Search

Activation of caspases results in the disruption of structural and signaling networks in apoptotic cells. Recent biochemical and cell biological studies have shown that components of the cadherin-catenin adhesion complex in epithelial adherens junctions are targeted by caspases during apoptosis. In epithelial cells, desmosomes represent a second type of anchoring junctions mediating strong cell-cell contacts. Using antibodies directed against a set of desmosomal proteins, we show that desmosomes are proteolytically targeted during apoptosis. Desmogleins and desmocollins, representing desmosome-specific members of the cadherin superfamily of cell adhesion molecules, are specifically cleaved after onset of apoptosis. Similar to E-cadherin, the desmoglein-3 cytoplasmic tail is cleaved by caspases. In addition the extracellular domains of desmoglein-3 and desmocollin-3 are released from the cell surface by a metalloproteinase activity. In the presence of caspase and/or metalloproteinase inhibitors, both cleavage reactions are almost completely inhibited. As reported previously, the desmosomal plaque protein plakoglobin is cleaved by caspase-3 during apoptosis. Our studies now show that plakophilin-1 and two other major plaque proteins, desmoplakin-1 and -2, are also cleaved by caspases. Immunofluorescence analysis confirmed that this cleavage results in the disruption of the desmosome structure and thus contributes to cell rounding and disintegration of the intermediate filament system.

Primary rat hepatocytes and two hepatoma cell lines have been used to study whether high mannose-type N-glycans of plasma membrane glycoproteins may be modified by the removal of mannose residues even after transport to the cell surface. To examine glycan remodeling of cell surface glycoproteins, high mannose-type glycoforms were generated by adding the reversible mannosidase I inhibitor deoxymannojirimycin during metabolic labeling with [3H]mannose, thereby preventing further processing of high mannose-type N-glycans to complex structures. Upon transport to the cell surface, glycoproteins were additionally labeled with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. This strategy allowed us to follow selectively the fate of cell surface glycoproteins. Postbiosynthetic demannosylation was monitored by determining the conversion of Man8-9GlcNAc2 to smaller structures during reculture of cells in the absence of deoxymannojirimycin. The results show that high mannose-type N-glycans of selected cell surface glycoproteins are trimmed from Man8-9GlcNAc2 to Man5GlcNAc2 with Man7GlcNAc2 and Man6GlcNAc2 formed as intermediates. It could be clearly shown in MH 7777 as well as in HepG2 cells that demannosylation affects plasma membrane glycoproteins after they are routed to the cell surface. As was determined for total cell surface glycoproteins in HepG2 cells, this process occurs with a half-time of 6.7 h. By analyzing the size of high mannose-type glycans of glycoproteins isolated from the cell surface at the end of the reculture period, i.e. after trimming had occurred, we were able to demonstrate that glycoproteins carrying trimmed high mannose glycans become exposed at the cell surface. From these data we conclude that cell surface glycoproteins can be trimmed by mannosidases at sites peripheral to N-acetylglucosaminyltransferase I without further processing of their glycans to the complex form. This glycan remodeling may occur at the cell surface or during endocytosis and recycling back to the cell surface.

Return of cell surface glycoproteins to compartments of the secretory pathway has been examined in HepG2 cells comparing return to the trans-Golgi network (TGN), the trans/medial- and cis-Golgi. Transport to these sites was studied by example of the transferrin receptor (TfR) and the serine peptidase dipeptidylpeptidase IV (DPPIV) after labeling these proteins with the N-hydroxysulfosuccinimide ester of biotin on the cell surface. This experimental design allowed to distinguish between glycoproteins that return to these biosynthetic compartments from the cell surface and newly synthesized glycoproteins that pass these compartments during biosynthesis en route to the surface. Reentry to the TGN was measured in that surface glycoproteins were desialylated with neuraminidase and were monitored for resialylation during recycling. Return to the trans-Golgi was traced measuring the transfer of [3H]fucose residues to recycling surface proteins by fucosyltransferases. To study return to the cis-Golgi, surface proteins were metabolically labeled in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM). As a result surface proteins retained N-glycans of the oligomannosidic type. Return to the site of mannosidase I in the medial/cis-Golgi was measured monitoring conversion of these glycans to those of the complex type after washout of dMM. Our data demonstrate that DPPIV does return from the cell surface not only to the TGN, but also to the trans-Golgi thus linking the endocytic to the secretory pathway. In contrast, no reentry to sites of mannosidase I could be detected indicating that the early secretory pathway is not or is only at insignificant rates accessible to recycling DPPIV. In contrast to DPPIV, TfR was very efficiently sorted from endosomes to the cell surface and did not return to the TGN or to other biosynthetic compartments in detectable amounts, indicating that individual surface proteins are subject to different sorting mechanisms or sorting efficiencies during recycling.

The metabolism of the cell surface glycoprotein dipeptidyl peptidase IV(DPPIV) was studied in cultured rat hepatocytes. In pulse-chase labelling experiments using L-[35S]methionine a 100-kDa high-mannose precursor polypeptide is converted into the mature complex-type 110-kDa glycoprotein. Digestion with exo- and endoglycosidases and metabolic labelling with radioactive sugars demonstrate that the 110-kDa form contains about 6 complex-type oligosaccharides which are fucosylated and sialylated. About 25 min after the beginning of the pulse-labelled glycoprotein appears in the sinusoidal membrane. Physiologically only the 110-kDa form is found in the cell surface. If cell surface DPP IV was desialylated by sialidase at 4 degrees C, it is resialylated during incubation at 37 degrees C. This oligosaccharide reprocessing indicates that the surface glycoprotein has been recycled to the cell compartment containing terminal glycosyltransferases (presumably the trans Golgi system). Two different methods demonstrate internalization of cell surface DPP IV: 1) The complex cell surface DPPIV -anti-DPP IV-antibody -L-[35S]methionine-labelled secondary goat-anti-mouse-antibody formed at 4 degrees C becomes less accessible to trypsin during incubation at 37 degrees C. 2) Part of the complex plasma membrane DPP IV-anti-DPP IV-antibody formed in the cold cannot be recognized by the radioactive secondary antibody after rewarming. Internalization is not blocked by inhibition of protein synthesis with cycloheximide. During internalization of plasma membrane DPP IV its concentration in the membrane remains constant.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of vitamin A deficiency on N-linked oligosaccharides of membrane glycoproteins was studied in rat liver in order to evaluate the suggested role of retinol in protein N-glycosylation. First, oligosaccharides of newly synthesized glycoproteins from rough endoplasmic reticulum of vitamin A deficient liver were compared with that of pair-fed controls. Oligosaccharides were metabolically labelled with D-[2-3H]mannose, released from the glycoproteins with endoglycosidase H, purified by reversed phase HPLC and ion exchange chromatography, and were reduced with sodium borohydride. HPLC fractionation of the oligosaccharide alditols showed that the glycoproteins carried mainly four oligosaccharide species, Glc1Man9GlcNAc2, Man9GlcNAc2, Man8GlcNAc2 and Man7GlcNAc2, in identical relative amounts in the vitamin A deficient and the control tissue. In particular, no increase in the proportion of short chain oligosaccharides was noted in vitamin A deficient liver. Second, the number of N-linked oligosaccharides was estimated in dipeptidylpeptidase IV (DPP IV), a major glycoprotein constituent of the hepatic plasma membrane, comparing the newly synthesized glycoprotein from rough endoplasmic reticulum and the mature form of DPP IV from the plasma membrane. No evidence was obtained that retinol deficiency caused incomplete glycosylation of this membrane glycoprotein. From these data, the suggested role of retinol as a cofactor involved in the synthesis of N-linked oligosaccharides of glycoproteins must be questioned.

Expression of six glycoproteins (Mr = 60,000 (gp 60), 80,000 (gp 80), 110,000 (gp 110), 120,000 (gp 120), 140,000 (gp 140), 160,000 (gp 160)) recently purified from rat liver plasma membranes (LPM) were compared in the liver, small intestine and kidney of the rat, rabbit and human. Immunoblotting studies with monospecific antisera showed that five of the six glycoproteins (gp 60, gp 80, gp 110, gp 120, and gp 140) were expressed not only in LPM of the rat but also in LPM from the rabbit and human with Mr corresponding to those of the glycoproteins isolated from the rat. In contrast, the glycoprotein gp 160 was only detected in rat liver. The same pattern of expression was found by immunofluorescence on isolated hepatocytes from the three species. In rat liver, the glycoproteins were localized primarily either in the bile canalicular domain (gp 80, gp 110, gp 120), or in the sinusoidal domain (gp 60, gp 140), or they were distributed over the whole hepatocellular surface (gp 160). In rat, but not in rabbit or human, the glycoproteins gp 110, gp 120 and gp 140 were also found in the small intestine localized either in the brush border membrane (gp 110, gp 120) or over the whole surface membrane of enterocytes (gp 140). Gp 120 was also detected in the luminal pole of tubular epithelial cells of rats kidney. The data show that LPM of different mammalian species share several common glycoprotein antigens. These glycoproteins, that are also partly expressed in extrahepatic tissues, may represent plasma membrane structures conserved among mammalian species.

The following procedure for the detection of non-acetylated amino sugars in the plasma membrane was established: i) derivatization of free amino groups with dansyl-chloride, ii) hydrolysis with 3 M HCl (for 4 h at 105 degrees C) to liberate the dansylated carbohydrate moieties from the plasma membrane, iii) purification of the dansylated amino sugars by paper chromatography and subsequent analysis by thin-layer chromatography. Using this procedure, plasma membranes from rat liver were analysed after injection of D-[14C]galactosamine. For this purpose, rats were divided into three groups: the first received D-galactosamine.HCl at a dose of 2 mg/kg b.w., the second at a dose of 75 mg/kg b.w. and the third at a hepatitis-inducing dose of 260 mg/kg b.w.. In all three groups the majority of the protein-bound radioactivity in the plasma membrane was not dansylated, thus representing N-acetylated amino sugars. At a dose of 2 mg/kg, only 0.34% of the protein-bound radioactivity in the plasma membrane reacted with dansyl-chloride. At a dose of 70 mg/kg this value increased to 1.9%. At 260 mg/kg the value was 3.6%. These results indicate that the incorporation of non-acetylated amino sugar into the plasma membrane was dose-dependent and reached 90 pmol per mg plasma membrane protein during galactosamine injury. However, this incorporation of non-acetylated amino sugars into the plasma membrane did not represent a pathological mechanism responsible for the onset of the galactosamine-induced liver injury.(ABSTRACT TRUNCATED AT 250 WORDS)

A method was developed to isolate cell surface proteins by a simple two-step procedure. Hepatocyte cell surface proteins were labeled by a cleavable biotin derivative in a covalent pulse reaction. Under the described conditions, NHS-SS-biotin proved to be an impermeant, cell surface-specific label which does not affect the impermeant, cell surface-specific label which does not affect the viability of rat hepatocytes. Biotinylated cell surface proteins could be selectively separated under non-denaturing conditions from non-biotinylated proteins and biotin-containing carboxylases by avidin affinity chromatography and sulfhydryl-mediated elution. Subsequent to alkylation of the eluted protein, individual cell surface proteins could be isolated by immunoprecipitation as shown for a selected Mr 120,000 glycoprotein gp120 of the hepatocyte plasma membrane. Using this technique, a transit time of gp120 from the endoplasmic reticulum to the cell surface of 2 h was determined. The results show that the combination of labeling with a cleavable biotin derivative, non-denaturing avidin affinity chromatography and immunoprecipitation is a useful method to isolate and study individual cell surface proteins.

In plasma membrane glycoproteins of rat liver L-fucose undergoes a rapid intramolecular turnover in that fucose residues are removed from the glycoproteins (Tauber, R., Park, C.S. & Reutter, W. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4026-4029). The present paper demonstrates that the intramolecular turnover of L-fucose is markedly decreased during liver regeneration. Turnover half-lives of L-fucose were measured in regenerating liver by pulse-chase experiments in five plasma membrane glycoproteins (Mr 60,000 (gp60), 80,000 (gp80), 120,000 (gp120), 140,000 (gp140), and 160,000 (gp160). The glycoproteins were isolated from plasma membranes by concanavalin A-Sepharose affinity chromatography and semipreparative NaDodSO4 polyacrylamide gel electrophoresis. L-Fucose turned over in the five glycoproteins with heterogeneous half-lives ranging from 22 h (gp160) to 49 h (gp120). The protein moieties of the glycoproteins were degraded with half-lives ranging from 56 h (gp80) to 107 h (gp140). Relative to the half-life of the protein backbone the half-live of L-fucose was increased in the five membrane glycoproteins by 70% (gp60), 150% (gp80), 182% (gp120), 60% (gp140) and 16% (gp160) during liver regeneration when compared to normal liver. The data show that L-fucose turns over in different membrane glycoproteins with individual rates, and that loss of L-fucose from plasma membrane glycoproteins is reduced in rapidly proliferating liver after partial hepatectomy.

During growth and differentiation the plasma membrane has a key role not only in the reception and transmission of extracellular signals such as hormones and growth factors, but also in communicating cellular response to the cellular microenvironment. Cellular response to trophic stimuli includes alterations of cell shape and cell surface antigenicity, of cell-cell recognition and cellular adhesion, of cell matrix binding and the adaptation of cell surface receptors. The plasma membrane is therefore regarded as a 'central agency' for the integration of a single cell into the complex system of a tissue or of an organism. The numerous functions of the plasma membrane are mainly mediated by membrane integrated glycoproteins or glycolipids both sharing the common feature of covalently bound oligosaccharide side chains. Specific alterations of oligosaccharide structure and metabolism associated with growth, differentiation and various pathologic conditions suggest a specific role for the oligosaccharide moieties in the regulation of cell surface functions (Table 1). This review intends to focus on the role of plasma membrane glycoproteins describing briefly principles of glycoprotein structure and function, and characteristics of their biosynthesis and degradation.