Arrangement of trichokeratin intermediate filaments and matrix in the cortex of Merino wool.

Tomograms of transverse sections of Merino wool fibers obtained from fleeces differing in fiber curvature were reconstructed from image series collected using a 300kV transmission electron microscope. Trichokeratin intermediate filaments (IFs) from the ortho-, para- and mesocortices were modeled from the tomograms. IFs were predominantly arranged in left-handed concentric helices with the relative angle of IFs increasing progressively from the center to the periphery of orthocortex macrofibrils. The median increase in IF angle between adjacent IFs between the center and periphery was 2.5°. The length of one turn of the helical path of an IF was calculated to be approximately 1µm for an IF tilted at 30° and positioned 100nm from the macrofibril center. With the exception of one paracortex macrofibril that weakly resembled an orthocortex macrofibril, all para- and mesocortex macrofibrils modeled had a parallel arrangement of the IFs, with a more ordered arrangement found in the mesocortex. Within the limited sample set, there appeared to be no significant relationship between IF angle and fiber curvature. We examined the matrix/IF ratio (in the form of proportion of matrix to one IF, calculated from IF center-to-center distance and IF diameter) for 28 macrofibrils used for modeling. The proportion of matrix was significantly different in the different cortex cell types, with paracortex having the most (0.61), orthocortex having the least (0.42), and mesocortex being intermediate (0.54). Fibers of different crimp type (high, medium or low crimp) were not significantly different from each other with respect to matrix proportion.

Protein expression in orthocortical and paracortical cells of merino wool fibers.

Crimp and bulk, important wool fiber properties, are thought to be related to differences in the protein composition of the orthocortex and paracortex. Fiber morphological studies have demonstrated that the paracortex has a higher proportion of matrix and cysteine than the orthocortex. While there is some evidence for the differential expression of genes between these cell types in the follicle, this has not been demonstrated satisfactorily in the mature fiber. Using proteolytic digestion of wool fibers, followed by ultrasonic disruption to obtain relatively pure fractions of both cell types, the KAP3 high sulfur protein family was found to be present in higher concentrations in the paracortex. This significant finding provides an explanation for the higher cysteine content reported in the paracortex. This represents an advance in our understanding of protein expression variation in the orthocortex and paracortex, and how this relates to key physical and mechanical properties of wool fibers.

Cortical cell types and intermediate filament arrangements correlate with fiber curvature in Japanese human hair.

Naturally straight and curved human scalp hairs were examined using fluorescence and electron microscopy techniques to determine morphological and ultrastructural features contributing to single fiber curvature. The study excluded cuticle and medulla, which lack known bilateral structural asymmetry and therefore potential to form curved fibers. The cortex contained four classifiable cell types, two of which were always present in much greater abundance than the remaining two types. In straight hair, these cell types were arranged annularly and evenly within the cortex, implying that the averaging of differing structural features would maintain a straight fiber conformation. In curved fibers, the cell types were bilaterally distributed approximately perpendicular to fiber curvature direction with one dominant cell type predominantly located closest to the convex fiber side and the other, closest to the concave side. Electron tomography confirmed that the dominant cell type closest to the convex fiber side contained discrete macrofibrils composed of helically arranged intermediate filaments, while the dominant cell type closest to the concave side contained larger fused macrofibrils composed of intermediate filament arrangements varying from helical to hexagonal arrays approximately parallel to the longitudinal fiber axis. These findings concur with the current hypothesis of hair curvature formation and behavior.

Investigations into charge heterogeneity of wool intermediate filament proteins.

Genomic studies have shown that there are four abundant type I and type II intermediate filament proteins (IFPs) in wool. When separated using 2D-PAGE, the type I IFPs separated into four clearly defined major rows. The type II IFPs separated into two distinct staggered rows. The large number of spots seen by 2D-PAGE has previously been attributed to charge heterogeneity caused by post-translational modification of the protein. However, analysis of wool IFPs by 2D-PAGE techniques and mass spectrometry suggested an absence of phosphorylation or glycosylation modifications. Investigations with both the type I and type II IFPs showed that when single protein spots from a 2D-PAGE separation are eluted, re-focused and re-electrophoresed, several spots are formed on both the acidic and basic side of the original spot. Amino acid analysis, mass spectrometry and Ellman's assay support the hypothesis that the proteins have the same sequence but vary in isoelectric charge, due to differences in exposure of charged residues on the molecular surface. The cause of IFP charge heterogeneity is thus proposed to be a conformational equilibrium between several different forms of the same protein in the rehydration solution used for the first dimension.

Two-dimensional gel electrophoresis of wool intermediate filament proteins.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) methods have been used to provide high-resolution separation of wool intermediate filament proteins (IFPs). An improved method of extraction was developed based on a previously published method. The improved method for extraction eliminates the use of dialysis and freeze-drying between the extraction and rehydration steps, allowing the extraction and rehydration for the first dimension gel to be achieved in one day. Improvements to the method for maintaining reducing conditions and chaotrope constitution, combined with low %T polyacrylamide gels, allowed the high-resolution separation of the two keratin IFP families and their individual family members. The IFPs were separated to produce a clearly defined spot pattern of higher intensity, with numerous minor spots not previously observed, and a marked improvement in the vertical resolution. Further work to analyse the composition of each of the protein spots has been made much easier by being able to separate the IFPs into discrete spots.

Characterization of the exocuticle a-layer proteins of wool.

The outermost protein layer of wool cuticle cells is known as the exocuticle a-layer. This layer is a resistant barrier to the degradation of the fibre and, as a result, little is known of its proteinaceous composition. Merino wool fibres were subjected to both proteolytic and chemical digestion and the resulting material was found by transmission electron microscopy to be highly enriched in a-layer. Amino acid analysis revealed a high cysteine and glycine content, with a close, but not exact, match to the Allwörden membrane. Subsequent digestion of the a-layer preparation by 2-nitro-5-thiocyano-benzoic acid produced a large number of short peptides, and analysis by mass spectrometry revealed peptides with strong homologies to cuticular ultra-high sulphur proteins of sheep wool and cuticular ultra-high and high-sulphur proteins of human hair, thus supporting other evidence for the presence of these sulphur-rich proteins in the a-layer.

The differential expression of proteins in the cortical cells of wool and hair fibres.

Three different cell types have been identified in the cortex of wool: orthocortex, mesocortex and paracortex. Fine wool fibres, particularly Merino sheep, are noted for their bilateral distribution of orthocortical and paracortical cells, with the latter following the concave side of the crimp wave. Furthermore, studies have indicated that the paracortex has a higher concentration of cysteine than the orthocortex. This has been supported by in situ hybridization studies in the follicle that have shown that sulphur-rich proteins are initially expressed on the paracortical side of the fibre, with some becoming more uniformly spread, laterally, over the entire fibre as the keratinization process progresses. In contrast, proteins high in glycine and tyrosine tend to be expressed initially on the orthocortical side of the follicle. While these in vitro studies have pointed to where specific proteins are located in the follicle, elucidating the situation for the mature fibre has been less easy. A range of approaches have been used to separate orthocortical and paracortical cells and these have only been able to provide evidence for a higher level of cysteine in the latter. Electrophoretic studies have found a number of differences in protein expression between the two sides but have not specifically identified which proteins. Thus, there appears to be good evidence for the paracortex containing a higher proportion of proteins in the ultra-high sulphur class but there is some uncertainty regarding the exact distribution of proteins high in glycine and tyrosine.

Determination of photo-oxidation products within photoyellowed bleached wool proteins.

Photo-oxidative processes occurring in wool can lead to significant photoyellowing of the fiber. In particular, wool that has been chemically bleached photoyellows more rapidly and to a greater degree than untreated wool. Direct identification of the chromophores responsible for such yellow discoloration in irradiated wool has proven to be elusive for many years. This article describes the characterization and location of yellow photo-oxidation products within the proteins of photoyellowed bleached wool fabric, using advanced protein chemistry techniques. The discolored fabric was enzymatically digested and chromatographed by high-pressure liquid chromatography, with monitoring at 400 nm, to select out fractions containing yellow chromophoric species. Thorough tandem mass spectrometric analysis was then used to sequence peptides and, in turn, to characterize modifications to key amino acid residues that had resulted in yellow chromophore formation. In total, 11 separate yellow chromophoric species were identified, ten derived from tryptophan residues and one from tyrosine. The tryptophan-derived modifications characterized included hydroxytryptophan, N-formylkynurenine, hydroxyformylkynurenine, kynurenine, hydroxykynurenine, carbolines, tryptophandiones and nitrotryptophan. The tyrosine-derived modification of tyrosine to dopa was also identified. The range of photomodifications we observed provides insight into the photo-oxidation pathways occurring within irradiated fibrous proteins leading to the formation of yellow chromophores.

The three-dimensional arrangement of intermediate filaments in Romney wool cortical cells.

The three-dimensional orientation and arrangement of intermediate filaments in Romney wool ortho-, meso-, and paracortical cells has been revealed using single axis high voltage electron tomography. Modelled tomograms confirm that intermediate filaments in orthocortical cells are arranged helically, with the helical angle progressively increasing from the centre to the periphery of macrofibrils. Intermediate filaments in meso- and paracortical cells display parallel arrangements differing mainly in packing density, with the mesocortex packed more tightly than the paracortex. The intermediate filament arrangements observed confirm expectations based on earlier two-dimensional transmission electron microscopy observations by the authors and other researchers. It is expected that these findings will contribute to a better understanding of the biological and structural basis of wool fibre curvature.

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 high sulphur proteins of wool: Towards an understanding of sheep breed diversity.

High sulphur proteins (HSPs) form part of the matrix surrounding the intermediate filaments in the cortical cells of the wool fibre. There are three known families of HSPs, comprising in excess of 40 components and their molecular weights range from 10-30 kDa. Here we report the use of the increased resolving power of isoelectric focusing in the first dimension of two-dimensional electrophoresis and modern gel comparison software to investigate the nature of within- and between-breed variations amongst the proteins of three breeds of sheep: Merino, Romney and Corriedale. In agreement with past studies we observed very little variation in the intermediate filament protein content in wool, both between and within these three sheep breeds. Instead, most of the observed variation occurred among the HSPs, along with some minor variation among the high glycine-tyrosine proteins. Breed-specific differences were observed in the HSP patterns in the wool proteome maps. Merino sheep were found to exhibit the simplest HSP expression patterns, with eight major spots linked to form four pairs. In contrast, the Romney and Corriedale HSP patterns exhibited more spots at lower isoelectric point values (around 4.8), while some of the lower molecular weight HSPs were less prevalent in Romney sheep and absent from the Corriedales.

Problems associated with the identification of proteins in homologous families: the wool keratin family as a case study.

The keratin proteins from wool can be divided into two classes: the intermediate filament proteins (IFPs) and the matrix proteins. Using peptide mass spectral fingerprinting it was possible to match spots to the known theoretical sequences of some IFPs in web-based databases, as enzyme digestion generated sufficient numbers of peptides from each spot to achieve this. In contrast, it was more difficult to obtain good matches for some of the lower molecular weight matrix proteins. Relatively few peaks were generated from tryptic digests of high-sulfur proteins because of their lower molecular weight and the absence of basic residues in the first two-thirds of the sequence. Their high sequence homology also means that generally only a few of these peptides could be considered to be unique identifiers for each protein. Nevertheless, it was still possible to uniquely identify some of these proteins, while the presence of two peptides in the matrix-assisted laser desorption/ionization time-of-flight mass spectrum allowed classification of other protein spots as being members of this family. Only one major peptide peak was generated by the high-glycine tyrosine proteins (HGTPs) and there were relatively few sequences available in web-based databases, limiting their identification to one HGTP family.