Keratin Proteins—A Brief History

The intermediate filaments (IF) of mammalian epithelial cells are obligate heteropolymers built from two classes of IF protein: type I and type II keratins. These type-specific distinctions were originally based on biochemical properties such as protein molecular weight and isoelectric point (^{Bowden et al, 1987}): type I keratins are smaller (40–65 kDa) and more acidic (pI 4.5–6.0) than type II keratins (50–70 kDa, pI 6.5–8.5). The early nomenclature (^{Moll et al, 1982}) listed eight type II epithelial keratins (K1–K8) and 11 type I epithelial keratins (K9–K19) using the accepted abbreviation for keratin proteins (K). In addition, size differences were detected in epidermal corneocyte keratins that could only be explained by post-translational modification, involving both cleavage of the N- and C-termini and phosphorylation (^{Bowden et al, 1984, 1987}). Furthermore, it was established that K11 in the original human keratin catalogue (^{Moll et al, 1982}) was a modified form of K10, so K11 does not appear to exist in the human genome. Investigations of the human hair follicle identified a new group of "trichocyte" or "hair-specific" keratins (Ha1-4 and Hb1-4) from which hair cortical and cuticle intermediate filaments are polymerized (^{Heid et al, 1986}). These proteins are also abundant in nail plate (^{Bowden et al, 1987}). Relating these findings to the early research on sheep wool keratins, however, proved difficult because of nomenclature differences and the different protein extraction and analysis methods employed. Because of the extreme difficulties in solubilizing wool structural proteins, much of the analysis was carried out after treatment with iodoacetate (S-carboxy-methylation), which alters the size of the extracted proteins (^{Powell and Rogers, 1986}).

Keratin cDNA and Gene Sequencing

Initial sequencing of the cDNA encoding keratin proteins showed that there was conservation of the -helical coiled-coil sequences within each type (^{Hanukoglu and Fuchs, 1983}), whereas the N- and C-terminal regions of specific keratin pairs were similar. For example, the differentiation-specific keratins of the epidermis (K1 and K10) both had glycine-rich terminal sequences (^{Bowden et al, 1987}), which made the filaments highly insoluble. Characterization of the first hair-specific keratin from mouse showed that the central -helical core was similar to the epidermal keratins but the N- and C-termini were cysteine-rich (^{Bertolino et al, 1988}), allowing them to form disulfide bridges with the high-sulfur matrix proteins of the hair fiber. Furthermore, at the level of cDNA and gene sequences, there was close homology among mouse hair, human hair, and sheep wool keratins, making cross-species comparisons much easier than at the protein level (^{Powell et al, 1992; Bowden et al, 1994}). More recently, a catalogue of human type I hair-specific keratins has been compiled (^{Langbein et al, 1999}), bringing the total of type I proteins in the hair to fiber nine. Further additions to the number of keratin genes have been made very recently with the finding that other specialized keratins exist in the hair follicle. This has been principally because of expansion in the number of known genes encoding similar K6 proteins. Initially, only two K6 proteins were identified (K6a and K6b), but evidence was then presented for up to six isoforms of K6 (^{Takahashi et al, 1995}), each having their own genes (KRT6a–6f). This was followed by the discovery of another distinct hair follicle K6 (K6hf) and the discovery of inner root sheath (IRS)-specific keratins (K6irs) in human and mouse hair follicles (^{Winter et al, 1998; Porter et al, 2001; Langbein et al, 2003}). Type I correlates of K6irs (termed IRSa1, IRSa2 and IRSa3.1) have also been identified in sheep and human hair follicles (^{Bawden et al, 2001}), as well as in the mouse (^{Porter et al, 2004}). Thus, the keratin gene family was still expanding.

Chromosomal Localization of Keratin Genes

Early attempts at chromosomal in situ hybridization showed that type I keratin genes were located at two loci (17p11–12 and 17q11–22) on chromosome 17 (^{Rosenberg et al, 1988}), whereas type II keratin genes localized to a single cluster (12q11–13) on chromosome 12 (^{Popescu et al, 1989}). The only exception to this general rule is the simple epithelial keratin (K18), a type I keratin gene (KRT18) that occurs in the type II cluster on chromosome 12, an observation made in early localization studies (^{Waseem et al, 1990}). In situ hybridization was later extended to include the human hair-specific keratin genes; these were found to be organized in the same type-specific clusters as the epithelial keratin genes (^{Rogers et al, 1995; Bowden et al, 1998}). These observations were of vital importance to investigating keratin-based genodermatoses, which ultimately linked to the two clusters on chromosomes 12 and 17, providing strong evidence for keratin mutation-based disease (see^{Irvine and McLean, 1999} for a review on diseases).

Keratin Clusters in the Human Genome

Early molecular genetics research showed that keratin genes were found in type-specific clusters (^{Powell et al, 1986}). These could be isolated by probing cosmid clones (40 kb in size) that contained 2–4 keratin genes and the intervening sequences (^{Bowden, 1993}). This approach was later replaced by screening larger genomic DNA fragments contained within P1 artificial chromosome and bacterial artificial chromosome clones (140 kb in size). This proved very effective in establishing the initial extent of the two human hair-specific keratin IF gene clusters (^{Rogers et al, 1998, 2000}), as well as establishing a cluster of human keratin-associated proteins (KAP) that are essential components of hair, wool, nail, and claw (^{Rogers et al, 2001}).

The human type I keratin gene cluster was found to be at least 140 kb and this contained one pseudogene () and nine functional genes (KRTHa6–KRTHa5–KRTHa2–KRTHa8–KRTHa7–KRTHaA–KRTHa1–KRTHa4–KRTHa3-II–KRTHa3-I) that encoded trichocyte "hard" keratins (^{Rogers et al, 1998}). The keratin genes in this cluster varied in size (4–7 kb), and were variably spaced (5–8 kb), but the direction of transcription was the same for all genes. A 300 kb portion of the human type II keratin gene cluster was also described a few years ago (^{Rogers et al, 2000}). This cluster contained four pseudogenes () and six functional hair-specific keratin genes that encoded trichocyte keratins (KRTHbA–KRTHb2–KRTHb4–KRTHb5–KRTHbB–KRTHb3–KRTHb6–KRTHb1–KRTHbC–KRTHbD). In addition, the margins of this larger cluster also contained epithelial keratin genes encoding KRT6b and KRT6hf upstream of the first pseudogene (KRTHbA) and KRT7 downstream of the last pseudogene (KRTHbD), providing a link between these functionally different keratins. In general, type II keratin genes were larger than the equivalent type I genes and ranged in size from 5 to 14 kb. They were also spaced more widely apart (5–19 kb), and some differed in the direction of transcription.

Keratin Bioinformatics

The completion of the human genome sequence and the use of bioinformatics have now allowed a final appraisal of the keratin gene clusters on chromosomes 12 and 17. Recent bioinformatics has identified a cluster of 26 functional type II keratin genes, one functional type I keratin gene (KRT18), five type II keratin pseudogenes, and three unrelated pseudogenes spanning 783 kb of human chromosome 12 (^{Hesse et al, 2004}). The boundaries of the type II keratin gene cluster are: TENC1–EIF4B–KRT18–KRT8 at one end and KRT7–KRTb20–FLJ11773–NR4A1 at the other. In addition, Hesse and colleagues have established that there are 27 functional type I keratin genes and four pseudogenes in the type I cluster on human chromosome 17. Furthermore, the human type I keratin gene cluster has been interrupted by a domain of multiple KAP genes (^{Rogers et al, 2001, Hesse et al, 2004}).

The report in this issue by Rogers and colleagues details their latest work on the type II keratin gene cluster, which in principle agrees with the findings of^{Hesse et al (2004)}. This may represent the final gene count for the type II cluster on human chromosome 12. The cluster spans approximately 830 kb and contains 27 functional keratin genes and eight pseudogenes. There does appear to be a slight difference of opinion, however, with regard to the nature and location of the pseudogenes. Rogers and colleagues have identified three keratin pseudogenes (KRTE, KRTF, and KRTG) between KRT2p and KRT1b, with another keratin pseudogene (KRTH) between KRT2e and KRT6irs3. In contrast,^{Hesse et al (2004)} have identified three non-keratin pseudogenes (Bart1, MGC13007, and prp28) and a keratin pseudogene (KRT19P) in the region between KRT2p and KRT1b, whereas they indicate that there are no genes between KRT2e and KRT6irs3. Both groups agree on the location of the other four pseudogenes and the location of the functional keratin genes. Thus, there is still some debate as to the final structure of this region even when the full sequence is available from the Human Genome Project.

Both groups agree on the identity and location of the "new" keratin genes found (KRT1b, KRT6l, KRT5b, and KRTb20). It should be noted, however, that KRT1b was cloned (^{Popescu et al, 1989}) and sequenced¹,² 15 y earlier under the guise of "HK?". At the time, it was not known whether this was a pseudogene or a functional gene, but Rogers and Langbein have now proved this to be functional and shown that it is expressed in the ducts of eccrine sweat glands.³ They have also shown that K5b and Kb20 are expressed in the tongue and K6l is expressed in scalp epidermis.

Another important contribution made by the current article from Rogers and colleagues revolves around the large number of K6 genes. Six isoforms of K6 were identified about 10 y ago (^{Takahashi et al, 1995}), but it has now been established that some of these differences probably represented polymorphisms in the human population. Thus, current bioinformatics work only provides evidence for three human K6 genes in the context of K6 isoforms (KRT6a, KRT6b, and KRT6h), and four of the originally designated genes (KRT6c, KRT6d, KRT6e, and KRT6f) do not exist in the human genome. Furthermore, Rogers and colleagues have found that KRT6c and KRT6d closely resemble KRT6a, whereas KRT6e and KRT6f closely resemble KRT6h, indicating these are polymorphic variants.

Finally, there is a nomenclature crisis in the keratin field, which is highlighted by the article in this issue and that by^{Hesse et al (2004)}. The currently accepted abbreviation for keratin proteins (including cytokeratins, an alternative term for epithelial keratins) is "K#" and "KRT#" for keratin genes (where # identifies the specific keratin). Owing to the "closed" nature of the original nomenclature (^{Moll et al, 1982}), and the subsequent discovery of over 50 functional keratin genes, however, a new nomenclature is sought. This is currently being reviewed by an international committee of experts in collaboration with the Human Genome Nomenclature Committee (HGNC) to obtain a unified "open" system that will clarify the current situation and allow any new genes discovered in any species to be subsequently added. There is a wish to preserve the original nomenclature but the need for an all-encompassing new keratin nomenclature is now an urgent matter.

Notes

¹ Bowden PE, Withers AP: Characterisation of a human keratin gene (HK?) linked to HK1. Br J Dermatol 125:478, 1991 (abstr).

² Bowden PE, Haley JL, Rothnagel JA: Characterisation of a new human type II keratin. J Invest Dermatol 106:907, 1996 (abstr).

³ Langbein L, Rogers MA, Praetzel S, Cribier B, Gassler N, Schweizer J: K1b—a new member of the human type II keratin gene family is specifically expressed in eccrine sweat gland ducts. J Invest Dermatol 123:A9, 2004 (abstr).

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