cell (Bio-Rad). Immunodetection (11) was performed using a polyclonal
antiserum raised against an NH2-terminal peptide common to
both the kidney subunits and urinary bladder content spiggin
(KTKEIQTYTCRTFGS-C) (Agri Sera AB, Umeå, Sweden) and an anti-rabbit
IgG horseradish peroxidase conjugate (Dako). Proteins were visualized
using the ECL System (Amersham Pharmacia Biotech).
Phylogenetic Analyses--
Amino acid sequences were aligned
using the Clustal W algorithm (version 1.7) (15) (gap opening penalty,
10.0; gap extension penalty, 0.2; gap separation distance, 4.0).
Phylogenetic trees were assembled in Tree View (version 1.6.2) (16)
using 1000 bootstrap replicates.
 |
RESULTS |
Urinary bladder spiggin was purified to homogeneity, and six
partial peptide sequences were determined by amino acid sequencing (Table I). Degenerate oligonucleotides
designed against the NH2-terminal sequence and peptide-4
were employed to amplify a partial cDNA sequence (636 base pairs)
encoding for a spiggin subunit from male stickleback total kidney RNA
by reverse transcriptase-polymerase chain reaction. The identity of
this clone was established as the translated protein sequence also
contained two peptides (SYYVR and IRDPVLRK) predicted from amino acid
sequencing (Table I). This nucleotide sequence was subsequently
employed for the generation of molecular probes.
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Table I
Partial peptide sequences and their determined location (amino acid
numbers) in the deduced spiggin subunit- protein sequence
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Isolation of Spiggin Subunit-
, -
, and -
--
Slot blot
analysis of spiggin spatial expression was performed on three separate
animals. In all analyses the expression was restricted to the kidney of
mature males (Fig. 1A) where a
predominant message of 4.2 kb and lesser mRNAs of 2.2 and 1.6 kb
were detected by Northern analysis. These transcripts were designated
spiggin subunit-
(4.2 kb), subunit-
(2.2 kb), and subunit-
(1.6 kb), respectively (Fig. 1B, lane 1). No
hybridization was detected in mature female kidney even after prolonged
exposure (Fig. 1B, lane 2). A total of 26 recombinant clones encoding for all transcripts were isolated from a
mature male stickleback kidney cDNA library. Sequencing of these
clones demonstrated that their untranslated regions were identical, and
thus only two recombinants encoding for each subunit were sequenced
entirely.

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Fig. 1.
A, slot blot analysis of spiggin
expression in selected tissues of stickleback. All determinations were
performed using 5 µg of total RNA from three separate animals. The
same results were obtained for all animals. B, Northern blot
analysis of spiggin expression in kidneys of mature male and female
sticklebacks. Lane 1, 5 µg of total male kidney RNA;
lane 2, 5 µg of total female kidney RNA. Positions of RNA
molecular mass markers (kb) (Life Technologies, Inc.) are given
in the left margin. The size (kb) of each spiggin subunit
transcript is indicated in the right margin.
|
|
The deduced open reading frame (ORF) of subunit-
encoded for a
910-amino acid protein with a predicted molecular mass of 103 kDa. Both subunit-
and -
encoded polypeptides of 613 and 472 residues with calculated molecular masses of 70 and 53 kDa, respectively (Fig. 2). The deduced
NH2-terminal amino acid sequence of each spiggin subunit
was located 25 amino acids upstream of the sequence obtained by
NH2-terminal sequencing of urinary bladder content spiggin.
The ORFs of each subunit were identical from the initiating
Met1 through to Gln466. This was
followed by six additional amino acid residues and a stop codon in
subunit-
, while the ORFs of both other peptides continued until
Trp606. At this point a further seven residues and a
termination codon were observed in subunit-
. The sequences of each
subunit exhibited a conserved identity at both the nucleotide and
protein levels that indicated that all subunits were derived from a
single locus by alternative splicing. The hybridization patterns
observed following Southern analysis of stickleback genomic DNA was
compatible with the existence of a single Spiggin gene (Fig.
3).

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Fig. 2.
The deduced amino acid sequences of spiggin
subunit- , subunit- ,
and subunit- . Spaces (-) are inserted to
allow the alignment of homologous amino acids. The positions of
conserved cysteine residues are indicated by asterisks.
Signal peptide sequences are underlined. Vicinal and
truncated cysteine motifs (CGLCG and GLCG) are shown by a
line above the sequence.
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Fig. 3.
Genomic Southern analysis of the
Spiggin locus. Stickleback genomic DNA (20 µg)
was digested with: BamHI (lane 1),
EcoRI (lane 2), HindIII (lane
3), KpnI (lane 4), NcoI
(lane 5), and XhoI (lane 6). The
positions of /HindIII molecular mass markers (kb)
(Promega) are given in the left margin.
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|
Regulation of Spiggin Subunit-
, -
, and -
Expression--
Spiggin mRNA was induced by 11-KA (Fig.
4A) whose in vivo
conversion into 11-ketotestosterone has been shown previously (17). A
dose-response induction was first observed at an implant concentration of 1 µg µl
1 (18 ± 8%), and this
was augmented 4- and 6-fold, respectively, with doses of 5 µg
µl
1 (75 ± 16%) and 25 µg
µl
1 (100 ± 10%). Kidney hypertrophy
followed a similar pattern as implantation of 1 µg
µl
1 11-KA induced intermediate hypertrophy,
while treatment with 5 and 25 µg µl
1
produced clear hypertrophy. Implantation of other steroids did not
induce spiggin expression or induce hypertrophy.

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Fig. 4.
Induction of spiggin in adult female
stickleback kidney. All values represent the mean (±S.D.) values
from three fish. The mean value of the maximal signal intensity was
arbitrarily set as 100%. % DPM, percent disintegrations
minute 1. ND, no signal detected.
A, dose-response and steroid specificity analysis of spiggin
induction. DHT, dihydrotestosterone. B, time
response analysis of spiggin induction by 25 µg
µl 1 11-ketoandrostenedione. h,
hours; d, days.
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|
Induction of spiggin mRNA by 25 µg
µl
1 11-KA over time was investigated (Fig.
4B). Expression was apparent 1 day after implantation (29 ± 14%) and increased following 3 days (43 ± 8%), 5 days (83 ± 8%), and 10 days (100 ± 23%) stimulation. The
absence of kidney stimulation in normal females was confirmed, as no
induction was detected in control groups (Figs. 4, A and
B).
Analysis of Spiggin Subunit-
, -
, and -
Proteins--
The
presence of spiggin within the kidney and urinary bladder content of
male sticklebacks was investigated by immunodetection (Fig.
5). Antiserum raised against an
NH2-terminal motif common to both kidney subunits and
urinary bladder content spiggin (KTKEIQTYTCRTFGS-C) recognized
predominant bands of 130 and 51 kDa and a faint 90-kDa signal in the
kidney (Fig. 5, lane 1). Conversely only a single distinct
protein band of 203 kDa was detected in the urinary bladder content
(Fig. 5, lane 2). This 203-kDa band was not detected in the
kidney even after prolonged exposure.

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Fig. 5.
Immunodetection of spiggin in the kidney and
urinary bladder content of mature male sticklebacks. Lane
1, 1 µg of kidney-soluble protein fraction; lane 2, 1 µg of bladder content-soluble protein fraction. The positions of
SDS-PAGE standards (kilodaltons) (Bio-Rad) are given in the left
margin. The size (kilodaltons) of each immunodetected protein is
indicated in the right margin.
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Structural and Phylogenetic Analyses--
Hydropathy analysis (18)
demonstrated that subunit-
, -
, and -
were overall hydrophobic
(Fig. 6). All subunits contained a signal
peptide with a predicted length of 19 residues at their amino terminus
(Table II) (19) and up to four
N-glycosylation sites and one O-glycosylation site (Table
II) (20). Each subunit exhibited a modular organization and could be
divided into different domains. Subunit-
was found to contain two
full (D1 and D2) and one truncated (D3) nontandem motifs that displayed
a high degree of sequence identity to the D domains found
within vWF. Similarly subunit-
was determined to contain two full
domains (D1 and D2), while subunit-
contained one full (D1) and one
truncated (D2) motif also arranged as nontandem repeats (Table II)
(21). In addition, conserved vicinal cysteine motifs (CGLCG) were
present in each D1 domain, while the D2 domains of subunit-
and -
contained an additional truncated version of this motif (GLCG) (Table
II). A second characteristic of each subunit was that each
D domain was bisected by regions exhibiting high cysteine
content (10-12%) (Fig. 7).

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Fig. 6.
Hydropathy profiles of spiggin
subunit- , subunit- ,
and subunit- . The hydropathy profiles
were predicted using the Kyte-Doolittle algorithm using an 11-amino
acid window. Plus (+) values indicate a hydrophilic character while
minus ( ) values represent a hydrophobic character.
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Table II
Predicted structural and functional motifs and their location (amino
acid numbers) in the deduced spiggin subunit- , subunit- , and
subunit- protein sequences
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Fig. 7.
Structural domains identified in spiggin
subunit- , subunit- ,
and subunit- . The location (amino acid
numbers) of identified motifs is given below each polypeptide chain.
The lengths of the polypeptides are not to scale.
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Similarities between the predicted amino acid sequences of each subunit
and vWF-related proteins were determined using the gapped BLAST
(version 2.0) algorithm (22). Each subunit exhibited the highest
similarity to Xenopus integumentary mucin B.1 (28%), rat
MUC2 (27%), human MUC5AC (27%), human vWF (26%), and murine otogelin
(25%), while lower similarities (<20%) were also exhibited to
diverse vWF-related proteins such as Xenopus kielin,
Drosophila hemolectin, and porcine zonadhesin. Phylogenetic
analysis further confirmed that each subunit exhibited an ancestral
relation to vertebrate mucins, vWF, and mouse otogelin (Fig.
8).

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Fig. 8.
Phylogenetic relations of spiggin
subunit- , subunit- ,
and subunit- to von Willebrand Factor-related
proteins. Bootstrap values (percent) supporting the position of
the assigned clades are given at each branch point. Branch lengths are
proportional to evolutionary distances. GenBankTM accession
numbers are as follows: AF323732 (G. aculeatus spiggin
subunit- ), AF323733 (G. aculeatus spiggin subunit- ),
AF323734 (G. aculeatus spiggin subunit- ), BAA95483
(Xenopus laevis kielin), T30886 (X. laevis
integumentary mucin B.1), AAA21655 (Rattus norvegicus mucin
MUC2), AAC15950 (Homo sapiens mucin MUC5AC), AAB59458
(H. sapiens von Willebrand Factor), NP_003881 (H. sapiens IgG Fc-binding protein), NP_038652 (Mus
musculus otogelin), NP_033373 (M. musculus
-tectorin), Q28983 (Sus scrofa zonadhesin precursor),
BAA88518 (Drosophila melanogaster hemolectin), and P980929
(Bombyx mori hemocytin precursor).
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 |
DISCUSSION |
This study presents the first molecular characterization of
spiggin, a novel extraorganismal adhesive protein synthesized by male
three-spined sticklebacks. Spiggin was expressed in the kidney as three
subunits (
,
, and
). Alternative splicing from one locus
generated each subunit. The expression of spiggin was only induced by
11-ketoandrogens. This is consistent with our previous findings that
11-oxygenated androgens were the most effective in stimulating
stickleback kidney hypertrophy (23), and 11-ketotestosterone is
regarded as the main androgen in male teleosts (5). Spiggin is
therefore the only currently known product being induced by 11-ketoandrogens in any species.
The amino acid sequence of subunit-
, -
, and -
exhibited an
overall hydrophobic character that is consistent with spiggins' role
as a water-insoluble adhesive (6) and contained signal peptides of a
predicted 19 residues at their amino termini (19). Interestingly, amino
acid sequencing of the NH2 terminus of urinary bladder
spiggin demonstrated that this translocation motif was absent and
suggests that the subunits are processed into mature protein by
signal peptide removal.
The ORFs of each subunit were predicted to encode for proteins with
molecular masses of 103 kDa (subunit-
), 70 kDa (subunit-
), and 53 kDa (subunit-
), respectively. However, immunodetection recognized predominant bands of 130 and 51 kDa and a faint 90-kDa signal in the kidney. The apparent differences between the predicted and observed molecular masses could be accounted for by
post-translational modifications as up to four
N-glycosylation sites and one O-glycosylation site were predicted (20) by their deduced amino acid sequences. This is
consistent with our previous investigation where spiggin assayed
positively for the presence of carbohydrate (6). Conversely, only a
single distinct protein band of 203 kDa was detected within the urinary
bladder of male sticklebacks that also concurred with our previous
investigations (6). These observations suggest that formation of
spiggin involves multimerization of the constituent kidney subunits in
the urinary bladder. A structural analysis of each subunit was
therefore conducted to identify motifs that could catalyze such a
mechanism. Each subunit exhibited a modular structure and could be
divided into distinct regions that displayed sequence similarity to the
D domains found within vWF (21) and other related proteins
such as vertebrate mucins, zonadhesin, and IgG Fc-binding proteins
(24-27). However, the D domains within each spiggin
subunit were organized into nontandem repeats bisected by several
cysteine-rich regions (10-12%). This is unlike that of vWF-related
proteins where D domains are arranged as repeating units
with a disulfide-rich region located at their carboxyl terminus (24,
28). D domains and disulfide elements in vWF and porcine submaxillary mucin are required for their assembly into large multimeric proteins (28-31). The structural similarities between particularly vWF and subunit-
, -
, and -
therefore suggest that the multimerization of spiggin is mediated by a conserved mechanism common to all vWF-related proteins.
Another interesting observation is that each subunit may have the
ability to self-catalyze multimerization. In vWF this autocatalysis was
dependent upon the presence of two sets of vicinal cysteine residues
(CGLCG) present in its D1 and D2 domains (32-35). Each spiggin subunit
possessed vicinal cysteine motifs in their D1 domains, while the
truncated version (GLCG) present in the D2 domains of subunit-
and
-
is also a characteristic of human MUC2 and porcine submaxillary
mucin (24, 28). Mutation of these motifs in porcine submaxillary mucin
resulted in proteins that were poorly secreted and suggested that the
D domains interacted to induce correct protein folding
(28). Such a process could therefore be envisioned for spiggin, as each
subunit possesses the necessary catalytic motifs required to induce multimerization.
The conserved domain structure between each spiggin subunit and
vWF-related proteins suggested an evolutionary relationship that was
confirmed by phylogenetic analysis. Spiggin subunit-
, -
, and -
were ancestrally related to vertebrate mucins, vWF, and mouse otogelin.
These results clearly demonstrate that spiggin shares structural
properties with vWF-related proteins. The presence of the D
domains and the vicinal cysteine motifs suggest that spiggin utilizes a
conserved multimerization mechanism for the formation of a viscous
agglutinate from its constituent subunits in the urinary bladders of
male sticklebacks. This novel extraorganismal structural protein is
therefore ideally suited to its function as an adhesive thread.
 |
FOOTNOTES |
*
This work was supported by the Swedish Forestry and
Agriculture Research Council (to P.-E. O.), the Swedish
Environmental Protection Agency (to P.-E. O.), and the Swedish
Natural Science Research Council (to B. B.).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) AF323732 (spiggin subunit-
), AF323733 (spiggin
subunit-
), and AF323734 (spiggin subunit-
).
To whom correspondence should be addressed: Dept. of Cell and
Molecular Biology, Unit of Physiology, Umeå University, SE-901 87 Umeå, Sweden. Tel.: 46-90-7869545; Fax: 46-90-7866691; E-mail: Per-Erik.Olsson@biology.umu.se.
Published, JBC Papers in Press, March 1, 2001, DOI 10.1074/jbc.M101142200
 |
ABBREVIATIONS |
The abbreviations used are:
vWF, von Willebrand
Factor;
11-KA, 11-ketoandrostenedione;
PAGE, polyacrylamide gel
electrophoresis;
kb, kilobase(s);
ORF, open reading frame..
 |
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