From the Laboratory of Skin Biology, NIAMS, National Institutes of
Health, Bethesda, Maryland 20892-2752
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.
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INTRODUCTION |
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
N
-(
-glutamyl)lysine or
bis(
-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
, 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.
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MATERIALS AND METHODS |
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
, 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.
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RESULTS |
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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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%.
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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).
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.
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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
) (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
31%), Cys (4.6%
versus
10%), and Lys (5.5% versus
11%),
but higher contents of Phe (6% versus 0%), Gly (5.9% versus 0%), and Thr (11.8% versus
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).
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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.
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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.
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 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).
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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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DISCUSSION |
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
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).
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We thank Drs. Eleonora Candi, Ulrike Lichti,
Michal Jarnik, and Edit Tarcsa for technical advice and many useful
discussions.