Cyclotides
Encyclopedia
Cyclotides are small disulfide
rich peptides isolated from plants. Typically containing 28-37 amino acids, they are characterized by their head-to-tail cyclised peptide
backbone and the interlocking arrangement of their three disulfide bond
s. These combined features have been termed the cyclic cystine knot
(CCK) motif (Figure 1). To date, over 100 cyclotides have been isolated and characterized from species of the Rubiaceae
, Violaceae
, and Cucurbitaceae
families. Recently, cyclotides have also been identified in the Fabaceae
or legumes, an agriculturally very important family .
sequence is required for this. In the case of kalata B1 the indicated glycine
(G) and asparagine
(N) amino acids are the terminal residues that are linked in a peptide bond to cyclise the peptide.
, insecticidal
, anti-tumour, antifouling, anti-microbial, hemolytic
, neurotensin
antagonism, trypsin
inhibition, and uterotonic activities. An ability to induce uterine contractions was what prompted the initial discovery of kalata B1.
The potent insecticidal activity of cyclotides kalata B1 and kalata B2 has prompted the belief that cyclotides act as plant host-defence agents (Figure 2). The observations that dozens or more cyclotides may be present in a single plant and the cyclotide architecture comprises a conserved core onto which a series of hypervariable loops is displayed suggest that, cyclotides may be able to target many pests/pathogens simultaneously.
studies in rats confirmed the uterotonic activity of the purified peptide, it was another 20 years before the cyclic cystine knot motif and structure of the purified peptide were elucidated.
The cyclotides fall into two main structural subfamilies. Moebius cyclotides, the less common of the two, contain a cis-proline in loop 5 that induces a local 180º backbone twist (hence likening it to a Möbius strip
, whereas bracelet cyclotides do not. There is smaller variation in sequences within these subfamilies than between them. A third subfamily of cyclotides are trypsin inhibitors and are more homologous to a family of non-cyclic trypsin inhibitors from squash plants known as knottins than they are to the other cyclotides.
It is convenient to discuss sequences in terms of the backbone segments, or loops, between successive cysteine
residues. The six cysteine residues are absolutely conserved throughout the cyclotide suite and presumably contribute to the preservation of the CCK motif. Although the cysteines appear essential to maintaining the overall fold, several other residues that are highly conserved in cyclotides are thought to provide additional stability.
Throughout the known cyclotides loop 1 is the most conserved. Apart from the six cysteine residues, the glutamic acid
and serine
/threonine
residues of loop 1 are the only residues to have 100% identity across the bracelet and Möbius subfamilies. Furthermore the remaining residue of this loop exhibits only a conservative change i.e. glycine
/alanine
. This loop is believed to play an important role in stabilizing the cyclotide structure through hydrogen bonding with residues from loops 3 and 5.
Loops 2-6 also have highly conserved features, including the ubiquitous presence of just a single amino acid in loop 4 that is likely involved in sidechain-sidechain hydrogen bonding. Other conserved residues include a hydroxyl-containing residue in loop 3, a glycine residue in the final position of loop 3, a basic and a proline
residue in the penultimate position in loop 5 of bracelet and Möbius cyclotides respectively, and an asparagine
(or occasionally aspartic acid
) residue at the putative cyclisation point in loop 6. It is of interest to note that not only are certain residues highly conserved, but the backbone and side chain angles are as well.
With recent screening programs suggesting that the number of cyclotide sequences may soon reach the thousands, a database, CyBase, has been developed that offers the opportunity for comparisons of sequences and activity data for cyclotides. Several other families of circular protein
s are known in bacteria, plants and animals and are also included in CyBase.
. The gene for the first such precursor is Oak1 (Oldenlandia affinis kalata clone number 1), which was shown to be responsible for the synthesis of kalata B1. Figure 4 illustrates the generic configuration of the precursor protein, which consist of an endoplasmic reticulum
signal sequence, a non-conserved pro-region, a highly conserved region known as the N-terminal repeat (NTR), the mature cyclotide domain and finally a short hydrophobic C-terminal tail. The cyclotide domain may contain either one cyclotide sequence, as in the case of Oak1, or multiple copies separated by additional NTR sequences as seen for Oak2 and Oak4. In precursor proteins containing multiple cyclotide domains these can either be all identical sequences, as is the case for Oak4, or they can be different cyclotides as in Oak2 which contains sequences corresponding to kalata B3 and B6.
and membrane permeability. A recent study on related cystine knot proteins as drug candidates showed that cystine knots do permeate well through rat small intestinal mucosa relative to non-cystine knot peptide drugs such as insulin
and bacitracin
. Furthermore, enzymatic digestion of cystine knot peptide drugs was associated with only a few proteases and it was suggested that this limitation may be overcome by mutating out particular cleavage sites. Thus, certain cystine knot proteins satisfy the basic criteria for drug delivery and represent exciting novel candidates as scaffolds for peptide drug delivery. The diverse range of intrinsic activities of cyclotides also continues to hold promise for a wide range of applications in the agricultural fields.
Disulfide
In chemistry, a disulfide usually refers to the structural unit composed of a linked pair of sulfur atoms. Disulfide usually refer to a chemical compound that contains a disulfide bond, such as diphenyl disulfide, C6H5S-SC6H5....
rich peptides isolated from plants. Typically containing 28-37 amino acids, they are characterized by their head-to-tail cyclised peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
backbone and the interlocking arrangement of their three disulfide bond
Disulfide bond
In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...
s. These combined features have been termed the cyclic cystine knot
Cystine knot
A cystine knot is a protein structural motif where two disulfide bridges are formed. The sections of polypeptide that occur between them then form a loop through which a third disulfide bond passes, forming a rotaxane substructure...
(CCK) motif (Figure 1). To date, over 100 cyclotides have been isolated and characterized from species of the Rubiaceae
Rubiaceae
The Rubiaceae is a family of flowering plants, variously called the coffee family, madder family, or bedstraw family. The group contains many commonly known plants, including the economically important coffee , quinine , and gambier , and the horticulturally valuable madder , west indian jasmine ,...
, Violaceae
Violaceae
Violaceae are a family of flowering plants consisting of about 800 species in 21 genera. It takes its name from the genus Viola, the violets and pansies.Older classifications such as the Cronquist system placed Violaceae in an order named after it, the Violales...
, and Cucurbitaceae
Cucurbitaceae
The plant family Cucurbitaceae consists of various squashes, melons, and gourds, including crops such as cucumber, pumpkins, luffas, and watermelons...
families. Recently, cyclotides have also been identified in the Fabaceae
Fabaceae
The Fabaceae or Leguminosae, commonly known as the legume, pea, or bean family, is a large and economically important family of flowering plants. The group is the third largest land plant family, behind only the Orchidaceae and Asteraceae, with 730 genera and over 19,400 species...
or legumes, an agriculturally very important family .
Cyclotide structure
Cyclotides have a well-defined three-dimensional structure as a result of their interlocking disulfide bonds and cyclic peptide backbone. Backbone loops and selected residues are labeled on the structure to help orientation. The amino acid sequence (single letter amino acid representation) for this peptide is indicated on the sequence diagram to the right. One of the interesting features of cyclic peptides is that knowledge of the peptide sequence does not reveal the ancestral head and tail; knowledge of the geneGene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
sequence is required for this. In the case of kalata B1 the indicated glycine
Glycine
Glycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...
(G) and asparagine
Asparagine
Asparagine is one of the 20 most common natural amino acids on Earth. It has carboxamide as the side-chain's functional group. It is not an essential amino acid...
(N) amino acids are the terminal residues that are linked in a peptide bond to cyclise the peptide.
Biological significance
Cyclotides have been reported to have a wide range of biological activities, including anti-HIVHIV
Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
, insecticidal
Insecticide
An insecticide is a pesticide used against insects. They include ovicides and larvicides used against the eggs and larvae of insects respectively. Insecticides are used in agriculture, medicine, industry and the household. The use of insecticides is believed to be one of the major factors behind...
, anti-tumour, antifouling, anti-microbial, hemolytic
Hemolysis
Hemolysis —from the Greek meaning "blood" and meaning a "loosing", "setting free" or "releasing"—is the rupturing of erythrocytes and the release of their contents into surrounding fluid...
, neurotensin
Neurotensin
Neurotensin is a 13 amino acid neuropeptide that is implicated in the regulation of luteinizing hormone and prolactin release and has significant interaction with the dopaminergic system...
antagonism, trypsin
Trypsin
Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when...
inhibition, and uterotonic activities. An ability to induce uterine contractions was what prompted the initial discovery of kalata B1.
The potent insecticidal activity of cyclotides kalata B1 and kalata B2 has prompted the belief that cyclotides act as plant host-defence agents (Figure 2). The observations that dozens or more cyclotides may be present in a single plant and the cyclotide architecture comprises a conserved core onto which a series of hypervariable loops is displayed suggest that, cyclotides may be able to target many pests/pathogens simultaneously.
A Serendipitous Discovery
During a Red Cross relief mission in the Democratic Republic of Congo during the 1960s, a Norwegian doctor, Lorents Gran, noted that during labor African women used a medicinal tea made from the leaves of the plant Oldenlandia affinis (Figure 3) to induce labor and facilitate childbirth. The active ingredient was later determined to be a peptide, named kalata B1, after the traditional name for the native medicine, kalata-kalata. Although in vivoIn vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
studies in rats confirmed the uterotonic activity of the purified peptide, it was another 20 years before the cyclic cystine knot motif and structure of the purified peptide were elucidated.
Cyclotide amino-acid sequences
Analysis of the suite of known cyclotides reveals many sequence homologies that are important for understanding their unique physico-chemical properties and bioactivities. Table 1 presents a selection of cyclotides.The cyclotides fall into two main structural subfamilies. Moebius cyclotides, the less common of the two, contain a cis-proline in loop 5 that induces a local 180º backbone twist (hence likening it to a Möbius strip
Möbius strip
The Möbius strip or Möbius band is a surface with only one side and only one boundary component. The Möbius strip has the mathematical property of being non-orientable. It can be realized as a ruled surface...
, whereas bracelet cyclotides do not. There is smaller variation in sequences within these subfamilies than between them. A third subfamily of cyclotides are trypsin inhibitors and are more homologous to a family of non-cyclic trypsin inhibitors from squash plants known as knottins than they are to the other cyclotides.
It is convenient to discuss sequences in terms of the backbone segments, or loops, between successive cysteine
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
residues. The six cysteine residues are absolutely conserved throughout the cyclotide suite and presumably contribute to the preservation of the CCK motif. Although the cysteines appear essential to maintaining the overall fold, several other residues that are highly conserved in cyclotides are thought to provide additional stability.
Throughout the known cyclotides loop 1 is the most conserved. Apart from the six cysteine residues, the glutamic acid
Glutamic acid
Glutamic acid is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates...
and serine
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...
/threonine
Threonine
Threonine is an α-amino acid with the chemical formula HO2CCHCHCH3. Its codons are ACU, ACA, ACC, and ACG. This essential amino acid is classified as polar...
residues of loop 1 are the only residues to have 100% identity across the bracelet and Möbius subfamilies. Furthermore the remaining residue of this loop exhibits only a conservative change i.e. glycine
Glycine
Glycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...
/alanine
Alanine
Alanine is an α-amino acid with the chemical formula CH3CHCOOH. The L-isomer is one of the 20 amino acids encoded by the genetic code. Its codons are GCU, GCC, GCA, and GCG. It is classified as a nonpolar amino acid...
. This loop is believed to play an important role in stabilizing the cyclotide structure through hydrogen bonding with residues from loops 3 and 5.
Loops 2-6 also have highly conserved features, including the ubiquitous presence of just a single amino acid in loop 4 that is likely involved in sidechain-sidechain hydrogen bonding. Other conserved residues include a hydroxyl-containing residue in loop 3, a glycine residue in the final position of loop 3, a basic and a proline
Proline
Proline is an α-amino acid, one of the twenty DNA-encoded amino acids. Its codons are CCU, CCC, CCA, and CCG. It is not an essential amino acid, which means that the human body can synthesize it. It is unique among the 20 protein-forming amino acids in that the α-amino group is secondary...
residue in the penultimate position in loop 5 of bracelet and Möbius cyclotides respectively, and an asparagine
Asparagine
Asparagine is one of the 20 most common natural amino acids on Earth. It has carboxamide as the side-chain's functional group. It is not an essential amino acid...
(or occasionally aspartic acid
Aspartic acid
Aspartic acid is an α-amino acid with the chemical formula HOOCCHCH2COOH. The carboxylate anion, salt, or ester of aspartic acid is known as aspartate. The L-isomer of aspartate is one of the 20 proteinogenic amino acids, i.e., the building blocks of proteins...
) residue at the putative cyclisation point in loop 6. It is of interest to note that not only are certain residues highly conserved, but the backbone and side chain angles are as well.
With recent screening programs suggesting that the number of cyclotide sequences may soon reach the thousands, a database, CyBase, has been developed that offers the opportunity for comparisons of sequences and activity data for cyclotides. Several other families of circular protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s are known in bacteria, plants and animals and are also included in CyBase.
Biosynthesis of cyclotides
Plants are a rich source of cyclic peptides, with the vast majority of these molecules being produced via non-ribosomal biosynthetic pathways. In contrast, the cyclotides are gene-coded products generated via processing of a larger precursor proteinProtein precursor
A protein precursor, also called a pro-protein or pro-peptide, is an inactive protein that can be turned into an active form by posttranslational modification. The name of the precursor for a protein is often prefixed by pro...
. The gene for the first such precursor is Oak1 (Oldenlandia affinis kalata clone number 1), which was shown to be responsible for the synthesis of kalata B1. Figure 4 illustrates the generic configuration of the precursor protein, which consist of an endoplasmic reticulum
Endoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...
signal sequence, a non-conserved pro-region, a highly conserved region known as the N-terminal repeat (NTR), the mature cyclotide domain and finally a short hydrophobic C-terminal tail. The cyclotide domain may contain either one cyclotide sequence, as in the case of Oak1, or multiple copies separated by additional NTR sequences as seen for Oak2 and Oak4. In precursor proteins containing multiple cyclotide domains these can either be all identical sequences, as is the case for Oak4, or they can be different cyclotides as in Oak2 which contains sequences corresponding to kalata B3 and B6.
Applications
The remarkable stability of cyclotides means that they have an exciting range of potential applications centred on either their intrinsic biological activities or the possibility of using the CCK motif as a scaffold for stabilizing biologically active epitopes. Interest in these has recently intensified with the publications of a chemical methodology capable of synthetically producing cyclotides with high yields, and the amenability of the CCK framework to amino-acid substitutions. But for molecules to be useful in a therapeutic setting they require useful biopharmaceutical characteristics such as resistance to proteolysisProteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...
and membrane permeability. A recent study on related cystine knot proteins as drug candidates showed that cystine knots do permeate well through rat small intestinal mucosa relative to non-cystine knot peptide drugs such as insulin
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
and bacitracin
Bacitracin
Bacitracin is a mixture of related cyclic polypeptides produced by organisms of the licheniformis group of Bacillus subtilis var Tracy, isolation of which was first reported in 1945....
. Furthermore, enzymatic digestion of cystine knot peptide drugs was associated with only a few proteases and it was suggested that this limitation may be overcome by mutating out particular cleavage sites. Thus, certain cystine knot proteins satisfy the basic criteria for drug delivery and represent exciting novel candidates as scaffolds for peptide drug delivery. The diverse range of intrinsic activities of cyclotides also continues to hold promise for a wide range of applications in the agricultural fields.
External links
- Cybase - Calculated orientations of cyclotides in membrane