Molecular Endocrinology, doi:10.1210/me.2003-0227
Molecular Endocrinology 18 (1): 1-12
Copyright © 2004 by The Endocrine Society
Comparative Genomic Analysis of the Eight-Membered Ring Cystine Knot-Containing Bone Morphogenetic Protein Antagonists
Orna Avsian-Kretchmer and
Aaron J. W. Hsueh
Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317
Address all correspondence and requests for reprints to: Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317. E-mail: aaron.hsueh{at}stanford.edu.
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ABSTRACT
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TGF-ß family proteins with a cystine knot motif serve as ligands for diverse families of plasma membrane receptors. Bone morphogenetic protein (BMP) antagonists represent a subgroup of these proteins, some of which bind BMPs and antagonize their actions during development and morphogenesis. Availability of completed genome sequences from diverse organisms allows bioinformatic analysis of the evolution of BMP antagonists and facilitates their classification. Using a regular expression algorithm (http://BioRegEx.stanford.edu), an exhaustive search of the human genome identified all cystine knot-containing BMP antagonists. Based on the size of the cystine ring, these proteins were divided into three subfamilies: CAN (eight-membered ring), twisted gastrulation (nine-membered ring), as well as chordin and noggin (10-membered ring). The CAN family can be divided further into four subgroups based on a conserved arrangement of additional cysteine residues—gremlin and PRDC, cerberus and coco, and DAN, together with USAG-1 and sclerostin. We searched for orthologs of human BMP antagonists in the genomes of model organisms and analyzed their phylogenetic relationship. New human paralogs were identified together with the verification of orthologous relationships of known genes. We also discuss the physiological roles of the CAN subfamily of BMP antagonists and the associated genetic defects. Based on the known three-dimensional structure of key cystine knot proteins, we postulated disulfide bondings for eight-membered ring BMP antagonists to predict their potential folding and dimerization.
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INTRODUCTION
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DISULFIDE BONDINGS FORMED by pairs of cysteine residues in proteins are essential for the formation of unique functional motifs and protein folds. Four half-cystine residues (the oxidized form of cysteine), which form two intrachain disulfide bonds, represent a unique framework for the formation of a cystine ring motif originally identified in mammalian endothelin and insect-derived neurotoxins (1, 2). This ring structure is conserved in diverse families of proteins including the cystine knot superfamily (3) that is characterized by the participation of a third pair of cysteine residues. For cystine knot proteins, the disulfide bonds are arranged so the third disulfide bond that threads back through the ring forms the characteristic knot motif (4), making the structure of these proteins exceptionally stable. Proteins with cystine knots exist in many unrelated species, and it has been hypothesized that this structural fold emerged multiple times as the result of convergent evolution (5). Proteins with a cystine knot motif are usually secreted by cells and play important roles in extracellular signaling in multicellular metazoans. In addition to the cystine knot-containing ligands, they include ion channel blockers, hemolytic agents, and molecules having antiviral and antibacterial activities (4, 6).
The cystine knot superfamily of ligands comprises many homodimeric and heterodimeric proteins that are involved in embryonic development, organogenesis, as well as tissue remodeling and repair. Included in this superfamily are the TGF-ß, growth differentiation factors (GDFs), bone morphogenetic proteins (BMPs), BMP antagonists, gonadotropins, and platelet-derived growth factors (7). In addition to the regulation of normal cell functions, many cystine knot-containing proteins have been implicated in tumorigenesis (8, 9, 10).
BMPs were first identified in the protein extracts of demineralized bone (11) and are involved in body patterning and morphogenesis (8). The developmental signaling pathway mediated by vertebrate BMPs and their fly ortholog Decapentaplegic is highly conserved during animal evolution. This pathway is required for dorsal-ventral patterning of the early embryo in both vertebrates and invertebrates (8), and the BMP family genes have undergone expansion during evolution leading to the generation of multiple paralogs in human (8). Several BMP paralogs were named as GDFs based on their roles in the growth and differentiation of diverse tissues (8, 12, 13). Loss- or gain-of-function mutations of the vertebrate BMP and GDF genes display a variety of developmental defects (8). Among the BMP proteins, the three-dimensional structure of BMP7 has been elucidated (Fig. 1A
) (14).
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Fig. 1. Known and Predicted Structure of Proteins with an Eight-Membered Ring Cystine Knot
Based on BMP7 structure (14 ), the fold prediction of the CAN family is illustrated. C1, C2, C3, C4, C5, and C6 represent the six cysteine residues that form the knot structure. G, Glycine residue; X, any residue that is not cysteine or glycine; C', additional cysteine residues that exist in the cystine knot domain but do not participate in basic knot formation; S:S, disulfide bond. A, BMP7 cystine knot structure. B, Model for the CAN subfamily of BMP antagonists.
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In addition to the tissue-specific expression of BMP ligands and their cell surface receptors, a crucial regulatory step of BMP signaling is modulation by specific BMP antagonists. Several groups of BMP antagonists were identified based on their ability to block the actions of BMP proteins through direct binding (15, 16). These antagonists include the CAN family of proteins (15, 17, 18), the twisted gastrulation protein (19, 20), the chordin family that includes chordin and ventroptin (21, 22), and noggin (23, 24, 25). Of particular interest, these BMP antagonists have a cysteine arrangement consistent with the formation of the cystine knot structure and represent a subfamily of cystine knot proteins. Few orthologs for the BMP antagonists are present in invertebrates and the BMP antagonist subgroup underwent major expansion during vertebrate evolution (7).
Recent availability of genome sequences from multiple vertebrates (26, 27) and the sea squirt (Ciona intestinalis) (28) provides the opportunity to perform bioinformatic searches of BMP antagonists in the GenBank based on their unique cysteine arrangements. Here, we report the phylogenetic relationship of different human BMP antagonists, divide them into three subfamilies based on the known or predicted size of their cystine rings, and unify the classification of these proteins from diverse species based on their evolutionary relationship. We focused on the CAN family of eight-membered ring BMP antagonists, identified their orthologs in model organisms, and predicted the three-dimensional fold of their cystine knot motif.
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THE USE OF REGULAR EXPRESSION TO IDENTIFY NEW CYSTINE KNOT- CONTAINING PROTEINS
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In the postgenomic era, there is an increasing need to develop bioinformatic tools for analyzing protein sequences. Regular expressions (RegExs) are powerful tools used in computer languages for identifying or searching patterns in strings. They are used in scripting languages to match one or more strings. They can be as simple as a word, which matches the word itself, or can be complex and made to match a large set of different words (29). Many characters—all letters, all digits, and many of the special symbols—can be matched literally using RegExs.
Because protein sequences can be described as strings, RegExs may be used for analyzing proteomic data. We developed the "BioRegEx," a web interface for pattern searches in the human proteome using regular expressions, http://BioRegEx.stanford.edu. As shown in Fig. 2, the human nonredundant protein sequences were downloaded from the International Protein Index at EMBL-EBI, http://www.ebi.ac.uk/index. html. To identify secreted proteins in the human proteome, subdatabases were built on the basis of the presence of a signal peptide and the absence of transmembrane domains. The signal peptide was determined using the signal peptide prediction server, http://www.cbs.dtu.dk/services/SignalP-2.0/ that predicts the presence and location of signal peptide cleavage sites in protein sequences (30). In addition, we used the SMART (simple modular architecture research tool) domain prediction server, http://smart.embl-heidelberg.de/ to identify transmembrane domains in protein sequences. The SMART algorithm allows the identification and annotation of genetically mobile domains and the analysis of domain architectures (31).
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Fig. 2. Regular Expression Searches for Novel Cystine Knot-Containing Proteins in the Human Proteome, and Analysis of Their Evolutionary Conservation
BioRegEx web interface at http://BioRegEx.stanford.edu. *, Smart prediction web server at http://smart.embl-heidelberg.de (31 65 ); signal peptide prediction server at http://www.cbs.dtu.dk/services/SignalP-2.0/ (30 ). **, In the cystine knot motif: Cys, cysteine residue; X, any residue; subscript numbers, number of repetitions; and n, any number of repetitions.
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As shown for BMP7 (Fig. 1A
) and for other eight-membered ring cystine knot proteins, the cysteine residues that form the cystine knot are numbered 1–6. Cysteine residues 2, 3, 5, and 6 form the ring and cysteine residues 1 and 4 form the knot. The cystine ring size is determined by the spacing between cysteine residues 2 and 3 (marked as C2 and C3 in Fig. 1) and cysteine residues 5 and 6 (marked as C5 and C6 in Fig. 1) (see also the gray shaded residues in Table 1).
After determining the conserved cysteine arrangement in the eight-membered ring cystine knot, we used the BioRegEx interface to perform a search in the human proteome database for all proteins with a cystine knot motif (Fig. 2). In addition to the known proteins, hypothetical and unknown proteins that contain an eight-membered ring cystine knot were detected and selected. We excluded all proteins with multiple cysteine residues that were not conserved in orthologs from other species. Among the hypothetical proteins, two new potential human BMP antagonists were identified. The first was found to be an ortholog of the rat USAG-1 that is preferentially expressed in the receptive rat endometrium (32), whereas the second was found to be an ortholog of the Xenopus coco gene (33). In contrast to RegEx searches for eight-membered ring cystine knot proteins, similar searches for nine- and 10-membered ring cystine knot proteins did not reveal novel proteins in the human proteome (Fig. 2). All sequence data retrieved by this study can be viewed at http://hormone.stanford.edu/ BMP-antagonists.
Human USAG-1 maps on chromosome 7p21 and shares a 98% homology with its newly identified mouse ortholog and 97% homology with the rat USAG-1. Recently, orthologs for USAG-1 have been found in Xenopus based on functional screening for developmental genes (34). Based on expressed sequence tag searches, human USAG-1 is widely expressed (kidney, skin, liver, mammary gland, aorta and vein, embryonic tissues, etc.). SOST, which encodes for sclerostin, is the closest human paralog of USAG-1; these two proteins share 54% homology overall, with 64% homology in the cystine knot domain. Coco is a new member of the CAN family of secreted BMP inhibitors recently identified in Xenopus (33). One hypothetical human protein (gi 22749329) was identified as human coco, and as in Xenopus, it is closely related to cerberus. The present searches also revealed the orthologous relationship between coco and the previously identified dante in mouse (17). The mouse dante gene represents a fragment of the predicted mouse coco, thus allowing the prediction of the full-length mouse coco protein as described below.
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CONSENSUS STRUCTURES OF THREE SUBFAMILIES OF BMP ANTAGONISTS WITH UNIQUE CYSTEINE ARRANGEMENTS
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We aligned the sequences of all the known and potential BMP antagonists and divided them into three subfamilies based on the predicted structure and spacing of the cysteine residues in the cystine rings—the eight-membered ring, the nine-membered ring, and two types of 10-membered ring motifs (Table 1, gray-shaded residues).
The first subfamily is the CAN family with a cystine knot comprised of an eight-membered ring. These proteins have a cystine knot with a ring structure similar to that of the BMPs (Fig. 1, Table 1) and glycoprotein hormone subunits (e.g. hCG-ß). The second subfamily is the twisted gastrulation protein with a carboxyl-terminal cysteine arrangement that could form a cystine knot comprising a nine-membered ring (Table 1). This putative cystine ring does not have a glycine residue between cysteine residue numbers 2 and 3 in the first half of the ring (Cys2-X-X-X-Cys3) but has an additional residue in the second half of the ring (Cys5-X-X-Cys6). This arrangement results in a total of nine residues in the ring. The third subfamily consists of two groups of BMP antagonists with a 10-membered ring. One group is the chordin family that, in addition to chordin and its fly ortholog SOG, includes the ventroptin protein. This group has four (in the case of chordin/SOG) or three (in the case of ventroptin) cysteine-rich domains. Analyzing the cysteine spacing and arrangement in each domain revealed a conserved arrangement (between the different domains in the same proteins and between the two human paralogs) that could form a 10-membered ring (Table 1). The first half of the ring has an additional cysteine residue (Cys2-X-Cys-X-Cys3), and the second half has two residues between cysteine residues 5 and 6 (Cys5-X-X-Cys6). The last group consists of the noggin protein with a 10-membered ring (Table 1). The three-dimensional structure of noggin is known (24).
Phylogenetic analyses (35) of representative members of the three BMP antagonist subfamilies in human (Fig. 3) show that the eight-membered ring CAN subgroups are more closely related to each other than to the nine- and 10-membered ring subfamilies. The nine- and 10-membered ring BMP antagonists form a second branch with the 10-membered ring proteins (noggin and chordin) closer to each other than to the nine-membered ring subfamily (twisted gastrulation). This phylogenetic analysis gives further support to the division of BMP antagonists into three subfamilies based on ring size in the cystine knot motif.
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Fig. 3. Phylogenetic Relationship of BMP Antagonists
Phylogenetic tree of human BMP antagonists based on the alignment of cystine knot sequences of representative members from each subfamily. Chordin has four cystine knot domains, all of which gave the same result—the fourth cystine knot domain for chordin is used here. The MultAlin server at http://prodes.toulouse.inra.fr/multalin/multalin.html was used for phylogenetic tree construction (66 ).
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CONSENSUS STRUCTURES OF THE EIGHT-MEMBERED RING SUBFAMILY OF BMP ANTAGONISTS—THE CAN FAMILY
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In addition to human CAN family proteins, we identified their orthologs in diverse vertebrates based on GenBank searches (Fig. 4A). All newly identified genes are underlined. Sequence comparison of all members of the human CAN family and their orthologs revealed a consensus cysteine arrangement signature (Fig. 4A). In addition to the six conserved cysteine residues that form the cystine knot, two extra cysteine residues (C’) (Fig. 4A and Table 1, dark background) are present in loop 1 and loop 2 of the cystine knot motif, respectively. This pair of cysteine residues could form a disulfide bond.
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Fig. 4. Sequence Alignment, Genomic Organization, and Phylogenetic Relationship of the Eight-Membered Ring Subfamily of BMP Antagonists
A, Sequence alignment of the CAN family members. The BCM search launcher at http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html was used for multiple sequence alignment. The six cysteine residues that form the knot are shown as white letters on a black background and are labeled as 1–6. The extra cysteine residues (labeled as C' and Cx) are shown on a dark gray background, and the conserved G residue between C2 and C3 is shown on a light gray background. The newly identified BMP antagonists were underlined. B, Genomic organization of the CAN subfamily of BMP antagonists. Genomic and mRNA sequences of each gene are compared using the SPIDEY tool of NCBI at http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey/index.html to predict the exon-intron junctions. Each box represents an exon and each line an intron. The number on top of each exon and intron is the size in base pairs. The checkerboard pattern represents the cystine knot location.
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Comparison of the cysteine residue arrangement between the CAN subfamily of BMP antagonists and known cystine knot-containing proteins allows the prediction of cystine knot folds for CAN family proteins (Fig. 1B) (7). Similar to BMP7, the cysteine residues that form the cystine knot are marked as 1–6. Cysteine residues 2, 3, 5, and 6 form the ring, and cysteine residues 1 and 4 form the knot. The additional cysteine residues (marked as C') located in the loops of the cystine knot are predicted to form an additional intrasubunit disulfide bond (Fig. 1B).
For several BMP antagonists of the CAN family, an additional cysteine residue, Cx (Table 1 and Fig. 4A), is present in the heel of the cystine knot two residues upstream of the Cys4 that forms the knot with Cys1. This extra cysteine residue could allow the formation of homodimers, but is missing in sclerostin and USAG-1. In contrast, the DAN protein has another cysteine two residues downstream of the Cys6 (Table 1 and Fig. 4A).
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DEVELOPMENTAL ROLES OF BMP ANTAGONISTS WITH AN EIGHT-MEMBERED RING CYSTINE KNOT
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All members of the CAN subfamily of BMP antagonists can be found in man and mouse in syntenic chromosomal regions, further confirming their orthologous relationship (Table 2). All of them are conserved proteins with a cystine knot motif in the carboxyl terminus (Table 3) (15, 17, 18, 36, 37, 38, 39, 40). Although the predicted proteins vary in length, they all have a signal peptide for secretion and putative N-linked glycosylation sites (Table 3). CAN family members are believed to regulate embryonic and organ development by selectively antagonizing the activities of different BMP ligands (15, 41) (Table 4). Together with USAG-1, there are a total of seven CAN family members.
Gremlin/DRM/IHG-2
Gremlin was first isolated from the neural crest of the Xenopus as an antagonist of BMP signaling (15, 42) and is an important BMP regulator for limb development that acts in a complementary fashion with other BMP antagonists (43). Gremlin binds directly to BMP2/4 and prevents them from interacting with their receptors (Table 4) (44). High levels of gremlin expression were found in nondividing and terminally differentiated cells such as neurons, alveolar epithelial cells, and goblet cells (18).
Gremlin also is known as DRM (down-regulated by v-mos) because it was identified as a gene that is down-regulated in mos-transformed cells. Another name for gremlin is IHG-2 (induced in high glucose 2) (45) because its expression in mesangial cells, derived from glomerular messangium of the kidney, is induced by high ambient glucose, mechanical strain, and TGF-ß. Gremlin was suggested as a modulator of mesangial cell proliferation and epithelial-mesenchymal transdifferentiation in a diabetic milieu (46). Increased expression of gremlin has recently been demonstrated in several models of diabetic nephropathy. Gremlin is involved in the pathophysiology of glomerulosclerosis and tubulointerstitial fibrosis, and represents a potential therapeutic target in progressive renal diseases (47).
PRDC (Protein Related to DAN and Cerberus)
PRDC was first described in mouse (48) and shares high homology with gremlin.
Cerberus
This gene is expressed in the anterior endomesoderm (38, 39, 49). Caronte, a chick ortholog, is involved in left-right asymmetry in the chick embryo (49). Cerberus functions as a multivalent growth factor antagonist in the extracellular space and inhibits signaling by BMP4, nodal, and Wnt (50). Mouse cerberus binds to BMP proteins and nodal via independent sites (39), whereas the Xenopus cerberus also binds Wnt proteins and inhibits their actions (50). Cerberus has the unique property of inducing ectopic heads in the absence of trunk structures (39). The expression of cerberus during gastrulation is activated by nodal-related signals in endoderm and by Spemann-organizer factors (51).
Coco
Studies in Xenopus indicated that coco functions as a blocker of BMP and TGF-ß signals in the ectoderm and regulates cell fate specification and competence before the onset of neural induction. Coco can also induce ectopic head-like structures in the neurula-staged embryos (33). Coco is expressed maternally in an animal to vegetal gradient, and its expression level declines rapidly after gastrulation. In contrast to other known BMP inhibitors, coco is broadly expressed in the ectoderm until the end of the gastrulation stage (33).
DAN (Differential Screening-Selected Gene Aberrative in Neuroblastoma)
DAN, also known as NO3 (40, 52, 53), is the founding member of the CAN family (15, 40). DAN interacts with GDF-5 in a frog embryo assay, suggesting that it may regulate signaling by the GDF-5/6/7 classes of BMPs (Table 4) (41). Like cerberus, DAN induces cement glands as well as markers of anterior neural tissues and endoderm in Xenopus animal cap assays, suggesting its role in the BMP signaling blockade. DAN is also expressed in the developing myotome. Overexpression of DAN in transformed cell lines suppresses the transformed phenotypes and reduces cell growth, thereby causing a retardation of the cell’s entry into the S phase (15, 40, 41, 53, 54). DAN may play a regulatory role during development and cell growth. Although originally proposed as a tumor suppressor gene for human neuroblastoma (55), this possibility was subsequently excluded (56).
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SOST
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Sclerostin, encoded by the SOST gene, is a secreted osteoclast-derived BMP antagonist (57). It was originally identified as the gene responsible for sclerosteosis (58) and is highly conserved across vertebrate species (59). Sclerostin binds to BMP6 and BMP7 with high affinity and to BMP2 and BMP4 with a lower affinity leading to the inhibition of BMP6 and BMP7 activities (57). High levels of sclerostin expression were detected in long bones, cartilage, kidney, liver, placenta, and fetal skin in human and mouse (59). Sclerostin could play an important role in bone remodeling, and links bone resorption and apposition (57). Based on its suppressive role in bone formation, SOST could be important in the development of therapeutic strategies for osteoporosis (58). Loss of function of the SOST gene product leads to a progressive bone overgrowth disorder known as sclerosteosis, an autosomal recessive disorder (57).
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EVOLUTION OF EIGHT-MEMBERED RING BMP ANTAGONISTS
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The identification of CeCan1 (emb Z74032.1 CEF35B12) as an ortholog of both gremlin and PRDC in the nematode Caenorhabditis elegans (17) and in Ciona intestinalis (Table 2) suggests that the CAN family is of ancient origin. As summarized in Table 2, gremlin and PRDC can be found in all model organisms examined except fly [only the DAN ortholog was found in fly (gi|24648796)]. Of interest, analyses of gene structures indicated that all proteins encoded by orthologous gremlin and PRDC genes are derived from a single exon (Fig. 4B). Five amino acids upstream of the cystine knot domain is a putative proteolytic cleavage site (RKY or RRY) that is highly conserved (Table 3).
Orthologs for cerberus and coco can be found in Xenopus tropicalis and Fugu rubripes but are missing in invertebrates (Table 2). The mouse dante (17) gene corresponds to a fragment of the mouse coco ortholog (Fig. 4A). In Fugu rubripes, there is only one ortholog for both cerberus and coco. All orthologous genes for cerberus and coco have two exons; the first eight amino acids of the cystine knot domain are encoded by the 3' end of the first exon and the remainder of the motif by the second exon (Fig. 4B). In some orthologs, a predicted proteolytic cleavage site can be found upstream of the beginning of the cystine knot domain (Table 3).
The DAN protein can be found in all model organisms except Caenorhabditis elegans (Table 3). This gene has three exons that encode the cystine knot domain (except for fly) (Fig. 4B). The human, mouse, and Xenopus orthologs have been cloned (15, 40, 60, 61) and share greater than 90% identity. A carboxyl-terminal proline-rich region in DAN could be involved in protein-protein interactions.
The USAG-1 and SOST genes are missing in fly and nematode. A single ortholog for both USAG-1 and SOST was found in Fugu rubripes and Ciona intestinalis (Table 2). All orthologous genes for USAG-1 and SOST have two exons that encode the cystine knot domain starting at six amino acids downstream of the second exon (Fig. 4B).
Based on their cystine knot structure (Table 1), overall sequence similarity (Fig. 4A), and conserved exon-intron arrangement (Fig. 4B), we divided the CAN family into four subgroups: 1) gremlin and PRDC; 2) cerberus and coco; 3) DAN; and 4) USAG-1 and SOST. This subdivision is consistent with the phylogenetic tree shown in Fig. 4C.
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THREE-DIMENSIONAL STRUCTURES OF THE EIGHT-MEMBERED RING BMP ANTAGONISTS
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The three-dimensional structures for BMP antagonists of the CAN subfamily are unknown. The region of homology shared by the CAN family genes closely resembles the cystine knot motif found in members of the TGF-ß superfamily. Crystallographic analysis of the hCG-ß structure suggested that this hormone subunit has six disulfide bonds; three form the cystine knot structure and the remaining three form intrasubunit bonds as shown (Fig. 5A) (62, 63). Based on the homology between the CAN family of BMP antagonists and hCG-ß (Fig. 5A), assisted by a three-dimensional structure prediction server http://www.sbg. bio.ic.ac.uk/
3dpssm/ (64), we analyzed the structure of three key members of the CAN family: gremlin, DAN, and USAG-1 (Fig. 5).
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Fig. 5. Three-Dimensional Structure Prediction for Key Members of the CAN Subfamily of BMP Antagonists
Protein sequences were analyzed using the three-dimensional fold recognition server at http://www.sbg.bio.ic.ac.uk/3dpssm/ (64 ) and the ICM Browser (Molsoft, L.L.C.) at http://www.molsoft.com/products/icm_browser.htm. Based on the known crystal structure of hCG-ß, structures of three members of the CAN family of BMP antagonists were predicted. Loop 1 of the cystine knot is marked in blue, loop 2 in red, and the heel in green. Cysteine residues forming the cystine ring (cysteine residues 2, 3, 5, 6) are shown as yellow spheres. Cysteine residues 1 and 4, forming the disulfide bond that penetrates the ring to form the knot, are indicated with a dashed yellow line. Cysteine residues that form a disulfide bond between loops 1 and 2 are marked as green spheres; cysteine residues that form intrasubunit disulfide bonds are marked as blue spheres. Additional cysteine residues likely involved in dimer formation are marked as white spheres and highlighted with an arrow. A, hCG-ß; B, gremlin; C, DAN; D, USAG-1.
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Similar to hCG-ß, all CAN family members have an additional cysteine residue in each loop with the potential to form a disulfide bond between loops 1 and 2, leading to a more stable and compact cystine knot (Fig. 5, B–D, dashed line between the two loops). Furthermore, gremlin has an additional cysteine residue near the cystine knot (Fig. 5B, arrow) that could be the basis for homodimerization. Further, DAN has two more cysteine residues that are close enough to form an additional intrasubunit disulfide bond (Fig. 5C, arrows). In contrast, USAG-1 does not have extra cysteine residues and, therefore, is unlikely to form a covalent dimer using disulfide bonds. Sclerostin is the closely related paralog of USAG-1, and the recombinant sclerostin protein was secreted as a monomer (57). Further studies are needed to reveal posttranslational modifications of these BMP antagonists.
Perspectives
Based on analyses of the genome sequences from several vertebrates and invertebrates, this study provides a comparative genomic view of BMP antagonists. These cystine knot-containing proteins play important roles during development, organogenesis, and tissue growth and differentiation. The present approach allows the identification of three subfamilies of the BMP antagonists and four CAN subgroups. Elucidation of the orthologous and paralogous relationships of genes in this family not only allows a unified nomenclature and classification for these proteins but also provides clues for the future analysis of their functions using model organisms. Regular expression analysis, coupled with comparative genomic approaches, represents a useful paradigm for the investigation of cystine knot-containing genes in published and emerging genome sequences.
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ACKNOWLEDGMENTS
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We thank Dr. Sheau Yu Hsu and the bioinformatic core of the Specialized Cooperative Centers Program in Reproduction Research for assistance in gene identification and helpful discussions, and to Caren Spencer for editorial assistance.
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FOOTNOTES
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This work was supported by the National Institute of Child Health and Human Development, NIH, through Cooperative Agreement U54-HD-31398 as part of the Specialized Cooperative Centers Program in Reproduction Research. Orna Avsian-Kretchmer was supported by the Stanford Endocrinology Training Grant DK10344.
Abbreviations: BMP, Bone morphogenetic protein; DAN, differential screening-selected gene aberrant in neuroblastoma; GDF, growth differentiation factor; RegEx, regular expression; SMART, simple modular architecture research tool.
Received for publication June 13, 2003.
Accepted for publication September 26, 2003.
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