Characterization of keratocalmin, a calmodulin-binding protein from human epidermis.

Using affinity-purified calmodulin-binding proteins from human epidermis we have developed a monoclonal IgM antibody, ROC 129.1, to a human desmosomal calcmodulin-binding protein. This antibody reacts with a submembranous 250-kD protein from human keratinocytes and stains human epidermis in a "cell-surface pattern". Permeability studies indicated that the epitope with which this monoclonal reacts is on the inner surface of the cell membrane. Immunoelectronmicroscopy localized the antigen to the desmosome. The epitope is restricted to stratified squamous epithelia and arises between 8-12 wk of fetal development. This desmosomal calmodulin-binding protein, which we have termed keratocalmin, may be involved in the calcium-regulated assembly of desmosomes.

Modulation of epidermal growth factor receptors by retinoic acid in ME180 cells.

Retinoic acid (RA) increases epidermal growth factor (EGF) receptors in many cells; in ME180 cells, a human epidermoid carcinoma, RA resulted in a dose- and time-dependent reduction of EGF binding. In RA-treated ME180 cells, binding was 41% of the control. The reduction of EGF binding was due to a decrease in the number of receptors, from 8.7 x 10(4) to 3.6 x 10(4) per cell. The difference was present 8 h after the addition of RA and was reversible 3 days after its removal. Scatchard analysis indicated that RA did not change the binding affinity of EGF (Kd = 1 nM). Also, RA did not alter the rate of EGF internalization or the down-regulation induced by exogenous EGF. Flow-cytometric analysis revealed that RA did not alter the cell cycle. Soluble cell membrane extracts were prepared in a Tris buffer with protease inhibitors, immunoprecipitated, electrophoresed, and immunoblotted with an antiserum to EGF receptors. The EGF receptor band of Mr 170,000 was decreased in RA-treated cells. These results suggest that RA reduces the synthesis of EGF receptors in ME180 cells.

Endogenous substrates for epidermal transglutaminase.

Potential in vivo substrates for epidermal transglutaminase have been isolated and partially characterized in human stratum corneum and new born rat epidermis. [14C]Putrescine and dansylcadaverine were incorporated into epidermal proteins in vitro. Two high molecular weight proteins incorporated the labels in both the rat ahd human homogenates. One of the proteins was too large to enter a 4% sodium dodecyl sulfate-polyacrylamide spacer gel; the other was seen at the interface between the spacer gel and a 10% sodium dodecyl sulphate-polyacrylamide running gel. These proteins were present in a buffer extract, sodium dodecyl sulphate-dithiothreitol extract and NaOH extract. The labels were also incorporated into protein in the insoluble pellet remaining after the afore-mentioned extractions. The incorporation of putrescine and dansylcadaverine was time dependent, and was inhibited by known inhibitors of epidermal transglutaminase. The two high molecular weight proteins had similar amino acid composition, characterized by high glycine, glutamic acid, serine and aspartic acid. The amino acid composition was similar to, although not identical with, the amino acid composition of alpha-keratin proteins. Epidermal homogenates incubated in the presence of transglutaminase showed progressive insolubilization of the protein. This cross-linking was inhibited by putrescine. [14C]Glycine, [14C]histidine and [4C]proline were incorporated into epidermal proteins in newborn rats in vivo. The glycine-labelled protein became progressively more insoluble when incubated in vitro in the presence of transglutaminase. In vitro incubation with transglutaminase had no effect on the histidine-and proline-labelled proteins.

Skin sulfhydryl oxidase. Purification and some properties.

A sulfhydryl-oxidizing enzyme has been found in skin of young rats and a method for purifying the enzyme over 600-fold has been developed. Enzymatic activity was assayed either by its ability to oxidize dithiothreitol of by measuring its ability to renature reductively denatured ribonuclease A. Skin sulfhydryl oxidase catalyzed the oxidation of various thiols: dithiothreitol, dithioerythritol, D-penicillamine, and L-cysteine. Glutathione and 2-mercaptoethanol were very poor substrates for the enzyme. The enzyme also reactivated reductively denatured ribonuclease A, with neither the presence of a thiol nor prior reduction of the enzyme being necessary. The molecular weight of the enzyme was estimated to be 66 000 +/- 2000, and the isoelectric point was determined to be at pH 4.65. Alkylating reagents alone had some inhibiting effect on skin sulfhydryl oxidase; when the enzyme was preincubated with thiols which were substrates, inhibition by alkylating reagents was greatly increased. After preincubation with dithiothreitol, treatment of the enzyme with alkylating reagents or N-ethylmaleimide caused significant inhibition; preincubation with a poor substrate, reduced glutathione, did not enhance inhibition by alkylating reagents or N-ethylmaleimide.

The polypeptide composition of epidermal prekeratin.

An alpha-fibrous protein, prekeratin, has been isolated from cow snout epidermis with citrate buffer, pH 2.65. Using acrylamide electrophoresis with 0.1% sodium dodecyl sulfate, prekeratin can be shown to contain three polypeptide chains of different molecular weights. The two faster migrating components are very similar with a mol. wt of about 47,000 while the slower one has a mol. wt of about 58,000. Chromatography on a number of molecular sieve and exchange resins does not separate the components, but use of Sepharose 2B with 0.1 M Tris, pH 9.0, containing 10% propanol gives two peaks of protein. The first and major peak contains all three components while the second has only the two with the faster mobility. The two more rapidly migrating components and the slower one were isolated by acrylamide electrophoresis, and the latter has an amino acid composition more compatible with a non-helical protein. Enzymatic digestion with tosyl-L-phenylalanine chloromethylketone-treated (TPCK-)trypsin shows that the component of mol. wt 58,000 is more susceptible to hydrolysis than the other two. These data suggest that prekeratin is not homogenous in composition and consists of several interacting polypeptide chains. One of these components would appear to be non-helical in structure.

Mutations of the gene encoding the transmembrane transporter protein ABC-C6 cause pseudoxanthoma elasticum.

We recently published the precise chromosomal localization on chromosome 16p13.1 of the genetic defect underlying pseudoxanthoma elasticum (PXE), an inherited disorder characterized by progressive calcification of elastic fibers in skin, eye, and the cardiovascular system. Here we report the identification of mutations in the gene encoding the transmembrane transporter protein, ABC-C6 (also known as MRP-6), one of the four genes located in the region of linkage, as cause of the disease. Sequence analysis in four independent consanguineous families from Switzerland, Mexico, and South Africa and in one non-consanguineous family from the United States demonstrated several different mis-sense mutations to cosegregate with the disease phenotype. These findings are consistent with the conclusion that PXE is a recessive disorder that displays allelic heterogeneity, which may explain the considerable phenotypic variance characteristic of the disorder.

A 500-kb region on chromosome 16p13.1 contains the pseudoxanthoma elasticum locus: high-resolution mapping and genomic structure.

We have recently mapped the genetic defect underlying pseudoxanthoma elasticum (PXE), an inherited disorder characterized by progressive calcification of elastic fibers in skin, eye, and cardiovascular system, to chromosome 16p 13.1. Here we report further data on the fine-mapping and genomic structure of this locus. Haplotype analysis of informative PXE families narrowed the locus to an interval of less than 500 kb located between markers D16B9621 and D16S764. Three overlapping YAC clones were found to cover this region through YAC-STS content mapping. An overlapping BAC contig was then constructed to cover this interval and the surrounding region. About 80% of this chromosomal region has been fully sequenced using the BAC shotgun technique. Gene content and sequence analysis predicted four genes (MRP1, MRP6, PM5, and a novel transcript) and two pseudogenes (ARA and PKDI) within this interval. By screening a somatic cell hybrid panel we were able to precision-map the breakpoint of Cy185 and the starting point of a chromosomal duplication within 20 kb of BAC A962B4. The present data further refine the localization of PXE, provide additional physical cloning resources, and will aid in the eventual identification of the genetic defect causing PXE.

Human epidermal transglutaminase.

Epidermal transglutaminase and its structural consequences were first described by studies of the Massachusetts General Hospital dermatology research laboratories in the early 1970s. The enzyme catalyzes an irreversible and necessary step in epidermal terminal differentiation. These features of the process catalyzed by the enzyme have generated great interest in the control mechanisms for enzyme activity. Like all transglutaminases, the human epidermal enzyme has strict requirements for calcium (or strontium) and for a free sulfhydryl group. It is similar to liver transglutaminase in not requiring proteolytic activation; plasma transglutaminase (factor XIII) requires proteolytic activation. Antibodies produced to human epidermal transglutaminases showed high species specificity and no cross-reaction with the human hair follicle transglutaminase. Purified human epidermal transglutaminase has several-fold increases in its activity after treatment with organic solvents, including dimethylsulfoxide, heating in the presence of calcium, and treatment with chaotropic reagents, such as NaSCN or Nal. The enzyme with enhanced activity has altered gel-filtration characteristics, although it exhibits no major molecular weight changes by SDS-electrophoresis or major immunologic differences with the conventional antibodies for human epidermal transglutaminases. A series of monoclonal antibodies to human epidermal transglutaminase is being prepared to allow detailed analysis of its structural activation and detection of common antigenic sites among transglutaminase that may be masked or not present in conventional antibodies to the enzyme. The ability of solvents, simple chemicals, and drugs to alter the function of transglutaminase allows one to consider safe methods for in vivo modulation of the enzyme and consequent modulation of altered function in human epidermal diseases.

Human epidermal transglutaminase.

Epidermal transglutaminase and its structural consequences were first described by studies of the Massachusetts General Hospital dermatology research laboratories in the early 1970s. The enzyme catalyzes an irreversible and necessary step in epidermal terminal differentiation. These features of the process catalyzed by the enzyme have generated great interest in the control mechanisms for enzyme activity. Like all transglutaminases, the human epidermal enzyme has strict requirements for calcium (or strontium) and for a free sulfhydryl group. It is similar to liver transglutaminase in not requiring proteolytic activation; plasma transglutaminase (factor XIII) requires proteolytic activation. Antibodies produced to human epidermal transglutaminases showed high species specificity and no cross-reaction with the human hair follicle transglutaminase. Purified human epidermal transglutaminase has several-fold increases in its activity after treatment with organic solvents, including dimethylsulfoxide, heating in the presence of calcium, and treatment with chaotropic reagents, such as NaSCN or NaI. The enzyme with enhanced activity has altered gel-filtration characteristics, although it exhibits no major molecular weight changes by SDS-electrophoresis or major immunologic differences with the conventional antibodies for human epidermal transglutaminases. A series of monoclonal antibodies to human epidermal transglutaminase is being prepared to allow detailed analysis of its structural activation and detection of common antigenic sites among transglutaminase that may be masked or not present in conventional antibodies to the enzyme. The ability of solvents, simple chemicals, and drugs to alter the function of transglutaminase allows one to consider safe methods for in vivo modulation of the enzyme and consequent modulation of altered function in human epidermal diseases.

Homeobox genes and skin development: a review.

Homeobox (HOX) genes are a gene family that encode information critical for the normal embryologic development of many different organisms, including vertebrates. HOX genes encode transcriptional regulatory factors that bind to multiple different genes and thereby determine the developmental fate of a cell. The role of HOX genes in the development of skin is undetermined but, based on information from other organisms and recent experimental data from skin models, it is likely that this class of genes is important for the normal development of skin adnexae, pigmentary system, and stratified epidermis during embryogenesis. The purpose of this review is to briefly summarize what is known about HOX genes and to familiarize the reader with recent insights into how HOX genes may function in skin development.

Monoclonal antibodies to anchoring fibrils for the diagnosis of epidermolysis bullosa.

Murine monoclonal antibodies to human anchoring fibrils reacted with human and monkey cervix, tongue, esophagus, and vagina. Rat, mouse, and guinea pig tissues were negative. In 11 patients with dystrophic recessive epidermolysis bullosa there was no reaction by immunofluorescence and immunoelectron microscopy. Other forms of epidermolysis bullosa had normal reactivity.

Antizyme release is an early event in ornithine decarboxylase induction by hair plucking.

Plucking of hair from the dorsal skin of rats resulted in a rapid decrease in ornithine decarboxylase (ODC) activity. A significant loss of activity did not occur in other skin enzymes under the same conditions and in vivo incorporation of [3H]-leucine in skin was not significantly decreased 60 min immediately following hair plucking. Treatment of ODC enzyme preparations with 10% (NH4)2SO4 resulted in recovery of approximately 75% greater ODC activity than in untreated samples, suggesting the presence of an inhibitor (antizyme). ODC inhibitor was detected in plucked skin; inhibitor levels increased after treatment of plucked skin extracts with 10% (NH4)2SO4.

Ornithine decarboxylase in rat skin.

Ornithine decarboxylase (ODC; E.C.4.1.1.17) activities can be stimulated 2-10 fold in rat epidermis and dermis by hair plucking. Stimulation does not involve the removal of a soluble ODC inhibitor. ODC activity in the dermis and whole skin decreased with aging, while the epidermis showed little change. The apparent Km for ornithine and the heat stability of ODC in plucked and unplucked skin were similar. ODC was assayed in plucked and unplucked skin of rats fed diets containing between 2 and 24% protein. Activities in both plucked and unplucked skin were higher in the animals fed diets with higher protein contents. ODC levels were positively correlated with the weight changes undergone by rats on controlled-protein diets. In animals restricted to 2% protein diets and rehabilitated with 16% protein diets, enzyme levels were increased after 2 days rehabilitation and peaked after 5 days rehabilitation. The responsiveness of ODC to changes in dietary protein may be useful in the diagnosis of protein malnutrition.

Human epidermal transamidase.

The possible presence of epsilon-(gamma-glutamyl) lysine covalent bonds in human epidermal proteins prompted a study of transamidase activity in human hair-free epidermis. Callus contains an enzyme which catalyzes the incorporation of radioactive putrescine into alpha-casein. The enzyme is active without prior treatment with exogenous proteolytic enzymes. The putrescine incorporation is calcium dependent and inhibited by iodoacetamide. The enzyme was partially purified (50-fold over starting material), and has an apparent molecular weight between 50,000 daltons and 55,000 daltons by agarose 0.5m gel filtration. The apparent molecular weight is unaltered by chromatography in the presence of 11 mMCaCl2, a condition known to dissociate plasma transglutaminase (Factor XIII) into its ultimate subunits. The enzyme is active over a wide pH range up to pH 10.4 The Km for putrescine varies by 1-fold over the pH range 6.0 to 10.2, although enzyme activity increases at least 20-fold over the same pH range. The human epidermal transamidase is similar to the guinea-pig hair follicle transglutaminase and cow snout transamidase in its ability to cross-link fibrin.

Human epidermal transglutaminase. II. Immunologic properties.

A monospecific antibody for human epidermal transglutaminase was prepared in rabbits. The antibody formed single immunoprecipitin lines with purified or crude human transglutaminases and quantitatively precipitated transglutaminase activity. There were no precipitin reactions between human factor XIII (zymogen or active enzyme) and antihuman epidermal transglutaminase or between human epidermal transglutaminase and antihuman factor XIII. Heating epidermal transglutaminase (56 degrees C, 15 min) in the presence of calcium increased the enzyme activity up to 10 times baseline levels. The heat-activated human epidermal transglutaminase was identical by immunodiffusion with the native enzyme, although slightly higher precipitation titers were detected following heating. There was no cross-reaction of antihuman epidermal transglutaminase with frog, rat, mouse, chicken, or human hair follicle transglutaminases.

Serine biosynthesis in human hair follicles by the phosphorylated pathway: follicular 3-phosphoglycerate dehydrogenase.

The phosphorylated pathway of serine biosynthesis was demonstrated in human hair bulbs and sheaths by the formation of phosphoserine and serine from (14C)3-phosphoglyceric acid. The initial and rate limiting enzyme of the pathway, 3-phosphoglycerate dehydrogenase (3-PGDH) was demonstrated by enzyme determinations in human and rat hair follicles, human epidermis, and chicken epidermis. Follicular 3-PGDH was characterized using a sensitive fluorometric assay with NADH as a co-substrate. Monovalent cations (Na+, K+, Li+, or NH4+) were necessary for full enzyme activity. p-Hydroxymercuribenzoate inhibited activity, and activity was 3 times higher with NADH as a co-substrate than with NADPH. The apparent Km for the substrate hydroxyphosphopyruvic acid was 32.8 muM, and the apparent Km for NADH 4.8 muM similar to the Kms for other mammalian 3-PGDHs. Enzyme activity was not altered by parenteral corticosteroids, a high carbohydrate diet, low protein diet, or starvation. Enzyme activity decreased over the first 12 days of life in newborn rats. The phosphorylated pathway of serine synthesis provides a potential nondietary and nonhepatic source of serine, glycine, and their products in keratinizing tissues.

Hepatic enzymes of tyrosine metabolism in tyrosinemia II.

A middle-aged adult male with a mild form of tyrosinemia II (Richner-Hanhart syndrome) is described. Treatment with a low-tyrosine diet caused a fall in plasma tyrosine and clearing of the hyperkeratosis of the soles. Liver biopsy of this patient revealed low but measurable levels of cytoplasmic tyrosine aminotransferase and elevated levels of the mitochondrial tyrosine-metabolizing enzyme aspartate aminotransferase. It is hypothesized that these enzymes have been induced in sufficient amounts to account for the mild clinical course.