Evidence that major 78-44-kD concanavalin A-binding glycopolypeptides in pig epidermis arise from the degradation of desmosomal glycoproteins during terminal differentiation.

The major concanavalin A (Con A)-binding component in urea/deoxycholate/mercaptoethanol extracts from pig ear epidermis had an apparent Mr of 78 kD. In indirect immunofluorescence affinity-purified polyclonal antibodies against this glycopolypeptide strongly stained the surface of suprabasal cells in the epidermis of pig and human skin. Immunocytochemical labeling with gold-labeled second antibody localized this staining to externally disposed, trypsin-sensitive components of desmosomes. Western blotting showed that the 78-kD glycopolypeptide was immunologically related to several other Con A-binding components in pig epidermis. Immunoreactive components with Mr of 115 and 100 kD were membrane-bound, appeared to be susceptible to trypsin in intact epidermis, and were absent from the stratum corneum. Immunoreactive components of lower Mr (78-44 kD) were not membrane-bound, were resistant to trypsin in intact tissue, and were present predominantly in the keratinized layers of pig epidermis. The 115-44-kD glycopolypeptides were also recognized by antisera raised against desmoglein II/desmocollin glycoproteins isolated from bovine spinous layer desmosomes. In addition, these antisera reacted with 120- and 105-kD bands that were apparently not recognized by the anti-78-kD glycopolypeptide antiserum in immunoblotting. In immune precipitation the anti-78-kD glycopolypeptide and antidesmoglein II/desmocollin antisera precipitated comparable amounts of the radioiodinated 78-44-kD components. Both antisera also precipitated the 120- and 105-kD components although the anti-78-kD glycopolypeptide serum was less effective. Little reaction with the 115- and 105-kD components was observed in immune precipitation with either serum. Proteolytic peptide mapping confirmed that the various immunoreactive glycopolypeptides were biochemically as well as immunologically related. The results suggest that terminal differentiation in pig epidermis is accompanied by the orderly degradation of desmoglein II/desmocollin glycoproteins resulting in the accumulation of 78-44-kD glycopolypeptides in the stratum corneum. These glycopolypeptides may represent functionally important nonmembranous domains of cell-adhesion molecules in desmosomes.

Characterization of epidermal glycosaminoglycans synthesized in organ culture.

1. Cellulose acetate electrophoresis together with specific enzymic and chemical degradation procedures indicated that hyaluronic acid (83%) and heparan sulphate (14%) were the major glycosaminoglycans synthesized by the epidermis when pig ear skin slices were cultured in the presence of D-[3H]-glucosamine and 35SO4 2-. 81% and 50%, respectively, of the total amount of each epidermal glycosaminoglycan was extracellular. 2. Total epidermal glycosaminoglycan synthesis decreased by 50% after 5 days in culture. 3. When the epidermis was cultured in the absence of the dermis the synthesis of hyaluronic acid was considerably reduced. The synthesis of sulphated glycosaminoglycans was essentially unaffected by the absence of the dermis. 4. 10(-5) M all-trans-retinoic acid stimulated the synthesis of hyaluronic acid and, to a lesser extent, of sulphated glycosaminoglycans whether the dermis was absent or present during culture. 5. The results suggest that hyaluronic acid may play an important role in some aspects of epidermal differentiation.

The dermis is required for the synthesis of extracellular glycosaminoglycans in cultured pig epidermis.

1. Slices of pig ear skin were cultured in the presence of D-[3H]glucosamine and the epidermis solubilised in 8 M urea/5% sodium dodecyl sulphate was analysed by polyacrylamide gel electrophoresis. A high molecular weight peak, previously shown to contain glycosaminoglycans, was a major labelled component of epidermis separated from dermis using either CaCl2, dispase or trypsin. This material was unlikely to represent dermal contamination of the epidermis since (a) it was present mainly in epidermis rather than dermis, and (b) both histology and comparison of protein compositions showed the epidermis to be essentially free of dermal components. 2. When the epidermis was separated from dermis before rather than after culture (using CaCl2, dispase, trypsin or suction) the labelled glycosaminoglycan peak was never observed. The labelling of other epidermal glycoconjugates was unaffected. Thus the dermis was necessary specifically for the synthesis of epidermal glycosaminoglycans. 3. All-trans-retinoic acid (1 x 10(-5) M) had a marked effect on the labelling of the epidermal but not the dermal glycosaminoglycan peak, indicating that the epidermal glycosaminoglycans were not synthesised in the dermis. 4. The results suggest that dermal influences in the epidermis could be mediated via dermal control of epidermal glycosaminoglycan synthesis.

Synthesis of cellular and extracellular glycoproteins by cultured human keratinocytes and their response to retinoids.

The glycoproteins synthesized by human keratinocytes cultured on 3T3 feeder layers were studied by metabolic labelling. Keratinocytes freed of feeder cells synthesized a complex pattern of cellular and extracellular glycoproteins that was distinct from that of 3T3 cells, dermal fibroblasts and epidermal melanocytes. The effect of low concentrations of all-trans-retinoic acid and arotinoid ethyl ester on glycoprotein synthesis was examined in keratinocyte cultures depleted of vitamin A. Treatment with either retinoid resulted in a 2-3-fold increase in the amount of D-[3H]glucosamine-labelled material in the culture medium. Gel electrophoresis revealed increased incorporation of D-[3H]glucosamine into extracellular glycoproteins of Mr 245,000, 170,000, 140,000, 130,000, 120,000 and 105,000 as well as into glycosaminoglycans in retinoid-treated cultures. The labelling of extracellular glycoproteins with L-[3H]leucine and L-[35S]methionine was also increased by retinoids suggesting increased synthesis of these components rather than an effect on their glycosylation. The Mr 245 000 glycoprotein was identified as keratinocyte-derived fibronectin by immunoblotting, immunoprecipitation and specific binding to gelatin. The results show that retinoids increase the synthesis of glycoprotein as well as glycosaminoglycan components of the extracellular matrix in human keratinocyte cultures. It is suggested that retinoids select for a population of cells that synthesize relatively large amounts of glycosaminoglycan, fibronectin and other as yet unidentified extracellular glycoproteins.

Antisense expression of a desmocollin gene in MDCK cells alters desmosome plaque assembly but does not affect desmoglein expression.

The desmocollins are one of two types of putative adhesive proteins present in the desmosome type of cell junctions, the other type being the desmogleins; both are members of the cadherin superfamily. Each type of desmosomal cadherin occurs as a number of isoforms which have differing tissue distribution; within stratifying epithelia some isoforms occur only suprabasally. We have sought to analyse desmocollin function by reducing the amount of protein using antisense gene expression in the widely studied Madin-Darby canine kidney (MDCK) cell line. Although this is a simple epithelial cell line, we show by Northern blot analysis that it expresses multiple isoforms of the desmosomal cadherins. Desmocollins DSC2 and DSC3 and desmogleins DSG2 and DSG3 (the pemphigus vulgaris antigen PVA) were detected, but DSC1 and DSG1, which are present exclusively in the suprabasal layers of the epidermis, were absent. The major desmocollin isoform was the type 2 (DSC2). A DSC2 clone isolated from a MDCK cDNA library had the same cell adhesion recognition sequence (Phe-Ala-Thr) as human, bovine and mouse type 2 isoforms. This sequence appears diagnostic for the three desmocollin isoforms. This cDNA clone was used to isolate a genomic DSC2 clone; antisense expression of this clone in MDCK cells resulted in a drastic reduction of desmocollin protein as judged by Western blots; Dsc3 was not upregulated to compensate for the loss of Dsc2. This antisense expression significantly altered desmosome assembly. There was a loss of punctate staining evident when using a desmosome plaque protein (desmoplakin) antibody. Electron microscopy revealed that there was a reduction in the number of desmosomes and a notable increase in the asymmetry of plaques between adjacent cells. Immunolabelling showed that similar levels of desmogleins and E-cadherin were present. Immunoelectron microscopy also showed that many vesicular structures were labelled, at intervals along the lateral membranes between cells. The distinctive loose organization of the remaining desmosomes may originate in modifications to the targeting and incorporation of proteins into fully assembled plaques. Other junctions were unaffected and the cells maintained their integrity as a confluent monolayer.

Electrophoretic and immunoelectrophoretic analysis of pig epidermal plasma membrane glycoproteins.

he glycoprotein components of a plasma membrane-enriched fraction from pig epidermis were isolated by deoxycholate extraction and affinity chromatography on concanavalin A (ConA)-Sepharose 4B. Reduction with 5% 2-mercaptoethanol, electrophoresis on 10% polyacrylamide slab gels, and periodic acid-Schiff (PAS) staining resolved the major glycoproteins into at least 5 components of Mr 180K, 150K, 130K, 100K and 85K. Neuraminidase removed essentially all the sialic acid whether or not the glycoproteins were solubilized with detergents. Neuraminidase treatment increased the electrophoretic mobility of most components on one-dimension polyacrylamide gels, indicating their sialoglycoprotein nature. An antiserum was raised in rabbits against isolated epidermal plasma membrane glycoproteins. Isolated immunoglobulins were used in crossed immunoelectrophoretic analysis of the glycoproteins and produced 5 major immunoprecipitates. The glycoprotein nature of the immunoprecipitates was shown by their susceptibility to neuraminidase. Crossed immunoelectrophoresis was used to examine the lectin binding specificity of isolated epidermal plasma membrane glycoproteins. The immunoprecipitation patterns were affected strongly by Ricinus communis agglutinin (RCA), moderately by wheatgerm agglutinin (WGA), and weakly by soybean agglutinin (SBA). Peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), and Ulex europaeus agglutinin (UEA) had little effect on the immunoprecipitation patterns, indicating little interaction between epidermal plasma membrane glycoprotein and these lectins. Other glycoproteins and/or glycolipids must therefore be responsible for the binding of these lectins by epidermal cells.

Desmoglein II-derived glycopeptides in human epidermis.

Antisera raised against a major 78 kD glycopeptide from pig epidermis were used to identify desmoglein II-derived glycopeptides in the conA-binding material isolated from human epidermis. In whole CaCl2-separated epidermis the antiserum recognized conA-binding components with apparent Mr of 115, 100, 82, 68, 50, 48, and 46 kD. The 82, 68, 48, and 46 kD immunoreactive bands were present in normal stratum corneum and plantar callus. Psoriatic scales contained significantly more of the 82 kD components and less of the 48 and 46 kD bands. Psoriatic scales also contained a major 50 kD conA-binding component unrelated to keratins or desmoglein II. Proteolytic peptide mapping showed that the major immunoreactive bands in normal stratum corneum and plantar callus were also chemically related. The 82 to 46 kD immunoreactive glycopeptides in plantar callus coincided with the major coomassie blue stained bands and were homogeneous on two-dimensional gels suggesting that this tissue may be a valuable source of human desmoglein II-derived glycopeptides. An antiserum directed against the electrophoretically co-purified 48/46 kD glycopeptides from plantar callus recognized the 82 to 46 kD bands in immunoblotting. In indirect immunofluorescence of frozen skin sections this antiserum stained the surface of epidermal cells in the spinous and granular layers of the tissue. In immunogold labeling of paraformaldehyde-fixed skin sections affinity-purified antibodies stained intact desmosomes in spinous and granular cells and desmosomal remnants in the stratum corneum. The results are consistent with our hypothesis that desmoglein II undergoes limited cleavage to stable fragments during terminal differentiation. Proteolytic degradation appears to be incomplete in psoriatic epidermis.

Expression of the "skin-type" desmosomal cadherin DSC1 is closely linked to the keratinization of epithelial tissues during mouse development.

Desmosomal junctions contain two classes of desmosomal cadherin, the desmocollins and the desmogleins, each of which occurs as three distinct isoforms. To investigate the role of the "skin-type" desmosomal cadherins (desmocollin 1 and desmoglein 1) in the formation of keratinized epithelial structures, we have now cloned full-length mouse desmocollin 1 complementary deoxyribonucleic acid and examined the expression of desmocollin 1 and desmoglein 1 and messages during murine embryonic development by in situ hybridization. In the general body epidermis, desmocollin 1 and desmoglein 1 transcripts both showed considerable upregulation at 15.5 d, which is after the onset of stratification and before the start of keratinization. Before this the epidermis expressed low levels of desmocollin 1 message, although the desmoglein 1 signal was always stronger and more extensive. In the tongue, expression of desmocollin 1 message occurred several days after desmoglein 1 and coincided with the formation of the keratinizing filiform papillae. Desmoglein 1 message was also detected in epithelial tissues in which desmocollin 1 was absent, suggesting that expression of the two "skin-type" desmosomal cadherins was not tightly coupled during embryonic development. Human desmocollin 1 monoclonal antibodies that cross-reacted with mouse skin and tongue indicated that desmocollin 1 protein was first expressed in those outermost epithelial cells destined to form the keratinized layers of the stratum corneum or the papillae. The results suggest that expression of desmocollin 1 is closely associated with the keratinization of epithelial tissues during mouse development.

The desmocollins of human foreskin epidermis: identification and chromosomal assignment of a third gene and expression patterns of the three isoforms.

A third human desmocollin, designated DSC3, was identified in foreskin epidermis by reverse transcriptase-polymerase chain reaction (PCR) using degenerate desmocollin primers. cDNA clones covering the entire coding sequence of the longer DSC3 splice variant were isolated and sequenced. Sequence comparisons indicated that this new desmocollin showed greater homology (67% amino acid identity) with the original human desmocollin (now designated DSC2) than with DSC1 (52% amino acid identity) although it had a unique potential cell adhesion recognition site (YAS). DSC3 was assigned to chromosome 18 by PCR analysis of rodent-human somatic cell hybrids, where it appears to be closely linked to all the other desmosomal cadherin genes. The expression of the three human desmocollins was examined in foreskin epidermis by in situ hybridization with 3'-untranslated riboprobes and by immunofluorescence with isoform-specific anti-peptide antibodies. DSC1 was present in the upper spinous/granular layers but not in the basal/lower spinous layers of the tissue. DSC2 and DSC3 were present in most of the living layers of the epidermis. DSC1 was not detected in any of the nonkeratinizing human epithelia examined (buccal mucosa, cervix, esophagus), indicating that it is specific for the keratinizing epithelium of the epidermis. However, all these internal epithelia expressed DSC2 and DSC3, and both were present in most of the living layers of the tissues including the basal layers.

Expression of distinct desmocollin isoforms in human epidermis.

Previous evidence suggested the presence of two distinct desmocollin isoforms in human epidermis. These isoforms have now been distinguished at the protein level using monoclonal and polyclonal antibodies against N-terminal fragments of desmosomal glycoprotein (DG) IV/V isolated from plantar callus and antibodies against a fusion protein containing the extracellular domain of DGII/III. Immune blotting of glycoprotein fractions from whole epidermis, plantar callus, psoriatic scales and cultured keratinocytes showed that intact DGIV/V and its proteolytic fragments consistently migrated faster than DGII/III during SDS-PAGE. The apparent Mr difference between the two isoforms was in the range 2-5 kD. DGIV/V was the predominant species in epidermal tissue but was much less prominent in cultured cells by immune-blotting and immune precipitation. This is consistent with the differentiation-related expression of desmocollins revealed by immunofluorescence. DGIV/V was strongly expressed in the upper spinous/granular layer of the epidermis whereas DGII/III was more prominent in the basal layers of the tissue. The DGIV/V monoclonal (LH50) recognized an N-terminal, Ca(++)-sensitive epitope, because its staining of unfixed epidermal tissue was markedly influenced by Ca++ levels. Ca++ inhibition was observed at concentrations as low as 50 microM, suggesting its possible physiologic significance. Ca++ inhibition of LH50 binding was also observed in an enzyme-linked immunosorbent assay system using denatured glycoproteins although higher concentrations were required. It remains to be seen whether direct effects of Ca++ on desmocollin conformation are involved in the regulation of keratinization by extracellular Ca++.

Basal cell glycoprotein in pig epidermis closely resembles the beta 1 subunit of the integrin family of cell adhesion molecules.

A 135-kD conA-binding glycoprotein isolated from pig epidermis was previously localized to the surface of basal cells in stratified epithelia using affinity-purified antibodies. Preembedding immunoperoxidase electron microscopy has now shown that this glycoprotein is concentrated on the lateral surfaces of basal cells but is not detectable on those surfaces adjacent to the basement membrane indicating a role in cell-cell rather than cell-substrate interactions. The basal cell glycoprotein was shown to resemble the beta 1 subunit of the integrin family following the generation of a specific monoclonal antibody (M5.25). The epidermal glycoprotein recognized by M5.25 and by antibodies against the beta 1 fibronectin receptor from human placenta co-migrated on SDS gels under both reducing and non-reducing conditions. Its response to disulphide reducing agents was characteristic of beta 1 integrin subunits. In addition, the basal cell glycoprotein was shown to bind to the 120-kD cell-binding fragment of fibronectin in a RGD-dependent manner. It was readily detected by immunoblotting whole cell lysates of cultured pig keratinocytes suggesting increased expression in cultured cells compared to fresh epithelial tissue. The results suggest that beta 1 integrin subunits may be involved in cell-cell interactions between basal keratinocytes in pig epidermis and that these receptors are lost from the cell surface during terminal differentiation. Thus modulation of beta 1 integrin subunit expression may play an important role in regulating differentiation in pig epidermis.

Incorporation of [3H]glucosamine into keratin-related polypeptides in pig epidermis.

Metabolic labelling studies have provided evidence for glycosylated keratins in cultured pig epidermis. [3H]Glucosamine was incorporated into five major particulate polypeptides of Mr 68 000, 61 000, 57 000, 53,000 and 48,000. Radioactivity was present in protein-bound carbohydrate. Non-enzymic glycation was excluded. Labelling was largely unaffected by tunicamycin indicating that radioactivity was incorporated mainly into O-linked oligosaccharides. These [3H]glucosamine-labelled components were closely related to keratins since they had a similar electrophoretic mobility to polypeptides of purified pig prekeratin, they were immunoprecipitated by anti-prekeratin serum and they were incorporated into reconstituted, intermediate-sized, keratin filaments.

Keratinization is associated with the expression of a new protein related to the desmosomal cadherins DGII/III.

Amino acid sequencing of a 48/46 kDa glycoprotein from human plantar callus, recognised by antisera raised against the desmosomal cadherins DGII/III, has revealed N-terminal homology to the DNA-derived sequence of human and bovine DGII/III. However, a tryptic fragment has homology only with a bovine clone. We propose that there are two classes of DGII/III-like molecule, that represented by the bovine cDNA clone and the 48/46 kDa protein, a monoclonal antibody against which stains mainly the suprabasal layers of human epidermis, and that represented by the human cDNA clone, identified by a monoclonal antibody which stains uniformly the living layers of the epidermis.

Increased epidermal hyaluronic acid synthesis caused by four retinoids.

Epidermal hyaluronic acid synthesis in pig skin is increased by all-trans-retinoic acid, all-trans-retinyl acetate, 13-cis-retinoic acid and etretinate.

Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro.

1. When pig ear skin slices were cultured for 18h in the presence of 1mug of tunicamycin/ml the incorporation of d-[(3)H]glucosamine into the epidermis, solubilized with 8m-urea/5% (w/v) sodium dodecyl sulphate, was inhibited by 45-55%. This degree of inhibition was not increased by using up to 5mug of tunicamycin/ml or by treating the skin slices with tunicamycin for up to 8 days. The incorporation of (U-(14)C)-labelled l-amino acids under these conditions was not affected by tunicamycin. Polyacrylamide-gel electrophoresis indicated that the labelling of the major glycosaminoglycan peak with d-[(3)H]glucosamine was unaffected, whereas that of the faster migrating glycoprotein components was considerably decreased in the presence of tunicamycin. 2. Subcellular fractionation indicated that tunicamycin specifically inhibited the incorporation of d-[(3)H]glucosamine but not of (U-(14)C)-labelled l-amino acids into particulate (mainly plasma-membrane) glycoproteins by about 70%. The labelling of soluble glycoproteins was hardly affected. Polyacrylamide-gel electrophoresis of the plasma-membrane fraction showed decreased d-[(3)H]glucosamine incorporation into all glycoprotein components, indicating that the plasma-membrane glycoproteins contained mainly N-asparagine-linked oligosaccharides. 3. Cellulose acetate electrophoresis of both cellular and extracellular glycosaminoglycans showed that tunicamycin had no significant effect on the synthesis of the major component, hyaluronic acid. However, the incorporation of both d-[(3)H]glucosamine and (35)SO(4) (2-) into sulphated glycosaminoglycans was inhibited by about 50%. This inhibition was partially overcome, at least in the cellular fraction, by 2mm-p-nitrophenyl beta-d-xyloside indicating that tunicamycin-treated epidermis retained the ability to synthesize sulphated glycosaminoglycan chains. Tunicamycin may affect the synthesis and/or degradation of proteoglycan core proteins or the xylosyltransferase. 4. Electron-microscopic examination of epidermis treated with tunicamycin for up to 4 days revealed no significant changes in cell-surface morphology or in epidermal-cell adhesion. Either N-asparagine-linked carbohydrates play little role in epidermal-cell adhesion or more probably there is little turnover of these components in epidermal adhesive structures such as desmosomes and hemidesmosomes during organ culture.

Isolation and characterization of plasma-membrane glycoproteins from pig epidermis.

1. Non-desmosomal plasma membranes enriched in plasma-membrane marker enzymes and in metabolically labelled glycoproteins were isolated on a large scale from up to 500g of pig ear skin slices. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and periodic acid/Schiff staining revealed the presence of four major glycosylated components in the apparent molecular-weight range 150000-80000. 2. A large proportion of the marker enzymes, the d-[(3)H]glucosamine-labelled glycoproteins and the periodic acid/Schiff-stained glycoproteins were solubilized by 1% (w/v) sodium deoxycholate. However, several non-glycosylated proteins, in particular those with mol.wts. 81000, 41000 and 38000 (possibly cytoskeletal components), were relatively resistant to solubilization. 3. The deoxycholate-solubilized membranes were fractionated by lectin affinity chromatography using both concanavalin A-Sepharose 4B and lentil lectin-Sepharose 4B. From 75 to 85% of the applied glycoprotein was recovered from the columns. From 30 to 40% of the recovered glycoprotein was specifically bound by the lectins and was eluted with 2% (w/v) alpha-methyl d-mannoside. The enrichment of labelled glycoproteins in the material bound by the lectins (2.5-fold) was similar with both lectins, although the yield was somewhat greater when lentil lectin was used. The glycoprotein-enriched fraction was also enriched in all the plasma-membrane marker enzymes, indicating their probable glycoprotein nature. 4. The glycoprotein-enriched fraction contained the four major periodic acid/Schiff-stained bands that were detected in the original plasma membrane. They had apparent mol.wts. 147000, 130500, 108000 and 91400. The higher-molecular-weight components contained relatively more d-[(3)H]glucosamine, indicating differences in the sugar composition or in the metabolic turnover of the individual glycoproteins in culture. The material bound by the lectins also contained a number of lower-molecular-weight Coomassie Brilliant Blue-stained components. These were weakly stained by periodic acid/Schiff reagent and were lightly labelled with d-[(3)H]glucosamine, indicating that they contained less carbohydrate than the four major glycoprotein bands. 5. Chloroform/methanol-extracted plasma membranes and isolated glycoproteins had a similar carbohydrate composition, containing sialic acid, hexosamine, fucose, xylose, mannose, galactose and glucose. Glucose was not enriched in the isolated glycoproteins, suggesting that it may be a contaminant. Xylose, however, was enriched in the isolated glycoproteins. It remains to be established whether this sugar, which is not usually found in plasma-membrane glycoproteins, is a genuine constituent of plasma-membrane glycoproteins in the epidermis.

Retinoids increase the incorporation of D-[3H]galactose into epidermal glycoproteins.

All-trans retinoic acid increased the incorporation of D-[3H]galactose into particulate and soluble glycoproteins in the epidermis of cultured pig skin slices nearly two-fold. Increased incorporation of D-[3H]galactose was not blocked by tunicamycin. This effect was specific for D-[3H]galactose since the incorporation of D-[3H]glucosamine and L-[14C]leucine into epidermal glycoproteins was unaffected by all-trans retinoic acid. All-trans retinoic acid and 13-cis retinoic acid had quantitatively similar effects on D-[3H]galactose incorporation. All-trans retinyl acetate and an aromatic retinoic acid analogue ('Etretinate') were less effective. SDS polyacrylamide gel electrophoresis and fluorography showed increased incorporation of D-[3H]galactose into all epidermal glycoproteins in the presence of all-trans retinoic acid. There was no evidence for synthesis of new glycoproteins such as mucins.