Bovine Epidermis Research Articles

Exfoliative toxin E, a new Staphylococcus aureus virulence factor with host-specific activity

Exfoliative toxins (ETs) are secreted virulence factors produced by staphylococci. These serine proteases specifically cleave desmoglein 1 (Dsg1) in mammals and are key elements in staphylococcal skin infections. We recently identified a new et gene in S. aureus O46, a strain isolated from ovine mastitis. In the present study, we characterized the new et gene at a genetic level and the enzymatic activity of the deduced protein. The S. aureus O46 genome was re-assembled, annotated and compared with other publicly available S. aureus genomes. The deduced amino acid sequence of the new et gene shared 40%, 53% and 59% sequence identity to those of ETA, ETB and ETD, respectively. The new et gene shared the same genetic vicinity and was similar in other S. aureus strains bearing this gene. The recombinant enzyme of the new et gene caused skin exfoliation in vivo in neonatal mice. The new et-gene was thus named ete, encoding a new type (type E) of exfoliative toxin. We showed that ETE degraded the extracellular segments of Dsg1 in murine, ovine and caprine epidermis, as well as in ovine teat canal epithelia, but not that in bovine epidermis. We further showed that it directly hydrolyzed human and swine Dsg1 as well as murine Dsg1α and Dsg1β, but not canine Dsg1 or murine Dsg1γ. Molecular modeling revealed a correlation between the preferred orientation of ETE docking on its Dsg1 cleavage site and species-specific cleavage activity, suggesting that the docking step preceding cleavage accounts for the ETE species-specificity. This new virulence factor may contribute to the bacterial colonization on the stratified epithelia in certain ruminants with mastitis.

Bioprospecting keratinous materials

The concept of bioprospecting for bioactive peptides from keratin-containing materials such as wool, hair, skin and feathers presents an exciting opportunity for discovery of novel functional food ingredients and nutraceuticals, while value-adding to cheap and plentiful natural sources. The published literature reports multiple examples of proline-rich peptides with productive bio-activity in models of human disease including tumour formation, hypertension control and Alzheimer’s disease. Bioactive peptides have been identified from food and other protein sources however the bioactivity of keratin-related proteins and peptides is largely unknown. Considering the high representation of proline-rich peptides among proven bioactive peptides, the proline-rich character of keratinous proteins supports current research. A selection of mammalian (cow epidermis, sheep wool) and avian (chicken feather) keratinous materials were subjected to enzymatic hydrolysis using established processing methods. A bio-assay of determining inhibition of early stage amyloid aggregation involved using a model fibril-forming protein – reduced and carboxymethylated bovine K-casein (RCMk-CN) and quantitation of fibril development with the amyloid-specific fluorophore, Thioflavin T (ThT). The assay was fully validated for analytical repeatability and used together with appropriate positive controls. Peptide library products derived from chicken feather (n=9), sheep wool (n=9) and bovine epidermis (n=9) were screened in the fibril inhibition assay based on K-casein. 3 of 27 products exhibited interesting levels of bio-activity with regard to fibril inhibition. HPLC profiles provide an indication of the complexity of the assemblage of peptides in the three active products. We conclude the bioprospecting research using keratinous materials shows promise for discovery of useful bioactive peptides.

High-Resolution Mapping of the Desmosomal Plaque and Adhesive Interface

A precise structural definition of the desmosome has so far remained elusive, likely in part because of its insolubility in aqueous buffers (Matoltsy, 1975). Despite this limiting factor, several key papers have attempted to address this question. What is becoming clear is that the desmosome is both a complex and dynamic structure whose different states may not be readily appreciated by its textbook presentation in single static images. Several approaches have been used to generate a structural map of the desmosome (Figure 1). In vitro binding assays, immunoprecipitation, immunofluorescent colocalization and yeast two-hybrid analyses have been useful in defining protein–protein interactions among desmosome components. Immunogold labeling with N-terminaland C-terminal-specific antibodies to known desmosome proteins was used by North et al. (1999) to generate a molecular map of desmosomes in ultrathin cryosections of bovine epidermis. Using this approach, they were able to generate quantitative measures of the distance of each label from the plasma membrane and thus define the orientation, intermolecular distance and ordering of desmosome protein components. Thus, desmoglein and desmocollin desmosomal cadherins are single-pass transmembrane proteins whose aminoterminal extracellular domains form the adhesive interface. The cytoplasmic tail of desmoglein binds to plakoglobin, which in turn binds to desmoplakin. Desmoplakin binds to keratin intermediate filaments and plakophilins bind to desmoplakin, plakoglobin, desmogleins and desmocollins. Complexes of this unit repeat structure localize into regions of the plasma membrane to form the desmosome, yielding a polyvalent adhesive interface. New specimen preparation techniques and imaging technologies are being applied to the study of desmosome structure. Vitrification, a process in which biological tissues are converted to solids free of crystalline water, facilitates preservation of native structures. Cryo-electron microscopy of vitreous sections is a technique that avoids specimen dehydration and heavy metal deposition of traditional electron microscopy, and thus offers the promise of significantly higher resolution images of the desmosome. Applying this technique to mouse skin, Al-Amoudi et al. (2004) generated an image of the desmosome in which the extracellular inter-desmosomal interface was characterized by highly ordered straight rod-like structures with a 5 nm periodicity. In electron microscopic tomography, multiple two-dimensional images are obtained by tilting a sample at angles to the electron beam; computer software is then used to reconstruct a

Interaction of entomopathogenic nematodes (Steinernematidae) with selected species of ixodid ticks (Acari: Ixodidae).

Entomopathogenic nematodes, currently used for biological control of various insect pests, were tested for their ability to penetrate and kill replete females of several species of ticks including Dermacentor variabilis (Say), Rhipicephalus sanguineus (Latreille), Amblyomma maculatum Koch, and A. cajennense (F.). These species were found to be susceptible to the entomopathogenic nematodes, Steinernema feltiae (Filipjev) or S. riobravus (Cabanillas & Poinar), shown in previous studies in our laboratory to be attracted to and kill replete A. americanum. S. riobravus killed D. variabilis (96%), R. sanguineus (89%), A. maculatum (24%), and A. cajennense (88%), and S. feltiae killed D. variabilis (91%) and R. sanguineus (71%). Of the ticks that survived mean egg mass weights were significantly lower than those of the unexposed controls. When nematode-exposed ticks were examined with light microscopy, nematodes were found to have entered ticks but did not multiply or produce subsequent generations of infective juveniles. The nematodes were separated from surrounding tissues by a clear space, suggesting that they produced protective compounds. Bacteria, thought to be symbiotes released from the nematodes, multiplied initially in the hemocoel of the tick and subsequently were found throughout the degenerating tick tissues. These bacteria eventually filled the tick and appeared to be the cause of tick death. Nematode guts were filled with the bacteria, suggesting that the bacteria were a food source. When ticks were exposed to nematodes while feeding on cattle, partially engorged females were most susceptible to the nematodes. Tick mortality and reduced egg production resulted when the ticks had fed 6 and 9 d before nematode exposure but not when ticks were exposed after 3 d of feeding. Exposure of feeding female ticks demonstrated that the nematodes were able to penetrate tick orifices other than via the hypostome, which was embedded in the bovine epidermis for the duration of the feeding process.

Pemphigus Foliaceus Sera Recognize an N-Terminal Fragment of Bovine Desmoglein 1

It has previously been demonstrated that sera from endemic and nonendemic pemphigus foliaceus patients recognize three immunoreactive fragments of 80, 62, and 45 kilodaltons (kD) from extracts of the envelope fraction of human and bovine epidermis. These polypeptides are also immunoprecipitated by approximately 50% of pemphigus vulgaris sera, but are unreactive with sera from bullous pemphigoid patients or normal controls. The 80-kDa antigen has been shown to be a glycoprotein with N-linked oligosaccharides. Complete removal of the carbohydrate moieties produced a 76-kD polypeptide that continued to react with pemphigus foliaceus autoantibodies in a Ca(++)-dependent manner. To further characterize this antigen/antibody system, the 80-kD pemphigus foliaceus antigen solubilized from a bovine epidermal envelope extract was purified by affinity chromatography using a pemphigus foliaceus patient's immunoglobulin (Ig)G immobilized on agarose. After elution with 0.2 M glycine/HCl, pH 2.8, 5 mM ethylene diaminetetraacetic acid, the polypeptide was mixed with a small amount of 125I-labeled 80-kD antigen, added as a tracer, fractionated by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and electrotransferred onto a polyvinylidene difluoride membrane. The 80-kD band detected by amido black staining and autoradiography was excised and characterized by amino acid sequence analysis. The resulting sequence, EXIKFAAAXREGEXNSKRNPIA, matched perfectly with the N-terminal 22 amino acids of the mature form of bovine desmoglein 1. These findings demonstrate that the 80-kD bovine autoantibody-reactive polypeptide is the glycosylated ectodomain of desmoglein 1, which may contain epitopes recognized by pathogenic autoantibodies.

A Non-desmoglein Component of Bovine Epidermis Reactive with Pemphigus Foliaceus Sera

A component from bovine epidermis has been purified and found to react with sera from patients with pemphigus foliaceus (PF). This component appears to have molecular weight characteristics similar to those of a previously reported component of human and bovine epidermis which was reactive with PF sera, but only detectable by radiolabeling. It is composed of an 80 kD and a 45 kD polypeptide which are thought to be covalently linked. By chromatographic data, the native form appears to be a noncovalently joined dimer of the 80 and 45 kD polypeptides. Amino acid analysis and sequence data indicate that the 80 kD component is not desmoglein (DG) nor any other known protein whose sequence is entered in data banks. Previous studies by Stanley indicate that DG is the major target of autoantibodies in PF sera. Our finding of another epidermal component reactive with PF antibodies raises some questions. It is not yet known if PF anti-DG antibodies cross-react with this new epidermal antigen or if these antibodies constitute separate and non-cross-reacting sets. Both possibilities and their ramifications are discussed.

Affinity immunoblotting studies on the restriction of autoantibodies from endemic pemphigus foliaceus patients.

Endemic pemphigus foliaceus or Fogo Selvagem (FS) is an autoimmune blistering skin disease mediated by autoantibodies directed against components on the surface of subcorneal keratinocytes. All patients have high titers of these autoantibodies in the IgG4 subclass as determined by indirect immunofluorescence on frozen skin sections. In addition, patients may also have autoantibodies in other IgG subclasses, particularly IgG1, but the titers in these subclasses are significantly lower than those found in the IgG4 subclass. We have now found that in addition to isotype preference, autoantibodies from FS patients show clonal restriction as evidenced by oligoclonal banding on isoelectric focusing after probing with extracts from both human and bovine epidermis. Both IgG1 and IgG4 exhibit oligoclonal banding, but the distribution of these bands in the pH gradient differs for these two subclasses. Whereas the IgG4 oligoclonal bands are distributed throughout the IgG4 pH range, IgG1 banding appears to be concentrated in the more basic region of the IgG1 pH range. This finding suggests that the IgG1 autoantibodies have undergone selective somatic mutation by a negatively charged autoantigen. Similar findings have been reported for pathogenic DNA autoantibodies associated with SLE. The wide distribution of IgG4 banding suggests that this response may have followed the IgG1 response and has not undergone selective mutation. Both IgG1 and IgG4 appear to be Ca++ dependent autoantibodies.

Structural analysis and expression of human desmoglein: a cadherin-like component of the desmosome

Desmosomes are adhesive cell junctions found in great abundance in tissues that experience mechanical stress. The transmembrane desmosomal glycoproteins have been proposed to play a role in cell adhesion; desmoglein I (DGI) is a major member of this class of desmosomal molecules. However, evidence supporting a role for DGI in cell adhesion or in the plaque is lacking. In order to begin to understand DGI function we have identified human cDNA clones encoding the entire mature polypeptide of 1000 amino acids. Our data suggest that like the bovine DGI molecule human DGI is highly related to the calcium-dependent class of cell adhesion molecules known as cadherins. Four related extracellular domains located in the amino-terminal domain of the molecule contain putative calcium binding sites originally identified in the cadherins. The highest degree of similarity between human N-cadherin and human DGI, and likewise between bovine DGI and human DGI, is greatest in the most amino-terminal extracellular domain. This suggests a conserved functional role for the extracellular domains, perhaps in calcium-mediated cell adhesion. The cytoplasmic portion of the molecule contains a cadherin-like region and, like bovine DGI, a carboxy-terminal tail that is not present in the cadherins, comprising three additional domains. One of these contains a novel repeating motif of 29 +/- 1 residues, first identified in bovine DGI. Each of the highly homologous repeating units is likely to consist of two beta-strands and two turns with special characteristics. Five amino acids that are identical in bovine and human DGI lie in the second of the two predicted beta-strands, and intriguingly contain putative target sites for protein kinase C. On the basis of structural analysis, a model predicting the disposition of human DGI domains in the desmosome is proposed. Northern analysis suggests that unlike bovine epidermis, which expresses a single mRNA of reported size approximately 7.6 kb, human foreskin and cultured keratinocytes display a complex pattern with bands of approximately 7.2, 4.0 and 3.0 kb. Each of these cross-hybridizing mRNAs is coordinately expressed in normal human keratinocytes in response to long-term culture and increased calcium.

Combined biochemical and immunochemical comparison of peptidylarginine deiminases present in various tissues

We have performed a combined biochemical and immunochemical study on the identity of peptidylarginine deiminases (EC 3.5.3.15) present in various mammalian tissues. First, we purified peptidylarginine deiminase from rat skeletal muscle. It gave a single band of molecular weight 83 000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Next we immunized rabbits with the purified enzyme. The resulting antibodies reacted specifically with the antigen in Western blot assay. Most of the enzyme activities present in rat skeletal muscle, brain, spinal cord, submaxillary gland and spleen could be characterized as the same muscle-type enzyme by immunoprecipitation and Western blot assay. The antibodies did not react with enzyme samples obtained from rat hair follicles and bovine epidermis. The lack of immunoreactivity of the epidermal enzyme could not be accounted for by the species difference, since the antibodies reacted with a 83 kDa polypeptide of bovine brain, which was thought to represent a bovine counterpart of the muscle-type enzyme. The epidermal enzyme could be distinguished from the other enzyme samples by its high activity towards benzoylarginine. These data suggest the existence of the least three types of peptidylarginine deiminase in mammalian tissues, i.e., a muscle type, a hair follicle type, and an epidermal type.

Immunocytochemical identification of bovine Langerhans cells by use of a monoclonal antibody directed against class II MHC antigens.

We undertook a study to develop a reliable light microscopic technique for identifying Langerhans cells (LC) in bovine epidermis. Monoclonal antibodies (MCA) detecting bovine class II MHC antigens were used in conjunction with an avidin-biotin-peroxidase complex (ABC) immunocytochemical staining method. The specificity of the MCA for LC was confirmed ultrastructurally by use of gold-labeled second antibody. Epidermal sheets and epidermal single-cell suspensions examined by light microscopy confirm that bovine epidermal LC express class II antigens. Anti-bovine class II MCA is a dependable reagent for identification of LC in normal bovine epidermis.

Identification of New Components of the Cornified Envelope of Human and Bovine Epidermis

Antibodies raised in rabbits to purified cornified envelopes (CEs) of cultured human keratinocytes reacted in a peripheral fashion with the granular and spinous layers of human, cow, rat, and mouse epidermis. This reaction could not be abolished by absorption of the antibody with purified human involucrin to which the antibody reacted by immunoblot, thus indicating the presence of an additional antigenic determinant(s). Antibodies raised to CEs of human epidermis stained the cytoplasm of epidermal cells and gave a strong reaction to cytokeratins and a weak one to involucrin, indicating that in tissue the keratins are also cross-linked. The antibody prepared to bovine CEs reacted with keratins, but when absorbed with prekeratin it gave a peripheral staining pattern with epidermis and reacted strongly with a 126 kD component of the neutral buffer extract of cow snout epidermis and weakly with 205 kD and 85 kD ones. This antibody reacted with human involucrin by immunoblot while an antibody to involucrin stained 143 kD, 119 kD, 113 kD, and 107 kD polypeptides in the bovine extract. These latter 3 bands were shown to be substrates of transglutaminase. Further, a monoclonal antibody to bovine CEs reacted with the 119 kD and 113 kD bands and gave a peripheral staining pattern in the epidermis. Proof that the 126 kD protein was a precursor of the envelope was obtained by preparing an antibody to it and demonstrating peripheral staining of epidermal cells. These results point out the value of preparing antibodies to CE as an additional approach to studying the composition of CEs and demonstrate previously undescribed components in human and bovine tissue.

Dissection of the bovine epidermal desmosome into cytoplasmic protein and membrane glycoprotein domains.

Epidermal desmosomes contain two main regions. The core consists of a pair of membranes, one on either side of a cross-striated intercellular space bisected by a denser midline. The cytoplasmic compartment comprises a dense plaque deposited on the cytoplasmic surface of each membrane and a diffuse layer occupying the zone between the plaque and attached alpha-keratin filaments. Analysis of isolated desmosomes by SDS-PAGE has shown the presence of four major protein (dpl-4) and three major glycoprotein (dgl-3) bands, which have been allocated to the cytoplasmic and core compartments, respectively. In the present paper, we report the use of urea to fractionate this complex structure, both in situ and following isolation with citrate buffer, pH2.6. Extraction of the living layers of bovine epidermis with 9M-urea, pH7.5, resulted in rapid removal of the dense desmosomal plaques, followed by separation and vesiculation of desmosomal membranes. The resistance of the plaque to urea increased abruptly at the transition between living epidermis and dead, dehydrated horny layer. A similar sequence of morphological changes accompanied the extraction of isolated desmosomes with urea. Analysis of residues and extracts of isolated desmosomes by SDS-PAGE confirmed the selectivity of 9 M-urea, pH7.5, for the cytoplasmic compartment. The four major desmosomal proteins, dpl-4 (Mr240, 215, 90 and 83 (X 10(3)), respectively) predominated in the extracts. Desmosomal membranes, both paired and vesiculated, consisted almost entirely of the three desmosomal glycoproteins dgl-3 (Mr150, 120 and 110 (X 10(3)), respectively). These results provide evidence that all three desmosomal glycoproteins are integral membrane proteins. The separation of desmosomal membranes by urea, which is not accompanied by additional loss of proteins, further suggests that desmosomal adhesion is based on interactions between membrane components with no separate extracellular molecules being involved. The dissection of the desmosome by urea into two topographically and biochemically distinct domains should facilitate further studies on the molecular basis of desmosomal adhesion and alpha-keratin filament binding.

Purification of desmoglein II: a method for the preparation and f fractionation of desmosomal components

We have developed rapid and efficient methods for the isolation of desmosomes and the fractionation of their components. These methods involve the use of 6 M guanidine HCl to isolate the desmosomes from bovine epidermis, followed by hydroxyapatite column chromatography in the presence of SDS to fractionate the desmosomal components. All of the desmoplakins and desmogleins were purified at least partially by these procedures, and desmoglein II was purified to apparent homogeneity. We expect these procedures to facilitate a detailed biochemical analysis of the molecular components of desmosomes. In addition, these methods may be applicable to the purification of other plasma membrane domains involved in cell adhesion.

The Occurrence and Immunology of Peptidylarginine Deiminase and Its Preparation from Bovine Epidermis by an Improved Method

Peptidylarginine deiminase activity has been found in some tissues from at least one representative of each vertebrate class, suggesting that the occurrence of the enzyme throughout the vertebrates is widespread. Using a three-step procedure including affinity chromatography on arginine agarose, a greatly improved purification of bovine epidermal peptidylarginine deiminase is presented. The purified enzyme preparation contains a 70-75 kD band and several minor components when examined by sodium dodecyl sulfate electrophoresis. A polyclonal antibody raised to the enzyme of bovine brain cross-reacts with human and newborn rat epidermis by indirect immunofluorescence. This cross-reactivity is markedly diminished by absorption of the antibody to the purified brain enzyme.

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology. We compare two polypeptides of the acidic cytokeratin subfamily, VIb ( M r 54,000) and VII ( M r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG S GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends. The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG S GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG S GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.

Lipid composition of the bovine epidermis

In contrast to skin surface or sebum lipid, cattle epidermis was found to contain a lower proportion of wax ester and unesterified fatty acid and a high proportion of triglyceride. The epidermal triglycerides differed significantly in their fatty acid composition from those of the skin surface and sebum. In particular they contained extremely low levels of linoleic acid. It is concluded that the skin surface triglycerides and most likely the other major lipid fractions of the bovine skin surface are derived mainly from sebum.

Significance of two desmosome plaque-associated polypeptides of molecular weights 75 000 and 83 000.

Isolated desmosomes from bovine epidermis contain two major polypeptides of mol. wts. 75 000 (D6) and 83 000 (D5) which, like the desmoplakins of mol. wt. greater than 200 000, are associated with the insoluble desmosomal plaque structure. We have characterized these two polypeptides and examined their significance by peptide map comparisons and translation of bovine epidermal mRNA in vitro. Polypeptide D5 is different from polypeptide D6 by its apparent mol. wt., its isoelectric pH (approximately 6.35, whereas D6 is a basic polypeptide isoelectric at pH approximately 8.5) and its peptide map. By all these criteria desmosomal polypeptides D5 and D6 are also different from cytokeratins, desmoplakins and the glycosylated desmosomal proteins. Both polypeptides are synthesized from different mRNAs separable by gel electrophoresis on agarose: mRNA coding for polypeptide D5 is approximately 3500 nucleotides long, that for D6 is significantly shorter (estimated to 3050 nucleotides), and both contain relatively large proportions of non-coding sequences. The translational products of these mRNAs co-migrate, on two-dimensional gel electrophoresis, with the specific polypeptides from bovine epidermis, indicating that they are genuine polypeptides and are not the result of considerable post-translational processing or modification of precursor molecules. The cell and tissue distribution of these two cytoskeletal proteins and possible functions are discussed.

Fibrous Proteins of Bovine Hoof

The matrix region of calf hoof was identified as the living precursor layer of the hardened hoof plate. Fibrous protein was isolated from the matrix with citrate buffer, pH. 2.65, while Tris buffer, pH. 9.5, with 8 M urea and a reducing agent was required to dissolve the cornified hoof. The purified matrix protein had a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern similar to hoof plate and different from bovine epidermis. When the s-carboxymethyl derivatives of matrix, hoof plate, and hair proteins was compared by urea-polyacrylamide gel electrophoresis, they were identical, and different from that of epidermal protein. The matrix protein reacted with an antibody to hair fibrous protein. Cultured matrix keratinocytes appeared to be identical to cultured epidermal cells, pointing to the importance of the dermis in epidermal cell differentiation.

Desmosomal Antigens Are Not Recognized by the Majority of Pemphigus Autoimmune Sera

Sera from 7 patients with pemphigus vulgaris and both mouse and rabbit antisera against bovine epidermal desmosomes contained antibodies that bound to cell surface components of the spinous layer of bovine epidermis. The antidesmosomal sera showed significant binding to purified desmosomal proteins in an enzyme-linked immunosorbent assay (ELISA). Two of 7 pemphigus sera bound to desmosomal protein-coated microtiter plates at low dilution titers. Two of 6 normal human sera also bound to desmosomal protein-coated microtiter plates at titers comparable to those of the pemphigus sera. Indirect immunofluorescent labeling of frozen sections of monkey esophagus revealed striking differences in the distribution of pemphigus antigens and desmosomal constituents. Pemphigus antisera produced rather uniform fluorescence around the borders of spinous cells of the esophageal epithelium, while anti-desmosomal antibodies bound in a punctate pattern. Anti-desmosomal antibodies labeled cells of the basal layer in a strongly punctate pattern. Only 1 pemphigus serum appreciably labeled basal cells. Two of 3 anti-desmosomal antisera bound avidly in the upper differentiating layers of the epithelium. Pemphigus antibodies did not. Pemphigus sera that reacted with desmosomal proteins in ELISA were absorbed by affinity chromatography on immobilized desmosomal proteins. This treatment did not alter the immunofluorescent labeling patterns produced by these sera. From these results we conclude that the pemphigus autoantibodies studied here bind to epithelial cell surface antigens which are distinguishable from the structural components of desmosomes.

The role of cross-linking in epidermal differentiation.

Cultured bovine keratinocytes contain two major classes of transglutaminase substrate proteins. These may be separated on Sephadex G-75 into a 30,000-to-50,000-dalton fraction and a fraction which is heavier than 75,000 daltons. By SDS-electrophoresis, the lighter fraction consists of two components of molecular weights 9,000 and 18,000 daltons, while the heavier fraction consists of a doublet of about 100,000 daltons. Bovine epidermis contains substrate proteins which apparently correspond to the two components of the lighter class. The 2 classes of substrate proteins may be analogous to two substrate proteins isolated from cultured human keratinocytes which are thought to be precursors of cornified envelope. The major transglutaminase substrate present in both newborn rat epidermis and cultured keratinocytes has a native molecular weight of greater than 75,000 daltons but an SDS-dissociated molecular weight of 22,000 daltons. The sera of 5 of 6 rabbits react immunologically with a protein present in cultured human keratinocytes which is a poor or minor substrate of transglutaminase. The cross-linking of keratins may play a role in epidermal differentiation since keratins from more differentiated areas of epidermis may contain a higher level of interchain isopeptide bonds. Keratin may be cross-linked itself or to stratum corneum basic protein, in vitro, to form both soluble and insoluble aggregates. No adducts between keratin and purified cornified envelope were formed when both were treated with active epidermal transglutaminase.