Cell, Vol. 61, 1103-l

112, June

15, 1990, Copyright

0 1990 by Cell Press

Identification of a Major Keratinocyte Cell Envelope Protein, Loricrin Thomas Mehrel:t Daniel Hohl,*§II Joseph A. Rothnagel,§ Mary A. Longley,§ Donnie Bundman,§ Christina Cheng: Ulrlke Lichti,’ Margaret E. Bisher,# Alasdair C. Steven,# Peter M. Steinert,* l l Stuart H. Yirspa,” and Dennis R. Roop’§ * Laboratory of Cellular Carcinogenesis + Dermatology Branch National Cancer Institute #Laboratory of Physical Biology National Institute of Arthritis, Musculoskeletal and Skin Diseases National Institutes of Health Bethesda, Maryland 20892 §Departments of Cell Biology and Dermatology Baylor College of Medicine Houston, Texas 77030

Summary During epidermal cell cornification, the deposition of a layer of covalently cross-linked protein on the cytoplasmic face of the plasma membrane forms the cell envelope. We have isolated and characterized cDNA clones encoding a major differentiation product of mouse epidermal cells, which has an amino acid composition similar to that of purified cell envelopes. *anscripts of this gene are restricted to the granular layer and are as abundant as the differentation-specific keratins, Kl and KIO. An antiserum against a C-terminal peptide localizes this protein in discrete granules in the stratum granulosum and subsequently at the periphery of stratum corneum cells. Immunofluorescence and immunoelectron microscopy detect this epitope only on the inner surface of purified cell envelopes. Taken together, these results suggest that it is a major component of cell envelopes. On the basis of its presumed function, this protein is named loricrin. Introduction Terminal differentiation of epidermial cells begins with the migration of cells from the basal layer, continues with the progression of cells through the spinous and granular layers, and terminates with the formation of mature epidermal cells (squames) of the stratum corneum. This process has been shown by morphological and biochemical studies to occur in stages (Matoltsy, 1975). Keratins K5 and K14 are major products of basal epidermal cells (Woodt Present Address: Department of Dermatology, New York University, Medical Center, New York, New York 10016. 11Present Address: Dermatologische Klinik, Universitatsspital, Gloriastrasse 31, CH-6091 Zurich, Switzerland. ** Present Address: Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20692.

cock-Mitchell et al., 1982). These proteins assemble into intermediate filaments and, together with microtubules (tubulin) and microfilaments (actin), compose the cytoskeleton of epidermal cells (Steinert and Roop, 1988). One of the earliest changes associated with the commitment to differentiation and migration into the spinous layer is the induction of a differentiation-specific pair of keratins, Kl and KlO. Intermediate filaments containing Kl and KlO replace those containing K5 and K14 as the major products of cells in the spinous layer (Fuchs and Green, 1980; Woodcock-Mitchell et al., 1982; Roop et al., 1983; Schweizer et al., 1984). In the granular layer of the epidermis, a non-intermediate filament protein, filaggrin, is synthesized as a high molecular weight precursor, which after processing, is thought to promote keratin filament aggregation and disulfide-bond formation (Dale et al., 1978; Steinert et al., 1981; Harding and Scott, 1983; Steinert, 1983). In addition, during late stages of epidermal cell maturation, a highly insoluble cornified envelope is assembled just beneath the plasma membrane, by the action of transglutaminases that catalyze the formation of s-(y-glutamyl)lysine cross-links (Rice and Green, 1977). The biochemical data predict that the progression of cells through various differentiation states would be correlated with changes in gene expression. Several laboratories have now made substantial progress toward confirming this prediction for the major keratins expressed in the epidermis (Steinert et al., 1983, 1985; Lehnert et al., 1984; Marchuk et al., 1985; Krieg et al., 1985; Rieger et al., 1985; Knapp et al., 1987; Lersch and Fuchs, 1988; Roop et al., 1988) as well as for filaggrin (Haydock and Dale, 1986; Rothnagel et al., 1987; McKinley-Grant et al., 1989). Less is known about components of the cross-linked envelope. Several proteins have been implicated as precursors of the cornified envelope on the basis of their reactivity to antibodies produced against purified cornified envelopes (Michei et al., 1987; Kubilus et al., 1987; Nagae et al., 1987) the reactivity of antibodies produced against specific proteins with the periphery of cells located in the granular layer and stratum corneum (Rice and Green, 1979; Lobitz and Buxman, 1982; Zettergren et al., 1984; Ma and Sun, 1986; Tezuka and Takahashi, 1987) and/or their ability to serve as substrates for transglutaminase (Rice and Green, 1979; Buxman et al., 1976; Goldsmith, 1977; Kubilus and Baden, 1982; Simon and Green, 1984). However, only one component, involucrin, has been studied in detail at the molecular level. lnvolucrin constitutes 5%-10% of soluble proteins (perhaps about 1% of total proteins) of cultured human keratinocytes and associates with envelopes under cross-linking conditions (Rice and Green, 1979). In experiments performed with cell-free components of cultured keratinocytes, involucrin is a preferred substrate in the cross-linking reaction catalyzed by an epidermal transglutaminase (Simon and Green, 1985). However, the unusual amino acid composition of involucrin (approximately 45% Glu and Gln), initially determined

Cdl 1104

A _ _ H

S /

/

S I

I

s 1

I

8 I

BE

Figure 1. Restriction quence, and Deduced of Mouse Loricrin

P

,

L-

pk 1005 / pk321

for the purified protein (Rice and Green, 1979) and more recently confirmed from the amino acid sequence deduced from the sequence of the gene (Eckert and Green, 1986) differs substantially from that of purified envelopes induced in cultured human keratinocytes (approximately 16% Glu and Gln; Rice and Green, 1979) and those isolated from human foreskin epidermis (approximately 9.0% Glu and Gln, see Table 1). This suggests the existence of other quantitatively important envelope components. Evidence is presented in this report for the isolation and

Map, Nucleotide SeAmino Acid Sequence

(A) Schematic of mouse loricrin cDNA showing the regions encoded by the four clones used to generate the full-length sequence. The coding region is shown as a box and noncoding regions as a solid line. The restriction sites used to generate specific probes are shown: Seal (S), BamHl (B), BstEll (BE), and Pstl (P). Clones pK 1005 and pK 321 were isolated from a cDNA library prepared in pBR322. Clone pg 8 was originally isolated from a hgtll cDNA library and later subcloned into pGEM7 (Promega). The 5’-most clone, pcrext, was generated from amplified products extended from an oligonucleotide sequence unique to the 5’ end of pg 8. (B) Nucleotide and amino acid sequence of mouse loricrin cDNA. The polyadenylation sig nal, AATAAA, is underlined. Nucleotides are numbered consecutively at the left-hand side and amino acids on the right-hand side. The unique carboxy-terminal sequence (HGTWKQAPTWPCK) was used for antibody production.

characterization of cDNA clones encoding a previously undescribed cell envelope protein. Several lines of evidence suggest that it is both a major product of terminally differentiating epidermal cells and quantitatively an important cell envelope component. Results Characterization of cDNA Clones In initial studies to isolate cDNA clones encoding

major

Loricrin: 1105

A Major Cell Envelope

Protein

proteins expressed at different differentiation states in mouse epidermis, three classes of clones were identified that were complementary to equally abundant mRNAs of 2.4, 2.0, and 1.6 kb (Roop et al., 1983). On the basis of hybridization selection analysis (Roop et al., 1983) and nucleotide sequence information (Steinert et al., 1963, 1985) the clones complementary to the 2.4 and 2.0 kb mRNAs were determined to correspond to the differentiation-specific keratins Kl and KlO, respectively. On the basis of preliminary hybridization selection data and partial nucleotide sequence analysis, the clones complementary to the 1.6 kb mRNA were presumed to encode a differentiation-specific 55 kd keratin (Roop et al., 1983). However, analysis of the complete nucleotide sequence of these original clones (pK 321 and pK 1005) (Roop et al., 1983) and additional clones (see Figure 1) failed to reveal a coiled-coil region characteristic of all keratin intermediate filament proteins (Steinert and Roop, 1968). Instead, the sequences possess numerous inexact peptide repeats containing glycine, serine, cysteine, and tyrosine residues reminiscent of the amino- and carboxy-terminal end domains of the keratins Kl (Steinert et al., 1985) and KlO (Steinert et al., 1983). However, the exact peptide repeat sequences are different from the keratins and extend for much longer stretches. Curiously, these peptide repeats are interrupted in two locations by sequences enriched in glutamines and, in addition, the amino- and carboxy-terminal ends also contain clusters of glutamines. The absence of methionine residues in the deduced amino acid sequence of this clone explains our failure to detect the true translation products in the initial hybridization selection assays performed with [Wlmethionine (Roop et al., 1983). From the sequence information in Figure 1, it is possible to calculate that this cDNA encodes a protein with a molecular weight of 37,828 and an estimated pl of 9.5. To date, an epidermal protein with these characteristics has not been described. However, Nischt et al. (1988) reported a sequence of a partial cDNA clone from mouse footpad epidermis that was extremely GC rich. The authors did not identify the protein encoded by this cDNA, presumably because of disruptions in the open reading frame of their sequence, but it has high homology (>98%) with pK 321 (nucleotides 1031-1734), suggesting that they are identical. To identify this protein, antibodies were produced against a synthetic peptide corresponding to 14 unique residues at the carboxyl terminus of this sequence (see Figure 1). This antiserum was purified by affinity chromatography with the synthetic peptide and immunoprecipitated a prominent protein of approximately 45 kd from a [35S]cysteinelabeled total protein extract of newborn mouse epidermis (Figure 2). In addition, [%]cysteine-labeled peptides resulting from the translation of mRNA selected by hybridization to a subcloned 3’ noncoding region of the cDNA (pK 1005, see Figure 1) were also immunoprecipitated with this antiserum (Figure 2). The difference in electrophoretie mobility of the in vitro synthesized translation products versus the in vivo labeled products (i.e., the in vitro products migrate as a broad band between 40-50 kd) may be due to the lack of posttranslational modification in the

3

4

68

Figure 2. lmmunoprecipitation teins and In Vitro Translation

oi Total [%]Cysteine-Labeled Products with Anti-pK 321 Serum

Pro-

Newborn mouse skin was minced and incubated with [%S]cysteine for 3 hr. Total protein was extracted in 5% SDS-20% P-mercaptoethanol at 100°C. Lane 1, total labeled protein. Total labeled proteins in lane 1 were immunoprecipitated with anti-p)< 321 (lane 2) and preimmune serum (lane 3). Lane 4, [%]cysteine-labeled proteins resulting from the in vitro translation of mRNA selected by hybridization to a 3’ noncoding clone, pK 1005 (see Figure l), were immunoprecipitated with anti-p)< 321 serum. The positions of %-labeled molecular size standards are shown on the right.

cell-free system. It should also be noted that the apparent molecular size of the in vivo labeled protein (approximately 45 kd) is greater than that predicted from the amino acid sequence (38 kd) and may result from the aberrant behaviorof this protein on SDS gels due to its unusual amino acid sequence. Evidence That pK 321 Encodes a Major Component of the Cell Envelope The carboxy-terminal-specific antiserum was shown by double-label indirect immunofluorescence to react with the granular layer of newborn mouse epidermis (Figure 3A). The epitope recognized by this antiserum was initially detected in discrete granules in the first layers of the granular compartment. Preincubation of the antiserum with the synthetic peptide eliminated this staining pattern and confirmed the specificity of the antiserum (data not shown). The staining in selected areas of newborn mouse epidermis suggested that the epitope was more abundant at the periphery of cells (data not shown). This prompted us to examine benign mouse skin tumors by indirect immunofluorescence, since these tumors contain many differentiated cell layers and the layers are less compact

Cdl 1106

Figure

3. Indirect

lmmunofluorescence

with Anti-pK

321 Serum

(A and B) Double-label immunofluorescence of newborn mouse epidermis. (A) Rabbit anti-p)90% are located on the side away from the backing layer, i.e., the envelope’s inner surface (Figure 4B), in agreement with the immunofluorescent staining pattern (Figure 4A). This labeling was not observed in control experiments, with rabbit IgGs from preimmune serum or protein A alone. Epidermal transglutaminase is the enzyme presumed to catalyze the formation of the s-(y-glutamyl)lysine crosslinks in cell envelopes (Thacher and Rice, 1985). Therefore, we attempted to determine if the protein encoded by pK 321 was a substrate for epidermal transglutaminase. Initial experiments demonstrated that the amount of immunoprecipitable protein present after a 3 hr labeling period with [35S]cysteine decreased after an 18 hr chase period (Figure 5A). This decrease may have resulted from cross-linking the labeled protein into insoluble complexes by transglutaminase. Therefore, a pulse-chase experi-

Loricrin: 1107

A Major

Cell Envelope

Protein

12345

1

2

34

18

B

A Figure

5. lmmunoprecipitation

of Pulse-Labeled

Protein

Extracts

(A) Effect of the transglutaminase inhibitor LTB-2 on cross-linking. Newborn mouse skin was minced and incubated with [%]cysteine for 3 hr. At that point, the medium was changed and cold cysteine was added for a chase period of 18 hr in the absence and presence of an inhibitor of epidermal transglutaminase (LTB-2; Syntex Corp.). Lane 1, total protein extract after a 3 hr labeling period. Labeled proteins in lane 1 were immunoprecipitated with anti-pK 321 (lane 2) or preimmune serum (lane 3). Lanes 4 and 5, immunoprecipitation of labeled protein extracted after the chase period in the absence (lane 4) and presence (lane 5) of the inhibitor. (B) lmmunoprecipitation of a human foreskin extract with anti-p)< 321 serum. Human foreskin was minced and incubated with $sS]cysteine. Lane 1, total protein extract after a 3 hr labeling period. Labeled proteins in lane 1 were immunoprecipitated with anti-pK 321 (lane 2) or preimmune serum (lane 3). An immunoprecipitation of mouse skin (the extract shown in [A], lane 1) was included on the gel for comparison. The positions of “C-labeled molecular size standards are shown between panels.

ment was carried out in the presence and absence of LTB2, a specific irreversible inhibitor of epidermal transglutaminase that blocks the formation of cross-linked envelopes (Killackey et al., 1989) (LTB-2 was kindly provided by Dr. Larry DeYoung, Syntex Research, Palo Alto, CA, and is identical to RS10823 in Killackey et al., 1989). The inhibitor was able to block substantially the decrease in immunoprecipitable labeled protein during the chase period and indicated this protein was a substrate for transglutaminase. Additional evidence suggesting that the protein encoded by pK 321 was a major component of cell envelopes was obtained by comparing the amino acid composition of the sequence deduced from the GDNA clone with that of envelopes isolated from newborn and adult mouse

epidermis (Table 1). The deduced sequence has a high Gly (55.1%) Ser (22.3%) and Cys (7.1%) content. These amino acids are also the most abundant in newborn and adult mouse cell envelopes. Thus, the protein encoded by pK 321 appears to be a major envelope component. This argument is further strengthened by including in the comparison data available for three other putative envelope components: involucrin, which was initially isolated from cultured human keratinocytes by Rice and Green (1979) and shown to have a high Glu/Gln content (46%); keratolinin, which was extracted from human and bovine tissue by Zettergren et al. (1984) and is enriched in Glu/Gln (13.5%), Ser (10.4%), and Thr (9.2%); and a cysteine-rich envelope protein recently isolated from human epidermis by Tezuka and Takahashi (1987), which is predominantly composed of Gly (18.5%) Glu/Gln (12.70/o), Ser (9.80/o), and, to a lesser extent, Cys (4.3%). The amino acid composition of human foreskin cell envelopes, included for comparison, also has a high Gly, Ser, and Cys content as observed for mouse envelopes. On the basis of its presumed function, we have called the protein encoded by pK 321 “loricrin” (from the Latin, lorica-a protective shell or cover). Detection of Loricrin in Human Epidermis In addition to involucrin and keratolinin, the high glycine (33.9%) and serine (18.9%) content of human foreskin cell envelopes (Table 1) predicts the existence of an envelope component similar in properties to the mouse envelope protein. To test this prediction, a [35S]cysteine-labeled total protein extract of human foreskin was analyzed by immunoprecipitation with the antiserum elicited against the carboxy-terminal peptide of mouse loricrin. A comparison with a similarly labeled extract from newborn mouse skin revealed a prominent band in the foreskin extract with an estimated molecular size of 30 kd (Figure 58). Localization of Loricrin Transcripts by In Situ Hybridization lmmunofluorescence data obtained with the carboxy-terminal-specific antiserum indicated that synthesis of loricrin was only occurring in the more differentiated layers of the epidermis (Figure 3). Transcripts of the loricrin gene were detected in RNA isolated from newborn epidermis, which is hyperplastic and contains a large percentage of terminally differentiated cells, but not in RNA isolated from primary cultures of mouse epidermal cells grown in low calcium medium, which is permissive for proliferation but not terminal differentiation (Roop et al., 1983). This indicated that loricrin transcripts were confined to terminally differentiated cells. To determine at which stage of differentiation transcription of the loricrin gene was occurring, we compared the in vivo distribution of loricrin transcripts with that of other genes expressed in mouse epidermis by in situ hybridization (Figure 6). Transcripts for keratin K14 are predominantly restricted to the proliferating basal layer of the epidermis and the outer root sheath of the hair follicle (Figure 6A; Roop et al., 1988). Transcripts for keratin Kl are mainly observed in the spinous layer and decrease in the upper spinous and the first granular layer

Cdl 1108

Table

Amino GIY Ser CYS Tyr Glx Thr Pro Aw LYS Val Ala His Leu lie Met Phe Asx Tv

1. Amino

Acid

Acid Composition

Newborn CEsa 45.1 23.4 8.1 4.3 4.1 1.4 3.7 1.1 2.5 3.0 0.8 0.5 0.7 0.3 0.3 0.2 0.5 ND

Mouse

(Moles

%) of Purified

Cell Envelopes

Adult Mouse CEsa

Mouse Loricrinb

Human CEsa

43.1 23.6 7.0 4.0 4.6 1.6 4.2 1.5 2.1 3.1 1 .o 0.7 1 .o 0.3 0.4 0.3 0.5 ND

55.1 22.3 7.1 5.2 3.2 1.5 1.7 1.9 1 .o 0.4 0.4 0.2

33.9 18.9 4.6 2.2 8.0 2.1 7.7 1.0 4.8 2.6 3.6 0.6 1.5 2.0 0.2 2.4 1.7 ND

and Putative

Cell Envelope

Components

Foreskin lnvolucrinC

Keratolinin”

Cysteine-Rich Proteine

6.5 1.2 0.3 0.3 45.5 1.4 7.4 0.5 7.7 4.1 1.2 5.0 15.2 0.2 1 .o 0.2 2.0 0.3

6.0 10.4 1 .o 1.6 13.5 9.2 4.5 2.5 5.7 6.3 a.4 1.1 6.2 3.1 0.6 2.7 8.7 0.7

10.5 9.8 4.3 12.7 5.4 6.3 9.0 5.3 5.3 2.3 6.8 3.7 2.9 8.3 ND

CEs Induced in Cell Culture’ 9.2 7.2 2.5 1.8 15.9 4.8 7.8 4.3 7.6 5.1 7.0 1.9 7.9 3.5 1.5 2.9 9.1 ND

B Determined as described in Experimental Procedures. b Deduced from nucleotide sequence (Figure 1). c Eckert and Green (1986). d Lobitz and Buxman (1982). B Tezuka and Takahashi (1987). f Rice and Green (1979). ND, not determined; CEs, cell envelopes.

Discussion

Figure 6. Detection bridization

of RNA Transcripts

in the Epidermis

by In Situ Hy-

Frozen sections of newborn mouse skin were hybridized with %i-labeled RNA probes corresponding to keratin K14 (A), keratin Kl (B), and loricrin (C). Arrows indicate the dermal-epidermal junction.

(Figure 6B; Roop et al., 1966). Transcripts for loricrin are initially detected in the upper spinous layer and continue to accumulate in the granular layer (Figure 6C). Thus, the transcription pattern of the loricrin gene is very similar to that of the filaggrin gene, which is also transcribed in the granular layer (Rothnagel et al., 1967; Fisher et al., 1967; McKinley-Grant et al., 1969).

In this study, we have identified a novel cDNA encoding a major keratinocyte cell envelope protein, which we have named loricrin. Immunological studies with antibodies elicited against a specific carboxy-terminal peptide indicate that loricrin is initially localized in discrete granules in the stratum granulosum of mouse epidermis and then accumulates at the periphery of cells in the upper strata of the epidermis. This antiserum was shown by immunofluorescence and immunoelectron microscopy to detect the carboxy-terminal epitope only on the inner surface of purified cell envelopes. We have used a specific inhibitor of epidermal transglutaminase to demonstrate that loricrin is a substrate for this enzyme. However, we do not know if it serves as an amine donor, amine acceptor, or both. In situ hybridization experiments suggest that loricrin is a major-late product of terminally differentiated epidermal cells since its transcripts are restricted to the granular layer and are as abundant as those of the differentiationspecific keratins, Kl and KlO, and filaggrin. Loricrin is not only expressed in the epidermis, but in all stratified squamous epithelia (data not shown). The carboxy-terminal epitope of loricrin has been evolutionarily conserved in humans and other vertebrates, although there is considerable variation in the size of the polypeptide in these species (Figure 56 and data not shown). Sequence analysis of human loricrin suggests that this is due to variability in the number of glycine-serine repeats (D. H., D. R. R., and l? M S., unpublished data). Thus, certain structural features of loricrin are highly conserved, and based on a

Loricrin: 1109

A Major

Cell Envelope

Protein

comparison of the amino acid composition of loricrin with purified cell envelopes, it appears to be quantitatively an important cell envelope component. The failure of previous attempts to identify this major envelope component can be attributed to several factors. First, most investigators have assumed that envelope components would initially be in the soluble (Kubilus et al., 1987; Lobitz and Buxman, 1982; Zettergren et al., 1984; Tezuka and Takahashi, 1987; Buxman et al., 1978) or membrane-bound fractions (Rice and Green, 1979; Simon and Green, 1985) and only become insoluble after crosslinking. Second, several investigators have attempted to isolate envelope components from keratinocyte cultures (Michel et al., 1987; Simon and Green, 1984, 1985). Loricrin is highly insoluble, even prior to cross-linking. It can only be isolated reproducibly by homogenization of tissue in 5% SDS-20% 2-mercaptoethanol followed by boiling, since it is inherently hydrophobic and appears to be highly disulfide bonded in differentiating cells. Furthermore, this protein is either not expressed or expressed at very low levels under the culture conditions employed by other investigators. Implications of the Structure of Loricrin The most striking feature of loricrin is its very high content of glycine and serine residues, which account for 77% of the total. They are configured with occasional tyrosines as quasipeptide repeats in the general form of G&G& that are bounded by cysteine residues (Figure 1). Although these repeats are clearly related, they are not exact repeats and, as such, do not lend themselves to a simple evolutionary analysis as performed for involucrin (Eckert and Green, 1986; Tseng and Green, 1988; Djian and Green, 1989). Interestingly, this same structural motif is found in the end domains of keratins Kl and KlO that are coexpressed in the epidermis (Steinert et al., 1983, 1985; Zhou et al., 1988) although loricrin is unique in the frequent occurrence of cysteine residues, which have a marked periodicity. We think that the extraordinary insolubility of loricrin is in part due to the presence of the high content of glycine and other hydrophobic residues and the formation of disulfide bonds. A third likely reason is the formation of isopeptide bonds, because of its content of glutamine and lysine residues that may serve as acceptor and/or donor residues for epidermal transglutaminase(s). At the present time, we do not know whether s-(Y-glutamyl)lysine cross-links involving loricrin are inter- or intramolecular, or both. Both lysine and glutamine residues are found in the distinctive amino-(residues l-20) and carboxy-terminal (residues 462-481) domains. Glutamine residues also occur in two non-glycine/serine islands in the carboxyterminal half of the protein (residues 268-273 and 351362), while lysine residues are found in a distinctive motif at four sites in the amino-terminal half of the protein (Figure 1). The motif (V/G)K(Y/T)S (residues 17-20; 72-75; 144-147; 201-204) may be a recognition sequence for transglutaminase. The polarity of transglutaminase amine donor/acceptor sites may be important in the formation

and subsequent stabilization of the cornified envelope. Since loricrin is synthesized at a very late stage of epidermal differentiation and is so readily detectable on the inner surface of cell envelopes, other envelope components may be expressed at earlier stages of differentiation and assembled into a scaffold upon which loricrin is crosslinked as a final step in envelope formation. In this regard, it will be of interest to analyze steps occurring during the assembly of the cell envelope and determine if loricrin becomes cross-linked to other components such as involucrin. Is Loricrin the Sulfur-Rich Component of Keratohyalin Granules? Results reported by several laboratories imply that cell envelopes contain a cysteine-rich component (for review see Goldsmith, 1983). The first amino acid analysis of cell envelopes was determined by Matoltsy and Matoltsy (1966), who reported a cysteine content of 4.9% for envelopes isolated from human plantar stratum corneum. Autoradiography performed by Fukuyama and Epstein (1975a, 197513) 6 hr after injection of [3H]cysteine showed heavy labeling near the cell envelope, and, in addition, certain dense homogeneous deposits were preferentially labeled. These deposits are identical to the single granule components that Jessen (1970) found were sulfur rich by X-ray microanalytical analysis. Jessen also suggested that these granules participate in formation of the cell envelope since they are preferentially localized along the periphery of granular cells and disappear as the envelope is formed in transitional cells (Jessen et al., 1976). Such data were confirmed by Goldsmith (1977) who found that the injection of radioactive cysteine into newborn rats resulted in significant incorporation into cell envelope fractions. Likewise, Tezuka and Hirai (1980) found that over 90% of incorporated [?S]cysteine was present in an insoluble fraction, suggesting that the radioactivity was in the cell envelope. Furthermore, techniques that depend on free disulfide bonds, using fluorescein-isothiocyanate (Christophers and Braun-Falco, 1971) or 7-(N-dimethylamino-4-methyl-3-coumarinyl) (Hirotani et al., 1981; Ogawa et al., 1979; Tezuka, 1982) stain the cell periphery of cornified epidermal cells. A cysteine-rich protein was isolated from keratohyalin granules, but it was only partially characterized (Matoltsy and Matoltsy, 1970; Matoltsy, 1975). More recently, Tezuka and Takahashi (1987) have reported the extraction of a cysteine-rich protein from the membrane region of stratum corneum cells from human sole epidermis with 50 mM Tris-HCI buffer (pH 7.3). The molecular weight of the protein was 16,000 and amino acid analysis of this protein revealed 4.3% cysteine, 9% lysine, 18.5% glycine, and 12.6% glutamic acid (Table 1). Antibodies produced against the, purified protein reacted with the periphery of cells from the stratum spinosum, stratum granulosum, and inner part of the stratum corneum. However, localization of this protein within granules was not reported, and its molecular weight and solubility in aqueous buffers clearly distinguish it from loricrin. We think that the spotted immunofluorescence images of Figure 3 depict granular de-

Cell 1110

posits of loricrin. Further evidence in support of the notion that loricrin is the sulfur-rich component of keratohyalin granules has recently been obtained by Steven et al. (1989). Electron microscopic immunocytochemical studies employing the affinity-purified carboxy-terminal peptide antiserum described in this study and a peptide antiserum against mouse filaggrin (Rothnagel et al., 1987) have been used to localize filaggrin to the large cytoplasmic granules and loricrin to small round granules in the stratum granulosum of mouse epidermis (Steven et al., 1989). Experimental

Procedures

Clonlng and Sequence Analysis of Loricrin mRNA The initial cDNA clones (pK 321 and pK 1005) were isolated while screening a cDNA library with cloned cDNAs for keratins Kl and KlO (Roop et al., 1983). This cDNA library was prepared by the insertion of double-stranded cDNA, synthesized from newborn mouse epidermis, into the Pstl restriction site of pBR322 (for details, see Roop et al., 1983). These cDNA clones were missing coding information at the 5’ end (see Figure 1A); therefore, we screened a hgtll expression cDNA library, which had also been prepared from total poly(A)+ RNA from newborn mouse epidermis (Hawley-Nelson et al., 1988). To ensure that clones containing 5’ sequences would be identified, we screened the expression library with a 32P-labeled riboprobe generated from the 5’ Pstl-BamHI fragment of pK 321 subcloned into pGEM3 (Promega; see Figure IA). To avoid cross-hybridization with keratin clones, filters were treated with RNAase A (300 &ml) and RNAase Ti (3 vglml) in 2x SSC at 37oC for 30 min followed by a 30 min wash in 0.1x SSC at 8oOC. Several clones were identified, and the largest one was subsequently subcloned into the EcoRl site of pGEM72 (Promega) for sequencing and designated pg 8 (see Figure IA). This clone encoded 150 nucleotides of additional 5’ information but still lacked all of the 5’ noncoding sequences as well as some coding sequences. Several attempts were made to obtain hgtll clones containing these missing sequences using a riboprobe made to the 5’-most EcoRI-Seal fragment of pg 8, but none were detected. The remainder of the Sinformation was obtained by polymerase chain reactions, which were used to amplify this region from cDNA synthesized from poly(A)+ RNA from newborn mouse epidermis using an oligonucleotide primer specific for the 5’ end of pg 8 (nucleotides 119-139) (Frohman et al., 1988). Sequencing was performed by double-strand sequencing methods employing pGEM3 vectors (Promega). Hybridization Seledion Assay The hybridization selection assay was performed essentially as described by Cleveland et al. (1980). Plasmid DNAs were linearized with EcoRI, and 10 bg was bound to (13 mm) nitrocellulose filters (BA85; Schleicher & Schuell). The filter-bound DNA was prehybridized for 2 hr at 41°C in 50% (v/v) formamide (Fluka), 0.4 M NaCI, 10 mM PIPES (pH 6.4), 5 mM EDTA. 250 wg of poly(A) per ml, 250 pg of yeast tRNA per ml, and 0.2% SDS. Hybridization was for 20 hr at 4i°C in the same buffer (150 pl per filter) containing 15 vg of epidermal poly(A)+ RNA. The filters were washed two times (5 min each) with lx SSC, 0.1% SDS at room temperature, three times (5 min each) with 0.1x SSC, 0.1% SDS at room temperature, and two times (5 min each) with 0.1x SSC, 0.1% SDS at 60°C. RNA was eluted from the filters in 300 PI of water at 100°C for 2 min. The RNA was collected by precipitation in the presence of 10 Kg of yeast tRNA and analyzed by translation in the reticulocyte system containing [35S]cysteine. lmmunoprecipitation was performed as described by Harper et al. (1986). Preparation and Characterization of the Antibodies Monospecific antibodies to loricrin were produced in rabbits by immunizing with a synthetic peptide, HQTQQKQAPTWPCK, corresponding to unique amino acid sequences at the carboxyl terminus of the sequence shown in Figure 1. The immunization protocol was as previously described (Roop et al., 1984). Collected sera were assayed for antibody activity and specificity by immunoblotting of epidermal extracts (Roop et al., 1984). The antibodies were purified by affinity chromatography using the synthetic peptides coupled to activated

Sepharose (Brinkley et al., 1980). Single- and double-label immunofluorescence was performed as previously described (Roop et al., 1987). For these studies, the antisera were diluted as follows: rabbit antiloricrin, 11500, and guinea pig anti-keratin 14 (K14), 112000. RNA Analysis For in situ hybridization experiments, 3’ noncoding regions of cDNA clones for loricrin (see Figure 1) and keratins Kl and K14 (Roop et al., 1988) were subcloned into pGEM3 vectors as described by the manufacturer (Promega). Labeled riboprobes were generated by either SP6 or T7 RNA polymerase in the presence of 35S-labeled ribonucleotides. The specificity of the riboprobes was confirmed by performing Northern hybridization on nitrocellulose blots of epidermal RNA as described (Roop et al., 1983). In situ hybridization was performed on frozen sections of newborn mouse skin as described previously (Roop et al., 1986). Purification of Cell Envelopes and Determination of Amino Acid Composition Cell envelope fragments were prepared according to the procedure of Nagae et al. (1987) from heat-separated (Marrs and Vorhees, 1971) human foreskin and mouse epidermis. Epidermis was heated with stirring in 2% SDS extraction buffer (0.1 mM X-is [pH 8.51, 20 mM DTT, 5 mM EDTA, 2% SDS) for IO min at 95OC. Envelopes, collected by centrifugation at 10,000 rpm (15 min), were reextracted once under the same conditions. Intact envelopes were obtained by flotation during centrifugation at 10,000 rpm (30 min) in extraction buffer containing 0.2% SDS and 2%-3% Ficoll (Pharmacia). The floating envelopes were washed once with 0.2% SDS extraction buffer and sonicated. The resulting fragments were collected by centrifugation, washed once in extraction buffer, and freed of any contaminating intact envelopes by centrifugation through 2%-30/o Ficoll in 0.2% SDS extraction buffer as above. The purified envelope fragment pellet was washed several times with 0.2% SDS extraction buffer. Fragments equivalent to 20 kg protein, as determined by the Bramhall dye absorption method (Bramhall et al., 1969), were centrifuged in glass tubes and freed of buffer by washing at least four times with water. lmmunoelectmn Microscopy of Isolated Cell Envelopes Purified cell envelopes (see above) were fragmented by sonication, washed extensively in 20 mM Tris-HCI, 2 mM EDTA, 5 mM DTT, and 0.2% SDS (pH 7.5) and finally resuspended in this buffer; 25 ~1 of this suspension (2.5 mglml) was mixed with 10 PI of anti-loricrin antibodies (at approximately 1 mglml) or preimmune serum in the negative control and 65 ~1 of buffer for 30 min. Envelope fragments were then separated from unbound antibodies by four cycles of centrifugation and resuspension in the same buffer. The final pellet was fixed in 2% glutaraldehyde for 3 hr at room temperature, rinsed in phosphatebuffered saline, also at room temperature, and left overnight at 4°C. These samples were postfixed in 2% osmium tetroxide for 30 min, serially dehydrated in ethanol, and transferred into propylene oxide for 30 min. Infiltration was accomplished by placing the samples in a 2:l mixture of propylene oxide and Epon for 45 min, then in a I:1 mixture overnight on ashaker, then in undiluted Epon for 4 hr, and finally transferred into flat molds. The resin was polymerized in a 60°C oven overnight. Thin sections, nominally 60 nm, were cut on a Reichert Ultracut E, picked up on uncoated 400 mesh copper grids, stained with Reynolds’ lead citrate and 5% uranyl acetate (Steer et al., 1984), and examined in either a Phillips EM40OT (Phillips, Mahwah, NJ) or a Zeiss EM902 (Carl Zeiss Inc., Thornwood, NY) transmission electron microscope. Labeling of Mouse Skin with [%]Cysteine in the PmenCe of the Transglutaminase Inhibitor LTB-2 Skin from newborn mice was minced and suspended in cysteine-free and serum-free Dulbecco’s modified Eagle’s medium (NIH Media Unit) (one skin equivalent per 3 ml) and incubated with [35S]cysteine (New England Nuclear) at 70 FCilml for 3 hr at 3PC with or without 0.1 mM LTB-2 (Syntex Research). Labeled skin fragments were washed twice with phosphate-buffered saline and processed for protein extraction or chased in complete medium containing 8% fetal Calf serum for 21 hr at 3pC. LTB-2 was added to the chase medium for skin that was labeled in the presence of LTB-2. Skin fragments were washed again with phosphate-buffered saline and heated in 1 ml of 2x upper gel

Loricrin: 1111

A Major

Cell Envelope

Protein

buffer, 5% SDS, and 10% mercaptoethanol at 95OC for 10 min and stored overnight at -20°Cc. Samples were dialyzed overnight against immunoprecipitation buffer. lmmunoprecipitation was performed as described by Harper et al. (1986). Acknowledgments We thank Suzanne Mascola for typing the manuscript, Dr. Larry DeYoung (Syntex Research, Palo Alto, CA) for providing the epidermal transglutaminase inhibitor, LTB-2, Dr. Rosalba Rothnagel for assistance with sequence analysis, and Ms. Maria Quintanilla for excellent technical assistance. This work was supported in part by a grant from the National Institutes of Health (AR40240) to D. Ft. R. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

February

1, 1990; revised

March

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Accession

The accession M34398.

number

Number for the sequence

reported

in this

paper

is