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Acrosomal status of mouse spermatozoa in the oviductal isthmus

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JEZ 0373D
EXPERIMENTAL ZOOLOGY 282:332–343 (1998)
Putative Multiadhesive Protein From the Marine
Sponge Geodia cydonium: Cloning of the cDNA
Encoding a Fibronectin-, an SRCR-, and a
Complement Control Protein Module†
Institut für Physiologische Chemie, Abteilung Angewandte
Molekularbiologie, Universität, D-55099 Mainz, Germany
Sponges (Porifera) representing the simplest metazoan phylum so far have been
thought to possess no basal lamina tissue structures. One major extracellular matrix protein that
is also a constitutive glycoprotein of the basal lamina is fibronectin. It was the aim of the present
study to identify the native protein from the marine sponge Geodia cydonium and to isolate the
corresponding cDNA. In crude extracts from this sponge protein(s) of Mr of ≈230 and ≈210 kDa
could be visualized by Western blotting using an anti-fibronectin [human] antibody. By PCR cloning from a cDNA library of G. cydonium we isolated a cDNA comprising one element of fibronectin,
the type-III (FN3) module.
The cDNA (2.3 kb long), encoding a 701 amino acid [aa] long putative “multiadhesive protein”
termed MAP_GEOCY, was found to contain (i) a fibronectin-, (ii) a scavenger receptor cysteinerich [SRCR]-, and (iii) a short consensus repeat [SCR] module. The 89 aa long fibronectin module
comprises the characteristic topology and conserved aa found in fibronectin type-III (FN3) elements. The SRCR module (101 aa) features the characteristics of group B SRCR molecules. The
predominant proteins belonging to this group are the mammalian WC1-, M130-, CD6- and CD5
antigens that probably are involved in immunological reactions. The SCR module (54 aa) shows
the characteristics of type III SCR modules found in complement receptors. Phylogenetic analyses
performed with all three building blocks of the “multiadhesive protein” showed that the respective
sponge modules form independent, possibly basal, lineages in trees that include the corresponding
modules from higher metazoan animals. In summary, these data demonstrate for the first time
that the phylogenetically oldest Metazoa, the sponges, contain protein modules seen in higher
animals in proteins of the extracellular matrix and in molecules involved in cell-mediated immune
reactions in vertebrates. J. Exp. Zool. 282:332–343, 1998. © 1998 Wiley-Liss, Inc.
Sponges (Porifera) are the most ancient phylum
of Metazoa; their body is surrounded by outer(pinacoderm) and inner epithelial cell sheets
(choanoderm) (Simpson, ’84) and considered to
have organ-like cell assemblies, e.g. the choanocyte chambers (Weissenfels cited in Mehlhorn, ’89).
Septate junctions in the epithelia of the sponge
Sycon ciliatum were described (Ledger, ’75).
Until now, no basal lamina has been identified
microscopically (Garrone, ’78); this supports the
assumption that sponges might have evolved from
Protozoa different than those that were the ancestors of the other metazoan phyla (Nielsen, ’95).
Therefore, biochemical and molecular biological
techniques were applied to identify receptors, adhesion molecules, and structural elements of the
basal lamina in sponges.
Recently a number of cDNAs and genes were
cloned from the marine sponge Geodia cydonium.
Among them are two cell-surface receptors: the
receptor tyrosine kinase (Schäcke et al., ’94a,b),
and the α subunit of integrin (Pancer et al., ’97a).
Furthermore, a galectin, whose protein has been
localized in situ (Pfeifer et al., ’93; WagnerHülsmann et al., ’96), as well as a protein featur-
Grant sponsor: Bundesministerium für Bildung und Technologie
(BMBT Verbundprojekt “TEPS”); Grant sponsor: International Human Frontier Science Program; Grant number: RG-333/96-M.
*Correspondence to: Prof. Dr. W.E.G. Müller, Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie,
Universität, Duesbergweg 6, 55099 Mainz, Germany. E-mail:
Received 7 January 1998; Accepted 13 April 1998
The sequence reported here is deposited in the EMBL data base
ing scavenger receptor cysteine-rich [SRCR] domains (Pancer et al., ’97b) that are prominent receptors known from vertebrates and one phylum
of invertebrates, the echinoderms (Resnick et al.,
’94). The gene for this protein is expressed in G.
cydonium in several membrane-bound forms and
as one soluble form (Pancer et al., ’97b). Surprisingly, among the membrane-bound species is one,
with an RGD-containing cell binding domain
(Ruoslahti, ’96; Müller et al., submitted). The
SRCR-rich sponge protein was determined immunohistologically as a cell surface and an intercellular protein (Müller, ’97). The existence of
collagen in marine and freshwater sponges first
was proven electron microscopically (Diehl-Seifert
et al., ’85; Garrone, ’85); later the corresponding
genes were cloned, a cDNA encoding a short-chain
collagen (Exposito and Garrone, ’90), and recently
a cDNA for collagen type IV, characteristic for
basal laminae (Boute et al., ’96).
Until now, no cDNAs encoding extracellular
matrix adhesion molecules (ECM) from sponges
have been published. Three lines of evidence suggest that sponges contain one major ECM protein, fibronectin. In 1981 Labat-Robert et al.
described that sponges contain a protein that
cross-reacts with antibodies raised against vertebrate fibronectin. Furthermore, the finding of
a cDNA from G. cydonium, which contains a putative RGD domain as well as the finding that G.
cydonium is provided with a cDNA for α subunit
of integrin, prompted us to search for fibronectin.
Until now a cDNA encoding a fibronectin-related
protein has been cloned only from Caenorhabditis
elegans [Nematoda] as the lowest metazoan phylum (EMBL accession number P46567).
In this study we report the isolation of a cDNA
from the marine sponge G. cydonium encoding a
protein consisting of three putative modules; a
fibronectin module type-III (FN3) (Bork et al., ’96),
a scavenger receptor cysteine-rich (SRCR) unit of
group B (Resnik et al., ’94), and a complement control protein module also termed short consensus
repeat (SCR) (Zipfel and Skerka, ’94). This whole
sponge protein is named “multiadhesive protein”.
Restriction endonucleases and other enzymes
for recombinant DNA techniques and vectors were
obtained from Stratagene (La Jolla, CA), QIAGEN
(Hilden; Germany), Boehringer (Mannheim; Germany), USB (Cleveland, OH), Amersham (Buck-
inghamshire, UK), and Promega (Madison, WI).
The monoclonal anti-fibronectin antibody [F0791], the secondary antibody, and the human
fibronectin [F-2518] were obtained from Sigma
(Deisenhofen, Germany).
Live specimens of Geodia cydonium (Porifera,
Demospongiae, Tetractinomorpha, Astrophorida,
Geodiidae) were collected near Rovinj (Croatia).
Immediately after collection from a depth of 25
m at 16°C, they were frozen in liquid nitrogen
until use.
Sponge tissue was homogenized in 1% Triton X100, 50 mM Na-phosphate [pH 11], centrifuged, neutralized, and the particle-free extract (Hayashi et
al., ’80) was analyzed for the presence of fibronectin.
PCR cloning of the putative sponge
MAP_GEOCY protein
Several consecutive rounds of PCR were employed to clone the complete cDNA of the “multiadhesive protein” MAP_GEOCY from G. cydonium
cDNA library in lambda ZAPII (Pfeifer et al., ’93).
The degenerate reverse (antisense) primer GSRCR-rd: 5´-(AG)TG(TC)A(AG)GCA(GA)CTG(AG)(AG)TGT-3´, designed toward the conserved region
of the SRCR domain in the sequence of group B
SRCR_GEOCY (Pancer et al., ’97b), was used in
conjunction with the vector-specific 5´-end primer
T3 (Stratagene) for PCR amplification from the
cDNA library. PCR reaction mixtures of 50 µl included 0.2 µM of GSRCR-rd and 0.2 µM of T3,
200 µM of each nucleotide, 1.5 µl of the cDNA library (approximately 109 pfu) and 2.5 units of Taq
DNA polymerase (Boehringer). PCR amplifications
were run on a GeneAmp 9600 thermal cycler
(Perkin Elmer) with the following cycling parameters: an initial denaturation of 3 min at 95°C,
then 35 cycles at 95°C for 45 sec, 57°C for 1 min,
74°C for 2 min, and a final extension step at 74°C
for 10 min. The amplification products were electrophoresed through 1% agarose gel and a 1.1
kb band was picked by a Pasteur pipette directly from the ethidum bromide-stained gel.
DNA was eluted from the agar plug in 20 µl
water, then 1 µl was PCR amplified again as
described above. The amplification products
were purified through a QIAquick Spin column
(QIAGEN), cloned into pGEM-T (Promega) and
one clone was sequenced.
Based upon the sequence of the final clone, forward (sense) primers corresponding to the nt positions in the final sequence at 603-622 and at
865-864, as well as reverse primers at nt 16461666 and at 1738-1758, were designed to complete the cDNA.
DNA sequencing was performed with an automatic DNA sequenator [Li-Cor 4000S].
Sequence analysis
Analyses of the sequence composition, structure,
and features were performed by the computer programs PC/GENE (’95). Homology searches and sequence retrieval were done via the E-mail servers
at the European Bioinformatics Institute, Hinxton Hall, UK ( and FASTA@, and the National Center for Biotechnology Information, National Institutes of
Health, MD ( Phylogenetic trees were constructed on the basis of aa sequence alignment by neighbour-joining, applying
the “Neighbor” program from the PHYLIP package (Felsenstein, ’93). The distance matrix was
calculated as described (Dayhoff et al., ’78). The
degree of support for internal branches further
was assessed by bootstrapping (Felsenstein, ’93).
The distance matrix was calculated as described
(Dayhoff et al., ’78). The graphic presentation was
composed with GeneDoc (Nicholas and Nicholas,
’96). The percentage aa similarity was calculated
by comparing aa’s of similar physico-chemical
properties including also identical aa’s (PC/GENEPhyschem, ’95).
acted with anti-fibronectin antibody. As seen in Fig.
1 (lane b), two main bands in the G. cydonium
sample with a size of ≈230 and ≈210 kDa could be
visualized by Western blotting; minor bands—Mr
75 kDa and 21 kDa—also are present, which are
apparently degradation products. A sample of human fibronectin was analyzed in parallel (lane a)
and found to display a similar protein staining pattern. In a control, the antibody was adsorbed with
purified fibronectin; this preparation did not react
with any protein on the membrane (not shown).
This result suggests that the interaction between
the anti-fibronectin antibody and the sponge
protein(s) is a specific one.
Cloning of the “multiadhesive protein”
from G. cydonium
In a first approach, the above mentioned antibody directed against the FN3 domain of fibronectin was used to identify the cDNA, encoding
sponge fibronectin, in an expression library from
G. cydonium. This was not successful, perhaps
due to cross-reactivity of the antibody with the
sponge lectin.
Therefore, we employed degenerate primers, designed against the conserved region of an SRCR
unit. This approach resulted in the detection of a
cDNA encoding a “multiadhesive protein”. Three
independent clones of 2,305 bp were isolated and
Western blot
Gel electrophoresis of the protein extracts was
performed in 5 to 15% gradient polyacrylamide
gel containing 0.1% NaDodSO4 (PAGE), according to Laemmli (’70). Semi-dry electrotransfer
was performed according to Kyhse-Andersen
(’84) onto PVDF-Immobilon. Membranes were
processed and incubated with anti-fibronectin
antiserum. The immune complexes were visualized by incubation with anti-mouse IgG (biotin
conjugated; dilution 1:100), followed by staining
with streptavidin-alkaline phosphatase conjugate/4-chloro-1-naphthol.
Identification of fibronectin in
tissue of G. cydonium
For the identification of fibronectin in the crude
extract from tissue of G. cydonium, the material
was size separated, blotted, and the proteins re-
Fig. 1. Testing for the presence of fibronectin in extract
from G. cydonium. Sponge tissue was solubilized and the particle-free extract was analyzed by gradient NaDodSO4-PAGE,
size separated, and analyzed by Western blotting using the
anti-fibronectin antiserum; 40 µg of protein was applied (lane
b). In parallel, a sample of human fibronectin was analyzed
(lane a).
termed GCMAP. Northern blot analysis with
GCMAP as a probe revealed a single band of 2.3
kb, confirming that the complete cDNA has been
cloned (data not shown). The open reading frame
in GCMAP is 2,103 bp long and predicts a 701 aa
long protein, MAP_GEOCY. The deduced Mr is
76,914 with a pI of 4.98 (PC/GENE-Physchem,
’95), Fig. 2. One typical polyadenylation signal site
AATAAA (Zarkower et al., ’86) is present from nt
2,230 to 2,235. No membrane associated helix has
been found (PC/GENE-Helixmem, ’95).
The deduced protein, MAP_GEOCY, consists of
the following three modules: Fibronectin (aa 254342) - SRCR unit (aa 343-443) - SCR module (aa
462-515); (Fig. 2).
Fibronectin module
The fibronectin module (aa 254-342) comprises the characteristic topology and aa found
in fibronectin type-III (FN3) elements (Bork et
al., ’96). As marked in the alignment (Fig. 3A)
the sequences from metazoan FN3 modules of
human (FINC_HUMAN), Drosophila melanogaster (FS21_DROME), Caenorhabditis elegans
(SR13_CAEEL) and G. cydonium (MAP_GEOCY)
have the consensus hydrophobic and aromatic
residues W, L and A (furthermore, the aa V and
I, preceding the aa W by 12 and 2 residues, are
conserved in most FN3 modules), and in addition the aa residues P and S; highlighted in Fig.
3A. The hydrophobic and aromatic aa are required to stabilize two β-sheets because no cysteine residues are present to form a disulphide
bridge (Bork et al., ’96).
FN3 modules are characterized by: (i) their typical β-sheet arrangement as well as by (ii) their
hydrophobic stretches within this unit (Bork et
al., ’96). The FN3 domain fold of the human and
the sponge sequence display the seven β strands,
as well as with the conserved aa at the same positions (not shown).
The distribution of the hydrophobicities within
the α helices, beta sheets, and turns were predicted (Novotny and Auffray, ’84; Fig. 4). A comparison between the human and sponge FN3
module clearly shows high similarity with respect to the conserved aa positions at corresponding sites.
No RGD-containing cell binding domain to
integrins (Ruoslahti, ’96) is present in FN3 module or in the whole MAP_GEOCY protein. Likewise, the second recognition site for integrins, the
peptide EILDV (Guan and Hynes, ’90), is absent.
However, the sequence KILDA, with the conserved
core aa residues and related flanking aa, is found
at the N-terminus of MAP_GEOCY (Fig. 2).
Phylogenetic analysis
FN3 modules have been primarily described in
Metazoa; in addition they are present in a related
sequence, perhaps due to horizontal gene transfer, in extracellular glycohydrolases from soil bacteria (Bork and Doolittle, ’92). Therefore, we have
aligned sequences according to the highest scores
found by homology searches using BLITZ and
FASTA systems, between the sponge FN3 and the
corresponding modules of Metazoa—deuterostomes, protostomes, pseudocoelomate—and one
The phylogenetic tree, with the bacterium
Thermoanaerobacter saccharolyticum as the outgroup composed from the deduced aa sequences
of FN3 modules, revealed that the FN3-related sequences from the sponge (G. cydonium) and the
pseudocoelomate (C. elegans) diverge earlier than
those from the deuterostomes (human and rat)
and the protostomes (D. melanogaster and Schistocerca americana) (Fig. 3B).
SRCR module
The scavenger receptor cysteine-rich (SRCR)
module is found primarily in receptor proteins
(Resnick et al., ’94). Molecules, featuring this domain comprise a superfamily, which includes one
invertebrate and several vertebrate protein members (reviewed by Resnick et al., ’94). The SRCR
domain consists of an approximately 100 aa-residue motif with conserved spacings of six to eight
cysteines, which apparently participate in intradomain disulphide bonds. They are classified according to the number of cysteines into two groups;
group A contains six C, while most of those in
group B possess eight C.
Most of the SRCR-containing proteins have been
described from vertebrates. Until now, only two
molecules from invertebrates were identified as
members of this superfamily, the sea urchin
(Strongylocentrotus purpuratus) speract [sperm
egg-peptide receptor] (Dangott et al., ’89), and the
scavenger-receptor found in G. cydonium (Pancer
et al., ’97b). The scavenger receptor from the sea
lamprey Pteromyzon marinus represents a member of the lowest vertebrate taxa (Mayer and
Tichy, ’95). All these SRCR-proteins belong to
group A (Pancer et al., ’97b).
The novel “multiadhesive protein” described
herein, MAP_GEOCY, contains one SRCR module spanning from aa 343 to 443 (Fig. 2). It is the
Fig. 2. Nucleotide sequence of G. cydonium cDNA,
GCMAP, and its deduced aa sequence, MAP_GEOCY. The
start ATG as well as the stop TAA nt triplets are underlined;
the potential polyadenylation signal site is double underlined.
The aa sequence is numbered beginning at the N-terminal
residue of the mature protein; the nt sequence starts with
+1, beginning with the open reading frame. The location of
the first degenerate reverse primer GSRCR-rd, designed to-
ward the conserved region of the SRCR domain in the sequence of group A SRCR_GEOCY (Pancer et al., ’97b) used
for the detection of the cDNA GCMAP in the library, is indicated as superscript and double underlined. The following
three modules: Fibronectin ([•FN3 :::: •FN3]) - SRCR unit
([•SRCR .... SRCR•]) - SCR module ([•SCR ~~~~ SCR•]) as
well as the putative recognition site for the integrin receptor
[•aa••aa•] are marked.
Fig. 3. Relationships of the FN3 module in the deduced
aa sequence MAP_GEOCY from G. cydonium. A. Alignment
of the aa sequences, comprising the fibronectin type III domain from human (FINC_HUMAN, accession no. P02751;
module 10 [coded by nt 1447-1723 of the sequence]), Drosophila melanogaster (FS21_DROME, P34082; module 1 [544754]), Caenorhabditis elegans (SR13_CAEEL, Z46343, one
module [14,114-14,363]), and the FN 3 module from G.
cydonium (MAP_GEOCY, [760-1027]). Residues conserved in
all sequences are shown in inverted type; those present in
at least two sequences are shaded. The conserved aa are
marked [•]. B. Unrooted phylogenetic tree composed from
the deduced aa sequences of FN3 modules found in I. Meta-
zoa, from the deuterostomes human (FINC_HUMAN) and
rat (FINC_RAT, P04937; module 6 [coded by nt 1085-1340
of the sequence]), from the protostomes FN 3 from D.
melanogaster (FS21_DROME), and the arthropode Schistocerca americana (FAS2_SCHAM, P22648; module one [548815]), the pseudocoelomate Caenorhabditis elegans
(SR13_CAEEL) and the sponge G. cydonium (MAP_GEOCY),
as well as the II. Bacterium Thermoanaerobacter saccharolyticum (APU_THESA, P36905; module one [735-999]). The
analysis was performed by neighbour-joining as described
under “Materials and Methods”; the bacterial sequence was
used as outgroup. Scale bar indicates an evolutionary distance of 0.1 aa substitutions per position in the sequence.
first invertebrate molecule displaying the cysteine
pattern for group B SRCR-receptors, eight C aa
residues [C1 to C8], with the characteristic spacing (Fig. 5A). As an example, this group B module is compared with the SRCR repeat nine,
belonging to group A, from the SRCR receptor of
G. cydonium published earlier (Pancer et al., ’97b;
Fig. 5A). The latter molecule lacks C1 and C4 and
displays in consequence longer stretches between
the cysteine residues.
CD6 antigen (Aruffo et al., ’91), the human CD5
surface glycoprotein (Jones et al., ’86), as well as
the human M130 antigen (Law et al., ’93), belong
to the group B scavenger receptors. SRCR modules from these sequences were aligned (Fig. 5B).
The percentage of identity [similarity] of the
sponge module MAP_GEOCY and the mammalian is highest for M130 with 44% [63%] and WC1
with 40% [50%] and lower for CD5 25% [32%] and
CD6 25% [36%].
The phylogenetic analysis, which is statistically
robust, shows that the sponge MAP_GEOCY
scavenger module again branches off first, while
the mammalian macrophage antigen M130 and the
Phylogenetic analysis
The mammalian WC1 surface antigens, e.g.
from bovine (Wijngaard et al., ’92), the human
Fig. 4. Comparison of the hydropathy plots of the derived
aa sequences for the human [top] and sponge FN3 module
[bottom]. Residues above the dotted lines represent hydrophobic residues; those below the lines are hydrophilic. Segments of nine residues were analyzed according to the
program of Novotny and Auffray (’84).
antigen WC1, expressed on gamma/delta T lymphocytes, as well as those of the CD6 and the CD5
antigens of lymphocytes, appear later (Fig. 5C).
SCR module
The short consensus repeats [SCR]-containing
proteins are modules that are abundant in complement regulatory proteins and in selectins (Pigott
and Power, ’93). However, in contrast to the
selectins that comprise six cysteine residues in
their domains, the complement proteins have only
four cysteines (Ahearn and Fearon, ’89). With respect to the latter group, the SCR repeats have
11 to 14 conserved aa residues, among them four
conserved cysteine residues have been classified
into four types (Nonaka et al., ’93): type I shows
the consensus aa pattern Cx2Gx5(G)...Cx3GWx3PxC, type II Cx2(G)x4Gx5Cx3GWx4PxC, type III
Cx 2(G)x4(G)x 5Cx 2GxWx nPxC and type IV Cx 2(G)...Cx2Gx4PxC.
In the sponge MAP_GEOCY protein, the SCR
module ranges from aa 462 to 515 (Fig. 2). It comprises the characteristic signature found in type
III SCR modules, which reads CNNGYVLVGSQRRICTATGAWSGEEPEC, (aa 488 to 515;
the conserved aa are underlined). The predicted
secondary structure of the SCRs displays short
segments of β-strands held together by two
disulphide bridges between C#461 and C#502 and
C#488 and C#515, spanning between β-strand 1 and
3, as well as 2 and 4, respectively (Bork et al.,
’96); Fig. 6A.
The following members of the type III SCRs
have been aligned with the sponge sequence:
human beta-2-glycoprotein I precursor; mouse
complement receptor type; D. melanogaster locomotion-related protein; Limulus clotting factor;
and host range protein precursor from the vaccinia virus strain LC16MO (Fig. 6A). The degree
of aa identity [similarity] to the selected SCRs varied between 30–33% [46–51%].
The phylogenetic tree built from the selected
SCRs revealed that the sponge SCR module is the
first branch. The two related SCRs from mammals, mouse complement receptor and human
beta-2-glycoprotein I precursor, and the two invertebrate sequences from the locomotion-related
protein of D. melanogaster and the Limulus clotting factor, appear later (Fig. 6B). The SCR from
the vaccinia virus is grouped together with the
mammalian sequences in one branch.
The isolation of cDNAs encoding structuraland regulatory proteins indicates that Porifera,
the lowest metazoan phylum, contain molecules
that, until recently, were considered to be constituent elements of higher Eu-Metazoa. Besides
adhesion molecules and -receptors (see Introduction), cDNAs encoding eumetazoan transcription
factors (Scheffer et al., ’97) or light sensory organs (Krasko et al., ’97) also have been isolated
from sponges, primarily from G. cydonium.
Based on these findings, it might be concluded
that all Metazoa are of monophyletic origin
(Gamulin et al., ’94; Müller, ’95, ’98) as suggested
by Morris (’93).
Now with this report on the existence of a
“multiadhesive protein”, comprising domains characteristic for the ECM (fibronectin) and for vertebrate cell surface receptors (SRCR-containing
proteins), as well as for cell surface antigens,
complement control moldules (SCR), a further
molecule typical for Metazoa is described.
Fibronectins are high molecular weight glycoproteins present in most ECM and also blood
plasma (Hynes, ’90). This class of adhesive ma-
Fig. 5. Relationships of the SRCR modules in MAP_GEOCY.
A. Alignment of the SRCR module from the G. cydonium
MAP_GEOCY protein, belonging to group B of the scavenger molecules, with the ninth repeat of the scavenger receptor of the same animal, SRCR_GEOCY (Pancer et al., ’97b;
aa segment 937-1036), belonging to the group A scavengers.
The positions of the cysteine residue (C1 through C8), as
well as the number of aa between these sites, are indicated.
B. Alignment of the group B SRCR modules from the human CD6 antigen (CD6_HUMAN, P30203; first module - aa
44-147), human CD5 surface glycoprotein (CD5_HUMAN,
P06127; first module - 275-368), human M130 antigen
(M130_HUMAN, Z22968; first module - 41-138) and bovine
WC1 surface antigen (WC1_HUMAN, P30205; first module
- aa 29-123) with the corresponding module from the sponge
MAP_GEOCY protein. Residues conserved among all five
sequences are shown in inverted type, while residues conserved in at least three of the sequences are shaded. The
consensus sequence is shown above the lines with totally
conserved aa in capital letters and partial conserved aa in
small letters. The location of six Cys residues characteristic
of group B is marked [•] and numbered. C. Phylogenetic
tree computed from the six scavenger molecules of group B
listed in B. The analysis was performed by neighbour-joining as described under “Materials and Methods”. The numbers at the nodes refer to the level of confidence as
determined by bootstrap analysis [1000 bootstrap replicates]. Scale bar indicates an evolutionary distance of 0.1
aa substitutions per position in the sequence.
Fig. 6. Relationships of the SCR module in MAP_GEOCY.
A. Alignment of the SCR module from MAP_GEOCY with
the corresponding type III SCRs from human beta-2-glycoprotein I precursor, activated protein C-binding protein
(APOH_HUMAN, P02749; aa 79-139), mouse CR2 complement receptor type 2 precursor (CR2_MOUSE, P19070; 401457), D. melanogaster locomotion-related protein (HIG_DROME,
Q09101; 814-890), Limulus clotting factor C precursor from
Tachypleus tridentatus (LFC_TACTR, P28175; 182-254), the
host range protein precursor from the vaccinia virus strain
LC16MO (VB05_VACCO, P24084; 140-240). The conserved
aa and the consensus are specified. The aa indicative for
type III SCR modules are marked [.]. The two predicted
disulphide bonds are inserted. B. Phylogenetic tree constructed from the six SCR modules, shown in A. The analysis was performed by neighbour-joining; the numbers at the
nodes refer to the level of confidence as determined by bootstrap analysis. Scale bar refers to the evolutionary distance.
trix proteins is subdivided into several types,
each composed of repeating modules of types I,
II and III (survey: Kreis and Vale, ’93). A typical
fibronectin molecule consists of >10 type I repeats, ≈2 type II repeats, and >15 type III (FN3)
modules (Hynes, ’90). In contrast to type I and
II modules, type III (FN3) modules contain no
disulphide bond. The “multiadhesive protein”
described here contains one FN3, which shows
all characteristic features of corresponding domains from members of higher metazoan phyla;
e.g. conserved aa, secondary structure, and distribution pattern of hydrophobic aa residues. Two
main binding sites to integrin receptors exist in
fibronectin molecules of higher animals, the tripeptide RGD and the pentapeptide EILDV (Guan
and Hynes, ’90). Only the latter one is found in
the sponge sequence as a related site, KILDA,
in the N-terminal region. The functional importance of this site is not yet known.
Most interestingly, the phylogenetic analyses
show that the sponge sequence forms with the C.
elegans molecule the basal lineage of the metazoan tree of FN3 elements. The presented finding
supports earlier immunochemical data on the
presence of fibronectin-like molecules in the freshwater sponge Ephydatia muelleri (Labat-Robert
et al., ’81). Applying the same approach, molecules
of Mr 230,000 and 210,000 have been identified
in extract from G. cydonium that cross-react with
anti-fibronectin antibodies (against human fibronectin), suggesting that fibronectin molecules
might be discovered in sponges that comprise a
more complex composition with respect to fibronectin modules than that in MAP_GEOCY. It
must be stressed that until now only one putative fibronectin module has been identified on the
level of cDNA, which cannot account for the large
fibronectin molecules, identified by the immunological technique.
Molecules comprising SRCR-repeats are widely
found on the cell surface of mammalian macrophages or lymphocyes, which, if known, bind to diverse ligands, lectins, or other cells (reviewed in:
Resnick et al., ’94). They are classified into groups
A and B, according to the number of conserved
aa C residues. Until recently, only two molecules
had been described from invertebrates, which be-
long to group A SRCR molecules: the speract
receptor from the sea urchin (S. purpuratus)
(Dangott et al., ’89), and a related molecule from
G. cydonium (Pancer et al., ’97b). Now it is reported that the protein MAP_GEOCY contains an
SRCR module typical for group B of those receptors. Molecules of that group, e.g. the WC1-,
M130-, CD6-, and CD5 antigen, are likely to be
involved in immunological reactions, e.g. WC1 in
mixed lymphocyte reactions (Okragly et al., ’95),
CD6 in antigen-specific cytotoxicity (Pauly et al.,
’95) or CD5 in hyperresponsivity of T lymphocytes
(Tarakhovsky et al., ’95).
Until now, only a few effector molecules, such
as hemolin in insects (Kanost and Zhao, ’96) or
the immunoglobulin-like molecule in tunicates
(Pancer et al., ’95) have been identified in invertebrates that might be involved in immune reactions. With the demonstration that SRCR modules
of group B exist already in the lowest invertebrates, the sponges, it is apparent that basic elements of cell-mediated immune molecules in
vertebrates have their roots in invertebrates. This
conclusion also is supported by the phylogenetic
tree demonstrating that the SCRC module of
MAP_GEOCY forms a possibly basal lineage for
the modules of higher Metazoa.
The finding that the complement control protein
module, also termed short consensus repeat (SCR)
or Sushi domain (Zipfel and Skerka, ’94), already
exists in a sponge molecule was unexpected. Among
invertebrates, a cDNA encoding a homolog of a
complement C3/C4 factor has been identified in sea
urchins (Smith et al., ’96). The sponge module belongs to type III of the SCR repeats, which are dominant modules in the complement receptor of type
1, -type 2, factor H but also in a few non-complement proteins, e.g. β2-glycoprotein I (reviewed by
Reid and Day, ’89). It is interesting that the SCR
module from vaccinia virus strain LC16MO displays
high homology to mammalian SCRs, suggesting
horizontal gene transfer from host to the virus
(Bishop, ’81).
Since there is no experimental evidence for a
complement system in invertebrates, it is not possible to speculate on respective functions in
sponges. However, such repeats also are found in
the initial activator of the clotting pathway in
horseshoe crabs, triggered by lipopolysaccharide,
the factor C (Nakamura et al., ’86). This molecule
shows similarities to the complement system in
higher mammalians (Smith, ’96). After knowing
these relationships and similarities, it was subsequently expected that the SCR repeat from the
G. cydonium MAP_GEOCY represents in the phylogenetic tree the base of the type III of the SCRs.
From homologous domains found in proteins of
vertebrates it is established that: (i) the FN3 module plays a role in dimerisation, e.g. between two
FN3 modules or two receptors (Walter et al., ’95);
(ii) the SRCR module mediates receptor-receptor
binding (Resnick et al., ’94); and (iii) the SCR
module promotes binding of natural or foreign
ligands (Lowell et al., ’89). Hence, it is reasonable to assume also that the sponge protein is an
adhesion molecule provided with prototypes of
metazoan binding modules. Future studies must
show whether the sponge MAP_GEOCY “multiadhesive protein” is expressed only as a soluble protein, as predicted here, or if this molecule is one
splice form of a gene also encoding a receptor-like
polypetide, as seen earlier for the group A SRCR
molecule from the same animal (Pancer et al., ’97b).
We thank Drs. Timpl and Mann, MPI für
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