Evolution and Classification of Cystine Knot-Containing Hormones and Related Extracellular Signaling Molecules
Ursula A. Vitt,
Sheau Y. Hsu and
Aaron J. W. Hsueh
Division of Reproductive Biology Department of Gynecology and
Obstetrics Stanford University School of Medicine Stanford,
California 94305-5317
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ABSTRACT
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The cystine knot three-dimensional structure is
found in many extracellular molecules and is conserved among divergent
species. The identification of proteins with a cystine knot structure
is difficult by commonly used pairwise alignments because the sequence
homology among these proteins is low. Taking advantage of complete
genome sequences in diverse organisms, we used a complementary approach
of pattern searches and pairwise alignments to screen the predicted
protein sequences of five model species (human, fly, worm, slime mold,
and yeast) and retrieved proteins with low sequence homology but
containing a typical cystine knot signature. Sequence comparison
between proteins known to have a cystine knot three-dimensional
structure (transforming growth factor-ß, glycoprotein hormone,
and platelet-derived growth factor subfamily members) identified new
crucial amino acid residues (two hydrophilic amino acid residues
flanking cysteine 5 of the cystine knot). In addition to the well known
members of the cystine knot superfamily, novel subfamilies of proteins
(mucins, norrie disease protein, von Willebrand factor, bone
morphogenetic protein antagonists, and slit-like proteins) were
identified as putative cystine knot-containing proteins. Phylogenetic
analysis revealed the ancient evolution of these proteins and the
relationship between hormones [e.g. transforming growth
factor-ß (TGFß)] and extracellular matrix proteins
(e.g. mucins). They are absent in the unicellular yeast
genome but present in nematode, fly, and higher species, indicating
that the cystine knot structure evolved in extracellular signaling
molecules of multicellular organisms. All data retrieved by this study
can be viewed at http://hormone.stanford.edu/.
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STRUCTURAL FEATURES OF KNOWN 10-MEMBERED CYSTINE KNOT PROTEINS
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Cysteine residues in amino acid chains are essential for disulfide
bonding and loop formation to establish functional motifs in the
tertiary structure of different proteins. A consensus cysteine
framework important for a homologous folding motif was originally
identified in mammalian endothelin and insect-derived neurotoxins (1, 2). This typical framework consists of four cysteine residues with a
cysteine spacing of Cys-(X)3-Cys and Cys-X-Cys,
important for a ring structure formed by eight amino acids. This ring
structure is conserved in many different groups of proteins including
the cystine knot superfamily (3). The cystine knot family members
contain two additional cysteines that form a third disulfide bond that
penetrates the ring structure, thus forming a cystine knot with 10
amino acids of which six are cysteine residues (4). The intrusion of
the additional disulfide bond through the cystine ring confines the
amino acid residue between the second and third cysteine to a glycine,
as any other amino acid at this position would imply severe steric
hindrance for the formation of the knot (5, 6). Thus, the consensus
sequence for the 10-membered cystine knot structure is:

Cysteines 2, 3, 5, and 6 form a ring by disulfide bonding between
cysteines 2 and 5 as well as 3 and 6. The third disulfide bond, formed
by cysteines 1 and 4, penetrates the ring, thus forming a knot (Fig. 1
).

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Figure 1. Schematic Drawing of the 10-Membered Cystine Knot
Structure
Arrows indicate the direction (N to C terminal) of the
amino acid chain. SS indicates disulfide bonds. The six cysteines
involved in knot formation are numbered consecutively and their spacing
is given in the lower panel. Cysteines 2 and 3 form
disulfide bonds with cysteines 5 and 6, respectively, thus
forming a ring. The ring is penetrated by the third disulfide bond
formed between cysteines 1 and 4. The amino acid chains between
cysteines 1 and 2 and between 4 and 5 typically form finger-like
projections, whereas the segment between cysteines 3 and 4 forms an
-helical structure and is designated as a heel. In some of the known
cystine knot proteins an additional cysteine is located in front of
cysteine 4, which was found to be essential for covalent dimer
formation.
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The three-dimensional structure common to the members of the cystine
knot superfamily is responsible for features shared by this family. To
date, all known 10-membered cystine knot proteins have been found to be
extracellular proteins, interacting with specific receptors and/or
other extracellular proteins. The cystine knot is formed
intracellularly and prevents the formation of a globular protein
structure commonly found for other extracellular peptides. The cystine
knot folding of these proteins leads to the formation of three distinct
domains. Two of them, between cysteines 1 and 2, as well as 4 and 5,
consist of antiparallel ß-strands that form finger-like projections,
whereas the third domain between cysteines 3 and 4 usually contains an
-helical structure (7, 8). Due to the arrangement of these domains,
the three-dimensional structure of these proteins has been referred to
as a "hand" containing two fingers and a middle heel.
Therefore, the cystine knot forces the protein to adapt a
three-dimensional arrangement that, in part, exposes hydrophobic
residues to the aqueous surrounding. These hydrophobic residues lead to
the formation of homo- or heterodimers that have been described for
selective members of all subfamilies (6, 7, 9). Some members of the
cystine knot superfamily have, in front of cysteine 4, an additional
cysteine residue that strengthens dimerization by forming a covalent
disulfide bridge between the two subunits of the dimer (10,
11).
The largest 10-membered cystine knot subfamily is the TGFß family
that consists of transforming growth factors, bone morphogenetic
proteins (BMP), growth differentiation factors, inhibins, and Mullerian
inhibiting substance (12, 13, 14, 15, 16). Most of these hormones play essential
roles during early embryonic development and, in adult life, have
diverse physiological roles in cell cycle regulation, modification of
the extracellular matrix, and the regulation of other growth factors
(16, 17, 18, 19, 20). All members of the TGFß subfamily have a stop codon at the
second position after the sixth cysteine of the cystine knot. Other
subfamilies, such as platelet- derived growth factors (PDGFs) and
glycoprotein hormones (GPHs) display an identical cystine knot
arrangement, but have a longer sequence after the sixth cysteine (21, 22). Several other proteins with low homology to the characterized
cystine knot proteins have been suggested to display a cystine knot
structure. These include BMP antagonists (23), the norrie disease gene
product (NDP) (24) as well as the von Willebrand factor (vWf) (25). In
addition to the well known 10-membered cystine knot structure, the
neurotrophic growth factor family has been described to adapt a similar
cystine knot arrangement even though nine and not three residues are
present between the second and the third cysteine, leading to the
formation of a 16-membered cystine knot (6).
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BIOINFORMATIC APPROACH TO TRACE THE EVOLUTION OF 10-MEMBERED
CYSTINE KNOT PROTEINS IN MODEL ORGANISMS
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Several members of the TGFß subfamily have been found to have
homologs in nematode and fly (26, 27, 28). However, no systematic analysis
of the evolution of all TGFß-like, 10-membered cystine knot proteins
has been made. With the completion of the sequencing of the human
genome, together with that of the nematode, Caenorhabditis
elegans, and the fly, Drosophila melanogaster, the
protein sequences from organisms belonging to three different animal
phyla (arthropods, nematodes, and chordates) can now be compared, which
provides an evolutionary perspective on this group of proteins. Studies
of sequence homology among these species could reveal the position of
crucial amino acid residues conserved in all orthologs. Furthermore,
the identification of paralogs and orthologs that share a common
ancestor assists in the unraveling of ligand-receptor relationships and
gene functions (29).
The proteins of the 10-membered cystine knot superfamily differ in size
and contain additional motifs, making their identification difficult by
traditional search methods such as pairwise alignments based on BLAST
tools (30). In this minireview, we present the results of a
pattern-search approach that exhaustively screened the sequenced human,
fly, and nematode genome to find all potential cystine knot-containing
proteins in the predicted protein sequences. Furthermore, the complete
genome of the yeast, Saccharomyces cerevisiae, and available
sequences of the slime mold, Dictyostelium discoideum, were
screened. We attempted to disclose the 10-membered cystine knot
signature in different proteins and revealed evolutionary relationships
of cystine knot-containing proteins from different species. Several
crucial amino acid residues in the cystine knot signature previously
not considered necessary for cystine knot formation were also
identified. The present approach allows the classification of
subfamilies of the cystine knot superfamily and the discovery of novel
homologs in worm and fly for some of the known subfamilies. All data
obtained in this study are searchable at the Cystine Knot Protein
Database [http://hormone.stanford.edu/cystine-knot].
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PROTEINS WITH A TYPICAL CYSTINE KNOT SIGNATURE
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More than 100,000 open reading frames from five (yeast, slime
mold, nematode, fly, human) model organisms were used to identify
proteins with a typical cystine knot signature (Fig. 2
, step 1). Each of these gene sets was
searched for a typical cystine knot signature using a regular
expression search. The regular expression used to identify the cystine
knot signature was C(X... . )CXGXC(X... . )C(X... .
)CXC. This placement of the six conserved knot-forming cysteines
corresponds to the 10-membered cystine knot structure of the TGFß,
GPH, and PDGF subfamilies as described by Sun and Davies (6). Because
the structurally similar cystine knot of the nerve growth factor
subfamily has a 16-membered knot structure, it is not closely
related to the other subfamilies and was excluded from the present
study. The TGFß subgroup is the largest subfamily consisting of a
wide variety of members, which all have a stop codon at the second
position after the sixth cysteine. Therefore, this subfamily was
extracted from the collected sets of data using a second regular
expression (CXCXstop). Redundant sequences were excluded manually
during analysis.

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Figure 2. Flow Diagram of the Methodological Steps Used
Step 1: The complete set of predicted protein sequences from
Saccharomyces cerevisiae, Caenorhabditis elegans, and
Drosophila melanogaster were downloaded from the
GenBank (http:// www.ncbi.nlm.nih.gov/), Sanger Center (http://
www.sanger.ac.uk/Projects/Celegans/), and Berkeley Flybase
(http://www.fruitfly.org/) containing 4,821 yeast, 22,942 worm, and
14,080 fly protein sequences, respectively. The human protein sequences
of 82,344 genes (available in August, 2000) were downloaded from
GenBank. Furthermore, all available slime mold, Dictyostelium
discoideum, sequences (1,672) were downloaded from the GenBank.
Step 2: Numbers of retrieved proteins with a cystine knot signature
after the pattern search approach. Step 3: Analysis of the
characteristics of the cystine knot-containing proteins and
their conservation among paralogs and orthologs. The occurrence of a SP
was tested by using a signal peptide server
(http://www.cbs.dtu.dk/services/ SignalP/#submission). The
potential for secretion of both SP-positive and SP-negative
sequences was verified using PubMed citations whenever possible.
This approach revealed approximately 3% false negative predictions of
SP in genes that were known to encode secreted proteins, and no false
positives. The sequences were analyzed for transmembrane regions
using publicly available servers (http://www.cbs.
dtu.dk/services/TMHMM-1.0/ and http://sosui.proteome.bio.tuat.
ac.jp/cgi-bin/sosui.cgi?/sosuisubmit.html) and verified using PubMed
citations whenever possible. Analyses of protein size and cystine knot
size as well as screening for EGF-like motifs (PROSITE:
http://expasy.cbr.nrc.ca/, PS00022 and PS01186) were done using Python
subroutines. In addition, the retrieved proteins were grouped according
to the number of amino acid residues between cysteines 2 and 6 and analyzed for the conservation of
cysteine residues not involved in knot formation. Furthermore, the
amino acid residues surrounding the cystine knot-forming cysteines were
analyzed for conservation among the known cysteine-knot proteins
(TGFß, GPH, and PDGF family members) from diverse species and
compared with the corresponding residues in the novel potential cystine
knot proteins. To group the genes into subfamilies, the human genes
containing the cystine knot signature were aligned using the BLAST
server. Genes with more than 40% positives over at least 60% of the
sequence length were considered potential paralogs. The function and
structure of these genes were further verified using PubMed records and
the Protein Data Bank, whenever available.
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The predicted protein sequences with cystine knot motifs were further
analyzed based on the protein size, the size of the cystine knot, the
presence of a signal peptide for secretion (SP) and/or transmembrane
helices. Of the 134 proteins with a typical cystine knot signature
found in humans, 84 had a signal peptide for secretion. Among them,
36% had a stop codon at the second position after the sixth cysteine
and were considered to be members of the TGFß subfamily. In the fly
and the worm, 38% and 68% of the selected proteins had a signal
peptide for secretion, but only 30% and 8% of them belonged to the
TGFß subfamily, respectively. The remaining set of human proteins
with a predicted signal peptide contained, as expected, the GPH (n
= 5) and PDGF subfamily members (n = 8) (Fig. 2
and Table 1
).
In addition to the well known cystine knot proteins mentioned above,
several human mucins, vWf, NDP, and several BMP antagonists (Gremlin,
DAN, and Cerberus) displayed the typical cystine knot signature.
Furthermore, the present search identified 31 novel potential cystine
knot proteins that had either one or two epidermal growth factor
(EGF)-like signatures. Other extracellular proteins retrieved included
tenascins, attractins, zonadhesins, thyroglobulins, insulin-like growth
factor binding proteins (IGFBPs), and many more. The subset of proteins
without a signal peptide for secretion contained the parkin gene, as
well as several enzymes and ion-carrying proteins, such as
arylsulfatase A, topoisomerase III-
, and the iron- responsive
element-binding protein 2.
Because the algorithms used for protein sequence prediction from
genomic sequences have been shown to predict only up to 70% of
the correct protein sequences (31), one cannot exclude the possibility
that additional cystine knot-containing members remain undetected. By
direct search of the genomic database we have indeed identified
potential fly PDGF and GPH homologs that are not present in the set of
predicted proteins.
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FROM CYSTINE KNOT SIGNATURE TO STRUCTURE: PROTEINS WITH A
POTENTIAL THREE-DIMENSIONAL CYSTINE KNOT STRUCTURE
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The cystine knot signature is crucial for the formation of the
three-dimensional protein structure and could influence the function of
other motifs (such as ligand binding or signaling) in the same protein.
Therefore, it is likely to be highly conserved in paralogous proteins
in the same species and in orthologs of diverse species. Because the
presence of the cystine knot signature does not necessarily lead to the
formation of a three-dimensional cystine knot structure, three criteria
were used to confirm the cystine knot structure and exclude false
positives: 1) The candidate protein belongs to a subfamily with similar
function, and the cystine knot arrangement is conserved in regions of
high sequence similarity; 2) the human, fly, or nematode orthologs have
conserved cystine knot signatures; and 3) a putative transmembrane
region does not overlap with the cystine knot signature as this could
disrupt cystine knot formation.
The cystine knot family members have conserved cystine knot signatures
but show low homology of their overall amino acid sequences. As a
result, some of the potential homologs could not be found based on
pairwise alignments alone. Therefore, an additional parallel approach
was applied to the sequences found to have the 10-membered cystine knot
signature. The cystine knot was analyzed according to the length of the
amino acid chain between cysteines 2 and 6. The sequences were
subsequently grouped and aligned according to the size of this
signature and the position of additional cysteine residues. Analysis of
the cystine knot size revealed that proteins in each subgroup had a
similarly sized cystine knot motif.
As expected, the TGFß, GPH, and PDGF subfamily
members showed a conservation of the cystine knot signature among
family members in the three model organisms (nematode, fly, and human)
analyzed (Fig. 3A
, subgroups 13 and
Table 1
). In addition to these proteins known to contain a cystine knot
structure, additional human proteins, which were proposed to have a
potential cystine knot structure, were also found. These include
proteins of the mucin-like, slit-like, and jagged-like subgroups. The
mucin-like subgroup includes the NDP as well as several BMP
antagonists, mucin-related proteins, and vWf.

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Figure 3. Alignments of Identified Subgroups of Cystine Knot
Orthologs
The cysteines involved in cystine knot formation are shown as
white letters on black background. The
consensus (cons.) sequence for all proteins in each subgroup is given
below each alignment. The stars at the
amino acid residues immediately flanking cysteine 5 indicate the
conserved hydrophilic residues in all known cystine knot proteins. Stop
codons are indicated by dollar signs. Species: w, worm;
f, fly; h, human. A, Known cystine knot proteins are divided into three
subgroups (TGFß, GPH, and PDGF subfamilies). Although several TGFß
members from a given species are very similar to each other, they only
share the cysteines as conserved residues when all sequences are
included in the alignments. B, Putative cystine knot proteins: Subgroup
4 containing proteins described in the literature as potential cystine
knot candidates and subgroup 5 containing novel cystine knot proteins.
Subgroup 4 includes the three paralogs of BMP antagonists found only in
vertebrates as well as the human NDP, the mucin-like genes, and the
human vWf with its fly ortholog hemolectin (hml). This subgroup of
proteins shows an additional conserved spacing of three cysteines in a
stretch of 18 amino acid residues that is not found in the other
subgroups (boxed). Subgroup 5: The worm homolog of the
slit-like proteins contains a stretch of 15 additional amino acids
inserted between cysteines 4 and 5 that is missing in the fly and human
orthologs (boxed). C, Probable cystine knot proteins
(subgroup 6). In contrast to the other subgroups, the
jagged-like proteins have up to 10 additional cysteine residues among
those potentially forming a cystine knot, thus rendering the analysis
of these proteins difficult. Note that the amino acid residue between
cysteines 5 and 6 in fly and human jagged orthologs are hydrophobic (I,
L), which is different from the preference of less hydrophobic residues
at this position in the members of other subgroups. In addition, the
sixth cysteine is followed by a proline, which is not found in any of
the other subgroups.
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Mucin-Like Subgroup
In addition to the cystine knot motif, the human mucins displayed
a conserved CXXCX{13}C signature at a comparable position (Fig. 3B
, gray shading), thus representing a new common functional
motif identified here. This motif was also present in vWf, NDP, and the
BMP antagonists, but not in the subgroups of the TGFß, GPH, and PDGF
subfamily members (Fig. 3A
). Due to their similar structure, the former
were grouped together in subgroup 4 as mucin-like proteins (Fig. 3B
).
Furthermore, the human mucin genes aligned well and showed conservation
of the cystine knot signature to two smaller fly proteins of unknown
function. The additional cysteines unique to this subgroup of proteins
are probably involved in intrachain folds of the subunits. As a result,
these proteins likely display loops and exposed residues different from
those of the TGFß, GPH, and PDGF subgroups.
Mucins were originally described as the
glycoprotein components of epithelial mucus secretions and defined by
their high content of O-linked oligosaccharides (32). Based on their
functions, mucins can be grouped into the membrane mucins, gel-forming
mucins, and small, soluble mucins. Four human gel-forming mucins were
identified as cystine knot proteins, with a high similarity to vWf and
NDP as well as the BMP antagonists. The C-terminal cystine knot in
these mucins has been described to be involved in the formation of
covalently linked dimers (33) and multimeric insoluble gels through
cross-linking of cysteine-rich domains in their C- and N-termini (34, 35).
Three BMP antagonists belonging to the mucin-like subgroup were
identified only in vertebrates. No receptor for this subgroup is known,
consistent with the hypothesis that these proteins inhibit BMP
signaling by binding to the TGFß subfamily ligands (36, 37). Thus,
the additional consensus cysteine motif in these proteins could be
important for BMP binding and/or inhibition of signaling. The
similarity between mucins and other members of this subgroup (NDP, vWf,
and BMP antagonists) and the potential of dimer formation in all these
proteins have been postulated (25, 38, 39, 40, 41).
The homology between NDP and other mucin-like proteins
indicates that NDP could be a component of the extracellular matrix.
Its mutations in the human, causing congenital blindness and often
sensineural hearing loss and mental retardation, might be due to a
disturbance in the extracellular matrix composition and the resultant
disruption of cell-cell interaction. This is underscored by the
phenotype of NDP mutant mice, which show snowflake-like opacities
within the vitreous (42). Alternatively, NDP might act as a functional
antagonist to other cystine knot superfamily signaling ligands.
Another member of the mucin-like subgroup that was identified
is the vWf. This factor acts as a blood-clotting agent by propagating
agglutination of platelets and their adhesion to the vessel surface.
Its function is highly dependent on the formation of complex
multi-mers. The carbohydrate portion of this glycoprotein seems to
be essential for its interaction with the platelets and the vessel wall
(43). The cystine knot region might be essential for dimerization of
these proteins, thus stabilizing the formation of multimers (25). Our
study identified hml as the fly homolog of vWf.
Slit-Like and Jagged-Like Subgroups
Two additional novel subgroups, previously not known to
form a cystine knot, were identified as potential candidates for
forming a cystine knot structure. These include the human homologs of
the fly slit protein and the jagged proteins. These proteins contain
EGF signatures and are cysteine rich, rendering the analysis of their
potential for forming a cystine knot structure difficult. The slit-like
proteins are characterized by a stop codon shortly after the sixth
cysteine and only two members were found in the human. The two human
jagged proteins contain several additional conserved cysteines and
other residues (Fig. 3
, B and C).
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PROTEINS EXCLUDED TO HAVE A POTENTIAL CYSTINE KNOT STRUCTURE
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Several proteins with a cystine knot signature were not
included as cystine knot-containing proteins because they showed a high
similarity to paralogs without conservation of the cysteine
arrangement. In the IGFBP superfamily, IGFBP-6 had a cystine knot
signature, but cysteines 2 and 3 were not conserved among family
members (Fig. 4A
, black
shading). However, several other adjacent cysteine residues were
highly conserved and the potential of forming a cystine knot structure,
different from that of the TGFß superfamily, cannot be ruled out
(gray shading).

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Figure 4. Proteins with a Cystine Knot Signature but Unlikely
to Contain a Cystine Knot Structure
A, Exclusion of proteins as potential cystine knot superfamily members
due to a lack of cysteine conservation among paralogs. Although IGFBP-6
has a characteristic cysteine arrangement, this signature is not
conserved in its paralogs, IGFBP-5 or IGFBP-3. The putative cysteines 2
and 3 involved in the 10-membered cystine knot formation of IGFBP-6 are
shown as white letters on a black background. However, a
different type of cystine knot might be formed by these proteins as
shown by the gray shading of conserved cysteine residues
among these paralogs. B, Exclusion of proteins as potential cystine
knot superfamily members due to a lack of cysteine conservation among
orthologs. Alignment of the human parkin protein and its fly ortholog.
The typical cystine knot signature found in the human parkin protein is
not conserved in its otherwise highly homologous fly ortholog as the
central glycine residue between cysteines 2 and 3 (boxed
area) is missing in the fly protein.
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Multiple other proteins with a cystine knot signature could be
excluded as potential cystine knot-forming proteins because their fly
and/or worm orthologs did not show conservation of the cystine knot
signature (e.g. human parkin, Fig. 4B
, boxed
area). Although dihydropyrimidine dehydrogenase showed an
unusually high homology (65%) between human, fly, and worm orthologs,
additional cysteine residues were not conserved and no human paralogs
could be found.
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UNIQUE FEATURES IDENTIFIED IN CYSTINE KNOT PROTEINS
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Due to the high number of known cystine knot proteins
studied here, it was possible to identify a new conserved pattern that
might be essential for cystine knot formation. Analysis of the amino
acid residues adjacent and between cysteines 2, 3, 5, and 6 of the
cystine knot signature among all known cystine knot proteins (subgroups
13), revealed that none of the highly hydrophobic amino acids, Trp,
Phe, Tyr, Ile, Leu, Val, and Met, are present at the residues before
and after cysteine 5 (Fig. 5
, shaded areas). Overall, amino acid residues surrounding the
knot-forming cysteines did not show high conservation among these
proteins. As more than 50 proteins known to form a cystine knot
structure were investigated, and the neighboring residues that were
studied varied highly, it is unlikely that the lack of hydrophobicity
flanking cysteine 5 is due to coincidence. Although it is unclear how
hydrophobic residues at these positions might interfere with cystine
knot formation, the present finding facilitates the analysis and search
for new cystine knot members.

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Figure 5. Conservation of Hydrophilicity at the Amino Acid
Residues Flanking Cysteine 5 of the Cystine Knot
AA, Amino acids; *, Hopp-Woods hydrophilicity values. The amino acids
are ranked in descending order of their hydrophilicity. The + indicates
whether this specific amino acid is found at this residue in any of the
more than 50 investigated known cystine knot proteins. None of the
highly hydrophobic amino acids can be found at the residues flanking
cysteine 5 (shaded areas).
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In proteins of both the mucin-like and the slit-like subgroup,
the lack of hydrophobicity at the residues before and after cysteine 5
was also found, consistent with a consensus property in the known
cystine knot proteins. However, in the jagged-like subgroup, the
residue between cysteines 5 and 6 is Ile or Leu. Furthermore, in this
subgroup, the residue after cysteine 6 is a conserved Pro, which cannot
be found in any of the other subgroups of proteins. As a result of the
presence of this Pro residue, the protein chain adapts a unique bend at
this position. Thus, the jagged subgroup is less likely to form a
cystine knot with structural features similar to that of the other
family proteins. Therefore, only the slit-like proteins were
classified as putative cystine knot hormones (Fig. 3B
, subgroup 5) and
the jagged-like proteins are grouped as probable cystine knot proteins
(Fig. 3C
, subgroup 6).
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THE CYSTINE KNOT SIGNATURE HAS A SIMILAR SIZE IN PROTEINS OF
VARIANT SIZES
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As expected, many of the proteins retrieved contained
cysteine-rich regions, which increases the likelihood of finding a
false-positive cystine knot. In addition, the chances of finding a
cystine knot-like signature in larger proteins are higher. Analysis of
total protein size revealed striking variations among the retrieved
proteins. Many had a size of about 400 amino acids, which is typical
for the TGFß subfamily, but others ranged from 55 to 5,400 amino
acids in length (Fig. 6
). Although
variable sizes for the cystine knot signature were found in some
proteins, all proteins selected as containing a cystine knot structure
based on the presented criteria had a similar size in their cystine
knot signature regardless of the protein size. The length between
cysteines 2 and 6 of the cystine knot varied from 42 amino acids in the
slit-like and mucin-like subgroup to 80 amino acids in the TGFß
family members.

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Figure 6. Comparison between the Length of Cystine Knot
Signature and the Total Protein Size for More Than 250 Proteins
Retrieved by the Pattern Search Approach
The circles indicate the cystine knot signature size for
each individual protein. The length of the cystine knot signature is
not related to the total protein size. The cystine knot signature
identified in all proteins selected as containing a cystine knot
structure (triangles), has a comparable size regardless
of the total protein size, thus suggesting the presence of a functional
motif rather than a random arrangement of cysteines.
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Figure 7
shows the position and size of
the cystine knot in relation to the entire protein of all the subgroups
under investigation. Except for the jagged-like subgroup, the cystine
knot signature is located in the C-terminal region of the protein. The
jagged-like proteins are the only subgroup with a transmembrane region
downstream of the cystine knot signature. The human mucins display a
cystine knot motif that aligns to two fly genes, but vary greatly in
size (ranging from 400 to 3,000 amino acids). These proteins also align
to the cystine knot region of the fly hml and human vWf protein. The
fly mucin-like genes are much smaller, sharing only the C-terminal
cystine knot region with the human mucins and lacking the
N-terminal carbohydrate-rich region characteristic of the human mucins.
The vWf and the fly hml both contain an extended N-terminal region. The
vWf is shorter than hml due to a deletion of sequences between the
conserved N-terminal region and the cystine knot motif.

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Figure 7. Position and Size of the Cystine Knot in Relation
to the Total Protein Size in Known and Putative Cystine Knot Proteins
Species: w, worm; f, fly; h, human. Except for the jagged-like
proteins, the cystine knot signature is found in the C-terminal segment
of all proteins. The jagged-like proteins have a transmembrane region
flanking the cystine knot signature. In the TGFß, as well as the PDGF
subfamilies, the cystine knot-containing C-terminal segment is cleaved
from a precursor. In one of the human mucins, the C-terminal cystine
knot is also cleaved from the N-terminal mucin-like region during
intracellular processing. Due to this cleavage the remaining C-terminal
segments with the cystine knot signature have a similar size among
members of different subgroups. Intracellular cleavage does not occur
in the larger proteins of the slit-like or jagged-like subgroups, or
fly hml and human vWf.
|
|
Because the size of the cystine knot signature in these proteins is not
related to the total protein size, this motif likely represents a
structural entity and not a random arrangement of cysteines. These
findings suggest that gene fusion events took place during evolution,
and the same motif was used in diverse proteins with different
functions.
 |
EVOLUTION OF THE 10-MEMBERED CYSTINE KNOT STRUCTURE
|
---|
Family trees were constructed using multiple alignments of
characteristic members of the known and putative 10-membered cystine
knot protein subfamilies in nematode, fly, and human. As shown in Fig. 8
, one distinct branch is formed by the
TGFß subfamily, which indicates early evolutionary divergence of this
large family. A second branch includes four subgroups: GPH,
PDGF, mucin-like, and slit-like. The inclusion of the mucin-like
sequences revealed that GPH as well as PDGF family members are more
closely related to the cystine knot-containing region of the
extracellular mucin-like proteins than to that of the TGFß subfamily.
This branch also contains NDP, vWf, and the BMP antagonists. The
slit-like subgroup forms a distinct subbranch and thus shows less
similarity to other subgroups. This phylogenetic relationship of
extracellular signaling molecules indicates a common origin for
hormones (e.g. TGFß) and extracellular matrix proteins
(e.g. mucins).

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Figure 8. Phylogenetic Tree of the Known and Putative Cystine
Knot Subgroups Together with Their Dimerization/Oligomerization and
Receptor Characteristics
Pairwise alignments of human genes with fly and nematode genes were
done by utilizing the BLAST servers of the Berkeley Flybase and the
Sanger Center using low stringency criteria (E<1,000, identity>10%).
To determine whether any of these potential homologs also contained the
above mentioned cystine knot signature, a subroutine was used to check
the presence of the cystine knot signature in subsets of genes. For
pairwise alignments of the human gene with the respective nonmammalian
homologs, the Baylor College of Medicine search launcher
(http://dot.imgen.bcm.tmc.edu:9331/seq-search/alignment.html) was used.
Multiple protein sequence alignments were done using a pattern
construction algorithm (51 52 ). Gaps in the alignments were modified
to minimize the number of mutations required to explain all differences
between the sequences. Representative sequences for each subgroup were
used to infer phylogenetic relationships using the protein sequence
parsimony method available in PHYLIP
(http://evolution.genetics.washington.edu/phylip.html) (53 ).
Furthermore, the phylogenies were analyzed using the Dayhoff PAM matrix
(54 55 ). Results of this matrix were further analyzed using the
Fitch-Margoliash algorithm (56 ). The degree of confidence for each
branchpoint was obtained using the bootstrap method (1,000
replications) (57 58 ). *, The fly PDGF and GPH orthologs were
identified by manual inspection of genomic data; they are not present
in the set of predicted proteins.
Two main branches can be identified. The first one includes the TGFß
subfamily and lefty, whereas the mucin-like proteins, GPH, PDGF
subfamily, and slit-like proteins form a separate branch. The NDP as
well as the BMP antagonists show close relationship to the mucin-like
genes and the hemolectins. Each subgroup was shown to bind to a
different type of receptor, which is given on the right,
except for proteins of the mucin-like branch, which has no known
receptors.
|
|
The low confidence probability found for some of the branches
is due to the low similarity between the different groups, which are
mainly conserved at the cysteine residues. This low homology and the
fact that the nematode has proteins present in most subgroups make it
difficult to trace the common ancestor for these proteins. It is
possible that convergent evolution led to the formation of a
similar knot structure in the different subfamilies. As shown in Fig. 9
, all major cystine knot protein groups,
including the TGFß, GPH, PDGF, and slit-like subgroups, with the
exception of the mucin-like subgroup, contain worm and fly homologs,
indicating that these proteins evolved before the separation of the
arthropod phylum about 1.2 billion years ago. The vWf and the fly hml
homolog could be derived by a gene fusion event before the emergence of
the fly, thus combining the cystine knot with other functional motifs.
This fusion event took place more than 1 billion, but less than 1.2
billion, years ago, as no ortholog can be found in the worm. A second
fusion event combined the cystine knot with the gel-forming N terminus
of the human mucin proteins. This fusion occurred later, because the
fly mucin-like genes of unknown function do not have the extended N
terminus typical for vertebrate mucins. These findings suggest that the
characteristic mucins evolved only in the vertebrate phylum. In
addition, no homolog of the human BMP antagonists was found in lower
species, suggesting the recent evolution of these genes in the chordate
phylum. On the other hand, the cystine knot of the TGFß, GPH, and
PDGF subfamilies is well conserved in worm indicating their origin to
be more ancient than that of the mucin-like proteins.

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Figure 9. Schematic Time Scale of the Evolution of Cystine
Knot-Containing Proteins in the Five Species Studied
On the right, the respective proteins found in each
animal phylum are shown. Mucin-like genes developed only in the
arthropod phylum whereas the BMP antagonists developed during
vertebrate evolution. All other subgroups are present in worm but no
cystine knot protein ortholog was found in available sequences from the
slime mold. As no cystine knot proteins were found in yeast, the
ancient cystine knot-containing proteins likely evolved in
multicellular organisms before the emergence of nematodes.
|
|
 |
THE ABSENCE OF CYSTINE KNOT PROTEINS IN YEAST
|
---|
As most identified cystine knot-containing proteins had
worm orthologs, proteins with a cystine knot signature were also
screened using sequences from lower species including the entire yeast
genome and the available slime mold data. Two candidate proteins were
found in yeastthe putative maltose permease (a transmembrane protein,
GenBank accession no. 1346493) and the metallothionein-like
protein CRS5 (an intracellular protein, accession no.
1169097). Because their cystine knot signatures were not conserved in
paralogs of the same species, they were excluded as cystine knot
proteins. As no cystine knot structure can be identified in yeast, it
is unlikely that this structure could have evolved in unicellular
organisms. However, the use of four cysteines to form a cystine
ring is widely practiced and has been described in protozoan as
well as metazoan species (44, 45).
The slime mold revealed three potential sequences, two
prestalk proteins and one predicted open reading frame, all with a
signal peptide and without transmembrane helices. One of the prestalk
proteins aligned well to other family members in the same species but
the cysteine residues were not conserved. Pairwise alignment of the two
remaining proteins with proteins from other species showed sequence
similarity and cystine knot conservation between DIF-induced prestalk
pDd63 precursor (accession no. 84105) and an as yet unnamed worm
protein (accession no. 6580331) with an EGF signature that was not
found to have a cystine knot. The size of the cystine knot signature in
this slime mold protein is 90 amino acids and therefore longer than
that in the other known cystine knot proteins (up to 80). Thus, it is
questionable that this protein represents the ancient ancestor of the
cystine knot proteins.
 |
MOST CYSTINE KNOT PROTEINS ARE EXTRACELLULAR LIGANDS: INTERACTIONS
WITH DIVERSE RECEPTORS
|
---|
The importance of the cystine knot signature for secreted proteins
has been demonstrated based on studies of cysteine mutants in different
family members (46, 47). The cystine knot constrains the folding of
these proteins to avoid a typical globular shape preferred by proteins
in aqueous solutions. The folding leads to a structure that exposes
specific hydrophobic residues to the exterior of the molecule, thus
facilitating protein-protein interactions. In addition to serving as
ligands for different cell surface receptors, these cystine knot
proteins are known to form homo- and heterodimers as well as multimers.
Of interest, the function of the cystine knot motif in human mucin-2
appears to be the facilitation of dimer formation because its cystine
knot-containing C terminus is cleaved away from the secreted protein
after dimerization (48).
All families identified in this study contained exclusively
extracellular proteins, which corroborates the importance of this
signature for extracellular signaling and cell-cell communication. Most
of the proteins identified are ligands to known receptors. The
phylogenetic grouping of the genes reflects their interaction with
different types of receptors (Fig. 8
). The TGFß family forms a
distinct subgroup and the members are known to interact with
serine/threonine kinase, single-transmembrane receptors. Of interest is
the alignment of the lefty gene, which is more closely related to the
TGFß subfamily than to the other subgroups. In contrast to the TGFß
subgroup, the sequence of lefty is not terminated at the second residue
after the sixth cysteine. The position of this stop codon is, however,
highly conserved among the TGFß members and seems to be
essential for receptor interaction as mutants with extended C terminus
display a dominant negative behavior (49). Although the functional
pathway of lefty is not known, it could constitute a potential
antagonist to the TGFs. The GPH and PDGF subfamily members signal
through seven-transmembrane G protein-coupled receptors and
tyrosine-kinase single-transmembrane receptors, respectively. Members
of the mucin-like subgroup are extracellular matrix proteins and have
not been found to interact with specific cell surface receptors.
Furthermore, the BMP antagonists likely interact with other ligands,
whereas members of the slit-like subgroup interact with
single-transmembrane protein receptors involved in axon guidance
(50).
 |
CONCLUSION
|
---|
A combination of bioinformatic approaches was used to exhaustively
survey the genomic data of five different model species and identify
potential members of the cystine knot superfamily. In addition to
demonstrating relationships between different family members, new
orthologs in different species were recognized. Furthermore, by a
comparison of the large number of known cystine knot members, new
consensus residues in the cystine knot signature were discovered, thus
improving future identification of cystine knot proteins. The spectrum
of members of the cystine knot superfamily was expanded by including
the mucin like subfamily and the slit-like proteins. Because all the
subfamilies that were found contained exclusively extracellular
proteins, the present findings underscore the importance of the cystine
knot structure for ligand-receptor interaction and cell-cell
communication. This is corroborated by the absence of cystine knot
structures in unicellular yeast and the presence of multiple subfamily
members in nematode, indicating that this ancient structure evolved
parallel with the development of multicellular life.
With the availability of the complete genomes of more than 60
organisms, including the human genome, bioinformatic analyses of
extracellular signaling molecules are essential to provide a global
perspective on the evolution and structural features of different
protein hormone families. The present minireview represents an initial
attempt in this direction to provide the basis for discovering new
human protein hormone paralogs and for understanding the structural
characteristics important for hormone function.
 |
ACKNOWLEDGMENTS
|
---|
We thank Caren Spencer for editorial support and John Carlsson
for assistance in computational tasks.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317. E-mail:
aaron.hsueh{at}stanford.edu
U.A.V. is supported by a fellowship from the Lalor Foundation.
Received for publication January 12, 2001.
Revision received February 12, 2001.
Accepted for publication February 20, 2001.
 |
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