Unique amino acid signatures that are evolutionarily conserved distinguish simple-type, epidermal and hair keratins

Abstract

Keratins (Ks) consist of central α-helical rod domains that are flanked by non-α-helical head and tail domains. The cellular abundance of keratins, coupled with their selective cell expression patterns, suggests that they diversified to fulfill tissue-specific functions although the primary structure differences between them have not been comprehensively compared. We analyzed keratin sequences from many species: K1, K2, K5, K9, K10, K14 were studied as representatives of epidermal keratins, and compared with K7, K8, K18, K19, K20 and K31, K35, K81, K85, K86, which represent simple-type (single-layered or glandular) epithelial and hair keratins, respectively. We show that keratin domains have striking differences in their amino acids. There are many cysteines in hair keratins but only a small number in epidermal keratins and rare or none in simple-type keratins. The heads and/or tails of epidermal keratins are glycine and phenylalanine rich but alanine poor, whereas parallel domains of hair keratins are abundant in prolines, and those of simple-type epithelial keratins are enriched in acidic and/or basic residues. The observed differences between simple-type, epidermal and hair keratins are highly conserved throughout evolution. Cysteines and histidines, which are infrequent keratin amino acids, are involved in de novo mutations that are markedly overrepresented in keratins. Hence, keratins have evolutionarily conserved and domain-selectively enriched amino acids including glycine and phenylalanine (epidermal), cysteine and proline (hair), and basic and acidic (simple-type epithelial), which reflect unique functions related to structural flexibility, rigidity and solubility, respectively. Our findings also support the importance of human keratin 'mutation hotspot' residues and their wild-type counterparts.

Fig. 1.

Amino acid composition differs significantly between the keratin head, rod and tail domains. To study the amino acid composition of keratin domains, the human keratin sequences K1–K10, K12–K14, K16–K20, K23, K24, K31, K35, K81, K85 and K86 were used. For each amino acid (denoted by the single letter abbreviation), the percentage found in each domain (A, head; B, rod and C, tail) and in the complete keratin sequence (D) was calculated. The various groupings of amino acids used here are based on standard nomenclature (Berg et al., 2007). Note that D, E, I, L, Q are more abundant in the rod domain than in the head or the tail (P<0.001 for all comparisons), whereas F and G are more frequent in the head than in rod and tail domains (P<0.05 for all comparisons). In the head domain L is rare but is more common in both the rod and tail, whereas S is infrequent in the rod domain but relatively abundant in the head and tail. (E) Distribution of the amino acids groups. Amino acids with aliphatic side chains: isoleucine (I), leucine (L), methionine (M), valine (V); amino acids with hydroxyl groups: serine (S), threonine (T); aromatic amino acids: phenylalanine (F), tryptophan (W), tyrosine (Y); basic amino acids: arginine (R), lysine (K); acidic amino acids: aspartate (D), glutamate (E); other amino acids: alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), histidine (H), proline (P).

Fig. 2.

Amino acid composition of human epidermal, simple-type epithelial and hair keratins. The percentage share of selected amino acid subgroups (A–F) within keratin domains and the whole keratin sequence was calculated. Grouping of amino acids as shown in Fig. 1. K1, K2, K5, K9, K10 and K14 sequences were analyzed for epidermal keratins, and K31, K35, K81, K85, K86 and K8, K9, K18, K19, K20 were used for hair and simple-type epithelial keratins, respectively. There are large differences between the amino acid composition of the head and tail domains, whereas the rods have largely identical compositions. Of note, amino acids with aliphatic side chains and those that are basic are the most and least abundant in heads and tails of simple-type epithelial and epidermal keratins, respectively. The difference in acidic amino acids and amino acids with aliphatic hydroxyl groups is most prominent in the tail domain (aliphatic: epidermal> hair; acidic: simple-type>hair/epidermal), whereas aromatic amino acids are most abundant in the heads and tails of epidermal keratins.

Fig. 3.

Percentage of selected amino acids in human keratins. The percentage share of (A) cysteine, (B) glutamic acid and (C) glycine was calculated for the whole keratin sequence. Note that epidermal, simple-type epithelial and hair keratins have different amino acid compositions. For example, cysteine residues are frequent in the hair keratins (i.e. K31, K35, K81, K85 and K86) but rare and almost never present in the epidermal and simple-type epithelial keratins, respectively (P<0.0001 for comparison between hair and simple-type epithelial or epidermal keratins). Compared with other sequence subgroups, glutamic acid is the least abundant, and glycine is the most abundant residue in epidermal keratins. K, keratin.

Fig. 4.

Distribution of selected amino acids within epidermal, simple-type epithelial and hair keratins. The percentage share of (A) alanine (B) cysteine, (C) glutamic acid, (D) phenylalanine, (E) glycine (F) proline and (G) tyrosine within keratin domains and the whole keratin sequence is shown. Chain designation is as given in Fig. 3. Of note, cysteines are abundant within hair keratins, but rare in epidermal keratins and almost never present in simple-type epithelial keratins. Compared with other keratin subgroups, the heads and tails of epidermal keratins are relatively alanine poor and glycine rich, whereas prolines are most abundant in the heads and tails of hair keratins. Also, phenylalanines are most abundant in the heads of epidermal keratins, whereas no striking differences are seen in other subdomains. Finally, tyrosines are particularly common in tails of epidermal keratins, but less frequent in tails of simple-type epithelial and hair keratins.

Fig. 5.

Cysteine content of epidermal, simple-type epithelial and hair keratins among multiple species. The average cysteine content within keratin domains (A–C) and the whole keratin sequence (D) in selected species. Chain designation is as shown in Fig. 3. Note that irrespective of the analyzed species, hair keratins have a high cysteine content, whereas cysteines are rarely seen in epidermal and almost never in simple-type epithelial keratins.

Fig. 6.

Glycine and proline content of epidermal, simple-type epithelial and hair keratins in a range of species. The average glycine (A) and proline (B) content within keratin domains and the whole keratin sequence in selected species. Chain designation is as shown in Fig. 3. Note that, independent of the analyzed species, glycines are most abundant in the heads and tails of epidermal keratins, and prolines are found most frequently in the heads and tails of hair keratins.

Fig. 7.

Summary of the major differences in amino acid composition of hair, epidermal and simple-type epithelial keratin structural domains. The schematic summarizes the major differences in amino acid content of hair, epidermal and simple-type epithelial keratins together with their respective P-values. Note that different amino acid content is seen predominantly in the keratin head (H) and tail (T) domains, whereas rods have a largely conserved amino acid composition. The different amino acid content is likely to affect keratin rigidity [through cysteine (Cys) crosslinking, which is facilitated by proline (Pro) residues], structural flexibility [through increased glycine (Gly) content], protein–protein interactions (through phenylalanine (Phe) content] as well as keratin solubility (through the presence of charged residues). The content of charged residues was determined as a sum of acidic (aspartic acid, glutamic acid) and basic (arginine, lysine) residues. In the first row, the first and second P-value refers to cysteine and proline residues, respectively, and in the last row, P-values refer to the differences in content of basic and acidic amino acids, respectively.

Fig. 8.

Overview of missense substitutions within selected human keratins and the resulting loss or gain of cysteine (A) and histidine (B) residues. Data are based on the Human Intermediate Filament database (www.interfil.org), which lists 184 and 169 unique missense substitutions for the analyzed type I and type II keratins, respectively.