1-Deoxynojirimycin Impairs Oligosaccharide Processing of al-Proteinase Inhibitor and Inhibits Its Secretion in Primary Cultures of Rat Hepatocytes*

(dry protein Elution glycerol eluted the quantification of the

1-Deoxynojirimycin was found to inhibit oligosaccharide processing of rat al-proteinase inhibitor. In normal hepatocytes al-proteinase inhibitor was present in the cells as a 49,000 M, high mannose type glycoprotein with oligosaccharide side chains having the composition MansGlcNAc and MansGlcNAc with the former in a higher proportion. Hepatocytes treated with 5 mM 1-deoxynojirimycin accumulated al-proteinase inhibitor as a 51,000 M , glycoprotein with carbohydrate side chains of the high mannose type, containing glucose as measured by their sensitivity against a-glucosidase, the largest species being GlcsMansGlcNAc. Conversion to complex oligosaccharides was inhibited by the drug. In addition, increasing concentrations of I-deoxynojirimycin inhibited glycosylation resulting in the formation of some al-proteinase inhibitor with two instead of three oligosaccharide side chains. 5 mM 1-deoxynojirimycin inhibited the secretion of al-proteinase inhibitor by about 50%, whereas secretion of albumin was unaffected. The oligosaccharides of al-proteinase inhibitor secreted from l-deoxynojirimycin-treated cells were characterized by their susceptibility to endoglucosaminidase H, incorporation of [3H]galactose, and [3Hlfucose and concanavalin A-Sepharose chromatography. It was found that 1deoxynojirimycin did not completely block oligosaccharide processing, resulting in the formation of alproteinase inhibitor molecules carrying one or two complex type oligosaccharides. Only these al-proteinase inhibitor molecules processed to the complex type in one or two of their oligosaccharide chains were nearly exclusively secreted. This finding demonstrates the importance of oligosaccharide processing for the secretion of al-proteinase inhibitor.
A series of enzymatic reactions is required for the biosynthesis of glycoproteins containing N-linked oligosaccharides of the complex type (for recent reviews see . In the endoplasmic reticulum a transfer of Glc3MangGlcNAc2 from *This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereiche 206 and 47) and grants from Stiftung Volkswagenwerk, the Fonds der Chemischen Industrie, and Justus-Liebig-Universitat Giessen. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''duertkement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Heisenberg Stipendium from the Deutsche Forschungsgemeinschaft.
(I Author to whom correspondence should be addressed. its dolichol derivative to the nascent polypeptide chain occurs. The three terminal glucosyl residues are essential for the transfer of the oligosaccharides to the protein backbone ( 5 ) .
Subsequently the glucose residues are rapidly removed in the endoplasmic reticulum by the sequential action of glucosidases I and I1 (6-10). The oligosaccharides are further processed in the Golgi apparatus where trimming of the mannose residues and attachment of the terminal sugars N-acetylglucosamine, galactose, sialic acid, and fucose occurs (1-4).
In order to study the function of the carbohydrate moiety in glycoproteins, specific inhibitors acting at different stages of the glycosylation process are useful. The most widely used inhibitor of glycosylation is tunicamycin, which blocks the formation of Glc3Man9GlcNAc2-pyrophosphoryldolichol (11)(12)(13). Inhibitors which block different enzymes in the trimming process have been described recently. 1-Deoxynojirimycin was found to inhibit glucosidases I and I1 (14). Bromoconduritol has been described to inhibit trimming of the innermost glucose residue of GlcsMangGlcNAcp (15). Swainsonine has been characterized as a potent inhibitor of mannosidase I1 (16).
We are interested in the secretion mechanisms of glycoproteins. As a model glycoprotein the secretion of al-proteinase inhibitor has been studied in primary cultures of rat hepatocytes. We found that inhibition of glycosylation by tunicamycin inhibited the secretion of al-proteinase inhibitor by 60-70% (17). In further studies we observed that inhibition of mannosidase I1 by swainsonine led to the formation of hybrid oligosaccharides in al-proteinase inhibitor (18). In spite of its effect on the carbohydrate part of al-proteinase inhibitor swainsonine did not impair the secretion of the incompletely processed glycoprotein.
In order to examine the inhibition of glycoprotein processing at an early stage, we studied the effect of l-deoxynojirimycin on the glycosylation and secretion of al-proteinase inhibitor. In the present paper we show that inhibition of glucose trimming leads to impaired secretion of al-proteinase inhibitor.
Preparation of Rat Hepatocyte Monolayers-Suspensions of rat hepatocytes were prepared as previously described by Bischoff et al. (19). After the cells were washed with Krebs-Henseleit buffer, they were suspended in a modified Waymouth medium (20) containing 10% fetal calf serum, 50 units/ml of penicillin, 50 pg/ml of streptomycin, M dexamethasone, and lo-' M insulin. Aliquots of 3 ml of cell suspension (4 X IO6 cells) were added to 55-mm contour bottom Falcon plastic tissue culture dishes. The dishes were incubated a t 37 "C in a humid atmosphere of 5% C 0 2 in air for 3 h. The plates were then washed with Krebs-Henseleit buffer and 3 ml of culture medium (Waymouth medium containing 5% fetal calf serum, penicillin, streptomycin, dexamethasone, and insulin in the same concentrations as mentioned above) were added. Confluent monolayers were formed after an overnight incubation a t 37 "C in a humid atmosphere of 5% CO, in air.
Labeling of Hepatocytes-Modified Waymouth medium without fetal calf serum, bovine serum albumin, and oleic acid was used for the radioactive labeling of the hepatocyte monolayers obtained after overnight incubation. 25 pCi of [:"S]methionine were added to 3 ml of methionine-free culture medium for the labeling of proteins. In order to label carbohydrates, 80 pCi of D-[2-'H]mannose, 50 pCi of ~-[4,5-:'H]galactose, or 50 pCi of ~-[6-:'H]fucose were added to each dish containing about 4 X lo6 cells. For the labeling with D-[2-%] mannose medium containing only %o of the normal glucose concentrations was used. If not otherwise stated, the incubation times were 2.5 h for [:"S]methionine and 4 h for the 'H-labeled sugars. After incubation a t 37 "C the media were separated from the cells. The cells of each dish were carefully washed with 0.15 M NaCI, 10 mM Tris/HCI, pH 7.6, homogenized in 1 ml of 25 mM Tris/HCl buffer, pH 7.5, 20 mM NaCI, 1% deoxycholate (Na'), and 1% Triton X-100 with a Potter-Elvehjem homogenizer a t 800 revolutions/min, and centrifuged for 10 min at 12,000 x g. The supernatant of this centrifugation and the culture medium were used for the immunoprecipitations.
Immunoprecipitation-For the immunoprecipitation 1.5-2.5 ml of medium or 0.4-0.8 ml of the supernatant obtained from the cell homogenate were added to 5 ml of buffer A (20 mM Tris/HCI, pH 7.6, 0.14 M NaCI, 5 mM EDTA, 1% Triton X-100) containing 1 mM phenylmethylsulfonyl fluoride and 0.1 mg of kallikrein trypsin inhibitor (kindly provided by Bayer AG, Wuppertal-Elberfeld). After addition of 7.5 pl of a specific antiserum against rat al-proteinase inhibitor (21) and incubation a t 0 "C overnight, the antigen-antibody complexes were bound to 7 mg (dry weight) of protein A-Sepharose and washed 4 times with buffer A and twice with 50 mM sodium phosphate buffer, pH 7.5. Elution was achieved by incubation with 0.1 M Tris/HCI, pH 6.8, 5% B-mercaptoethanol, 5% sodium dodecyl sulfate, and 10% glycerol at 95 "C for 5 min. The eluted proteins were analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide slab gels (22) and by fluorography (23). For the quantification of the radioactivity incorporated into nl-proteinase inhibitor or rat serum albumin, the corresponding bands identified by fluorography were cut from the gels, solubilized with Protosol/water (91, v/v) at 45 "C overnight, and counted in a liquid scintillation spectrophotometer.
Treatment of nl-Proteinase Inhibitor with Endoglucosaminidme H-The tu,-proteinase inhibitor IgG complexes eluted from the protein A-Sepharose were dialyzed exhaustively against 50 mM sodium phosphate buffer, pH 6.0,0.01% sodium dodecyl sulfate and incubated in a total volume of 0.1 ml with 5 milliunits of endoglucosaminidase H at 37 "C for 16 h.
Rio-Gel P-4 Chromatography of Olig~saccharides-[~H]Mannoselabeled oligosaccharides of nl-proteinase inhibitor obtained after digestion with endoglucosaminidase H followed by gel filtration on Sephadex G-25 were subjected to Bio-Gel P-4 chromatography. Bio-Gel P-4 (-400 mesh) columns (1 X 150 cm), eluted with water containing 0.02% sodium azide, were used. The void volume marker was bovine serum albumin (24). The volume of the fractions was 0.35 ml. Determination of radioactivity was done using a Packard Tri-Carb liquid scintillation spectrophotometer model 460 C. Treatment of oligosaccharides with n-glucosidase was carrired out according to Grinna and Robbins (9) modified by addition of 10 mM EDTA to inhibit residual mannosidase as recently described (15).
Concanavalin A-Sepharose Chromatography of Glycopeptides from crl-Proteinuse Inhibitor-After immunoprecipitation the nl-proteinase inhibitor IgG complexes were eluted from the protein A-Sepharose by incubation with 20 mM Tris/HCI, pH 7.5, 2% sodium dodecyl sulfate at 95 "C for 5 min. Glycopeptides were prepared by digestion with 1 mg/ml of proteinase K in 20 mM Tris/HCl, pH 7.5, 0.15 M NaCI, and 0.2% sodium dodecyl sulfate a t 37 "C for 18 h. The reaction was stopped by the addition of 1 mM phenylmethylsulfonyl fluoride and subsequent treatment at 95 "C for 10 min. The incubation mixture diluted with water to a final sodium dodecyl sulfate concentration of 0.02% was then added onto a concanavalin A-Sepharose column (3-ml bed volume) equilibrated with 10 mM Tris/HCl, pH 7.5, 0.3 M NaCI, 1 mM CaCI,, 1 mM MgCI,, and 1 mM MnCI,. The column was washed with equilibration buffer followed by elution with 9.75 ml of 10 mM a-methylglucoside and subsequent 9.75 ml of 500 mM amethylmannoside. Fraction volumes of 0.65 ml were collected and their radioactivity was determined.
Rocket Immunoelectrophoresis of al-Proteinase Inhibitor-Rocket immunoelectrophoresis was carried out as described by Laurell (25). For the calibration we used al-proteinase inhibitor purified from rat serum to homogeneity by a 50% and a subsequent 80% ammonium sulfate precipitation, followed by affinity chromatography on activated thiol-Sepharose 4B as described by Laurell et al. (26), by affinity chromatography on concanavalin A-Sepharose according to Saklatvala et al. (27). and by gel filtration on Sephacryl S-200.

RESULTS
1-Deoxynojirimycin was used to inhibit the trimming of the carbohydrate side chains of al-proteinase inhibitor in primary cultures of rat hepatocytes. Fig. 1 shows the effect of increasing concentrations of 1-deoxynojirimycin on the synthesis and secretion of a,-proteinase inhibitor. As described previously (17, 28), two forms of al-proteinase inhibitor exist in control hepatocyte cultures: an intracellular 49,000 M, precursor of the high mannose type and an extracellular 54,000 M, glycoprotein of the complex type ( Fig. 1, lanes I and 2). A small amount of the complex type al-proteinase inhibitor is also present in the cells. Incubation of hepatocytes with increasing concentrations of 1-deoxynojirimycin ranging from  1 and 2) or with 1.25 mM (lanes 3 and 4), 2.5 mM (lanes 5 and 6). 5 mM (lanes 7 and 8), and 10 mM (lanes 9 and IO) 1-deoxynojirimycin for 1 h. The medium was changed and 25 pCi of ["S]methionine were added to each dish. Incubation was done a t 37 "C for 2.5 h in the presence of the above mentioned concentrations of l-deoxynojirimycin. nl-Proteinase inhibitor was immunoprecipitated from the cells (lanes I , 3, 5, 7, and 9) and their media (lanes 2, 4, 6, 8, and IO) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography as described under "Materials and Methods." Molecular weight standards were conalbumin (86,000), bovine serum albumin (68,000), catalase (60,000), ovalbumin (43,000). and alcohol dehydrogenase from yeast (37,000).

Effect of 1 -Deoxynojirimycin on Glycosylation 12205
1.25 to 10 mM leads to the formation of an intracellular form of a,-proteinase inhibitor with an apparent molecular weight of about 51,000 which is higher than that found in control cells. Furthermore, an additional protein band with an apparent molecular weight of about 47,000 is found. The intensity of this band increases with higher 1-deoxynojirimycin concentrations (lanes 3, 5, 7, and 9). At 10 mM 1-deoxynojirimycin (lune 9) a faint band with an even smaller apparent molecular weight (44,000) occurs. The incubation of hepatocytes with 1- deoxynojirimycin prevents the formation of the 54,000 M, complex type at-proteinase inhibitor normally secreted into the medium. Instead, a form of smaller molecular weight was observed. The secretion of this form was markedly inhibited compared to that of the 54,000 M, al-proteinase inhibitor secreted by control hepatocytes. In none of the experiments with 1-deoxynojirimycin was total protein synthesis inhibited by more than 10-15%.
In order to study the effect of 1-deoxynojirmycin on the size of the oligosaccharide chains of al-proteinase inhibitor endoglucosaminidase H digests of ["H]mannose-labeled aIproteinase inhibitor were analyzed on Bio-Gel P-4 columns. It can be seen in Fig. 2A that the majority of the radioactive material from control cells coelutes with the marker MangGlcNAc. With the exception of radioactivity under the small peak ahead of MangGlcNAc these oligosaccharides were resistant against treatment with a-glucosidase (Fig. 2B). Thus, in the absence of 1-deoxynojirimycin oligosaccharide chains with 8 and 9 mannose residues are found on atproteinase inhibitor. It is documented in  (lanes 1-4) or with (lanes 5-8) 5 mM 1-deoxynojirimycin for 1 h followed by labeling with ["S]methionine (25 pCi/dish) for 2.5 h. n,-Proteinase inhibitor was immunoprecipitated from control cells (lanes 3 and 4 ) and their respective media (lanes 1 and 21, from 1-deoxynojirimycin treated cells (lanes 7 and 8) and their respective media (lanes 5 and 6 ) and incubated without (lanes I , 3 , 5 , and 7) or with (lanes 2 , 4 , 6 , and 8) 5 milliunits of endoglucosaminidase H at 37 "C for 16 h as described under "Materials and Methods." Lane 9 shows the intracellular form of nl-proteinase inhibitor obtained after tunicamycin treatment of hepatocytes. Tunicamycin was given to the culture medium at a concentration of 3 pg/ml. After 1 h of incubation the medium was removed and fresh medium containing 25 pCi of ["S]methionine and tunicamycin a t a final concentration of 3 pg/ml was added. After a labeling period of 3 h cu,-proteinase inhibitor was immunoprecipitated from the cells. Molecular weight markers were the same as those given in the legend to Fig. 1. tions which correspond to a lower molecular weight, mainly Man9GlcNAc.
To further characterize the a,-proteinase inhibitor species synthesized and secreted in the presence of 5 mM l-deoxynojirimycin, digestions with endoglucosaminidase H have been carried out. Whereas the 54,000 molecular weight form of a,-proteinase inhibitor found in the medium of control cells was resistant to the action of endoglucosaminidase H (Fig. 3,  lanes 1 and 2), the 49,000 intracellular form was found to be sensitive to endoglucosaminidase H (Fig. 3, lanes 3 and 4). The weak band above the 54,000 M, a,-proteinase inhibitor (Fig. 3, lanes 1 and 2 ) is an artefact, which rarely occurs during immunoprecipitation and is not related to al-proteinase inhibitor.
When al-proteinase inhibitor was immunoprecipitated from the medium of 1-deoxynojirimycin treated hepatocytes, 2 bands corresponding to apparent molecular weights of about 52,000 and 48,000 were observed (lane 5 ) . Incubation with endoglucosaminidase H led to the formation of two major bands with apparent molecular weights of 49,000 and 46,000 and a minor one with an apparent molecular weight of 43,000 (lane 6). The intracellular al-proteinase inhibitor from 1deoxynojirimycin-treated hepatocytes could be deglycosylated by endoglucosaminidase H, resulting in a protein with an apparent molecular weight of about 43,000 (lanes 7 and 8). For comparison lane 9 shows al-proteinase inhibitor isolated from tunicamycin-treated hepatocytes. a,-Proteinase inhibitor cleaved by endoglucosaminidase H exhibits a slightly higher apparent molecular weight than the unglycosylated form obtained after tunicamycin treatment (M, = 41,000).
This difference is very likely due to the fact that endoglucosaminidase H leaves the first GlcNAc molecule of each oligosaccharide chain attached to asparagine.
From experiments where glycosylation of rat al-proteinase inhibitor was incompletely inhibited by low doses of tunicamycin, we have concluded that rat a,-proteinase inhibitor carries three oligosaccharide chains (17). This agrees with the conclusions of Carlson and Stenflo (29) drawn from experiments where the high mannose type a,-proteinase inhibitor was incompletely digested with endoglucosaminidase H. Furthermore, the carbohydrate composition of rat serum alproteinase inhibitor (30)  Effect of 1 -Deoxynojirimycin on Glycosylation 12207 of three oligosaccharide chains. As shown in Fig. 3 (lanes 7 and 8) the two intracellular forms of n,-proteinase inhibitor found in l-deoxynojirimycintreated hepatocytes are both susceptible to endoglucosaminidase H. Therefore, we conclude that the two forms are different with respect to their number of oligosaccharide chains (3 chains for the upper, and 2 chains for the lower band) and not to their carbohydrate type. This might be due to an inhibitory effect of 1-deoxynojirimycin on the glycosylation of newly synthesized glycoproteins. In the medium of 1deoxynojirimycin-treated hepatocytes also two different forms of nl-proteinase inhibitor of slightly higher apparent molecular weights than the intracellular forms are present (Fig. 3, lane 5). From the fact that the majority of the medium forms of nl-proteinase inhibitor isolated from l-deoxynojirimycin-treated cells cannot be completely deglycosylated by endoglucosaminidase H, we conclude that the secreted forms of n,-proteinase inhibitor contain oligosaccharides of both the high mannose and the complex type. A minute amount of the secreted nl-proteinase inhibitor contains only high mannose type oligosaccharides (see faint band corresponding to an apparent molecular weight of 43,000). The results show that the oligosaccharide trimming is not completely inhibited by 1-deoxynojirimycin. They further suggest that the nl-proteinase inhibitor molecules which have one or two complex type digosaccharide chains are preferentially secreted.
In order to characterize the oligosaccharide side chains of x,-proteinase inhibitor secreted from l-deoxynojirimycin-  (lanes 1, 3, 5, 7, and 9) and from the media (lanes 2, 4, 6, 8, and IO) 10 min (lanes I and 2), 30 min (lanes   3 and 4 ) . 60 min (lanes 5 and 6 ) , 90 min (lanes 7 and 8), and 120 min (lanes 9 and IO) after the chase. treated hepatocytes, the carbohydrate part of al-proteinase inhibitor was labeled with ['HHJmannose and analyzed by affinity chromatography on concanavalin A-Sepharose as described by Reitman et al. (31). Fig. 4A shows that the ['HI mannose-labeled glycopeptides obtained after proteinase K digestion of the sodium dodecyl sulfate-denatured al-proteinase inhibitor from the medium of control cells are found essentially in the flow-through fractions and the 10 mM amethylglucoside eluate. The glycopeptides of al-proteinase inhibitor secreted by 1-deoxynojirimycin-treated hepatocytes were found partially in the flow-through fractions and in the 10 mM a-methylglucoside eluate; the largest amount, however, was found in the 500 mM a-methylmannoside eluate. The results of the concanavalin A-Sepharose chromatography suggest that control cells secrete nl-proteinase inhibitor with mainly bi-and some probably tri-or tetra-antennary oligosaccharide chains. 1-Deoxynojirimycin-treated cells, on the other hand, secrete al-proteinase inhibitor containing complex type (flow-through and 10 mM a-methylglucoside eluate) and high mannose type oligosaccharides (500 mM a-methylmannoside eluate). T o further demonstrate the existence of complex type oligosaccharides in al-proteinase inhibitor secreted from 1-deoxynojirimycin-treated hepatocytes, cells were labeled with ['H]galactose or ['H]fucose. The ['Hlgalactose-or ['HHJfucose-labeled glycopeptides were subjected to concanavalin A-Sepharose affinity chromatography (Fig. 4B). The elution profiles show that the ["Hlgalactose or ['HI fucose-labeled oligosaccharides are of the complex type.
T o study the inhibitory effect of 1-deoxynojirimycin on the secretion of al-proteinase inhibitor (already presented in Fig.  1) in greater detail, pulse-chase experiments were carried out. For comparison, albumin, an unglycosylated secretory protein, has been examined. Hepatocytes were pulse-labeled with ["'S]methionine for 10 min followed by a chase with unlabeled methionine. When al-proteinase inhibitor (Fig. 5A) and albumin (Fig. 5C) were immunoprecipitated from the cells and from the medium at different times ranging from 10 to 180 min, a continuous decrease of radioactivity in the cells and an increase of radioactivity in the medium was found. When the same pulse-chase experiments were carried out under conditions, where oligosaccharide trimming was blocked by 1-deoxynojirimycin, it was found that the secretion of alproteinase inhibitor (Fig. 5B) was markedly inhibited, whereas the secretion of albumin (Fig. 50) was not affected.
In Table I the data of the pulse-chase experiments (Fig. 5) are quantitated and show that 1-deoxynojirimycin inhibits the secretion of al-proteinase inhibitor but does not affect albumin secretion.

TABLE I
Effect of 1 -deoxynojirimycin on the secretion of cul-proteinuse inhibitor and rat serum albumin The radioactive bands containing ['ZsS]methionine-labeled al-proteinase inhibitor and rat serum albumin from the pulse-chase experiments presented in Fig. 5 were cut from the gels and their radioactivity was measured. For each protein the ratio of extracellular to intra-plus extracellular radioactivity is given for the various times after chase. The amount of a,-proteinase inhibitor secreted during 24 h by cultured rat hepatocytes was determined by rocket immunoelectrophoresis. Normal rat hepatocytes secreted 4.39 pg of a,-proteinase inhibitor/mg of cellular protein. In the presence of 5 mM 1-deoxynojirimycin only 2.15 pg of a,-proteinase inhibitor/mg of cellular protein/24 h was secreted. DISCUSSION 1-Deoxynojirimycin has been found to inhibit glucosidase activities (32, 33), including the glucosidases involved in the processing of Glc3MangGlcNAc2 oligosaccharides from Streptomyces cereuisiae (14). In this paper we report that incubation of hepatocytes with 1-deoxynojirimycin results in the formation of an intracellular a'-proteinase inhibitor form (apparent M, 51,000) larger than that found in normal cells (apparent Mr 49,000). The analysis of the oligosaccharides obtained after endoglucosaminidase H digestion showed that a,-proteinase inhibitor synthesized in the absence of I-deoxynojirimycin carries high mannose oligosaccharides containing 9 and 8 m a n n o s e r e s i d u e s , t h e p r e d o m i n a n t s p e c i e s b e i n g MangGlcNAc. With the exception of a small peak ahead of the marker MangGlcNAc (see "Results") oligosaccharides are insensitive against treatment with a-glucosidase. The elution profile obtained upon analysis of oligosaccharides which have been synthesized in the presence of 1-deoxynojirimycin is clearly different. Most of the radioactivity elutes in three peaks before MangGlcNAc, the largest coeluting with GlcsMangGlcNAc, the predominant species being one hexose unit larger than MangGlcNAc. After treatment with a-glucosidase virtually all of the radioactivity elutes in positions which correspond to Man,GlcNAc (x = 9, 8, 7), the predominant species being MangGlcNAc. This result suggests that the three peaks ahead of the Man9GlcNAc standard (Fig. 2C) contain Glc3MangGlcNAc, Glc2MangGlcNAc, and GlclMangGlcNAc and, in addition, some glucose-containing oligosaccharides which have been trimmed with respect to their mannose residues, resulting in the formation of species containing 8 or 7 mannoses. In a study with another derivative of nojirimycin (N-methyl-1-deoxynojirimycin), it has been found that oligosaccharides of the composition GlcaMan,GlcNAc (x = 9 , 8 , 7 ) were synthesized in the presence of the inhibitor.' In addition to the glucosidase-inhibiting effect of l-deoxynojirimycin, there is evidence for an inhibition of glycosylation. It was found that 10 mM 1-deoxynojirimycin ("Results," Fig. 1) led to the formation of two additional forms of a,proteinase inhibitor with smaller apparent molecular weights, both being susceptible to endoglucosaminidase H. An inhibition of glycosylation was also found for a,-acid glycoprotein which showed six different molecular weight forms after treatment of hepatocytes with 10 mM 1-deoxynojirimycin (not shown). This agrees well with recent findings of Yoshima et al. (34) and Baumann and Jahreis (35) who found 6 asparagine-linked sugar chains per 1 molecuie of a,-acid glycoprotein. The reason for the inhibitory effect of l-deoxynojirimycin on glycosylation is presently unknown.
1-Deoxynojirimycin does not completely block oligosaccharide processing in &,-proteinase inhibitor. This has been demonstrated by endoglucosaminidase H digestion and affinity chromatography of glycopeptides on concanavalin A-Sepharose. In these experiments we found endoglucosaminidase H-resistant oligosaccharides and glycopeptides which did not bind to concanavalin A-Sepharose or could be eluted by 10 mM a-methylglucoside.
1-Deoxynojirimycin inhibits the secretion of al-proteinase Romero, P. A., Datema, R., and Schwarz, R. T. (1983) Virology, inhibitor. This inhibition is not due to an inhibitory effect of 1-deoxynojirimycin on glycosylation, since a,-proteinase inhibitor molecules with 2 or 3 oligosaccharide side chains are present in the same ratio intra-and extracellularly. Analysis of the carbohydrate part of al-proteinase inhibitor secreted in the presence of 1-deoxynojirimycin showed that nearly only those molecules which carry one or two complex type oligosaccharides are secreted. This finding demonstrates the importance of oligosaccharide processing for the secretion of a,proteinase inhibitor. Interestingly, it seems to be sufficient for the secretion when 1 or 2 of the 3 carbohydrate side chains of al-proteinase inhibitor are processed to the complex type. From the experiments presented in this paper it is not possible to say where within the hepatocyte the unprocessed a'-proteinase inhibitor accumulates and which mechanisms lead to this accumulation. In order to answer this question, an inhibitor which blocks the glucosidase reaction completely would be desirable. Even by another approach-using a mutant cell line-a similar "escape" phenomenon has been observed by Reitman et al. (31). They studied a lectin-resistant mouse lymphoma cell line which showed a nearly complete lack (<0.3% of parent) of glucosidase 11, but still made complex type oligosaccharides in an amount of 25% of that found in the parent cells.