Mapping the accessibility of the disulfide crosslink network in the wool fiber cortex

The disulfide bond network within the cortex of mammalian hair has a critical influence on the physical and mechanical characteristics of the fiber. The location, pattern, and accessibility of free and crosslinked cysteines underpin the properties of this network, but have been very difficult to map and understand, because traditional protein extraction techniques require the disruption of these disulfide bonds. Cysteine accessibility in both trichocyte keratins and keratin associated proteins (KAPs) of wool was investigated using staged labeling, where reductants and chaotropic agents were used to expose cysteines in a stepwise fashion according to their accessibility. Cysteines thus exposed were labeled with distinguishable alkylation agents. Proteomic profiling was used to map peptide modifications and thereby explore the role of KAPs in crosslinking keratins. Labeled cysteines from KAPs were detected when wool was extracted with reductant only. Among them were sequences from the end domains of KAPs, indicating that those cysteines were easily accessible in the fiber and could be involved in forming interdisulfide linkages with keratins or with other KAPs. Some of the identified peptides were from the rod domains of Types I and II keratins, with their cysteines positioned on the exposed surface of the α‐helix. Peptides were also identified from keratin head and tail domains, demonstrating that they are not buried within the filament structure and, hence, have a possible role in forming disulfide linkages. From this study, a deeper understanding of the accessibility and potential reactivity of cysteine residues in the wool fiber cortex was obtained. Proteins 2015; 83:224–234. © 2014 Wiley Periodicals, Inc.     

Sheep wool (glycine + tyrosine)-rich keratin genes. A family of low sequence homology

We report the isolation and complete nucleotide sequence of genes encoding the two major high-(glycine + tyrosine) (HGT) keratins of sheep wool in separate genomic clones. The genes have negligible sequence homology except for an 18-bp conserved element immediately preceding the initiation codon. The same element has been found in the corresponding position of a number of co-expressed high-sulphur (HS) keratin genes and may therefore represent a common element between these wool intermediate-filament-associated proteins. As seen for the HS keratin genes, the HGT keratin genes also lack introns. Southern blot data show that both HGT genes are unique in the sheep genome, indicating that the observed heterogeneity of the HGT protein class may not be as complex as previously suggested.     

Ovine keratome: identification,localisation and genomic organisation of keratin and keratin‐associated proteins

Wool is composed primarily of proteins belonging to the keratin family. These include the keratins and keratin‐associated proteins (KAPs) that are responsible for the structural and mechanical properties of wool fibre. Although all human keratin and KAP genes have been annotated, many of their ovine counterparts remain unknown and even less is known about their genomic organisation. The aim of this study was to use a combinatory approach including comprehensive cDNA and de novo genomic sequencing to identify ovine keratin and KAP genes and their genomic organisation and to validate the keratins and KAPs involved in wool production using ovine expressed sequence tag (EST) libraries and proteomics. The number of genes and their genomic organisation are generally conserved between sheep, cattle and human, despite some unique features in the sheep. Validation by protein mass spectrometry identified multiple keratins (types I and II), epithelial keratins and KAPs. However, 15 EST‐derived genes, including one type II keratin and 14 KAPs, were identified in the sheep genome that were not present in the NCBI gene set, providing a significant increase in the number of keratin genes mapped on the sheep genome.     

The cDNA sequence of a Type II cytoskeletal keratin reveals constant and variable structural domains among keratins

We present the cDNA and amino acid sequences of a cytoskeletal keratin from human epidermis (Mr = 56K) that belongs to one of the two classes of keratins (Type I and Type II) present in all vertebrates. In these two types of keratins the central approximately 300 residue long regions share approximately 30% homology both with one another and with the sequences of other IF proteins. Within this region, all IF proteins are predicted to contain four helical domains demarcated from one another by three regions of beta-turns. The amino and carboxy termini of the Type II keratin are very different from those of microfibrillar keratins and other nonkeratin IF proteins. However, they contain unusual glycine-rich tandem repeats similar to the amino terminus of the Type I keratin. Thus the size heterogeneity among keratins appears to be a result of differences in the length of the terminal ends rather than the structurally conserved central region.     

The effect of oxidation or alkylation on the separation of wool keratin proteins by two-dimensional gel electrophoresis

Comparative two-dimensional electrophoretic (2-DE) studies were performed over a time-course to examine the effect of oxidation or alkylation on the separation of wool keratin proteins. The effect of oxidation was followed by treating scoured wool fibres with increasing levels of hydrogen peroxide, ranging from 0-12 g/L, using conditions mimicking the industrial wool bleaching process. Peroxide treatment was found to have only a minor effect on the 2-DE separation of the intermediate filament protein (IFP) class. Conversely, peroxide treatment of the 24-28 kDa high sulphur protein (HSP) class, which contains up to 40 cysteine residues per protein, resulted in the gradual disappearance of the major HSP spots correlated with the appearance of a few discrete spots at lower isoelectric point (pI). This suggested that only a few specific cysteine residues were being oxidized to cysteic acid by treatment with hydrogen peroxide. Peroxide treatment also appeared to have affected a discrete number of cysteine residues among proteins in the high glycine-tyrosine protein (HGTP) class, reducing the intensity of the high pI spots, while correspondingly increasing the intensity of those at lower pI. In a separate study, wool proteins were alkylated with iodoacetamide (1 M, pH 8) for periods ranging from 10 min to 48 h. In contrast to treatment with peroxide, the pI values of the HSP spots were unaffected by alkylation, irrespective of the length of this treatment. Alkylation resulted in a shift to lower pI and a loss of resolution of individual spots in the Type I and II IFP trains, to the extent that after 24 h alkylation individual spots in these trains merged. In addition after 1 h the intensity of the high pI Type II IFPs decreased until they were no longer visible on the 2-DE map after 24 h. Similarly as alkylation time increased, the major, high pI HGTP spots decreased in intensity. In unison with their decrease, some of the lower pI spots increased in intensity, while new spots appeared at more acidic pIs. Mass spectral studies indicated that cysteine alkylation was relatively fast, with 70-95% of the cysteines in the keratin proteins being alkylated within the first 10 min, while in the case of the HGTPs there was evidence for noncysteine alkylation occurring within this period. Alkylation of proteins for periods of up to 6 h prior to electrofocusing is being promoted as a better alternative to the current 2-DE protocol of the inclusion of a reductant in the immobilized pH gradient rehydration solution. This study has clearly demonstrated that long alkylation times do not suit all protein types or classes.     

The cDNA sequence of a human epidermal keratin: divergence of sequence but conservation of structure among intermediate filament proteins

We have determined the DNA sequence of a cloned cDNA that is complementary to the mRNA for the 50 kilodalton (kd) human epidermal keratin. This provides the first amino acid sequence for a cytoskeletal keratin. Comparison of this sequence with those of other keratins reveals an evolutionary relationship between the cytoskeletal and the microfibrillar keratins, but shows no homology to matrix or feather keratins. The 50 kd keratin shares 28%-30% homology with partial sequences of other intermediate filament proteins, which suggests that keratins may be the most distantly related members of this class of fibrous proteins. Our computer analyses predict that the 50 kd keratin contains two long alpha-helical domains separated by a cluster of helix-inhibitory residues in the middle of the protein. These findings indicate that despite major sequence divergence among intermediate filament proteins, they retain sequences compatible with secondary structural features that appear to be common to all of them.     

Proteomic evaluation of the biodegradation of wool fabrics in experimental burials

Woollen textiles recovered from archaeological excavations are usually fragile, discoloured, mineralised, or highly biodeteriorated fragmentary remains. The nature and extent of preservation is highly dependent on the site of burial and factors such as soil composition, pH, temperature, oxygen content, and contact with a wood coffin or metals. Understanding the particular biodegradation in archaeological sites is important for biomolecular studies of textiles, and to assist in the conservation of these finds. Wool fabrics dyed and buried for up to 8 yr in bog-type soils in Denmark (Lejre) and Norway (Rørmyra), and in marine sediments in Sweden (Marstrand) were evaluated by proteomics analysis. Wool degradation was found to occur through a range of differing mechanisms, mainly due to the complex nature of wool itself with its many families of proteins (keratin and keratin-associated proteins) and structures. Microbial activity was a large contributory factor to the physical deterioration of the wool fabrics at Lejre and Marstrand, and might result in faster loss of keratin-associated proteins over keratins. Additional hydrolysis took place at Marstrand, influenced by the environmental conditions of the sediment, and in particular the alkaline pH, contributing to the degradation of keratins. However, cross-linking was associated with the long-term preservation of the fabrics at Rørmyra, where pH, temperature, and vegetative composition of the bog prevented microbial activity, and sphagnum moss might preserve wool by binding with keratins.     

Monoclonal antibody studies of alpha-keratin low-sulfur proteins

Monoclonal antibodies to the two families of low-sulfur proteins from wool were produced. Selection was applied to identify those hybridomas secreting antibody that was effective in the Electro-blot system. The specificities of eight different monoclonal antibodies were investigated by their binding to alpha-keratin low-sulfur proteins which had been subjected to electrophoresis from wool, goat hair, porcupine quill, rat hair and echidna quill, using the Electro-blot procedure. Considerable cross-reactivity was found both within the low-sulfur protein components of individual keratins from a particular species, and also between the keratins of the different species. Some antibodies were found to bind selectively to components of one family of low-sulfur proteins in wool, while others recognized determinants in both families, indicating some homology between the two families.     

In vitro assembly and structure of trichocyte keratin intermediate filaments: a novel role for stabilization by disulfide bonding

Intermediate filaments (IF) have been recognized as ubiquitous components of the cytoskeletons of eukaryotic cells for 25 yr. Historically, the first IF proteins to be characterized were those from wool in the 1960s, when they were defined as low sulfur keratins derived from "microfibrils." These proteins are now known as the type Ia/type IIa trichocyte keratins that constitute keratin IF of several hardened epithelial cell types. However, to date, of the entire class of >40 IF proteins, the trichocyte keratins remain the only ones for which efficient in vitro assembly remains unavailable. In this paper, we describe the assembly of expressed mouse type Ia and type IIa trichocyte keratins into IF in high yield. In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins. However, when oxidized in vitro, several intermolecular disulfide bonds form and the molecular alignments rearrange into the pattern shown earlier by x-ray diffraction analyses of intact wool. We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF. Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.     

Structural analysis of alpha-helical proteins from wool using cysteine labelling and mass spectrometry

A simple reduction/labelling/extraction protocol has been developed to fractionate cortical matrix proteins from filament proteins in wool. Through differential labelling of cysteine residues their relative accessibility in the wool fibre has been investigated. This has allowed the preliminary development of a map of the chemical functionality that is accessible within wool fibres under native conditions. Protein analyses of wool subjected to mechanical action, wet chemical permonosulphate/sulphite treatment and dry argon plasma treatment revealed that none of these detectably improved the accessibility of functional groups at the wool cortex. It is anticipated that this analytical method can be extended to improve the sensitivity and scope with which chemical functionality within native fibres can be mapped and lead to a better understanding of the potential limits/opportunities for fibre modification.     

Cornification in reptilian epidermis occurs through the deposition of keratin‐associated beta‐proteins (beta‐keratins) onto a scaffold of intermediate filament keratins

The isolation of genes for alpha‐keratins and keratin‐associated beta‐proteins (formerly beta‐keratins) has allowed the production of epitope‐specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha‐keratin range (40–70 kDa) or beta‐protein range (10–30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha‐proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin‐associated beta‐proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin‐associated beta‐proteins is typical for keratin‐associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine‐rich or sulfur‐rich proteins). In turtles and crocodilians epidermis, keratin‐associated beta‐proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine‐rich versus cysteine‐glycine‐rich keratin‐associated beta‐proteins in cells sequentially produced from the basal layer and not from the alternation of beta‐ with alpha‐keratins. The process gives rise to Oberhäutchen, beta‐, mesos‐, and alpha‐layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin‐associated proteins (KAPs) of the epidermis, the keratin‐associated beta‐proteins of sauropsids are capable to form filaments of 3–4 nm which give rise to an X‐ray beta‐pattern as a consequence of the presence of a beta‐pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

The structural relation between intermediate filament proteins in living cells and the alpha-keratins of sheep wool.

Although not complete, the available sequence data on smooth muscle desmin, a prototype of 10 nm filaments present in living vertebrate cells, and two wool alpha-keratin components indicate a common structural motif . A similarly sized rod-like middle domain based mainly on alpha-helices probably able to form coiled-coils is flanked by differently sized terminal domains of non-alpha-helical nature. Within the middle domain there seem to be at least two regions where wool keratins and 10 nm filament proteins show a noticeable degree of sequence homology. In general, however, the proteins have diverged to an astonishing degree. Although the analysis seems to support, in general terms, a separation of the rod into two nearly equally long coiled-coils it raises doubts about additional aspects of current models of 10 nm filament organization. We propose that the terminal domains are directly involved in filament assembly making this process permanent in wool alpha-keratins because of the many disulfide bonds present in these regions. The 10 nm filaments of most living cells seem to avoid this frozen state and lack a similar wealth of cysteine residues.     

Cytokeratin immunoreactivity in lobular intraepithelial neoplasia.

Eighteen commercially available antibodies reactive against different cytokeratin proteins were tested on classic examples of lobular intraepithelial neoplasia (LIN) and of ductal intraepithelial neoplasia (DIN) of the breast. About 90% of higher-grade DIN (AIDH and DCIS) show no or substantially diminished reaction with clone 34betaE12 (specified as reactive against keratins 1, 5, 10, and 14 as determined by the manufacturer), while the cells of LIN were found to express the antigen reactive with this antibody. To determine which of these four keratins are present in the cells of LIN, antibodies reactive against these individual four keratins were tested. None of the four antibodies to keratins 1, 5, 10, or 14 reacted with the cells of LIN. To investigate this further, 13 additional monoclonal antibodies to various other keratin proteins were tested on the cells of LIN. Those that successfully reacted with the cells of LIN were further tested on the cells of DIN. All of the individual antibodies reactive with the cells of LIN were also reactive with the cells of DIN to a degree, with clone RCK108 (reactive against keratin 19) coming the closest to demonstrating the reactivity seen with 34betaE12. We conclude that the reactivity seen in the cells of LIN with 34betaE12 is due to either (a) a crossreaction with keratin 19 that is slightly less prominent than the reaction of the individual clone RCK108, (b) a crossreaction with a keratin protein that was not tested (3, 11, 12), (c) a crossreaction with a protein closely resembling keratin in formalin-fixed, paraffin-embedded tissue, or (d) the detection of a mutated or truncated form of keratin 1, 5, 10, or 14 that cannot be detected by the individual monoclonal antibody.     

The role of β-sheets in the structure and assembly of keratins

X-ray diffraction, infrared and electron microscope studies of avian and reptilian keratins, and of stretched wool and hair, have played a central role in the development of models for the β-conformation in proteins. Both α- and β-keratins contain sequences that are predicted to adopt a β-conformation and these are believed to play an important part in the assembly of the filaments and in determining their mechanical properties. Interactions between the small β-sheets in keratins provide a simple mechanism through which shape and chemical complementarity can mediate the assembly of molecules into highly specific structures. Interacting β-sheets in crystalline proteins are often related to one another by diad symmetry and the data available on feather keratin suggest that the filament is assembled from dimers in which the β-sheets are related by a perpendicular diad. The most detailed model currently available is for feather and reptilian keratin but the presence of related β-structural forms in mammalian keratins is also noted.     

Monoclonal antibody analysis of bovine epithelial keratins. Specific pairs as defined by coexpression

We have characterized the keratin proteins of various bovine epithelial tissues by one- and two-dimensional gel electrophoresis, coupled with the immunoblot technique using AE1, AE2, AE3, AE5, CA20, BE14, and 6.11 monoclonal antikeratin antibodies. The results indicate that all known bovine keratins can be divided into two subfamilies. The "acidic" (Type I) subfamily consists of 41-, 43-, 45-, 46-, 50-, 54-, 56-, and 56.5-kDa keratins, all of which have a pI of less than 5.6, and most of them are recognized by our AE1 antibody, whereas the "neutral-to-basic" (Type II) subfamily consists of 55-, 57-, 58-, 62-65-, 66-, and 67-kDa keratins, all of which have a pI of greater than 6.0 and are recognized by our AE3 antibody. Tissue distribution data and cell culture studies show that, within the two subfamilies, keratins with similar "size ranks" form a "pair" as defined by frequent co-expression. Furthermore, within most "keratin pairs," the basic keratin is larger than the acidic one by 8-10 kDa. These results provide further support for the concepts of "keratin subfamilies" and keratin pairs and are consistent with the possibility that the acidic and basic members of at least some keratin pairs may interact specifically during in vivo tonofilament assembly and/or function. Immunoblotting data derived from the use of several monospecific antibodies show that although the size, charge, and pattern of expression of most bovine keratins are similar to those of the human counterparts, there are important exceptions to this rule.     

Biochemical identification and tissue-specific expression patterns of keratins in the zebrafish Danio rerio

We have identified a number of type I and type II keratins in the zebrafish Danio rerio by two-dimensional polyacrylamide gel electrophoresis, complementary keratin blot-binding assay and immunoblotting. These keratins range from 56 kDa to 46 kDa in molecular mass and from pH 6.6 to pH 5.2 in isoelectric point. Type II zebrafish keratins exhibit significantly higher molecular masses (56–52 kDa) compared with the type I keratins (50–48 kDa), but the isoelectric points show no significant difference between the two keratin subclasses (type II: pH 6.0–5.5; type I: pH 6.1–5.2). According to their occurrence in various zebrafish tissues, the identified keratins can be classified into “E” (epidermal) and “S” (simple epithelial) proteins. A panel of monoclonal anti-keratin antibodies has been used for immunoblotting of zebrafish cytoskeletal preparations and immunofluorescence microscopy of frozen tissue sections. These antibodies have revealed differential cytoplasmic expression of keratins; this not only includes epithelia, but also a variety of mesenchymally derived cells and tissues. Thus, previously detected fundamental differences in keratin expression patterns between higher vertebrates and a salmonid, the rainbow trout Oncorhynchus mykiss, also apply between vertebrates and the zebrafish, a cyprinid. However, in spite of notable similarities, trout and zebrafish keratins differ from each other in many details. The present data provide a firm basis from which the application of keratins as cell differentiation markers in the well-established genetic model organism, the zebrafish, can be developed.     

Homo‐ and heteropolymer self‐assembly of recombinant trichocytic keratins

In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues. However, during extraction from natural sources, such as hair and wool fibers, natural keratins are subject to extensive processing conditions that lead to formation of unwanted by‐products. Additionally, natural keratins suffer from limited sequence tunability. Recombinant keratin proteins can overcome these drawbacks while maintaining the desired chemical and physical characteristics of natural keratins. Herein, we present the bacterial expression, purification, and solution characterization of human hair keratins K31 and K81. The obligate heterodimerization of the K31/K81 pair that results in formation of intermediate filaments is maintained in the recombinant proteins. Surprisingly, we have for the first time observed new zero‐ and one‐dimensional nanostructures from homooligomerization of K81 and K31, respectively. Further analysis of the self‐assembly mechanism highlights the importance of disulfide crosslinking in keratin self‐assembly.     

Evidence of linkage between high-glycine-tyrosine keratin gene loci and wool fibre diameter in a Merino half-sib family

Candidate genes for quantitative trait loci have been studied in a Medium Peppin Merino flock. Obvious candidates for effects on wool production traits are genes for the major proteins expressed in the wool fibre, the keratin and keratin-associated protein genes. Two keratin-associated protein loci, KRTAP6 and KRTAP8, have previously been shown to be linked. The results of analyses between these two loci and production traits gave significant evidence of linkage with wool fibre diameter in one out of eight halfsib groups tested. High-glycine-tyrosine proteins (KRTAP6, 7 and 8) are known to vary considerably in abundance in wool fibres and it is possible that a gene for major effect on fibre diameter is located within the same chromosomal region as KRTAP6 and KRTAP8.     

Localization of low-sulfur keratin proteins in the wool follicle using monoclonal antibodies

Monoclonal antibodies that recognize components of the low-sulfur keratin proteins extracted from Merino wool have been used to locate these components within the wool follicle. Immunoblotting procedures showed that all of the monoclonal antibodies bound more than one of the eight low-sulfur protein components, indicating that these proteins have antigenic determinants in common. Immunofluorescence studies showed that those antibodies specific for the component 7 family of the low-sulfur proteins bound to the developing wool fiber, whereas those antibodies recognizing the component 8 family bound to areas throughout the wool follicle, particularly the inner and outer root sheaths, but also to the fiber, the cuticle, and the epidermis. One of the monoclonal antibodies also bound to intermediate filament networks of cultured human epithelial cells.