Structure and assembly of desmosome junctions: biosynthesis, processing, and transport of the major protein and glycoprotein components in cultured epithelial cells.

Abstract

Abstract. Extracts of metabolically labeled cultured epithelial cells have been analyzed by immunoprecipitation followed by SDS-PAGE, using antisera to the major high molecular mass proteins and glycoproteins (>100 kD) from desmosomes of bovine muzzle epidermis. For nonstratifying cells (Madin-Darby canine kidney [MDCK] and Madin-Darby bovine kidney), and A431 cells that have lost the ability to stratify through transformation, and a stratifying cell type (primary human keratinocytes) apparently similar polypeptides were immunoprecipitated with our antisera. These comprised three glycoproteins (DGI, DGII, and DGIII) and one major nonglycosylated protein (DPI). DPII, which has already been characterized by others in stratifying tissues, appeared to be absent or present in greatly reduced amounts in the nonstratifying cell types.The desmosome glycoproteins were further characterized in MDCK cells. Pulse-chase studies showed all three DGs were separate translation products. The two major glycoprotein families (DGI and DGII/III) were both found to be synthesized with co-translational addition of 2-4 high mannose cores later processed into complex type chains. However, they became endo-13-N-acetylglucosaminidase H resistant at different times (DGII/III being slower). None of the DGs were found to have O-linked oligosaccharides unlike bovine muzzle DGI. Transport to the cell surface was rapid for all glycoproteins (60-120 min) as demonstrated by the rate at which they became sensitive to trypsin in intact cells. This also indicated that they were exposed at the outer cell surface. DGII/III, but not DGI, underwent a proteolytic processing step, losing 10 kD of carbohydrate-free peptide, during transport to the cell surface suggesting a possible regulatory mechanism in desmosome assembly.

Cloning and sequence analysis of desmosomal glycoproteins 2 and 3 (desmocollins): cadherin-like desmosomal adhesion molecules with heterogeneous cytoplasmic domains.

“…Two of the three correspond to sites in N-cadherin in EC1 and EC4. A reduction in the relative molecular mass of dg2 and 3 by ,,o10,000 D in tunlcamycin treated MDCK cells suggests three to four N-linked glycan side chains in dg2 and 3 (Penn et al, 1987). This is in good agreement with the three possible glycosylation sites of bovine dg3.…”

High Throughput Quantitative Glycomics and Glycoform-focused Proteomics of Murine Dermis and Epidermis

“…Given that these tissue-specific glycoproteins are dominantly modified with high mannose glycans, it may also prove beneficial to increase the relative proportion of high mannose glycans in epidermis. Although desmogleins and desmocollins are reported to be glycoproteins containing mainly N-glycans, detailed information about oligosaccharide structures has been so far limited (33)(34)(35). The current study also raises an interesting query: why desmosomes require high mannose glycans particularly when it is possible that any type of oligosaccharides could protect desmosomal proteins from protease degradation and may prevent premature desquamation as suggested by Walsh and Chapman (36).…”

A study of desmosomes in colorectal carcinoma

“…The presence of dp2 in colorectal specimens is of interest since a number of groups have been unable to detect dp2 in simple epithelia (Cowin et al, 1985;Penn et al, 1987) and it has been suggested that dp2 may be restricted to stratified epithelia (Cowin et al, 1985). In this study we have consistently found both dpl and 2 in all preparations derived from benign, malignant and cultured colorectal specimens indicating that dp2 is a consistent component of this simple epithelium in agreement with others (Suhrbier & Garrod, 1986;Pasdar & Nelson, 1988).…”

“…out of four carcinoma tissues and a 120,000 polypeptide in normal tissue. The minor band at 120,000 may represent one of the precursors of the 115,000 and 107,000 proteins, since in MDCK cells precursors of slightly higher molecular weight are proteolytically processed to form the mature dg2/3 glycoproteins (Penn et al, 1987).…”

“…Figure 6 shows blotting of colorectal tissues and cells with dg2/3 antiserum. Two bands were common to all with relative mobilities of 115,000 and 107,000 and are homologous to dg2 and 3 of similar Mr in other tissues and cells (Skerrow & Matoltsy, 1974b;Gorbsky & Steinberg, 1981;Mueller & Franke, 1983;Cowin & Garrod, 1983;Suhrbier & Garrod, 1986;Penn et al, 1987). An additional band of 120,000 (Mr) was found in the various non-malignant mucosal specimens (Figure 6a 120,000 band was absent but an additional band of 140,000 was present (Figure 6b).…”