The effect of α-mannose-terminal oligosaccharides on the survival of glycoproteins in the circulation

ARCItlVES OF BIOCHEMISTRY AND BIOPHYSICS Vgl. 188, No. 2, June, pp. 418-428, 1978

The Effect of ~x-Mannose-Terminal O l i g o s a c c h a r i d e s on the Survival of Glycoproteins in the Circulation Rapid Uptake and Catabolism of Bovine Pancreatic Ribonuclease B by Nonparenchymal Cells of Rat Liver 1 TERRY L. BROWN, 2 LEE A. HENDERSON,* SUZANNE R. THORPE,* JOHN W. BAYNES 3

AND

Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, Minnesota 55108, and *Department of Chemistry, College of Science and Mathematics and School of Medicine, University of South Carolina, Columbia, South Carolina 29208 Received October l, 1977; revised March l, 1978 ~'I-Labeled ribonuclease B (RNase B), injected intravenously, is rapidly cleared from the rat circulation (he ~ 15 min). Clearance of this glycoprotein depends on specific recognition of the a-mannosyl residues of the attached oligosaccharide and is inhibited by the coinjection of an excess of unlabeled RNase B or yeast mannan, but not by RNase A, dextrans, galactan, or asialofetuin. Following L~I-labeled RNase B injection into nephrectomized rats, radioactivity accumulated primarily in the lysosomal subcellular fraction in the liver and was rapidly degraded to low molecular weight iodinated products. Free iodide began to accumulate in the stomach and intestines after 30 min. Liver, spleen, and bone marrow were most active, on a weight basis, in the clearance of RNase B. At 15 min after the injection of ~'~I-labeled RNase B, parenchymal and nonparenchymal cells were isolated from liver following perfusion with dilute formalin and collagenase. More than 90% of the radioactivity was localized in the nonparenchymal cell fraction. The evidence indicates that mannose-dependent glycoprotein clearance from the circulation occurs primarily in tissues characteristic of the reticuloendothelial system.

Recently Baynes and Wold (1) determined that the simple, a-mannosyl-terminal oligosaccharide of RNase B 4 was the signal for rapid and selective clearance of J This work was supported in part by USPHS Research Grants GM 15053, to Professor Finn Wold at the University of Minnesota, and AM 19971, to SRT and JWB at the University of South Carolina. JWB was recipient of American Cancer Society Postdoctoral Fellowship PF-1147 ~it the University of Minnesota. 2 This work was submitted, in part, by TLB in partial fulfillment of the requirements for the B.S. degree, Summa Cum Laude, Biochemistry, from the University of Minnesota. :~To whom correspondence should be addressed at Department of Chemistry, University of South Carolina, Columbia, South Carolina 29208. Abbreviations used: RNase B, ribonuclease B; CMF, complete perfusion buffer free of calcium and magnesium; ICS, initial cell suspension; PC, parenchymal cells; NPC, nonparenchymal cells. 418 0003-9861/78/1882-0418502.00/0 Copyright 9 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

this isozyme from the circulation of nephrectomized rats. The half-life of RNase B was about 15 min, while that of RNase A (no carbohydrate) was 9-10 h and that of RNases C and D (complex, sialyl-terminal oligosaccharide) was 12-15 h. Enzymatic removal of the a-mannosyl residues of RNase B resulted in an increase in its halflife to 10-11 h, similar to that of the other RNase isozymes. The observations indicated that a mechanism exists in the rat for removing glycoproteins from the circulation based on specific recognition of terminal a-mannosyl residues. Since the initial observations of Ashwell and Morell (2) on the selective, hepatic uptake of galactose-terminal glycoproteins, there has been considerable interest in the potential use of carbohydrate tags to direct proteins or therapeutic agents to specific organs or cell types (3). Our observations

HEPATIC U P T A K E AND CATABOLISM OF RNase B

on the mannose-dependent uptake of RNase B provided evidence for an additional carbohydrate-specific uptake process in mammals; the present studies were designed to identify the tissue site(s) of uptake of RNase B and to evaluate a possible role for the protein portion of the RNase B molecule in the recognition process. In addition, by studying the metabolic fate of RNase B after tissue uptake, we hoped to gain insight into the physiological function of this clearance pathway. MATERIALS AND METHODS

Enzyme assays. Ribonuclease B enzymatic activity was assayed using cytidine 2':3'-phosphate or yeast RNA as substrate, as previously described (1). RNase A (Sigma Type I-A) was used as the standard for measurement of RNase concentration. Iodination of RNase and purification of labeled product. Both RNase A and RNase B were labeled with '~'~Ias described below for RNase B only. Results with the two isozymes were essentially identical. Iodine-125 was obtained from New England Nuclear. RNase B (Sigma Type XII-B) was purified by affinity chromatography on insolubilized concanavalin A-agarose (1). This RNase B was previously shown to contain an oligosacchraide consisting of five mannose and two N-acetylglucosamine residues (1). Iodination was carried out by the technique of Davidson et al. (4, 5) using equimolar amounts of RNase B and NaI (initial specific activity -~ 2 Ci/mmol), with N-bromosuccinimide as the oxidizing agent. After quenching the reaction with excess NaI and Na2S20:~, the solution was chromatographed on Sephadex G-75 (Fig. 1, top) to isolate monomeric '2'~I-labeled RNase B. Radioactivity was quantitated using a Searle Analytic Model 1195 gamma counter. Recovery of RNase B enzymatic activity at that point was 70%. Labeling efficiency was approximately 60%, i.e., approximately 0.6 mol of '2'~I/mol of RNase B. Appropriate fractions were pooled for chromatography on a ribonuclease affinity column made from N-(6-aminohexyl)cytidine-2'(3')monophosphoric acid coupled to activated CH-Sepharose 4B, according to Scofield et al. (1, 6). Both enzyme activity and radioactivity were quantitatively bound to, and coeluted from, the affinity column (Fig. 1, bottom). Active fractions were pooled, neutralized with ammonium hydroxide, lyophilized, and stored frozen in the 0.9% saline solution used for injections. The specific enzymatic activity of the recovered, radioiodinated RNase B was approximately 95% of the specific activity of the native enzyme. The '~aI-labeled RNase B could be quantitatively bound to concanavalin A-agarose and eluted by 1 M a-methylmannoside (1). '2aI-Labeled RNase A passed through this column unretarded.

419

Animal injection experiments. Experiments were performed using nephrectomized male albino rats, 300 + 30 g, unless otherwise indicated. Bilateral nephrectomies were performed as previously described (1). Proteins and polysaccharides were dissolved in physiological saline solution and warmed to 37~ prior to intracardial injection. When precise measurements of injected radioactivity were desired, the syringe was rinsed after injection, and the true amount injected was estimated as the difference between the amount originally withdrawn into the syringe and the amount in the rinse. Blood samples were taken at various times postinjection from lateral tail veins using heparinized capillary tubes. Following centrifugation, plasma aliquots were assayed for radioactivity and/or RNase activity. '2aI-Labeled RNase B was distinguished from lower molecular weight radioactive materials by acid precipitation; 20 #l of plasma was mixed with an equal volume of 2.5 mg/ml bovine serum albumin and precipitated by the addition of 200 td of a 12.5% trichloroacetic acid, 2.5% uranyl acetate solution. To determine the site of uptake of '2'~I-iabeled RNase B, the animals were sacrificed at various times after injection by overanesthetizing with ether. The chest cavity was opened and the carcass was perfused with 100 ml of iced physiological saline solution containing 250 units of sodium heparin (Riker Laboratories). The saline was injected through a needle inserted in the left ventricle of the heart, and the perfusate exited through a cut in the right atrium. Following perfusion, selected organs were removed, rinsed, blotted, and weighed, and small pieces (_<1 g) were counted directly in the gamma counter. Stomach and intestines were opened and rinsed free of contents. The contents were dispersed by sonication, and aliquots were assayed for radioactivity. For estimation of radioactivity in the bone marrow, both femurs were removed, weighed, and extracted with 6% aqueous KOH at 60~ for 24 h. The KOH extract was assayed for radioactivity, and residual bone fragments were washed in 80% ethanol, dried, and weighed. The marrow weight in the femurs was estimated by subtracting the weight of bone fragments from the original femur weight. Total marrow radioactivity was estimated by multiplying total femur radioactivity by 5.6, according to Keene and Jandl (7). Liver cell fractionation. The surgical procedure followed that of Wagle and Ingebretsen (8). The complete perfusion buffer contained 8 g of NaC1, 0.4 g of KC1, 0.2 g of MgSO4- 7H20, 0.4 g of CaCl2.2H~O, 0.06 g of Na2HPO4 92H20, 0.06 g of KH2PO4 92H~O, and 4.76 g of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) in 1 liter of water, adjusted to pH 7.5 with 10% NaOH as described by Nilsson and Berg (9). Perfusion was initiated in situ in the above buffer free of calcium and magnesium (CMF buffer), followed by a recirculating perfusion in vitro in CMF buffer with 0.1% formaldehyde for 10 min (10). The CMF buffer

420

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FIG. 1. Preparation and characterization of ~2"~I-labeledRNase B. Top: RNase B (10 rag) was labeled with 2 mCi of ~ I , as described. The iodination reaction mixture was chromatographed on a Sephadex G-75 column (36 x 1.5 cm) in 0.2 M ammonium acetate. One-milliliter fractions were collected. Bottom: Affinity chromatography of pooled ~2'~I-labeledRNase B. Fractions 36-46 from the Sephadex G-75 column were pooled, lyophilized, dissolved in 5 ml of 0.1 N sodium acetate, pH 5.2, and then chromatographed on a RNase affinity column (5 • 1.5 cm}. Elution with 0.1 N acetic acid resulted in quantitative recovery of the starting material. One-milliliter fractious were collected. was then replaced with complete buffer containing collagenase (Worthington Biochemicals; 50 mg/150 ml) and perfusion was continued for 15-20 min. The weight of the liver was determined in a tared beaker containing cold CMF buffer, and a small portion was removed for determination of total liver radioactivity. The remaining liver was minced with scissors and passed through two layers of cheesecloth to yield the initial cell suspension (ICS). The cell types were then

separated by differential centrifugation according to Tolleshaug et al. (11). An initial low-speed spin (1.5 min at 40g, v) separated a pellet composed predominately of parenchymal cells (PC) and a supernatant containing mainly nonparenchymal cells (NPC). The PC were resuspended in CMF buffer and the centrifugation step was repeated two or three times to obtain the final purified PC preparation. The original supernatant was treated with an additional low-speed spin

421

HEPATIC UPTAKE AND CATABOLISM OF RNase B to remove the majority of residual PC. NPC were sedimented by centrifugation for 5 rain at 220 g ~ and washed two or three times to remove cellular debris. A final low-speed spin (1.5 rain at 40g, D removed any remaining hepatocytes. Cell counts were performed with a hemocytometer. Despite the formalin treatment, less than 5% of the cells at all steps in the isolation procedure were stained by trypan blue, indicating that cellular membranes were intact. The yields of purified PC and NPC by this method were 60-80 and 25-35%, respectively. These recoveries are comparable to reported values (9-11). RESULTS

Clearance of 12~I-labeled RNase B.

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FIG. 2. Clearance of '2~I-labeled RNase B from the plasma of nephrectomized rats. Approximately 1 • 107 cpm, or about 200 #g, of '2'~I-labeled RNase B was injected. Plasma aliquots were assayed for both enzymatic activity and radioactivity. The data represent the average and range of observed values for three rats, expressed as a percentage of the 100-s time point.

When ]25I-labeled RNase B was injected into nephrectomized rats, there was good correlation between the kinetics and extent of clearance from the blood of RNase enzymatic activity and total radioactivity (Fig. 2). In this experiment, 107 cpm ]25Ilabeled RNase B (~ 1 #g) was mixed with 200 #g of native RNaseB, and the observed circulating half-life of both the enzymatic activity and the radioactivity was approximately 15 min, in agreement with earlier observations on the kinetics of clearance of unlabeled RNase B from the rat circulation (1). In samples taken after 1 h postinjection, it was observed that plasma radioactivity FIG. 3. Determination of high and low molecular was consistently greater than would be preweight radioactivity in plasma at times following the dicted from the residual RNase enzymatic injection of '~SI-labeled RNase B. Approximately 1 x activity. However, as shown in Fig. 3, this 10~ cpm of '~I-labeled RNase B was injected, and is explained by the appearance of low mo- plasma samples were acid-precipitated for the mealecular weight radioactivity in the blood surement of high (acid-precipitable) and low (acidstream after 20-30 min and thus does not soluble) molecular weight radioactivity. Note the difrepresent intact RNase B molecules. By 2 ference in scales used for high and low molecular h, 60-80% of the residual plasma radioactiv- weight radioactivity. ity was acid soluble. The essentially identical kinetics of clearance of the native and beled RNase B, 3 mg of RNase B was radioactive enzymes suggests that both are sufficient to slow the initial clearance being cleared by the same mechanisms. slightly and 10 mg of RNase B had a strong Competition experiments. The. role of a- inhibitory effect. The same amounts of mannosyl residues in the rapid clearance of RNase A had no effect. The specificity of the RNase B receptor RNase B was further investigated in the competition experiments presented below. site for a-mannosyl residues was also indiAs shown in Fig. 4, when tracer amounts of cated by the inhibition of RNase B clear~25I-labeled RNase B were injected with ance by yeast mannan (Sigma). As shown large excesses of unlabeled RNase A or B, in Fig. 5a, RNase B clearance was inhibited only RNase B was effective in inhibiting in a dose-dependent fashion by the simulthe clearance of radioactivity from the cir- taneous injection of as little as 1-2 mg of culation. Although the addition of 1 mg of mannan. With coinjections of 5 or 10 mg of competing RNase B had no detectable ef- mannan, the kinetics of clearance of RNase fect on the kinetics of removal of ]2~I-la- B during the first hour postinjection were 0

TIME

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422

BROWN E T AL.

not alter the rapid kinetics of clearance of ~2~I-labeled asialofetuin, indicating that the mannan inhibition was specific for mannose-terminal glycoproteins. In addition, the possibility that RNase B clearance was retarded because of complex formation between enzyme and mannan was considered unlikely since the elution volume of RNase B on Sephadex G-75 was unaffected by preincubation with mannan. That this competition was not a general effect due to the injection of polysaccharide was ruled out by the demonstration that dextrans T-10, T-80, and T-200 (Sigma) and Lupinus albus galactan (Calbiochem) had no competitive effect on the clearance of ]25I-labeled RNase B (Fig. 5b). Organ sites of uptake of RNase B. Table I shows the tissue distribution of ]25I-labeled RNase B radioactivity recovered at various times after injection in a series of weight-matched rats. Significant uptake of ~25I-labeled RNase B in liver and marrow was apparent within 15 min, but the radioactivity in these organs started to decline

not significantly different from that of RNase A (1). In control experiments, it was shown that mannan coinjection (10 mg) did ioo -

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HEPATIC UPTAKE AND CATABOLISMOF RNase B

423

TABLE I TISSUE DISTRIBUTION OF ~25I-LABELED RNase B RADIOACTIVITY AT VARIOUS TIMES POSTINJECTION" Tissue

Liver Marrow Mesentery s Spleen Lung Thymus Stomach Stomach contents Total stomach Intestines Intestinal c o n t e n t s T o t a l intestines

T o t a l radioactivity (cpm x 10 -:~) at: 15 m i n

30 m i n

60 min

135 m i n

300 m i n

1320 263 82 49 44 11 55 32 87 109 96 205

939 212 150 35 38 19 49 47 96 126 101 227

462 180 120 31 27 12 64 220 284 101 298 399

245 109 65 17 17 12 129 745 874 67 415 481

I97 80 36 12 10 9 138 863 1010 132 433 565

" Individual nephrectomized rats (150-165 g) were injected with 5 x lff ~ c p m of ~2'~I-labeled R N a s e B a n d sacrificed at indicated times. Following perfusion, ~rgans a n d tissues were processed as described u n d e r Materials a n d M e t h o d s . h Includes pancreas.

by 30 min, concurrent with an accumulation of radioactivity in the stomach and intestines. This decline was accompanied by the appearance of low molecular weight radioactivity in plasma, as shown in Fig. 3. After 2 h, 80-90% of the stomach and intestinal radioactivity was recovered in the lumen or contents of these organs (Table I, columns 4 and 5) and more than 90% was low molecular weight, acid-soluble material. The 2-h stomach contents were extracted with 0.2 M acetic acid and further characterized by chromatography on a Sephadex G-25 column in 0.2 N acetic acid, according to LaBadie et al. {12). As shown in Fig. 6, the low molecular weight radioactivity chromatographed with free iodide at the salt volume of the column. When a 2-h plasma sample was mixed with an equal volume of 0.4 N acetic acid, centrifuged, and chromatographed as in Fig. 6, about 5% of the radioactivity cnromatographed with iodinated tyrosine derivatives and 75% with free iodide. From a comparison of the specific activities of tissues at various times postinjection (Fig. 7), the liver and spleen were clearly the most active organs, on a weight basis, in the uptake of 125I-labeled RNase B. The decline in radioactivity in these tissues, and in marrow and lung, followed a similar temporal pattern. There is an apparent delay

between the release of radioactivity from liver and its accumulation in the gastrointestinal tract, but this delay is accompanied by a transient increase in radioactivity in muscle and hide (Table II). As shown in Table II, injections of iodinated RNase A and RNase B resulted in distinctly different patterns of tissue radioactivity. The liver and spleen, which were most active per gram of tissue in the uptake of RNase B, showed little radioactivity after RNase A injection. The stomach and intestines, in which the degradation products of RNase B accumulate, contained significantly less radioactivity several hours after RNase A injection, consistent with the longer, 9-h half-life of RNase A in the circulation. Recovery of radioactivity from the perfused carcass in these experiments was essentially quantitative [96 • 6% (SD)]. About 45-65% of the radioactivity was localized in the internal organs, and an additional 35-55% in muscle and hide. Radioactivity in the perfusate accounted for 30 _+ 5% of the injected dose at 15 and 30 min and 24 _+ 3% thereafter. Doubling the amount of perfusing solution did not significantly alter these results. When liver was fractionated into subcellular fractions at 15 min postinjection, using the procedure of Gregoriadis et al. (14), 12~I-

BROWN ET AL.

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FIG. 6. Chromatography of stomach (O O) and intestinal (@------@}extract on Sephadex G-25. Organ contents were extracted by stirring with 3 vol of 0.2 N acetic acid for 1 h at room temperature. After centrifugation at 2000g for 10 min, supernatants were concentrated by lyophilization and redissolved in 0.2 N acetic acid; then 5-10 absorbance units (280 nm) of 3-iodo-Ltyrosine and 3,5-diiodo-L-tyrosinewas added as internal standards. Aliquots of the extract were applied to the column and 1-mlfractions were collected.More than 90%of the applied radioactivity was recovered in the eluted fractions. labeled RNase B radioactivity was localized predominantly in the lysosome-enriched fraction (Table III). In the control experiment in which t25I-labeled RNase B was added to the buffer prior to homogenization, 90% of the radioactivity was recovered in the supernatant fraction. Thus, the radioactivity accumulating in liver after the 125I-labeled RNase B injection is clearly intraceUular and not the result of nonspecific interaction of RNase B and lysosomes. Subcellular localization of RNase B in lysosomes is consistent with the observed rapid rate of degradation.

Uptake of 125I-labeled RNase B by liver cells in vivo. Rats were injected with 2 x 107 cpm of ]25I-labeled RNase B and liver perfusion was started at 15 min postinjection. Formaldehyde (0.1%) was included during the initial 10-min perfusion to fix the tissue and inhibit protein degradation (10). Measurements of radioactivity in aliquots of liver and perfusing solutions indicated that less t h a n 10% of the radioactivity was lost from liver during the perfusion. In the initial cell suspension, 80% of the radio-

activity was recovered in the pellet following centrifugation for 10 min at 220g. T h e remaining 20% was either freely soluble or enclosed in unsedimentable debris. When liver was separated into PC and NPC fractions, radioactivity was closely correlated with the concentration of N P C during fractionation, both after the initial separation into PC- and NPC-enriched fractions and during the intermediate purification steps. From the data shown in Table IV, it can be estimated that NPC would account for about 72% of the total radioactivity in the ICS, and PC for an additional 5%. T h e incomplete recovery of cellular radioactivity probably reflects leakage of radioactivity from cells resulting from damage during fractionation. In this and similar experiments, the specific activity (counts per minute per cell) of the NPC fraction was consistently 10-25 times t h a t of the PC fraction. T h e data clearly indicate that sinusoidal-lining cells (NPC) rather than hepatocytes (PC) were responsible for the a-mannose-dependent uptake of l~I-labeled RNase B in liver.

425

H E P A T I C U P T A K E AND C A T A B O L I S M OF RNase B

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In this study we have identified the liver as the primary visceral site for a-mannosedependent uptake of RNase B from the circulation of nephrectomized rats. We have also demonstrated that this clearance process is a function of nonparenchymal cells in liver and that the 125I-labeled RNase B is sequestered in a lysosome-enriched subceUular fraction, where it is rapidly catabolized. We found that more than 90% of the low molecular weight radioactivity released into the plasma from ~25I-labeled RNase B is identifiable as free iodide, similar to the observations of LaBadie et al. (12) on the degradation of 125I-labeled asialofetuin in liver parenchymal cells. Rhodes (16) and LaBadie et al. (12) previously reported on iodide transfer from the circulation to the stomach, and this pathway appears to predominate in the nephrecto-

mized rats used here. Unlike RNase B, 125Ilabeled RNase A is not concentrated in the liver after injection, confirming that there is no inherent signal on the polypeptide surface marking the RNase protein for rapid hepatic uptake. In contrast, the effective competition of mannan and unlabeled RNase B for clearance of '25I-labeled RNase B indicates that the carbohydrate moiety serves as the recognition signal. When liver cells were separated into parenchymal and nonparenchymal fractions, the uptake of RNase B was shown to occur almost exclusively in the nonparenchymal, sinusoidal-lining cells, in contrast to the uptake of galactose-terminal glycoproteins, which occurs in the parenchymal or hepatocyte fraction (2, 10). The sinusoidal cell fraction of liver contains at least two subpopulations (11, 17, 18), but it is not clear at present whether the uptake of RNase B

426

BROWN E T A L . TABLE II

RAI)IOACTIVITY IN TISSUES AT VARIOUS TIMES POST~NJECTION:" COMPARISON OF RNase A AND RNase B h Tissue

RNase B at:

RNase A at:

15 min

30 min

60 min

120 min

300 min

30 min

180 min

40 1.4 2.3 7.6 1.3 0.4 2.5 5.9 19 18

30 1 4.8 6.8 1.2 0.4 3.1 7.2 25 19

12.4 1 3.2 4.8 0.7 0.3 7.6 10.7 32 26

6.2 0.5 1.6 2.8 0.4 0.3 22 12.2 24 22

5.1 0.3 0.5 2.0 0.2 0.2 30 15.5 20 21

2.2 0.4 1.2 5.7 0.9 0.3 1.3 5.4 35 39

1.5 0.3 1.2 4.5 1.0 0.3 5.8 8.9 33 36

Liver Spleen Mesentery Marrow Lung Thymus Stomach' Intestine' Muscle ~t Hide '1

" Percentage of total radioactivity remaining in carcass after perfusion. ~'Average values for two or three rats at each time point. ' Includes organ contents. d Calculated from the specific activity of muscle and hide samples. Muscle mass estimated to be 41% of body mass, and hide 18% (13}. TABLE III HEPATIC SUBCEIJ.ULAR DISTRIBUTION OF ~25ILABELED RNase B RADIOACTIVITYAND fl-NACETYLHEXOSAMINIDASE ENZYMATIC ACTIVITY Fraction" Radioactivity (%) Hexosaminidase Control h Experimen- activity, extal perimental" (%) Nuclear M + L Microsomal Supernatant

3 4 4 88

12 68u 5 15~

18 70 6 7

" Subcellular fractionation was performed according to Gregoriadis et al. (14), yielding nuclear (600g, I0 min), crude mitochondrial and lysosomal (M + L; 14,000g, 30 rain), microsomal (100,000g, 2 h), and supernatant fractions. h A 160-g nephrectomized rat was sacrificed 15 min after the injection of 5 • l0 (~cpm of '~I-labeled RNase B. A control rat liver was homogenized in buffer containing I • I0~ r of ~'~I-labeled RNase B. ' Assayed according to Barrett (15) using 4-methylumbelliferyl-fl-N-acetylglucosamine as substrate. ,/Seventy-seven percent of the radioactivity in this fraction was acid precipitable. Thirty-six percent of the radioactivity in this fraction was acid precipitable. occurs in the Kupffer or the endothelial cells, o r b o t h . T h e o v e r a l l t i s s u e d i s t r i b u tion of radioactivity, with the highest specific a c t i v i t y i n l i v e r , s p l e e n , a n d m a r r o w , does suggest, however, that the uptake and c a t a b o l i s m o f R N a s e B is o c c u r r i n g i n e l e ments of the reticuloendothelial system.

TABLE IV UPTAKE OF ~2'~I-LABEI,EDRNase B BY

PARENCHYMAL AND NONPARENCHYMAL CELLS IN RAT LIVER in Vivo ~ Percentage Specific' activity PC NPC Radio(cpm x activity h 10~/cell) Initial cell suspension Initial supernatant Initial pellet Purified PC Purified NPC

100

100

100

23

13

76

86

57

87 59 1

24 2 25

14 3 18

4.7 1.8 49

" '2'~I-Labeled RNase B, 20 • 106 cpm, was injected into a 150-g rat. The animal was sacrificed 15 min later. An aliquot of ICS which contained 8.5 x 107 PC and 4.4 • l07 NPC was separated into PC and NPC fractions as described under Materials and Methods. Initial intracellular radioactivity was 3 • 10"~cpm, or 80% of the total radioactivity in the ICS (see test). Percentage initial intracellular radioactivity. The kidneys were removed from rats used in these experiments in order to prevent t h e r a p i d (tl/2 ----- 2 m i n ) , n o n s p e c i f i c r e n a l c l e a r a n c e o f t h e l o w m o l e c u l a r w e i g h t (13,000-16,000) RNase i s o z y m e s (1). U s i n g larger proteins, however, other workers have shown that the kidney may also function in mannose-dependent protein uptake f r o m t h e c i r c u l a t i o n (19, 20). I n 1976, S t a h l e t a l . (21) r e p o r t e d t h a t the rapid clearance of lysosomal enzymes

HEPATIC UPTAKE AND CATABOLISM OF RNase B

from the circulation was inhibited by agalacto(N-acetylglucosamine-terminal)orosomucoid. Achord et al. (22) later presented evidence, based on competition experiments with yeast mannans and mannose, that the mannose recognition system was at least partially responsible for the clearance of agalacto-orosomucoid, suggesting that both terminal and subterminal mannose may trigger the rapid clearance of lysosomal enzymes. These reports argue strongly that the mannose recognition system functions in the uptake of injected lysosomal enzymes from the circulation. Achord et al. (23) recently presented evidence that clearance of human placental fl-glucuronidase from the rat circulation is accomplished primarily in liver and spleen in" normal rats and in bone, lung, and kidney in eviscerated rats. As is the case with RNase B, clearance of this lysosomal enzyme occurs in tissues characteristic of the reticuloendothelial system. The studies presented here, and those of Achord et al. (22, 23), suggest that a-mannose residues are elements of a carbohydrate code recognized by the reticuloendothelial system. It seems that it should be possible to chemically modify proteins by attaching mannose derivatives or mannose-terminal oligosaccharides in order to direct proteins specifically to the reticuloendothelial system. Such a chemical modification of enzymes may be especially useful in enzyme therapy of lysosomal storage diseases with significant reticuloendothelial involvement (24}. Based on observations of decreased liver neutral (pancreatic-type) RNase levels following pancreatectomy in rats, Bartholeyns et al. (25) suggested that neutral RNase activity in liver may be of pancreatic origin. Alpers and Isselbacher (26), in fact, reported on the transport of low levels of bovine RNase A across the rat intestinal wall into the portal circulation, following intraduodenal injection of the enzyme. These observations, coupled with our own on the selective hepatic clearance of RNase B, suggest that the glycosylated enzyme could be more efficiently delivered to the liver by this enterohepatic circulation. Studies on the carbohydrate structures of

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ovine (27) and porcine (28) RNase also reveal the presence of simple, mannose-terminal oligosaccharides, suggesting that the enterohepatic circulation of glycosylated pancratic RNase could be metabolically significant in a number of mammalian species. Further study of the cell types involved in the clearance of RNase B, and possibly of other pancreatic glycoprotein enzymes, will be necessary in order to appreciate the possible physiological significance of this pathway. ACKNOWLEDGMENTS The authors wish to express their deep gratitude to Professor Finn Wold at the University of Minnesota for his generous and thoughtful advice and encouragement during the course of the studies conducted in his laboratory. We also thank Professor William Ingebretsen at the University of South Carolina for his excellent instruction in methods for the isolation of rat liver cells. REFERENCES 1. BAYNES, J. W., AND WOLD, F. (1976) J. Biol. Chem. 251, 6016-6024. 2. ASHWELL, G., AND MORELL, A. G. (1974) Advaa. Enzymol. 41, 99-128. 3. ROGERS, J. C., AND KORNFELD, S. (1971) Biochem. Biophys. Res. Commun. 45, 622-626. 4. DAVIDSON,S. J., HUGHES, W. L., AND BARNWELL, A. (1971) Exp. Cell Res. 67, 171-187. 5. DAVIDSON, S. J. (1973) J. Cell Biol. 59, 213-222. 6. SCOFIELD, R. E., WERNER, R. P., AND WOLD, F. (1977) Anal. Biochem. 77, 152-157. 7. KEENE, W. R., AND JANDL, J. H. (1965) Blood 26, 157-175. 8. WAGLE, S. R., AND INGEBRETSEN, W. R. (1974) Methods Enzymol. 35B, 579-594. 9. NILSSON, M., AND BERG, T. (1977) Biochim. Biophys. Acta 497, 171-182. 10. BISSELL, D. i . , HAMMAKER,L., AND SCHMID, R. (1972) J. Cell Biol. 54, I07-119. 11. TOLLESHAUG, H., BERG, T., NILSSON, M., AND NORUM, K. R. (1977) Biochim. Biophys. Acta 499, 73-84. 12. LABADIE, J. H., CHAPMAN, K. P., AND ARONSON, N. N. (1975) Biochem. J. 152, 271-279. 13. DONALDSON, H. H. (1924) The Rat, 2nd ed., Wistar Institute, Philadelphia. 14. GREGORIADIS, G., MORELL, A. G., STERNLIEB, I., AND SCREINnnnC,, I. H. (1970) J. Biol. Chem. 245, 5833-5837. 15. BARRETT, A. J. (1972) in Lysosomes, A Laboratory Handbook (Dingle, J. T., ed.), pp. 117-118, American Elsevier, New York. 16. Rhodes, B. A. (1968) Acta Endoerinol. (Suppl.)

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