High-mannose Oligosaccharide Chains Research Articles

Analysis of recombinant Duffy protein-linked N-glycans using lectins and glycosidases.

Duffy antigen is a glycosylated blood group protein acting as a malarial and chemokine receptor. Using glycosylation mutants we have previously demonstrated, that all three potential glycosylation sites of the Duffy antigen are occupied by N-linked oligosaccharide chains. In this study, wild-type Duffy glycoprotein and three mutants, each containing a single N-glycan, were used to characterize the oligosaccharide chains by lectin blotting and endoglycosidase digestion. The positive reaction of all the recombinant Duffy forms with Datura stramonium and Sambucus nigra lectins showed that each Duffy N-linked glycan contains Galbeta1-4GlcNAc units terminated by (alpha2-6)-linked sialic acid residues, typical of complex oligosaccharides. The reactivity with Aleuria aurantia and Lens culinaris lectins suggested the presence of (alpha1-6)-linked fucose at the N-glycan chitobiose core. The failure of the Galanthus nivalis and Canavalia ensiformis lectins to bind to any of the Duffy mutants or to the wild-type antigen indicated that none of the three Duffy N-glycosylation sites carries detectable levels of high-mannose oligosaccharide chains. Digestion of Duffy samples with peptide N-glycosidase F and endoglycosidase H confirmed the presence of N-linked complex oligosaccharides. Our results indicate that Duffy antigen N-glycans are mostly core-fucosylated complex type oligosaccharides rich in N-acetyllactosamine and terminated by (alpha2-6)-linked sialic acid residues.

Differential expression of two cathepsin Es during metamorphosis-associated remodeling of the larval to adult type epithelium in Xenopus stomach.

Cathepsin E (CE) was purified from the foregut of Xenopus laevis tadpoles as a mature dimeric form. The purified enzyme was a typical CE among aspartic proteinases with respect to pH dependence of proteolytic activity, susceptibility to pepstatin, and having N-linked high-mannose type oligosaccharide chains. We isolated two cDNAs for the CE (CE1 and CE2) from adult stomach. The amino acid sequence of the N-terminal region of the purified CE coincided with the corresponding sequence predicted from CE1. Northern blot analysis and in situ hybridization were performed. The CE1 mRNA was highly expressed in surface mucous cells and gland cells constituting the larval epithelium of the foregut of pro-metamorphic tadpoles. As metamorphosis began and progressed, CE1 mRNA drastically decreased in amount, and subsequently both CE1 and CE2 mRNAs gradually increased. The increase in CE2 mRNA was detected shortly after the increase in CE1 mRNA. The decrease in CE1 expression correlated with degeneration of the larval type epithelium, while the increases in both CE1 and CE2 expression correlated with formation of the adult type epithelium. Thus, cathepsin E gene expression was differentially regulated during metamorphosis-associated remodeling of the larval to adult type epithelium in stomach.

Biosynthesis, Intracellular Targeting, and Degradation of the EAAC1 Glutamate/Aspartate Transporter in C6 Glioma Cells

Rat C6 glioma cells were used as a model system to study the biosynthesis, intracellular targeting, and degradation of the EAAC1 transporter, a sodium-dependent glutamate/aspartate transport protein that encodes System X(-)A,G activity. At steady state, nearly 70% of the EAAC1 transporter was located at the cell surface. The newly synthesized EAAC1 protein was co-translationally N-glycosylated with high mannose oligosaccharide chains that were processed into complex-type sugar chains as the protein matured. The final maturation steps for EAAC1 protein coincided with its plasma membrane arrival, which was first detected at about 45 min after the initial synthesis. The newly synthesized EAAC1 protein was protected from degradation during the maturation and targeting process, as well as during the first 5 h after plasma membrane arrival. After this initial lag period, both the newly synthesized transporter and the total cellular EAAC1 pool were degraded by first order kinetics with a half-life of 6 h. These results represent the first analysis of the synthesis and degradation of the EAAC1 amino acid transporter.

Suppression of Integrin Expression and Tumorigenicity by Sulfation of Lactosylceramide in 3LL Lewis Lung Carcinoma Cells

To investigate the cellular functions of sulfated glycosphingolipids, we introduced the cerebroside sulfotransferase (CST) gene into J5 cells, a subclone of 3LL Lewis lung carcinoma cells. The J5 cells lack acidic glycosphingolipids but accumulate their common biosynthetic precursor, lactosylceramide. We established the stable CST transfectants, J5/CST-1 and J5/CST-2 clones, highly expressing sulfated lactosylceramide (SM3). Both clones exhibited more spherical morphology in comparison to mock transfectant, and their adhesiveness to fibronectin and laminin was significantly lower. The loss of cell-substratum interactions in these SM3-expressing cells could be attributed to decreased expression of integrins (alpha(5), alpha(6), and beta(1)) on the cell surface and their whole cellular levels. However, the levels of H-2K(b) and H-2D(b) antigens remained unchanged. Reverse transcriptase-polymerase chain reaction and Northern blot analyses for these integrins exhibited significant decrease of beta(1) gene expression in J5/CST-1 and 2, but there was no change in the levels of alpha(5) and alpha(6) transcripts. Deglycosylation by endoglycosidase H treatment clearly demonstrated that the precursor form of beta(1) integrin, possessing high mannose oligosaccharide chains, was preferentially decreased in the CST transfectants. These results demonstrate that endogenous SM3 negatively regulates beta(1) integrin expression at the transcriptional level, and the decrease of alpha integrin proteins in the CST transfectants was due to the post-transcriptional modification. We suggest the putative importance of the intracellular pre-beta(1) integrin pool for normal integrin maturation and subsequent function. Although the rates of cell proliferation in vitro for mock and CST transfectants were similar, tumorigenicity of J5/CST-1 and -2 cells inoculated into syngeneic C57/BL6 mice was greatly decreased or even absent. This was probably due to global loss of the efficient cell-matrix interactions, which are essential for the development of malignant tumors in vivo. Thus, we showed the evidence that cellular SM3 negatively regulates the cell-substratum interaction, resulting in the loss of tumorigenicity.

Identification of a 374 amino acid protein encoded by RNA segment 6 of influenza C virus.

Unspliced mRNA from RNA segment 6 of influenza C virus contains a single open reading frame that potentially encodes a polypeptide of 374 amino acids. This polypeptide, which has not been identified as yet, is predicted to contain the complete amino acid sequence of the matrix protein, M1, as well as that of a small integral membrane protein, CM2. Here, we found that small amounts of two previously unrecognized proteins with apparent molecular masses of 42 (P42) and 44 kDa (P44) were immunoprecipitated with immune serum against CM2. The electrophoretic mobilities of P42 and P44 varied depending on virus strain, indicating that they are virus-coded. Treatment of infected cells with tunicamycin and digestion of immunoprecipitated proteins with various endoglycosidases revealed that P42 is modified by the addition of a high-mannose oligosaccharide chain to generate P44. A monoclonal antibody against M1, like anti-CM2 serum, was able to immunoprecipitate both the P42 and P44 proteins. Furthermore, the tryptic peptide map of either P42 or P44 was indistinguishable from the map of the mixture of M1 and CM2. These results, taken together, suggest strongly that P42 and P44 correspond to the 374 amino acid protein encoded by unspliced RNA segment 6 mRNA and its N-glycosylated form, respectively.

Structure of asparagine-linked oligosaccharides of an aspartic proteinase from the zygomycete fungus Rhizomucor pusillus.

The zygomycete fungus Rhizomucor pusillus (previously called Mucor pusillus) secretes an aspartic proteinase containing two asparagine-linked, high-mannose type oligosaccharide chains at Asn79 and Asn188. For structural elucidation of the carbohydrate moieties, the protein was divided into two portions, an N-terminal portion containing Asn79 and a C-terminal portion containing Asn188, by a specific autocatalytic cleavage under alkaline conditions. Each of the asparagine-linked oligosaccharides was then released by peptide-N-glycosidase F digestion and pyridylaminated with a fluorescent reagent, 2-aminopyridine, at the reducing end. High-performance liquid chromatography analyses showed that the structure of the asparagine-linked oligosaccharide chain attached to residue Asn79 was Man5GlcNAc2, and that bound to residue Asn188 was Man5GlcNAc2 and Man6GlcNAc2. These observations suggest that the processing of mannose residues in asparagine-linked oligosaccharides in the Golgi apparatus of Rhizomucor resembles that in mammalian cells.

The Cytoplasmic Tail of the Mouse Brown Locus Product Determines Intracellular Stability and Export from the Endoplasmic Reticulum

The Cytoplasmic Tail of the Mouse Brown Locus Product Determines Intracellular Stability and Export from the Endoplasmic Reticulum

Abnormal Glycosylation of Procathepsin L Due to N-terminal Point Mutations Correlates with Failure to Sort to Lysosomes

A single point mutation in the lysosomal proenzyme receptor-inhibiting sequence near the N terminus of mouse procathepsin L can result in glycosylation of a normally cryptic site near its C terminus. When alanine replaced His36, Arg38, or Tyr40, the nascent chain of the mutant protein cotranslationally acquired a high mannose oligosaccharide chain at Asn268. In contrast, when alanine replaced Ser34, Arg37, or Leu39, this second carbohydrate chain was not added. This alternating pattern of abnormal glycosylation suggested that propeptide residues 36-40 normally assume an extended conformation having the side chains of residues 36, 38, and 40 facing in the same direction. When tyrosine conservatively replaced His36 or lysine replaced Arg38, Asn268 was not glycosylated. But the procathepsin L mutant having phenylalanine in place of Tyr40 was glycosylated at Asn268, which indicates that the hydrogen bond between the hydroxyl group of Tyr40 and the carboxylate group of Asp82 is necessary for normal folding of the nascent proenzyme chain. Mutation of the adjacent alpha2p (ERININ) helix of the propeptide or addition of a C-terminal epitope tag sequence to procathepsin L also induced misfolding of the proenzyme, as indicated by addition of the second oligosaccharide chain. In contrast, the propeptide mutation KAKK99-102AAAA had no effect on carbohydrate modification even though it reduced the positive charge of the proenzyme. Misfolded mutant mouse procathepsin L was not efficiently targeted to lysosomes on expression in human HeLa cells, even though it acquired phosphate on mannose residues. The majority of the mutant protein was secreted after undergoing modification with complex sugars. Similarly, epitope-tagged mouse procathepsin L was not targeted to lysosomes in homologous mouse cells but was efficiently secreted. Since production of mature endogenous protease was not reduced in cells expressing the tagged protein, the tagged protein did not compete with endogenous procathepsin L for targeting to lysosomes.

Mutational analysis of the ligand-binding domain of M-T2 protein, the tumor necrosis factor receptor homologue of myxoma virus.

The myxoma virus-encoded M-T2 protein shares extensive sequence homology with the ligand-binding domains of the TNF receptors (TNFRs) and has been shown to bind and inhibit rabbit TNF-alpha with affinities similar to those of TNF-alpha with cellular receptors. Here we show that M-T2 protein is secreted from infected cells as an N-linked glycoprotein, with both complex and hybrid or high mannose oligosaccharide chains. Since amino acid homology between M-T2 and cellular TNF receptors is limited to the four N-terminal cysteine-rich domains (CRDs), various M-T2 C-terminal truncations were created in recombinant vaccinia virus vectors. C-terminal deletions that include truncations up to the middle of the fourth CRD effectively bound and inhibited rabbit TNF-alpha. In contrast, removal of any one of the first three CRDs resulted in a mutant M-T2 protein incapable of binding or inhibiting rabbit TNF-alpha. The C-terminal portion of M-T2, which is not homologous to the cellular TNFRs, appears to be important for efficient secretion of M-T2 from infected cells, since all the C-terminal truncations, including a truncation removing only the last 24 amino acids, were effectively retained as intracellular proteins that were still capable of binding and inhibiting rabbit TNF-alpha. We conclude that the first three CRDs of M-T2 fulfill the same ligand-binding function as the cellular TNFRs, and the nonhomologous C-terminal region participates in protein trafficking of M-T2 in virus-infected cells.

Cloning and expression of glucosidase I from human hippocampus.

Glucosidase I, the first enzyme in the N-linked oligosaccharide processing pathway, cleaves the distal alpha 1,2-linked glucose residue from the Glc3-Man9-GlcNAc2 oligosaccharide precursor highly specifically. A human hippocampus cDNA library was screened against oligonucleotide probes, generated by PCR using primers derived from the amino acid sequences of tryptic peptides of pig liver glucosidase I. Two independent lambda clones were isolated which allowed the construction of a full-length glucosidase I cDNA of 2881 bp. This cDNA construct encodes, in a single open reading frame, a polypeptide of 834 amino acids corresponding to a molecular mass of 92 kDa. The 92-kDa protein contains a single N-glycosylation site of the Asn-Xaa-Thr/Ser type at Asn655, as well as a strongly hydrophobic sequence close to its N-terminus (amino acids 38-58) which, most likely, functions as a transmembrane anchor. The amino acid sequences of all tryptic peptides of the pig liver enzyme were found, with little deviation, within the coding sequence. This demonstrates the authenticity of the cDNA construct and the close evolutionary relationship between the enzymes from human hippocampus and pig liver. In contrast, the nucleotide and amino acid sequence revealed no homology with other processing enzymes cloned so far. Transfection of COS 1 cells with the glucosidase I cDNA construct resulted in overexpression (about fourfold) of enzymic activity, which was inhibited strongly by 1-deoxynojirimycin or N,N-dimethyl-deoxynojirimycin. The expressed enzyme, with a molecular mass of 95 kDa, is degraded by endoglycosidase H to a 93-kDa form, indicating that it carries a high-mannose oligosaccharide chain at Asn655. The presence of this glycan is in line with the localization of glucosidase I in the lumen of the endoplasmic reticulum, shown by immunofluorescence microscopy. The hydrophobicity profile as well as the removal by trypsin of an approximately 4-kDa polypeptide from the membrane-associated glucosidase I in intact microsomal structures, supports the view that the enzyme is a type-II transmembrane glycoprotein, which contains a short cytosolic tail of approximately 37 amino acids, followed by a single transmembrane domain and a large C-terminal catalytic domain located on the luminal side of the endoplasmic reticulum membrane.

An extracellular domain of the β subunit is essential for processing, transport and kinase activity of insulin receptor

The extracellular portion of the insulin receptor (IR) beta-subunit has four cysteine and four asparagine residues which are potentially involved in disulphide bond formation between the alpha- and beta-subunits and N-linked glycosylation respectively. However, the function of this portion is not fully understood. In order to investigate the role of the extracellular domain of beta-subunit, we created a deletion mutant of IR cDNA which lacked 47 amino acid residues encoded by 141 bp corresponding to exon 13 of the IR gene. Insulin binding and surface labelling of COS 7 cells transiently expressing the mutant insulin receptors (IR delta Ex13) showed that the mutated receptors were not expressed on the cell surface. However, immunoblot analysis showed that uncleaved form (190 kDa) of the mutant receptors were intracellularly expressed. Deglycosylation with endoglycosidase H showed that the mutant receptors had mainly high-mannose oligosaccharide chains. The mutant IRs bound with high affinity to lentil lectin but with low affinity to wheat germ agglutinin. Therefore, it is suggested that misfolding of the mutant receptors inhibits transport to the Golgi apparatus where processing of oligosaccharide chains, as well as proteolytic cleavage into subunits, takes place. The binding affinity of the mutant receptors for insulin was 50% of normal. Furthermore, insulin-stimulated autophosphorylation of IR delta Ex13 was markedly impaired. These data provide the evidence for a critical role of the extracellular domain of IR beta-subunit for processing and transport as well as the intramolecular signal transduction to activate IR tyrosine kinase.

Biosynthesis and processing of lysosomal cathepsin D in primary cultures of rat hepatocytes.

To investigate the intracellular transport and maturation of lysosomal cathepsin D, we carried out an in vivo pulse-chase analysis with [35S]methionine in the primary cultures of rat hepatocytes. Cathepsin D was initially synthesized as a proenzyme of 45 kDa. The proenzyme was subsequently processed, becoming a mature enzyme of 43 kDa. The proenzyme and mature enzyme showed complete susceptibility to endoglycosidase H treatment, suggesting the presence of high-mannose type oligosaccharide chains. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, the 42.5-kDa unglycosylated precursor polypeptide appeared in the cell, and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results support the notion that the oligosaccharide moieties are of importance in addressing the lysosomal hydrolases to the lysosomes. However, in the presence of chloroquine, proteolytic processing of the proenzyme was prevented, and the enhanced release of proenzyme from the cells was observed. These results indicate that the processing of proenzyme to mature enzyme would take place in the lysosomes.

Crystal structure of endo-beta-N-acetylglucosaminidase F1, an alpha/beta-barrel enzyme adapted for a complex substrate.

Endo-beta-N-acetylglucosaminidase F1 (Endo F1) is an endoglycosidase, secreted by Flavobacterium meningosepticum, that cleaves asparagine-linked oligosaccharides after the first N-acetylglucosamine residue. The enzyme is selective for high-mannose oligosaccharide chains. The crystal structure of Endo F1 has been determined at 2.0-A resolution. The molecular fold consists of a highly irregular alpha/beta-barrel, a commonly observed motif consisting of a cyclic 8-fold repeat of beta-strand/loop/alpha-helix units with an eight-stranded parallel beta-barrel at the center. Endo F1 lacks two of the alpha-helices, those of units 5 and 6. Instead, the links after beta-strands 5 and 6 consist of a short turn followed by a section in an extended conformation that replaces the helix and a long loop at the bottom of the molecule. The absence of any excursion on top of the molecule following beta-strands 5 and 6 results in a pronounced depression in the rim of the barrel. This depression forms one end of a shallow cleft that runs across the surface of the molecule, over the core of the beta-barrel to the area between the loops of units 1 and 2. The active site residues, Asp130 and Glu132, are located at the carboxyl end of beta-strand 4 and extend into this cleft. These residues are surrounded by several tyrosine residues. The cleft area formed by loops 1 and 2 is lined with polar residues, mainly asparagines. The latter area is thought to be responsible for oligosaccharide binding and recognition while the protein moiety of the substrate would be located outside the molecule but adjacent to the area of loops 5 and 6.(ABSTRACT TRUNCATED AT 250 WORDS)

Overexpression of Uteroferrin, a Lysosomal Acid Phosphatase Found in Porcine Uterine Secretions, Results in its High Rate of Secretion from Transfected Fibroblasts1

Lysosomal enzymes can, under certain circumstances, be secreted in large amounts. One example is uteroferrin (Uf), an iron-containing, purple-colored acid phosphatase secreted by the uterus of the pig during pregnancy. Uf is identical to the intracellular tartrate-resistant acid phosphatase of pig spleen, yet is the major protein component of uterine secretions. To investigate possible regulatory mechanisms that might direct Uf along a secretory pathway, we expressed Uf in Chinese hamster ovary (CHO) cells under the control of the SV40 early promoter using an expression construct, pX/Uf. The proportion of Uf secreted into the medium relative to the amount retained intracellularly increased as total Uf expression was increased. At transfection doses of 15 micrograms pX/Uf per 10(6) cells, over 80% of the Uf produced in 48 h was secreted. A parallel situation was observed when human cathepsin D was overexpressed in CHO cells. Thus, high production of Uf, as occurs in the uterus in response to progesterone, may overwhelm the intracellular enzymatic and receptor systems that are normally employed to target acid hydrolases to lysosomes, resulting in secretion. Both Uf and cathepsin D secreted by CHO cells possess N-linked, phosphorylated high-mannose oligosaccharide chains. However, the phosphate groups on the oligosaccharide chains of Uf, unlike those on cathepsin D, cannot be readily removed by alkaline phosphatase treatment. These results suggest that the phosphate groups on Uf are masked at least partially by covering N-acetylglucosamine residues and that two mechanisms may contribute to hypersecretion of Uf in the uterus: 1) very high rates of synthesis and 2) partial masking of the mannose 6-phosphate recognition signal.

Direct Demonstration of the Essential Role of the Intramolecular High-Mannose Oligosaccharide Chains in the Folding and Assembly of Soybean (Glycine max) Lectin Polypeptides

We investigated the role of the intramolecular high-mannose oligosaccharide chains in the folding and assembly of soybean lectin polypeptides. Soybean lectin, dissociated into subunits and completely denatured in 6 M guanidine hydrochloride, was quantitatively reconstituted to the active tetrameric structure by simple dilution. However, neither the activity nor the tetrameric structure was regained in the presence of 100 microM of an asparagine-linked oligosaccharide, Man9GlcNAc2Asn, having the same structure as that of the sugar chains of soybean lectin. Besides, the same concentration of this glycopeptide even dissociated, although only gradually, the native lectin into subunits. On the other hand, the deglycosylated subunits had no ability to regain the activity or the tetrameric structure. The present study provided for the first time direct evidence of the essential role of the intramolecular high-mannose oligosaccharide chains in the proper folding and assembly of glycopolypeptides.

The presence of N-glycosylated proteins in cell nuclei

The protein-DNA crosslinking capability of cis-dichloro diammineplatinum has been exploited to check the intranuclear location of N-glycosylated proteins. When intact liver cells were treated with this reagent, a number of glycoproteins, recognized by Concanavalin A, have been shown to become crosslinked to DNA; many of them have been recognized as nuclear matrix components. The recognition by this lectin was abolished by treatment with N-glycosidase F, showing the presence of N-glycosidic bonds between the sugar moiety and the protein. Most of the glycoproteins appeared to have high mannose oligosaccharide chains, but sialic acid containing oligosaccharides were also identified.

The 43-kDa glycoprotein from the human pathogen Paracoccidioides brasiliensis and its deglycosylated form: Excretion and susceptibility to proteolysis

Biochemical properties of the concanavalin A-binding 43-kDa glycoprotein (gp43) of Paracoccidioides brasiliensis and its deglycosylated form were compared. Deglycosylation was achieved by treatment with trifluoromethanesulfonic acid, endoglycosidase H, N-glycanase, or metabolically, by growing cells with tunicamycin. The resulting antigen in all cases had M r 38,000, and probably derived from the gp43 by loss of N-linked high-mannose oligosaccharide chains. The presence of galactopyranose units in the carbohydrate chains was sugested by antigen binding to peanut lectin. Pulse and chase experiments using [ 35S]methionine metabolic labeling of P. brasiliensis growing in the presence of tunicamycin showed that the N-linked chains of gp43 are not required for antigen secretion. The 38-kDa antigen was more susceptible than the native antigen to the action of papain and pronase, thus indicating a protective role of the carbohydrate moiety against proteolysis. Both forms are equally resistant to endogenous proteases at neutral pH. The gp43, itself, has a proteolytic activity at pH 5–6, but not at neutral pH. Deglycosylation with endoglycosidase H or tunicamycin preserved epitopes in the 38-kDa molecule reactive with (a) antibodies from patients with paracoccidioidomycosis, or rabbit immunized with the gp43 and (b) mouse monoclonal antibodies against the gp43 antigen. The present results provide a basis for the understanding of diagnostic reactions and fungal virulence involving the gp43 exocellular antigen of P. brasiliensis.

Biosynthesis of the precursor of a soluble human insulin receptor ectodomain in insect Sf9 cells infected with a recombinant baculovirus

In contrast to transfected mammalian cells, insect Sf9 cells infected with a recombinant Baculovirus inefficiently process and secrete a soluble derivative of the extracellular domain of the human insulin receptor. The high-mannose form of the receptor precursor that accumulates intracellularly is not grossly aberrant or malfolded, as its interaction with a diverse panel of monoclonal antibodies are comparable to secreted precursor and proteolytically processed receptor, both of which bear partially trimmed oligosaccharide chains. Thus the inefficient step in the biosynthesis of this protein in Sf9 cells is either at, or just preceding, the trimming of its high-mannose oligosaccharide chains.

Synthesis and processing of the major envelope glycoprotein of murine cytomegalovirus

We have characterized the synthesis and processing pathway of the major envelope glycoprotein complex of murine cytomegalovirus (gp52/105/150). We have demonstrated that it belongs to the “late” kinetic class of MCMV proteins, and is initially synthesized as a 128K glycoprotein (gpl 28) which contains N-linked, high-mannose type oligosaccharide chains and is phosphorylated predominantly at serine residues. A fraction of the gp128 molecules also contains O-linked GaINAc residues. The majority of the gpl28 molecules appears to be retained in the endoplasmic reticulum as evidenced by their sensitivity to endoglycosidase H digestion. The formation of disulfide linkages and dimerization allow the transport of gpl28 to the Golgi compartments where modification of N-linked carbohydrate structures and extension of O-linked oligosaccharide chains take place, cumulating in the appearance of the mature gpl50. The final processing step involves the cleavage of gpl50 into gp52 and gpl05. By blocking the transport of the glycoprotein precursor to the trans-Golgi compartments with the ionophore monensin, the cleavage process is inhibited, suggesting that the trans-Golgi compartment is the site where gpl50 is cleaved. However, the cleavage process is incomplete, resulting in the formation of multiple disulfide-linked complexes made up of different combinations of gp52, gpl05, and gp150. Therefore, the processing of the major envelope glycoprotein complexes of murine cytomegalovirus resembles that of the gcl/gp55–116 complex of human cytomegalovirus.

Increased cytotoxicity of ricin in a putative Golgi-defective mutant of Chinese hamster ovary cell

We have studied the cytotoxicity of ricin in a monensin-resistant mutant (Mon R-31) of Chinese hamster ovary (CHO) cell line which is presumably altered in Golgi functions/structures. The cytotoxicity of ricin was increased in Mon R-31 mutant cells compared with that in its parental CHO cells. In wild-type CHO cells, the cytotoxicity of ricin was enhanced by NH 4Cl, bafilomycin A 1, or nigericin. The enhancement of ricin cytotoxicity by these compounds was greatly reduced in Mon R-31 mutant cells. Brefeldin A (BFA), which disrupts the structure of the Golgi apparatus, inhibits the cytotoxicity of ricin in both CHO and Mon R-31 cells. We have also examined the effects of glycosylation inhibitors and the removal of high mannose oligosaccharide chains in ricin on the ricin hypersensitivity in Mon R-31 cells. The hypersensitivity of Mon R-31 cells to ricin is apparently not due to any difference in glycosylation between CHO and Mon R-31 cells or in the processing of oligosaccharides on ricin by the target cells. Nigericin at low concentration (10 n M), which has no effect on the cytotoxicity of diphtheria toxin, enhances the ricin cytotoxicity, but inhibits the modeccin cytotoxicity. Our results suggest that important step(s) in the intoxication process of CHO cells by ricin and modeccin take place in the Golgi region.