By: Klaus D. Linse
Collagen mimetic peptides, or CMPs, are typically made of 30 or fewer amino acids. These types of peptides are usually composed of multiple helix promoting peptide trimers. Collagens are integral structural proteins which are among the most diverse and abundant proteins found in the animal kingdom where they play key functional roles in cellular modulation. Therefore these proteins have attracted scientists in the research fields of supramolecular chemistry, biomedical and materials science in recent years as a guide for the design of unique synthetic biomolecules. In the last three decades CD4 α-turn mimetic peptides that inhibit human immunodeficiency virus envelope glycoprotein gpl20 binding and infection of human lymphocytes have been designed and synthesized. In addition the design of similar peptide mimics has blossomed as well. Collagen mimetic peptides were initially developed and used by biochemists for the investigation and elucidation of the structures and stability of natural collagens. Biologists and polymer chemists followed soon to produce nanostructured fibrous scaffolds using collagen mimetic peptides as the building blocks. The design of CMPs is based on ProProGly and ProHypGly trimer sequence motifs. The best characterized CMPs to date contain the collagen-like triple-helical structure within their peptide sequence and show reversible melting characteristics that are well documented in the literature. Over the years modern synthesis methods have been developed employing techniques such as ligation chemistries based on activated esters, click chemistry, carbodiimide chemistry or other ligation chemistries. These methods now provide synthetic scientists versatile strategies to prepare collagen-polymer conjugates. Furthermore, researchers observed that these collagen mimetic peptide conjugates can spontaneously assemble when stimulated accordingly. Development of engineered tissue and organ replacement therapies has increased in recent years, promoting a emand for new approaches to immobilize components derived from outside cells or tissue to natural collagen.
These peptide mimics have similar behavior as amphiphilic peptides that are known to form defined nanostructures such as molecular wires, well defined nanotubes as well as nanovesicles. Amphiphilic peptides have been used as scaffolds for the synthesis of defined nanometer structures in recent years and studies of biological systems on the molecular level in the 20th century revealed that molecular self-assembly is a fundamental process in all living systems.
Wang et al. in 2004 developed an alternative to the conventional “covalent” modification methods called a “physical” modification technique that is based on collagen’s native ability to associate into a triple-helical molecular architecture. Chemical coupling of synthetic moieties to amino acid side chains such as lysines (K, Lys) or glutamines (E, Glu) is a routinely used technique for such purposes. Unfortunately, these types of coupling reactions are difficult to control when used on large proteins and generally are not easy to control when modifying integrated collagen scaffolds that contain live cells and tissues. To circumvent this the research group synthesized collagen mimetic peptides containing the sequence -(Pro-Hyp-Gly)- multiple times. The scientists report that these peptides exhibit a strong affinity to both native and gelatinized type I collagen under controlled thermal conditions. Furthermore, they show that the cell adhesion characteristics of collagen can be readily altered by applying a poly (ethylene glycol)-CMP conjugate to a prefabricated collagen film. The next table shows the melting behavior of selected peptides.
Table 1: Melting Transition Temperatures of Collagen Mimetic Peptide Derivatives Determined by Circular Dichroism Spectroscopya (Source: Wang et al., 2005).
a Measured in 57.5 µM acetic acid solution.
b 5CF-GGGGPPPHPHGPGGG PPHPPHGPHGPPHPGPHPHPGGPHPHPP, (PH:Hyp).
c mPEG2000, CH3O-(CH2-CH2-O)n-OH, 2250 Da.
The researchers demonstrated the binding of the synthetic CMP to natural acid soluble, bovine type I collagen or denatured gelatin collagen by treating collagen films with solutions of a fluorescently labeled CMP. Results from rinsing the treated collagen films and measuring the fluorescence intensity of the exposed film suggested that 5-carboxy fluorescein (5CF)-labeled-Gly3-(ProHypGly)10- tightly attached to partially denatured collagen when it is introduced as a single strand. The ability of the peptide to assemble into a triple helix appears to be essential for the attachment to happen. The researchers argue that the ability to control the organization of cells in collagen matrices may provide a new pathway for engineered tissues and that the affinity between the CMP and collagen could be used to immobilize therapeutic drugs to collagens in the living tissues and biomaterials that incorporate natural collagens.
In 2007 Rele et al. designed and synthesized collagen-mimetic triple helix promoting peptides that self-assembled into a fibrous structure with well-defined periodicity as visualized by transmission electron microscopy (TEM). The researchers used a Xaa-Yaa-Gly triad sequence to create sequence specific peptides containing three different Xaa-Yaa-Gly domains, including a central core of Pro-Hyp-Gly repeat sequences flanked by distinct sets of peptide repeats, containing either negatively (Glu) or positively (Arg) charged amino acid residues. The Pro-Hyp-Gly peptide sequence was reported to form the structurally critical hydrophobic core of the assembly, responsible for maintaining the thermodynamic stability of the collagen triple-helical structure. Furthermore, the researchers reasoned that the synthesis of collagen-mimetic triple helix peptide protomers (THPs) that display the capacity to form triple helices with improved stability and that exhibit a propensity to form linear assemblies through a process of axially oriented alignment will prove to have a number of important practical applications in the design of novel biomaterials. These types of material may lend themselves for the development of collagen-based biomaterials for wound healing.
In 2008 Cejas et al. synthesized collagen model peptides that form triple helices and self-assemble into supra-molecular fibrils exhibiting collagen-like biological activity without the need for preorganizing the peptide chains by covalent linkages. The researchers accomplished this by placing aromatic groups on the ends of a representative 30-mer CMP, (GPO)10, by using L-phenylalanine and L-pentafluorophenylalanine in 32-mer. The use of atomic force microscopy topographical imaging indicated that some of these peptides self-organized into microfibrillar species. In addition, two peptides, 1a and 1b, where reported to induce the aggregation of human blood platelets with a potency similar to type I collagen.
Su et al. in 2010 demonstrated that treatment with the apoA-I mimetic peptides, L-4F, D-4F (the peptide Ac-D-W-F-K-A-F-Y-D-K-VA-E-K-F-K-E-A-F-NH2 synthesized from all L- or all D-amino acids, respectively), or L-5F (Ac-D-W-L-K-A-F-Y-D-K-V-F-EK-F-K-E-F-F-NH2, synthesized from all L-amino acids) decreases tumor burden in mice injected with ID8 cells.
Yu et al. in 20011 reviewed progress made in the field of collagen mimetic peptides that are useful for the design and synthesis engineered collagen-like materials for potential biomedical use. The scientists report that the collagen triple helix has become a promising structural motif for engineering self-assembled, hierarchical constructs similar to natural tissue scaffolds. Further, they discuss various CMPs and collagen-like proteins that mimic either structural or functional characteristics of natural collagens. This paper provides helpful information to bioengineers and biomaterials scientists interested in collagen engineering.
Li et al. in 2012 reported the synthesis and use of collagen mimetic peptides (CMPs) that can be phototriggered to fold into triple helix and bind to collagens denatured by heat or by matrix metalloproteinase (MMP) digestion. The peptide binding assays that were used by this research group indicated that the binding is primarily driven by stereo-selective triple-helical hybridization between monomeric CMPs of high triple-helical propensity and denatured collagen strands. Furthermore the scientists showed that photo-triggered hybridization allows specific staining of collagen chains in protein gels as well as photo-patterning of collagen and gelatin substrates. Their in vivo experiments demonstrated that systemically delivered CMPs can bind to collagens in bones, and in articular cartilages and tumors characterized by high MMP activity. They further showed that CMP-based probes can detect abnormal bone growth activity in a mouse model of Marfan syndrome. This approach allowed the researcher targeting the microenvironment of abnormal tissues.
He et al. in 2013 reviewed modern synthesis methods that were developed for the synthesis of collagen mimetic peptide conjugates used in polymer science. These methods employ particular ligation chemistries basing on activated ester, click chemistry, carbodiimide chemistry or other ligation chemistries allowing the preparation of collagen-polymer conjugates. Furthermore, the researchers point out that these conjugates made with collagen mimetic peptides as the building blocks show exciting stimuli responsive or spontaneously assembly behavior.
All these findings have let researchers in the tissue engineering and biomedical field now to speculate that the ability to control the organization of cells in collagen matrices may provide new pathways to engineer new types of tissues. Furthermore, the affinity between the CMPs and collagen could be used to immobilize therapeutic drugs to collagens in living tissues and biomaterials that incorporate natural collagens.
To conclude, highly helical peptides can now be made using a variety of peptide sequences. These types of peptide mimics may have many applications in experimental biology, biomedicine and tissue engineering. Since many peptides can retain ligand-binding properties of proteins from which they are derived they may act as inhibitors of antigen-antibody reactions or of hormone-receptor interactions, which would make them good starting molecules for the design of new types of biomaterials.
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