Oligosaccharide Side Chains of Glycoproteins that Remain in the High-Mannose Form Are Not Accessible to Glycosidases

Glycoproteins present in the soluble and organelle fractions of developing bean (Phaseolus vulgaris) cotyledons were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, affinoblotting, fractionation on immobilized concanavalin A (ConA), and digestion of the oligosaccharide side chains with specific glycosidases before and after protein denaturation. These studies led to the following observations. (a) Bean cotyledons contain a large variety of glycoproteins that bind to ConA. Binding to ConA can be eliminated by prior digestion of denatured proteins with α-mannosidase or endoglycosidase H, indicating that binding to ConA is mediated by high-mannose oligosaccharide side chains. (b) Bean cotyledons contain a large variety of fucosylated glycoproteins which bind to ConA. Because fucose-containing oligosaccharide side chains do not bind to ConA, such proteins must have both high-mannose and modified oligosaccharides. (c) For all the glycoproteins examined except one, the high-mannose oligosaccharides on the undenatured proteins are accessible to ConA and partially accessible to jack bean α-mannosidase. (d) Treatment of the native proteins with α-mannosidase removes only 1 or 2 mannose residues from the high-mannose oligosaccharides. Similar treatments of sodium dodecyl sulfate-denatured or pronase-digested glycoproteins removes all α-mannose residues. The results support the following conclusions: certain side chains remain unmodified as high-mannose oligosaccharides even though the proteins to which they are attached pass through the Golgi apparatus, where other oligosaccharide chains are modified. The chains remain unmodified because they are not accessible to processing enzymes such as the Golgilocalized α-mannosidase.     

The removal of carbohydrates from ricin with endoglycosidases H,F and D and α-mannosidase

Recently, several investigators have explored the possibility of targetting ricin to designated cell types in animals by its linkage to specific antibodies. There is evidence, however, that the mannose-containing oligosaccharide chains on ricin are recognised by reticuloendothelial cells in the liver and spleen and so cause the immunotoxins to be removed rapidly from the blood stream. In the present study we analysed the carbohydrate composition of ricin and examined enzymic methods for removing the carbohydrate. The carbohydrate analysis ricin A-chain revealed the presence of one residue of xylose and one of fucose in addition to mannose and N-acetylglucosamine which had been detected previously. The B-chain contained only mannose and N-acetylglycosamine. Ricin A-chain is heterogeneous containing two components of molecular weight 30 000 and 32 000. Strong evidence was found that the heavier form of the A-chain contains an extra carbohydrate unit which is heterogeneous with respect to concanavalin A binding and sensitivity to endoglycosidase H. The lower molecular weight form of A-chain did not bind concanavalin A and was insusceptible to endoglycosidases. Only one of the two high mannose oligosaccharide units on the isolated B-chain could be removed by endoglycosidases H or F, whereas both were removable after denaturation of the polypeptide by SDS. Both the isolated A- and B-chains were sensitive to α-mannosidase. Intact ricin was resistant to endoglycosidase treatment and was only slightly sensitive to α-mannosidase. The addition of SDS allowed endoglycosidase H to remove both of the B-chain oligosaccharides from intact ricin and increased the toxin's sensitivity to α-mannosidase. In conclusion, extensive enzymic deglycosylation of ricin may only be possible if the A- and B-chains are first separated, treated with enzymes and then recombined to form the toxin.     

Substrate Specificities of N-Acetylglucosaminyl-, Fucosyl-, and Xylosyltransferases that Modify Glycoproteins in the Golgi Apparatus of Bean Cotyledons

As part of their posttranslational maturation process, newly synthesized glycoproteins that contain N-linked oligosaccharide side chains pass through the Golgi apparatus, where some of their oligosaccharides become modified by carbohydrate processing reactions. In this paper, we report the presence of Golgi-localized enzymes in plant cells (Phaseolus vulgaris cotyledons) that transfer GlcNAc, fucosyl, and xylosyl residues to the oligosaccharide side chains of glycoproteins. All three enzyme activities are involved in the transformation of high mannose side chains into complex glycans. As judged by acceptor specificity studies, at least two GlcNAc residues can be added to the nonreducing side of high mannose oligosaccharides, which have been trimmed by α-mannosidase(s). A Man₅(GlcNAc)₂-peptide serves as the acceptor for the first GlcNAc added. The second GlcNAc can be added only after the prior removal of two additional mannose residues, ultimately yielding (GlcNAc)₂Man₃(GlcNAc)₂-peptide. Fucosyltransferase can transfer fucose to GlcNAcMan₅(GlcNAc)₂Asn, GlcNAcMan₃(GlcNAc)₂Asn, and (GlcNAc)₂Man₃(GlcNAc)₂Asn; xylosyltransferase exhibits significant activity toward the latter two substrates only. These results suggest an overlapping sequence of oligosaccharide modification in the Golgi apparatus that, in regard to GlcNAc and fucose additions, is analogous to pathways of oligosaccharide processing reported for animal cells. To our knowledge, this is the first report characterizing a xylosyltransferase involved in N-linked oligosaccharide modification, an activity that is apparently absent in most animal cells.     

Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with α-methylmannoside, constitute about 25–30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chrolide columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with α-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.     

Observations on the carbohydrate moiety of bovine pancreatic deoxyribonuclease A

The carbohydrate side chain of bovine pancreatic deoxyribonuclease A, which is attached to asparagine residue 18, contains two residues of N-acetylglucosamine proximal to the peptide chain followed by a variable number of mannose residues (4–10). The oligosaccharide structure bears a similarity to that in bovine pancreatic ribonuclease B. The present sequence studies have made use of α-mannosidase chromatographically purified from jack bean meal.     

Selective Yolk Deposition and Mannose Phosphorylation of Lysosomal Glycosidases in Zebrafish

The regulation and function of lysosomal hydrolases during yolk consumption and embryogenesis in zebrafish are poorly understood. In an effort to better define the lysosomal biochemistry of this organism, we analyzed the developmental expression, biochemical properties, and function of several glycosidases in zebrafish eggs, embryos, and adult tissues. Our results demonstrated that the specific activity of most enzymes increases during embryogenesis, likely reflecting a greater need for turnover within the embryo as yolk-derived nutrients are depleted. Analysis of glycosidase activity in zebrafish and medaka eggs revealed selective deposition of enzymes required for the degradation of N-linked glycans, including an abundance of acidic mannosidases. Treatment of zebrafish embryos with the α-mannosidase inhibitor swainsonine resulted in the accumulation of glycosylated vitellogenin fragments and demonstrated a function for maternally deposited acid α-mannosidase in yolk consumption. Surprisingly, we also found that, unlike mammals, acid α-glucosidase from zebrafish and medaka does not appear to be modified with mannose 6-phosphate residues. We further showed these residues were not acquired on human acid α-glucosidase when expressed in zebrafish embryos, suggesting unique differences in the ability of the human and zebrafish N-acetylglucosamine-1-phosphotransferase to recognize and modify certain lysosomal glycosidases. Together, these results provide novel insight into the role of acidic glycosidases during yolk utilization and the evolution of the mannose 6-phosphate targeting system in vertebrates.     

Monosaccharide Sequence of Protein-Bound Glycans of Uukuniemi Virus

Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells in the presence of [³H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [³H]mannose-labeled glycopeptides of the mixture of the two envelope glycoproteins G1 and G2 were characterized by degrading the glycans with specific exo-and endoglycosidases, by chemical methods, and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into three categories: complex, high-mannose type, and intermediate. The complex glycopeptides probably contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core (Man)₃(GlcNAc)₂ peptide. The high-mannose-type glycans were estimated to contain at least five mannose units attached to two N-acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The results suggested that the intermediate glycopeptides contained a mannosyl core. In about half of the molecules, one branch appeared to be terminated in mannose, and one appeared to be terminated in N-acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. Electron microscopic data suggest that the virions bud into smooth-surfaced cisternae in the Golgi region. The relative amounts of [³H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62, and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated [³⁵S]methionine-labeled G1 and G2 proteins suggested that most of the complex and intermediate chains were attached to G1 and that most of the high-mannose-type chains were attached to G2.     

Mechanism of Inhibition of Jack Bean alpha-Mannosidase by Swainsonine

The indolizidine alkaloid, swainsonine, was previously shown to be a potent inhibitor of lysosomal and jack bean α-mannosidase (Dorling, Huxtable, Colegate 1980 Biochem J 191: 649-651). We examined the effects of various concentrations of this alkaloid on a number of commercially available glycosidases and found swainsonine to be quite specific for α-mannosidase (50% inhibition at 1-5 × 10⁻⁷ molar). Optimum inhibition was observed after a 2-minute preincubation of enzyme and inhibitor. Lineweaver-Burk plots of substrate concentration versus velocity in the presence of various amounts of swainsonine showed considerable curvature at high substrate concentrations, suggesting that swainsonine may be a competitive inhibitor that binds tightly to the enzyme and is only slowly removed. Periodate oxidation of swainsonine completely destroyed its inhibitory activity.     

2-Acylamido Analogues of N-Acetylglucosamine Prime Formation of Chitin Oligosaccharides by Yeast Chitin Synthase 2

Chitin, a homopolymer of β1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N′-diacetylchitobiose (GlcNAc₂) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc₂ and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc₂ as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues.     

Purification and Properties of a Glycoprotein Processing alpha-Mannosidase from Mung Bean Seedlings

The microsomal fraction of mung bean seedlings contains mannosidase activities capable of hydrolyzing [³H]mannose from the [³H]Man₉GlcNAc as well as for releasing mannose from p-nitrophenyl-α-d-mannopyranoside. The glycoprotein processing mannosidase was solubilized from the microsomes with 1.5% Triton X-100 and was purified 130-fold by conventional methods and also by affinity chromatography on mannan-Sepharose and mannosamine-Sepharose. The final enzyme preparation contained a trace of aryl-mannosidase, but this activity was inhibited by swainsonine whereas the processing enzyme was not. The pH optimum for the processing enzyme was 5.5 to 6.0, and activity was optimum in the presence of 0.1% Triton X-100. The enzyme was inhibited by ethylenediaminetetraacetate while Ca²⁺ was the most effective cation for reversing this inhibition. Mn²⁺ was considerably less effective than Ca²⁺ and Mg²⁺ was without effect. The processing mannosidase was inhibited by α1,2- and α1,3-linked mannose oligosaccharides (50% inhibition at 3 millimolar), whereas free mannose and α1,6-linked mannose oligosaccharides were ineffective. Mannosamine was also an inhibitor of this enzyme. The aryl-mannosidase and the processing mannosidase could also be distinguished by their susceptibility to various processing inhibitors. The aryl-mannosidase was inhibited by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol but not by deoxymannojirimycin or other inhibitors, while the processing mannosidase was only inhibited by deoxymannojirimycin. The processing mannosidase was incubated for long periods with [³H]Man₉GlcNAc and the products were identified by gel filtration. Even after a 24 hour incubation, the only two radioactive products were Man₅GlcNAc and free mannose. Thus, this enzyme appears to be similar to the animal processing enzyme, mannosidase I, and is apparently a specific α1,2-mannosidase.     

Effects of the α-mannosidase inhibitors, 1,4-dideoxy-1,4-imino-d-mannitol and swainsonine,on glycoprotein catabolism in cultured macrophages

Thioglycollate-stimulated murine peritoneal macrophages were cultured for eight days in the presence of swainsonine, or 1,4-dideoxy-1,4-imino-d-mannitol (DIM), or both of these competitive -mannosidase inhibitors together. Analysis of accumulated high-mannose oligosaccharides by reversed phase HPLC after perbenzoylation revealed that DIM- and DIM-plus swainsonine-treated macrophages contained larger amounts of Man₇GlcNAc, Man₈GlcNAc and Man₉GlcNAc, while swainsonine-treated macrophages contained relatively more Man₃GlcNAc and Man₅GlcNAc. These results are consistent with the known inhibitory effects of DIM and swainsonine on Golgi mannosidases I and II, respectively, and on lysosomal -mannosidase. Depletion of stored oligosaccharides to control values was complete within seven days of terminating swainsonine treatment.     

The periodate oxidation of bovine bone sialoprotein, and some observations on its structure

1. Bovine bone sialoprotein (mol.wt. 23000) contains N-acetylneuraminic acid and N-glycollylneuraminic acid, fucose, galactose, mannose, N-acetylgalactosamine and N-acetylglucosamine residues in the form of a very small number, perhaps one, of highly branched oligosaccharide structures linked covalently to peptide. 2. Periodate oxidation of the sialoprotein results in quantitative destruction only of the sialic acid and fucose residue consistent with the earlier findings of their positions as terminal groups. 3. Terminal sialic acid residues are attached to galactopyranose residues by 2,3-linkages, and to some N-acetylgalactosamine residues (at C-6). 4. Sequential Smith degradation indicates that N-acetylgalactosamine residues may be present as points of branching (linked in C-1, C-3 and C-6) and N-acetylglucosamine residues are located in the inner part of the structure, adjacent to the carbohydrate–peptide bond(s). 5. Mannose residues appear to be linked in the 1,3-positions.     

Partial purification and characterization of the glucosidases involved in the processing of asparagine-linked oligosaccharides

A glucosidase preparation with activity toward certain glucose-containing oligosaccharides was partially purified from calf liver membranes by Triton X-100 solubilization and DEAE-cellulose and hydroxylapatite chromatography. The enzyme preparation hydrolyzed the glucose residues from (glucose)₁,(mannose)₉(N-acetylglucosamine)₁, and (glucose)₂(mannose) ₉(N-acetylglucosamine)₁ but was totally inactive toward (glucose)₃(mannose)₉(N-acetylglucosamine) ₁. In contrast, crude membrane preparations of the calf liver were active toward all three substrates. The partially purified enzyme had a pH optimum of 6.7 and was very unstable in the absence of added 20% glycerol. The rate of glucose release from the one-and two-glucose-containing oligosaccharides was significantly decreased when four or five of the mannose residues were first removed from the substrate. The release of glucose from (glucose)₁(mannose)₉(N-acetylglucosamine)₁ was inhibited by p-nitrophenyl-α-d-glucoside much more effectively than by p-nitrophenyl-β-d-glucoside, suggesting that this glucose residue may be linked α to the mannose residue. We conclude that during oligosaccharide processing at least two different glucosidases are involved in glucose removal.     

The high mannose oligosaccharide of phytohemagglutinin is attached to asparagine 12 and the modified oligosaccharide to asparagine 60

Phytohemagglutinin, the lectin of the common bean Phaseolus vulgaris, has a high mannose and a modified (fucosylated) oligosaccharide on each polypeptide. Fractionation by high performance liquid chromatography of tryptic digests of [³H]fucose or [³H]glucosamine labeled phytohemagglutinin, followed by amino acid sequencing of the isolated glycopeptides, shows that the high mannose oligosaccharide is attached to Asn¹² and the modified oligosaccharide to Asn⁶⁰ of the protein. In animal glycoproteins, high mannose chains are rarely found at the N-terminal side of complex chains.     

The position of the oligosaccharide side-chains of phytohemagglutinin and their accessibility to glycosidases determines their subsequent processing in the Golgi

Phytohemagglutinin (PHA), the glycoprotein lectin of Phaseolus vulgaris has two types of asparagine-linked oligosaccharides per polypeptide: a high-mannose chain with the formula (Man)8-9(GlcNAc)2 on Asn12 and a modified chain with fewer mannose residues and additional fucose and xylose residues on Asn60. Glycosylation of PHA is a cotranslational process, which occurs in the endoplasmic reticulum, and newly synthesized PHA has two high-mannose chains. Transport of PHA to the protein bodies via the Golgi complex is accompanied by the modification of one of the two high-mannose chains. Why is only one chain modified, while the other remains in the high-mannose configuration? By determining the effect of digestion with various glycosidases (alpha-mannosidase, endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F) on native and denatured PHA we obtained evidence consistent with the interpretation that the accessibility of oligosaccharide chains to modifying enzymes is of major importance in determining whether a high-mannose chain becomes modified or not. The high-mannose chain of mature undenatured PHA is only partially accessible to glycosidases, while PHA obtained from the endoplasmic reticulum has one high-mannose chain, which is readily accessible to alpha-mannosidase and endoglycosidases H and F. We show that this readily accessible chain is in the same position on the polypeptide (Asn60) as the modified oligosaccharide on mature PHA. Thus, accessibility of the oligosaccharide side-chains to processing enzymes in the Golgi determines whether a particular oligosaccharide side-chain is processed or not.     

Determination of the structure of the carbohydrate chains of acid α-glucosidase from human placenta

Acid α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) from human placenta (70 and 76 kDa) was found to contain 4 N-glycosidic carbohydrate chains per molecule. Sugar analysis of purified enzyme revealed the presence of mannose, N-acetylglucosamine and fucose at a molar ratio of 5.0:2.0:0.6. In addition, trace amounts of galactose and N-acetylneuraminic acid were detected. The sugar chains were liberated from the polypeptides by the hydrazinolysis procedure and subsequently fractionated by gel filtration and HPLC. Purified compounds were investigated by 500-MHz ¹H-NMR spectroscopy. Oligomannoside-type chains of intermediate size, e.g., Man₅GlcNAcGlcNAc-ol and Man₇GlcNAcGlcNAc-ol, and N-type chains of smaller size e.g., Man_2–3GlcNAc[Fuc]_0–1GlcNAc-ol, were demonstrated to be present at a ratio of 2:3. In addition, a small amount of sialylated N-acetyllactosamine-type chains has been found. The possible biosynthetic route of the fucose-containing small-size chains is discussed.     

Structure of the "keratosulfate-like" material in liver from a patient with G M1 -gangliosidosis ( -D-galactosidase deficiency)

Large amounts of a glycopeptide containing galactose, $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and threonine in the ratio 4:3:1:1, together with smaller amounts of mannose, fucose, sialic acid, sulfate, serine, and other amino acids were isolated from the liver of a patient with G_M1-gangliosidosis. Treatment with mild alkali and sodium borohydride indicated an $\text{O}$-glycosidic linkage between $\text{N}$-acetylgalactosamine and threonine. All the hexosamine residues were resistant to sodium metaperiodate whereas 2 out of 4 D-galactose residues were destroyed. Further studies indicated that one of the galactose residues was 1→3 linked to $\text{N}$-acetylgalactosamine (as in G_M1) and the other 1→4 linked to $\text{N}$-acetylglucosamine as found in skeletal keratosulfate.     

Glycan-Dependent Immunogenicity of Recombinant Soluble Trimeric Hemagglutinin

Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA₃) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA₃ glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH5₃) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH5₃ preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man₉GlcNAc₂ moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man₅GlcNAc₂ moieties derived from HEK293S GnTI(−) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(−) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.     

Effect of swainsonine, an inhibitor of glycoprotein processing, on cultured mammalian cells

Swainsonine is an indolizidine alkaloid that inhibits glycoprotein processing by inhibiting mannosidase II. Thus, cells grown in the presence of this alkaloid exhibit a decreased amount of complex types of oligosaccharides at their cell surface, and instead have hybrid types of structures. Since this compound could be useful for studying functional roles of glycoproteins, it was important to determine whether it affected the growth of mammalian cells in culture, and whether it was cytotoxic to these cells. At levels of up to 1 μg/ml, swainsonine did not affect the growth rate of Madin-Darby canine kidney (MDCK) cells, Chinese hamster ovary (CHO), simian virus-181 (SV-101), B-16 melanoma, or intestine 407 cells, as measured by the increase in cell numbers over a 5-day period. There was also no apparent change in cell size or cell shape in cells grown in the presence of this inhibitor. Swainsonine also did not appear to be cytotoxic, nor to cause alterations in cell morphology, as evidenced by comparison of thin sections of normal and swainsonine-grown cells in the electron microscope. Since alterations in the oligosaccharide chains of cell surface glycoproteins could greatly affect cell surface properties, we examined the binding of various lectins and bacteria to cells grown in swainsonine as a measure of changes in their cell surface carbohydrates. Thus, when MDCK cells, CHO cells, or B-16 melanoma cells were grown for several days in the presence of swainsonine (100–500 ng/ml), these cells showed a 50–100% increase in their ability to bind [³H]concanavalin A, and a substantial decrease in the binding of [³H]wheat germ agglutinin. These alterations suggested an increase in high-mannose (or hybrid) types of receptors and a decrease in the complex types. The adhesion of E. coli B-886, a bacterium that binds to high-mannose glycoproteins, was also increased 1.5-to twofold, in cells grown in swainsonine. However, the binding of E. coli SS-142, another bacterial strain that does not bind to high-mannose receptors, was not altered by growth in swainsonine. In addition to the decrease in wheat germ agglutinin binding, another indication of a decrease in complex chains was the finding that CHO cells grown in swainsonine were more resistant to the toxic effects of the lectin, ricin. This increased resistance could be measured microscopically by the decrease in the number of cells remaining attached to the plates, or by the inhibition of amino acid incorporation, at various ricin concentrations. The effect of swainsonine on the incorporation of amino acids and sugars into protein was also examined. When MDCK cells were grown overnight in swainsonine (1 μg/ml), or were incubated in the alkaloid for several hours before the start of the experiment, there was no alteration in the incorporation of [³H]leucine or [³H]proline into protein. There was, however, a significant inhibition in the incorporation of [³H]fucose, [³H]glucosamine, and [³H]galactose caused by this alkaloid. Fucose incorporation was decreased by about 40%, glucosamine by about 40 or 50%, and galactose by about 50%. In many cases (but not all), the incorporation of mannose was enhanced about 20–30% in cells grown in swainsonine.     

Synthesis and processing of cellulase from ripening avocado fruit

The biosynthesis and processing of cellulase from ripening avocado fruit was studied. The mature protein is a glycoprotein, as judged by concanavalin A binding, with a molecular weight of 54,200. Upon complete deglycosylation by treatment with trifluoromethane sulfonic acid the mature protein has a molecular weight of 52,800 whereas the immunoprecipitated in vitro translation product has a molecular weight of 54,000. This result indicates that cellulase is synthesized as a large molecular weight precursor, which presumably possesses a short-lived signal peptide. A membrane-associated and heavily glycosylated form of the protein was also identified. This putative secretory precursor was enzymically active and the carbohydrate side chains were sensitive to endoglycosidase H cleavage. Results of partial endoglycosidase H digestion suggest that this precursor form of the mature glycoprotein possesses two high-mannose oligosaccharide side chains. The oligosaccharide chains of the mature protein were insensitive to endoglycosidase H cleavage, indicating that transport of the membrane-associated cellulase to the cell wall was accompanied by modification of the oligosaccharide side chains. The presence of a large pool of endoglycosidase H-sensitive membrane-associated cellulase (relative to an endoglycosidase H-insensitive form) suggest that transit of this protein through the Golgi is rapid relative to transit through the endoplasmic reticulum.