Substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases and effects of swainsonine suggest a role of the cytosolic enzyme in glycoprotein catabolism.

Swainsonine is a potent inhibitor of lysosomal alpha-D-mannosidase, causes the production of hybrid glycoproteins, and is reported to produce a phenocopy of hereditary alpha-mannosidosis. We now report that the effects of swainsonine administration in the rat are different in two respects from those found in other animals thus far studied. Swainsonine caused the accumulation of oligosaccharide in kidney and urine but not in liver or brain. The accumulated oligosaccharides were mainly Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, Man(alpha 1-3)[Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4) GlcNAc, and Man(alpha 1-3)[Man(alpha 1-6)]Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc. Analogous branched Man4 and Man5 structures are found in pig and sheep tissues, but they are N, N'-diacetylchitobiose derivatives. The substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases were investigated because in one type of hereditary alpha-mannosidosis, that occurring in man, the major storage products are linear rather than branched oligosaccharides. The lysosomal enzyme showed much greater activity toward linear oligosaccharides than toward the branched oligosaccharides induced in the kidney by swainsonine. On the other hand, cytosolic alpha-D-mannosidase preferred the branched oligosaccharides, a result suggesting that this mannosidase might be inhibitable by swainsonine and that the enzyme might play a normal role in glycoprotein catabolism. Swainsonine was indeed found to inhibit this enzyme at relatively high concentrations (I50 at 100 microM swainsonine), and concentrations of this magnitude were in fact found in the cytosol of kidney of swainsonine-fed rats. The kidney cytosolic alpha-D-mannosidase levels were reduced in these rats and, more important, the accumulated oligosaccharides were present mainly in the cytosol rather than in lysosomes. These results point to possible involvement of cytosolic alpha-D-mannosidase in glycoprotein degradation in the rat.

Substrate Specificities of Rat Kidney Lysosomal and Cytosolic a-D-Mannosidases and Effects of Swainsonine Suggest a Role of the Cytosolic Enzyme in Glycoprotein Catabolism* (Received for publication, February 13,1986) Daulat Ram P. Tulsiani and Oscar TousterS From the Department of Molecular Bwbgy, Vanderbilt University, NashviUe, Tennessee 37235 Swainsonine is a potent inhibitor of lysosomal a-Dmannosidase, causes the production of hybrid glycoproteins, and is reported to produce a phenocopy of hereditary a-mannosidosis. We now report that the effects of swainsonine administration in the rat are different in two respects from those found in other animals thus far studied. (i) Swainsonine caused the accumulation of oligosaccharide in kidney and urine but not in liver or brain. (ii) The accumulated oligosaccharides were mainly Man(a1-3)[Man (al-6)IMan-(Bl-Q)GlcNAc, Man(a1-3 (6) Man (al-6)
The substrate specificities of rat kidney lysosomal and cytosolic a-D-mannosidases were investigated because in one type of hereditary a-mannosidosis, that occurring in man, the major storage products are linear rather than branched oligosaccharides. The lysosomal enzyme showed much greater activity toward linear oligosaccharides than toward the branched oligosaccharides induced in the kidney by swainsonine. On the other hand, cytosolic a-D-mannosidase preferred the branched oligosaccharides, a result suggesting that this mannosidase might be inhibitable by swainsonine and that the enzyme might play a normal role in glycoprotein catabolism. Swainsonine was indeed found to inhibit this enzyme at relatively high concentrations (Is0 at 100 p~ swainsonine), and concentrations of this magnitude were in fact found in the cytosol of kidney of swainsonine-fed rats. The kidney cytosolic a-D-mannosidase levels were reduced in these rats and, more important, the accumulated oligosaccharides were present mainly in the cytosol rather than in lysosomes. These results point to possible involvement of cytosolic a-D-mannosidase in glycoprotein degradation in the rat.
Investigation of glycoprotein metabolism and of the functioning of a-D-mannosidases has been facilitated by the avail-* This investigation was supported by Grant GM 26430 and by Biomedical Research Support Grant S07-RR07201 from the National Institutes of Health, United States Public Health Service. The costa of 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.
4 To whom correspondence should be addressed Dept. of Molecu-TN 37235. lar Biology, Vanderbilt University, Box 1820, Station B, Nashville, ability of the plant toxin, swainsonine (1-9), which is found in plants of the genus Swainsona and in spotted locoweed (Astragalus lentiginosus). It is a potent inhibitor of liver lysosomal a-D-mannosidase (4,lO) and Golgi mannosidase I1 (4). The inhibition of the latter enzyme explains the ability of the alkaloid to induce the formation of glycoproteins containing N-linked oligosaccharides of the hybrid type in place of those of the complex type (4-8). Moreover, the ingestion of swainsonine-containing plants produces, in grazing animals, a neurological condition that has been characterized as a phenocopy of the hereditary lysosomal storage disease, amannosidosis (1, 10, ll). These animals accumulate mannoserich oligosaccharides in their tissues. In the pig, both swainsonine and locoweed induced the accumulation of branched oligosaccharides in all tissues examined (12).
The structures of the oligosaccharides that accumulate in a-mannosidosis patients and in swainsonine toxicosis are different. In human a-mannosidosis the major storage oligosaccharides contain linearly linked mannosyl residues (13-18). The origin of the linear compounds is unclear at present, since the specificities of known lysosomal enzymes do not explain their formation. Also unclear is the role of cytosolic a-D-mannosidase. We report herein substrate specificity studies on rat kidney lysosomal and cytosolic a-D-mannosidases and swainsonine-induced changes in rat kidney which strongly suggest an important role of the cytosol in the metabolism of glycoproteins in the rat.

EXPERIMENTAL PROCEDURES
Materials and Methods-Swainsonine, isolated from Rhizoctonia leguminicola as previously described (2) rides that accumulate in human mannosidosis (e.g. Refs. [13][14][15][16][17][18], were prepared from Gl~([~H]Man)gGlcNAc. This oligosaccharide (Fig. lA), when extensively digested with jack bean mannosidase and resolved on a high resolution Bio-Gel P-4 column, yielded one product (Fig. lB, Peak I), other than free mannose, which eluted at the position of MaQGlcNAc and presumably has the structure of GlcMan,GlcNAc. The terminal glucose residue was removed by incubating the oligosaccharide with rat liver microsomal fraction at pH 7.0 (20) in the presence of 5 p~ swainsonine. The resulting oligosaccharides resolved into two peaks on a Bio-Gel P-4 column (Fig. IC). The oligosaccharides present in Peak I and Peak I1 did not bind to a ConA-Sepharose  ([~H]M~~)~GICNAC, -600,000 cpm), obtained from a Bio-Gel P-4 column (1 X 212 cm, -400 mesh, panel A), was mixed with 100 mM sodium acetate buffer, pH 4.5,2 units of jack bean mannosidase, and 2 drops of toluene, and the mixture (0.1 ml) was incubated at 37 "C. Additional enzyme (1 unit) was added every 16 h. After incubation for 96 h, the mixture was resolved on the above Bio-Gel P-4 column equilibrated with 0.1 M acetic acid (panel B). The oligosaccharide fractions (panel B, Peak I) were pooled, dried in a Bio-Dryer, and incubated with rat liver microsomal fraction at pH 7.0 for 4 h (20) in the presence of 5 p~ swainsonine. The resulting oligosaccharides were resolved on the above Bio-Gel P-4 column (panel C). Fractions (1.2 ml) were collected at a flow rate of 1.8 ml/h and the radioactivity was measured in the aliquots as described (19). The column exclusion volume was determined using bovine serum albumin. The standards are the same as described (12). 4B column (12). In addition, when the oligosaccharide alcohols were subjected to acetolysis and then resolved on a high resolution Bio-Gel P-4 column (4, 8), more than 95% of the radioactivity eluted at the position of original oligosaccharides and less than 5% eluted at the position of free [3H]mannose (data not shown). The manner in which oligosaccharides in Peak I and Peak I1 are formed (by the treatment of GlcMansGlcNAc, an oligosaccharide of known structure, with enzymes of known specificity), the failure of the oligosaccharides to bind ConA-Sepharose 4B, and their insensitivity to acetolysis all indicate that the structures of the oligosaccharides in Peak I and tively. The oligosaccharide in Peak I appears to be a single specie. However, minor.
contamination of ManaGlcNAc (Peak 11) by ManzGlcNAc cannot be ruled out. The formation of ManaGlcNAc and perhaps a little ManzGlcNAc is due to the combined activities on Man,GlcNAc (Peak I) of endoplasmic reticulum a-D-mannosidase (21), Golgi mannosidase IA (19,22), and Golgi mannosidase IB (19) present in the liver microsomes. All of these enzymes are al,a-specific and are rather insensitive to swainsonine (4,21).
Swainsonine Administration-Male Wistar rats (170-200 g body weight) from Harlan Industries Inc. were administered swainsonine in their drinking water as described (23). In all experiments in which the effect of swainsonine on enzyme and oligosaccharide levels was determined, the swainsonine-fed animals were compared to the agematched control rata given tap water.
Preparation of Kidney Extracts and Organelles-The following preparations were done according to Shibko and Tappel (24) except that the animals were not fasted. Rats were stunned by a blow to the head and killed by decapitation. The kidneys were quickly excised, cut into small pieces, and homogenized in 0.45 M sucrose containing 0.68 mM EDTA, pH 7.0. The mitochondrial-lysosomal fraction obtained as described (24) was suspended in 1 volume of detergent solution (10 mM potassium phosphate buffer, pH 7.0, containing 0.5% Triton X-100 and 0.25 M NaCl)/g of the initial kidney weight. Cytosolic fraction was prepared by homogenizing the kidney in 1 volume of the above sucrose solution. The homogenate was centrifuged at 105,000 X g for 60 min and the supernatant (cytosolic fractions) was removed by aspiration. Subcellular fractions, namely mitochondrial-lysosomal, microsomal, and cytosolic fractions, from the kidneys of controls and rats administered swainsonine were prepared as described (24).
Enzyme Assays-PNP-mannosidase activity was assayed in 100 mM sodium acetate with 4 mM p-nitrophenyl a-D-mannosidase as substrate at pH 4.4 (lysosomal) or pH 6.0 (cytosolic) as described (23) by incubating 5 pl of the kidney fraction (mitochondrial-lysosomal suspension or cytosolic fraction) with the appropriate buffer and substrate in a total volume of 0.5 ml. Following incubation for 30 min at 37 "C, the reaction was stopped by adding 1.0 ml of stopping buffer (25). One unit is the amount of enzyme which catalyzed the release of 1 pmol of p-nitrophenollh at 37 "C.
Oligosaccharide-cleaving activity ( [3H]Man-mannosidase activity) was assayed by measuring the hydrolysis of [3H]mannose-labeled oligosaccharide in 100 mM sodium acetate buffer at pH 5.0 (lysosomal) or pH 6.0 (cytosolic). In method A, 5 pl of the kidney fraction was incubated with the buffer and 3000 cpm of the labeled oligosaccharide in a total volume of 0.1 ml. Following incubation for 2 h at 37 "C, the reaction was stopped by heating the samples at 100 "C for 5-7 min. In method B, 25 pl of the kidney fraction was incubated as above in a total volume of 0.05 ml.
The reaction was stopped (as above) following incubation at 37 "C for 30 min.
Free [3H]mannose was separated from oligosaccharide by gel filtration on a column of Bio-Gel P-2 and quantitated as described (19). One unit of [3H]Manmannosidase activity is the amount of enzyme which catalyzes the release of 1000 cpm of [3H]mannose/h. Protein was assayed by the fluorometric method (26) using bovine serum albumin as standard.
tained from the kidney of a rat administered the alkaloid was quan-Swainsonine Assay-Swainsonine in the subcellular fractions obtitated by comparing the inhibition of Golgi mannosidase I1 (27) produced by a known volume of heat-inactivated (5-7 min at 100 "C) kidney fraction with that of standard swainsonine solutions.

Urine Collection and Preparation of Urinary Oligosacchuride-
Twenty-four-hour urine samples were obtained from rats kept in metabolic cages. A few drops of toluene were added in the collecting beakers to prevent growth of microorganisms. The clear supernatant obtained by centrifuging the urine at 1,600 X g for 30 min was deproteinized by mixing with 3 volumes of ethanol followed by centrifugation. The crude oligosaccharide from the deproteinized  Urine 11,565 15,540 134 140 145 102 The animal groups are: 1, swainsonine for 1 week; 2, swainsonine for 2 weeks; 3, swainsonine for 4 weeks; 4, swainsonine for 4 weeks, and then no swainsonine for 1 week. The dose of swainsonine was 5 pg/ml of drinking water. urine was precipitated with 2 volumes of ether (12).
Preparation and Churacterization of Tissue Oligosaccharide-Crude oligosaccharide from whole tissues as well as from kidney subcellular fractions was prepared as described from our laboratory (12). In brief, crude oligosaccharide was precipitated by ethanol/ether, dried, and then suspended in 0.3 ml of 0.1 M acetic acid (12). The suspension was assayed for total hexose by the phenol/sulfuric acid method (28), scaled down ti-fold, with mannose as standard. The crude oligosaccharide was fractioned by gel filtration on Bio-Gel P-4 and purified by ConA-Sepharose 4B chromatography and high resolution Bio-Gel P-4 colum chromatography (12). The purifed oligosaccharides were analyzed for neutral sugars and hexosamine (29, 30). The oligosaccharides were labeled by reduction with NaB[3H]4 (31), and the labeled products were isolated on a Bio-Gel P-4 column (8).
The labeled oligosaccharide alcohols were subjected to acetolysis (33). The resulting labeled products were separated by high resolution Bio-Gel P-4 colum chromatography (8) and characterized by (a) size of the oligosaccharide, and (b) ConA-Sepharose column chromatography (12).

FIG. 2. Fractionation of oligosaccharide from tissues and urine of control and swainsonine-fed rats.
Male Wistar rats (200 g body weight) were administered swainsonine (5 pg/ml drinking water for 4 weeks) as described under "Experimental Procedures." The ethanol/ether-precipitated oligosaccharide was prepared from 1.5 g of tissues (A, liver; B, kidney; and C, brain) and 24 h urine ( D ) from the control ( 0 ---0) and the swainsonine-fed rat (M). The oligosaccharide was suspended in 0.3 ml of 0.1 M acetic acid and applied to a Bio-Gel P-4 column (0.8 X 136 cm, -400 mesh) equilibrated with 0.1 M acetic acid Fractions (0.5 ml) were collected at a flow rate of 2.5 ml/h. Aliquots from each fraction were assayed for hexose by the phenol/sulfuric acid method

RESULTS
Effect of Swainsonine Administration on Oligosaccharide Content of Rat Tissues and Urine-Comparison of the total hexose levels in the deproteinized ethanol/ether precipitate showed that only kidney, and perhaps urine, but not liver or brain, from swainsonine-fed rats contained higher levels of total hexose than the age-matched control rats. As shown in Table I, total hexose is essentially unchanged in the swainsonine-fed rat liver and brain, but the kidney of experimental animals contained nearly &fold higher oligosaccharide content than the control kidney. The ethanol/ether precipitate from the experimental rat urine showed somewhat higher levels of total hexose (134-146%) than the control rat urine ( Table I).
Fractionation of the ethanol/ether-precipitated crude oligosaccharide from the urine and tissues of control and swainsonine-fed rats on a Bio-Gel P-4 column showed that only kidney and urine, but not liver or brain, of experimental animals showed accumulation of oligosaccharides (Fig. 2).
Three oligosaccharides were separated and purified from the experimental rat kidney fractions 90-115 ( Fig. 2) by ConA-Sepharose 4B and then high resolution Bio-Gel P-4 column chromatography. When the pooled oligosaccharides were applied to a ConA-Sepharose 4B column (1.5 X 18 cm) as previously described (12), less than 10% of the oligosaccharide passed unadsorbed into the effluent. This unadsorbed oligosaccharide, when applied to the Bio-Gel P-4 column, eluted as a sharp peak at the position of standard MaaGlcNAc (Fig. 3A) and had the composition of MaaGlcNAc (oligosaccharide B) ( Table 11). The nearly 90% of the original oligosaccharide that adsorbed to the ConA-Sepharose column, when eluted with 250 mM a-methylmannoside and applied to the Bio-Gel P-4 column, separated into two peaks (Fig. 3B). Sugar analyses of Peak I (oligosaccharide C) and Peak I1 (oligosaccharide A) yielded the composition Man,GlcNAc and Man,GlcNAc, respectively (Table 11).
It was observed that the total amount of crude oligosaccharide in kidney is dependent on the amount of swainsonine administered. The rats fed 5 pg of swainsonine/ml of drinking water showed 3-and &fold higher oligosaccharides in kidney

Sugar ~o m~s i t w n
of o~~o s~c~~e s isolated from swainsonine-treated rat kidney The oligosaccharides were isolated from the kidney of a rat administered swainsonine (5 pg/ml drinking water for 4 weeks) as described under "Experimental Procedures." The purified oligosaccharides were analyzed for neutral s u m and hexosamine (29.30). M~~SGICNAC "Gas liquid chromatographic analysis for the neutral sugars showed the presence of only mannose. N-acetylglucosamine (GlcNAc) was arbitrarily taken as 1.00.
*The behavior of the three oligosaccharides on Bio-Gel P-4 agreed with the composition shown in this table (see "Results"). after 2 and 5 days, respectively; the rats fed 20 or 50 pg of swa~sonine/ml showed 5-and 15-fold increases (Fig. 4). On the other hand, administration of higher concentration of the alkaloid had no effect on the liver or brain oligosaccharide (data not shown). It may be noted here that we have previously shown (9) that the relative amounts of the three oligosaccharides accumulating in the experimental kidney are de- Male Wistar rata were fed various concentrations of swainsonine (0-50 pg/ml drinking water) for 2 days (0---0) or 5 days (U). The ethanol/ether precipitated oligosaccharide was prepared from 1.5 g of kidney as described under "Experimental Procedures" and assayed for total hexose by the phenol/sulfuric acid method (28). pendent on the concentration of swainsonine administered.
Structures of Kidney Oligosaccharides A, B, and C-The structures of the three oligosaccharides isolated from the kidneys of swainsonine-fed rats were established by first reducing them with NaB[3H]4 and then subjecting the labeled oligosaccharide alcohols to various treatments which yielded the following results. (i) The three oligosaccharides were sensitive to jack bean a-D-mannosidase treatment, resulting in the production of a substance which eluted from a Bio-Gel P-4 column at the position of Man-GlcNAc~. (ii) Unlike the two major oligosaccharides accumulating in the brain and kidney of swainsonine-and locoweed-fed pigs (12), the rat kidney oligosaccharides were resistant to both endo-8-Nacetylglucosaminidase H and endo-P-N-acetylglucosaminidase D. The resistance to these endoglycosidases agrees with analyses indicating that each oligosaccharide contained only 1 GlcNAc residue (Table 11). (iii) Rat liver Golgi m a n n o s i~ I1 had no effect on these oligosaccharides. However, oligosaccharides B and C, but not A, were sensitive to Golgi mannosidase I1 after they were N-acetylglucosaminylated (S), a result indicating that B and C are Man4 or Mans derivatives. (iv) Following acetolysis, which rather selectively cleaves 1,6 linkages, the labeled products from oligosaccharides A, B, and C eluted from a Bio-Gel P-4 column at the position of Man2GlcNAcoT. Together with the compositional data, these results indicate that the three oligosaccharides accumulating in the kidney of the swainsonine-fed rat have the structures:  (Fig. 5). It is noteworthy that oligosaccharides A and C bind to a ConA-Sepharose 4B column, in conformance with their assigned structures, which have 2 interacting mannose residues attached to a single mannose residue (34,35), whereas oligosaccharide B does not bind to this adsorbent. Oligosaccharides B and C are similar to the oligosaccharides isolated from swainsonine-and locoweed-fed pig brain and kidney (12) Table 111. Surprisingly, the a-D-mannosidase present in the mitochondriallysosomal fraction showed low activity toward high mannose oligosaccharides (MansGLcNAc to Man6GlcNAc). Even the branched oligosaccharides which accumulate in the kidney of rats administered swainsonine, namely ManaGlcNAc, ManlGlcNAc and Man,GlcNAc, were poor substrates. However, the linear oligosaccharides were good substrates for this enzyme. There was complete hydrolysis of both al-2 and al-3 linkages in linear Man,GlcNAc, whereas there was much less cleavage of al-2 linkages in high mannose oligosaccharides and of the a1-3 linkage in branched Man3GlcNAc. On the other hand, with cytosolic a-D-mannosidase the high mannose oligosaccharides (M-GlcNAc to Man6GlcNAc) and two of the three oligosaccharides accumulating in swainsonine toxicity were better substrates than the linear oligosaccharides ( Table 111). The difference in the substrate specificities of the rat kidney lysosomal a-D-mannosidase and cytosolic a-D-mannosidase is also evident from the time course of the hydrolyses of the smaller oligosaccharides (Fig.  6). Although the limited supply of mannose-labeled substrates available for these specificity studies did not permit variations in substrate concentrations, the results obtained do show substantial differences with the two enzymes, especially in activity toward Man6GlcNAc, which was cleaved only by the cytosolic enzyme.

6)]Man(al-6)[Man(~l-3)]Man(~l-4)GlcNAc
It was of interest to determine the structures of products formed by the action of cytosolic a-mannosidase on oligosaccharide. Table IV gives the results of an experiment on MansGlcNAc. After extensive action of kidney cytosolic fraction on this substance, the reaction products were isolated by gel filtration on Bio-Gel P-2 and then fractionated on a ConA-Sepharose 4B column. The purified oligosaccharide products were subjected to acetolysis, and the degradation products   Effect of Swainsonine on Cytosolic a-D-Mannosidase-We have previously reported that swainsonine is a potent inhibitor of rat liver lysosomal a-D-mannosidase and Golgi mannosidase I1 (4). Although the alkaloid caused 50% inhibition of these two enzymes at a concentration of 0.2 p~, it had little or no effect on liver cytosolic a-D-mannosidase even at a concentration of 10 p~ (4). We now report that at a much higher concentration the alkaloid inhibits both the PNPmannosidase and [3H]Man-mannosidase activities present in the kidney cytosol. As shown in Fig. 7, 50% inhibition of the cytosolic a-D-mannosidase activity is caused by 100 pM swainsonine, a concentration that might have biological relevance in spite of its being 500 times higher than required to cause FIG. 6

. The time course of hydrolysis of [8H]Man-oligosaccharides by kidney lysosomal and cytosolic a-Dmannosidase.
Approximately 3000 cpm of each of the five oligosaccharides was incubated at 37 "C with aliquots from a rat kidney mitochondrial-lysosomal suspension (pH 5.0) or cytosolic fraction (pH 6.0), each containing 1.5 units of PNP-mannosidase activity, in a total volume of 0.1 ml.
The reaction was stopped at the indicated time by heating the samples in boiling water (5-7 min). Free [3H]mannose was quantitated after separation from labeled oligosaccharide on a column of Bio-Gel P-2 (19). M, Man; N , GlcNAc.

Products formed from action of rat kidney cytosol on (pH]Man)5GlcNAc
Approximately 60,000 cpm of uniformly labeled ( [3H]Man)5GlcNAc (8) was incubated at 37 "C and pH 6.0 with rat kidney cytosol (containing 1 unit of PNP-mannosidase activity) in a total volume of 0.1 ml as described under "Experimental Procedures." Additional enzyme (1 unit) was added after 16 and 35 h. After 48 h, the reaction mixture was heated at 100 'C for 5-7 min and applied to a Bio-Gel P-2 column (19). Nearly 48% of the radioactivity eluted in fractions 28-37 (oligosaccharides), the remainder eluting as free [3H]mannose. The oligosaccharidecontaining fractions were pooled, dried in a Bio-Dryer, and fractionated on a concanavalin A-Sepharose 4B column (12). The unadsorbed material was then fractionated by a high resolution Bio-Gel P-4 column (8), yielding Man3GlcNAc and ManZGlcNAc. The adsorbed material was eluted with 0.5 M a-methylmannoside (a-MeMan) and purified on the Bio-Gel P-4 column. It was Man3GlcNAc.
The three purified oligosaccharides, as well as the original ([3H]Man)6GlcNAc substrate, were reduced and then subjected to acetolysis aa described under "Experimental Procedures." The acetolysis products were fractionated on a high resolution Bio-Gel P-4 column (8) If acetolysis were completely specific for 1,6 linkages, the amount of labeled cleavage products obtained would allow 1520% of Man(al-6)Man(@1-4) to be present in the fraction analyzed. Since acetolysis is not that specific, considerably less is present with the Man(al-B)Man(a1-6)Man(@1-4)GlcNAc. similar inhibition of the other two mannosidases.

Effect of Swainsonine Administration on a-D-Mannosidme Levek in Rat
Kidney-Since the swainsonine inhibition of lysosomal a-D-mannosidase (27) and cytosolic ff-D-mannosidase' is largely reversible, it occurred to us that, if enzyme assays were carried out at different dilutions, the levels of enzymatic activities found might give evidence for the presence of inhibitory amounts of swainsonine in the kidney fractions. Table V shows the results obtained in assaying D. R. P. Tulsiani and 0. Touster,unpublished results. oligosaccharide cleavage by cytosolic fractions from swainsonine-fed and control animals. Method A, employing the more diluted extracts (20-fold dilution), gave higher enzymatic levels only for the swainsonine-fed animals than did Method B (2-fold dilution). The data strongly suggest that the cytosolic mannosidase is in fact inhibited in the intact kidney and that the inhibition is due to swainsonine.

Subcellular Localization of Oligosaccharides and Swainsonine in the Kidney of the Swainsonine-fed
Rat-The kidney of the swainsonine-fed rat shows a rapid and massive accumulation of mannose-rich oligosaccharides (9). Since lysosomal i 0 FIG. 7. Effect of swainsonine on kidney cytosolic a-D-maIInosidase. Rat kidney cytosolic fraction was assayed for a-D-mannosidase activity using 4 mM p-nitrophenyl a-D-mannoside or -3000 cpm of [3H]Ma~GlcNAc in 100 mM sodium acetate buffer, pH 6.0, the total volumes being 0.5 and 0.1 ml, respectively. The enzyme was preincubated (15 min at 0-4 "C) with varying concentrations of swainsonine in the buffer before addition of substrate and incubation at 37 "C for 60-120 min. Releasedp-nitrophenol or [3H]mannose was quantitated as described (19).  "The animal groups are: 1, swainsonine (5 pg/ml drinking water) for 3 weeks; 2, swainsonine (20 pg/ml drinking water) for 1 week.
The values are calculated for control (C) and swainsonine-fed animals (S) analyzed at the same time. Values in parentheses are percentages. a-D-mannosidase did not show appreciable activity toward these oligosaccharides in vitro (see Table I11 and Fig. 6) and since the experiments discussed above (Table V) gave evidence that the cytosolic mannosidase might be inhibited in intact kidney, it appeared possible that the lysosomal enzyme might not be involved in the catabolism of the accumulating oligosaccharides in uiuo. Support for this possibility was obtained by studying the subcellular localization of the oligosaccharides and swainsonine, as well as lysosomal marker enzymes, in the kidney of the swainsonine-fed rat. These studies showed that the bulk of oligosaccharide (over 60%) was present in the cytosolic fraction, whereas only 31 and 7% of the oligosaccharides sedimented with the mitochondrial-lysosomal and microsomal fractions, respectively (Table VI). The two lysosomal glycosidases, on the other hand, showed their expected organellar localization.
The amount of swainsonine found in the mitochondriallysosomal fraction is adequate to cause a marked inhibition of lysosomal a-mannosidase (Table VII). However, 30 times more swainsonine was found in the cytosol than in the mitochondrial-lysosomal fraction (Table VII); the higher concentration of the alkloid found for group 2 agrees with the greater reduction in enzymatic activity shown in Table V. The swainsonine concentration in the cytosol is far higher than could be explained by leakage from the organelles, which in fact TABLE VI Total oligosaccharide and lysosomal enzymes in rat kidney subceUular fractions following swainsonine administration Subfractions were prepared from the kidney of rats administered swainsonine (20 pg/ml drinking water) for 5 days as previously described (24) except that the rats were not fasted.  The animal groups are 1, swainsonine (5 pg/ml drinking water) for 3 weeks; and 2, swainsonine (20 pg/ml drinking water) for 1 week.
* Subcellular fractions were obtained as described under "Experimental Procedures." Swainsonine was assayed as described under "Experimental Procedures." The values reported are averages of two or three animals with f indicating the range in values.
were not washed during their preparation. Although the level of swainsonine varied with the dosage, its concentration in the cytosol in both experiments in Table VI1 appears to be in the proper range to inhibit cytosolic a-D-mannosidase. If the volume of cytosol in kidney cells averages 25% of the cells, then the cytosolic swainsonine concentrations in Table VI1 are 34 ~L M for group 1 and 122 p~ for group 2. In Fig. 7 the cytosolic mannosidase was shown to be 50% inhibited at 100 p~ swainsonine. DISCUSSION It has long been assumed that lysosomal a-D-mannosidase, together with hydrolases in this organelle for other glycosidic linkages, has a central role in the catabolism of glycoproteins containing asparagine-linked oligosaccharides. The assumption is based on the accepted role of lysosomes in catabolism and on the fact that oligosaccharides accumulate in the lysosomes of animals and humans with an inherited deficiency of a-D-mannosidase. At the same time, there has been no clue as to the function of cytosolic a-D-mannosidase. Substrate specificity studies on the two enzymes have been limited (36, 37) and, perhaps in part for this reason, it has been difficult to determine why particular oligosaccharides are found in mannosidosis tissues and urine (13)(14)(15)(16)(17)(18).
In the present discussion, the term "cytosol" is used operationally, designating the high speed supernatant solution from a tissue homogenate prepared by a mild homogenization procedure. Nonetheless, the presence of uncharacterized microvesicles is possible. Alternatively, some type of fragile vesicles (other than lysosomes) may have been extensively ruptured during homogenization. Perhaps the best approach to investigating these possibilities would be to carry out electron microscopic immunocytochemical analysis. This point is especially important because current views of glycoprotein compartmentalization and transport would not permit access of cytosolic enzymes to glycoproteins.
It has been suggested (21, 37) that similarities between cytosolic a-D-mannosidase and a recently discovered endoplasmic reticulum mannosidase raise the possibility that the former is a proteolytic product of the latter. While this possibility cannot be precluded at the present time, it is noteworthy that, whereas the endoplasmic reticulum mannosidase is inactive toward GlcNAcManSGlcNAc (21), the cytosolic enzyme readily hydrolyzes this substrate (Table 111).
Since the present work has relevance to the abnormal oligosaccharide catabolism in hereditary a-mannosidosis, some brief comments on this disease are in order. In the amannosidosis of man, the accumulated tissue oligosaccharides are predominantly linear in structure and contain one GlcNAc (16,18). Bovine a-mannosidosis is accompanied by the excretion of oligosaccharides, half of which appear to be branched and half of which are Man2GlcNAc2 (38). Very recently the excreted oligosaccharides in feline a-mannosidosis have been reported to be branched in structure, Man3GlcNAc2 being the predominant urinary substance (39). Three points should be made. (i) The species differences make enzymatic interpretations more difficult to reach. (ii) Since urinary oligosaccharides may result from subsequent modification of accumulated tissue oligosaccharides, their structures may not be reliable indicators of the nature of the enzymatic deficiencies in tissues. (iii) The feline storage disease appears to be more complex than the others. In addition to the deficiency of lysosomal a-D-mannosidase, the liver and kidney cytosolic mannosidase were depressed to one-third of normal (40). Tissue analyses in the cat were not reported.
In the present work we show that swainsonine administration causes accumulation of oligosaccharides in rat kidney and urine, but not in liver or brain. The three oligosaccharides isolated from the kidneys of swainsonine-fed animals were shown to be branched-chain structures of the composition Man,GlcNAc, Man,GlcNAc, and Man,GlcNAc. The pentamannosyl compound is probably derived from the hybrid type glycoproteins produced as a result of the inhibition of Golgi mannosidase I1 by swainsonine, with the other compounds presumably resulting from residual mannosidase action. Man,GlcNAc and Man,GlcNAc have the same oligomannose structure as in the two major oligosaccharides found in the brain and kidney of pigs fed swainsonine or locoweed, the oligosaccharides from this species containing a diacetylchitobiose residue in place of the single GlcNAc (12). This difference is undoubtedly due to differences in amount, or action, of mammalian endoglycosidase which, until recently, has been found to be localized in the cytosol (41-45), not the lysosomes. A lysosomal di-N-acetylchitobiase with substrate specificity different from that of the cytosolic enzyme has recently been reported (46). The cytosolic localization of the longer known enzyme obviously raises the question as to whether glycoprotein degradation occurs in the cytosol, which contains all of the required enzymes, namely neuraminidase (47, 48), @-galactosidase (49, 50), N-acetyl-@hexosaminidase (51), a-D-mannosidase (52, 53), and a /3-mannosidase (54), as well as endoglycosidase. Therefore, we examined the substrate specificity of both lysosomal and cytosolic a-D-mannosidase of rat kidney, the only tissue in the rat which showed oligosaccharide accumulation during swainsonine administration.
The comparative study of the two mannosidases disclosed sharp and unexpected differences in substrate specificity. Whereas the lysosomal enzyme readily hydrolyzed linear oli-gomannose compounds, it had little activity toward high mannose oligosaccharides or toward the three branched oligosaccharides which accumulated during swainsonine administration. While these results are consistent with the accumulation of linear compounds in human mannosidosis, they raise the question as to how the high mannose, branched oligosaccharides are hydrolyzed normally during catabolism. These compounds were found to be better substrates for the cytosolic a-D-mannosidase, which was rather ineffective in cleaving the linear oligosaccharides. These results also suggested a normal role for the cytosol in oligosaccharide degradation and that swainsonine might be inhibiting cytosolic a-D-mannosidase, thereby causing accumulation of the three storage compounds.
Although we had earlier found that, as compared to the lysosomal enzyme and mannosidase 11, cytosolic mannosidase is relatively insensitive to swainsonine (4), a new test with comparatively high alkaloid concentrations showed that it is 50% inhibited by 100 PM swainsonine. Moreover, the level of cytosolic mannosidase activity in the kidney of swainsoninefed rats was depressed, particularly when concentrated tissue samples were used for assays to counteract the reversibility of inhibition by dilution. This experiment suggested that swainsonine was in fact the cause of the depressed enzyme levels. Furthermore, the concentration of swainsonine required to produce the depressed levels of cytosolic mannosidase in kidney was reasonably similar to that actually found in kidney cytosol, in which the administered swainsonine is highly concentrated. Whether the decreased cytosolic enzymatic activity is sufficient to cause oligosaccharide accumulation cannot be answered at this time, since it would depend in part on the load of oligosaccharide and its structure as well.
Finally, as predicted from the above findings, subcellular localization of the accumulated oligosaccharide in the kidney of the swainsonine-fed rat disclosed that the oligosaccharide was present mainly in the cytosol, rather than in a fraction containing lysosomes. The concentration of both oligosaccharide and swainsonine in the cytosol, the inhibitory effect of swainsonine on cytosolic a-D-mannosidase, the depressed levels of cytosolic mannosidase in the swainsonine-fed rat, and the structures of the accumulated oligosaccharides all suggest that the swainsonine-induced storage in rat kidney is a result of events occurring in the cytosol. The presence of substantial endoglycosidase in the cytosol (41-45) is consistent with this conclusion, and it is relevant that this enzyme is reported to show a marked preference for branched as compared to linear oligosaccharides (45).
What, then, are the different roles of the cytosol and lysosomes in glycoprotein breakdown? One possibility is that each compartment has a different enzymatic system, the cytosolic system involving branched oligosaccharides and the lysosomal one involving the linear oligosaccharides. Among the possible mechanisms for the production of the linear compounds within lysosomes are the following: (i) action of the newly discovered lysosomal endoglycosidase followed by cleavage of high mannose oligosaccharides by unknown exoand/or endo-a-D-mannosidase; (The existence of a new a -~mannosidase in lysosomes has, in fact, been suggested to be present in fibroblasts, this enzyme being inhibited by swainsonine but not absent in hereditary mannosidosis (55).) (ii) cleavage by a-D-mannosidase(s) of high mannose oligosaccharides which are temporarily blocked, perhaps by Glc, at the 1,3-antenna; and (iii) catabolism of glycoproteins containing linear oligosaccharides (e.g. rat liver cathepsin B (56)).
It is conceivable that oligosaccharide hydrolysis generally begins in the cytosol and that linear products are then trans-ferred to lysosomes for further cleavage. In one experiment bearing on this question, we found that cytosolic mannosidase produced from branched MansGlcNAc a mixture containing only 29% branched Man3GlcNAc, the other products being linear Man3GlcNAc and Man2GlcNAc. No transport system is currently known by which the latter two substances might enter lysosomes. However, there is now evidence for transport systems in lysosomal membranes, for example, for cystine (57) and N-acetylneuraminic acid (58).
It is particularly noteworthy that in human mannosidosis branched Mans derivatives are only very minor excretory products and are not found in the tissues of patients (13). If these Man, products of the breakdown of complex glycoproteins, the most abundant N-linked glycoproteins of mammalian tissues (59), were normally handled by lysosomal a-Dmannosidase, they should be major storage products. In fact, neither the lysosomal nor cytosolic mannosidases studied in the present work, or even a new brain a-mannosidase recently reported (60), utilize branched Man3GlcNAc very well.
It is evident that the major degradative pathways for asparagine-linked oligosaccharides have not as yet been clearly delineated. The results of our study of rat kidney indicate that subsequent investigations should deal with possible involvement of cytosolic as well as lysosomal enzyme systems. In particular, the substrate specificities of the glycosidases in the two cellular compartments are of particular relevance.