ORIGINAL ARTICLE
Characterization of Human Keratin-Associated Protein 1
Family Members
Yutaka Shimomura, Noriaki Aoki, Michael A. Rogers,w Lutz Langbein,n Jˇrgen Schweizer,w and Masaaki Ito
Department of Dermatology, Niigata University School of Medicine, Niigata, Japan; nThe Divisions of Cell Biology and wBiochemistry of Tissue Speci¢c
Regulation, German Cancer Research Center, Heidelberg, Germany
Keratin-associated proteins are involved in the forma-
tion of the cross-linked network of the keratin-inter-
mediate ¢lament proteins that support hair ¢bers. In
recent years, several keratin-associated protein genes have
been identi¢ed and become an attractive topic in hair
research. More recently, we isolated two cDNA encod-
ing novel members of the human keratin-associated
protein 1 family (human keratin-associated protein
1.6 and human keratin-associated protein 1.7), and
described their expression in the hair follicle by RNA
in situ hybridization. A comparison of human keratin-
associated protein 1.6 and human keratin-associated
H
uman hair ¢bers share a common structural orga-
nization, in which a multicellular cortex is encased
in a cuticular layer of £attened cells, often with a
medulla layer centrally placed in the cortex. Hair
¢bers are composed primarily of keratin proteins
that are derived from several multigene families. These hair-spe-
ci¢c keratins have been classi¢ed into the keratin intermediate-
¢lament proteins (KIF) and their associated proteins (KAP) (Powell
et al, 1991). KAP are a major component of the hair ¢ber matrix and
participate in the formation of a rigid hair shaft by cross-linking
the KIF to form a meshwork (Powell and Rogers, 1997). Based on
their amino acid composition, KAP have been originally classi¢ed
into three groups: (i) high-sulfur KAP (16^30 mol% cysteine;
KAP1-3, 10^16 and 23 families) (Swart and Haylett, 1973; Swart
et al, 1976; Powell et al, 1983; Frenkel et al, 1989; Zhumbaeva et al,
1992; Huh et al, 1994; Powell and Rogers, 1997; Aoki et al, 1998; Cole
and Reeves, 1998; Mitsui et al, 1998; Takaishi et al, 1998; Kuhn et al,
1999; Rogers et al, 2001; Rogers et al, 2002, Shimomura et al, 2002a);
(ii) ultra-high-sulfur KAP (430 mol% cysteine; KAP4, 5, 9, and 17
families) (McNab et al, 1989; MacKinnon et al, 1990; Fratini et al,
1994; Jenkins and Powell, 1994; Powell et al, 1995; Perez et al, 1999;
Rogers et al, 2001); and (iii) high-glycine/tyrosine KAP (KAP6^8
families) (Kuczek and Rogers, 1987; Fratini et al, 1993; Aoki et al, 1997;
Rogers et al, 2002). To date, KAP have been subdivided into a total
of 23 families (KAP1.n^KAP23.n), based on their cysteine/tyrosine-
glycine content as well as the degree of amino acid homology
within each family and the nature of repeat structures often
found in these molecules, according to an uni¢ed nomenclature
Manuscript accepted for publication February 1, 2003
Reprint requests to: Yutaka Shimomura, Department of Dermatology,
Niigata University School of Medicine, Asahimachi-dori, Niigata 951-8510,
Japan. Email: yshimo@med.niigata-u.ac.jp
Abbreviations: KAP, keratin-associated protein; KIF, keratin intermediate
¢lament protein.
0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc.
96
protein 1.7 with other human keratin-associated protein
1 members revealed that keratin-associated protein 1
proteins are fundamentally composed of ¢ve distinct
domains, and that they can be classi¢ed primarily by
a striking variation in double cysteine-containing
pentapeptide repeats in the repetitive I domain. The
sum of the data analyzed suggests that human keratin-
associated protein 1 family genes may have arisen
mainly through gene duplication of the cysteine-repeat
motifs during evolution. Key words: human gene/
in situ hybridization/keratin/keratin-associated protein. JID
Symposium Proceedings 8:96 ^99, 2003
proposed by Rogers and Powell (1993). Of these KAP families,
the KAP1 family has been de¢ned as a group of high-sulfur pro-
teins expressed in the cortical layer of the hair follicle (Rogers
and Powell, 1993), and so far, eight KAP1 members have been re-
ported from sheep, rabbits, and humans (Elleman and Dopheide,
1972; Powell et al, 1983; Zhumbaeva et al, 1992; Mitsui et al, 1998).
In humans, two KAP1 genes, hB2A and hB2B, were initially iso-
lated from a single genomic clone (Zhumbaeva et al, 1992), and
later renamed hKAP1.1A and hKAP1.2, respectively (Rogers et al,
2001). In a recent study, we isolated two cDNA encoding new
hKAP1 members during the analysis of the hKAP1.1A gene in
patients with a congenital fragile hair disorder, which we have
named hKAP1.6 and hKAP1.7 (Shimomura et al, 2002). hKAP1.6
and hKAP1.7 show high nucleotide sequence homology with
hKAP1.1A. In particular, the three genes share a completely iden-
tical sequence in their 30 -noncoding regions. At the amino acid
sequence level, the degree of identity between hKAP1.1A,
hKAP1.6, and hKAP1.7 is striking (Fig 1). A comparison of hKA-
P1.1A and hKAP1.6 revealed a single amino acid change between
the proteins as well as a 46 -residue deletion in the amino term-
inal region of hKAP1.6. Similarly, comparison of hKAP1.1A with
hKAP1.7 revealed complete identity, except for a 92-residue ami-
no acid deletion in hKAP1.7. In addition, the multialignment of
hKAP1.6 and hKAP1.7 with sheep and rat KAP1 members de-
monstrated the structural features of the KAP1 family at the ami-
no acid sequence level (Shimomura et al, 2002a).
Unexpectedly, as we identi¢ed hKAP1.6 and hKAP1.7, a large
cluster of human high/ultra-high-sulfur KAP genes was found
on chromosome 17q12^21, resulting in the identi¢cation of
four additional novel hKAP1 genes, termed hKAP1.1B, hKAP1.3,
hKAP1.4, and hKAP1.5 (Rogers et al, 2001). In particular,
hKAP1.1B is nearly identical to hKAP1.1A in DNA sequence.
The multialignment of eight human KAP1 members showed
that these proteins are composed of ¢ve individual domains: an
N-terminal domain, a repetitive I domain, a central nonrepetitive
VOL. 8, NO. 1 JUNE 2003 CHARACTERIZATION OF HKAP1 MEMBERS 97

Figure 1. Multi-alignment of hKAP1 family members. Residues that are identical are colored light blue. Dashes denote gaps in the sequence to max-
imize alignment. The pentapeptide repeats are boxed with black lines. Brackets show the ¢ve KAP subdomains. The unique nonrepetitive sequences in the
repetitive I domain are boxed with red lines. The accession numbers for the respective protein sequences are: hKAP1.1A (hB2A), X63337; hKAP1.1B
(AC007455, nt 8801^9334); hKAP1.2 (hB2B), X63338; hKAP1.3 (AC007455, nt 15377^15880); hKAP1.4 (AC007455, nt 20120^20485); hKAP1.5 (AC007455,
nt 23043^23568); hKAP1.6 (AB052868); hKAP1.7 (AB055057).

domain, a repetitive II domain, and a C-terminal domain (Fig 1).
The N-terminal, central nonrepetitive, and repetitive II domains
appear to be signi¢cantly conserved among the hKAP1 members.
Likewise, these domains are well conserved in the KAP1 mem-
bers of other species (Shimomura et al, 2002a), indicating that
they may play an essential part in KAP1 family function. The C-
terminal domain, which is the smallest region, is composed of
four to seven amino acid residues and generally contains a unique
element, ‘‘EPTC’’, although this is not found in hKAP1.2, and
shows high similarity across the family (Fig 1). An additional re-
gion of interest in the KAP1 family members is the repetitive I
domain, which is striking in its high degree of repetitiveness
and size variability. The repeat structures in this domain appear
to be composed essentially of amino acid variations on a double
cysteine-containing pentapeptide, CCQ(P/T)S and CCETS. In
addition, hKAP1 members have a curious nonrepetitive sequence
‘‘FCGF(P/R)SFST(G/S)GTC(D/S)SS’’or ‘‘FCDFLASQLVDLQLS’’
between these pentapeptide repeats in the repetitive I domain
(Fig 1). These sequences could not be detected in the repetitive I
domains of the KAP1 members of other species already reported
(Shimomura et al, 2002). The comparisons of the hKAP1 mem-
bers showed high homologies at the N-terminal, central nonre-
petitive, repetitive II, and C-terminal domains, but not in the
repetitive I domain. Therefore, the distinction between hKAP1
members obviously appears to be dependent on the number of
pentapeptide repeats and the arrangement of the repeat segments
98 SHIMOMURA ET AL JID SYMPOSIUM PROCEEDINGS

Figure 2. Transcripts of hKAP1 genes are expressed in the cortex of the hair follicle. (A) hKAP1.1A/B, hKAP1.6, and hKAP1.7. (B) hKAP1.3. (C)
hKAP1.5. Note that due to the high DNA sequence homology of the hKAP1.1A/B, hKAP1.6, and hKAP1.7 genes, the in situ probe used for (A) recognizes
four transcripts. The red arrows demarcate the area of major KAP expression. No transcripts were detected in the medulla (data not shown) or in the ORS,
IRS, and CU. ORS, outer root sheath; IRS, inner root sheath; CU, cuticle of the hair shaft; CO, cortex; DP, dermal papilla. Scale bar: 150 mm.

and nonrepetitive sequences in the repetitive I domain, indicating
that this domain is likely to de¢ne the individual functional char-
acteristics of each hKAP1 member.
At present, eight hKAP1 genes have been isolated from hu-
mans. It is intriguing to consider how these hKAP1 genes have
arisen in the human genome. The presence of various patterns
of pentapeptide repeat structures in the repetitive I domain raises
the possibility that the hKAP1 genes arose by gene duplication,
especially the duplication or deletion of cysteine-repeat segments,
during the course of evolution (Rogers and Powell, 1993; Rogers
et al, 2001). Furthermore, additional mutations and/or small dele-
tion events occasionally seem to have occurred in other domains,
such as the N-terminal domain in hKAP1.4 and the C-terminal
domain in hKAP1.2.
In situ hybridization studies of human scalp hair using a
probe against the 30 -noncoding region of hKAP1.6 revealed
strong signals, predominantly in the cortical layer, throughout
the keratogenous zone (Fig 2A). This ¢nding indicated that
hKAP1.1A/B and hKAP1.7 mRNA are also expressed in the same
region, because hKAP1.1A/B, hKAP1.6, and hKAP1.7 have a
completely identical nucleotide sequence in the 30 -noncoding
region. Similarly, hKAP1.3 (Fig 2B) and hKAP1.5 (Fig 2C)
mRNA were detected in the cortical layer as well, using
speci¢c probes, and their region of expression was consistent
with that of the hKAP1.1A/B, hKAP1.6, and hKAP1.7 mRNA.
The relative quantities of these mRNA expressed in this region
remain to be analyzed. Expression studies of all the hKAP1 mem-
bers, as well as of other hKAP family members, at both the
mRNA and protein levels, may give us some insight into the
characteristics that distinguish the hair of di¡erent individuals,
and provide additional impetus to discover the complete set of
hKAP genes.
hKAP gene clusters are located on chromosomes 17q12^21
(Rogers et al, 2001) and 21q22.1 (Hattori et al, 2000; Rogers et al,
2002); however, the hKAP1.6 and hKAP1.7 genes are not detect-
able at these gene loci, indicating that there may be a further clus-
ter of hKAP genes elsewhere. Alternatively, the presence of high
sequence identities among hKAP1.1A/B, hKAP1.6, and hKAP1.7
in the 50 - and 30 -noncoding regions as well as in the open read-
ing frame suggests the possibility that hKAP1.6 and hKAP1.7 are
the polymorphic alleles of either hKAP1.1A or hKAP1.1B created
by intragenic deletions during evolution. So far, we have per-
formed further analysis of hKAP1 family members concerning
these problems and have got some interesting results (Shimomura
et al, 2002).
Mutations in the hair keratin genes, hHb1 or hHb6, have pre-
viously been shown to cause the congenital hair disease monile-
thrix, which is characterized by beaded and fragile hairs (Winter
et al, 1997a, b; Korge et al, 1999). Along with the keratins, hKAP
are the major component of human hair, in which mutations are
likely to cause hereditary hair genodermatoses. Therefore, the
analysis of hKAP genes in patients with hair abnormalities will
provide meaningful information. In addition, the production of
transgenic mice expressing mutated KAP is likely to provide in-
sight into the function of the KAP in hair follicles, and may pro-
vide a model for human hair disorders, as well.
In this study, we gave only one example out of many hKAP
families. The hKAP1 family, however, shares similar characteris-
tics with the other hKAP families. Thus, a detailed study of hKAP1
members will be a good guide for understanding all the hKAP.
VOL. 8, NO. 1 JUNE 2003
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