Swainsonine Causes the Production of Hybrid Glycoproteins by Human Skin Fibroblasts and Rat Liver Golgi Preparations*

The synthesis of glycoproteins containing N-linked complex oligosaccharides is blocked by swainsonine at the step catalyzed by Golgi mannosidase I1 (Tulsiani, D. R. P., Harris, T. M., and Touster, 0. (1982) J. Biol Chem. 257, 7936-7939). Accordingly, hybrid glycoproteins might be produced in the presence of swain- sonine. In this report, we demonstrate that swainsonine causes human skin fibroblasts to synthesize such glycoproteins. In control fibroblasts, there were ap- proximately equal amounts of complex and high mannose glycoproteins. In the presence of swainsonine (10 wg/ml), most of the complex glycoproteins were re- placed by hybrid types. The principal oligosaccharide had the following structure: A smaller amount of the asialo hybrid was also pro- duced. The structure of the hybrid was established by Bio-Gel P-4 fractionation of oligosaccharides produced by endoglycosidase H treatment of pronase-derived glycopeptides, followed by examination of the suscep- tibility of the oligosaccharide to glycohydrolases and by its adsorbability to serotonin-Sepharose 4B. The same hybrid oligosaccharide was produced efficiently by 8 h at 37 "C. Neuraminidase digestions were carried out for 24 h at 37 "C in a total volume of 50 pl containing 100 mM sodium acetate buffer, pH 5.5, and 0.25 unit of enzyme. Protein was assayed by the fluorometric method of Anderson and Desnick (26) using bovine serum albumin as standard.

A smaller amount of the asialo hybrid was also produced. The structure of the hybrid was established by Bio-Gel P-4 fractionation of oligosaccharides produced by endoglycosidase H treatment of pronase-derived glycopeptides, followed by examination of the susceptibility of the oligosaccharide to glycohydrolases and by its adsorbability to serotonin-Sepharose 4B. The same hybrid oligosaccharide was produced efficiently by rat liver Golgi membranes in the presence of (['HI Man),GlcNAc, UDP-GlcNAc, UDP-Gal, CMP-NeuAc, and swainsonine. Golgi mannosidase I1 had no action on the hybrid oligosaccharide, and little action on asialo hybrid, but both were converted to the mannosidase I1 substrate, GlcNAcManaGlcNAc, by appropriate treatment with neuraminidase and &galactosidase. Jack bean a-D-mannosidase gave the expected yields of free mannose from the various oligosaccharides studied in this work. Swainsonine should be useful in investigating the role of oligosaccharide structure of glycoproteins because of its ability to alter the oligosaccharide, The biosyntheses of glycoproteins containing asparaginelinked high mannose and complex oligosaccharides initially * This investigation was supported in part by Grant GM 26430 and by Biomedical Research Support Grant S07-RR07201 from the National Institutes of Health, United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. follow a common pathway which then diverges to produce the two classes of glycoproteins (1)(2)(3)(4)(5)(6). When Mane derivatives have been converted to Man5 derivatives by al-2-mannosidases (7, 8), the latter are N-acetylglucosaminylated to form GlcNAcMan5 species. Golgi mannosidase I1 then removes the two terminal a1-3-and al- 6-mannosyl residues to yield GlcNAcMan3 derivatives (7-101, which are then converted by several glycosyltransferases to complex oligosaccharides. We recently reported that swainsonine is a potent inhibitor of Golgi mannosidase I1 and blocks mannosyl cleavage at the GlcNAcMan, stage (11). Since this oligosaccharide possesses one branch theoretically susceptible to galactosyl-and sialyltransferases, oligosaccharides of the hybrid type could be produced. Oligosaccharides of this type have been isolated from ovalbumin (12)(13)(14), bovine rhodopsin (15), mouse myeloma IgM (16, 171, the F, region of human immunoglobulin D (18), avian myeloblastosis-associated viruses (19), and Prague C Rous sarcoma glycoproteins (20).
We now report that human skin fibroblasts partially synthesize glycoproteins containing oligosaccharides of the hybrid type instead of complex oligosaccharides when the cells are cultured in the presence of swainsonine. Similar products are produced in vitro when rat liver Golgi membranes are incubated with appropriate substrates in the presence of the inhibitor.
Swainsonine-Cells were grown to a density of 1.0 to 1.2 mg of cell protein/flask. They were then carefully rinsed with 5 ml of Waymouth MAB 87/3 medium, as formulated in the catalogue of GIBCO (Grand Island, NY), with added 5% dialyzed calf serum and 5 mg of glucose/ ml medium. The flasks were divided into two groups. The medium in control flasks was replaced with 5 ml of fresh medium (see above). The medium in experimental flasks was replaced by 5 ml of fresh medium supplemented with 10 pg of swainsonine/ml of medium. After 24 h at 37 "C, the culture medium from control and experimental flasks was removed by aspiration, and the cells were rinsed twice with 5-ml portions of glucose-free medium. Each flask of the control cells was mixed with 5 ml of the above Waymouth medium containing 5% dialyzed fetal calf serum, 0.1 mg of glucose/ml of medium, and 0.1 mCi of ~-[2-~H]mannose/ml of medium. Each flask of the experimental cells was mixed with 5 ml of the same medium per flask supplemented with 10 pg of swainsonine/ml of medium. The cells were incubated for 24 h at 37 "C.
Harvesting of Cells-The cells from control and experimental culture flasks were detached by trypsinization and collected in tubes by centrifugation at 400 X g for 3 min. The cells were washed with 5and 2.5-ml portions of cold 0.9% NaCl containing 1% bovine serum albumin and twice with 5-ml portions of 0.25 M sucrose in 10 mM Tris-C1 buffer, pH 8.0.
Preparation and Pronase Digestion of Cellular Glycoproteins-The cell pellets after the second sucrose wash were dried in a Bio-Dryer and extracted three times with 1.5-ml portions of chloroform/methanol/water (1010:3, v/v/v). The insoluble residue, dried under NP, contained labeled glycoproteins (25). The residue from each flask was suspended in 0.2 ml of 100 mM Tris-C1, pH 7.2, containing 10 mM CaCIZ. Pronase-CB protease (2 mg) was added to each tube, and the mixtures were incubated at 37 "C under a few drops of toluene for 3 days, with additions of pronase (1 mg) after 24 h and 48 h. The pronase was inactivated by heating the mixture at 100 'C for 5-7 min, and the labeled glycopeptides were fractionated on a column of Bio-Gel P-4.
Endoglycosidase H Treatment of Glycopeptides-The [3H]mannoselabeled glycopeptide fractions were pooled, evaporated to dryness, suspended in 1 ml of distilled water, and dried again. Finally, the glycopeptides were suspended in 25 pl of 100 mM sodium citrate buffer, pH 5.5, containing 0.27% sodium azide and incubated with 0.025 unit of Endo H in a total volume of 50 pl. After 24 h at 37 "C, additional Endo H (0.025 unit) was added, and the incubation was carried out for an additional 24 h at 37 "C. At the end of the incubation, the reaction mixture was inactivated by heating at 100 "C for 5-7 min, and the oligosaccharides were fractionated on a column of Bio-Gel P-4.
Enzymatic Digestion of Oligosaccharides-Jack bean exo-b-N-acetylglucosaminidase and a-D-mannosidase as well as Golgi mannosidase I1 digestions were carried out as described previously (8). Jack bean 8-D-galactosidase digestion was carried out in a total volume of 50 pl containing 100 mM sodium citrate buffer, pH 3.5, and 0.25 unit of the enzyme. Additional enzyme (0.25 unit) was added after 16 h at 37 "C, and the incubation was carried out for an additional 8 h at 37 "C. Neuraminidase digestions were carried out for 24 h at 37 "C in a total volume of 50 pl containing 100 mM sodium acetate buffer, pH 5.5, and 0.25 unit of enzyme.
Protein was assayed by the fluorometric method of Anderson and Desnick (26) using bovine serum albumin as standard.  (11). In the studies reported below, ([3H]Man)6GlcNAc was (a) incubated with Golgi membrane suspension in the presence and absence of 10 /IM swainsonine and nucleotide sugars for various time periods, (b) the reaction mixtures were fractionated on Bio-Gel P-4 columns, and (c) the structures of isolated oligosaccharide products inferred from their elution positions were subjected to confirmation by digestion of the oligosaccharides with specific glycohydrolases.

Production of Hybrid Oligosaccharides by Rat Liver Golgi
When ([3H]Man)aGlcNAc was incubated with Golgi membrane suspension in the absence (Fig. lA) and presence of 10 FM swainsonine ( Fig. 2 A ) , nearly all the oligosaccharide was recovered unchanged from the P-4 column. Swainsonine had no effect on the processing of this oligosaccharide unless UDP-GlcNAc was also present. In the absence of swainsonine, this nucleotide caused this production of free [3H]mannose and G~C N A C~( [~H ] M~~)~G~C N A C (Peak 1, Fig. 1B). In the presence of swainsonine, the release of free [3H]mannose was blocked due to inhibition of Golgi mannosidase II; this inhibition caused the accumulation of GlcNAc( [3H]Man)5GlcNAc (Peak 1, Fig. 2B). The  Man)sGlcNAc (-20,000 cpm), 100 mM Na cacodylate buffer, pH 6.2, 5 mM MgCl,, 5 pl of Golgi membrane suspension (8.0 mg of membrane protein/ml of 0.3% Triton X-loo), and additions as indicated below. After incubation for 8 h at 37 "C, the reactions were stopped by heating at 100 "C for 5-7 min, and each mixture was applied to a Bio-Gel P-4 (1 X 214 cm, -400 mesh) equilibrated with 0.1 M acetic acid. The column was eluted with 0.1 M acetic acid at a flow rate of 1.8 ml/h. Fractions (0.6 ml) were collected, and the radioactivity was measured in aliquots as described (8) Fig. 2C), is sensitive to jack bean mannosidase. However, unlike the latter, it is resistant to Golgi mannosidase I1 (Table I), a result suggesting that terminal GlcNAc is required for an oligosaccharide to be cleaved by Golgi mannosidase 11. Treatment of Peak 1, Fig.  2C, oligosaccharide with jack bean @-galactosidase resulted in its elution from a P-4 column at the position expected of GlcNAc( [3H]Man)5GlcNAc. This modified oligosaccharide is now sensitive to both jack bean mannosidase and Golgi mannosidase I1 ( Table I).
The presence of CMP-NeuAc in the incubation mixture of Fig. 2C caused the production of three oligosaccharides (Fig. 20). Peak 1 oligosaccharide has the expected hybrid structure, NeuAcGalGlcNAc( [3H]Man)5GlcNAc, as indicated by (a) its elution position from a P-4 column, (b) its susceptibility to jack bean mannosidase but resistance to Golgi mannosidase I1 (Table I), and (c) study of its degradation by neuraminidase and P-galactosidase. These enzymes cleaved sialyl and galactosyl residues, respectively, and the resulting oligosaccharide eluted from P-4 column at the position expected of GlcNAc( [3H]Man)sGlcNAc. This oligosaccharide was hydrolyzed by both jack bean mannosidase and Golgi mannosidase I1 ( Table I) (Table I). Table I1 shows effect of swainsonine on the in vitro processing of high mannose oligosaccharide as a function of time. It is interesting that when ( [3H]Man)5GlcNAc was incubated mannose was quantitated after separation from 3H-labeled oligosaccharide on a column of Bio-Gel P-2 as described (8) (Table 11). Therefore, under the conditions used, GlcNAc-and sialyltransferases act more rapidly than Galtransferase.
The processing of ([3H]Man)5GlcNAc in the absence of swainsonine but presence of UDP-GlcNAc and UDP-Gal (Fig.  IC) caused the oligosaccharide products to elute in several peaks. These oligosaccharides have not been extensively identified but appear to be complex type, since jack bean mannosidase treatment released a negligible amount of free mannose (-5%). Similarly, the further addition of CMP-NeuAc ( Fig.  1 0 ) caused the ( [3H]Man)5GlcNAc products to elute in several peaks which have not been characterized. However, their resistance to jack bean mannosidase suggests that they are also complex-type oligosaccharides.

G~c N A c (~H ] M~~)~G~~N A c ,
and G~N A C ( P H I M~~)~G~~N A~ by Jack Bean Mannosidme and Golgi Mannosidme ZI-Jack bean mannosidase cleaves all three oligosaccharides efficiently (Fig. 3A). After 24-h incubation at 37 "C, nearly 50% of [3H] mannose (of 60% theoretically possible) was released, a result suggesting that jack bean mannosidase cleaves 3 of the 5 mannosyl residues from the 3 oligosaccharides. Rat liver Golgi   [3H]mannose-labeled processing intermediates produced by rat h e r Golgi membranes The oligosaccharides obtained from the peak fractions shown in Fig. 2 (indicated by the bars) were pooled and dried in a Bio-Dryer; the residues were suspended in HzO and redried. The residues were suspended in 25 p1 of H20 and aliquots were used for the enzymatic digestions. * If the oligosaccharide was incubated with jack bean p-galactosidase (0.5 unit) for 24 h at 37 "C and applied to a P-4 column, the product eluted from the column at the position expected of GlcNAcMandIcNAc. The modified oligosaccharide is susceptible to Golgi mannosidase 11.
The oligosaccharide binds to the serotonin-Sepharose 4B affinity column (see Table IV). When treated with neuraminidase for 24 h at 37 "C and applied to a P-4 column, it elutes from the column at the position expected of GalGlcNAcMan6GlcNAc. The modified oligosaccharide does not bind to the serotonin-Sepharose 4B column.
~~ mannosidase I1 showed negligible activity toward the sialylated and asialo hybrids, but, as expected, this enzyme removed 2 mannosyl residues from GlcNAc( [3H]Man)6GlcNAc (-34% [3H]mannose of 40% theoretically possible (Fig. 3B)). This experiment again indicated the substrate specificity of GoIgi mannosidase 11. Treatment of sialylated hybrid with neuraminidase and then with P-galactosidase, or treatment of asialo hybrid with /3-galactosidase, yielded oligosaccharide which eluted from a P-4 column at the position expected of GlcNAc( [3H]Man)5GlcNAc. As expected, this oligosaccharide was sensitive to both the jack bean mannosidase and Golgi mannosidase 11. Production of Hybrid Glycoprotein by Human Skin Fibroblasts in the Presence of Swainsonine-Fibroblasts were labeled with [3H]mannose and their glycopeptides prepared and fractionated as described under "Experimental Procedures." The control and swainsonine-treated cells incorporated similar amounts of [3H]mannose. The elution profiles of L3H] mannose-labeled glycopeptides of the control (Fig. 4A) and swainsonine-treated cells (Fig. 40) were quite similar, with the exception of a larger radioactive peak between fractions 120 and 140 of the swainsonine-treated cells. However, when the glycopeptides were treated with Endo H and oligosaccharide products were fractionated on a Bio-Gel P-4 column, the oligosaccharide profiles of control cells (Fig. 4A) and swainance to Endo H was also demonstrated by treating fractions sonine-treated cells (Fig. 40) were quite different. Nearly 35% 101-140, Fig. 4 A , with the enzyme and observing that the of the label in glycopeptides from control was unchanged by position of elution of radioactivity from a P-4 column was Endo H treatment (fractions 101-140, Fig. 4A) and therefore unchanged (Fig. 4B). As expected for complex oligosaccharide, apparently contained complex-type oligosaccharide. Resistthe eluted material was resistant to jack bean mannosidase

TABLE I1
Effect of incubation time on the pmduction of oligosaccharides by Golgi membranes in the presence of swainsonine and various nucleotide sugars The standard incubation mixture contained ([3H]Man)sGlcNAc ("20,000 cpm), 100 mM Na cacodylate buffer, pH 6.2,5 mM MgC12, swainsonine (10 PM), 5 pl of rat liver Golgi membrane suspension (8 mg of Golgi membrane protein/ml of 0.4% Triton X-lOO), and nucleotides (10 mM of each) as indicated, in a total volume of 50 pl. After incubation at 37 "C for the time indicated, the reactions were stopped by heating at 100 "C for 5-7 min and each mixture was applied to a calibrated Bio-Gel P-4 column (1 X 214 cm, -400 mesh) equilibrated with 0.1 M acetic acid. The column was eluted with 0.1 M acetic acid at a flow rate of 1.8 ml/h. Fractions (0.6 ml) were collected and the radioactivity was measured in aliquots. The total radioactivity in the fractions containing each oligosaccharide (as in Fig. 2) was calculated.  Susceptibility to jack bean a-smnnosidase of PHlmnnose-labeled processing intermediates from fibroblasts The peak fractions from the control and swainsonine-treated fibroblasts (Fig. 4) were pooled and dried in a Bio-Dryer. The dried oligosaccharides were suspended in water and redried. The residues were finally suspended in 50 ul of water and aliauots containing -3000 cDm were used for each mannosidase digestion.

Experimental (Fig. 4F) % [ s H ] m a n~s e released
Oligosaccharide from Fig. 4 Fig. 4E 48. 8 (60.0) GalGlcNAcMaQGlcNAc Incubations were carried out at 37 "C for 24 h with jack bean mannosidase as described (11). The theoretical Lower levels of [3H]mannose released than theoretically expected is perhaps in part due to some contamination No results are given for control fibroblasts because hybrid oligosaccharides were not observed (see Fig. 4B). dIf the oligosaccharide is incubated with neuraminidase as described under "Experimental Procedures" and then applied to a Bio-Gel P-4 column, the product eluted from the column at the position expected of GalGlc-NAcMan6GlcNAc. This oligosaccharide as well as the oligosaccharide of Peak 2 (Fig. 4E), when incubated with jack bean 8-D-galactosidase as described under "Experimental Procedures" and applied to a Bio-Gel P-4 column, eluted from the column at the position expected of GlcNAcMansGlcNAc. Both sialylated hybrid (Peak 1, Fig. 4E) and desialylated hybrid (Peak 2, Fig. 4E) were not cleaved by Golgi mannosidase 11. However, when sialyl and galactosyl residues were removed by neuraminidase and @-galactosidase treatment, the resulting oligosaccharide (GlcNAcMaGGlcNAc) becomes an excellent substrate for Golgi mannosidase 11. Fig. 4E' value for releasable 13H]mannose for each oligossaccharide is shown in parentheses.

Oligosaccharide from
with GlcMmGlcNAc, which would also be eluted at the position of Peak 1.
On the other hand, the swainsonine-treated cells showed less than 10% of the label eluting between fractions 101 and 140, compared with 35% from control cells (Fig. 4, D uersus  A). When glycopeptide fractions 101-140 of Fig. 4 0 were pooled and treated with Endo H, and the oligosaccharide products were fractionated on a P-4 column, the elution profile ( Fig. 4E) was quite different from the corresponding elution profile observed from control cells (Fig. 4B). The new distinct peaks (Peak 1 and Peak 2, Fig. 4E) were produced by the Endo H treatment, a result indicating that the two peaks are oligosaccharides and not glycopeptides.
The distinctive oligosaccharides from the swainsoninetreated cells (Peaks I and 2, Fig. 4E) were characterized. Peak 1 oligosaccharide was identified as sialylated hybrid with the structure NeuAcGalGlcNAc( [3H]Man)5GlcNAc on the basis of its elution from the P-4 column and treatment with specific glycohydrolases. This oligosaccharide was sensitive to jack bean mannosidase, which cleaved 49% of the 60% theoretically susceptible [3H]mannose (Table 111). However, Golgi mannosidase 11, which cleaves the exposed a1-3and a1-6mannosyl residues from G~C N A C ( M~~)~G I C N A C (8, 11), failed to hydrolyze this oligosaccharide, which contains similarly linked mannosyl residues. Treatment of the oligosaccharide with neuraminidase and @-galactosidase resulted in the elution of oligosaccharide from a P-4 column at the position expected of GlcNAc(Man),GlcNAc (11). This product was sensitive to both jack bean mannosidase and Golgi mannosidase I1 (Table 111). Peak 2, Fig. 4E, was identified as Gal- based upon the same type of evidence as given above. It may be also noted that both control and swainsoninetreated cells showed several high mannose oligosaccharides (Fig. 4, C and F), which were identified by their position of elution from the P-4 column and digestion with jack bean mannosidase ( Table 111). The high mannose oligosaccharides ([3H]Man)9.5GlcNAc made up nearly 70% of total radiolabel, and no difference in the total amount of these oligosaccharides  (-3,500 cpm) in 0.2 ml of water were applied to a serotonin-Sepharose 4B column (0.5 X 6.2 cm) equilibrated with water. After washing the column with 7 ml of water (flow rate, 60 ml/h), it was eluted with 100 mM Tris-C1 buffer, pH 7.2. Fractions (0.5 ml) were collected, and the radioactivity was determined (8). Recovery of the applied radioactivity in the wash (unadsorbed) and eluate (adsorbed) was auantitative. Standard oligosaccharides. See ''Experimental Procedures." When the oligosaccharide was treated with neuraminidase before it was applied to the column, nearly 84% of the oligosaccharide was not adsorbed. The remainder was eluted with Tris-C1. ~~ was observed between control and swainsonine-treated cells (Fig. 4, C and F).
Serotonin-Sepharose 4B Column Chromatography-That the hybrid oligosaccharide produced by both Golgi membranes (Peak I, Fig. 20) and fibroblasts (Peak 1, Fig. 4E) contained terminal sialic acid was further substantiated by the binding of these oligosaccharides to serotonin-Sepharose 4B, an affinity resin recently reported to bind sialic acid-containing glycoproteins (22). As shown in Table IV, only the two oligosaccharides presumed to be sialic acid-containing hybrids ad-sorbed to the resin. Prior treatment of the two oligosaccharides with neuraminidase led to loss of affinity for the adsorbent, as expected from the lack of adsorbability of Galterminal oligosaccharide (Table IV).

DISCUSSION
The inhibition by swainsonine of the synthesis of glycoproteins containing asparagine-linked complex oligosaccharides, first demonstrated by Elbein and his co-workers (27, 28), was subsequently shown in our laboratory to be a consequence of the inhibition of Golgi mannosidase I1 (11). We now demonstrate that, in the presence of swainsonine, human skin fibroblasts and rat liver Golgi membranes produce hybrid-type oligosaccharides. These biantennary oligosaccharides possess only on one branch the additional sugars (GlcNAc, Gal, NeuAc) often found in complex oligosaccharides, the other mannose-containing branch remaining unsubstituted. Similar hybrid-type oligosaccharides have been reported in several glycoproteins (12)(13)(14)(15)(16)(17)(18)(19)(20). However, all but one of these oligosaccharides contain a bisecting GlcNAc on the innermost man-nosy1 residue (12)(13)(14)(15)(16)(17)(18)(19). Only the glycoproteins of Prague C Rous sarcoma virus (20) contain hybrid-type oligosaccharides identical with the one reported herein, in which a bisecting GlcNAc has not been added.
In the experiments reported in this paper, oligosaccharides of the glycoproteins of control fibroblasts were approximately 56% complex type and 44% high mannose type, whereas the swainsonine-treated cells contained 16% complex, 23% sialylated hybrid, and 9% asialo hybrid. These estimates are based upon the [3H]mannose found in the various fractions ( Fig. 4) and the differing mannose content of the different types of oligosaccharide. The main forms of high mannose oligosaccharide in both types of cells were MansGlcNAc and Man7GlcNAc. With the i n uitro Golgi membrane system, the total yield of the two hybrids was over 70% (Table 11).
The evidence for the production of hybrid glycoproteins was primarily based on the properties of the oligosaccharides produced from glycopeptides by Endo H treatment. The position of elution of the oligosaccharides from P-4 columns, the responses of the oligosaccharides to digestion with jack bean a-D-mannosidase, and the use of serotonin-Sepharose 4B affinity resin indicated that the principal new species produced by swainsonine-treated cells is hybrid-type oligosaccharide possessing one branch with the structure NeuAc-GalGlcNAcMan, and another branch containing terminal al-3-and al-6-mannosyl residues.
Although the hybrid oligosaccharide contains 2 terminal mannosyl residues, it was resistant to Golgi mannosidase I1 unless sialyl and galactosyl residues were first removed by neuraminidase and &galactosidase treatment. It therefore appears that a terminal GlcNAc residue on one branch is required for recognition of substrate by the enzyme. Two additional observations may be noted. First, although the sialylated hybrid possesses the structure of complex oligosaccharide on one branch, it behaved like high mannosetype oligosaccharide in its sensitivity to Endo H, an observation similar to that reported for Rous sarcoma virus glycoproteins (20). Second, the studies demonstrated the usefulness of the affinity resin serotonin-Sepharose 4B (22), which is highly specific for the adsorption of sialic acid-containing oligosaccharide. The sialylated hybrids produced in vitro and in viuo bind to the resin, but the asialo oligosaccharides do not.
Swainsonine has been isolated from Swainsona sp. (29-31) and locoweed (32), plants that induce in livestock a condition resembling the lysosomal storage disease mannosidosis. The causes of the neurological symptoms, the numerous intracel-lular vacuoles, and the accumulation of mannose-rich oligosaccharides have not bee'n established, although the fact that swainsonine is a potent inhibitor of lysosomal a-D-mannosidase has led to the supposition that the alkaloid is the specific cause of the mannosidosis (30, 31, 33). However, since the administration of swainsonine to rats induces large increases in lysosomal a-mannosidase of the liver and brain of rats,2 there is question as to whether swainsonine is in fact primarily responsible for all of the major symptoms of animals ingesting swainsonine-containingplants. Although swainsonine administration to rats raises the level of lysosomal a-mannosidases, it markedly lowers the activity of Golgi mannosidase I1 but not of other Golgi enzymes tested.' This effect of swainsonine on mannosidase I1 in uiuo, which is consistent with the in uitro effects of the alkaloid on this enzyme (ll), obviously is the cause of the formation of hybrid glycoproteins.
The biological consequences of the production of hybrid glycoproteins in place of complex glycoproteins are difficult to predict at this time. The function of the oligosaccharides of glycoproteins is a subject of considerable contemporary interest, with suggestive evidence on functions deriving in part from the discovery of several carbohydrate-specific receptors on cells (34). The chemical and biological properties of glycoproteins are, in many cases, affected by inhibitors which cause alterations in the structure of the oligosaccharide moieties (35). In a study involving swainsonine, Elbein et al. (28) reported that influenza virus produced in chick embryo fibroblasts in the presence of the alkaloid has normal infectivity in spite of the abnormality of its glycoprotein. Tunicamycin, on the other hand, prevents the production of infectious virus particles (36). Since swainsonine induces the formation of hybrid oligosaccharides in both human fibroblasts and in the Golgi membrane system described herein, the substance should have wide applicability in investigations of glycoprotein processing, transport, and function. It also seems likely that the pathological effects of swainsonine will in part be explained by its effect on glycoproteins.