Collagen hybridizing peptide

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Schematic of a CHP strand (labeled with an "X" tag) hybridizing to denatured collagen chains and forming a collagen triple helix. During disease progression, tissue development, or ageing, collagen can be extensively degraded by collagenolytic proteases, causing its triple helix to unfold at the physiological temperature due to reduced thermal stability. X may represent a biotin or fluorescent tag. CHP strand schematic.jpg
Schematic of a CHP strand (labeled with an "X" tag) hybridizing to denatured collagen chains and forming a collagen triple helix. During disease progression, tissue development, or ageing, collagen can be extensively degraded by collagenolytic proteases, causing its triple helix to unfold at the physiological temperature due to reduced thermal stability. X may represent a biotin or fluorescent tag.

A collagen hybridizing peptide (CHP) 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. [1] [2] 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. [1] [3] In the single-stranded (monomeric) status, the peptide can recognize denatured collagen strands in tissues by forming a hybridized triple helix with the collagen strands. [2] 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. [4] The binding does not depend on a specific sequence or epitope on collagen, enabling CHPs to target denatured collagen chains of different types. [5] [6]

Contents

Collagen, CHP, CMP, and CLP

Schematic showing relationship between the CMP and CHP. Triple helical CMPs can be heated (above a defined temperature) to dissociate into monomeric CHPs; upon cooling, CHP strands can re-assemble into a triple helix over time. CMP vs CHP.jpg
Schematic showing relationship between the CMP and CHP. Triple helical CMPs can be heated (above a defined temperature) to dissociate into monomeric CHPs; upon cooling, CHP strands can re-assemble into a triple helix over time.

Collagen is the main component of the extracellular matrix (ECM). [7] The collagen superfamily consists of 28 different types of collagen. [7] Although the function and hierarchical structure of these collagens may vary, they all share the defining structural feature known as the triple helix, [1] where three left handed polyproline II-type (PPII) helices assemble to form a right-handed supercoiled helical motif. [1] [8] Short synthetic peptides known as collagen mimetic peptides (CMPs) or collagen-like peptides (CLPs) 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. [3] [9] [10] [11] 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. [2]

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 (collagen triple helix) for folding and chain assembly, as opposed to specific epitopes binding that is seen for monoclonal antibodies (mAbs), for example. [12] Due to their unique targeting mechanism, CHPs have a high binding specificity towards denatured collagen chains but have almost no affinity for intact (triple helical) collagen. [13] CHPs can broadly target collagen chains that have been denatured by thermal, [13] chemical, [14] mechanical, [15] or enzymatic processes, [13] as well as multiple collagen types (e.g., Col I, II, IV). [5] [6] Studies also showed CHPs and their fluorophore conjugates have superior stability in contact with serum. [16]

Denatured collagen as a biomarker for tissue remodelling and damage

A fluorescence image of an axial cross section of a mouse heart at day 14 post myocardial infarction, stained with Hoechst 33342 (blue) and biotin-labeled CHP (detected with AlexaFluor647-streptavidin, red). Scale bar: 1 mm. CHP mouse heart section.jpg
A fluorescence image of an axial cross section of a mouse heart at day 14 post myocardial infarction, stained with Hoechst 33342 (blue) and biotin-labeled CHP (detected with AlexaFluor647-streptavidin, red). Scale bar: 1 mm.

Controlled collagen turnover is crucial for embryonic development, organ morphogenesis, as well as tissue maintenance and repair. [17] 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. [17] 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 (MMP) activity or quantifying collagen peptide fragments in urine, serum, or synovial fluid. [18] [19] [20] Using conventional methods for directly targeting collagen, researchers have to relied on collagen binding peptides selected by phage display, [21] derived from collagen binding proteins, [22] 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. [2] [23] In contrast, CHPs, in principle, can bind to all types of denatured collagens. [4] [5] [6]

Applications

Tissue staining

A fluorescence image of a sagittal section of an 18 d.p.c. mouse embryo double stained with biotinylated-CHP (detected by AlexaFluor647-streptavidin, orange) and an anti-collagen I antibody (detected by AlexaFluor555-labeled donkey anti-rabbit IgG H&L, cyan). mx, maxilla; md, mandibular bone; bp, basisphenoid bone; bo, basioccipital bone; vc, vertebral column; rb, rib; h, hipbone; d, digital bones. Scale bar: 3 mm. CHP mouse embryo image.jpg
A fluorescence image of a sagittal section of an 18 d.p.c. mouse embryo double stained with biotinylated-CHP (detected by AlexaFluor647-streptavidin, orange) and an anti-collagen I antibody (detected by AlexaFluor555-labeled donkey anti-rabbit IgG H&L, cyan). mx, maxilla; md, mandibular bone; bp, basisphenoid bone; bo, basioccipital bone; vc, vertebral column; rb, rib; h, hipbone; d, digital bones. Scale bar: 3 mm.

Fluorophore- or biotin-labeled CHPs are used as a staining agent for detecting collagen degradation and denaturation via immunofluorescence and immunohistochemistry applications. [5] CHPs can stain frozen tissue sections, formalin-fixed paraffin embedded (FFPE) sections, [5] as well as fresh tissues. [14] [15] CHP is applicable to tissue specimens from multiple species and a range of diseases, such as myocardial infarction, arthritis, nephritis, and fibrosis. [5]

In vivo imaging

CHPs can also be labelled with near-infrared fluorophores for in vivo fluorescent imaging. [13] [24]

Collagen identification

CHPs can be used for visualizing many different types of collagen bands in SDS-PAGE gels. [6] Collagen is denatured by heating in the presence of SDS prior to loading the gel. The collagen bands are visualized through CHP-collagen hybridization when the gels are stained by fluorescently-labeled CHPs. [6]

Detecting mechanical damage to connective tissue

Collagen offers mechanical strength in load bearing tissues in the body such as tendons, ligaments, and bone. As forces are applied to these tissues, the collagen triple helix can be damaged and unwind, and CHPs allow for molecular level detection of mechanical damage in such connective tissues. [15] [25]

Related Research Articles

<span class="mw-page-title-main">Collagen</span> Most abundant structural protein in animals

Collagen is the main structural protein in the extracellular matrix found in the body's various connective tissues. As the main component of connective tissue, it is the most abundant protein in mammals, making up from 25% to 35% of the whole-body protein content. Collagen consists of amino acids bound together to form a triple helix of elongated fibril known as a collagen helix. It is mostly found in connective tissue such as cartilage, bones, tendons, ligaments, and skin. Vitamin C is vital for collagen synthesis, and Vitamin E improves the production of collagen.

<span class="mw-page-title-main">Collagen helix</span> Main protein structure of fibrous collagen

In molecular biology, the collagen triple helix or type-2 helix is the main secondary structure of various types of fibrous collagen, including type I collagen. In 1954, Ramachandran & Kartha advanced a structure for the collagen triple helix on the basis of fiber diffraction data. It consists of a triple helix made of the repetitious amino acid sequence glycine-X-Y, where X and Y are frequently proline or hydroxyproline. Collagen folded into a triple helix is known as tropocollagen. Collagen triple helices are often bundled into fibrils which themselves form larger fibres, as in tendons.

<span class="mw-page-title-main">Immunofluorescence</span> Technique used for light microscopy

Immunofluorescence(IF) is a light microscopy-based technique that allows detection and localization of a wide variety of target biomolecules within a cell or tissue at a quantitative level. The technique utilizes the binding specificity of antibodies and antigens. The specific region an antibody recognizes on an antigen is called an epitope. Several antibodies can recognize the same epitope but differ in their binding affinity. The antibody with the higher affinity for a specific epitope will surpass antibodies with a lower affinity for the same epitope.

<span class="mw-page-title-main">Z-DNA</span> One of many possible double helical structures of DNA

Z-DNA is one of the many possible double helical structures of DNA. It is a left-handed double helical structure in which the helix winds to the left in a zigzag pattern, instead of to the right, like the more common B-DNA form. Z-DNA is thought to be one of three biologically active double-helical structures along with A-DNA and B-DNA.

A coiled coil is a structural motif in proteins in which 2–7 alpha-helices are coiled together like the strands of a rope. They have been found in roughly 5-10% of proteins and have a variety of functions. They are one of the most widespread motifs found in protein-protein interactions. To aid protein study, several tools have been developed to predict coiled-coils in protein structures. Many coiled coil-type proteins are involved in important biological functions, such as the regulation of gene expression — e.g., transcription factors. Notable examples are the oncoproteins c-Fos and c-Jun, as well as the muscle protein tropomyosin.

<span class="mw-page-title-main">Classical complement pathway</span> Aspect of the immune system

The classical complement pathway is one of three pathways which activate the complement system, which is part of the immune system. The classical complement pathway is initiated by antigen-antibody complexes with the antibody isotypes IgG and IgM.

<span class="mw-page-title-main">Single-domain antibody</span> Antibody fragment

A single-domain antibody (sdAb), also known as a Nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, single-domain antibodies are much smaller than common antibodies which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments and single-chain variable fragments.

<span class="mw-page-title-main">Gp41</span> Subunit of the envelope protein complex of retroviruses

Gp41 also known as glycoprotein 41 is a subunit of the envelope protein complex of retroviruses, including human immunodeficiency virus (HIV). Gp41 is a transmembrane protein that contains several sites within its ectodomain that are required for infection of host cells. As a result of its importance in host cell infection, it has also received much attention as a potential target for HIV vaccines.

<span class="mw-page-title-main">Collagen, type III, alpha 1</span>

Type III Collagen is a homotrimer, or a protein composed of three identical peptide chains (monomers), each called an alpha 1 chain of type III collagen. Formally, the monomers are called collagen type III, alpha-1 chain and in humans are encoded by the COL3A1 gene. Type III collagen is one of the fibrillar collagens whose proteins have a long, inflexible, triple-helical domain.

<span class="mw-page-title-main">Complement component 1q</span> Protein complex

The complement component 1q is a protein complex involved in the complement system, which is part of the innate immune system. C1q together with C1r and C1s form the C1 complex.

Collagen IV is a type of collagen found primarily in the basal lamina. The collagen IV C4 domain at the C-terminus is not removed in post-translational processing, and the fibers link head-to-head, rather than in parallel. Also, collagen IV lacks the regular glycine in every third residue necessary for the tight, collagen helix. This makes the overall arrangement more sloppy with kinks. These two features cause the collagen to form in a sheet, the form of the basal lamina. Collagen IV is the more common usage, as opposed to the older terminology of "type-IV collagen". Collagen IV exists in all metazoan phyla, to whom they served as an evolutionary stepping stone to multicellularity.

FACIT collagen is a type of collagen and also a proteoglycan that have two or more triple-helical domains that connect to collagen fibrils and share protein domains with non-collagen matrix molecules. FACIT collagens derive their name from their association and interaction with fibrillar collagens. Unlike fibrillar collagens, which form long fibers.

<span class="mw-page-title-main">Heat shock protein 47</span> Protein-coding gene in the species Homo sapiens

Heat shock protein 47, also known as SERPINH1 is a serpin which serves as a human chaperone protein for collagen.

<span class="mw-page-title-main">Collagen, type XVII, alpha 1</span> Mammalian protein found in humans

Collagen XVII, previously called BP180, is a transmembrane protein which plays a critical role in maintaining the linkage between the intracellular and the extracellular structural elements involved in epidermal adhesion, identified by Diaz and colleagues in 1990.

<span class="mw-page-title-main">Immunolabeling</span> Procedure for detection and localization of an antigen

Immunolabeling is a biochemical process that enables the detection and localization of an antigen to a particular site within a cell, tissue, or organ. Antigens are organic molecules, usually proteins, capable of binding to an antibody. These antigens can be visualized using a combination of antigen-specific antibody as well as a means of detection, called a tag, that is covalently linked to the antibody. If the immunolabeling process is meant to reveal information about a cell or its substructures, the process is called immunocytochemistry. Immunolabeling of larger structures is called immunohistochemistry.

<span class="mw-page-title-main">Collagen, type IV, alpha 3</span> Protein found in humans

Collagen alpha-3(IV) chain is a protein that in humans is encoded by the COL4A3 gene.

<span class="mw-page-title-main">Collagen, type IV, alpha 1</span> Protein found in humans

Collagen alpha-1(IV) chain (COL4A1) is a protein that in humans is encoded by the COL4A1 gene on chromosome 13. It is ubiquitously expressed in many tissues and cell types. COL4A1 is a subunit of the type IV collagen and plays a role in angiogenesis. Mutations in the gene have been linked to diseases of the brain, muscle, kidney, eye, and cardiovascular system. The COL4A1 gene also contains one of 27 SNPs associated with increased risk of coronary artery disease.

<span class="mw-page-title-main">Collagen, type XV, alpha 1</span> Protein found in humans

Collagen alpha-1(XV) chain is a protein that in humans is encoded by the COL15A1 gene.

<span class="mw-page-title-main">Triple helix</span> Set of three congruent geometrical helices with the same axis

In the fields of geometry and biochemistry, a triple helix is a set of three congruent geometrical helices with the same axis, differing by a translation along the axis. This means that each of the helices keeps the same distance from the central axis. As with a single helix, a triple helix may be characterized by its pitch, diameter, and handedness. Examples of triple helices include triplex DNA, triplex RNA, the collagen helix, and collagen-like proteins.

<span class="mw-page-title-main">Yu-Shan Lin</span> Taiwanese chemist

Yu-Shan Lin is a computational chemist. She is an associate professor of chemistry at Tufts University in the United States. Her research lab uses computational chemistry to understand and design biomolecules, with topics focusing on cyclic peptides, protein folding, and collagen.

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