How many fibrous proteins are there?

Fibrous Proteins

Fibrous proteins contain polypeptide chains organized approximately in parallel along a single axis, producing long fibers or large sheets. Such proteins tend to be mechanically strong and resistant to solubilization in water. Fibrous proteins often play a structural role in nature. For example, α-keratin is composed of α-helical segments of polypeptides and is the predominant constituent of claws, fingernails, hair, and horn in mammals. The structure of the α-keratin is dominated by α-helical segments of polypeptide. The structure of the central rod domain of a typical α-keratin is shown in Fig. 4.12. Pairs of right-handed helices wrap around each other to form a left-twisted coiled coil.

Collagen is a rigid, inextensible fibrous protein that is composed of three intertwined polypeptide chains and is a principal constituent of connective tissue in animals, including tendons, cartilage, bones, teeth, skin, and blood vessels. Collagen is the most abundant protein in vertebrates. It is organized in water-insoluble fibers of great strength. A collagen fiber consists of three polypeptide chains wrapped around each other in a ropelike twist, or triple helix (Fig. 4.13). The three individual collagen chains are themselves helices that differ from the α-helix. They are twisted around each other in a superhelical arrangement to form a stiff rod, and the three strands are held together by hydrogen bonds involving the hydroxyproline and hydroxylysine residues.

1.1 Technological Interest in Natural Protein Fibers

Fibrous proteins have been exploited directly for hundreds—in some cases thousands—of years. Examples include silk and wool harvested for textile applications, the use of intestinal collagen to stitch wounds or to “string” instruments of music and sport, and hair serving as the moisture sensor in hygrometers. However, it was late in the twentieth century before materials characterization technology became sufficiently advanced to reveal detailed links between structure and function in these complex materials.

Studies of natural fibers promise a number of potentially useful lessons for materials chemistry and processing. Nature’s range of functional materials represents the success stories of up to four billion years of research and development. Nature has optimized supramolecular self-assembly mechanisms, hierarchical microstructures, property combinations, and in-service durability. Nature’s fibrous materials are not only damage-tolerant but often self-repairing. They offer the attractions of biosynthesis (they are produced from renewable resources), benign processing conditions (they are assembled and shaped in an aqueous environment and at mild temperatures), and biodegradability (they break down into harmless components when exposed to some specific natural environments).

The techniques of molecular biology can be used to genetically engineer host cells or even multicellular organisms that are capable of synthesizing economic quantities of protein for possible processing into fiber. Proteins that already exist in Nature, as well as entirely new materials, can be produced in this way.

Fibrous Proteins

Since fibrous proteins generally have periodical amino acid sequences and higher-order structure, the clarification of their fine structure in the solid state becomes very important not only when discussing the physical and chemical properties, but when obtaining information about the molecular design of synthetic polypeptides. The conformation-dependent 13C CP MAS NMR chemical shifts are particularly useful for the determination of the conformational features of fibrous proteins such as silk fibroin, collagen and wool keratin. For example, the conformational transition of S-carboxymethyl keratin that has low-sulfur fractions (SCMK low-sulfur) has been estimated. For SCMK low-sulfur heated at 200 °C for 3 hr under vacuum, the 13C MAS NMR spectrum shows that each signal becomes broader than those of other treated specimens. This indicates the existence of various conformations and/or different microenvironments in the heated SCMK low-sulfur. Thus, it can be said that the random coil form appears by heating. On the other hand, from X-ray diffraction, the α helix form completely vanishes in SCMK low-sulfur under the same conditions. The difference between the results from X-ray diffraction and NMR spectroscopy suggests that only the packing of the ordered structure (α helix form) in SCMK low-sulfur is disrupted by heating, while the secondary structure is retained.

Fibrous Proteins

Fibrous proteins are the most abundant proteins in the ECM and include the collagen and elastin families. Collagens form between 25% and 35% of the total mammalian protein content. The mammalian genome contains genes for over 40 different collagen subunits that are located on over a dozen different chromosomes [5]. The defining molecular characteristic of collagen subunits is a Gly-X-Y triplet repeat, where X is frequently proline and Y is frequently 4-hydroxyproline. Collagen subunits are synthesized by fibroblasts. After transcription of collagen mRNA on the ribosomes of the rough endoplasmic reticulum, the precursor polypeptides undergo extensive posttranslational modifications in the lumen of the endoplasmic reticulum. These posttranslational modifications include hydroxylation of proline and lysine residues, which requires vitamin C as cofactor. Three collagen subunits subsequently join to form a triple helix, known as the procollagen molecule. Procollagen is secreted by vesicles of the Golgi apparatus into the ECM. In the extracellular space, the procollagen are cleaved to form tropocollagen. Tropocollagen molecules then spontaneously aggregate to form higher order 3-D collagen structures that can be classified into different categories [6].

Fibrillar collagens include types I, II, III, V, XI, XXIV, and XXVII. These collagens are characterized by long triple-helical segments with continuous Gly-X-Y repeats over their full length. This allows them to assemble into cross-striated fibers.

The network-forming collagens include types IV, VIII, and X. These collagens contain sections of Gly-X-Y repeats that are interrupted by other motifs that allow them to assemble into supercoiled net-like structures [7]. Fibril-associated collagens with interrupted triple helices (FACIT) include types IX, XII, XIV, XVI, XX, and XXI. These collagens have characteristic triple-helical segments that are interspersed with nonhelical domains. These collagens attach to the surface of fibrillar collagens [8]. Collagens with transmembrane domains include types XIII, XVII, XXIII, and XXV. These collagens participate in cellular adhesions and are found in hemidesmosomes of the skin, for example [9]. Multiplexin collagens include types XV and XVIII. These collagens have highly interrupted triple helices and large globular domains at their terminal ends. Finally, filamentous and anchoring fibrillar collagens include Types VI, VII, XXVI, and XXVIII. The functions of these collagens include linking the basement membrane to the underlying ECM. The most abundant types of collagens found in the ECM are types I, II, III, IV, and V [6]. Table 13.1 provides a summary of these collagens.

Table 13.1. List of the Most Abundant Types of Collagens [10]

The function of collagen molecules is to provide structural strength to the ECM. Collagen fibers are organized in different patterns depending on the functional requirements of the tissues. For example, type I collagen fibrils in human tendons are arranged in a linear fashion to maximally resist longitudinal tension. In contrast, type IV collagen in renal glomeruli is arranged as networks to withstand the mechanical force of capillary blood flow and to participate in filtration of urine. These functions are reflected in the diverse disorders caused by genetic defects of collagen genes. These disorders range from brittle bone disease in osteogenesis imperfecta [11] to end-stage renal disease in Alport syndrome [12].

Another type of fibrous protein in the ECM is elastin. In contrast to collagen, elastin is encoded in a single gene localized to chromosome 7. The elastin gene has 36 exons that allow extensive alternative splicing of different isoforms [13]. Elastin mRNA is translated by ribosomes of the rough endoplasmic reticulum into elastin precursor polypeptides called tropoelastin. This molecule undergoes posttranslational modifications including hydroxylation. Tropoelastin is then secreted into the ECM [14]. Similar to collagen, several elastin precursors then aggregate to form a 3-D mature elastin molecule. The elastin precursors are covalently cross-linked to each other. It is thought that elastin molecules adopt a coiled confirmation that allows it to expand and recoil in response to tensile stress [15]. This allows elastin to fulfill its function of endowing the ECM with elastic recoil. The function of elastin is reflected in its abundance in connective tissues that require elastic expansion and contraction, including the conducting arteries. Genetic defects of the elastin gene thus result in defective function of these tissues. For example, supravalvular aortic stenosis causes a significant narrowing of the large conduction arteries [16].

Collagen Triple Helix

Collagen is a fibrous protein which is present in abundance in the human body, being a major constituent of skin, bones as well as various connective tissues. It helps in forming a scaffold to provide strength and structure. Collagens have a unique tripeptide repeat sequence with Glycine at every third position and the iminoacids Proline and Hydroxyproline often being present at the other two positions (Bowes and Kenten, 1948; Eastoe, 1955). The first triple helical structure of collagen had two inter chain H-bonds for every three residues (Ramachandran and Kartha, 1954, 1955). This was slightly modified to a one hydrogen-bonded structure (Rich and Crick, 1955). However a second water mediated hydrogen bond is also found to stabilize the triple helix (Ramachandran and Sasisekharan, 1961). Each helix in collagen has left handed screw sense and the major helix formed by three such helices is right handed. Each helix has 10 residues in 3 turns resulting in a pitch of 85.8 Å. The twist between two neighboring helices was found to be −108.8° and rise of ~2.86 Å (Bhattacharjee and Bansal, 2005; Ramachandran and Kartha, 1955; Ramachandran and Sasisekharan, 1961). Molecular defects in biosynthesis of collagen can lead to many rare genetic diseases involving connective tissues like Ehlers-Danlos syndrome (De Paepe, 1998; Gaisl et al., 2017; Miyake et al., 2017; Prockop et al., 1979). Collagen has been shown to have wide range of applications in the medical and cosmetic field as it is biodegradable, biocompatible, easily available and weak antigenic (Cheng et al., 2017; Chvapil, 1977; Chvapil et al., 1973; Lee et al., 2001; Sheikh et al., 2017). In the field of tissue engineering, collagen based biomaterials are used to improve tissue functions (Parenteau-Bareil et al., 2010).

III Relation of Glial Filaments to Neurofilaments and Microtubules

Studies of the fibrous proteins have been hampered by their insolubility in aqueous solvents and once solubilized, their tendency to self-aggregate and co-aggregate with other proteins during purification. Immunologic methods have been essential in resolving problems in isolation and identification of the filament proteins; however, inaccurate interpretations have been proposed due to the lack of understanding of the limitations and insufficient controls in some immunologic methods employed. Until recently, considerable confusion existed concerning the chemical and immunological relationships between the glial filament, neurofilament, and neurotubule proteins (Eng, 1980) (Fig. 1). Previously neurotubule protein had been reported to have similar chemical properties to glial filaments and GFA protein (Johnson and Sinex, 1974; Dahl, 1976a,b; Dahl and Bignami, 1976b; Chan et al., 1977). Neurotubules had also been reported to share common properties with neurofilaments (Wisniewski et al., 1968, 1971; Gaskin and Shelanski, 1976; Iqbal et al., 1977; Dahl and Bignami, 1977). Numerous other studies have suggested that neurofilament proteins have similar chemical and immunologic properties with the GFA protein and glial filaments (Dahl and Bignami, 1976a; Yen et al., 1976; Davison, 1975; Davison and Hong, 1977; Day, 1977; Goldman et al., 1978; Lee et al., 1977). The most likely subunits of mammalian neurofilaments were discovered in the slow component of axonal transport (Hoffman and Lasek, 1975). This protein complex, which consists of three polypeptides with 70,000, 150,000, and 200,000 MW, has been designated the “Lasek” neurofilament triplet. For more details, see the recent reviews (Schlaepfer, 1979; Shelanski and Liem, 1979; Norton and Goldman, 1980). Presently it is generally agreed that the GFA protein is chemically, morphologically, and antigenically distinct from neurotubule and neurofilament proteins. Our early reports (DeVries et al., 1976; Eng et al., 1976b), which indicated that the 50,000-MW protein in the intermediate preparation was GFA protein from astrocytes and not related to neurofilaments, were in contention but have now been confirmed by many laboratories (Bignami and Dahl, 1977; Liem et al., 1978; Schachner et al., 1978; Dahl and Bignami, 1979; Schlaepfer et al., 1979; Chiu et al., 1980).

2.2.1.4 Silk

Silks represent naturally occurring fibrous protein polymer materials created by several insects and spiders for a variety of functions (e.g., web construction, capture of prey, reproduction).41 The filament core of silk is comprised of a protein known as fibroin, a naturally occurring block copolymer of alternating hydrophobic and hydrophilic regions.42 The hydrophobic segments are highly repetitive, consisting primarily of glycine and alanine residues. Hydrophilic segments have more complex sequences and contain charged amino acids including serine and aspartic acid. The hydrophobic portions self-assembled into stable crystalline regions primarily comprised of β-sheets through hydrophobic interactions and hydrogen bonding. Hydrophilic regions limit some of these interactions, rendering these tough fibroins moderately elastic. Coating this core and enabling fibroin fibers to stick to one another is a glue-like mixture of sericin proteins. This unique structure gives rise to silk's unique physical properties: high strength-to-weight ratio, toughness, and elasticity.43

Silk is most often harvested from the Chinese silkworm Bombyx mori. This material can be woven or electrospun into fibrous net membranes, or further processed to generate film, hydrogels, and sponges for stem cell culture. The resulting scaffolds are relatively biocompatible and biodegradable, while providing structural support to encapsulated cells.44

General information

Collagen is a natural fibrous protein found in human cartilage, connective tissue, and bone. Glutaraldehyde cross-linked bovine collagen is a sterile, biocompatible, biodegradable, purified bovine dermocollagen cross-linked with glutaraldehyde, mixed in a phosphate buffered saline solution.

Purified solubilized bovine collagen is used as bio-material for the treatment of soft tissue defects and has been used for the treatment of stress urinary incontinence since the late 1980s [1]. Injected material precipitates at body temperature, forming a matrix allowing fibroblastic infiltration and formation of new tissue. It has been used for cosmetic purposes by injection in the dermis to correct scars and other contour deformities of the skin.

Urethral injection of bovine collagen under local anesthesia is considered safe and effective, with minimal complications [2,3]. It has low antigenicity and is associated with a minimal inflammatory response, although foreign body reactions have occasionally been reported [4]. However, antibody formation is not impossible [5]. Patients with a history of allergic reactions to bovine collagen-derived products should be investigated because of the widespread use of collagen-derived therapeutic devices, the potential for immunological cross-reactivity with dietary collagen (gelatin), and the potential for anaphylaxis.

Artecoll® is a permanent synthetic cosmetic filler substance, composed of 80% bovine collagen and 20% polymethylacrylate. It is used for augmentation of deep wrinkles and is injected subdermally. The collagen is biodegradable with 2–4 months; however, the polymethylacrylate microspheres are non-biodegradable and long-lasting cosmetic effects are achieved.

Gelatin is an important constituent of many drug formulations, including capsules and suppositories. It is also found in some volume replacement solutions (for example Gelofusine). Gelofusine is a colloidal plasma volume expander used in the treatment of hypotension. It is prepared from bovine collagen. Severe non-IgE-mediated anaphylactic (anaphylactoid) reactions after gelofusine occur with an incidence that has been reported at between 0.066% and 0.345% of cases. They are more common in those with known drug allergy and in men. The mechanism may be triggering of non-immune complement C3 activation by colloid particles in the gelatine formulation or by combination with components in the patient’s blood [6].

11.1 Introduction

Collagen is an insoluble fibrous protein of chief importance for proper structure of bones, tendons, skin, among other tissues, and organs. It is abundantly distributed in the extracellular matrix (ECM) and in connective tissue of mammalians, and is the most abundant protein in animals, as it is responsible for maintaining the structural integrity of organs and tissues. Besides its function as a structural protein, collagen has several other biological functions, such as regulation of cell adhesion, migration, and differentiation [1].

Collagen three-dimensional structure is a very well-known one, as it is the most famous prototypical example of a fibrous triple-helix protein. There are at least 20 types of collagen, according to particular structural features; yet, in the human body, 80%–90% collagen belongs to types I, II, and III, which are fibrillar collagens about 300-nm long. Type I [α1(I)]2[α2(I)] is abundant in skin, tendon, bone, ligaments, dentin, and interstitial tissues, type II [α1(II)]3 is mainly found in cartilage, and vitreous humor, and type III [α1(III)]3 is preferentially located in skin, muscle, and blood vessels. The remaining 20%–10% of human collagens belong to, type V [α1(V)]3, a fibrillar collagen similar to type I, and to types VI [α1(VI)][α2(VI)] and IX [α1(IX)][α2(IX)][α3(IX)], both of which are fibril-associated collagens. While type VI is abundant in most interstitial tissues, type IX is mainly found in cartilage, and vitreous humor. Finally, type IV [α1(IV)]2[α2(IV)] is a component of all basal laminae forming two-dimensional reticula, in contrast with the other collagen types, whose molecules pack together to form long and thin fibrils of similar structure [2].

Due to their three-dimensional structure, collagen fibrils have enormous tensile strength; as such, the different collagens and the structures they form all serve the same purpose, which is helping tissues endure stretching while remaining unharmed. Depending on their specific type, collagens differ in their ability to form fibers and to organize those fibers into networks, which underlies the fact that some types of collagens are preferentially located in certain tissues than in others. For example, as mentioned previously, type II collagen is the major collagen in cartilage; its fibrils are smaller in diameter than those from, e.g., type I collagen, and are oriented randomly in the viscous proteoglycan matrix. This provides type II collagen-containing matrices a strength and compressibility that affords high resistance to large deformations in shape, making such matrices suitable to allow joints to absorb shocks [2].

Collagen chains are biosynthesized as longer precursors called procollagens, where some specific proline and lysine residues are hydroxylated by membrane-bound hydroxylases. Procollagens are glycosylated on addition of galactose and glucose to hydroxylysine residues, then long oligosaccharides are added to specific asparagine residues in the C-terminal propeptide, a segment at the C-terminus of procollagen that is absent from mature collagen. Finally, intra-chain disulfide bonds between the N- and C-terminal propeptide sequences align the three chains before the triple helix is formed. The central portions of the chains zipper from C- to N-terminus to form the triple helix. Procollagen is finally cleaved into the mature form, tropocollagen or, simply, collagen, which almost entirely consisting of a triple-stranded helix and is secreted into the extracellular space. In the process of collagen biosynthesis, posttranslational hydroxylations of procollagen are crucial for the assembly of fully functional mature collagen. The activity of prolyl hydroxylases leading to conversion of proline into hydroxyl-proline residues requires an essential cofactor, ascorbic acid (vitamin C); as such, when cells are deprived of ascorbate, as in the disease scurvy, there is a deficient hydroxylation pattern of procollagen chains, which become unable to form stable triple helices or normal fibrils at normal body temperature [3].

Despite the considerable progress regarding in-depth elucidation of collagen triple helices, and of the physicochemical features that are critical for its structural stability, there is still a long way ahead regarding use of collagens or their mimetics, to deal with significant clinical issues, such as, for example, healing of large bone defects. Current treatments for impaired bone regeneration and remodeling mainly settle with the use of auto- or xeno-grafts, which are limited by donor supply and morbidity, insufficient biological viability, and risk of infection. One approach to circumvent this problem has been proposed by Parmar et al. [4] where recombinant bacterial collagens were used as well-defined biological “blank templates” that can be modified to incorporate bioactive and biodegradable peptide sequences within a precise three-dimensional system mimicking articular cartilage. However, appropriate methods for production of recombinant human collagens have not been established yet, mainly due to the specificities of posttranslational hydroxylation of proline residues. Moreover, due to its size and structural complexity, natural collagens are yet an unmet synthetic goal for peptide chemists.

Over the past decade, efforts have been made toward development of collagen-like peptides (CLP), which have been proposed as, for example, synthetic scaffold coatings potentially able to promote bone formation in critically sized segmental defects in rats [5]. CLP have been mainly used for elucidating structural features of the collagen triple helix and of the key factors for its stabilization. Although a large portion of such research is still at an early stage, peptides able to mimic the collagen triple helix constitute promising structural templates for engineering self-assembled, hierarchical constructs similar to natural tissue scaffolds expected to exhibit unique or enhanced biological activities [6]. In this connection, artificial collagen-like fibrils that exhibit some, but not all, the properties of natural collagen have been made accessible through chemical synthesis; despite such mimics are still far from fully functional collagen surrogates, they contribute to an increasing understanding of the mechanical and structural properties of native collagen fibrils [7]. Latest advances in CLP research, toward better understanding of the collagen triple helix are reviewed herein, hopefully contributing to guide future development of artificial collagenous materials for biomedicine and bionanotechnology.