A highly conserved lysine residue on the head domain of type II keratins is essential for the attachment of keratin intermediate filaments to the cornified cell envelope through isopeptide crosslinking by transglutaminases

Abstract

We have addressed the question of how keratin intermediate filaments are associated with the cell envelope at the periphery of cornified epidermal cells. Many peptides from human epidermal cell envelopes containing isopeptide crosslinks inserted by transglutaminases in vivo have been characterized. A major subset involves the type II keratin chains keratin 1, 2e, 5, or 6 crosslinked to several protein partners through a lysine residue located in a conserved region of the V1 subdomain of their head domains. This sequence specificity was confirmed in in vitro crosslinking experiments. Previously the causative mutation in a family with diffuse nonepidermolytic palmar-plantar keratoderma was shown to be the loss in one allele of the same lysine residue of the keratin 1 chain. Ultrastructural studies of affected palm epidermis have revealed abnormalities in the organization of keratin filaments subjacent to the cell envelope and in the shape of the cornified cells. Together, these data suggest a mechanism for the coordination of cornified cell structure by permanent covalent attachment of the keratin intermediate filament cytoskeleton to the cell envelope by transglutaminase crosslinking. Furthermore, these studies identify the essential role of a conserved lysine residue on the head domains of type II keratins in the supramolecular organization of keratin filaments in cells.

The terminal differentiation program of stratified squamous epithelia includes the formation of a cell envelope (CE) structure on the intracellular surface of the plasma membrane, which it eventually replaces (1–3). In the case of human epidermis, the CE consists of an ≈15-nm-thick layer of insoluble crosslinked protein with an ≈5-nm-thick layer of attached lipids (3–7). This insoluble CE is vitally important in barrier function for the tissue and organism (1–7). The bulk of the terminally differentiated epidermal corneocyte consists of a cytoskeletal keratin intermediate filament (KIF)–filaggrin complex that is retained within this CE structure (4).

In nucleated epidermal cells, the KIF cytoskeleton is mechanically integrated with the cell periphery primarily at desmosomal junctions, which in turn provide structural continuity throughout the tissue (8–11). Although the exact details remain unclear, the connection of KIF to desmosomes involves a large number of proteins, including the major intracellular desmosomal protein desmoplakin (8–11). However, the terminal differentiation program in the epidermis also involves major changes in morphology, the formation of a lipid-enriched barrier, loss of most “housekeeping” components, including the cytoskeletal connections to the cell periphery, and controlled cell death (4). As the keratinocyte becomes a terminally differentiated corneocyte, the structural integrity of desmosomes decreases and, indeed, most epitopes of proteins such as desmoplakin are no longer detectable as a result of degradation or masking (11). Thus, the question arises as to how the KIF cytoskeleton is mechanically integrated with the CE after the major cellular remodeling events involved in terminal differentiation.

We have characterized the protein composition of CEs obtained from foreskin epidermal corneocytes and have shown that a variety of structural proteins are crosslinked together by the action of transglutaminases (TGases) to form the CE (refs. 12–15; P.M.S. and L.N.M., unpublished data). TGases form N^ɛ-(γ-glutamyl)lysine crosslinks between protein-bound Gln and Lys residues, which result in stable, permanent macromolecular assemblies (1–3). The major proteins are loricrin admixed with lesser amounts of small proline-rich (SPR) proteins and elafin (14). These proteins constitute about 80% of CE protein and occupy the most intracellular aspect of the CE, that is, that which corresponds to the later stages of CE assembly. After saponification of some of the protein-bound ceramides from the extracellular surface of the CE, we identified by immunogold electron microscopy (15) and sequencing (P.M.S. and L.N.M., unpublished data) certain proteins presumably involved in the earlier stages of CE assembly, including involucrin and the carboxyl termini of desmoplakin and envoplakin. In addition, we identified many peptides involving the keratin 1, 2e, 5, or 6 chains, crosslinked by isopeptide bonds to a variety of these proteins, both in the untreated and saponified CE samples. Similar peptides have been isolated and characterized from immature foreskin keratinocytes (15), as well as from cultured keratinocytes induced to terminally differentiate in submerged serum-free cultures (P.M.S. and L.N.M., unpublished data). In this paper, we have characterized these keratin-containing crosslinked peptides and report that KIF are crosslinked by isopeptide bonds to the CE primarily through a single Lys residue located on the head domain of the type II keratin chains. The importance of this anchorage of the KIF cytoskeleton to the CE is demonstrated in a family with Unna–Thost disease, autosomal dominant diffuse nonepidermolytic palmar-plantar keratoderma (NEPPK), in which a mutation involving loss of this essential lysine residue in one allele (16) results in structural changes in the epidermis. These data identify a critical function of the end domains of the epidermal keratins in the supramolecular coordination of cell structure.

MATERIALS AND METHODS

Sequencing of CE Peptides.

Experimental details for the isolation, sequencing, sorting, and characterization of peptides containing isopeptide crosslinks have been described from: foreskin cornified tissue (14); immature foreskin epidermis and saponified cornified tissue (15); and from 3-day and 7-day cultured normal human epidermal keratinocytes grown in serum-free submerged cultures in the presence of high (1.2 mM) Ca²⁺ medium (P.M.S. and L.N.M., unpublished data). In this paper, only those crosslinks involving keratin chains are discussed.

Expression of Wild-Type and Mutated Forms of Keratin 5 and 14 Chains and Assembly of KIF in Vitro.

Full-length human keratin 5 and 14 cDNAs were constructed by PCR amplification by using DNA from a human keratinocyte library as template and confirmed by DNA sequencing. These were assembled into the pET11a vector for protein expression in bacteria (17). Two mutant forms of the keratin 5 chain (Lys-71 → Ile and Leu-174 → Pro) were generated by using the QuikChange site-directed mutagenesis kit (Stratagene). The keratins were expressed in very high yields. Bacterial pellets were dissolved in SDS gel buffer, and the proteins were resolved on preparative PAGE gels. The desired keratin bands were cut out, eluted into SDS gel buffer overnight, freed of bound SDS by ion-pair solvent extraction (18), and finally dissolved into urea buffer. We were unable to express the human keratin 1 chain, presumably because of toxicity of its glycine-loop sequences (19, 20).

Keratin 1 and 10 proteins were extracted from human foreskin epidermis (21) and dialyzed through solutions of decreasing urea concentration into assembly buffer of 10 mM Tris⋅HCl (pH 7.6) and 1 mM DTT to give a final protein concentration of 0.5–1 mg/ml (≈4–8 nmol/ml of keratin 1 and 10 heterodimer) (21). Equimolar amounts of the expressed wild-type keratin 14 chain and either the wild-type keratin 5 chain, or the Lys-71 → Ile or Leu-174 → Pro mutant forms of it were dissolved and mixed in a buffer of 8 M urea/50 mM Tris⋅HCl, pH 7.6/1 mM EDTA/1 mM DTT and dialyzed into KIF assembly buffer as above. KIF were examined by electron microscopy after negative staining with 0.7% uranyl acetate.

In Vitro Transglutaminase Crosslinking Assays.

A synthetic peptide of sequence Val-Ser-Ser-Gln-Gln-Val-Thr-Gln-Ser-Cys-Ala, corresponding to residues 210–223 of human loricrin (22), was synthesized, partially labeled by acetylation of its amino terminus with ³H-labeled acetic anhydride (23), and purified by HPLC, with a resultant specific activity of 83 dpm/pmol. Activated guinea pig epidermal TGase 3 enzyme (24) was used, and its specific activity was measured by incorporation of ³H-labeled putrescine into succinylated casein (17). Analytical crosslinking reactions contained (in 100 μl reaction volume) 0.4 nmol of KIF containing the keratin 1 and 10 (1/10) or 5/14 chains, a 0.5- to 5-fold molar excess of labeled loricrin peptide, in a buffer of 0.1 M Tris-acetate, pH 8.3/1 mM DTT/5 mM CaCl₂, and sufficient enzyme to incorporate 1 pmol/min of putrescine into casein (10–20 pmol of enzyme in different experiments). This was determined in control assays to be sufficient enzyme to drive the crosslinking reactions to completion. After 1 h at 37°C, reactions were terminated by addition of EDTA (to 15 mM). Aliquots of 25 μl were spotted onto 3-mm filter papers, washed in trichloroacetic acid, and counted (17, 20). In preparative-scale experiments, 8 nmol of keratin 1/10 KIF was mixed with 15 nmol of the ³H-labeled loricrin peptide, reacted as above, and terminated. The reactions were digested to completion within 3 h with trypsin (Sigma, bovine, sequencing grade, 1% wt/wt). The tryptic digests were resolved by HPLC on a reverse-phase ultrasphere ODS column (4.5 × 250 mm) at a flow rate of 1 ml/min with a gradient of 0–100% acetonitrile containing 0.08% trifluoroacetic acid, collected into 0.5-min fractions, of which half was counted for radioactivity. Aliquots of selected fractions were covalently attached to a support (Sequelon-AA, Millipore) for amino acid sequencing (14, 15).

Electron Microscopy Procedures.

Punch biopsies (ca. 3 mm diameter) were surgically removed from the previously untreated sides of the palms of one member of a previously described (16) family with diffuse NEPPK and from an age- and sex-matched donor with no known palmar-plantar involvement, fixed in glutaraldehyde, and washed by established procedures (25). Samples were then postfixed with OsO₄ and processed for electron microscopy (7, 25).

RESULTS AND DISCUSSION

Keratin Chains Are Crosslinked by Isopeptide Bonds in Vivo to Many CE Proteins Through a Common, Highly Conserved Lysine Residue.

We have reported immunogold localization and protein sequencing data that suggests that the assembly of the CE of foreskin epidermis occurs in at least two discrete steps (refs. 13–15; P.M.S. and L.N.M., unpublished data). These involve first, the accretion at or near desmosomes of early proteins such as involucrin, envoplakin (and variants), and cystatin α, which together form a scaffold for the second step: the accumulation of late “reinforcement” proteins including elafin, SPRs, and large amounts of loricrin. During the course of characterization of peptides from several epidermal CE preparations (refs. 14 and 15; P.M.S. and L.N.M., unpublished data), we identified 256 that included a keratin chain directly crosslinked to one of these proteins (Table 1). Of these, 249 (97%) involved a type II keratin, either keratins 1, 2e, 5, or 6. Significantly, all 249 peptides involved a crosslink formed through a Lys residue in a conserved sequence region of the head domain, encompassing the V1 subdomain, of the keratin chains (Table 2). This Lys residue is present only in the keratins expressed in stratified squamous epithelia that form CE structures (16). Only seven peptides involved either lysines 9 or 32 located in the E1 subdomain of the keratin 10 chain.

Table 1.

Occurrences of proteins directly crosslinked to keratins

CE experiment number	Number of peptides	Cystatin α	Elafin	Envoplakin	Involucrin	Loricrin	SPRs 1 and 3
Keratin 1
1	6					6  (70)
2	8		1  (15)	3  (50)	1  (40)	3  (250)
3	31	1  (10)	1  (10)	19  (240)	1  (50)	4  (600)	5  (190)
4	7			1  (10)	4  (80)	1  (110)	1  (25)
5	19	1  (15)	1  (15)	2  (25)	9  (140)	4  (220)	2  (40)
Keratin 2e
1	1					1  (5)
2	4			2  (20)	1  (20)	1  (50)
3	3			1  (5)	1  (10)	1  (50)
4	1					1  (10)
5	2					2  (40)
Keratin 5 (or 6)
1	0
2	12			7  (160)	4  (60)	1  (10)
3	33	1  (10)	2  (20)	22,1  (340)	1  (10)	5  (60)	1  (10)
4	67	1  (5)	1  (10)	34,3  (650)	21,3  (270)	1  (5)	2  (10)	1  (10)
5	55	2  (20)	3  (50)	18  (250)	14,3  (240)	8,1  (110)	5  (40)	1  (5)
Keratin 10
1	1					1  (10)
2	2					2  (20)
3	1					1  (20)
4	0
5	3		1  (10)			2  (30)

The numbers in parenthesis are yields in pmol. CE experiment numbers are: 1, foreskin stratum corneum (14); 2, saponified foreskin stratum corneum (15); 3, “immature” foreskin epidermis (15); 4, 3-day NHEK cultures (P.M.S. and L.N.M., unpublished data); 5, 7-day NHEK cultures (P.M.S. and L.N.M., unpublished data). In most cases, the short sequences of the keratin branch did not permit distinction between the keratin 5 or 6 chains. The second number listed defines known keratin 6 sequences; e.g., 22,1 (340) means that of a total of 23 peptides (totaling 340 pmol), 22 involved the keratin 5 or 6 chains and 1 was confirmed from keratin 6. 

Table 2.

Sequences involved in direct crosslinks with keratin chains

Chain	Sequence	Keratin chains yield, pmol
		1	2e	5/6	6
Keratin chain
Keratin 1 (Lys-73)	NLGGSKSISISVAR
Keratin 2e (Lys-69)	GLGGTKSISISVA
Keratin 5 (Lys-71)	NLGGSKRISISTR
Keratin 6 (Lys-68)	GLGGSKRISI
Crosslinked partner
Cystatin α (Gln-45)	TQVVAGTNY	25		35
Elafin (Gln-2)	AQEPVKGPV	40		80
Envoplakin* (Gln-1970)	AQLLQDESSFEKDL	235	25	600	40
Envoplakin (Gln-1973)	AQLLQDESSFEKDL	80		460	30
Involucrin (Gln-88/525)	HLEQQEGQLK	120	5	110
Involucrin (Gln-288)	YLEQQEGQLK	170	20	130	20
Involucrin (Gln-328)	HLEQQEGQLEQL	20	5	110	5
Involucrin (Gln-436)	HLEEQEGQLK			40
Involucrin (Gln-445)	HLEQQQGQLEV			90	15
Involucrin (Gln-446)	HLEQQQGQLEV			50	10
Involucrin (Gln-455)	PEQQVGQPKNL	40		65	10
Involucrin (Gln-456)	PEQQVGQPKNL			10
Loricrin (Gln-6)	QKKQPTPQPPV	40		10
Loricrin (Gln-153)	SGQAVQCQSY	70	10
Loricrin (Gln-215)	YVSSQQVTQTSCA	570	105	65
Loricrin (Gln-216)	YVSSQQVTQTSCA	360	20	20
Loricrin (Gln-219)	SSQQVTQTSCA	180	20
Loricrin (Gln-303)	CHQTQQKQA			30
Loricrin (Gln-305)	HQTQQKQA	20		40
Loricrin (Gln-308)	HQTQQKQTW	10		10
SPR1 (Gln-5)	QQQKQPC			10
SPR1 or 3 (Gln-19)	QQQQV			10
SPR1 or 3 (Gln-83)	QQKTKQK	90		10
SPR1 or 3 (Gln-87)	QQKTKQK	170		15
SPR3 (Gln-5)	QQKQTF	10
SPR2 (Gln-6)	QQQQCKQPCQPPPV			5
SPR2 (Gln-9)	QQQQCKQPC	5

Single-letter code is used for amino acids. Residues involved in crosslinks are shown in bold. 

^*Note that three envoplakin variants have been described (15), but the data are combined here because the same Gln residues were used. 

Analysis of the data shows that most crosslinks involved keratins 5 and/or 6 with the early CE protein components involucrin and envoplakin (ref. 15; P.M.S. and L.N.M., unpublished data). Although the molar yield of each was relatively low, the total yield was about 60% of all keratin-containing crosslinks. Another 40% of the total yield involved the later CE protein components elafin, SPRs, and loricrin, with some of the highest yields recovered for peptides having crosslinks between keratin 1 and three favored donor Gln residues of loricrin, Gln-215, Gln-216, and Gln-219 (20). The keratin 5 (and keratin 6 from CEs made in cell cultures) chains are typically expressed in basal epidermal cells, although they are retained into the differentiating layers (26), where keratins 1 and 2e are expressed in larger amounts (26, 27).

The Conserved Lys-71 of the Keratin 5 Chain Is Not Essential for Efficient KIF Assembly in Vitro.

We expressed in bacteria the wild-type keratin 5 and 14 chains, as well as the Lys-71 → Ile and Leu-174 → Pro-substituted forms of keratin 5. After isolation from a preparative SDS/polyacrylamide gel, equimolar mixtures of keratin 14 and either the wild-type (Fig. 1a) or the Lys-71 → Ile-substituted (Fig. 1b) keratin 5 chains assembled in vitro into long (L_av > 20 μm) KIF. This Ile substitution is comparable to that of the keratin 1 chain (Lys-73 → Ile) seen in disease (16). These data suggest this Lys residue is not required for KIF assembly in vitro. However, a negative control substitution of Leu-174 → Pro, corresponding to position 7 of the 1A rod domain of the keratin 5 chain, failed to assemble (Fig. 1c), as expected, because it interferes with KIF structure (28).

Figure 1.

The conserved Lys residue 71 of the keratin 5 chain is not essential for KIF assembly in vitro. KIF were assembled from equimolar mixtures of: wild-type keratins 5 and 14 (a); the Lys-71 → Ile-substituted form of keratin 5 and wild-type keratin 14 (b); and the Leu-174 → Pro-substituted form of keratin 5 and wild-type keratin 14 (c). (Bar = 100 nm.)

Crosslinking of KIF and a Synthetic Peptide in Vitro with the TGase 3 Enzyme Involves the Same Conserved Lys Residue.

We further explored in vitro the specificity of crosslinking of KIF by TGases. In initial experiments, we found that the TGase 3 enzyme did not use keratin 1/10 or 5/14 KIF as a complete substrate in vitro, because <2% of KIF chains became oligomerized as judged on SDS/PAGE gels (data not shown). We made and labeled a synthetic peptide corresponding to sequences of human loricrin that efficiently donate Gln residues for TGase crosslinking both in vivo and in vitro (14, 20). Only about 1 mol of peptide was incorporated per mol into KIF assembled from wild-type keratins 5/14 (Fig. 2, solid bars) or wild-type keratins 1/10 KIF (not shown) with peptide:KIF ratios ≤1:3. At similar ratios, only trace amounts were incorporated into KIF assembled from the wild-type keratin 14 and Lys-71 → Ile-substituted keratin 5 chains (Fig. 2, open bars). However, at higher molar ratios, >1 mol of peptide/mol of KIF was incorporated into both wild-type and substituted KIF (Fig. 2).

Figure 2.

Stoichiometry of crosslinking of a synthetic loricrin peptide to KIF in vitro. Wild-type keratin 5/14 KIF (solid bars) or with KIF assembled from the keratin 5 chain with Lys-71 → Ile substitution and wild-type keratin 14 (open bars) were mixed with the synthetic peptide in various molar ratios as shown, crosslinked with the TGase 3 enzyme, and assayed.

In a preparative crosslinking experiment with keratin 1/10 KIF, followed by tryptic digestion, three labeled tryptic peptide peaks were identified by HPLC (Fig. 3 Lower) and isolated. By amino acid sequencing (Table 3), we could identify four novel peptides, although mixed with unlabeled tryptic peptides. From the incorporated radioactivity, 94% involved Lys-73 of the keratin 1 chain. Small amounts were also incorporated into Lys-9 and Lys-32 of the keratin 10 chain, as also seen in the in vivo data. Together, these data confirm the specificity of the conserved Lys residue of the type II keratin chains for TGase crosslinking.

Figure 3.

Recovery by HPLC of tryptic peptides containing crosslinks introduced by the TGase 3 enzyme in vitro between keratin 1/10 KIF and a synthetic loricrin peptide. (Lower, Inset) The labeled peptides shown were isolated for quantitation and sequencing (Table 3).

Table 3.

Labeled peptides from in vitro crosslinking of keratin 1/10 KIF

Peptide	Yield, mol	Sequence	Origin
1	0.02	VSSQQVTQTSCA	Gln-216
		:
		YSSSSKQFSSSR	Keratin 10 Lys-9
2A	0.55	VSSQQVTQTSCA	Gln-215
		:
		SLVNLGGSKSISISVAR	Keratin 1 Lys-73
2B	0.48	VSSQQVTQTSCA	Gln-219
		:
		SLVNLGGKSISISVAR	Keratin 1 Lys-73
3	0.05	VSSQQVTQTSCA	Gln-215
		:
		ISSSKGSLGGGF	Keratin 10 Lys-32

Single-letter code of amino acids is used. Molar amounts of crosslinked peptides were determined from the recovered amount of labeled peptide of known specific activity, because in the Edman degradation sequencing reactions, each peak from Fig. 3 also contained other tryptic peptides from the keratin chains. 

Significant Morphological Changes in Human Epidermis Are Due to the Loss of the Conserved Lys Residue in a Family with Diffuse NEPPK.

Previously, one case of this disease was reported to be caused by a mutation in the KRT1 gene of one allele that resulted in a substitution of the Lys residue by Ile in the keratin 1 chain (16), which is the same residue identified in the above in vivo and in vitro crosslinking studies. This disease was manifested as hyperkeratosis of the epidermis of the palms and soles and, to a mild degree, other limited body sites. In view of the foregoing data on the importance of this Lys residue in crosslinking, we performed ultrastructural studies on skin samples. We could not detect any abnormality in the organization of KIF bundles in basal or spinous keratinocytes (not shown). However, in granular cells where assembly of the CE is initiated, KIF bundles appeared to detach and retract abnormally from beneath desmosomes and along much of the cell periphery, often forming microclefts or vacuolar inclusions (Fig. 4b). This feature is not present in palmar epidermis from a normal individual (Fig. 4a). Moreover, the boundary between the uppermost granular layer and the first cornified cell layer was irregular in shape with deep interdigitations between the two layers (Fig. 4c), which was also not evident in normal palmar epidermis (Fig. 4d). At high magnification, the retraction of the KIF from the cell periphery at and between desmosomal sites is evident (Fig. 4e), which is not present in normal epidermis (Fig. 4d). The outer cornified layers appeared similar to the control (not shown). Epidermis from other uninvolved body sites appeared normal (not shown).

Figure 4.

Ultrastructural analysis of palmar epidermis from a normal individual (a and d) and an affected patient with diffuse NEPPK disease (b, c, and e). In the patient, the basal and lower spinous layers appeared normal (data not shown). However, in cell layers extending from the upper spinous to granular layers, where CE assembly is initiated, separations of KIF tonofibrils from desmosomes and the cell periphery occur in the patient, leading to microclefts, which are marked by asterisks in b. In addition, at the interface between the uppermost granular and first cornified cell, a highly convoluted interface is evident in the patient (c), which is absent in normal epidermis (d). At higher resolution, the KIF appear withdrawn from the desmosomal and interdesomomal regions (asterisk) in many places in the patient (e), unlike in normal (d). [Bars = 2.5 μm (a and b), 1 μm (c), 0.5 μm (d and e)]. SC, stratum corneum; G, granular layer; KH, keratohyalin granule.

These data suggest that the loss of the Lys-73 residue in about 50% of the keratin 1 chains expressed in the cells could cause discoordination between the KIF cytoskeleton and periphery of granular cells, apparently resulting in a distortion in the shape of the keratinocyte. The increased surface area of the corneocytes and their abnormal shape may prevent efficient distribution of lamellar body lipid material, which in turn results in reduced barrier formation, and thus a hyperkeratotic response (5).

The Role of the Conserved Lysine Residue: A Model for the Coordination of KIF and the CE.

There are more than 50 Lys residues in the keratin 1 and 10 (or 5 and 14) chains, yet our data reveal that only one of these, located within a highly conserved region of the V1 subdomain of the head domain of type II chains, is utilized with high specificity for crosslinking to many protein partners by TGases in vivo and in vitro. In addition, this residue is not essential for KIF assembly in vitro (Fig. 1). That this residue may not be essential for normal KIF assembly in vivo comes from the ultrastructural data of Fig. 4: there was no abnormal KIF clumping or cell lysis typically seen in other keratin disorders because of mutations in rod domain sequences that are known to be essential for KIF assembly (28, 29). Therefore, this residue position is likely to be more important for the function or supramolecular organization of the KIF in cells.

Specifically, our new data suggest that this Lys residue plays an essential role in the normal crosslinking of KIF to the cell periphery and desmosomes in the formation of the CE. We propose that in this way the KIF cytoskeleton of cornified or other terminally differentiated stratified squamous epithelial cells is structurally attached permanently to the CE of the cornified cell.

Our data suggest that this may occur in two types of interaction complexes (Fig. 5). The first is at desmosomal anchorage sites where KIF containing the keratin 5 chain (and keratin 6 in palmar-plantar epidermis and in cultured cells) and later, the keratin 1 chain, interact with envoplakin, involucrin, desmoplakin, and probably other proteins. The data in Table 1 imply these may represent up to 60% (molar basis) of all keratin-containing crosslinks. A second or concurrent interaction complex is where KIF cytoskeleton containing predominantly the keratin 1, 2e, and 10 chains in the cornified cells contacts the intracellular surface of the CE, which is predicted to be composed mostly of loricrin and SPRs (14) (Fig. 5). Our data from Table 1 imply these constitute at least 40% of the total.

Figure 5.

Model showing two different modes of association of KIF with the foreskin epidermal CE. At left is the possible organization of KIF near the vicinity of a desmosomal remnant including the cytoplasmic domain of desmoplakin with envoplakin and involucrin. At right is shown the possible association of KIF with the intracellular surface of the CE, which is enriched in loricrin and SPRs. This model is not drawn to scale, because the molecular dimensions of no CE structural proteins are known. Note that although few direct data are available, it is to be expected that foreskin and palmar-plantar epidermal CEs will be generally similar, especially because they both contain high amounts of loricrin and have similar masses per unit area (30); however, they are different from other internal stratified squamous epithelia, which contain little or no loricrin.

Existing data allow an estimate of the degree of crosslinking of KIF to the CE. These calculations assume a model in which KIF of 15 nm in diameter containing 16 heterodimer molecules/46 nm (29) line the intracellular surface of the CE (ref. 4; see also Fig. 4d) and assume that: (i) keratin-containing crosslinks represent about 0.1% of the total isopeptide crosslink in foreskin epidermis (14, 15); (ii) there are 89 nmol of crosslinks/mg of foreskin CE proteins (14, 15, 22); and (iii) CEs from a variety of murine tissues including the footpad, which is analogous to human palmar-plantar epidermis, are remarkably consistent in thickness of about 7.2 kDa [=7.2 × 10³ atomic mass units (amu)]/nm² (30). Thus, there are [(89 × 10⁻⁹) × (1 × 10⁻³) × (6 × 10²³)] keratin crosslinks/6 × 10²⁰ atomic mass units (amu) of CE mass = 1.1 × 10⁸ amu/keratin crosslink = one keratin crosslink/1,500 nm² of CE surface = one crosslink/100 nm of KIF length, or about 0.5 crosslink/unit molecule length of KIF. This means that 2–3% of available keratin 1 Lys-73 residues participate in crosslinking. Accordingly, irrespective of the exact molecular model of association with the CE, the data indicate that KIF are frequently attached to the CE, providing a tight, permanent anchor. Therefore, it could be reasonably expected that reductions in crosslinking caused by a mutation may have significant consequences for the structural integrity of the cornified cell.

A question thus arises as to why pathology in the case of NEPPK disease affects primarily sites of thickened epidermis of the palms and soles. One possible explanation lies with the abundance of total crosslinks in CE samples from different body sites, which range from 89 nmol/mg in foreskin epidermis (3, 14, 20), to 75 nmol/mg in human trunk epidermis (31), but 45 nmol/mg in palmar callus epidermis (32). It is possible that the loss of 50% of the potential crosslinks through the keratin 1 chain may reduce the amount necessary for normal structure and function below a critical threshold level in the palms and soles.

In summary, our data reveal a mechanism by which the KIF cytoskeleton is structurally integrated with the cell periphery in terminally differentiated stratified squamous epithelia such as the epidermis. This system utilizes TGase crosslinking to build a permanent, stable structure essential for normal epidermal structure and function. In addition, the present identification of in vivo crosslinks, coupled with this case of diffuse NEPPK, has provided valuable new information of the role of the highly conserved Lys residue on head domain of type II keratin chains in the supramolecular organization and function of KIF. Also, sequences near this lysine residue have been implicated in in vitro assays in the association of KIF with desmoplakin (33, 34).

ABBREVIATIONS

KIF

keratin intermediate filaments

NEPPK

nonepidermolytic palmar-plantar keratoderma

SPR

small proline-rich (class of proteins)

TGase

transglutaminase

CE

cell envelope