Genome Analysis of the Unicellular Green Alga Chlamydomonas reinhardtii Indicates an Ancient Evolutionary Origin for Key Pattern Recognition and Cell-Signaling Protein Families

Abstract

The evolution of specific cell signaling and adhesion domains may have played an important role in the transition to a multicellular existence in the metazoans. Genomic analysis indicates that several signaling domains predominately found in animals are also present in the unicellular green alga, Chlamydomonas reinhardtii. A large group of proteins is present, containing scavenger receptor cysteine-rich (SRCR) and C-type lectin domains, which function in ligand binding and play key roles in the innate immune system of animals. Chlamydomonas also contains a large family of putative tyrosine kinases, suggesting an important role for phosphotyrosine signaling in the green algae. These important signaling domains may therefore be widespread among eukaryotes and most probably evolved in ancestral eukaryotes before the divergence of the Opisthokonts (the animal and fungal lineage).

THE evolution of multicellularity requires the development of complex signaling mechanisms to regulate intercellular interactions such as tissue differentiation and pathogen defense. Comparative genomics approaches suggest that certain cell-signaling and adhesion protein domains are unique to animals (Arabidopsis Genome Initiative 2000; Hynes and Zhao 2000; Rubin et al. 2000). Many of these protein domains may have evolved as a result of specialization within multicellular animals, although others may have been present early in a unicellular ancestor of the metazoans and contributed to primitive cell–cell interactions. As a result, much interest has focused on the cell-signaling and adhesion proteins present in the unicellular relatives of the metazoa, the choanoflagellates (King et al. 2003, 2008; Steenkamp et al. 2006).

The completion of the genome of the biflagellated chlorophyte alga, Chlamydomonas reinhardtii, represents an important addition to the array of sequenced eukaryotes (Merchant et al. 2007). We present an analysis of the Chlamydomonas genome, indicating that many proteins are present that contain scavenger receptor cysteine-rich (SRCR) domains, C-type lectin domains, and putative tyrosine kinase domains. These protein domains are largely associated with the metazoa (Muller 2001). Chlamydomonas is a member of the chlorophyte algae, which diverged from the land plants >1000 million years ago (MYA), following the earlier divergence of the Opisthokonts, containing the animal and fungal lineages (1600 MYA) (Yoon et al. 2004). Genes shared between chlorophytes and animals were presumably present in ancestral eukaryotes, indicating a truly ancient origin for these cellular processes.

SRCR and C-type lectins:

The SRCR and C-type lectin domains are both important in the recognition of pathogen-associated molecular patterns (PAMPs) by the metazoan innate immune system (Gordon 2002). The SRCR domain is a highly conserved cysteine-rich domain, first defined during the analysis of the type I macrophage scavenger receptor (Freeman et al. 1990), but subsequently identified in many different proteins with diverse functions and domain organizations. The sea urchin, Strongylocentrotus purpuratus, contains an extraordinarily large gene family of SRCR-domain-containing proteins, many of which may function in the immune response (Pancer 2000; Rast et al. 2006). SRCR proteins perform a variety of other cellular functions, including the well-characterized speract receptor in the flagella of sea urchin sperm that mediates chemotactic responses to the egg (Dangott et al. 1989; Cardullo et al. 1994; Resnick et al. 1994). The C-type lectins were initially defined as animal-type lectins displaying Ca²⁺-dependent carbohydrate-binding activity (Drickamer 1988), although the C-type lectin domain (CTLD) has since been identified in many diverse proteins, many of which may not bind carbohydrate or Ca²⁺ (Zelensky and Gready 2005). CTLD proteins such as the mannose receptor (MR) and Dectin-1 have a well-characterized role in the mammalian innate immune systems (Brown and Gordon 2001; East and Isacke 2002; McGreal et al. 2005).

The Chlamydomonas genome contains 22 gene models containing SRCR domains, 4 gene models with CTLDs, and a further 7 gene models that contain both domains (Figure 1 and Table 1). Between 1 and 11 SRCR domains were identified in each protein, in combination with a range of other domains associated with signaling and adhesion such as PAN, WSC, and epidermal growth factor (EGF)-like (supplemental Table T1). The majority of the CTLDs were found within a subgroup of four homologous proteins that each contain 2 SRCR domains in addition to 13–14 CTLDs (SRR15, SRR16, SRR24, and SRR28). These proteins are absent from the closely related alga, Volvox carterii, suggesting their function may be specific to Chlamydomonas. As the Chlamydomonas SRCR domains and CTLDs possess the conserved cysteines required for intradomain disulphide bridge formation, these proteins may be involved in ligand binding and endocytosis as observed in animals. However, 14 of the putative Chlamydomonas proteins exhibit a unique domain organization, suggesting they may perform novel cellular roles. In particular, while metazoan genomes contain many SRCR and CTLD proteins, there are very few examples of proteins containing both domains (supplemental Table T1). Comprehensive experimental characterization of the function and localization of these proteins is required before we can determine their function. In unicellular organisms, cell adhesion domains may function in chemoreception, self/nonself recognition, bacterial interactions, and cell wall attachment (Harwood and Coates 2004; Boehm 2006).

Figure 1.—

SRCR and C-type lectin domains from Chlamydomonas. (A) Multiple-sequence alignment of SRCR domains from Chlamydomonas with group A and group B SRCR domains from metazoans. Cysteines are numbered according to group B nomenclature. Cysteines were not present in positions C1 and C4, suggesting that Chlamydomonas does not possess group B SRCR domains. The sequences of two truncated SRCR domains from Chlamydomonas lacking C2 and C7 are also shown. (B) Multiple-sequence alignment of Chlamydomonas CTLDs displaying mannose (EPN) and galactose (QPD) binding motifs (boxed). The presence of these conserved motifs is a reliable prediction of Ca²⁺-dependent carbohydrate-binding activity, although there are several exceptions (Zelensky and Gready 2005). Conserved cysteine residues are also displayed (C1–C4). Sequence analysis of the CTLDs in the predicted Chlamydomonas proteins indicates that 18 CTLDs possess conserved amino acid motifs associated with Ca²⁺-dependent carbohydrate binding.

TABLE 1.

The Chlamydomonas genome contains multiple protein domains associated with important signaling and adhesion roles in animals

Organism	Taxonomic group	Kingdoma	SRCR	CTLD	TKb
Chlamydomonas reinhardtii	Chlorophyta	Plantae	29 (74)	11 (67)	28
Ostreococcus tauri	Chlorophyta	Plantae	0 (0)	0 (0)	2
Arabidopsis thaliana	Streptophyta	Plantae	0 (0)	2 (2)	2
Cyanidioschyzon merolae	Rhodophyta	Plantae	0 (0)	0 (0)	0
Thalassiosira pseudonana	Bacillariophyta	Chromalveolata	0 (0)	0 (0)	0
Homo sapiens	Metazoa	Opisthokonta	16 (81)c	78 (115)	93
Caenorhabditis elegans	Metazoa	Opisthokonta	1 (3)c	128 (183)d	94

A comparison of the protein families containing scavenger receptor cysteine-rich (SRCR) domains, C-type lectin domains (CTLDs), and putative tyrosine kinase (TK) domains in green algae (Chlamydomonas reinhardtii, Ostreococcus tauri), land plants (Arabidopsis thaliana), red algae (Cyanidioschyzon merolae), diatoms (Thalassiosira pseudonana), and animals (Homo sapiens, C. elegans) is shown. Protein number is displayed with the total number of domains in parentheses. SRCR domains and CTLDs were identified in plant and algal genomes, using a combination of BLAST searches and hits to the Superfamily, Pfam, and SMART databases. TKs were identified using a multilevel HMM library for kinase classification (Miranda-Saavedra and Barton 2007). Full details are provided in the supplemental information.

^aSimpson and Roger (2004).

^bMiranda-Saavedra and Barton (2007).

^cRast et al. (2006).

^dDrickamer and Dodd (1999).

Examination of the distribution of SRCR domain proteins among plant and algal genomes revealed that they are absent from the genomes of the land plants (including the moss Physcomitrella patens), the marine chlorophyte algae Ostreococcus tauri and O. lucimarinus, the red alga Cyanidioschyzon merolae, the Oomycetes Phytophora ramorum and P. sojae, and the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana, although SRCR proteins are present in Volvox. The SRCR domain may have been associated with an ancestral cellular function that has been repeatedly lost in these lineages. The CTLDs exhibit a wider distribution, being absent from the Ostreococcus and diatom genomes but present in low numbers in multicellular plants (Arabidopsis, Oryza sativa, and the moss Physcomitrella) (Zelensky and Gready 2005). CTLDs have also been described in a number of proteins from parasites and viruses, although it is likely that these CTLDs arose by lateral gene transfer from the host and we will not include them in our further analysis of the evolution of this domain. The presence of SRCR domains and CTLDs in the chlorophyte algae suggests that these important protein domains evolved in ancestral eukaryotes, before the divergence of the Opisthokont lineage. As the eukaryotes diverged, these signaling domains may have been repeatedly lost or become divergent beyond recognition. The functional diversity of both domains results from a fold-forming core maintained by a few highly conserved residues interspersed with freely variable regions, allowing substantial diversification in ligand-binding specificity. During the evolution of the metazoa, domain shuffling and the functional diversity conferred by SRCR domains and CTLDs may have resulted in their expansion and adaptation for the complex pattern recognition processes associated with innate immunity.

Tyrosine kinases:

Given that the Chlamydomonas genome contains both SRCR domains and CTLDs, we searched for further homologs of animal cell-adhesion proteins. We did not find homologs of the cadherins, laminins, type-II fibronectins, or fibrillar collagens in support of the hypothesis that these protein domains are probably unique to metazoans and the closely related choanoflagellates. In addition to these cell adhesion mechanisms, the development of specific signaling mechanisms may also have contributed to the evolution of multicellularity in the ancestors of metazoans (King et al. 2003, 2008). In particular, metazoans possess an extensive collection of tyrosine kinases (TKs) that specifically phosphorylate tyrosine residues and are therefore distinct from dual-specificity kinases that phosphorylate serine/threonine residues in addition to tyrosine (Hanks and Hunter 1995). TKs have not been characterized from yeasts, plants, and algae, suggesting that TKs may have evolved in the metazoan lineage (Muller 2001). However, tyrosine phosphorylation has been reported in a wide range of plants and algae (Zhang et al. 1996; Corellou et al. 2000; Kameyama et al. 2000) and a comprehensive phylogenetic analysis identified putative TKs in the genomes of Chlamydomonas [Joint Genome Institute (JGI) version 1], Phytophora, and Entamoeba (Shiu and Li 2004). We recently identified putative TKs in Entamoeba histolytica and the plants Arabidopsis and O. sativa (Miranda-Saavedra and Barton 2007).

To search for the presence of TKs, we examined the genome of Chlamydomonas, using a multilevel hidden Markov model (HMM) library of the protein kinase superfamily. We identified 355 protein kinases, indicating that the Chlamydomonas kinome is larger than those of the other unicellular algae O. tauri (104 kinases), O. lucimarinus (107 kinases), and C. merolae (62 kinases). The kinome of Chlamydomonas harbors 28 putative TKs, comparable to the TK complement of Drosophila (33 TKs) (Miranda-Saavedra and Barton 2007). The closely related alga, V. carteri contains 31 putative TKs. Twenty-six of the 28 putative TKs of Chlamydomonas contain conserved motifs found in catalytically active kinases (supplemental Table T2). The remaining two putative TKs that do not possess these conserved motifs are likely to be pseudokinases (Boudeau et al. 2006). Thus, Chlamydomonas contains many more putative TKs with predicted catalytically active kinase domains than the land plants, Arabidopsis (0 of the 2 TKs are predicted to be catalytically active) and O. sativa ssp. Indica (3 of 6 predicted active) (Miranda-Saavedra and Barton 2007).

Tyrosine phosphorylation plays an essential signaling role during the mating of Chlamydomonas gametes. Sexual fusion of Chlamydomonas gametes of opposite mating types is initiated by flagellar adhesion through mating-type-specific adhesion molecules (agglutinins). Tyrosine phosphorylation of a cGMP-dependent serine/threonine kinase (CrPKG) within the flagella occurs immediately after flagellar adhesion and is inhibited by the TK inhibitor genistein, which results in the inhibition of fertilization (Wang and Snell 2003; Wang et al. 2006). Biochemical evidence is necessary to determine whether members of the putative Chlamydomonas TK family phosphorylate tyrosine residues in vivo and are responsible for the well-characterized flagella-signaling processes. Clues to cellular function from domain organization are scarce as the majority of TKs were found in proteins without accessory domains. Four putative TKs contain a single predicted transmembrane domain, although none of these proteins contain an extracellular ligand-binding domain typical of animal receptor tyrosine kinases or plant receptor-like kinases. The putative TKs were not present in the flagellar proteome (Pazour et al. 2005).

A protein tyrosine phosphatase (PTP) has been characterized from C. eugametos (Haring et al. 1995) and several PTPs were recently identified in the genome of C. reinhardtii (Kerk et al. 2008). A search for phosphotyrosine-binding domains identified a single SH2 domain-containing protein in the Chlamydomonas genome (SHD1) and a homologous protein in Volvox (protein ID: 116796) (supplemental Figure S1). SH2 domains bind phosphotyrosine residues and therefore function specifically in protein tyrosine kinase pathways (Machida and Mayer 2005). Chlamydomonas therefore contains a full complement of the phosphotyrosine-signaling tool kit as found in metazoans and choanoflagellates (King et al. 2008). The discovery of TKs, SH2 domains, and PTPs in chlorophyte algae and land plants suggests that phosphotyrosine signaling mediated by TKs is of general importance in photosynthetic organisms and represents an ancestral mode of cellular signaling (Williams and Zvelebil 2004; Miranda-Saavedra and Barton 2007; Kerk et al. 2008). In metazoans, the cellular roles of TKs have diversified through the combinations of accessory domains and the evolution of the receptor tyrosine kinases. In contrast, receptor and cytoplasmic serine/threonine kinases predominate in the land plants and the complement of putative TKs appears to be minimal, although there is widespread evidence for tyrosine phosphorylation. The identification of such a large family of putative TKs in Chlamydomonas supports the hypothesis that phosphotyrosine signaling appeared early in eukaryote evolution before the divergence of the Opisthokont lineage. The characterization of their cellular roles will provide important information on the factors driving the evolution of cellular signaling among the different eukaryote lineages.