Search

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.

Most protein in hair and wool is of two broad types: keratin intermediate filament-forming proteins (commonly known as keratins) and keratin-associated proteins (KAPs). Keratin nomenclature was reviewed in 2006, but the KAP nomenclature has not been revised since 1993. Recently there has been an increase in the number of KAP genes (KRTAPs) identified in humans and other species, and increasingly reports of variation in these genes. We therefore propose that an updated naming system is needed to accommodate the complexity of the KAPs. It is proposed that the system is founded in the previous nomenclature, but with the abbreviation sp-KAPm-nL*x for KAP proteins and sp-KRTAPm-n(p/L)*x for KAP genes. In this system “sp” is a unique letter-based code for different species as described by the protein knowledge-based UniProt. “m” is a number identifying the gene or protein family, “n” is a constituent member of that family, “p” signifies a pseudogene if present, “L” if present signifies “like” and refers to a temporary “place-holder” until the family is confirmed and “x” signifies a genetic variant or allele. We support the use of non-italicised text for the proteins and italicised text for the genes. This nomenclature is not that different to the existing system, but it includes species information and also describes genetic variation if identified, and hence is more informative. For example, GenBank sequence JN091630 would historically have been named KRTAP7-1 for the gene and KAP7-1 for the protein, but with the proposed nomenclature would be SHEEP-KRTAP7-1*A and SHEEP-KAP7-1*A for the gene and protein respectively. This nomenclature will facilitate more efficient storage and retrieval of data and define a common language for the KAP proteins and genes from all mammalian species.

Keratin-associated proteins (KAPs) are a major component of wool and other keratin-containing tissues. While four KAP1-n proteins have been identified in sheep, only three genes have been described encoding KAP1-1, KAP1-3 and KAP1-4. Here, we used a sequence conserved across the known KAP1-n genes to search the inaugural Ovine Genome Sequence (v1.0) and identified a new KAP1-n sequence on chromosome 11. PCR amplification of this sequence revealed an open reading frame of 474-bp that putatively encodes a polypeptide sequence very similar to the previously described ovine KAP1-2 protein and suggests that the newly identified sequence represents the previously unidentified KAP1-2 gene (KRTAP1-2). Its expression in skin was confirmed by PCR, and the mRNA was localized to the cortex of the mid-keratinization zone of a growing wool fibre using a gene-specific probe and in situ hybridization. PCR-SSCP analysis of KRTAP1-2 revealed nine unique banding patterns representing nine different DNA sequences. One sequence was identical to, and the other eight were homologous to, the sequence identified above, suggesting that they were allelic variants of ovine KRTAP1-2. There were ten single nucleotide substitutions identified, although only three of these were non-synonymous and would potentially result in amino acid changes. The variation identified here may influence the expression or protein structure of KAP1-2 and consequently wool structure and wool traits.

Keratin IF (KRT) and keratin-associated protein genes encode the majority of wool and hair proteins. We have identified cDNA sequences representing nine novel sheep KRT genes, increasing the known active genes from eight to 17, a number comparable to that in the human. However, the absence of KRT37 in the type I family and the discovery of type II KRT87 in sheep exemplify species-specific compositional differences in hair KRT genes. Phylogenetic analysis of hair KRT genes within type I and type II families in the sheep, cattle and human genomes revealed a high degree of consistency in their sequence conservation and grouping. However, there were differences in the fibre compartmentalisation and keratinisation zones for the expression of six ovine KRT genes compared with their human orthologs. Transcripts of three genes (KRT40, KRT82 and KRT84) were only present in the fibre cuticle. KRT32, KRT35 and KRT85 were expressed in both the cuticle and the fibre cortex. The remaining 11 genes (KRT31, KRT33A, KRT33B, KRT34, KRT36, KRT38-39, KRT81, KRT83 and KRT86-87) were expressed only in the cortex. Species-specific differences in the expressed keratin gene sets, their relative expression levels and compartmentalisation are discussed in the context of their underlying roles in wool and hair developmental programmes and the distinctive characteristics of the fibres produced.

The catalogue of hair keratin intermediate filaments (KIFs) and keratin-associated proteins (KAPs) present in wool follicles is incomplete. The full coding sequences for three novel sheep KIFs (KRT27, KRT35 and KRT38) and one KAP (KRTAP4-3) were established in this study. Spatial expression patterns of these and other genes (KRT31, KRT85, KRTAP6-1 and trichohyalin) were determined by in situ hybridisation in wool follicles at synchronised stages of growth. Transcription proceeded in the order: trichohyalin, KRT27, KRT85, KRT35, KRT31, KRT38, KRTAP6-1 and KRTAP4-3, as determined by increasing distance of their expression zones from the germinal matrix in anagen follicles. Expression became gradually more restricted to the lower follicle during follicle regression (catagen), and ceased during dormancy (telogen). Some genes (KRT27, KRT31, KRT85 and KRTAP6-1), but not others, were expressed in cortical cells forming the brush-end, indicating specific requirements for the formation of this anchoring structure. The resumption of keratin expression was observed only in later stages of follicle reactivation (proanagen). KIF expression patterns in primary wool follicles showed general resemblance to their human homologues but with some unique features. Consistent differences in localisation between primary and secondary wool follicles were observed. Asymmetrical expression of KRT27, KRT31, KRT35, KRT85 and trichohyalin genes in secondary follicles were associated with bulb deflection and follicle curvature, suggesting a role in the determination of follicle and fibre morphology.