Biochemical Evidence That Small Proline-rich Proteins and Trichohyalin Function in Epithelia by Modulation of the Biomechanical Properties of Their Cornified Cell Envelopes*

Peter M. SteinertDagger , Tonja Kartasova, and Lyuben N. Marekov

From the Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-2752

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The cornified cell envelope (CE) is a specialized structure involved in barrier function in stratified squamous epithelia, and is assembled by transglutaminase cross-linking of several proteins. Murine forestomach epithelium undergoes particularly rigorous mechanical trauma, and these CEs contain the highest known content of small proline-rich proteins (SPRs). Sequencing analyses of these CEs revealed that SPRs function as cross-bridgers by joining other proteins by use of multiple adjacent glutamines and lysines on only the amino and carboxyl termini and in functionally non-polar ways. Forestomach CEs also use trichohyalin as a novel cross-bridging protein. We performed mathematical modeling of amino acid compositions of the CEs of mouse and human epidermis of different body sites. Although the sum of loricrin + SPRs was conserved, the amount of SPRs varied in relation to the presumed physical requirements of the tissues. Our data suggest that SPRs could serve as modifiers of a composite CE material composed of mostly loricrin; we propose that increasing amounts of cross-bridging SPRs modify the structure of the CE, just as cross-linking proteins strengthen other types of tissues. In this way, different epithelia may use varying amounts of the cross-bridging SPRs to alter the biomechanical properties of the tissue in accordance with specific physical requirements and functions.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

One of the major roles of stratified squamous epithelia is to provide a physical and chemical barrier against the environment in order to protect the underlying tissues (1, 2). A large body of data suggests that a major component of this barrier function is the cornified cell envelope (CE),1 which is a specialized structure formed just beneath the plasma membrane of the terminally differentiating epithelial cells (3-6). In the case of "dry" epithelia such as foreskin epidermis and the hair cuticle for example, the CE consists of two parts, a protein envelope (about 15 nm or 5 nm thick, respectively) and a lipid envelope about 5 nm thick (7-9). Internal "wet" epithelia assemble a protein but often not a lipid envelope, although an exception appears to be the CE of the rodent forestomach (10).

The mechanical attributes of the CE are afforded by its rigidity and extraordinary insolubility. These properties are a result of extensive cross-linking of the constituent proteins by disulfide bonds as well as Nepsilon -(gamma -glutamyl)lysine or bis(gamma -glutamyl)spermidine isopeptide bonds formed by the action of transglutaminases (TGases) (1-6, 11-15). To date, three distinct TGase enzymes are known to be present in the epidermis (3-6), of which TGases 1 and 3 seem to be the most important and may have complementary or perhaps overlapping roles in the cross-linking of various CE structural proteins (16). A growing number of proteins, including cystatin alpha , desmoplakin, elafin, envoplakin, filaggrin, involucrin, five keratin chain types, loricrin, and several individual members of the small proline-rich (SPR) family, are now known to be components of the epidermal CE and are cross-linked together by isopeptide bonds (17-19), of which loricrin is by far the most abundant protein (17-22). Certain other calcium-binding proteins and desmosomal proteins appear to be components of CEs (23), but their mode of covalent attachment is not yet resolved.

Emerging data suggest that certain proteins are common components of the CEs formed by many if not all mammalian epithelial cell types, including in particular involucrin and perhaps the desmosomal-related proteins desmoplakin and envoplakin (19, 23), which together function as early "scaffold" components of the CE. On the other hand, a variety of data have suggested that CEs from different epithelia are not the same. Early studies showed that CEs formed in cultured keratinocytes or in abnormal psoriatic epidermis are "fragile" in comparison to those from normal cornified epidermis (24-26). More recent analyses of the amino acid compositions of CEs from a variety of tissues have suggested that these physical properties are probably a result of significant differences in the amounts of the several structural proteins listed above (10, 20, 21). Thus, a current view holds that CEs formed in different stratified squamous epithelia may be built from a common scaffold, but the proteins selected for the subsequent reinforcement of this may vary widely (4, 18-23). For example, whereas loricrin is the major component of epidermal CEs, those from cultured epidermal keratinocytes in contrast contain much less loricrin (21, 27); moreover, loricrin is not expressed at all in most internal epithelia (28, 29). Similarly, the amounts of SPR proteins were estimated to vary significantly in the CEs of different epithelia (30-40). Direct sequencing data of cross-linked peptides reveal they constitute about 5% of the CEs of foreskin epidermis, admixed with the far more abundant loricrin (18, 19). By both in situ hybridization and immunohistochemical methods, SPR1 proteins are present in the CEs of fetal periderm, are essentially absent from those of newborn and adult interfollicular epidermis, but seem to be abundant components of those of mouse epidermis of the lip, snout, foot pad, and hair follicle (33, 34, 36, 38-40). Notably, they are especially abundant in rodent forestomach CEs (10, 36, 41). SPR2 and SPR3 proteins are rare in the CEs of many epithelia, but are more abundant in the CEs of oral and esophageal epithelia, or in response to chemical insult, or in tumors (30, 33, 35-40). Together, these observations have led to the suggestion that the physical and mechanical attributes of the CE and thus the entire epithelium may be determined in part by the selection of proteins, including the SPRs, used to reinforce the CE structure.

Our preliminary data have revealed that mouse forestomach CEs contain about 95 nmol of cross-link/mg of total protein, which is one of the highest content of any TGase cross-linked product known to date, and corresponds to about one cross-link/100 amino acid residues. Accordingly, it is a useful structure to examine in order to better understand the physical barrier properties of CEs in general. In the present report, we have characterized the cross-linked proteins of mouse forestomach CEs to determine whether novel proteins might be involved in the cross-linking of this particularly toughened CE, whether loricrin and SPRs are cross-linked together in the same way and by the same residue positions as is known to occur in foreskin epidermal CEs, and to explore the role of the SPRs in CE structure. Our data offer novel insights into the roles of cross-bridging SPRs; the amounts present may modulate the biomechanical properties of the CEs and their epithelial tissue of origin.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Preparation of CEs-- Forestomach epithelial tissue from BALB/c mice was dissected out, washed in phosphate-buffered saline, and then extracted by exhaustive boiling in SDS buffer (10). The same procedure was used to isolate CEs from human trunk epidermis, newborn mouse epidermis, and adult epidermis from the lip, footpad, and trunk of nude mice. The resultant CE fragments were pelleted through 20% Ficoll in phosphate-buffered saline to remove contaminating (i.e. not cross-linked) proteins (21, 22).

Protein Chemistry Procedures-- Protein and peptide amounts were determined by amino acid hydrolysis (110 °C for 22 h in 5.7 N HCl in vacuo). Amounts of the isodipeptide cross-link were determined by amino acid analysis following complete enzymatic digestions of CE samples (16). Suspensions of CE samples (1 mg/ml in 0.1 M N-ethylmorpholine acetate, pH 8.3) were digested at 37 °C with trypsin (Sigma, sequencing grade, 1% by weight) for 6 h, followed by proteinase K (Life Technologies, Inc., 3% by weight) for 3 h. The solubilized peptides were recovered by centrifugation at 14,000 × g, dried, and resolved by HPLC as described previously (18, 19). Peptides eluted by >30% acetonitrile were covalently attached to a solid support and sequenced. Empirically, these peptides contained >= 15 residues, and on sequencing, two or more amino acids were released at each cycle, giving rise to multiple peptide "branches" adjoined by one or more cross-links. Sequences assignments were generally straightforward (18, 19), since the sequences of most mouse CE protein constituents are known. The exact position of the cross-link(s) in some cases could not be determined.

Mathematical Modeling Analyses-- These were performed on amino acid composition data of isolated and purified CEs exactly as described previously, using 13 amino acids (Asx, Thr, Ser, Glx, Pro, Gly, Ala, Val, Cys, Leu, Tyr, Lys, and Arg), and using the proteins cystatin alpha , desmoplakin, elafin, envoplakin, filaggrin, involucrin, keratin (averages of keratin 1 and keratin 10), loricrin, and SPR1-SPR3 (10, 21, 22). The principal criterion of robustness of these calculations comes from the root mean squared discrepancy value; values of <= 0.3 are well within the standard error of the amino acid analyses. In addition, the data generated predicted composition values close to 100% without difficulty. Thus, these analyses generated highly constrained fits, suggesting the presence of few if any other proteins of differing compositions in significant amounts.

Cloning and Characterization of Mouse SPR3-- Based on comparative analyses of nucleotide sequences of the SPR1 from different species (36), and the SPR3 genes of human (31) and rabbit (37), we designed two primers, forward (5'-TACCAGC-AGAAGAACCCTTT-3') and backward (5'-ACCAAGGTCCCTGAGTCAGG-3'), to amplify mouse (BALB/c) genomic DNA, using the conditions described previously (36). A fragment of 0.2 kilobase pairs was generated after 35 polymerase chain reaction cycles, purified using a GeneClean II kit (BIO 101, Inc., Vista, CA), cloned into a pTA vector (Invitrogen Corp., San Diego, CA), and sequenced. Its sequence was highly homologous to human and rabbit SPR3 genes, and provided information for designing specific nested sets of primers that were used with a mouse GenomeWalker kit (CLONTECH). This resulted in about 1.2 kilobases of sequence information (recorded as GenBankTM entry Y09227), and includes the entire coding sequence of 720 nucleotides, 292 nucleotides upstream of the initiation codon, and 196 nucleotides of 3'-noncoding sequences.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation and Characterization of Cross-linked Peptides from Mouse Forestomach CEs-- Tryptic digestion of purified forestomach CEs released about 30% of the total protein and 20% of the total isopeptide cross-link content. A subsequent 3-h digestion with proteinase K solubilized an additional 65% of protein and 70% of the cross-link. The remaining 5% of protein and cross-link was only poorly solubilized after an additional 6-h digestion, but was estimated by mathematical modeling of amino acid analyses to be highly enriched in loricrin and involucrin. This resistance to proteolysis may be a result of adherent lipids (10, 19). The solubilized trypsin and proteinase K peptides contained 86 nmol of a total of about 95 nmol of cross-link/mg of total CE protein. Following fractionation by HPLC, >200 peptide peaks were recovered (Fig. 1). As had been found in earlier experiments (18, 19), peptides eluted by <30% acetonitrile generally were <15 residues long and contained only traces of cross-link, and thus presumably consisted largely of non-cross-linked portions of CE proteins. However, 166 peptides eluted by >30% acetonitrile were sequenced, of which 146 yielded two or more residues/cycle, i.e. they contained two or more peptide branches connected by one or more isopeptide cross-links. These data generated 458 peptide branches (Table I), of which 429 could be identified and the exact location of the isopeptide cross-links assigned. The unknown 29 sequences either were too short for unequivocal identification or had no match in data bases. More than 83% of total CE cross-link could be accounted for in these peptides; the remainder was presumably eluted in peptides that were not sequenced or lost in experimental manipulations. Finally, the 20 peptides that yielded only one sequence may have originated from non-cross-linked sequences of the CE proteins, or may have been attached to the CEs through other bonds, including polyamines (15).


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Fig. 1.   Fractionation of tryptic/proteinase K peptides of forestomach CEs by HPLC.

                              
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Table I
Occurrences of sequences of known proteins in cross-linked peptides from mouse forestomach CEs
Number of peptides: 146; number of peptide branches: 458; total amount of cross-link: 95 nmol/mg; recovery yield: 83%.

Mouse and Human Loricrins Are Functionally Equivalent-- Analysis of the data (Table I) revealed that loricrin was the major component of mouse forestomach CEs, and it was cross-linked to every other protein species identified (Table II). Loricrin constituted 62% of the molar amount of peptides; together with the 5% insoluble remnant that may consist largely of loricrin, the total content of loricrin compares favorably with our previous estimates of 65% by mathematical modeling (10).

                              
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Table II
Frequency of cross-linking between protein partners in mouse forestomach CEs

Mouse and human loricrins are built with a common plan. They contain three glycine loop domains, but those of mouse loricrin are considerably larger than those of human loricrin, and the first domain is broken into four segments by Lys residues. Moreover, there is a high degree of homology in the locations and sequences of the amino- and carboxyl-terminal Lys-Gln-rich sequences, as well as in internal Gln-rich regions (17). Examination of the residue positions used for cross-linking of mouse loricrin in vivo in forestomach CEs revealed a pattern consistent with previous in vivo (18, 19) and in vitro (16) data for human loricrin (Fig. 2). Few cross-links involved amino-terminal Gln or Lys residues (18% of all cross-links for mouse versus 10% human); most of the Gln and Lys residues of the proteins were used for cross-linking, at least to some degree (91% versus 86%); the most frequently used Gln residues for cross-linking involved a pair of residues located between glycine loop domains 2 and 3 (56% versus 52%); the most commonly cross-linked Lys residue was at the carboxyl terminus (25% versus 22% of all cross-links); and the most common Gln-Lys partners for cross-linking involved one of the pair of the internal Gln residues and the terminal Lys residue (33% versus 38%), suggesting that the loricrins become more compact upon cross-linking (16). These data affirm transgenic mice studies, which showed that human and mouse loricrins are functionally identical (20).


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Fig. 2.   Utilization of Gln and Lys residues for cross-linking in mouse and human loricrins has been conserved. The numbers of occurrences of cross-links involving Gln and Lys residues of mouse loricrin (upper panel) generated in the present work and human loricrin (lower panel) taken from Refs. 18 and 19. The horizontal scale for the residue numbers is not linear; sequences are aligned to maximize homologies with the internal and terminal Gln-Lys-rich sequences. Open bars, Lys residues. Closed bars, Gln residues. The glycine loop domain motifs are illustrated at top.

Identification, Cloning, and Sequence of the Mouse SPR3 Protein-- The second most abundant protein constituents of mouse forestomach CEs were the SPRs (21.9% compared with our previous estimate of 18%) (Table I). However, we recovered seven times a sequence, KQKTKQK, that was similar but not identical to the carboxyl-terminal sequence of mouse SPR1 (36) and rabbit SPR3 (cornifin beta ) (37) proteins. On the assumption that it originated from a novel mouse SPR protein, we determined its complete sequence (Fig. 3A). The data confirm an SPR protein. The sequence is most closely related to the human and rabbit SPR3 proteins, rather than SPR1 proteins, on the basis of the presence of two conserved Phe residues at position 9 and 33 in the head domain, and Gly and Thr residues in positions 4 and 6 of the consensus central peptide repeat (Fig. 3B). In addition, in comparison to SPR1 proteins, the deduced amino acid composition shows characteristically lower contents of Pro (20.6% versus approx 31%), Cys (4.6% versus approx 10%), and Lys (5.5% versus approx 11%), but higher contents of Phe (6% versus 0%), Gly (5.9% versus 0%), and Thr (11.8% versus approx 3%). Therefore, since its carboxyl-terminal sequence is identical to that seen in the cross-links, these studies represent the first report of the participation of the SPR3 protein in CE structure and confirm earlier predictions (30, 31, 37). Three other peptides were recovered from the amino-terminal sequence of SPR3, of which one is shown in Table III (item 9). The Gln and Lys residues identified in cross-links are shown in bold in Fig. 3.


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Fig. 3.   Sequence of mouse SPR3. A, the sequence is arranged to the designate separate domains, the head and tail domains involved in cross-linking, and central domain, which consists of numerous generally conserved eight-residue peptide repeats. The repeats are aligned to maximize homologies. Residues involved in cross-linked peptides recovered in this work are shown in bold. B, a comparison with other SPR sequences is also aligned to maximize homologies. The distinctive Phe residues in SPR3 proteins are shown in bold. The different residues of positions 4 and 6 of the central repeating domain of SPR3 proteins in comparison to SPR1 proteins are shown in bold. C, amino acid compositions of SPR1 and SPR3 sequences. The Cys, Phe, Gly, Lys, Pro, and Thr residues, which show characteristic amounts in the proteins, are shown in bold.

                              
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Table III
Examples of peptides in which SPRs function as cross-bridging proteins
Single-letter codes for amino acids are used. Amounts released at each Edman degradation cycle are given in picomoles in parentheses. Data are corrected for carryover between cycles. Peptides had been bound to a solid support by water-soluble carbodiimide through carboxyl groups, so that the amount of PTH derivative released at an expected Glu or Asp residue position, or at the carboxy-terminus is substoichiometric. X, PTH isodipeptide cross-link. Note that, in the case of SPR1 and 2 proteins, due to the variable numbers of peptide repeats of their central domain of the two or more members likely to be expressed in forestomach tissue, the terminal residues used in cross-linking are denoted from the carboxyl-terminal end (E).

Exclusive and Near-equivalent Use of the Head and Tail Domains of SPRs for Cross-linking-- Another 111 peptides were clearly recovered from mouse SPR1 and SPR2 proteins (Table I). Most of these involved cross-links with loricrin (Table II). Interestingly, examination of the distributions of the Lys and Gln residues used for cross-linking revealed that all resided in amino- or carboxyl-terminal sequences (Fig. 4, a and b). Note that there are either two (SPR1) or about eight (SPR2) individual gene products; SPR1a2 and some SPR22 are known to be expressed in the mouse forestomach. The various members differ in the numbers of their central peptide repeats, whereas the sequences of the amino- and carboxyl termini have been conserved. Accordingly, we have indicated the sequence positions from the carboxyl terminus, designated here as E. From the sequences of the SPR proteins available in data bases to date, we can calculate that 45-55% of the total Lys and Gln residues of the SPRs are located in the central peptide repeat domain. However, our data show that none of these residues was utilized for cross-linking. In both SPR1 and SPR2, approximately equal numbers of cross-links involved the amino and carboxyl termini, which implies that these two domains are functionally equivalent in cross-linking. There is minor evidence for asymmetry since 55% of the Gln residues were located on the amino terminus, whereas 60% of Lys residues, including the most commonly used Lys, were located on the carboxyl terminus (Fig. 4, a and b).


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Fig. 4.   Exclusive and conserved utilization of Gln and Lys residues on the amino and carboxyl termini of mouse and human SPR1 and SPR2 proteins. The numbers of occurrences of cross-links involving Gln and Lys residues in SPR1 (a) and SPR2 (b) are listed. Mouse forestomach data are from the present study; data for human proteins (foreskin tissue) are from Refs. 18 and 19. Mouse SPR2 sequences are from unpublished data (see Footnote 2). Because of the variable numbers of peptide repeats on some of the proteins, yet conserved carboxyl termini, actual residue numbers are unknown; accordingly, the carboxyl termini are numbered from the terminal residue, designated as E. Locations of the protein domains are designated by the arrows illustrated at top. Open bars, Lys residues; closed bars, Gln residues. Note that in both SPR1 and SPR2 proteins the amounts of cross-linking of the head and tail domains is approximately equal, although there is evidence of asymmetry in Gln and Lys usage.

Mouse and Human SPR1 and SPR2 Proteins Are Functionally Equivalent-- In addition, in Fig. 4, we have compared the in vivo utilization of Lys and Gln residues for cross-linking of mouse SPRs with the data accumulated from human foreskin epidermal CEs (18, 19) and from cultured foreskin keratinocytes.3 The interspecies comparisons are possible because the termini of the SPR1 and SPR2 classes have been conserved. The data are remarkably consistent, which, as was the case for loricrin, establishes that the SPR proteins are functionally equivalent in the two species as well as in two different CE structures.

Evidence That SPRs Function as Cross-bridging Proteins in CEs-- Furthermore, examination of the 52 peptides recovered in the present work, which involved three or more peptide branches of which at least one was an SPR sequence, we found that, in all cases, the SPR protein formed an interchain cross-bridge between other proteins (Tables III and IV). Although most involved loricrin, the SPRs were also used to link many other CE proteins, such as envoplakin, involucrin, keratin 1, or trichohyalin (THH) (see also Tables IV and VII). Rarely (two occurrences) were the SPR1 proteins cross-linked to themselves (Table III and IV). In the first of these, the same SPR1 sequences were utilized (Table III, item 34), indicating interchain cross-bridging by separate molecules. However, in the second case (item 35), two cross-links involved nearby Gln and Lys residues of the head domain, and a third of the tail domain of SPR1, i.e. it is possible the three SPR1 sequences were contributed by the same molecule, so that the cross-links may have been intrachain in a single molecule, interchain between separate molecules, or a combination of both. Likewise, in the case of items 9 and 29, head and tail sequences of SPR1 were utilized, so that it is possible these were intrachain cross-bridges contributed by a single molecule, or interchain.

                              
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Table IV
SPRs function as cross-bridging proteins in mouse forestomach CEs

Together, these data indicate that the SPRs function as interchain, and perhaps intrachain, cross-bridging proteins in the forestomach CE structure, by utilization of multiple Gln or Lys residues simultaneously on either the head or tail domain, or both.

Summed Amount of SPR Proteins and Loricrin in Epidermal CEs of Different Body Locations Is Constant-- The above observations for the CE of forestomach epithelium, which is an especially toughened and subject to considerable mechanical trauma or abrasion, reminded us of previous observations on the widely varying abundance of SPR1a and SPR1b proteins in different epithelial tissues and the presumed physical requirements of the tissues (10, 36). To explore this further, we isolated CEs from several mouse and human epidermal sources of varying degrees of thickness and physical requirements, performed amino acid analyses (Table V), and then used mathematical modeling on those data to estimate the amounts of various known proteins (10, 21, 22), including in particular loricrin and SPRs (Table VI). Based on actual values ascertained by sequencing of peptides derived from foreskin epidermal CEs (18, 19) and the present mouse forestomach CEs (Table I), these estimates provided data that are highly consistent. Moreover, the small root mean residual discrepancy values indicate highly constrained fits, suggesting that: (i) the CE preparations contain little, if any, keratin from other contaminants and (ii) the proteins and amino acids used for the calculations closely account for the actual compositions (although the presence of other proteins with similar amino acid compositions to those used in the calculations cannot be excluded). Together, these data confirm the validity of these estimates and allow for the wider applicability of the method. As expected, the new data show that loricrin is always the major component in all epidermal CE samples. However, the amount of SPRs varied widely, from <1% in newborn mouse or human trunk epidermis, to 22% in mouse forestomach. Furthermore, the total amounts of loricrin + SPRs in all tissues examined remained constant at 83-85%. Moreover, the ratio of loricrin:SPRs varied from >100:1 in trunk or newborn epidermis, to 4.5-7.5:1 in mouse footpad or lip, and human callus, to as low as 3:1 in the forestomach CEs (Table VI). These data reveal a clear correlation between the amount of SPRs likely to be present and the presumed physical properties of different epithelial tissues.

                              
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Table V
Amino acid compositions of CEs from different sources (mol/100 mol)

                              
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Table VI
Total amounts of loricrin and SPRs are conserved in CEs: correlation with epithelial function
Percentages may not add to 100 because of rounding to the nearest whole number. Percentages in bold type were determined in sequencing experiments of CEs from human foreskin epidermis (18, 19) and mouse forestomach (Table I).

Trichohyalin Is a Cross-linked Component in Mouse Fore- stomach CEs-- A novel component of these CEs was THH, which accounted for about 5% of the total and was cross-linked to a variety of other structural proteins (Tables II and VII). This is the first report of its participation in any CE structure and confirms our earlier predictions of its role in cells (42). Although the sequence of mouse THH is not known, the several peptides recovered here show significant homology to the human (42) and sheep (43) proteins, indicating that it possesses a similar domain structure, as defined by various peptide repeating motifs. Cross-links were found to involve several domains, indicating that there are multiple cross-linking sites along THH, as we have discovered in a recent in vitro study (44). Interestingly, many of these peptides also contained citrulline (Table VII), which arises from modification of arginines by the enzyme peptidylarginine deiminase (44-47). The predominant protein partners of THH were loricrin and SPRs, although others also involved envoplakin and involucrin. Moreover, examination of the multibranch peptides suggests that THH functions to join other protein components in the forestomach CEs (Table VII, items 11-16), primarily between the more abundant loricrin and SPRs.

                              
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Table VII
Cross-links involving trichohyalin

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Rodent forestomach is used as a primary storage of food prior to initiation of digestion. It is an especially flexible yet toughened epithelium, which has evolved to withstand vigorous mechanical abrasion as well as large expansions on ingestion and then contraction on digestion of food. It is a cornifying epithelium, which shares certain biochemical features with the epidermis, including the abundant expression of the keratin intermediate filament chains 1 and 10, profilaggrin, loricrin, SPR proteins, and the TGase 1-3 enzymes (10, 41).2 Furthermore, like the epidermis, these epithelial cells are bound by a CE consisting of a highly cross-linked amalgam of several shared proteins, and perhaps covalently bound lipids (10). In this report, we have identified a correlation between the SPR content of the CEs and the tough physical barrier requirements of the forestomach. The data presented here provide novel evidence for the role of SPRs, and perhaps THH, in the determination of the biomechanical properties of the forestomach epithelium, which has important significance for the epidermis as well as all other epithelia where SPRs are expressed.

The SPRs Function as Cross-bridging Proteins by Use of Only the Head and Tail Domains for Cross-linking-- SPRs consist of a heterogeneous population of proteins for which the primary functions identified to date are as CE structural proteins. However, the way in which they function and the consequences of their presence are not yet understood. In mouse (Fig. 3), as in other mammalian species, the SPR3 class consists of one protein and contains Lys- and Gln-rich amino- and carboxyl-terminal domains, separated by a central domain containing about 23 conserved eight-residue peptide repeats. The SPR1 class consists of two known members in mouse, which have Lys- and Gln-rich termini that are homologous to those of SPR3 and differ by having a central domain composed of 13 (SPR1a) or 14 (SPR1b) eight-residue repeats of sequence rather different from those of SPR3 (36). The mouse SPR2 class consists of at least eight different genes that also have Lys- and Gln-rich termini, which are generally homologous to the other SPRs, but their central domains contain a different nine-residue proline-rich motif repeated 3.5-9 times.2 In all SPRs, the central peptide repeats contain multiple Gln and Lys residues. Several earlier reports have documented that SPRs are good substrates for cross-linking by TGases, and that at least some members can be oligomerized by membrane fractions containing the TGase 1 or TGase 2 enzyme in vitro (32, 34, 37-40). The present analyses of a large number of cross-linked peptides involving the SPRs have provided direct and novel information on the way in which they are utilized in vivo.

Notably, the present mouse (Fig. 4) and earlier human (18, 19) data on peptides derived from CEs reveal that only those Gln and Lys residues located on the termini are used for cross-linking in vivo. On this basis, we propose the use of the terms "head" and "tail" domains for the amino- and carboxyl termini, respectively, to designate these functional sequences (Figs. 3 and 4).

Moreover, by careful adjustment of the degree of proteolytic digestion of the forestomach CEs, we were fortunate to obtain many multibranched peptides adjoined by two or more isopeptide cross-links, which have provided more information on their macromolecular associations and functions. First, we found that SPRs were cross-linked to virtually every other protein component of the CEs. Second, multiple adjacent Gln and Lys residues were utilized simultaneously on the same head or tail domain for inter- and perhaps intrachain cross-linking. Third, the head and tail domains were utilized approximately equally in cross-linking, i.e. the SPRs are functionally equivalent or directionally non-polar. Fourth, in every case involving an SPR protein in the multibranched peptides, the SPR functioned as a cross-bridge through either the head or tail domain (Table IV), or most commonly both. Thus, the available sequencing evidence indicates that SPRs function in vivo mainly as promiscuous bridging proteins spanning between other proteins (including themselves) in the CE structure, linked together through multiple isopeptide bonds.

CEs Are a Composite Material: Modulation of Biomechanical Properties by the Cross-bridging SPR Proteins-- We show here that the protein portion of forestomach CE contains similar early scaffold proteins, including involucrin, envoplakins, and desmoplakin, and in similar amounts, as are found in foreskin epidermal CEs (Table I; cf. Ref. 19). However, it differs primarily in the mix of proteins which contribute the reinforcement components of CE structure. In the foreskin epidermal CE, loricrin + SPRs comprised about 85% of the total CE protein mass with a relative molar ratio of about 20:1 (Table V; Refs. 18, 19, and 22). In the forestomach CE, although a similarly large amount of 88% was composed of loricrin + SPRs + THH, the relative molar ratios were about 3:1:0.2 (Table I), a variation made striking by the marked net increases in the SPRs and the first reported occurrence of THH. Furthermore, we have estimated the relative amounts of loricrin and SPRs in epidermal CEs recovered from a variety of body sites in mouse and human (Table VI). Again, we note that the sum of loricrin + SPRs was conserved at about 85%; however, notably, the ratio of loricrin:SPRs varied from >100:1 in thinner trunk epidermis to 4.5-7:1 in the thickened and toughened epidermis of the lip, callus, and footpad.

Together, these data document a correlation between the amounts of SPRs in CEs of forestomach epithelium and epidermis from different body sites, and in the presumed physical properties and requirements of these tissues. Specifically, the forestomach of rodents and the epidermis of palm/sole, footpad, lip, etc., are subjected to considerably more mechanical abrasion and trauma during normal functions than other body sites such as the trunk.

These data afford biochemical support for our earlier hypothesis on the role of SPRs as modifiers of a composite CE structure (10). Composite materials consist largely of a ground substance (in the present case, loricrin) and minor amounts of a cross-linking matrix substance (SPRs). For example, the properties of composites such as polyacrylamide gels can be widely varied by modest changes in the amount of cross-linking bis-acrylamide (48). Generally, increases in the cross-linker result in a more strengthened and/or toughened composite material (49). In the case of CE structures, loricrin is thought to provide an insoluble (through inter- and intrachain isopeptide and disulfide bonds) yet flexible (because of the glycine loop sequence motifs) texture to the CE (17, 50). By analogy with the materials science composite concept, inclusions of cross-bridging SPR proteins, linked by isopeptide (and perhaps disulfide) bonds to the abundant loricrin component, would be expected to significantly modify the biomechanical properties of the CE. Specifically, we propose that the high contents of cross-bridging SPRs are utilized to provide toughness and strength, yet retain marked flexibility characteristics, to the CEs of the forestomach and specialized epidermal sites.

To an extent, such biomechanical properties would also be affected by the protein density as well as the ratio of components of the composite. However, recent mass measurements using scanning transmission electron microscopy of CEs recovered from a variety of mouse epithelia including trunk epidermis and forestomach reveal the same mass density of about 7 kDa/nm2 of approx 15-nm-thick CE fragments (51). This strongly suggests that the higher content of SPRs in the latter is the likely cause for the change in its properties.

There are many other examples of composite materials in biology, including in specialized epithelial tissues. For example, hair fiber cortical cells contain large numbers of aligned keratin intermediate filaments (KIF) embedded in a matrix of cysteine-rich proteins, and the two components are extensively cross-linked together by disulfide bonds. In many fibers, this composite is not uniform; a paracortex may exist on half of the fiber on one side and an orthocortex on the other side. This is caused by a relatively larger matrix protein:KIF ratio of about 1:3 in the former as compared with the latter (about 1:1, calculated in volume) (52-54). The paracortex is thus somewhat weaker, manifests as a concave bend, and resulting in a kinked fiber, a feature which is of important survival value in many animals. In another example, the inner root sheath forms an especially hardened structure early in hair follicle development. Its purpose is to provide a rigid sheath to constrain the differentiating hair fiber cortical cells internal to it (55). The sheath is made rigid by extensive cross-linking of the KIF with the matrix protein THH by isopeptide bonds (44, 55, 56). In this case, the matrix:KIF ratio is about 1:2 (44, 56). Additionally, this tissue has by far the highest content of isopeptide cross-links than any other tissue identified to date (about 1 residue in 30 is a cross-link, compared with 1 in 100 in the forestomach CE; Ref. 44). These cases offer cogent examples of the way in which the biomechanical properties of tissues may be varied by alterations in the amounts of cross-linking or -bridging proteins in composites.

Cross-linking of SPRs and THH to KIF at the CE Interface Affects the Biomechanical Properties of the Entire Epithelium-- We have demonstrated previously that the KIF cytoskeleton of terminally differentiated epidermal keratinocytes is covalently attached to the CE by isopeptide cross-links inserted by TGases, through a specific Lys residue located on the head domain of type II keratin (mostly keratins 1 and 2e, but also keratins 5 and 6) chains (57). We estimated that 2-3% of these Lys residues were involved in this linkage in foreskin epidermal CEs. Loss of the Lys residue in one allele by mutation of the keratin 1 chain resulted in irregularly shaped cells, a scaling hyperkeratotic phenotype, and reduced skin barrier function (57, 58), primarily in the palms/soles and the epidermis of other toughened body sites. Accordingly, the structure of the CE has a significant impact upon the structural organization, properties, and function of the cell that it bounds, and thus on the structural integrity of an entire stratified squamous epithelial tissue.

We note in the forestomach CEs studied here the presence of a significant number of cross-links between type II keratin chains and several CE protein components, including the SPRs and THH (Tables II-IV and VII), which thus describe a similar covalent connection between the CE and the KIF cytoskeleton in this tissue as well. In fact, the degree of cross-linking of keratins (1590 pmol/mg of CE proteins; Table I) is more than 10 times greater in the forestomach CEs than in foreskin epidermis (Table I; Refs. 18, 19, and 57), indicating that at least one-third of the available specific Lys residues are used for cross-linking. This would thus create a very tight and permanent connection indeed between the CE and the KIF cytoskeleton of this tissue. On this basis, therefore, we conclude that the combination of this extensive connection to the CE structure and the CE, which itself is toughened as a result of its high content of SPRs, affords a novel molecular explanation for the particular biomechanical properties of the forestomach epithelium, as well as for selected specialized sites of the epidermis.

Cross-bridging by THH May Further Contribute to the Biomechanical Properties of Epithelia-- As mentioned, THH is used in tissues such as the inner root sheath of the hair follicle to form a cross-linked toughened composite with KIF by isopeptide cross-linking (44, 55, 56). Notably, both THH and SPRs are also abundantly co-expressed in sheep ruminal epithelium, which is another internal lining epithelium analogous to rodent forestomach that is subject to significant mechanical trauma (43, 59). Likewise, SPRs and THH are co-expressed in the specialized hardened tissues of the hoof epithelium, nail bed, and filiform ridges of the tongue (36, 59, 60). Although direct data on the CEs of these tissues are not yet available, we suggest that inclusion of THH together with SPRs in their CEs could contribute significantly to their toughened biomechanical properties.

In summary, our biochemical data on the protein composition of the mouse forestomach CE provide new insights on the role of SPRs and THH in the determination of the physical properties of the CEs of forestomach and epidermis (and perhaps of other tissues), and thus these entire specialized epithelial tissues (Fig. 5). The strengthened and toughened properties we predict to be imparted by the cross-bridging role of these proteins are consistent with the especially rigorous physical requirements of the tissues. Further work will now be needed to explore the TGase enzyme(s) responsible for the cross-linking of the SPRs in their possible role as modulators of the biomechanical properties of different stratified squamous epithelia.


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Fig. 5.   Model of the structure of the CE of forestomach epithelium. The model shows that the cytoplasmic surface of the CE consists largely of loricrin admixed with the three classes of SPR proteins (pink ovoids) that function as cross-bridgers between primarily loricrin. The model also shows THH (elongated blue ovoids) that may have a similar function. In addition, the cross-link data of Tables II-IV indicate there are numerous close associations between the loricrin/SPR/THH phase of the CE with the inner structural proteins including envoplakin, involucrin, and desmoplakin. Additionally, cross-links were recovered between KIF and several other CE proteins, suggesting two modes of association of the keratin cytoskeleton with the CE as described previously (57).

    ACKNOWLEDGEMENTS

We thank Drs. Eleonora Candi, Ulrike Lichti, Michal Jarnik, and Edit Tarcsa for technical advice and many useful discussions.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom all correspondence should be addressed: NIAMS, National Institutes of Health, Bldg. 6, Rm. 425, 9000 Rockville Pike, Bethesda, MD 20892-2752. Tel.: 301-496-1578; Fax: 301-402-2886; E-mail: pemast{at}helix.nih.gov.

1 The abbreviations used are: CE, cornified cell envelope; HPLC, high performance liquid chromatography; KIF, keratin intermediate filaments; SPR, generic for small proline-rich (SPR1a is small proline-rich protein 1a, etc.); TGase, transglutaminase (TGase 1 is transglutaminase 1, etc.); THH, trichohyalin.

2 T. Kartasova and P. M. Steinert, unpublished data.

3 P. M. Steinert and L. N. Marekov, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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