Collagen hybridizing peptide
A collagen hybridizing peptide is a synthetic peptide sequence with typically 6 to 10 repeating units of the Gly-Xaa-Yaa amino acid triplet, which mimics the hallmark sequence of natural collagens. A CHP peptide usually possesses a high content of proline and hydroxyproline in the Xaa and Yaa positions, which confers it a strong propensity to form the collagen's unique triple helix conformation. In the single-stranded status, the peptide can recognize denatured collagen strands in tissues by forming a hybridized triple helix with the collagen strands. This occurs via the triple helical chain assembly and inter-chain hydrogen bonding, in a manner similar to primers binding to melted DNA strands during PCR. The binding does not depend on a specific sequence or epitope on collagen, enabling CHPs to target denatured collagen chains of different types.
Collagen, CHP, CMP, and CLP
Collagen is the main component of the extracellular matrix. The collagen superfamily consists of 28 different types of collagen. Although the function and hierarchical structure of these collagens may vary, they all share the defining structural feature known as the triple helix, where three left handed polyproline II-type helices assemble to form a right-handed supercoiled helical motif. Short synthetic peptides known as collagen mimetic peptides or collagen-like peptides have played a major role in elucidating the 3D structure of the collagen triple helix, its folding kinetics, and thermal stability as small triple helical models. CMPs, CLPs, and CHPs are all very similar in terms of their amino acid sequences but only when CMPs or CLPs are heated above their melting temperatures, do they exist in the dissociated, single-stranded state and can be considered as CHPs.Binding mechanism
Single-stranded CHPs bind to denatured collagen chains and gelatin in a manner that is unique from other targeting mechanisms, in that they specifically recognize a unique structural motif for folding and chain assembly, as opposed to specific epitopes binding that is seen for monoclonal antibodies, for example. Due to their unique targeting mechanism, CHPs have a high binding specificity towards denatured collagen chains but have almost no affinity for intact collagen. CHPs can broadly target collagen chains that have been denatured by thermal, chemical, mechanical, or enzymatic processes, as well as multiple collagen types. Studies also showed CHPs and their fluorophore conjugates have superior stability in contact with serum.Denatured collagen as a biomarker for tissue remodelling and damage
Controlled collagen turnover is crucial for embryonic development, organ morphogenesis, as well as tissue maintenance and repair. However, changes of collagen homeostasis are associated with numerous diseases and pathological conditions. Excessive collagen degradation may be associated with cancer metastasis, skin ageing, arthritis, and osteoporosis. CHPs can target tissues undergoing remodelling based on their ability to bind to degraded and unfolded collagen strands through triple helix formation. As a targeting moiety, CHPs offer great potential in histopathology, diagnostics, and drug delivery for a wide range of diseases.Most methods for the evaluation of collagen denaturation in disease states are indirect, such as detecting matrix metalloproteinase activity or quantifying collagen peptide fragments in urine, serum, or synovial fluid. Using conventional methods for directly targeting collagen, researchers have to relied on collagen binding peptides selected by phage display, derived from collagen binding proteins, or antibodies raised against collagens. Unfortunately, these compounds cannot target denatured collagens which are unstructured and do not present a defined 3D epitope. In addition, antibodies that were reported to distinguish specific degraded collagen fragments can only recognize one or few collagen types. In contrast, CHPs, in principle, can bind to all types of denatured collagens.