Molecular Biology Reports 16: 39-47, 1992. 9 1992 Kluwer Academic Publishers. Printed in Belgium.
39
Structure and site of expression of a murine type II hair keratin Edda Tobiasch, Jtirgen Schweizer & Hermelita Winter Institute of Biochemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 6900 Heidelberg, Germany Received 19 September 1991; accepted in revised form 11 October 1991
Key words: mouse epithelia, keratin expression, in situ hybridization, hair keratin
Abstract
We present here a 1770 bp-long cDNA which encodes a murine type II keratin. Sequence comparisons of the keratin with those of various type II keratins expressed in mouse epidermis and internal stratified epithelia reveal that the new keratin is unrelated to epithelial keratins. Rather the structural organization of its amino- and carboxyterminal domains and the high content of cysteine and proline residues in these regions suggest that the keratin represents a murine type II hair keratin. This assumption was confirmed by in situ hybridization which localized the mRNA of the keratin in upper cells of the hair cortex and in suprabasal cells of the central core unit of filiform papillae of the tongue. Hybrid selection analyses revealed that the keratin has a molecular weight of 58 kD. It remains to be seen whether the keratin corresponds to MHb 3 or MHb 4.
Introduction
The keratin multigene family consists of about 30 individual, however, structurally related members which can be grouped into epithelial-type keratins ('soft' ~-keratins) and wool- and hairtype keratins ('hard' ~-keratins) [1-3]. The great majority of these proteins belongs to the epithelialtype of keratins. As a rule, the mammalian hair follicle contains only a set of eight major 'hard' ~-keratins [ 1-6]. Four of them, Hbl, Hb2, Hb3 and Hb4, belong to the basic to neutral, type II subfamily of keratins, whereas the remaining four keratins, Hal-Ha4, represent acidic, type I keratins [ 1-6]. The occurrence of an equal number of type II and type I hair keratins indicates that their synthesis follows the principle of pairwise expression, although the exact composition of the sixteen possible hair keratin pairs has not yet been
elucidated. Comparisons of the presently available amino acid sequence data of both wool and hair keratins with that of epithelial keratins have revealed that 'hard' and 'soft' keratins are rather homologous in their central s-helical rod domain, however, differ substantially in their non s-helical head and tail portions [3, 6-8]. Typically, the 'hard' keratins show an accumulation of cysteine and proline residues in their amino- and carboxyterminal domains [6-8] and the high content of cysteine is thought to essentially account for the rigidity and physical strength of the mature hair and wool fiber by extensive S-S-bridging of the constituent proteins. Immunohistochemical studies and protein investigations in different laboratories have shown that the eight hair type keratins are not only expressed in the hair follicle, but also occur in nail-forming cells, the filiform papillae of the tongue and the thymic reticulum [ 1, 2, 4, 5, 9].
40 It is evident that the knowledge of the amino acid squences of the individual hair keratins would essentially contribute to the understanding of the development and formation of hard, keratinized structures in these different anatomical locations. However, at present sequence information for wool and hair type keratins is relatively sparse and to our knowledge limited to that of a sheep wool type II keratin 7c [7] and to two murine type I hair keratins, M H a l and MHa4 [6, 8]. In the course of our studies on keratin expression in various stratified and keratinized epithelia of the mouse, we have recently constructed a cDNA library with polyA+RNA from adult mouse tail epidermis. This library was aimed to serve for the isolation of cDNA clones for keratins which are supposed to be expressed in the parakeratotic scale epidermis of this morphologically complex epithelium [ 10]. The screening of the library for type II keratin cDNAs yielded, besides several new keratin clones, one clone which turned out to encode a murine type II hair keratin. In this paper we present the almost complete amino acid sequence of this keratin and, by in situ hybridization, provide data on its tissue-specific expression.
Materials and methods
(final wash with 0.1 x SSC, 0.1~o SDS). The resulting positive phage clones were further hybridized with a mixture of [3ap]-labeled specific 3' c D N A fragments of type II keratin clones pke70 [14], pktl-1 and pktl-5 [unpub. results]. Hybridization was performed for 18 h at 42 ~ (50~o formamide, 5 x SSPE) and the final wash was at 68 ~ (0.1 • SSC, 0.1~o SDS).
RNA slot blot hybridization Phage clones which did not react with the mixture of specific 3'-fragments of the clones coding for K1, K5 and the 70 kd keratin were subjected to R N A slot blot hybridization. R N A was isolated as described above from the epidermis of different skin sites, various internal stratified epithelia, epidermal tumors and cell lines. Slot blots were performed with Gene screen plus membranes (DuPont, Dreieich, Germany) using a minifold I system (Schleicher & SchOll, Dassel, Germany). Hybridization was carried out for 18 h at 42 ~ (50~o formamide, 1 M NaC1, 1~/o SDS, 5 0 m M Tris-C1, pH7.6, 10~o dextrane sulfate) with [32p]-labeled phage inserts. Membranes were washed stringently (0.1 • S SC, 1 ~o SDS at 68 ~o) and exposed at -80 ~C to Kodak X-Omat films. One phage clone, termed 2ktlII-4 was chosen for further characterization.
cDNA cloning and screening procedures PolyA + RNA of adult mouse tail epidermis, obtained after a short incubation of tail skin in 60 ~C hot water [10], was isolated according to the method of Gough [ 11 ] and used for the construction of a cDNA library in 2gtl0 [ 12]. The library was screened by hybridization with a [32p]_ labeled 535 bp ClaI/KpnI fragment of the previously described c D N A clone pkt57 which encodes the type II keratin K4 of internal stratified mouse epithelia [13]. The fragment spans f r o m position 601 of 1136 of pkt57 and thus contains a portion of the sequence coding for the s-helical domain of K4 [13]. Hybridization was carried out for 18h at 42 ~ (35~o formamide, 5 x SSPE) and subsequent washing was at 52 ~
Subcloning and DNA sequencing The insert of 2ktllI-4, attached to Eco RI adaptors, was cloned into the transcription vector Bluescript II KS + (Strategene; La Jolla, USA). Sequencing of both strands of this plasmid clone pktlII-4 was performed according to the dideoxy sequencing method of Sanger et al. [15], using first M13 and T3 primers and subsequently 17mer synthetic oligonucleotides as walking primers. To prepare a specific probe of 3' noncoding region of clone pktlII-4, a 262 bp Pst I fragment was subcloned into Bluescript II KS +. This subclone was designated pktlII-4-3'.
41
In situ hybridization The protocol used for in situ hybridization of frozen tissue sections (nominally 6 #m thick) was essentially as described previously [14, 16, 17], however, with some modifications [ 18, 19]. Upon linearization ofpktllI-4-3' with Sma I, riboprobes were obtained that were labeled with [35S]-UTP by in vitro transcription with T3 RNA polymerase. A sense probe was used as negative control. Slides were dipped in Kodak NTB2 photoemulsion, developed after 2 to 5 days and stained with hematoxylin-eosin.
Hybrid selection analysis and translation in vitro PolyA § RNA obtained by oligo (dT) cellulose chromatography of total RNA of tail epidermis was hybridized to filter bound DNA of pktllI-43' and the selected m R N A was translated in vitro in the presence of [ 35S ]-methionine. PolyA + RNA from both footsole epidermis and tongue epithelium was also translated in vitro. One dimensional SDS PAGE of the in vitro translation products and autoradiography were performed as described [20].
the remaining clones, these were subjected to slot blot hybridization analysis with RNA from epidermis of different skin sites, various internal stratified epithelia, benign and malignant epidermal tumors and a murine epidermal cell line. This analysis resulted in the identification of three clones which exhibited a strong hybridization signal with polyA + RNA of tail epidermis, however, also showed a weak, but significant reaction with polyA + RNA of tongue epithelium (Fig. 1). This intriguing expression characteristics prompted us to sequence the corresponding clones. Two of them turned out to be identical and were found to encode the desired type II 65 kD keratin whose properties will be reported elsewhere [Tobiasch et al., in prep.]. The nucleotide sequence of the 1770 bp insert of the third clone, pktlII-4, is shown in Fig. 2. It
Results and discussion
In an attempt to isolate a cDNA clone for a particular type II 65 kD keratin of adult mouse tail epidermis [ 10], we have screened a 2gtl0 library, constructed with polyA + RNA of the epidermis of this skin site for the presence of type II keratin cDNAs. To this purpose we used an appropriately taylored DNA probe which was derived from the region coding for the a-helical domain of murine keratin K4 [ 13 ]. From the resulting bulk of keratin cDNAs, we first eliminated those clones which hybridized with the specific 3'noncoding ends of the previously isolated cDNAs of the 70 kD keratin [14] and of keratins K1 and K5 [unpub. data]. These keratins represent the major type II keratins expressed in adult mouse tail epidermis [ 10]. In order to preliminary characterize
Fig. 1. RNA slot blot hybridization. Total RNA from (a) neonatal mouse epidermis, (b)adult mouse footsole epidermis (e)a DMBA/TPA-induced epidermal squamous cell carcinoma, (d) a papilloma-derived epidermal cell line, SP1 [26], (e) mouse tail epidermis, ( f ) mouse tongue epithelium, (g) mouse palate epithelium and (h) mouse brain was spotted onto a nylon membrane in concentrations of 1 #g, 2/~g and 4/~g each and hybridized with the phage clone )tktl II-4 insert. Note the strong reaction with RNA of tail epidermis and the weak reaction with RNA of tongue epithelium (e, f ) .
42 I 1
Cga aar gac ag9 tca tgg tgg agg aga age cac 9cg cga aga gga gag aag t t g aca gig t i t 9ga act gga aac R N D ~ S # N ~ H A ~ R G E K L T V F G T G N
~6 2, 1SI S 226
~6
ttc r
egc t g c gcc t e a g e t t g c ggg ccc egg c o t ggc r s 9 * s 9 9 c ~ ~ ~ G ~
tgr I
~
~9c t g c a t e t o t ~ca g e t cr 9 9 i s , A ~
taC cgJ gga c~r tCa ggg g9r t t C ggc agC ca 9 a g t g t r t g t Y ~ G L S G G F G S O S V ~ G A
age ate ggg tic
cga t o t
gga ggc a r c t g c ggg ccc age e t a
s
R
~
r
301 C t g t i c 1 0 1 L
o
Y
s
G
:
~
~
~
S
~
a c a c e c c t g a a c e t 9 ga 9 a t e 9 a e c c c a a t 9ca eaq t g t L T ~ L N L s 1 6 3 1 6 3
t a r a99 ggr &to t r 1 6 2 150 y ~ a x s so
99~ gcc ~ t c cgc t c c 9qr t c c F R S G S ~ G R
ccc t g c a t e
~
Q
:
9S 2S
tgt
gga CgC
225 ~5
ace act
9tc tot
gtc aac gag a9r
300
T
v
v
100
9t9 aag cat
T
S
.
t
S
gag gag aaa gag tag arc aag
37S 12S
3~6 t g t CtC a a c a g e a g g ~ t c 9 c g g e t t t C a t e g a r a a g g a g ego t t C c t g g a g c&g t a g a a c a a g c t g c t 9 g a g a c e ~ 2 6 C L N S R F A A F Z D ~ V R F L s
4SO 150
451 aag tgg tag t i c tac tag aac ego aa9 t9r t g t gag age aac atg gag cct ctg t i t I ~ I K W O F Y O N ~ K C C s 1 6 3 1 6 3
52S 195
gag 9gc tac ate gag 8cg
$26 c~g a g g e g g g a g g e t g a g t g t g a g g a g 9ca g a c a g e g99 a g g c t g g c t 9ca g i g c t c a a c c a t 9 c g t a g g a g t c c I ~ 6 L ~ R E A E C V s 1 6 3 1 6 3
600 200
601 atg gag gge tac aag aag agg t a t gaa gaa gaa g t t gca ere cgg 9cc aca gca gag aat gag t t t ~ 0 1 M s 1 6 5
6~S 225
6~6 a a g a a g g a t g a g g a c t g t g c c t a c c t 2 2 6 K K D V D C A Y
gtg qc[ eta
9 c g c a a g a c e ~a~ e t g g a g 9cc a a c ~ca g a g 9 c a c t g a c e ca~ g a g L R X S D L E A N A E A L T O E ~
~$1 gaC t~C c t g agg aga a t g t a t 9 a t gag gag a c t ego a r c c t c 2 5 1 D F L R R M Y D s
act
~S0 250
ca~ ~cc cac a r c t e a gac aca o c t g t c a t e ~ c
82S 2?5
626 aag atg gac aac age egg gac ctg aac atg gac t g t gtc gig get gag ate sag get tag t a t gat gac a r t gcc 2 9 6 ~ M D N S R D L N K D C V V A s
900 300
901 age cgc agC c g t g e t gag gCC gag t c c t g g t a c ccr a c t aa~ ~ g t gag ga 9 a t g aag gcc aca gcg a~c egg c a t 3 0 1 S ~ S ~ A s 1 6 3 1 6 3 1 6 3
S~5 325
9?6 9 g a g a g ac~ c t 9 c g r cgc a c t a~a g a g g a g a t e a&~ g a g c t g a a c a g a a t g a t e 3 2 6 G E T L R ~ T H s 1 6 3
1050 350
10~1 g a g a a t 3 5 1 s
g c c a a g t g c t a g a a c a c e s a g c~g g a g g c t g e t g a g a c t
caa t o t
tag agg ctg act
g c t gag a~c
gag t a g t a g 9ga gag g e t gcc ~ t
1125 3~5
1126 g c t g a t g e t c g c a g e a a g c t g g e t g a g t t 9 g a g ~ g t 9co c t g t a g a a g g c t a a g c a g g a c a t g g c r t g c c t g c t c ~ 6 A D A ~ C K L ~ s
1200 400
1201 aag gag tac tag gag gig atg aac tcc aag ctg ggg ctg 9ac gig gag ate ate ace tac agg cgc ctg ctg gag 401K s u 0 s V ~ K S K k G L D v s ~ ~ 9 u . . L L ~
12~S 42S
12~6 99C g a g g a g t a g a g g c t g t g t g a a g~c g t 9 g g a g c t g a g a a t 4 2 6 G s 1 6 3 1 6 9 1 6 3 1 6 9
g
13S0 450
13~1 tgc 999 gac ctc t g t gig t o t ggc t~a c?~ cot gig ac~ gqc ag~ ~tc tgc agt gcc eta tgc agc ggg aa~ ~ g
142S
1426 gca 9ta age ac~ ggr ctg t g t 9c9 tee t ~ t
~SO0
gtc tgt
gqa age 9gc c o t t g t
g~: age age tcc
c=c ccg gg9 agg t g t
cgt ggt gga get
tag ~ag a c a aga ggg
iSOl
age Cag gaa gag gee t 9 9 act aca agg c~a a9r atg 9ta get tea agg act get gcc cat gag ate tga gaa tac
15~5
15~6
art ccr cat Ccc cag cag ctg cca c~e c a t tCa gct acc t~c t g 9 caa 9gg gc~ ~gc age tga tag ate agc ctc
16SO
I~51
ctg cot tag r
1~25
1926 ~ t
ca~ ccc tg9 g~a tac cca g~g c~g t~t cct gag oct ct~ gc~ act agg cct gtt gag Caa tat
gag Ctg act CtC aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa
1790
Fig. 2. Nucleotide sequence of the pktl 11-4 insert and deduced amino acid sequence of the encoded type II keratin. The nucleotide sequence is shown in the 5' to 3' direction of the mRNA. The stop codon is marked by an asterisk, the polyadenylation signal is underlined. Arrows denote the region coding for the s-helical domain. In the non ~-helial domains, cysteine residues are encircled and proline residues are boxed.
43 contains the complete 3' noncoding region and almost the complete coding region of the m R N A of a keratin whose amino acid sequence is also indicated in Fig. 2. Sequence alignments with various type II murine keratins expressed in the epidermis and in internal stratified epithelia (shown here for keratin K4 in Fig. 3) readily reveal that the keratin encoded by pktllI-4 is unrelated to these keratins. The a-helical rod domains of the two keratins exhibit a sequence homology of about 55 ~o. However, there is evidence for an only poorly conserved H1 subdomain [21] in the aminoterminal region of the new keratin, whereas a distinct H2 subdomain in its carboxyterminus cannot be detected. In addition, although both the head and tail regions of the new keratin are relatively rich in serine residues, the absence of the typical accumulation of G G G and G G X motifs (with X being predominantly serine) [21] in the central part of both regions, does not allow to delineate distinct V1 and V2 subdomains [21]. Instead both, the amino- and the carboxyterminus of the new keratin contain a high percentage of cysteine and proline residues (10 cysteines, 8 prolines in the aminoterminus, 10 cysteines and 5 prolines, in the carboxyterminus). Especially in the carboxyterminus, these amino acids frequently appear as PC motifs (Fig. 2). Interestingly, all these properties are also a typical feature of the sheep wool type II keratin 7c [7] and of two murine type I hair keratins, M H a l and MHa4, recently described by Bertolino et al. [6, 8]. It therefore appears that clone pktllI-4 encodes a type II murine hair keratin. This assumption is confirmed by sequence comparison of the carboxyterminal regions of the new keratin and sheep wool type II keratin 7c (Fig. 4). Except for the penultimate parts of the carboxytermini, such an alignment demonstrates an almost complete homology of the two sequences. Moreover, in situ hybridization with the insert of subclone pktllI-4-3' to frozen sections of adult mouse tail epidermis and newborn mouse back epidermis reveals hybridization signals specifically over hair follicles. In longitudinal sections of hair follicles (Fig. 5a), it can be seen that the hybridization signals occur only over upper
cortex cells, whereas cells of the outer and inner root sheaths and matrix ceils in the bulbar region are free of label. This particular distribution of the m R N A of the new keratin could also be confirmed in cross sections through different levels of the hair follicle (Fig. 5b-d). The site of expression of the m R N A of the new hair keratin is therefore in agreement with numerous studies in which the localization of hair keratin proteins in the follicles of different species has been investigated by indirect immunofluorescent staining techniques [ 1, 2,4,5,6,8]. With the demonstration that clone pktllI-4 encodes a type II hair keratin, the positive reaction with tongue epithelial polyA + R N A in the slot blot hybridization analysis of Fig. 1 becomes understandable. Recent investigations in different laboratories have shown that hair type keratins are expressed in the central core unit of the filiform tongue papillae [5, 9]. In this compartment of the morphologically complex murine filiform papillae [22-24] the synthesis of these keratins is thought to be responsible for the formation of the posteriorly inclined, hook-shaped solid spine which consists of an extremely condensed keratin material [9]. As shown in Fig. 6, the m R N A of the new type II hair keratin is clearly expressed in the central core unit of the filiform papillae. The label is concentrated over living, suprabasal cells, whereas basal cells are free of label (Fig. 6b). We have previously shown that basal cells of the illiform papillae express keratins K5 and K14 [ 17]. The finding that clone pktllI-4 encodes a type II hair keratin, requires an explanation for its strong reaction with m R N A of tail epidermis and the negative reaction with m R N A of both newborn mouse epidermis and adult mouse footsole epidermis in the slot blot hybridization analysis of Fig. 1. We have previously shown that by gently lifting tail epidermis from the dermis of tail skin incubated for a short period in hot water, hair follicles quantitatively remain in the dermis [25]. In the present investigation, however, tail epidermis was scraped off from the dermis with forceps after incubating the skin in hot water. By this manipulation, large quantities of hair follicles are removed together with the epidermis. In contrast
20
30
40
50
60
70
80
90
.
.
.
S
~
FG
1%*
.
.
.
.
S
*
*
r-
GG G
**
Lt---I
*
G F
1%
G
1%
"
G CONSENSUS
1%
MK4 MHb4
1%
*1%1%W1%*
~
N SLLTPL
****
EIDP
*
9
Q
~1%*1%1%
o
E EQIK
*1%
LN
1%W
*1%1%*******1%1%*
9
~
FA F I D K V R F L E Q Q N K
LETKW
****1%
9
1%
Q
S
W
9
1%
*1%
1%
P FE Y C O N S E N S U S
LR
D GRL
-
EL
9
Q S E
L2
K
YEEE
.
R
.
.
AEN FV L K K D V D
.
AY
K
.
LEA
E L
.
E
.
F R
Y
E
9
*
D
It
~1%
AE
~
*1%~
AQY
*1%1%
DIA
"
~1%
1%1%*1%
S AE ESWY
1%
~
*
K
~
1%*
HG
1%
L
V
t
*
T
**
**~****1%1%
~
EI E L N R M I Q R L
*~*~*
9
AEIEN
*
*
K Q
~
1%
~
A V
**
~
MK4
EQ GE C O N S E N S U S
MK4 MHb4
CONSENSUS
K AELE ALQKAK
D A LL
G
YQ
MN KL LDVEI
TYR L L E G E E
R
E
AV
V
G
SG
GS
CONSENSUS
Fig. 3. Comparison of the amino acid sequences of the murine type II epithelial keratin K4 (first row) and the keratin encoded by clone pktl 11-4 (second row). Also indicated is the consensus sequence between the two keratins (fourth row). The arrowheads denote the cental or-helical domains in which the non coiled-coil linker regions are indicated.
G
501 G G G I Y G G S G S K I T S S A T I T K R S P R 472 S G N V A V S T G L C A P C G S G P C H P G R C
AL DA
AALADARCKLAELEGALQKAKQDMACLLKEYQEVMNSKLGLDVEIIT•RRLLEGEEQR-LCEGVGAVNVCVSSSRGGVVCGDLCVSGLRPVTGSVCSAP• MHb4
401 L A L K D A Y S K R A E L E T A L Q K A K E D L A R L L R D Y Q A L M N V K L A L D ~ E I A T Y R K L L E G E E ~ R ~ S G E C K S A V S I ~ w G G ~ Q H W R S G L G L G ~ G F C S G S G ~ G S G F G F 373
*
*1%1%
MDN R L
9
V
*1%
H SDTS
301 V V L S M D N N R N L D L D G I I A E V R A Q Y E D I A R K ~ I E V ; ~ S ' ~ W Q I K V Q Q L O M S A D Q H G D S L K T T K N E I S E L N R M I Q R L R A E I E N I K K Q S Q T P Q A S V A D A E Q R G E 273 V I V K M D N S R D L N M D C w A E I K A • Y D D I A S R S R A E A E S W Y P T K C E E M K A T V I R H G E T L R R T R E E I N E L N R M I Q R L T A E I E N A K C Q N T K L E A A V T Q S E Q Q G E
--Llt2--"i
I
MHb4
~
1%
V
MK4
9
I
1%
173 IETLRREAECVEADSGRI.~I~I%ELNHAQESMEGYKKR YEEEVALI:IATAENEFVALKKDVDcAYLRKSDLEANAEALTQETDFLRItNYDEETRILHSHISDTS
W
G
1%
G
SFNGRGGPGFPV~PAGG~QEVT~NQSLLTPL~VE~DPE~QK~TAEREQ~KTLNNKF~F~DKVRFLEQQNKVLETKWNLLQ~J~TTTT~PK~LDPFFETY MK4 R~FGYRSGGI~GPSPP~ITTVSVNESLLTPLNLEIDPNAQCVKHEE~EQIKCLNSRFAAFIDKVRFLEQQNKLLETKWQFY~.NRK~CES--NMEPLFEGY MHb4
.
R
1%
RNDRSWWRRRHARRGEKLTVFGTGNFSCASACGPRPGRCCISAAPYRGISCYRGLSGGFGSQSVCGAFRSGSCG
i00
201 INALRKNLDTLSNDKGRL~ELKMM~DSVEIAFKTKYEEEINKRTAAENDFwLKKDVDAAYMIKVELEAKMESLKDEINFTRVLYEAELAQMQTVS~DTS
i01 75
1 ..........................
1 MIAR••SVRGASPGFTSGSAIAGGV•CRVAFSSGSMSGGAGRC•SGGFGSRSLYNLGGHKSISMSVAGSCQGGGYGGAGGFGVGGYGAGFGAGGFGGGFGG
I0
45 LCEGVGAVNVCVSSSRGGVVCGDLCVSGLRPVTGSVCSAPCSGNVAVSTG
llLilll11111ilLli11111Ltltli
LILIIIIIBITIILi
~'V~
LIIII
LCEGVGAVNVCVSSSRGGVVCGDLCVSGSRPVTGSVCSAPCSGNLAVSTG
S~
L C A P C G S G P C H P G R C .......
LILLLL
i
LCAPCGQLNTTCGGGSCSLGRC Fig. 4. Sequence comparison of the carboxyterminal regions of the new murine type II keratin encoded by clone pktl II-4 (MK) and sheep wool type II keratin 7c (SK) [7].
Fig. 5. In situ hybridization of the [35S]-labeled specific riboprobe of subclone pktl 1I-4-3' to frozen sections of (a) adult mouse tail skin and (b-d) neonatal (3 days old) mouse back skin. In (a) a longitudinal section and in (b-d), cross sections of hair follicles are shown, ors, outer root sheath; irs, inner root sheath; c, cortex; hm, hair matrix; ct, cuticle; h, hair; dp, dermal papillae. Bar = 100/~m throughout.
newborn mouse epidermis was gently peeled off from the dermis and in the case of footsole epidermis, only the glabrous skin portion was used for the heat isolation of epidermis. It is thus con-
ceivable that these differences in tissue separation may account for the presence of hair keratin mRNAs in tail epidermis. Finally, in order to investigate to which of the
46
Fig. 7. Hybrid selection analysis. ThemRNA-species selected by hybridization of polyA + RNA of tail epidermis to filterbound DNA of subclone pktl II-4-3' was translated in vitro in the presence of [3sS]-methionine. The translation product was resolved by one dimensional SDS-PAGE on a 9% gel (lane c). Lanes a and b represent the keratin profiles obtained by in vitro translation of polyA + RNA of footsole epidermis (a) and tongue epithefium (b). The molecular weight of the keratin in lane c was estimated relative to the molecular weights of keratins KI (67 kD), K10 (60 kD), K14 (52 kD) in lane a and K4 (57 kD) and K13 (47 kD) in lane b.
Fig. 6. In situ hybridization of the [35S]-labeled specific riboprobe of subclone pktl 1I-4-3' to frozen sections of mouse tongue (a, b). Note the specific localization of hybridization signals in suprabasal cells of the central core unit of the fillform papillae (b). fp, filiform papillae; ac, anterior compartment; cu, central core unit; ipe, interpapillary epithelium; dp, dermal papillae. Bar = 100/~m in (a) and (b).
four type II murine hair keratins the new keratin might correspond, we performed a hybrid selection experiment with polyA + RNA of tail epidermis. As shown in Fig. 7, subclone pktlII-4-3' selected a mRNA which on release and in vitro translation yielded a single protein (Fig. 7, lane c). Using the in vitro translated keratins of footsole epidermis and tongue epithelium polyA + RNA as a reference, the size of the protein could be assessed to 58 kD. Calculation of the molecular weight of the presented part of the pktlII-4 encoded keratin yields a value of 55 kD, indicating that the sequence of the keratin lacks only few
47 amino acids of the aminoterminal domain. Previous experiments h a d s h o w n that b o t h murine type II hair keratins M H b 3 and M H b 4 migrate in the 58 k D molecular weight range [6, 8]. Further experiments are required to definitely assign our new keratin within the type I I murine hair keratin subfamily.
Acknowledgement We t h a n k M. M a c L e o d for typing the manuscript.
Note After submission o f our paper, Yu et al. published a full length c D N A clone encoding a murine type I I hair keratin. By m e a n s of Western blotting with a monospecific antibody directed against the last 18 amino acids of the c arboxyterminus o f the encoded keratin, the authors showed that the keratin c o r r e s p o n d s to M H b 4 (Yu et al., J. Invest. Dermatol. 97, 354-361, 1991). Except for a short sequence area coding for the penultimate aminoterminal region, the clone published by Yu et al. and our clone are completely identical. The b r e a k between the two sequences occurs u p s t r e a m f r o m p o s i t i o n 6 1 (triplet ttt in Fig. 2). W e have re-examined the sequence area 1-61 o f our clone and found it to be correct. The calculated molecular weight o f the keratin encoded by the clone of Yu et al. is 52 k D . This value is far below the S D S - P A G E size estimates of about 5 7 - 5 8 k D for M H b 4 (Bertolino et al., J. Invest. Dermatol. 94, 297-303, 1990). As a rule molecular weights o f keratins calculated f r o m cloned sequences are slightly larger than those estimated from S D S P A G E . This is also true for type I hair keratins (Bertolino et al., J. Invest. Dermatol. 91, 5 4 1 546, 1988). It, therefore, appears that the aminoterminal d o m a i n o f the clone published by Yu et al. m a y be too short.
References 1. Heid W, Werner E & Frank WW (1986) Differentiation 32:101-119 2. Lynch MH, O'Guin WM, Hardy C, Mak L & Sun T-T (1986) J. Cell Biol. 103:2593-2606 3. Conway JF & Parry DAD (1988) Int J. Biol. Macromol 10:79-98 4. Heid W, Moll I & Franke WW (1988) Differentiation 37: 137-157 5. Heid W, Moll I & Franke WW (1988) Differentiation 37: 215-230 6. Bertolino AP, Checkla DM, Notterman R, Sklaver I, SchiffTA, Freedberg IM & DiDona GJ (1988) J. Invest. Dermatol 91:541-546 7. Sparrow LG, Robinson CP, MacMahon DTW & Rubira MR (1989) Biochem. J. 261:1015-1022 8. Bertolino AP, Checkla DM, Heitner S, Freedberg IM & Yu DW (1988) J. Invest. Dermatol 94:297-303 9. Dhouailly D, Xu C, Manabe M, Schermer A & Sun T-T (1989) Exp. Cell Res. 1818:141-158 10. Schweizer J, Fflrstenberger G & Winter H (1986) J. Invest. Dermatol. 89:125-131 11. Gough NM (1988) Anal. Biochem. 173:93-95 12. Maniatis T, Fritsch EF & Sambrook J (1989) In: Molecular Cloning (A Laboratory Manual). Cold Spring Harbor Laboratory 13. Knapp B, Rentrop M, Schweizer J & Winter H (1986) Nucleic Acids Res. 14:751-763 14. Rentrop M, Nischt R, Knapp B, Schweizer J & Winter H (1987) Differentiation 34:189-200 15. Sanger P, Nicklen S & Coulson AR (1977) Proc. Natl. Acad. Sci. (USA) 74:5463-5467 16. Rentrop M, Knapp B, Winter H & Schweizer J (1986) Histochem. J. 18:271-276 17. Rentrop M, Knapp B, Winter H & Schweizer J (1986)J. Cell Biol. 103:2583-2591 18. Angerer LM, Cox KH & Angerer RC (1987) Methods in Enzymol. 152:649-661 19. Angerer LM & Angerer RC (1989) DuPont Biotech. Update 4:3-6 20. Knapp B, Rentrop, Schweizer J & Winter H (1987)J. Biol. Chem. 262:938-945 21. Steinert PM, Steven AC & Roop DR (1985) Cell 42: 411-419 22. Cameron IL (1966) J Exp. Zool. 163:271-284 23. Cane AK & Spearman RIC (1969) Arch. Oral Biol. 14: 829-814 24. Farbman AI (1979) J. Anat. 106:233-242 25. Schweizer J & Marks F (1977) Cancer Res. 37: 41954201 26. Strickland JE, Greenhalgh DA, Koceva-Chyla A, Hennings H, Restrepo C, Balaschak M & Yuspa SH (1988) Cancer Res. 48:165-169
39
Structure and site of expression of a murine type II hair keratin Edda Tobiasch, Jtirgen Schweizer & Hermelita Winter Institute of Biochemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 6900 Heidelberg, Germany Received 19 September 1991; accepted in revised form 11 October 1991
Key words: mouse epithelia, keratin expression, in situ hybridization, hair keratin
Abstract
We present here a 1770 bp-long cDNA which encodes a murine type II keratin. Sequence comparisons of the keratin with those of various type II keratins expressed in mouse epidermis and internal stratified epithelia reveal that the new keratin is unrelated to epithelial keratins. Rather the structural organization of its amino- and carboxyterminal domains and the high content of cysteine and proline residues in these regions suggest that the keratin represents a murine type II hair keratin. This assumption was confirmed by in situ hybridization which localized the mRNA of the keratin in upper cells of the hair cortex and in suprabasal cells of the central core unit of filiform papillae of the tongue. Hybrid selection analyses revealed that the keratin has a molecular weight of 58 kD. It remains to be seen whether the keratin corresponds to MHb 3 or MHb 4.
Introduction
The keratin multigene family consists of about 30 individual, however, structurally related members which can be grouped into epithelial-type keratins ('soft' ~-keratins) and wool- and hairtype keratins ('hard' ~-keratins) [1-3]. The great majority of these proteins belongs to the epithelialtype of keratins. As a rule, the mammalian hair follicle contains only a set of eight major 'hard' ~-keratins [ 1-6]. Four of them, Hbl, Hb2, Hb3 and Hb4, belong to the basic to neutral, type II subfamily of keratins, whereas the remaining four keratins, Hal-Ha4, represent acidic, type I keratins [ 1-6]. The occurrence of an equal number of type II and type I hair keratins indicates that their synthesis follows the principle of pairwise expression, although the exact composition of the sixteen possible hair keratin pairs has not yet been
elucidated. Comparisons of the presently available amino acid sequence data of both wool and hair keratins with that of epithelial keratins have revealed that 'hard' and 'soft' keratins are rather homologous in their central s-helical rod domain, however, differ substantially in their non s-helical head and tail portions [3, 6-8]. Typically, the 'hard' keratins show an accumulation of cysteine and proline residues in their amino- and carboxyterminal domains [6-8] and the high content of cysteine is thought to essentially account for the rigidity and physical strength of the mature hair and wool fiber by extensive S-S-bridging of the constituent proteins. Immunohistochemical studies and protein investigations in different laboratories have shown that the eight hair type keratins are not only expressed in the hair follicle, but also occur in nail-forming cells, the filiform papillae of the tongue and the thymic reticulum [ 1, 2, 4, 5, 9].
40 It is evident that the knowledge of the amino acid squences of the individual hair keratins would essentially contribute to the understanding of the development and formation of hard, keratinized structures in these different anatomical locations. However, at present sequence information for wool and hair type keratins is relatively sparse and to our knowledge limited to that of a sheep wool type II keratin 7c [7] and to two murine type I hair keratins, M H a l and MHa4 [6, 8]. In the course of our studies on keratin expression in various stratified and keratinized epithelia of the mouse, we have recently constructed a cDNA library with polyA+RNA from adult mouse tail epidermis. This library was aimed to serve for the isolation of cDNA clones for keratins which are supposed to be expressed in the parakeratotic scale epidermis of this morphologically complex epithelium [ 10]. The screening of the library for type II keratin cDNAs yielded, besides several new keratin clones, one clone which turned out to encode a murine type II hair keratin. In this paper we present the almost complete amino acid sequence of this keratin and, by in situ hybridization, provide data on its tissue-specific expression.
Materials and methods
(final wash with 0.1 x SSC, 0.1~o SDS). The resulting positive phage clones were further hybridized with a mixture of [3ap]-labeled specific 3' c D N A fragments of type II keratin clones pke70 [14], pktl-1 and pktl-5 [unpub. results]. Hybridization was performed for 18 h at 42 ~ (50~o formamide, 5 x SSPE) and the final wash was at 68 ~ (0.1 • SSC, 0.1~o SDS).
RNA slot blot hybridization Phage clones which did not react with the mixture of specific 3'-fragments of the clones coding for K1, K5 and the 70 kd keratin were subjected to R N A slot blot hybridization. R N A was isolated as described above from the epidermis of different skin sites, various internal stratified epithelia, epidermal tumors and cell lines. Slot blots were performed with Gene screen plus membranes (DuPont, Dreieich, Germany) using a minifold I system (Schleicher & SchOll, Dassel, Germany). Hybridization was carried out for 18 h at 42 ~ (50~o formamide, 1 M NaC1, 1~/o SDS, 5 0 m M Tris-C1, pH7.6, 10~o dextrane sulfate) with [32p]-labeled phage inserts. Membranes were washed stringently (0.1 • S SC, 1 ~o SDS at 68 ~o) and exposed at -80 ~C to Kodak X-Omat films. One phage clone, termed 2ktlII-4 was chosen for further characterization.
cDNA cloning and screening procedures PolyA + RNA of adult mouse tail epidermis, obtained after a short incubation of tail skin in 60 ~C hot water [10], was isolated according to the method of Gough [ 11 ] and used for the construction of a cDNA library in 2gtl0 [ 12]. The library was screened by hybridization with a [32p]_ labeled 535 bp ClaI/KpnI fragment of the previously described c D N A clone pkt57 which encodes the type II keratin K4 of internal stratified mouse epithelia [13]. The fragment spans f r o m position 601 of 1136 of pkt57 and thus contains a portion of the sequence coding for the s-helical domain of K4 [13]. Hybridization was carried out for 18h at 42 ~ (35~o formamide, 5 x SSPE) and subsequent washing was at 52 ~
Subcloning and DNA sequencing The insert of 2ktllI-4, attached to Eco RI adaptors, was cloned into the transcription vector Bluescript II KS + (Strategene; La Jolla, USA). Sequencing of both strands of this plasmid clone pktlII-4 was performed according to the dideoxy sequencing method of Sanger et al. [15], using first M13 and T3 primers and subsequently 17mer synthetic oligonucleotides as walking primers. To prepare a specific probe of 3' noncoding region of clone pktlII-4, a 262 bp Pst I fragment was subcloned into Bluescript II KS +. This subclone was designated pktlII-4-3'.
41
In situ hybridization The protocol used for in situ hybridization of frozen tissue sections (nominally 6 #m thick) was essentially as described previously [14, 16, 17], however, with some modifications [ 18, 19]. Upon linearization ofpktllI-4-3' with Sma I, riboprobes were obtained that were labeled with [35S]-UTP by in vitro transcription with T3 RNA polymerase. A sense probe was used as negative control. Slides were dipped in Kodak NTB2 photoemulsion, developed after 2 to 5 days and stained with hematoxylin-eosin.
Hybrid selection analysis and translation in vitro PolyA § RNA obtained by oligo (dT) cellulose chromatography of total RNA of tail epidermis was hybridized to filter bound DNA of pktllI-43' and the selected m R N A was translated in vitro in the presence of [ 35S ]-methionine. PolyA + RNA from both footsole epidermis and tongue epithelium was also translated in vitro. One dimensional SDS PAGE of the in vitro translation products and autoradiography were performed as described [20].
the remaining clones, these were subjected to slot blot hybridization analysis with RNA from epidermis of different skin sites, various internal stratified epithelia, benign and malignant epidermal tumors and a murine epidermal cell line. This analysis resulted in the identification of three clones which exhibited a strong hybridization signal with polyA + RNA of tail epidermis, however, also showed a weak, but significant reaction with polyA + RNA of tongue epithelium (Fig. 1). This intriguing expression characteristics prompted us to sequence the corresponding clones. Two of them turned out to be identical and were found to encode the desired type II 65 kD keratin whose properties will be reported elsewhere [Tobiasch et al., in prep.]. The nucleotide sequence of the 1770 bp insert of the third clone, pktlII-4, is shown in Fig. 2. It
Results and discussion
In an attempt to isolate a cDNA clone for a particular type II 65 kD keratin of adult mouse tail epidermis [ 10], we have screened a 2gtl0 library, constructed with polyA + RNA of the epidermis of this skin site for the presence of type II keratin cDNAs. To this purpose we used an appropriately taylored DNA probe which was derived from the region coding for the a-helical domain of murine keratin K4 [ 13 ]. From the resulting bulk of keratin cDNAs, we first eliminated those clones which hybridized with the specific 3'noncoding ends of the previously isolated cDNAs of the 70 kD keratin [14] and of keratins K1 and K5 [unpub. data]. These keratins represent the major type II keratins expressed in adult mouse tail epidermis [ 10]. In order to preliminary characterize
Fig. 1. RNA slot blot hybridization. Total RNA from (a) neonatal mouse epidermis, (b)adult mouse footsole epidermis (e)a DMBA/TPA-induced epidermal squamous cell carcinoma, (d) a papilloma-derived epidermal cell line, SP1 [26], (e) mouse tail epidermis, ( f ) mouse tongue epithelium, (g) mouse palate epithelium and (h) mouse brain was spotted onto a nylon membrane in concentrations of 1 #g, 2/~g and 4/~g each and hybridized with the phage clone )tktl II-4 insert. Note the strong reaction with RNA of tail epidermis and the weak reaction with RNA of tongue epithelium (e, f ) .
42 I 1
Cga aar gac ag9 tca tgg tgg agg aga age cac 9cg cga aga gga gag aag t t g aca gig t i t 9ga act gga aac R N D ~ S # N ~ H A ~ R G E K L T V F G T G N
~6 2, 1SI S 226
~6
ttc r
egc t g c gcc t e a g e t t g c ggg ccc egg c o t ggc r s 9 * s 9 9 c ~ ~ ~ G ~
tgr I
~
~9c t g c a t e t o t ~ca g e t cr 9 9 i s , A ~
taC cgJ gga c~r tCa ggg g9r t t C ggc agC ca 9 a g t g t r t g t Y ~ G L S G G F G S O S V ~ G A
age ate ggg tic
cga t o t
gga ggc a r c t g c ggg ccc age e t a
s
R
~
r
301 C t g t i c 1 0 1 L
o
Y
s
G
:
~
~
~
S
~
a c a c e c c t g a a c e t 9 ga 9 a t e 9 a e c c c a a t 9ca eaq t g t L T ~ L N L s 1 6 3 1 6 3
t a r a99 ggr &to t r 1 6 2 150 y ~ a x s so
99~ gcc ~ t c cgc t c c 9qr t c c F R S G S ~ G R
ccc t g c a t e
~
Q
:
9S 2S
tgt
gga CgC
225 ~5
ace act
9tc tot
gtc aac gag a9r
300
T
v
v
100
9t9 aag cat
T
S
.
t
S
gag gag aaa gag tag arc aag
37S 12S
3~6 t g t CtC a a c a g e a g g ~ t c 9 c g g e t t t C a t e g a r a a g g a g ego t t C c t g g a g c&g t a g a a c a a g c t g c t 9 g a g a c e ~ 2 6 C L N S R F A A F Z D ~ V R F L s
4SO 150
451 aag tgg tag t i c tac tag aac ego aa9 t9r t g t gag age aac atg gag cct ctg t i t I ~ I K W O F Y O N ~ K C C s 1 6 3 1 6 3
52S 195
gag 9gc tac ate gag 8cg
$26 c~g a g g e g g g a g g e t g a g t g t g a g g a g 9ca g a c a g e g99 a g g c t g g c t 9ca g i g c t c a a c c a t 9 c g t a g g a g t c c I ~ 6 L ~ R E A E C V s 1 6 3 1 6 3
600 200
601 atg gag gge tac aag aag agg t a t gaa gaa gaa g t t gca ere cgg 9cc aca gca gag aat gag t t t ~ 0 1 M s 1 6 5
6~S 225
6~6 a a g a a g g a t g a g g a c t g t g c c t a c c t 2 2 6 K K D V D C A Y
gtg qc[ eta
9 c g c a a g a c e ~a~ e t g g a g 9cc a a c ~ca g a g 9 c a c t g a c e ca~ g a g L R X S D L E A N A E A L T O E ~
~$1 gaC t~C c t g agg aga a t g t a t 9 a t gag gag a c t ego a r c c t c 2 5 1 D F L R R M Y D s
act
~S0 250
ca~ ~cc cac a r c t e a gac aca o c t g t c a t e ~ c
82S 2?5
626 aag atg gac aac age egg gac ctg aac atg gac t g t gtc gig get gag ate sag get tag t a t gat gac a r t gcc 2 9 6 ~ M D N S R D L N K D C V V A s
900 300
901 age cgc agC c g t g e t gag gCC gag t c c t g g t a c ccr a c t aa~ ~ g t gag ga 9 a t g aag gcc aca gcg a~c egg c a t 3 0 1 S ~ S ~ A s 1 6 3 1 6 3 1 6 3
S~5 325
9?6 9 g a g a g ac~ c t 9 c g r cgc a c t a~a g a g g a g a t e a&~ g a g c t g a a c a g a a t g a t e 3 2 6 G E T L R ~ T H s 1 6 3
1050 350
10~1 g a g a a t 3 5 1 s
g c c a a g t g c t a g a a c a c e s a g c~g g a g g c t g e t g a g a c t
caa t o t
tag agg ctg act
g c t gag a~c
gag t a g t a g 9ga gag g e t gcc ~ t
1125 3~5
1126 g c t g a t g e t c g c a g e a a g c t g g e t g a g t t 9 g a g ~ g t 9co c t g t a g a a g g c t a a g c a g g a c a t g g c r t g c c t g c t c ~ 6 A D A ~ C K L ~ s
1200 400
1201 aag gag tac tag gag gig atg aac tcc aag ctg ggg ctg 9ac gig gag ate ate ace tac agg cgc ctg ctg gag 401K s u 0 s V ~ K S K k G L D v s ~ ~ 9 u . . L L ~
12~S 42S
12~6 99C g a g g a g t a g a g g c t g t g t g a a g~c g t 9 g g a g c t g a g a a t 4 2 6 G s 1 6 3 1 6 9 1 6 3 1 6 9
g
13S0 450
13~1 tgc 999 gac ctc t g t gig t o t ggc t~a c?~ cot gig ac~ gqc ag~ ~tc tgc agt gcc eta tgc agc ggg aa~ ~ g
142S
1426 gca 9ta age ac~ ggr ctg t g t 9c9 tee t ~ t
~SO0
gtc tgt
gqa age 9gc c o t t g t
g~: age age tcc
c=c ccg gg9 agg t g t
cgt ggt gga get
tag ~ag a c a aga ggg
iSOl
age Cag gaa gag gee t 9 9 act aca agg c~a a9r atg 9ta get tea agg act get gcc cat gag ate tga gaa tac
15~5
15~6
art ccr cat Ccc cag cag ctg cca c~e c a t tCa gct acc t~c t g 9 caa 9gg gc~ ~gc age tga tag ate agc ctc
16SO
I~51
ctg cot tag r
1~25
1926 ~ t
ca~ ccc tg9 g~a tac cca g~g c~g t~t cct gag oct ct~ gc~ act agg cct gtt gag Caa tat
gag Ctg act CtC aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa
1790
Fig. 2. Nucleotide sequence of the pktl 11-4 insert and deduced amino acid sequence of the encoded type II keratin. The nucleotide sequence is shown in the 5' to 3' direction of the mRNA. The stop codon is marked by an asterisk, the polyadenylation signal is underlined. Arrows denote the region coding for the s-helical domain. In the non ~-helial domains, cysteine residues are encircled and proline residues are boxed.
43 contains the complete 3' noncoding region and almost the complete coding region of the m R N A of a keratin whose amino acid sequence is also indicated in Fig. 2. Sequence alignments with various type II murine keratins expressed in the epidermis and in internal stratified epithelia (shown here for keratin K4 in Fig. 3) readily reveal that the keratin encoded by pktllI-4 is unrelated to these keratins. The a-helical rod domains of the two keratins exhibit a sequence homology of about 55 ~o. However, there is evidence for an only poorly conserved H1 subdomain [21] in the aminoterminal region of the new keratin, whereas a distinct H2 subdomain in its carboxyterminus cannot be detected. In addition, although both the head and tail regions of the new keratin are relatively rich in serine residues, the absence of the typical accumulation of G G G and G G X motifs (with X being predominantly serine) [21] in the central part of both regions, does not allow to delineate distinct V1 and V2 subdomains [21]. Instead both, the amino- and the carboxyterminus of the new keratin contain a high percentage of cysteine and proline residues (10 cysteines, 8 prolines in the aminoterminus, 10 cysteines and 5 prolines, in the carboxyterminus). Especially in the carboxyterminus, these amino acids frequently appear as PC motifs (Fig. 2). Interestingly, all these properties are also a typical feature of the sheep wool type II keratin 7c [7] and of two murine type I hair keratins, M H a l and MHa4, recently described by Bertolino et al. [6, 8]. It therefore appears that clone pktllI-4 encodes a type II murine hair keratin. This assumption is confirmed by sequence comparison of the carboxyterminal regions of the new keratin and sheep wool type II keratin 7c (Fig. 4). Except for the penultimate parts of the carboxytermini, such an alignment demonstrates an almost complete homology of the two sequences. Moreover, in situ hybridization with the insert of subclone pktllI-4-3' to frozen sections of adult mouse tail epidermis and newborn mouse back epidermis reveals hybridization signals specifically over hair follicles. In longitudinal sections of hair follicles (Fig. 5a), it can be seen that the hybridization signals occur only over upper
cortex cells, whereas cells of the outer and inner root sheaths and matrix ceils in the bulbar region are free of label. This particular distribution of the m R N A of the new keratin could also be confirmed in cross sections through different levels of the hair follicle (Fig. 5b-d). The site of expression of the m R N A of the new hair keratin is therefore in agreement with numerous studies in which the localization of hair keratin proteins in the follicles of different species has been investigated by indirect immunofluorescent staining techniques [ 1, 2,4,5,6,8]. With the demonstration that clone pktllI-4 encodes a type II hair keratin, the positive reaction with tongue epithelial polyA + R N A in the slot blot hybridization analysis of Fig. 1 becomes understandable. Recent investigations in different laboratories have shown that hair type keratins are expressed in the central core unit of the filiform tongue papillae [5, 9]. In this compartment of the morphologically complex murine filiform papillae [22-24] the synthesis of these keratins is thought to be responsible for the formation of the posteriorly inclined, hook-shaped solid spine which consists of an extremely condensed keratin material [9]. As shown in Fig. 6, the m R N A of the new type II hair keratin is clearly expressed in the central core unit of the filiform papillae. The label is concentrated over living, suprabasal cells, whereas basal cells are free of label (Fig. 6b). We have previously shown that basal cells of the illiform papillae express keratins K5 and K14 [ 17]. The finding that clone pktllI-4 encodes a type II hair keratin, requires an explanation for its strong reaction with m R N A of tail epidermis and the negative reaction with m R N A of both newborn mouse epidermis and adult mouse footsole epidermis in the slot blot hybridization analysis of Fig. 1. We have previously shown that by gently lifting tail epidermis from the dermis of tail skin incubated for a short period in hot water, hair follicles quantitatively remain in the dermis [25]. In the present investigation, however, tail epidermis was scraped off from the dermis with forceps after incubating the skin in hot water. By this manipulation, large quantities of hair follicles are removed together with the epidermis. In contrast
20
30
40
50
60
70
80
90
.
.
.
S
~
FG
1%*
.
.
.
.
S
*
*
r-
GG G
**
Lt---I
*
G F
1%
G
1%
"
G CONSENSUS
1%
MK4 MHb4
1%
*1%1%W1%*
~
N SLLTPL
****
EIDP
*
9
Q
~1%*1%1%
o
E EQIK
*1%
LN
1%W
*1%1%*******1%1%*
9
~
FA F I D K V R F L E Q Q N K
LETKW
****1%
9
1%
Q
S
W
9
1%
*1%
1%
P FE Y C O N S E N S U S
LR
D GRL
-
EL
9
Q S E
L2
K
YEEE
.
R
.
.
AEN FV L K K D V D
.
AY
K
.
LEA
E L
.
E
.
F R
Y
E
9
*
D
It
~1%
AE
~
*1%~
AQY
*1%1%
DIA
"
~1%
1%1%*1%
S AE ESWY
1%
~
*
K
~
1%*
HG
1%
L
V
t
*
T
**
**~****1%1%
~
EI E L N R M I Q R L
*~*~*
9
AEIEN
*
*
K Q
~
1%
~
A V
**
~
MK4
EQ GE C O N S E N S U S
MK4 MHb4
CONSENSUS
K AELE ALQKAK
D A LL
G
YQ
MN KL LDVEI
TYR L L E G E E
R
E
AV
V
G
SG
GS
CONSENSUS
Fig. 3. Comparison of the amino acid sequences of the murine type II epithelial keratin K4 (first row) and the keratin encoded by clone pktl 11-4 (second row). Also indicated is the consensus sequence between the two keratins (fourth row). The arrowheads denote the cental or-helical domains in which the non coiled-coil linker regions are indicated.
G
501 G G G I Y G G S G S K I T S S A T I T K R S P R 472 S G N V A V S T G L C A P C G S G P C H P G R C
AL DA
AALADARCKLAELEGALQKAKQDMACLLKEYQEVMNSKLGLDVEIIT•RRLLEGEEQR-LCEGVGAVNVCVSSSRGGVVCGDLCVSGLRPVTGSVCSAP• MHb4
401 L A L K D A Y S K R A E L E T A L Q K A K E D L A R L L R D Y Q A L M N V K L A L D ~ E I A T Y R K L L E G E E ~ R ~ S G E C K S A V S I ~ w G G ~ Q H W R S G L G L G ~ G F C S G S G ~ G S G F G F 373
*
*1%1%
MDN R L
9
V
*1%
H SDTS
301 V V L S M D N N R N L D L D G I I A E V R A Q Y E D I A R K ~ I E V ; ~ S ' ~ W Q I K V Q Q L O M S A D Q H G D S L K T T K N E I S E L N R M I Q R L R A E I E N I K K Q S Q T P Q A S V A D A E Q R G E 273 V I V K M D N S R D L N M D C w A E I K A • Y D D I A S R S R A E A E S W Y P T K C E E M K A T V I R H G E T L R R T R E E I N E L N R M I Q R L T A E I E N A K C Q N T K L E A A V T Q S E Q Q G E
--Llt2--"i
I
MHb4
~
1%
V
MK4
9
I
1%
173 IETLRREAECVEADSGRI.~I~I%ELNHAQESMEGYKKR YEEEVALI:IATAENEFVALKKDVDcAYLRKSDLEANAEALTQETDFLRItNYDEETRILHSHISDTS
W
G
1%
G
SFNGRGGPGFPV~PAGG~QEVT~NQSLLTPL~VE~DPE~QK~TAEREQ~KTLNNKF~F~DKVRFLEQQNKVLETKWNLLQ~J~TTTT~PK~LDPFFETY MK4 R~FGYRSGGI~GPSPP~ITTVSVNESLLTPLNLEIDPNAQCVKHEE~EQIKCLNSRFAAFIDKVRFLEQQNKLLETKWQFY~.NRK~CES--NMEPLFEGY MHb4
.
R
1%
RNDRSWWRRRHARRGEKLTVFGTGNFSCASACGPRPGRCCISAAPYRGISCYRGLSGGFGSQSVCGAFRSGSCG
i00
201 INALRKNLDTLSNDKGRL~ELKMM~DSVEIAFKTKYEEEINKRTAAENDFwLKKDVDAAYMIKVELEAKMESLKDEINFTRVLYEAELAQMQTVS~DTS
i01 75
1 ..........................
1 MIAR••SVRGASPGFTSGSAIAGGV•CRVAFSSGSMSGGAGRC•SGGFGSRSLYNLGGHKSISMSVAGSCQGGGYGGAGGFGVGGYGAGFGAGGFGGGFGG
I0
45 LCEGVGAVNVCVSSSRGGVVCGDLCVSGLRPVTGSVCSAPCSGNVAVSTG
llLilll11111ilLli11111Ltltli
LILIIIIIBITIILi
~'V~
LIIII
LCEGVGAVNVCVSSSRGGVVCGDLCVSGSRPVTGSVCSAPCSGNLAVSTG
S~
L C A P C G S G P C H P G R C .......
LILLLL
i
LCAPCGQLNTTCGGGSCSLGRC Fig. 4. Sequence comparison of the carboxyterminal regions of the new murine type II keratin encoded by clone pktl II-4 (MK) and sheep wool type II keratin 7c (SK) [7].
Fig. 5. In situ hybridization of the [35S]-labeled specific riboprobe of subclone pktl 1I-4-3' to frozen sections of (a) adult mouse tail skin and (b-d) neonatal (3 days old) mouse back skin. In (a) a longitudinal section and in (b-d), cross sections of hair follicles are shown, ors, outer root sheath; irs, inner root sheath; c, cortex; hm, hair matrix; ct, cuticle; h, hair; dp, dermal papillae. Bar = 100/~m throughout.
newborn mouse epidermis was gently peeled off from the dermis and in the case of footsole epidermis, only the glabrous skin portion was used for the heat isolation of epidermis. It is thus con-
ceivable that these differences in tissue separation may account for the presence of hair keratin mRNAs in tail epidermis. Finally, in order to investigate to which of the
46
Fig. 7. Hybrid selection analysis. ThemRNA-species selected by hybridization of polyA + RNA of tail epidermis to filterbound DNA of subclone pktl II-4-3' was translated in vitro in the presence of [3sS]-methionine. The translation product was resolved by one dimensional SDS-PAGE on a 9% gel (lane c). Lanes a and b represent the keratin profiles obtained by in vitro translation of polyA + RNA of footsole epidermis (a) and tongue epithefium (b). The molecular weight of the keratin in lane c was estimated relative to the molecular weights of keratins KI (67 kD), K10 (60 kD), K14 (52 kD) in lane a and K4 (57 kD) and K13 (47 kD) in lane b.
Fig. 6. In situ hybridization of the [35S]-labeled specific riboprobe of subclone pktl 1I-4-3' to frozen sections of mouse tongue (a, b). Note the specific localization of hybridization signals in suprabasal cells of the central core unit of the fillform papillae (b). fp, filiform papillae; ac, anterior compartment; cu, central core unit; ipe, interpapillary epithelium; dp, dermal papillae. Bar = 100/~m in (a) and (b).
four type II murine hair keratins the new keratin might correspond, we performed a hybrid selection experiment with polyA + RNA of tail epidermis. As shown in Fig. 7, subclone pktlII-4-3' selected a mRNA which on release and in vitro translation yielded a single protein (Fig. 7, lane c). Using the in vitro translated keratins of footsole epidermis and tongue epithelium polyA + RNA as a reference, the size of the protein could be assessed to 58 kD. Calculation of the molecular weight of the presented part of the pktlII-4 encoded keratin yields a value of 55 kD, indicating that the sequence of the keratin lacks only few
47 amino acids of the aminoterminal domain. Previous experiments h a d s h o w n that b o t h murine type II hair keratins M H b 3 and M H b 4 migrate in the 58 k D molecular weight range [6, 8]. Further experiments are required to definitely assign our new keratin within the type I I murine hair keratin subfamily.
Acknowledgement We t h a n k M. M a c L e o d for typing the manuscript.
Note After submission o f our paper, Yu et al. published a full length c D N A clone encoding a murine type I I hair keratin. By m e a n s of Western blotting with a monospecific antibody directed against the last 18 amino acids of the c arboxyterminus o f the encoded keratin, the authors showed that the keratin c o r r e s p o n d s to M H b 4 (Yu et al., J. Invest. Dermatol. 97, 354-361, 1991). Except for a short sequence area coding for the penultimate aminoterminal region, the clone published by Yu et al. and our clone are completely identical. The b r e a k between the two sequences occurs u p s t r e a m f r o m p o s i t i o n 6 1 (triplet ttt in Fig. 2). W e have re-examined the sequence area 1-61 o f our clone and found it to be correct. The calculated molecular weight o f the keratin encoded by the clone of Yu et al. is 52 k D . This value is far below the S D S - P A G E size estimates of about 5 7 - 5 8 k D for M H b 4 (Bertolino et al., J. Invest. Dermatol. 94, 297-303, 1990). As a rule molecular weights o f keratins calculated f r o m cloned sequences are slightly larger than those estimated from S D S P A G E . This is also true for type I hair keratins (Bertolino et al., J. Invest. Dermatol. 91, 5 4 1 546, 1988). It, therefore, appears that the aminoterminal d o m a i n o f the clone published by Yu et al. m a y be too short.
References 1. Heid W, Werner E & Frank WW (1986) Differentiation 32:101-119 2. Lynch MH, O'Guin WM, Hardy C, Mak L & Sun T-T (1986) J. Cell Biol. 103:2593-2606 3. Conway JF & Parry DAD (1988) Int J. Biol. Macromol 10:79-98 4. Heid W, Moll I & Franke WW (1988) Differentiation 37: 137-157 5. Heid W, Moll I & Franke WW (1988) Differentiation 37: 215-230 6. Bertolino AP, Checkla DM, Notterman R, Sklaver I, SchiffTA, Freedberg IM & DiDona GJ (1988) J. Invest. Dermatol 91:541-546 7. Sparrow LG, Robinson CP, MacMahon DTW & Rubira MR (1989) Biochem. J. 261:1015-1022 8. Bertolino AP, Checkla DM, Heitner S, Freedberg IM & Yu DW (1988) J. Invest. Dermatol 94:297-303 9. Dhouailly D, Xu C, Manabe M, Schermer A & Sun T-T (1989) Exp. Cell Res. 1818:141-158 10. Schweizer J, Fflrstenberger G & Winter H (1986) J. Invest. Dermatol. 89:125-131 11. Gough NM (1988) Anal. Biochem. 173:93-95 12. Maniatis T, Fritsch EF & Sambrook J (1989) In: Molecular Cloning (A Laboratory Manual). Cold Spring Harbor Laboratory 13. Knapp B, Rentrop M, Schweizer J & Winter H (1986) Nucleic Acids Res. 14:751-763 14. Rentrop M, Nischt R, Knapp B, Schweizer J & Winter H (1987) Differentiation 34:189-200 15. Sanger P, Nicklen S & Coulson AR (1977) Proc. Natl. Acad. Sci. (USA) 74:5463-5467 16. Rentrop M, Knapp B, Winter H & Schweizer J (1986) Histochem. J. 18:271-276 17. Rentrop M, Knapp B, Winter H & Schweizer J (1986)J. Cell Biol. 103:2583-2591 18. Angerer LM, Cox KH & Angerer RC (1987) Methods in Enzymol. 152:649-661 19. Angerer LM & Angerer RC (1989) DuPont Biotech. Update 4:3-6 20. Knapp B, Rentrop, Schweizer J & Winter H (1987)J. Biol. Chem. 262:938-945 21. Steinert PM, Steven AC & Roop DR (1985) Cell 42: 411-419 22. Cameron IL (1966) J Exp. Zool. 163:271-284 23. Cane AK & Spearman RIC (1969) Arch. Oral Biol. 14: 829-814 24. Farbman AI (1979) J. Anat. 106:233-242 25. Schweizer J & Marks F (1977) Cancer Res. 37: 41954201 26. Strickland JE, Greenhalgh DA, Koceva-Chyla A, Hennings H, Restrepo C, Balaschak M & Yuspa SH (1988) Cancer Res. 48:165-169
