Identification and characterization of a novel component of the cornified envelope, cornifelin

Psoriasis is recognized as a chronic inflammatory disease characterized by epidermal hyperproliferation. To identify psoriasis-related genes, we compared the mRNA populations of normal and psoriatic skin. We identified one gene, designated as cornifelin, which showed increased expression in psoriatic skin. Human cornifelin contains 112 amino acids and is expressed in the uterus, cervix, and skin. In situ hybridization analysis demonstrated the presence of human cornifelin in the granular cell layer of the epidermis. To investigate the function of cornifelin, we established a transgenic mouse line overexpressing human cornifelin. Using these mice, we have shown that cornifelin is directly or indirectly cross-linked to at least two other cornified envelope proteins, loricrin and involucrin, in vivo. Overexpression of human cornifelin correlated with decreased loricrin expression and increased involucrin expression in the transgenic mouse. However, abnormality of epidermal differentiation was not observed in the transgenic mouse.     

Systematic repeat addition at a precise location in the coding region of the involucrin gene of wild mice reveals their phylogeny

The involucrin gene encodes a protein of terminally differentiated keratinocytes. Its segment of repeats, which represents up to 80% of the coding region, is highly polymorphic in mouse strains derived from wild progenitors. Polymorphism includes nucleotide substitutions, but is most strikingly due to the recent addition of a variable number of repeats at a precise location within the segment of repeats. Each mouse taxon examined showed consistent and distinctive patterns of evolution of its variable region: very rapid changes in most M. m. domesticus alleles, slow changes in M. m. musculus, and complete arrest in M. spretus. We conclude that changes in the variable region are controlled by the genetic background. One of the M. m. domesticus alleles (DIK-L), which is of M. m. musculus origin, has undergone a recent repeat duplication typical of M. m. domesticus. This suggests that the genetic background controls repeat duplications through trans-acting factors. Because the repeat pattern differs in closely related murine taxa, involucrin reveals with greater sensitivity than random nucleotide substitutions the evolutionary relations of the mouse and probably of all murids.     

Molecular Characterization and Evolution of the SPRR Family of Keratinocyte Differentiation Markers Encoding Small Proline-Rich Proteins

SPRR genes (formerly SPR) encode a novel class of polypeptides (small pr oline rich proteins) that are strongly induced during differentiation of human epidermal keratinocytes in vitro and in vivo. Recently we found that the N- and C-terminal domains of these proteins show strong sequence homology to loricrin and involucrin, suggesting that SPRR proteins constitute a new class of cornified envelope precursor proteins. Here we show that SPRR proteins are encoded by closely related members of a gene family, consisting of two genes for SPRR1, approximately seven genes for SPRR2, and a single gene for SPRR3. All SPRR genes are closely linked within a 300-kb DNA segment on human chromosome 1 band q21-q22, a region where the related loricrin and involucrin genes have also been mapped. The most characteristic feature of the SPRR gene family resides in the structure of the central segments of the encoded polypeptides that are built up from tandemly repeated units of either eight (SPRR1 and SPRR3) or nine (SPRR2) amino acids with the general consensus *K*PEP**. Sequencing data of the different members, together with their clustered chromosomal organization, strongly suggest that this gene family has evolved from a single progenitor gene by multiple intra- and intergenic duplications. Analysis of the different SPRR subfamilies reveals a gene-specific bias to either intra- or intergenic duplication. We propose that a process of homogenization has acted on the different members of one subfamily, whereas the different subfamilies appear to have diverged from each other, at the levels of both protein structure and gene regulation.     

The involucrin gene of the owl monkey: origin of the early region

A large part of the coding region of the hominoid involucrin gene is of recent origin. This part of the gene, which we have called the modern segment, contains numerous repeats of a sequence of 10 codons, created by multiple duplications some of which consist of 3-12 repeats. We have sequenced two alleles of the involucrin gene in the owl monkey and found that the involucrin gene of this species also possesses a modern segment. By comparing the modern segment of the owl monkey with that of the hominoids, we find that only a part of this segment is shared by the two species. We call this part the early region because it must have originated in a common ancestor of the anthropoids. The rest of the hominoid modern segment does not correspond to any groups of repeats in the owl monkey and was therefore created after divergence of the two lineages. As in the hominoids, the latest additions to the modern segment of the owl monkey have been in its 5' half, which possesses different duplication patterns in the two alleles. Lineage divergences within the anthropoids can be detected at different sites within the modern segment.      

The involucrin genes of the mouse and the rat: study of their shared repeats

The involucrin genes of the mouse (Mus musculus) and the rat (Rattus norvegicus) have been cloned and sequenced. The coding region of each gene contains, at site P, a segment of repeats homologous to that of other nonanthropoid mammals. In contrast to the repeats of species belonging to different mammalian orders, many individual repeats of the mouse and the rat can be matched. Both before and after the divergence of the two species, these repeats have been the site of systematic alterations in nucleotide sequence. One of the alterations is the correction of nucleotides of one repeat by those of another. Corrected nucleotides may be closely linked to flanking nucleotides that are uncorrected; the systematic correction process therefore appears to be due to gene conversion. There is a stretch of 18 reiterated CAGs in the segment of repeats of the Mus gene; most of these reiterations were introduced recently, supporting the idea that the gene was generated originally from poly CAG. An antiserum to a synthetic peptide encoded by the segment of repeats of the Mus gene reveals differentiation- specific expression of the gene in the epidermis.      

Initiation of assembly of the cell envelope barrier structure of stratified squamous epithelia

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.     

Polymorphism due to variable number of repeats in the human involucrin gene.

The coding region of the involucrin gene in higher primates contains a segment consisting of numerous tandem repeats of a 10-codon sequence. The process of repeat addition began in a common ancestor of all higher primates and subsequent repeats were added vectorially. As a result, the principal site of repeat addition has moved in the 3' to 5' direction and the most recently generated repeats (the late region) are close to the 5' end of the segment of repeats. In the human, most of the late region is made up of two different blocks, each consisting of nearly identical repeats. We describe here five polymorphic forms resulting from the addition of differing numbers of repeats to each block. As the variety and nature of the polymorphic alleles are different in different human populations, we postulate that the process of repeat addition is genetically determined.     

The involucrin gene of the orangutan: generation of the late region as an evolutionary trend in the hominoids

In the evolutionary line leading to the higher primates, the coding region of the involucrin gene evolved a segment consisting of numerous repeats of a 10-codon sequence. Additions to this segment of repeats have been made successively, thus generating regions that can be defined as early, middle, and late. The involucrin gene of the orangutan (Pongo pygmaeus abelii) possesses a segment of repeats whose early region has the same repeat structure as that in other anthropoids. The middle region is not similar in repeat structure to that of all anthropoids but is similar to that of other hominoids. The late region is unique to the species; it does not correspond at all in its repeat structure to that of the human or gorilla and is much larger. The late region of the orangutan was generated by duplications of blocks of older repeats clearly belonging to the middle region. Continued duplications extending the late region are an evolutionary trend in the hominoids. The process of addition of repeats at a particular location is a more significant aspect of the evolution of involucrin than are random nucleotide substitutions; in addition, it has proceeded more rapidly.      

Remodeling of the involucrin gene during primate evolution

The protein involucrin is a product of terminal differentiation in the epidermal cell and related cell types. By comparing the nucleotide sequence of the involucrin gene of the lemur with that of the human, it is clear that the gene has undergone unusual evolution in the primates. The coding region of the gene contains an ancestral segment, most of which is common to the lemur and the human, and a species-specific segment of repeats derived from the ancestral segment. Instead of the modern segment of repeats found in the human gene, the lemur gene possesses repeats derived from another sequence at a different location in the ancestral segment. The two kinds of segments of repeats probably represent alternative ways of creating a repeat structure in the involucrin molecule. The modern segment of repeats must have been created after divergence of the higher primates from the prosimians.     

Molecular characterization of MRJP3, highly polymorphic protein of honeybee (Apis mellifera) royal jelly.

Major proteins of honey bee (Apis mellifera) royal jelly are members of the MRJP protein family. One MRJP protein termed MRJP3 exhibits a size polymorphism as detected by SDS-PAGE. In this report we show that polymorphism of the MRJP3 protein is a consequence of the polymorphism of a region with a variable number of tandem repeats (VNTR) located at the C-terminal part of the MRJP3 coding region. We present the characterization of five polymorphic alleles of MRJP3 by DNA sequencing. By PCR analyses, at least 10 alleles of distinct sizes were found in randomly sampled bees. Studies with nurse bees from a single honeybee colony revealed both Mendelian inheritance and very high variability of the MRJP3 genomic locus. The high variability and simple detection of the MRJP3 polymorphism may be useful for genotyping of individuals in studies of the honeybee.     

Origin of the polymorphism of the involucrin gene in Asians.

The involucrin gene, encoding a protein of the terminally differentiated keratinocyte, is polymorphic in the human. There is polymorphism of marker nucleotides a two positions in the coding region, and there are over eight polymorphic forms based on the number and kind of 10-codon tandem repeats in that part of the coding region most recently added in the human lineage. The involucrin alleles of Caucasians and Africans differ in both nucleotides and repeat patterns. We show that the involucrin alleles of East Asians (Chinese and Japanese) can be divided into two populations according to whether they possess the two marker nucleotides typical of Africans or Caucasians. The Asian population bearing Caucasian-type marker nucleotides has repeat patterns similar to those of Caucasians, whereas Asians bearing African-type marker nucleotides have repeat patterns that resemble those of Africans more than those of Caucasians. The existence of two populations of East Asian involucrin alleles gives support for the existence of a Eurasian stem lineage from which Caucasians and a part of the Asian population originated.     

The involucrin gene of the gibbon: the middle region shared by the hominoids

The evolution of the anthropoid involucrin gene has resulted largely from a process of vectorial addition of short tandem repeats. The coding region of the involucrin gene of the gibbon (Hylobates lar), including the segment of repeats, has been cloned and sequenced, and its repeat structure can now be compared with that of the other hominoids. In the gibbon, as in the others, repeat additions in the past can be assigned to early, middle, and late regions of the present-day segment of repeats. All 10 repeats of the gibbon early region were completed in a common anthropoid ancestor. All 17 repeats of the gibbon middle region were completed in a common hominoid ancestor. After divergence of the gibbon lineage, eight repeats were added to the middle region of the great ape-human lineages. Seven of these are shared by two to four species, according to the order of their divergences from each other. After its divergence, the gibbon lineage added a short species-specific late region. The gibbon also possesses an incomplete repeat just 3' of the early region, the only addition in this region in any hominoid. Comparison of the number of repeats added with the number of nucleotides substituted shows an inconstant relation between the two.     

A minisatellite and a microsatellite polymorphism within 1.5 kb at the human muscle glycogen phosphorylase (PYGM) locus can be amplified by PCR and have combined informativeness of PIC 0.95.

We sequenced a genomic clone (pMCMP1), previously reported to detect a VNTR polymorphism at the PYGM locus, and found a dinucleotide repeat segment (CA)14(GA)25 and a complex (AT)-repeat-rich segment containing 63 repeats spanning 160 bp. Resolution of PCR-amplified genomic DNA from the (CA)(GA) repeat region on DNA sequencing gels revealed a highly informative polymorphism with alleles differing by 2-bp intervals and ranging in size from 156 to 190 bp. Among three racial groups, a total of 18 alleles were observed. Fourteen alleles were observed in Caucasians (PIC 0.89), 12 alleles in American Blacks (PIC 0.89), and 9 alleles in Pima Indians (PIC 0.73). PCR amplification of the (AT) repeat region and resolution of the products on DNA sequencing gels revealed a complex variable length polymorphism with alleles distributed in size from 367 to 970 bp. Twenty-eight alleles were found in American Blacks (PIC 0.94), 6 alleles in Pima Indians (PIC 0.70), and 11 alleles in Caucasians (PIC 0.71). Comparison of the previously described VNTR RFLP alleles visualized by Southern hybridization to the PCR products described in this report demonstrated that the polymorphism described in both assays was identical. However, a larger number of alleles could be detected from the PCR-amplified products. Combined informativeness, PIC 0.95, for the two polymorphisms was determined from haplotype analysis of 100 Caucasian chromosomes. Therefore, for genotyping purposes, informativeness is maximized from using both polymorphisms.     

The involucrin gene of the tree shrew: recent repeat additions and the relocation of cysteine codons

The coding region of the involucrin gene of Tupaia glis has been cloned and sequenced. It resembles the involucrin coding region of other non-anthropoid mammals in possessing a segment of related, short tandem repeats at a defined location, but in Tupaia, there has been recent serial duplication of a repeat into which a cysteine codon had earlier been introduced. As a result of the duplication, there is a total of as many as six cysteine codons in the segment of repeats, a number larger than for any other species yet examined. In Rattus there has been a comparable but independent addition of cysteine codons, and both Tupaia and Rattus have eliminated an otherwise conserved cysteine codon 75 located close to but outside the segment of repeats. In Tupaia, this elimination probably occurred by gene conversion. Also independently, the gene of Canis has added cysteine codons to the segment of repeats but has not yet lost cysteine 75. It is proposed that the gain and the loss of cysteine codons are parts of a multi-stage program of cysteine relocation.     

Consecutive actions of different gene-altering mechanisms in the evolution of involucrin.

During the evolution of primates from nonprimates, the gene for involucrin was greatly altered by changes in the short tandem repeats that are present in some form in the gene of each of 17 species examined. The evolution of involucrin was not the result of a single continuum of more or less random changes, and it was not confined to the process of nucleotide substitution, the most commonly studied evolutionary change in DNA. Instead, the evolution of this gene took place through different mechanisms that shortened the length of the repeats, increased their number, and changed their codon sequence. As part of this trend, one entire segment of repeats was replaced by another located elsewhere in the coding region. To bring about these changes, specific mechanisms have been activated, deactivated, and replaced by other mechanisms. The resulting serial revisions in the involucrin gene must depend on gene-altering machinery whose synthesis or activity can be controlled.     

PCR-analyzed microsatellites of the mouse genome—additional polymorphisms among ten inbred mouse strains

Eighty sequences from the mouse genome database containing microsatellites (simple sequence repeats) have been analyzed for size variation among ten different inbred strains of mice; 62/80 (77.5%) showed polymorphism of at least three alleles. We have been able to detect all the polymorphims by agarose gel electrophoresis, often running the gels for up to 3 h. Between individual pairs of mouse strains to be used in chromosomal mapping studies in our laboratory, 35–60% polymorphism occurred. There are potentially enough microsatellites within the mouse and human genome to have a marker at every 1-cM distance. This simple approach will, therefore, continue to be useful in genome mapping studies, leading eventually to high-resolution maps of both the mouse and human genomes; this should allow for physical mapping and cloning of specific genes.     

A unique AT-rich hypervariable minisatellite 3' to the ApoB gene defines a high information restriction fragment length polymorphism

A DNA restriction fragment length polymorphism has been found immediately 3' to the human apoB gene. Digestion of many different human DNAs at sites flanking the region and Southern blotting analysis reveal that this region can vary in length by approximately 300 base pairs with five alleles readily distinguishable. The length polymorphism is due to a unique AT-rich minisatellite that consists primarily of a 30-base pair tandem repeat with two structurally related subunit sequences, x (ATAATTAAATATTTT) and y (ATAATTAAAATATTT). In general, the sequences repeat in an x-y order. The AT-rich region also contains variant x and y sequences that result from C or G for A substitution. Sequence analysis of one large allele revealed the expected increased number of xy repeats. In addition, similar analysis of three different smaller alleles with the same apparent size on Southern blotting analysis showed that all were of slightly different size due to minor differences in the number of xy repeats. The heterogeneity of this AT-rich minisatellite provides the basis for a highly informative restriction fragment length polymorphism of the apoB gene and should be very useful in association and linkage analysis studies of the contribution of this locus to atherosclerosis susceptibility.     

Distribution of keratin and associated proteins in the epidermis of monotreme,marsupial, and placental mammals

The expression of acidic and basic keratins, and of some keratinization marker proteins such as filaggrin, loricrin, involucrin, and trichohyalin, is known for the epidermis of only a few eutherian species. Using light and high-resolution immunocytochemistry, the presence of these proteins has been studied in two monotreme and five marsupial species and compared to that in eutherians. In both monotreme and marsupial epidermis lamellar bodies occur in the upper spinosus and granular layers. Development of the granular layer varies between species and regionally within species. There is great interspecific variation in the size (0.1-3.0 microm) of keratohyalin granules (KHGs) associated with production of orthokeratotic corneous tissues. Those skin regions lacking hairs (platypus web), or showing reduced pelage density (wombat) have, respectively, minute or indiscernible KHGs, associated with patchy, or total, parakeratosis. Ultrastructural analysis shows that monotreme and marsupial KHGs comprise irregular coarse filaments of 25-40 nm that contact keratin filaments. Except for parakeratotic tissues of platypus web, distribution of acidic and basic proteins in monotreme and marsupial epidermis as revealed by anti-keratin antibodies AE1, AE2, and AE3 resembles that of eutherian epidermis. Antibodies against human or rat filaggrins have little or no cross-reactivity with epidermal proteins of other mammals: only sparse areas of wombat and rabbit epidermis show a weak immunofluorescence in transitional cells and in the deepest corneous tissues. Of the available, eutherian-derived antibodies, that against involucrin shows no cross-reactivity with any monotreme and marsupial epidermal tissues and that against trichohyalin cross-reacts only with cells in the inner root sheath and medulla of hairs. These results suggest that if involucrin and trichohyalin are present throughout noneutherian epidermis, they may have species-specific molecular structures. By contrast, eutherian-derived anti-loricrin antibodies show a weak to intense cross-reactivity to KHGs and corneous tissues of both orthokeratotic and parakeratotic epidermis in monotremes and marsupials. High-resolution immunogold analysis of loricrin distribution in immature keratinocytes of platypus parakeratotic web epidermis identifies labeled areas of round or irregular, electron-pale granules within the denser keratohyalin component and keratin network. In the deepest mature tissues, loricrin-like labeling is diffuse throughout the cytoplasm, so that cells lack the preferential distribution of loricrin along the corneous envelope that characterizes mature eutherian keratinocytes. Thus, the irregular distribution of loricrin in platypus parakeratotic tissues more resembles that which has been described for reptilian and avian keratinocytes. These observations on the noneutherian epidermis show that a stratum granulosum is present to different degrees, even discontinuous within one tissue, so that parakeratotic and orthokeratotic areas may alternate: this might imply that parakeratotic monotreme epidermis reflects the primitive pattern of amniote alpha-keratogenesis. Absent from anamniote epidermis and all sauropsid beta-keratogenic tissues, the ubiquitous presence of a loricrin-like protein as a major component of other amniote corneous tissues suggests that this is a primitive feature of amniote alpha-keratogenesis. The apparent lack of specific regionalization of loricin near the plasma membranes of monotreme keratinocytes could be an artifactual result of the immunofluorescence technique employed, or there may be masking of the antigenicity of loricrin-like proteins once they are incorporated into the corneous envelope. Nevertheless, the mechanism of redistribution of such proteins during maturation of monotreme keratinocytes is different from, perhaps more primitive, or less specialized, than that in the epidermis of eutherian mammals.