The Pyrrolidine Alkaloid, 2,5-Dihydroxymethyl-3,4-dihydroxypyrrolidine, Inhibits Glycoprotein Processing*

2 , 5 - 3 ,4 - dihydroxypyrrolidine (DMDP) is a pyrrolidine alkaloid that was isolated from the plant, Lonchocarpus sericeus. In the present study, DMDP was tested as an inhibitor of glycoprotein processing. MDCK cells were infected with influenza virus and the virus was raised in the presence of various amounts of DMDP. The glycoproteins were by the of or the The virus was isolated by differ- ential centrifugation and treated with Pronase to obtain glycopeptides. These glycopeptides were isolated by chromatography on Bio-Gel P-4, then digested with endoglucosaminidase H (Endo H) and rechromato- graphed on the Bio-Gel P-4 column. In the control virus, more than 70% of the glycopeptides were resist- ant to Endo H and were previously characterized as complex types of oligosaccharides. The remaining 20- 26% are sensitive to Endo H and are of the high-mannose type. However, in the presence of (250 pg/ml), more than 80% glycopeptides digestion by Endo H. The oligosaccharide released this like a hexosell-lzGlcNAc a calibrated column of Bio-Gel P-4, and only slightly susceptible to a-mannosidase treatment. This oligosaccharide was also labeled in the glucose moieties by the virus in [ l-’H]galactose in the presence of DMDP. Following isolation, the oligosaccharide was subjected to complete methylation. Acid hydrolysis of the methylated oligosaccharide gave three methylated glucose derivatives, corresponding to 2,3,4,6-tetra-methylglucose, 3,4,6-trimethylglucose, and 2,4,6-tri-methylglucose in almost equal amounts. These data indicate that the oligosaccharide is a GlcsManGa- GlcNAc and that DMDP inhibits glucosidase 1. Similar results were obtained with the cellular glycoproteins. DMDP did not inhibit the incorporation of [‘Hlleucine into protein in MDCK cells, nor did it inhibit virus production as measured by plaque counts or hemagglutination assays. DMDP did cause some inhibition of mannose incorporation into the lipid-linked monosaccharides, but incorporation into lipid-linked oligosac- charides was not greatly affected, and incorporation into protein was stimulated. These results suggest that the pyrrolidine alkaloids are a new class of processing inhibitors.

2 , 5 -Dihydroxymethyl-3 , 4dihydroxypyrrolidine (DMDP) is a pyrrolidine alkaloid that was isolated from the plant, Lonchocarpus sericeus. In the present study, DMDP was tested as an inhibitor of glycoprotein processing. MDCK cells were infected with influenza virus and the virus was raised in the presence of various amounts of DMDP. The glycoproteins were labeled by the addition of [2-'H]mannose or [l-'Hlgalactose to the medium. The virus was isolated by differential centrifugation and treated with Pronase to obtain glycopeptides. These glycopeptides were isolated by chromatography on Bio-Gel P-4, then digested with endoglucosaminidase H (Endo H) and rechromatographed on the Bio-Gel P-4 column. In the control virus, more than 70% of the glycopeptides were resistant to Endo H and were previously characterized as complex types of oligosaccharides. The remaining 20-26% are sensitive to Endo H and are of the highmannose type. However, in the presence of DMDP (250 pg/ml), more than 80% of the glycopeptides are susceptible to digestion by Endo H. The oligosaccharide released by this treatment sized like a hexosell-lzGlcNAc on a calibrated column of Bio-Gel P-4, and was only slightly susceptible to a-mannosidase treatment. This oligosaccharide was also labeled in the glucose moieties by growing the virus in [ l-'H]galactose in the presence of DMDP. Following isolation, the oligosaccharide was subjected to complete methylation. Acid hydrolysis of the methylated oligosaccharide gave three methylated glucose derivatives, corresponding to 2,3,4,6-tetramethylglucose, 3,4,6-trimethylglucose, and 2,4,6-trimethylglucose in almost equal amounts. These data indicate that the oligosaccharide is a GlcsManGa-GlcNAc and that DMDP inhibits glucosidase 1. Similar results were obtained with the cellular glycoproteins. DMDP did not inhibit the incorporation of ['Hlleucine into protein in MDCK cells, nor did it inhibit virus production as measured by plaque counts or hemagglutination assays. DMDP did cause some inhibition of mannose incorporation into the lipid-linked monosaccharides, but incorporation into lipid-linked oligosaccharides was not greatly affected, and incorporation into protein was stimulated. These results suggest that the pyrrolidine alkaloids are a new class of processing inhibitors.
of Health Grants HL-17783 and GM-31355 to A. D. E. The costa of * The studies reported here were supported by National Institutes publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The biosynthesis of the complex types of N-linked oligosaccharides involves a series of processing reactions whereby sugars are trimmed from the high-mannose types of structures, and other sugars are added to the oligosaccharide chains (1). Thus, the initial oligosaccharide that is transferred to protein is a GlcsMansGlcNAcz, and this structure is rapidly trimmed by the removal of all three glucose residues by at least two membrane-bound glucosidases, called glucosidase I and glucosidase I1 (2)(3)(4)(5)(6). Following the removal of glucose, mannosidase I may remove all four of the al,2-linked mannoses to give the Mana1,3(Manal,6)Manal,6(Manal,3)-ManpGlcNAc2-protein (7-10). A GlcNAc is then added to the mannose that is linked a1,3 to the &linked mannose (11,12), and this GlcNAc appears to be the signal for mannosidase I1 to remove the d , 3 -and al,6-linked mannoses (13,14). These reactions result in the formation of a GlcNAc-Manal,3(Manal,6)Man@GlcNAc2-protein, which can be further elongated to complex structures by the addition of GlcNAc, galactose, sialic acid, and fucose (15).
One mechanism for studying the biosynthesis and function of the N-linked complex chains is with the use of inhibitors that block specific steps in the processing pathway. A number of such inhibitors are now known and these have proven to be valuable tools in a variety of studies (16,17). In this report, we describe some studies with 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, an analog of 8-D-fructofuranose, that demonstrate that this compound (DMDP') is an inhibitor of glycoprotein processing. DMDP was first isolated from the leaves of Derris elliptica (18). In the present study, DMDP was isolated from the seeds o f the closely related plant, Lonchcarpus sericeus, and shown by NMR studies to be identical to the compound from Derris elliptica.2 In the studies described here, we tested DMDP as a processing inhibitor of the influenza virus hemagglutinin, and found that it prevented the formation of complex chains and gave rise to a new glycopeptide(s) having GlcaMan7-9GlcNAc2 structures. These results indicate that DMDP is an inhibitor of glucosidase I. Thus, DMDP represents one member of a new class of processing inhibitors, the pyrrolidine alkaloids. Such compounds may be valuable tools for functional and biosynthetic studies on N-linked glycoproteins. The structure of DMDP is shown in Structure 1.
Comparisons of the compound from the leaves of Derris elliptica (18) with that from Lonchocarpus sericeus by NMR analysis showed that they were identical and had the structure 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine. We thank Dr. G. W. Fleet, Oxford University for these analysis. This data will be published elsewhere.

DMDP Inhibits Glycoprotein
Processing L H STRUCTURE 1

EXPERIMENTAL PROCEDURES
MDCK cells were grown in modified Eagle's medium containing 10% fetal calf serum, and the confluent monolayers were infected with the NWS strain of influenza virus (19). After infection, the cells were placed in 2% minimal Eagle's medium, and various amounts of DMDP were added, from 1 pg/ml to 250 pg/ml. The cultures were allowed to incubate for 2 to 3 h to allow the inhibitor to take effect, and then either [2-3H]mannose or [l-3H]galactose was added to label the viral glycopeptides. In some experiments, the cells were harvested after 3 h of incubation in the isotopes in order to determine the effect of DMDP on the formation of lipid-linked saccharide intermediates. In these cases, the monolayers were removed from the plates by scraping and the cells were isolated by centrifugation and washed with CHC13:CH30H:H20 (1:l:l) and with CHCkCH30H:HzO several times with saline. The cell pellets were extracted sequentially (10103) to isolate the lipid-linked monosaccharides and the lipidlinked oligosaccharides (20). Aliquots of these lipids were counted to determine their radioactive content. The cell pellets, remaining after these extractions, were digested with Pronase to obtain the cellular glycoproteins.
However, in most experiments, the infected cells were allowed to incubate with the isotope and inhibitor for 48 h in order to produce mature virus. The presence of virus in the medium was determined by hemagglutination assays, and the virus was isolated by differential centrifugation. In these cases, the cell pellets were also examined for their glycoprotein content and structure.
Virus, labeled with either [2-3H]mannose or [l-3H]galactose, was digested exhaustively with Pronase to produce glycopeptides, and these glycopeptides were isolated initially on columns of Bio-Gel P-4 (1.5 X 150 cm; 200-400 mesh). Since these columns did not completely resolve the complex from the high-mannose structures, the entire glycopeptide peak from these initial runs was pooled, concentrated to dryness, and digested with endoglucosaminidase H, as described previously (21). After digestion, the samples were rechromatographed on the Bio-Gel P-4 column. Aliquots of every other fraction were removed for the determination of radioactivity.
Susceptibility of the various oligosaccharides and glycopeptides to digestion by a-mannosidase, or to a combination of p-galactosidase and P-N-acetylhexosaminidase, was also examined. These incubations were done in 50 mM acetate buffer, pH 5.0, under a toluene atmosphere. For a-mannosidase, 0.1 units of enzyme were added initially and again after 18 h. These incubations were done for a total of 36 h and also contained 0.05 mM ZnClz. For the other digestions, 50 milliunits of each enzyme were added initially and again at 18 h, for a total incubation of 36 h. Susceptibility of the various oligosaccharides to digestion was determined by chromatography of the digestion mixtures on columns of Bio-Gel P-4.

RESULTS AND DISCUSSION
In order to determine the effects of DMDP as a glycoprotein processing inhibitor, MDCK cells were infected with the NWS strain of influenza virus, and various amounts of DMDP were added to the cultures. Following an incubation of 3 h to allow the inhibitor to act, [2-3H]mannose was added to label the viral glycoproteins. The cultures were allowed to incubate for about 40 h to produce mature virus, and the presence of viral particles in the medium was measured by hemagglutination assays. The virus was isolated from the medium by differential centrifugation, and the viral pellets were digested with Pronase to obtain the glycopeptides. Fig. 1 (profiles A-C) show the glycopeptide profiles from control and DMDP-treated (50 and 250 pg/ml) virus. It can be seen that there was a considerable alteration in the profiles obtained in the presence of DMDP especially at 250 pg/ml. However, these columns did not completely resolve the complex types of glycopeptides from the high-mannose (and hybrid) structures.
In order to completely separate the various types of glycopeptides, each glycopeptide peak (fractions 30-50) was pooled, concentrated to a small volume, and digested exhaustively with endoglucosaminidase H. These digests were rechromatographed on the Bio-Gel P-4 column as shown in profiles D- Fig. l. This treatment clearly resolved the control viral glycopeptides into two peaks (D). The first and major radioactive peak remained in the original position and was resistant to Endo H (complex types), while the second peak now emerged at later fractions (50-54), indicating susceptibility to this enzyme (high-mannose types). The identity of these peaks as complex and high-mannose structures was also confirmed by chromatography on columns of concanavalin A-Sepharose (data not shown). In the control virus, about 75% of the radioactivity was in the complex chains and about 25% in the high-mannose types. On the other hand, in the presence of DMDP at 250 pg/ml, more than 80% of the radioactive glycopeptides were susceptible to Endo H, and only a small peak of radioactivity remained in the original position (profile F ) . In addition, the new peak resulting from this digestion appeared to be larger in size than the high-mannose structures of the control virus, since it emerged earlier from the Bio-Gel columns. Although the resolution on this column was not sufficient for an accurate size determination, this oligosaccharide appeared to be a hexosellGlcNAc. Table I summarizes the percentage of the total radioactivity in the complex (Endo H-resistant) and high-mannose (Endo H-sensitive) structures at various concentrations of DMDP. It can be seen that the percentage of radioactivity in the complex chains continually decreased with increasing amounts of DMDP, with a corresponding increase in the percentage of [3H]mannose in the high-mannose types (Endo H-sensitive).
To determine the exact size of the DMDP-induced oligosaccharide, it was sized on a long, calibrated column of Bio-Gel P-4 as shown in Fig. 2A. On this column, the oligosaccharide migrated mostly near the hexosellGlcNAc standard, but there was some heterogeneity with larger (hexoselpGlcNAc), and smaller (hexoseloGlcNAc) oligosaccharides. This heterogeneity is not surprising since these studies were done in cell culture, and some processing of the oligosaccharides would be expected. The oligosaccharide peak from Fig. 2A was pooled (fractions 158-176), concentrated to a small volume, and treated exhaustively with a-mannosidase. These digestions were then chromatographed on the same Bio-Gel P-4 column as shown in Fig. 2B. It can be seen that the oligosaccharide was only partially susceptible to this enzyme and the major peak was shifted to the hexoseloGlcNAc area. These results indicated removal of one or two mannose residues by the amannosidase treatment. On the other hand, the oligosaccharide was not susceptible to digestion by @-galactosidase or p-N-acetylhexosaminidase, either sequentially or in combination. These data suggested that the original oligosaccharide contained glucose residues that blocked or capped one or more of the mannose branches, making this structure only slightly susceptible to a-mannosidase digestion.
To demonstrate that this oligosaccharide did contain glucose and to determine the number of glucose residues present, infected MDCK cells were incubated with DMDP and labeled with [ l-3H]galactose. Viral glycopeptides were isolated as described above, treated with Endo H, and rechromatographed on the Bio-Gel column. In the presence of DMDP, a radioactive oligosaccharide was obtained that migrated in the same position as the mannose-labeled oligosaccharide described above (data not shown). This oligosaccharide also migrated on the calibrated Bio-Gel column in the same position as the mannose-labeled oligosaccharide. The label was   shown to be present in glucose by complete acid hydrolysis and paper chromatography. The number of glucose residues present in this oligosaccharide was determined by complete methylation and isolation of the methylated glucose derivatives by thin-layer chromatography. As shown in Fig. 3, three methylated glucose derivatives were obtained, corresponding to 2,3,4,6-tetramethylglucose, 3,4,6-trimethylglucose, and 2,4,6-trimethylglucose. These three peaks were present in the approximate ratio of l:l:l, based on their radioactive content (actual ratio 1.0:0.75:0.86). Thus, the DMDP-induced oligosaccharide appears to contain three glucose residues and is a Gl~~Man~-~GlcNAc. These data would be consistent with the inhibition of glucosidase I by this inhibitor. Preliminary experiments with a rat liver microsomal fraction incubated with [3H]glucose-labeled Glc3MansGlcNAcz indicated that DMDP did inhibit glucosidase I.
In order to determine whether DMDP had any effect on the formation of lipid-linked saccharide intermediates, infected MDCK cells were incubated in the presence of 10, 100, or 250 pg/ml of DMDP, and then labeled with either [2-3H] mannose or [l-3H]galactose. In one experiment, the cells were   harvested after a 3-h incubation in isotope, and the lipidlinked monosaccharides and lipid-linked oligosaccharides were isolated by extraction with various mixtures of CHC13:CH30H:Hz0. After these extractions, the cell residues were digested with Pronase to obtain the glycopeptides. In other experiments, the infected cells were incubated for 48 h with isotope and then extracted as described above. Each of the lipid fractions and the glycopeptide fractions were counted to determine their radioactive content. The data from this experiment are presented in Table 11. It can be seen that increasing amounts of DMDP did cause some inhibition of mannose incorporation into the lipid-linked monosaccharides, at both 3 and 48 h. The incorporation of mannose into the lipid-linked oligosaccharides was somewhat inhibited at 3 h, but at 48 h it was either unchanged or slightly stimulated. On the other hand, mannose incorporation into protein (both viral and cellular) was stimulated by DMDP, especially at 48 h. This is probably not surprising since DMDP prevents trimming of mannose residues and would cause the accumulation of high-mannose types of structures. DMDP had relatively little effect on the incorporation of galactose into the various lipid and glycoprotein fractions, although it may have stimulated incorporation into the 1:l:l material. It is not known at this stage whether this lipid material represents lipid-linked monosaccharides (ie. glucosyl-P-dolichol) or other glycolipids (ie. cerebrosides).
The effect of DMDP on the incorporation of [3H]leucine into protein in MDCK cells was examined to determine whether this compound affected protein synthesis. Confluent monolayers were preincubated with DMDP for several hours in 2% minimal Eagle's medium, and then [3H]leucine was added to each culture. At the times shown in Fig. 4, the monolayers were washed well with saline, and the cells were removed from the dish by scraping. The cells were extracted with 5% trichloroacetic acid, and the trichloroacetic-acid insoluble residue was suspended in Protosol and counted to determine its radioactive content. Fig. 4 shows that even at 250 pg/ml of DMDP, there was no change in the amount of radioactivity incorporated into protein over a 2-h incubation. In addition, we examined the yield of virus produced at various concentrations of DMDP, and the infectivity of these particles. Virus yields were determined by hemagglutination assays on the medium, and infectivity was measured by plaque counts. DMDP had no effect on either of these parameters (data not shown). Based on these studies, we assume that DMDP is not affecting protein synthesis or virus production. However, since the virus assays were only done at one time point, it is possible that this inhibitor might affect the rate of virus production.
The results reported here indicate that DMDP is an inhibitor of glycoprotein processing by virtue of the fact that it or 250 pg/ml ( M ) of DMDP. At the end of this time, 10 pCi of 13H]leucine were added to each well, and the plates were incubated for the times shown. After incubation, the monolayers were washed several times with saline and the cells were removed from the plates by scraping. The cell residues were suspended in 5% trichloroacetic acid and allowed to remain overnight in the cold. The residues were isolated by centrifugation, washed with 5% trichloroacetic acid, and suspended in Protosol for the determination of radioactivity. inhibits glucosidase I. Thus, the major oligosaccharide produced in the presence of inhibitor was susceptible to Endo H and migrated on calibrated Bio-Gel columns like a hex-osellGlcNAc. This oligosaccharide was only partially susceptible to a-mannosidase, but was resistant to both P-galactosidase and &hexosaminidase, suggesting that it contained blocking glucose units. The oligosaccharide was synthesized in the presence of [3H]galactose to label the glucose moieties, and this oligosaccharide was subjected to methylation analysis. Since three methylated glucose derivatives were obtained, corresponding to terminal, 3-linked, and 2-linked glucose, the oligosaccharide must contain three glucoses and be a Glc3MansGlcNAc. Since DMDP did not affect protein synthesis and also had no effect on virus production or release, it should be a useful inhibitor for studies on glycoprotein biosynthesis and function. Perhaps the most interesting aspect of these studies is that DMDP represents a member of a new class of processing inhibitors, the pyrrolidine alkaloids. Based on studies with other processing inhibitors, it seems quite likely that other pyrrolidine alkaloids with different chirality will prove to be inhibitors of other glycosidases, and perhaps other processing enzymes.
There are a number of processing inhibitors that have now been described (16,17). All of these compounds are glycosidase inhibitors and share several common features, i.e. 1) they all contain a nitrogen in the ring, and 2) they all contain three or more assymetric hydroxyl groups. Thus, swainsonine (23) was the first processing inhibitor to be described (19), and this compound has been shown to inhibit mannosidase I1 (24). In the presence of swainsonine, cells in culture produce hybrid types of oligosaccharides (25)(26)(27)(28)(29). Deoxynojirimycin (29)(30)(31)(32)(33) and castanospermine (21) inhibit glucosidase I and lead to the accumulation in cell culture of G l~~M a n~-~G l c N A c~ structures on the protein. Recently, the mannose analog of deoxynojirimycin (deoxymannojirimycin) was synthesized chemically (34), and shown to be an inhibitor of mannosidase I (35). This inhibitor causes the accumulation of MansGlcNAca structures. The important point here is that all of the above inhibitors contain the nitrogen in a 6-membered ring. DMDP is the first 5-membered ring compound that has been found to be an inhibitor of glycoprotein processing. Such a structure may be of considerable importance in understanding the mechanism of glycosidase inhibition. In addition, &membered ring structures are likely to be easier to synthesize chemically and therefore it should be feasible to prepare various isomers as inhibitors of other glycosidases. In fact, another pyrrolidine alkaloid that inhibits a-mannosidase has been synthesized chemically?