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Cloning and Characterization of Integrin Subunits from the Solitary Ascidian, Halocynthia roretzi¹

Seita Miyazawa^*, Kaoru Azumi and Masaru Nonaka^2,*

^* Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan; and Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent molecular and biochemical analysis has revealed the presence of an opsonic complement system in the solitary ascidian, Halocynthia roretzi, composed of at least C3, two mannan binding protein-associated serine proteases, and factor B. To elucidate further the structure and function of this apparently primitive complement system in the urochordates, we looked for the ascidian complement receptor type 3 (CR3), or type 4 (CR4), which are members of the leukocyte integrin family in mammals. Using degenerate primers, we isolated two integrin subunits (_Hr1 and _Hr2) from the hemocyte mRNA of H. roretzi, by RT-PCR, and the entire coding sequence of _Hr1 was determined from cDNA clones. _Hr1 contains an I domain, the inserted domain characteristic of a subset of mammalian subunits, including the leukocyte integrin family. A phylogenetic tree constructed for the subunits also supports the ancestral position of _Hr1 in the monophyletic cluster of I domain-containing integrins. The _Hr1 gene shows hemocyte-specific expression on Northern blot analysis. Western blot analysis and immunocytochemical staining of the hemocytes of H. roretzi using anti-_Hr1 Ab showed that _Hr1 subunits exist on the surface of a subpopulation of phagocytic hemocytes. Furthermore, anti-_Hr1 Ab inhibited C3-dependent phagocytosis, but not basic phagocytosis, of yeast cells by ascidian hemocytes. These observations strongly suggest that _Hr1 constitutes an integrin molecule on the hemocytes of H. roretzi that functions as an ancestral form of CR3 and CR4 and mediates phagocytosis in the primitive complement system of the ascidian.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Integrins are a family of cell surface receptors that have diverse functions in cell-cell and cell-extracellular matrix interactions (1). In vertebrates, at least 20 different integrins function in both developmental and immunological processes, such as embryonic morphogenesis, regulation of cell proliferation and differentiation, leukocyte migration, and complement receptor-mediated phagocytosis. All integrins are heterodimers composed of a noncovalently associated pair of and subunits. Each subunit has a large extracellular domain that forms a globular head and stalk, a transmembrane region, and a short cytoplasmic tail.

Because of their involvement in many biologically important adhesion processes, integrins are conserved across a wide range of multicellular animals. In invertebrates, integrins have been identified to date from six phyla: Porifera, Cnidaria, Nematoda, Arthropoda, Mollusca, and Echinodermata (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). These invertebrate integrins have been most effectively characterized in their morphogenetic context (7, 8, 10, 12). There are no data to suggest any immunological functions for the invertebrate integrins, although one subunit was isolated from crayfish hemocytes (9).

The recent discovery of complement components from the sea urchin and the ascidian demonstrated that the complement system, one of the most sophisticated innate immunities, had emerged at an early stage of deuterostome evolution. Molecular analyses have to date identified several components, such as C3 and factor B (Bf)³ from the sea urchin (15, 16, 17) and the ascidian (18, 19), and mannan binding protein-associated serine proteases (MASPs) from the ascidian (20). These findings suggest that the primitive complement system has activating pathways similar to mammalian alternative and lectin pathways (21). On the other hand, complement receptors and terminal components, both of which are important for functional activities, have not yet been identified in any invertebrates, and the physiological functions of the primitive complement system remain largely undocumented. Our previous study (18) suggested that one physiological role of the ascidian complement system was the enhancement of the phagocytic activities of hemocytes through C3 deposition on invading microbes. In mammals, C3-dependent phagocytosis is mediated by complement receptor type 1 (CR1), type 3 (CR3), and type 4 (CR4) (22). CR3 and CR4 are members of the leukocyte integrin family, each comprised of the common ₂ subunit and a distinct subunit. The subunits of the leukocyte integrins all contain a characteristic inserted domain, the I domain (23). The I domain plays a significant role in the recognition and binding of various ligands, including iC3b, the degradation product of C3, through the metal ion-dependent adhesion site (MIDAS) motif (24, 25, 26). The I domain also exists in the C3-binding complement proteins, C2 (27) and Bf (28), and even in the ancient Bf of the sea urchin (17) and the ascidian (19). Thus, recognition of C3 (or iC3b) via the I domain might be a conserved mechanism in the evolution of the immune system.

The solitary ascidian, Halocynthia roretzi, is one of the most suitable animal species for both structural and biochemical studies of the primitive complement system, because of the ease with which its body fluid is collected and the plethora of previous studies of its hemocytes (29, 30, 31, 32, 33).

In this report, we document the presence of CR3 or CR4 in the primitive complement system of the ascidian. We cloned two integrin subunits from hemocyte cDNA libraries of H. roretzi and characterized them from an immunological viewpoint.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

Solitary ascidians, H. roretzi, were obtained from the Asamushi Marine Biological Station of Tohoku University (Aomori, Japan). Ascidian hemocytes were collected as described previously (31). In brief, coelomic fluid was collected from the coelomic cavity by cutting the tunic matrix without injuring internal organs and was immediately added to an equal volume of EDTA solution (0.56 M NaCl and 2.7 mM EDTA, pH 5.2). After centrifugation, precipitated hemocytes were washed with Ca²⁺- and Mg²⁺-free Herbst’s artificial sea water (F-HASW; 450 mM NaCl, 9.4 mM KCl, 32 mM Na₂SO₄, and 3.2 mM NaHCO₃, pH 7.6).

RNA extraction

Total RNA was isolated from hemocytes and other organs by ultracentrifugation in 5.7 M cesium chloride after homogenization in 4 M guanidinium thiocyanate or using ISOGEN (Nippongene, Toyama, Japan). Poly(A)⁺ RNA was prepared on an oligo(dT)-cellulose column.

RT-PCR amplification of integrin cDNAs

Consensus-degenerate hybrid oligonucleotide primers (34) for PCR amplification of ascidian subunits were designed to a highly conserved region of previously described vertebrate and invertebrate subunits. The forward primer, 5'-GATGGATACAATGATGTTGCTrtnggngcncc-3', corresponds to the amino acid sequence DGYNDVA[VIM]GAP, and the reverse primer, 5'-GTCAGGTGGACGATTTCTTTTraaraancc-3', corresponds to the sequence GFFKRNRPPD. Each primer consists of a short 3' degenerate core region (lower case) and a longer 5' consensus clamp region (upper case). Double-stranded cDNA synthesized from hemocyte total RNA was used as the template for PCR, with 40 cycles of amplification (denaturing, 94°C for 30 s; annealing, 55°C for 30 s; extension, 72°C for 2 min). Two bands of the expected sizes (2 and 1.8 kb) were gel-purified and subcloned into the pCR2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Three and six clones, obtained from each band, respectively, were sequenced using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 377 DNA sequencer (Applied Biosystems). All three clones that had the longer (2-kb) inserts were identical, and the corresponding cDNA was named _Hr1. Two of the shorter (1.8 kb) clones corresponded to a part of _Hr1, and the other four shorter clones represented a second cDNA species, named _Hr2.

Construction and screening of cDNA libraries

Oligo(dT)-primed double-stranded cDNA was synthesized from 5 µg of hemocyte poly(A)⁺ RNA using the TimeSaver cDNA synthesis kit (Amersham Pharmacia Biotech, Aylesbury, U.K.), ligated into ZAPII (Stratagene, La Jolla, CA), and packaged in vitro using MaxPlax (Epicentre Technologies, Madison, WI). A total of 3.5 x 10⁵ plaques of the unamplified library were transferred to Biodyne nylon membranes (Pall, Portsmouth, U.K.). DNA was denatured in 1.5 M NaCl and 0.5 M NaOH, neutralized in 1.5 M NaCl and 0.5 M Tris-HCl (pH 7.5), and rinsed in 0.2 M Tris-HCl (pH 7.5) and 2 x SSC. After cross-linking, membranes were prehybridized in 10x Denhardt’s solution, 1 M NaCl, 50 mM Tris, 10 mM EDTA, 0.1% SDS, and 0.1 mg/ml denatured salmon sperm DNA at 65°C for 30 min. ³²P-labeled probes prepared from EcoRI-digested inserts of the subcloned PCR fragments using the RediPrime kit (Amersham Pharmacia Biotech) were added to the hybridization buffer and incubated at 65°C overnight. Membranes were washed twice in 0.1x SSC and 0.1% SDS at 65°C for 30 min. Positive clones were replated, rescreened, and isolated for in vivo excision to pBluescript plasmids using ExAssist helper phage (Stratagene).

To screen for _Hr2 cDNA, another cDNA library was constructed from 1.8 µg of hemocyte poly(A)⁺ RNA using the SMART PCR cDNA library kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. Individually amplified aliquots of the SMART library were prescreened by PCR amplification using the gene-specific primers for the cDNA fragment of _Hr2. The PCR-positive aliquot of the library representing 2 x 10⁵ plaques was transferred to membranes. Membranes were processed and hybridized with the _Hr2 probe, and positive clones were isolated as described above. Isolated phage clones were conversed to pTriplEx2 plasmids.

DNA sequence analysis

DNA sequence analysis was performed by the dideoxy chain termination method using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and an ABI 377 DNA sequencer (Applied Biosystems).

Northern blot analysis

Total RNA from ascidian hemocytes, mantle, gill, digestive gland, gonad, and hepatopancreas were denatured by glyoxal at 50°C for 1 h and separated electrophoretically on a 1% agarose gel. The gel was blotted onto a Hybond-N⁺ nylon membrane (Amersham Pharmacia Biotech) using 20x SSC, cross-linked with UV light, and baked at 80°C for 2 h. Membranes were prehybridized in 10x Denhardt’s solution, 1 M NaCl, 50 mM Tris, 10 mM EDTA, 0.1% SDS, and 0.1 mg/ml denatured salmon sperm DNA at 65°C for 30 min. The same probes used for screening were added to the hybridization buffer and incubated at 50°C overnight. Membranes were washed twice in 0.2x SSC and 0.1% SDS at 50°C for 30 min.

Preparation of antiserum

The whole extracellular region of _Hr1 (nucleotides -21 to 3777, numbering nucleotides from the first methionine) was expressed as a recombinant protein (_Hr1EC) in the Sf9 insect cell line using the pVL1392 transfer vector (PharMingen, San Diego, CA) and BaculoGold baculovirus DNA (PharMingen). For detection and purification of the expressed recombinant protein, a V5 epitope sequence and a 6x histidine tag sequence were attached to the 3' end of the _Hr1EC insert using pcDNA3.1/V5 vector (Invitrogen). Recombinant _Hr1EC proteins were purified on nickel-nitrilotriacetic acid agarose (Qiagen, Tokyo, Japan) under nonreducing conditions. Anti-_Hr1 serum was prepared in a New Zealand White rabbit by injection of purified recombinant _Hr1EC protein. An IgG fraction was purified from serum using protein A agarose (Bio-Rad, Hercules, CA) and was dialyzed against F-HASW.

Western blot analysis

Hemocytes of H. roretzi were lysed in 1 ml of lysis buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 10 mM EDTA, and 0.1% Triton X-100) containing 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A. The lysate was centrifuged at 500 rpm for 5 min to remove debris, and the supernatant was recentrifuged at 15,000 rpm for 30 min. The pellet (membrane fraction) was subjected to SDS-PAGE using a 4% stacking gel and a 7.5% separating gel. Proteins were electrophoretically transferred onto polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked with 5% skim milk and TBS containing 0.1% Tween 20 (TBST) at room temperature for 1 h and washed three times with TBST. Anti-_Hr1 serum was diluted 1/5,000 in TBST and incubated with the membranes for 2 h. Preimmune rabbit serum and anti-_Hr1 serum preabsorbed with recombinant _Hr1 proteins were used as controls. After washing three times with TBST, membranes were incubated for 1 h with HRP-conjugated donkey anti-rabbit IgG Ab (Amersham Pharmacia Biotech) diluted 1/10,000 in TBST. ECL detection reagents (Amersham Pharmacia Biotech) were used for visualization according to the manufacturer’s instructions.

Immunocytochemistry

Hemocytes were placed on coverslips (3 x 10⁵ cells each), incubated on ice for 10 min to allow cells to attach, then fixed in 10% formalin/F-HASW for 15 min. After washing twice with wash buffer (F-HASW containing 0.1% gelatin and 0.05% Tween 20), coverslips were blocked with 10% normal goat serum in F-HASW for 30 min and washed twice. Coverslips were overlaid with primary antiserum (anti-_Hr1 serum or control preimmune rabbit serum) diluted 1/100 in F-HASW containing 3% normal goat serum and incubated at room temperature for 30 min. After washing three times, coverslips were incubated with Cy3-conjugated goat anti-rabbit IgG (H+L; Jackson ImmunoResearch Laboratories, West Grove, PA; 1/400) for 30 min, washed four times, and then placed onto slides for observation under a fluorescence microscope (Carl Zeiss, Thornwood, NY).

Phagocytosis assay

The body fluid of H. roretzi was freshly collected and centrifuged at 3000 rpm for 5 min to exclude hemocytes. Yeast cells (2 x 10⁷) were incubated with 1 ml of H. roretzi hemocyte-free body fluid or control F-HASW at 20°C for 30 min and washed three times with F-HASW. Freshly collected hemocytes (4 x 10⁵) were resuspended in 20 µl of F-HASW and incubated with 100 µg/ml anti-_Hr1 IgG at 4°C for 1 h. Normal rabbit IgG (100 µg/ml) or F-HASW was used as the control. Hemocytes were then mixed with body fluid-treated or untreated yeast cells (2 x 10⁶) and incubated at 20°C for 30 min. Sodium diethyldithiocarbamate (0.1 M) was added to terminate phagocytosis. Hemocytes were stained with Nile Blue. Those that had ingested one or more yeast cells were counted as positive. The degree of phagocytosis was expressed as the ratio of the number of positive hemocytes to the total number of hemocytes.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of integrin subunits from H. roretzi

Using consensus-degenerate hybrid primers (34) designed to highly conserved amino acid sequences of vertebrate and invertebrate integrin subunits, two DNA fragments of the expected size (2.0 and 1.8 kb) were successfully amplified by RT-PCR from hemocyte total RNA of H. roretzi. Cloning and sequence analysis identified two ascidian integrin genes, termed _Hr1 and _Hr2. The deduced amino acid sequences of these cDNAs showed similarity (40% identity) to vertebrate subunits. H. roretzi hemocyte cDNA libraries were constructed and screened using the cDNA fragments of _Hr1 and _Hr2 as probes. Eleven and two independent clones were isolated, respectively. The clones of _Hr1 and _Hr2 that contained the longest inserts (A1–17 and A2–1, respectively) were sequenced, and one long open reading frame (ORF) was identified in each clone. Comparison of the deduced amino acid sequence of the A1–17 clone with vertebrate and invertebrate subunit sequences indicated that the coding sequence for approximately 200 aa residues at the N-terminus was not included in this clone. Therefore, PCR amplification of the 5' cDNA end was undertaken using the ZAPII cDNA library together with the vector primers and a gene-specific antisense primer designed to the 5' terminal region of the A1–17 clone. Using the amplified PCR product as a probe, the same cDNA library was rescreened. The clone containing the longest insert (A1'-12, 1.7 kb) included the 5' terminal region of _Hr1 and an approximately 350-bp region overlapping the A1–17 clone with complete identity. The combined sequences from clones A1–17 and A1'-12 included an ORF of 3996 bp encoding a novel integrin subunit, _Hr1 (GenBank accession no. AB048261). The ORF is flanked by 62- and 844-bp untranslated regions (UTR) at the 5' and 3' ends, respectively. The 5' UTR contains a termination codon, TAG, at nucleotides -9 to -7. The 3' UTR has a possible polyadenylation signal, AATAAA, at nucleotides 4810–4815, and contains a poly(A) tail. The A2–1 clone has an ORF of 2520 bp (AB048262), but lacks both the 5' and 3' terminal sequences. The complete sequence of _Hr2 has yet to be determined.

Coding sequence analysis of _Hr1 and _Hr2 and comparison with other integrin subunits

The deduced amino acid sequence of _Hr1 consists of 1332 residues. The putative signal peptide of _Hr1 comprises 30 aa, and the m.w. of the mature _Hr1 is calculated to be 142,567. The mature _Hr1 protein has a large extracellular domain of 1,238 aa, a potential transmembrane domain (residues 1,239–1,261), and a cytoplasmic domain of 41 residues. There are 18 potential N-linked glycosylation sites in the extracellular domain of _Hr1. The deduced partial amino acid sequence of _Hr2 consists of 840 residues, probably lacking about 200 N-terminal amino acids and several C-terminal residues. The amino acid sequences of _Hr1 and _Hr2 were aligned with those of the human integrin subunits and of various invertebrate species, using ClustalX, with some corrections made by eye. (Only part of this alignment, containing _Hr1, human _E, _M, _X, _L, and ₁, is shown in Fig. 1). Based on this alignment, a phylogenetic tree was constructed for the subunits by the neighbor-joining method (Fig. 2).

View larger version (97K): [in this window] [in a new window]   FIGURE 1. Multiple alignment of Hr1 and human E (SwissProt Databank accession no. P38570), M (P11215), X (P20702), L (P20701), and 1 (P56199) integrin subunits. The entire amino acid sequences of these subunits were aligned using ClustalX. The signal peptide cleavage site is indicated by an arrowhead. Cysteine residues are shown in bold, and conserved cysteine residues are numbered. The I domains are boxed. The FG-GAP sequences of seven homologous repeats are boxed and numbered, and the three divalent cation binding regions are underlined. The transmembrane region is indicated by a box with TM above it. The GFFKR sequence is indicated (#).

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Phylogenetic tree for Hr1, Hr2, and other human and invertebrate integrin subunits. The unrooted tree was constructed using the neighbor-joining method based on the alignment of amino acid sequences. Numbers indicate the bootstrap percentage (only >50% are shown). I domain-containing subunits are indicated.

Some mammalian subunits have an extra region of approximately 200 aa, known as the I domain (23), which is inserted before the third FG-GAP repeat and contains the MIDAS motif that is important for the recognition of various ligands (24, 25, 26). The I domain-containing human subunits form a cluster in the phylogenetic tree, suggesting that the acquisition of the I domain occurred only once during the evolution of the integrin subunits (37). Ascidian _Hr1 has an I domain of about 209 residues and is located at the base of the clade of I domain-containing human subunits on the neighbor-joining tree (Fig. 2). This is the first report of an I domain-containing integrin from a nonmammalian species, suggesting that the I domain was inserted into the integrins before the divergence of the chordates. Furthermore, the basal location of _Hr1 in the clade of I domain-containing subunits also indicates that the multiplicity of I domain-containing subunits found in humans arose by gene duplications in the vertebrate lineage after the divergence of the urochordates. On the other hand, _Hr2 has no I domain and belongs to the cluster consisting of Drosophila _PS2, Caenorhabditis elegans _F54F2.1, sea urchin _SU2 and _P, and human _V, ₅, ₈, and _IIb (12). The subunits that have no I domains are often post-translationally cleaved to give a 25- to 30-kDa transmembrane peptide disulfide-bonded to a larger extracellular chain (2). A potential proteolytic cleavage site (KRD) was found in _Hr2.

Most of the subunits analyzed to date have a consensus membrane-proximal sequence, GFFKR. Although _Hr2 also retains this sequence, in _Hr1 the arginine residue has been replaced by a serine.

Hemocyte specific expression of _Hr1

Northern blot analysis of total RNA from several tissues of the adult H. roretzi showed highly specific expression of the _Hr1 transcript in hemocytes (Fig. 3). The _Hr1 transcript is about 5.1 kb long, in close agreement with the length of the cDNA sequence. The expression of _Hr2 was not detected in any tissue tested (data not shown), probably implying stage-specific expression of the _Hr2 gene during development or metamorphosis.

View larger version (38K): [in this window] [in a new window]   FIGURE 3. Northern blot analysis of Hr1. Five micrograms of total RNA from each tissue was denatured with glyoxal, separated electrophoretically on a 1% agarose gel, and transferred to nylon membrane. The Hr1 probe hybridized to a hemocyte mRNA of 5.1 kb. The 18S ribosomal RNA bands are presented to show the amount and quality of RNA loaded in each lane.

View larger version (50K): [in this window] [in a new window]   FIGURE 4. Western blot analysis of Hr1. The hemocyte membrane fraction was separated by SDS-PAGE (7.5%) under reducing and nonreducing conditions and transferred to polyvinylidene difluoride membranes. Lane 1, Preimmune rabbit serum; lane 2, anti-Hr1 serum; lane 3, anti-Hr1 serum plus recombinant Hr1EC.

View larger version (135K): [in this window] [in a new window]   FIGURE 5. Immunocytochemical staining of H. roretzi hemocytes. Hemocytes were fixed on a coverslip and stained using anti-Hr1 serum and Cy3-conjugated secondary Ab. The left and right panels show fluorescent and light microscopic views, respectively. A, Fifty to 60% hemocytes were stained with anti-Hr1 serum. B, No hemocytes were stained with preimmune rabbit serum. Bar = 10 µm.

To test the possible contribution of _Hr1 to C3-dependent phagocytic activities of ascidian hemocytes, a phagocytosis assay was performed. In controls, 10–30% of H. roretzi hemocytes ingested at least one untreated yeast cell. When yeast cells were pretreated with ascidian body fluid, the phagocytic activities of hemocytes were enhanced almost 2-fold (Fig. 6). Our previous study showed that this enhancement of phagocytosis was completely abolished by depletion of C3 from the body fluid using anti-ascidian C3 IgG, indicating that this enhanced phagocytosis is C3 dependent (18). In this experiment the C3-dependent enhancement of phagocytosis was also abolished by the addition of anti-_Hr1 IgG, while no significant reduction was observed for basic phagocytosis. These results strongly suggest that _Hr1 constitutes part of an integrin molecule that functions as an ascidian C3 receptor (i.e., the ancestral form of CR3 and CR4) on hemocytes and mediates phagocytic activities.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. Phagocytosis assay of H. roretzi hemocytes. Yeast cells were treated with fresh ascidian body fluid () or control F-HASW (). Hemocytes were incubated with yeast cells after treatment with anti-Hr1 IgG (100 µg/ml), normal rabbit IgG (100 µg/ml), or F-HASW alone. Hemocytes ingesting one or more yeast cells were counted as phagocytosis positive. The degree of phagocytosis of body fluid-untreated yeast cells by IgG-untreated hemocytes was defined as 100%. Data are presented as a mean percentage of the control value ± SEM of five individuals. *, p < 0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The presence of ascidian CR3/CR4 indicates that the ancestors common to the ascidians and the vertebrates had already developed mechanisms for CR-mediated phagocytosis as a central part of the physiological function of their primitive complement systems. In the primitive complement system of ascidians, the activating pathway composed at least of C3 (18), Bf (19), and MASPs (20) has been demonstrated, and corresponds to the mammalian alternative and lectin pathways (21). Thus, the primitive complement system already had both activating and functional mechanisms similar to those of mammals when the chordates emerged 600 million yr ago, well ahead of the emergence of adaptive immunity. Since mannan-binding protein has recently been identified from a different species of ascidian (38), it is likely that H. roretzi also uses mannan binding protein as a recognition molecule. Furthermore, there should be other components, especially regulatory factors, still to be isolated. Therefore, the ascidian complement system could be one of the most complicated innate immune systems analyzed to date in invertebrates.

In the phylogenetic tree constructed for the integrin -chains, _Hr1 clusters with mammalian I domain-containing subunits. The basal location of _Hr1 in that clade suggests that _Hr1 has arisen directly from a common ancestor of the I domain-containing subunits before the multiplication of I domain-containing -chains that resulted in the functional diversity of mammalian leukocyte and other integrins. In the clade of I domain-containing subunits, mammalian _M and _X subunits and the most divergent ascidian _Hr1 show hemocyte-specific expression and mediate C3-dependent phagocytosis. This leads us to infer that the ancestor of I domain-containing integrins originated as a leukocyte-type integrin, with C3-recognizing functions. The vertebrate I domain-containing laminin and collagen receptor group (₁, ₂, ₁₀, ₁₁) probably arose by gene duplication from the leukocyte-type integrin in the vertebrate lineage.

It remains unclear when in the evolutionary process the I domain was inserted into the integrins. Recently published Drosophila (39) and C. elegans (40) genomes appear to have no I domain-containing integrins. The ascidian has now been shown to express an I domain-containing integrin, indicating that the insertion predated the divergence of the ascidians from the vertebrates. The I domain also exists in the vertebrate C3-binding complement protein C2 (27) and Bf (28), and even in the ancient Bf of the sea urchins (17) and the ascidians (19). It is tempting to infer that the recruitment of the I domain played a critical role in establishing the complement system. The emergence of the complement system and the subsequent formation of complement receptors probably reinforced and refined the phagocytic activities of the innate immune system of higher invertebrates. Further study of I domain-containing integrins from other animal species, especially the echinoderms, will give insight into the evolution of the complement system as well as into the evolution of the I domain-containing integrins.

	Acknowledgments

 
We thank Seiichi Tamura, Asamushi Marine Biological Station, Tohoku University, for a continuous supply of fresh ascidians.

	Footnotes

 
¹ This work was supported by Grants-in-Aid 11236205 and 10558089 from the Japanese Ministry of Education, Science, Sports, and Culture (to M.N.).

² Address correspondence and reprint requests to Dr. Masaru Nonaka, Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

³ Abbreviations used in this paper: Bf, factor B; MASP, mannan binding protein-associated serine protease; MIDAS, metal ion-dependent adhesion site; F-HASW, Ca²⁺- and Mg²⁺-free Herbst’s artificial sea water; TBST, TBS containing 0.1% Tween 20; ORF, open reading frame; UTR, untranslated region.

Received for publication September 13, 2000. Accepted for publication November 1, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Hynes, R. O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11.[Medline]
2. Burke, R. D.. 1999. Invertebrate integrins: structure, function, and evolution. Int. Rev. Cytol. 191:257.[Medline]
3. Brower, D. L., S. M. Brower, D. C. Hayward, E. E. Ball. 1997. Molecular evolution of integrins: genes encoding integrin subunits from a coral and a sponge. Proc. Natl. Acad. Sci. USA 94:9182.[Abstract/Free Full Text]
4. Pancer, Z., M. Kruse, I. Muller, W. E. Muller. 1997. On the origin of metazoan adhesion receptors: cloning of integrin subunit from the sponge Geodia cydonium. Mol. Biol. Evol. 14:391.[Abstract]
5. Gettner, S. N., C. Kenyon, L. F. Reichardt. 1995. Characterization of pat-3 heterodimers, a family of essential integrin receptors in C. elegans. J. Cell Biol. 129:1127.[Abstract/Free Full Text]
6. Davids, B. J., X. J. Wu, T. P. Yoshino. 1999. Cloning of a integrin subunit cDNA from an embryonic cell line derived from the freshwater mollusc, Biomphalaria glabrata. Gene 228:213.[Medline]
7. Zusman, S., Y. Grinblat, G. Yee, F. C. Kafatos, R. O. Hynes. 1993. Analyses of PS integrin functions during Drosophila development. Development 118:737.[Abstract]
8. Yee, G. H., R. O. Hynes. 1993. A novel, tissue-specific integrin subunit, , expressed in the midgut of Drosophila melanogaster. Development 118:845.[Abstract]
9. Holmblad, T., P. O. Thornqvist, K. Soderhall, M. W. Johansson. 1997. Identification and cloning of an integrin beta subunit from hemocytes of the freshwater crayfish Pacifastacus leniusculus. J. Exp. Zool. 277:255.[Medline]
10. Brown, N. H.. 1993. Integrins hold Drosophila together. BioEssays 15:383.[Medline]
11. Susan, J. M., M. L. Just, W. J. Lennarz. 2000. Cloning and characterization of P integrin in embryos of the sea urchin Strongylocentrotus purpuratus. Biochem. Biophys. Res. Commun. 272:929.[Medline]
12. Hertzler, P. L., D. R. McClay. 1999. SU2, an epithelial integrin that binds laminin in the sea urchin embryo. Dev. Biol. 207:1.[Medline]
13. Marsden, M., R. D. Burke. 1998. The _L integrin subunit is necessary for gastrulation in sea urchin embryos. Dev. Biol. 203:134.[Medline]
14. Marsden, M., R. D. Burke. 1997. Cloning and characterization of novel integrin subunits from a sea urchin. Dev. Biol. 181:234.[Medline]
15. Smith, L. C., L. Chang, R. J. Britten, E. H. Davidson. 1996. Sea urchin genes expressed in activated coelomocytes are identified by expressed sequence tags: complement homologues and other putative immune response genes suggest immune system homology within the deuterostomes. J. Immunol. 156:593.[Abstract]
16. Al-Sharif, W. Z., J. O. Sunyer, J. D. Lambris, L. C. Smith. 1998. Sea urchin coelomocytes specifically express a homologue of the complement component C3. J. Immunol. 160:2983.[Abstract/Free Full Text]
17. Smith, L. C., C. S. Shih, S. G. Dachenhausen. 1998. Coelomocytes express SpBf, a homologue of factor B, the second component in the sea urchin complement system. J. Immunol. 161:6784.[Abstract/Free Full Text]
18. Nonaka, M., K. Azumi, X. Ji, C. Namikawa-Yamada, M. Sasaki, H. Saiga, A. W. Dodds, H. Sekine, M. K. Homma, M. Matsushita, et al 1999. Opsonic complement component C3 in the solitary ascidian. Halocynthia roretzi. J. Immunol. 162:387.[Abstract/Free Full Text]
19. Ji, X., C. Namikawa-Yamada, M. Nakanishi, M. Sasaki, M. Nonaka. 2000. Molecular cloning of complement factor B from a solitary ascidian: unique combination of domains implicating ancient exon shufflings. Immunopharmacology 49:43.
20. Ji, X., K. Azumi, M. Sasaki, M. Nonaka. 1997. Ancient origin of the complement lectin pathway revealed by molecular cloning of mannan binding protein-associated serine protease from a urochordate, the Japanese ascidian. Halocynthia roretzi. Proc. Natl. Acad. Sci. USA 94:6340.[Abstract/Free Full Text]
21. Smith, L. C., K. Azumi, M. Nonaka. 1999. Complement systems in invertebrates: the ancient alternative and lectin pathways. Immunopharmacology 42:107.[Medline]
22. Ross, G. D.. 1986. Opsonization and membrane complement receptors. G. D. Ross, ed. Immunobiology of the Complement System 87. Academic Press, New York.
23. Dickeson, S. K., S. A. Santoro. 1998. Ligand recognition by the I domain-containing integrins. Cell. Mol. Life. Sci. 54:556.[Medline]
24. Lee, J. O., P. Rieu, M. A. Arnaout, R. Liddington. 1995. Crystal structure of the A domain from the subunit of integrin CR3 (CD11b/CD18). Cell 80:631.[Medline]
25. Diamond, M. S., J. Garcia-Aguilar, J. K. Bickford, A. L. Corbi, T. A. Springer. 1993. The I domain is a major recognition site on the leukocyte integrin Mac-1 (CD11b/CD18) for four distinct adhesion ligands. J. Cell Biol. 120:1031.[Abstract/Free Full Text]
26. Zhang, L., E. F. Plow. 1999. Amino acid sequences within the alpha subunit of integrin _M2 (Mac-1) critical for specific recognition of C3bi. Biochemistry 38:8064.[Medline]
27. Bentley, D. R.. 1986. Primary structure of human complement component C2: homology to two unrelated protein families. Biochem. J. 239:339.[Medline]
28. Campbell, R. D., R. R. Porter. 1983. Molecular cloning and characterization of the gene coding for human complement protein factor B. Proc. Natl. Acad. Sci. USA 80:4464.[Abstract/Free Full Text]
29. Fuke, T. M.. 1979. Studies on the coelomic cells of some Japanese ascidians. Bull. Mar. Biol. St. Asamushi Tohoku Univ. 16:143.
30. Sawada, T., Y. Fujikura, S. Tomonaga, T. Fukumoto. 1991. Classification and characterization of ten hemocytes types in the tunicate Halocynthia roretzi. Zool. Sci. 8:939.
31. Azumi, K., N. Satoh, H. Yokosawa. 1993. Functional and structural characterization of hemocytes of the solitary ascidian. Halocynthia roretzi. J. Exp. Zool. 265:309.
32. Ohtake, S., T. Abe, F. Shishikura, K. Tanaka. 1994. The phagocytes in hemolymph of Halocynthia roretzi and their phagocytic activity. Zool. Sci. 11:681.
33. Dan-Sohkawa, M., M. Morimoto, H. Mishima, H. Kaneko. 1995. Characterization of coelomocytes of the ascidian Halocynthia roretzi based on phase-contrast, time-lapse video and scanning electron microscopic observation. Zool. Sci. 12:289.
34. Rose, T. M., E. R. Schultz, J. G. Henikoff, S. Pietrokovski, C. M. McCallum, S. Henikoff. 1998. Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res. 26:1628.[Abstract/Free Full Text]
35. Springer, T. A.. 1997. Folding of the N-terminal, ligand-binding region of integrin -subunits into a -propeller domain. Proc. Natl. Acad. Sci. USA 94:65.[Abstract/Free Full Text]
36. Oxvig, C., T. A. Springer. 1998. Experimental support for a -propeller domain in integrin -subunits and a calcium binding site on its lower surface. Proc. Natl. Acad. Sci. USA 95:4870.[Abstract/Free Full Text]
37. Hughes, A. L.. 1992. Coevolution of the vertebrate integrin - and -chain genes. Mol. Biol. Evol. 9:216.[Abstract]
38. Nair, S. V., S. Pearce, P. L. Green, D. Mahajan, R. A. Newton, D. A. Raftos. 2000. A collectin-like protein from tunicates. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 125:279.[Medline]
39. Adams, M. D., S. E. Celniker, R. A. Holt, C. A. Evans, J. D. Gocayne, P. G. Amanatides, S. E. Scherer, P. W. Li, R. A. Hoskins, R. F. Galle, et al 2000. The genome sequence of Drosophila melanogaster. Science 287:2185.[Abstract/Free Full Text]
40. The C. elegans Sequencing Consortium. 1998. Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282:2012.[Abstract/Free Full Text]

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