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Synthesis and intracellular processing of sucrase-isomaltase in rat jejunum
(;ASTKOEn’TEROt,OGY
1985;88:531-8
Synthesis and Intracellular Processing of Sucrase-Isomaltase in Rat Jejunum RICHARD J. GRAND, ROBERT K. MONTGOMERY, and ARTURO PEREZ Division of Gastroenterology and Nutrition, Department of Medicine, The Children’s Hospital Medical Center; Division of Pediatric Gastroenterology and Nutrition, New England Medical Center, Floating Hospital; and Departments of Pediatrics. Harvard Medical School and Tufts Universitv School of Medicine, Boston, Massachusetts
To examine directly the synthesis and intracellular processing of rat intestinal sucrase-isomaltase, [“HIIeucine-labeled enzyme was solubilized from purified microsomal and microvillus fractions, immunoprecipitated, and analyzed. Immunologic identity between the microsomal and microvillus forms of the enzyme was demonstrated. Time-course studies using both intravenous and intraluminal labeling demonstrated an initial peak of microsomal sucrase-isomaltase labeling at 30 min followed by a sharp decline, and a subsequent rise in microvillus sucrase-isomaltase labeling, suggesting a precursorproduct relationship. Fluorography and quantitative analysis of electrophoresed immunoprecipitates confirmed these results, and revealed the presence of two high molecular weight proteins in the microsomes. Susceptibility of the smaller high molecular weight precursor to cleavage with endo-P-N-acetylglucosaminidase H indicated that this was the initially synthesized form of the glycoprotein. These data are consistent with the membrane flow hypoth-
Received September 17. 1981. Accepted August 29, 1984. Address requests for reprints to: Dr. Richard J. Grand, Chief, Division of Pediatric Gastroenterology and Nutrition, New England Medical Center, 171 Harrison Avenue, Box 213, Boston, Massachusetts 02111. This study was supported by U.S. Public Health Service Research Grants HD-14498, AM-14523, and AM-32658, and Training Grants AM-07183, AM-07333. and AM-07471 from the National lnstitute of Arthritis, Digestive and Kidney Diseases. This work was presented in part at a meeting of the American Gastroenterological Association, Las Vegas, Nevada, May 1978 (GASTROENTEROLOGY 1978;74:1126). The authors thank Ezio Merler. Ph.D. for valuable consultations; Mrs. Mariana Sybicki, Ms. Ann Forcier, and Mrs. Stephanie I. Ryan for expert technical assistance; Ms. Cynthia Richardson and Ms. Karen Cox for preparations of electron micrographs; Gabriel Goldberger, M.D. for ovalbumin; and Ms. Lisa LoPresti for preparation of the manuscript. 0 1985 by the American Gastroenterological Association 0016~5085/85/$3.30
esis and the single polypeptide sucrase-isomaltase synthesis.
model suggested for
Sucrase-isomaltase is an intrinsic protein of the intestinal microvillus membrane. This protein can only be removed by detergents (1) or by controlled proteolytic digestion (2). Extensive evidence has demonstrated conclusively that sucrase-isomaltase is located on the luminal side of the microvillus membrane (3,4). When isolated from the microvillus membrane, sucrase-isomaltase is a dimer consisting of two subunits (sucrase-a-glucohydrolase, EC. 3.2.1.48, and isomaltase, E.C. 3.2.1.10) which may be readily separated from one another by reduction (5) or citraconylation (6). The functional dimer is attached to the membrane by a hydrophobic anchor sequence which is part of the isomaltase subunit (4). Although the location and structure of mature sucrase-isomaltase have been well established, the mechanisms by which the enzyme is synthesized and transported to the cell surface remain to be completely elucidated. Most recent evidence demonstrates that sucraseisomaltase is synthesized as a single high molecular weight polypeptide (3,4,7-g), although there are data suggesting other mechanisms (10,ll). The Golgi form of the enzyme has been well studied (7,8), but not much is known of the earlier synthetic forms. Accordingly, the present studies were undertaken to provide direct evidence for microsomal synthesis of sucrase-isomaltase, and to demonstrate transfer of the newly synthesized enzyme from microsomes to microvillus membrane. The data also provide additional evidence in support of the single-polypeptide synthetic mechanism.
Abbreviations used in this paper: Endo H. endo+N-acetylglucosaminidase H; SDS, sodium dodecyl sulfate; OVA, ovalbumin.
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Materials and Methods Experimental procedures were similar to those described previously (12). All experiments were performed using adult, male, albino rats (Charles River Breeding Laboratories, Wilmington, Mass.]. The animals weighed 125-150 g and were fed standard laboratory Chow [Ralston Purina Co., St. Louis, MO.) and water ad libitum. Studies were always begun by 9 AM. Radiolabeling of intestinal mucosal proteins was performed with animals maintained under light ether anesthesia. When intravenous labeling was used, 2 &i/g body wt of [“Hlleucine (40-60 Ci/mmol, New England Nuclear, Boston, Mass.) was administered by tail vein injection. When intraluminal labeling was used, the segment of intestine to be used was identified, the proximal end was ligated, and enough warm phosphate-buffered saline was administered via a plastic catheter to fill the required segment. The segment was then filled with -95 &i/g mucosa of [3H]leucine (40-60 mCi/mmol, New England Nuclear] in 0.9% NaCl solution. The radioisotope was allowed to remain in the intestinal lumen for 20 min; there was no observable loss of volume from the lumen during this period. This was followed by a chase of 5 mM cold leucine in phosphate-buffered saline. At appropriate times after labeling, animals were killed, and the jejunum, defined as the middle third of the small intestine (measured from the ligament of Treitz to ileocecal valve), was quickly excised and rinsed with 50 ml of ice-cold 0.9% NaCl solution. Scraped mucosa was subjected to fractionation techniques as described below.
Preparation
of Subcellular
Fractions
Jejunal microsomes were prepared by differential centrifugation according to a modification of previously published techniques (12). Mucosa was homogenized in 6 vol of 0.25 M sucrose containing 1 mM MgClz and 0.5 mM KC1 (pH 7.0). The final microsomal pellet (100,000 g for 1 h, Beckman Ultracentrifuge model L8-55) was suspended in isotonic sucrose (0.25 M), dialyzed overnight against the same solution, and then stored at 4°C for further processing, or frozen (-20°C). To obtain microsomal sucrase-isomaltase free of contamination by other membranous forms of the enzyme, intestinal microsomes were subjected to further purification. As sucrase-isomaltase is known to be located external to the microvillus membrane, it was assumed that the microsomal form of the enzyme would be found exclusively on the inside of the microsomal vesicles. This allowed the use of controlled protease digestion to remove contaminating microvillus forms of the enzyme without affecting the microsomal vesicles (I 3). The total microsomal protein was determined by the method of Lowry et al. (14); 50 pg of elastase (porcine pancreas type III) per milligram of microsomal protein was added and incubated at 30°C for 30 min (13). At the end of the incubation period, the microsomes were suspended in the original volume of isotonic sucrose, and the microsomal pellet was recovered as before and resuspended in
isotonic sucrose (pH 7.0). Enrichment of the microsomal fraction was verified using the specific activity of nicotinamide adenine dinucleotide phosphate, reduced form, cytochrome C reductase as a marker (15), and by electron microscopic demonstration of a lack of contamination by recognizable fragments of other organelles. Microvillus membranes were prepared from mucosal scrapings suspended in 50 mM mannitol and 2.0 mM TrisHCl, pH 7.1, by the method of Kessler et al. (16). Sucrase specific activity (12,14) was used as a marker for microvillus membrane purification. Final preparations had sucrase activities comparable to those reported by other groups using this and other methods of microvillus membrane isolation (1.2-1.5 pmol of glucose per milligram of protein per minute). Immunoprecipitation
Techniques
To measure the incorporation of radioactivity into newly synthesized sucrase-isomaltase in microsomal and microvillus fractions, a quantitative immunoprecipitation assay was developed. Highly purified rat jejunal sucraseisomaltase was prepared by the method of Cogoli et al. (17), which yielded a LOO-to 400-fold purification. Further purification was obtained by polyacrylamide disk gel electrophoresis in a nondenaturing system on which suerase-isomaltase migrated as a single band. The sucraseisomaltase band identified by correlation of enzyme activity and Coomassie Blue staining was sliced from untreated gels using calculated Rf value, and sufficient slices were pooled to yield 60 pg of sucrase-isomaltase. Gel slices were homogenized in equal volumes of complete Freund’s adjuvant, and this preparation was injected into rabbits injections at 3-wk intervals). (three subcutaneous Immunoglobulin G was purified from antisera by ammonium sulfate fractionation and chromatography on Whatman diethylaminoethanol-cellulose (18). The resulting antibody was shown to be monospecific by radial diffusion and by immunoelectrophoresis (data not shown) (19). Quantitative immunoprecipitation using this antibody and purified sucrase-isomaltase showed that, at equivalence, >95% of the sucrase activity was precipitated. This antibody was used throughout the experiments reported. Subcellular fractions were prepared for immunoprecipitation as described previously. Each fraction was sonicated (Ultrasonic I, Plainview, N.Y.) at half-maximal setting for 15 s in 6 bursts [with 15 s between each burst). LubrolPX [Sigma Chemical Co., St. Louis, MO.) was added to a final concentration of 0.6%. After 1 h at 2O"C,fractions were certrifuged at 100,000 g for 1 h. The clarified supernatants were then used in the immunoprecipitation assay. Quantitative immunoprecipitation curves were constructed for each sample using a fixed concentration of antisucrase-isomaltase immunoglobulin G and varying quantities of sample. Equivalence was estimated for these initial curves from the enzyme activity of the sample compared with the activity of purified sucrase precipitated at equivalence (-0.0843 U of sucrase activity per 25 ~1 of antisucrase immunoglobulin G). Reaction mixtures were incubated first at 37°C for 1 h and then at 4°C for at least 2 h. After equivalence was determined, immunoprecipita-
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SUCRASE-ISObIALTASE INTRACELLULAR PROCESSING 533
tion was repeated with larger volumes of sample and antibody to assure sufficient pellet for measurement of radioactivity. Precipitates were recovered by centrifugation at 12,000g for 5 min in an Eppendorf Microfuge (Brinkman Instruments, Inc., Westbury, N.Y.). The supernatants were assayed for sucrase activity, and any remaining activity was subtracted from the original units used. The pellets were washed once in saline, dissolved in 0.1 N NaOH, and aliquots were assayed for radioactivity. Nonspecific precipitation of radioactivity was determined from reaction mixtures containing solubilized microsomal or microvillus fractions, purified ovalbumin (OVA, 0.5 mgiml, Schwartz-Mann, Mannheim, F.R.G.), and rabbit anti-OVA (22.5 mg/ml generously provided by Dr. Gabriel Goldberger and Dr. Harvey Colten, Division of Cell Biology, Children’s Hospital Medical Center, Boston, Mass.). Immunoprecipitates from these mixtures were treated exactly as described previously. Radioactivity precipitated by the OVA-anti-OVA complex was subtracted from the radioactivity of the original precipitate to yield net radioactivity precipitated by antisucrase immunoglobulin G. Correction for nonspecific precipitation of radioactivity was also achieved by reprecipitation through the remaining supernatant, using added unlabeled sucraseisomaltase and antibody at equivalence, which gave the same pattern of labeling as described.
Electron
Microscopy
Microsomal pellets prepared as described previously were fixed (201, processed to Spurr’s plastic (21), cut on the LKB Ultratome III with a diamond knife (sections 800 A), and examined at 60 KV in a Philips 300 electron microscope (Philips Electronic Instruments, Inc., Mahwah, N.J.).
Sodium Dodecyl Electrophoresis
Sulfate-Polyacrylamide
Gel
Immunoprecipitates of labeled microsomal suerase-isomaltase were prepared as described previously. Precipitates were solubilized by heating to 100°C in a boiling water bath for 5 min in 0.056 M Tris buffer containing 1% sodium dodecyl sulfate (SDS) and 5% pmercaptoethanol. All other samples were solubilized in the same manner. Electrophoresis was performed on SDSpolyacrylamide slab gels consisting of a 3% stacking gel and a 7.5% separating gel made according to the method of Neville (22). After running, gels were simultaneously fixed and stained in a 0.006% solution of Coomassie Blue in acetic acid. methanol, and water. Gels were prepared for fluorography according to the method of Laskey and Mills (23), except that En”Hance (New England Nuclear) was used as the scintillant. Fixed and destained gels were soaked in En3Hance, washed in tap water, and dried on a filter paper backing. The dried gels were enclosed in an x-ray cassette against a sheet of preflashed Kodak X-Omat film (Eastman Kodak Co., Rochester, N.Y.) and stored in a -70°C freezer for 4 wk. When quantification of radioactivity on the gels was required,
the appropriate bands were cut from the gels, eluted in 5% Protosol in Econofluor at 37°C overnight [New England Nuclear Applications Note 22), and counted as described previously.
Treatment of Microsomal Preparations Endo-P-N-Acetylglucosaminidase H
With
Equal aliquots of solubilized microsomal fractions were incubated with and without 20 mU of endo+Nacetylglucosaminidase H (Endo H, Miles Laboratories, Elkhart, Ind.) prepared as described by the manufacturer. After 30 min of incubation at 37°C. equal amounts of antibody were added to each sample and immunoprecipitates were prepared as described previously. The resulting immunoprecipitates were solubilized, electrophoresed, and fluorograms were prepared as described for all samples.
Liquid
Scintillation
Counting
Radioactivity of immunoprecipitates was determined in a liquid scintillation spectrometer model LS 8000 (Beckman Instrument Co., Irvine, Calif.). Disintegrations per minute were calculated using a commercial quench series (New England Nuclear) and the Compton edge shift measure of counting efficiency (24). Data presented are mean + SE of at least three triplicate experiments at each time point described in the figure legends.
Results Characterization
of the Microsomal
Fraction
Enrichment of this fraction was confirmed by a lo-fold increase in the specific activity of nicotinamide adenine dinucleotide phosphate, reduced form, cytochrome C reductase, compared with the specific activity in whole mucosal homogenate, as reported also by others (25,26). Similar values have been obtained for hepatic microsomes (27,283. There was no enrichment of sucrase activity in this fraction, which, as reported previously, was 3.7% ? 0.05% of that in the homogenate (12). Examination of the microsomal pellets by electron microscopy revealed rough and smooth endoplasmic reticulum vesicles without evidence of extraneous organelle fragments.
Labeling
of Sucrase-lsomaltase
The incorporation of intravenously administered [3H]leucine into immunoprecipitable sucraseisomaltase from microsomes and microvillus membranes is shown in Figure 1. In the microsomal fractions, there was a peak of labeling of sucraseisomaltase at 30 min, followed by an abrupt fall and plateau at low levels of radioactivity. In the microvil-
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Vol. 88, No. 2
MINUTES
Figure
Figure
I. Specific radioactivity in sucrase-isomaltase immunoprecipitated from microsomal (closed circles) and microvillus (open circles] fractions prepared at the indicated times after intravenous label injection as described in Materials and Methods.
lus fraction, radioactivity of the enzyme increased slowly, with a maximal value established by 240 min. This fraction showed a distinct lag in maximal radioactivity in comparison with that in the microsomal fraction, suggesting a precursor-product relationship between the sucrase-isomaltase in the two fractions. The pattern of labeling obtained when [3H]leucine was introduced intraluminally is shown in Figure 2. In the microsomal fraction, a peak in the incorporation of radioactivity into sucrase-isomaltase was achieved by XI min, with a rapid reduction and plateau thereafter. Labeling of sucrase-isomaltase in the microvillus fraction showed a steady increase over time, with a maximal value occurring at 240 min. Comparing these patterns with those obtained by the intravenous route (Figure l),microsomal sucrase-isomaltase again appeared as a biosynthetic precursor to the microvillus form of enzyme. In addition, as noted previously by Alpers (29), the intraluminal labeling techniques yielded greater than a lo-fold increase in radioactivity in the fractions in comparison with that obtained by the intravenous route. Fractional incorporation of Label Into Sucrase-Isomaltase To compare the labeling of sucrase-isomaltase with the labeling of total proteins in the microsomal and microvillus fractions, relative specific activity or fractional incorporation was calculated according to the following formula: Fractional
incorporation
=
in sucrase-isomaltase immuno2. Specific radioactivity precipitated from microsomal (closed circles) and microvillus (open circles] fractions prepared at the indicated times after intraluminal label injection as described in Materials and Methods.
where MVM is the microvillus membrane and DPM is disintegrations per minute. In the microsomal fraction, sucrase-isomaltase represented 2.4% +-0.5% of the newly labeled proteins, whereas in the microvillus fractions sucraseisomaltase accounted for 9.3% -I- 1.3% of the newly labeled proteins. The difference in the fractional incorporation in microsomal and microvillus fractions is compatible with the concept that numerous rapidly labeled microsomal proteins are not destined for insertion into the microvillus membrane. Hence, the proportion of rapidly labeled microsomal suerase-isomaltase is less than that in the microvilli. Electrophoresis
and Fluorography
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated sucrase-isomaltase from microsomal fractions followed by fluorography revealed two high molecular weight protein bands as the earliest labeled form of sucraseisomaltase (Figure 3, lane 1). On the other hand, immunoprecipitation of sucrase-isomaltase from microvilli labeled for the same length of time, followed by identical electrophoresis and fluorography, showed no label incorporation (data not shown], although, at 120 min, the same procedure revealed label in both microvillus subunits (Figure 3, lane 2). To quantify the distribution of radioactivity in the gels at various times after labeling, stained bands were cut out of the gels and counted as described previously. The data are shown in Table 1. At 30 min, >99% of the radioactivity in microsomal SUerase-isomaltase was in the high molecular weight
milligrams
of lactase
in MVM x DPM/milligram
of lactase
milligrams
of protein
in MVM x DPM/milligram
of protein
x 100,
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1985
SUCRASE-ISOMALTASE
INTRACELLULAR
specific and characteristic in the fraction.
PROCESSING
535
of the form of the enzyme
Treatment of Microsomal Precursor With Endo-P-N-Acetylglucosaminidase H
Figure
3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated sucrase-isomaltase. Lane 1 shows a fluorogram of sucrase-isomaltase immunoprecipitated from solubilized microsomes that were prepared after a 30-min period of labeling with [3H]leucine. For comparison, lane z shows a fluorogram of sucrase-isomaltase immunoprecipitated from solubilized brush borders that were prepared after a 120-min period of labeling with [3H]leucine. Solubilization, electrophoresis, and fluorography of both were carried out as described in Materials and Methods. Lane 3 shows a sample of papain-purified brush border sucrase-isomaltase run as marker on the same gel system and stained with Coomassie Blue. Whereas the sucrase corn&rates in lanes 2 and 3, isomaltase in the papain-purified sample in lane 3 runs slightly more rapidly than that in the detergentsolubilized sample in lane 2, due to the cleavage of the anchor sequence which is the first step in the purification procedure. All three samples were aligned by internal markers in order to compare relative migration distance, and demonstrate that the only proteins immunoprecipilabeled at 30 min in the microsomal tates were the two high molecular weight forms.
Further characterization of the two high molecular weight proteins in the microsomes was carried out by treating solubilized microsomes with Endo H before immunoprecipitation. As shown in Figure 4A, Endo H digestion did not affect the larger of the two proteins, but increased the mobility of the smaller of the two. Figure 4B demonstrates also that short-term labeling sometimes resulted in incorporation of label into only the smaller of the two high molecular weight forms, suggesting that it is the earliest synthesized protein. The Endo H effect indicates that the smaller band is an incomplete glycoprotein, containing only the core carbohydrate structure that is susceptible to Endo H cleavage, whereas the larger protein band, on the other hand, is not susceptible to such cleavage, and represents a more complete glycoprotein. Since these experiments were performed, Miles Laboratories has advised that their Endo H may be contaminated with protease. Our microsome fraction has, in effect, an internal control, because it displays two protein bands, one with mobility that is changed by Endo H treatment, and one with mobility that is not changed. The differential effect on the two protein bands mitigates against the result being due to nonspecific proteolytic cleavage. Because this initial glycosylation step is known to occur concurrent with or immediately after translation of the protein message, these data also demonstrate that this form
Table
1. Quantification Isomaltase
Fraction Microsomes
form. Subsequently, the counts in the gels fell rapidly, and no other peaks were identifiable. In the microvillus fractions at the time points examined, there were negligible counts in the high molecular weight region, but radioactivity in the sucrase and isomaltase subunits increased over time. These results confirm those from liquid scintillation counting of the total sucrase-isomaltase immunoprecipitates (Figures 1 and 2). In addition, they show that the microsomal sucrase-isomaltase label is actually in two high molecular weight proteins, and that the labeling of sucrase-isomaltase in each fraction is
Microvilli
Time (min) 15 30 60 240 15 30 60 240
of Radioactivity in Fractions
Sucrase-
in Subcellular DPM applied to gel 101
18,167 2,737 971 82 100 229 27.300
DPM distribution
in gel Ab
SI
PSI 0
18,100 2,140 308 o 0 3 351
0
362 84 589 0 26 23 18,830
0
36 58 119 0 3 43 131
As described in Materials and Methods, immunoprecipitates containing comparable quantities of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel bands were cut out and counted as described. PSI refers to high molecular weight forms; SI refers to counts in the sucrase band plus the isomaltase band; Ab is the radioactivity migrating with the antibody: DPM is disintegrations per minute.
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of sucrase-isomaltase is in fact located in the rough endoplasmic reticulum.
Discussion
Figure
4. Treatment
of microsomal sucrase-isomaltase with Endo-P-N-acetylglucosaminidase H (Endo H]. A. Coomassie Blue-stained gel showing the effect of Endo H on the slower (PJ and faster (PI) migrating precursor forms of microsomal sucrase-isomaltase. Absence or presence of Endo H indicated by (-) or (+). Arrows indicate the increase in mobility of the Endo H-treated precursor (P,). Precursor P, is unresponsive to Endo H, suggesting more extensive glycosylation. B. Fluorogram of two samples of microsomal sucrase-isomaltase labeled for 30 min with [“Hlleucine. immunoprecipitated, and electrophoresed after incubation without (-1 and with (+) Endo H. Arrows indicate the change in mobility of the sucrase-isomaltase precursor.
These studies provide information concerning both the time-course and localization of synthesis and intracellular processing of sucrase-isomaltase. The time-course of leucine incorporation shows an early peak in the microsomal form of the enzyme, followed by a rapid decline, and a succeeding progressive rise in labeling of the microvillus form, suggesting a precursor-product relationship. This pattern is consistent with the hypothesis that membrane derived from the microsomes, containing this intrinsic protein, ultimately merges with the microvillus membrane, thereby transporting sucrase-isomaltase to the cell surface. Because the sucraseisomaltase that is immunoprecipitated from the microsomal vesicles is first solubilized by detergent treatment to disrupt the membranes with which it sediments, these data are consistent with an attachment of the newly synthesized enzyme protein to the endoplasmic reticulum membrane, presumably similar to the attachment demonstrated for the microvillus enzyme (5). These data also eliminate the possibility that the early microsomal peak is due to contamination of the microsomal fraction with microvillus fragments because, at the time of maximal microsomal labeling, microvillus labeling is negligible. This is further corroborated by both fluorography and quantification of electrophoresed immunoprecipitates, which demonstrates that only the high molecular weight forms of sucrase-isomaltase are rapidly labeled, whereas the microvillus form is not. These findings are in agreement with the membrane flow hypothesis described by Palade (30). Taken together, our labeling and electrophoretic data indicate that sucrase-isomaltase is initially synthesized in the microsomal fraction as a single high molecular weight protein. This evidence provides direct confirmation of the hypothesis proposed by Semenza’s group that sucrase-isomaltase is originally synthesized as a single polypeptide which is subsequently processed to the dimeric form found on the microvillus membrane (31).Thus, there is accumulating evidence that the intracellular form of sucrase-isomaltase is a single high molecular weight protein (8,9,32). Studies of glycoprotein synthesis in a number of systems indicate that a major class of glycoproteins, which have mannose and N-acetylglucosamine cores attached to the peptide via an N-glycosidic bond between asparagine and N-acetylglucosamine, are first glycosylated with a core sugar structure via a dolichol-linked intermediate located on or in the
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1985
membrane of the endoplasmic reticulum (33). This core carbohydrate structure is then modified by additional glycosylation in the endoplasmic reticulum and finally undergoes excision of some residues and terminal glycosylation in the Golgi apparatus (34). A uniquely specific probe for this core glycosylation was discovered with the isolation of Endo H. This enzyme specifically cleaves between the two Nacetylglucosamine residues of incomplete glycoproteins, whereas the mature form of the glycoprotein is not affected (35). Susceptibility to Endo H is thus a specific and sensitive probe for a particular stage in the synthesis of this kind of glycoprotein. Our data indicate that the first identifiable precursor form of sucrase-isomaltase is an immature form of glycoprotein of this family, and that the higher molecular weight form of the precursor has had the glycoprotein modified so that it is now insensitive to Endo H. Thus, both are precursor forms at different stages of maturity. This is supported by our finding that the briefest exposure to radioactivity labels predominantly the smaller of the microsomal precursors, showing that it is the initially synthesized form. The sensitivity to Endo H demonstrates that the smaller precursor form is indeed an immature glycoprotein of the high mannose core structure. Our data on Endo H susceptibility of the microsomal sucraseisomaltase precursor are consistent with the findings of other investigators (36-38) that there is a group of mannose-rich glycoproteins in isolated intestinal epithelial cells that are Endo H degradable and, from changes both in this susceptibility and in kinetics of labeling, appear to be precursors of the group of complex glycoproteins. It is also consistent with the data of Kelly and Alpers (39) that rat sucrase-isomaltase has more mannose than any other sugar and the report of Cezard et al. (10) that sucrase-isomaltase binds to concanavalin A, a lectin that specifically binds mannose residues. Glycoproteins of this structure are members of the group synthesized via a dolichol intermediate, suggesting that sucrase-isomaltase is also a member of this group. Finally, as the formation of the core glycoprotein is known to occur in the endoplasmic reticulum (40,41), the Endo H digestion indicates that this early labeled high molecular weight sucrase-isomaltase is in fact located in the endoplasmic reticulum of the enterocytes. It is generally accepted that the subcellular “microsome” fraction contains a spectrum of cellular membrane vesicles that sediment together and includes vesicles of endoplasmic reticulum membrane, as well as vesicles of Golgi apparatus membrane. The presence of two protein bands in our preparation is consistent with a combination of
SUCRASE-ISOMALTASE
INTRACELLIJLAR PROCESSING
537
immature high molecular weight endoplasmic reticulum precursor, and a Golgi-derived higher molecuSuch a situation has been lar weight precursor. reported also for aminopeptidase, sucrase-isomaltase, maltase-glucoamylase, and lactase-phlorizin hydrolase from pig intestinal “microsomes” prepared by calcium precipitation (42). These authors also showed that in each case the faster migrating band in electrophoresis was susceptible to Endo H digestion, whereas the slower was not. Our data thus identify the early precursor form of sucrase-isomaltase present in the microsomal fraction of the enterocyte. The structure of this precursor remains to be elucidated in detail, as does the question of whether there is an even earlier precursor form incorporating a subsequently removed signal or whether the anchor protein of isomaltase represents a retained signal sequence.
References 1. Sigrist H, Ronner P, Semenza
G. A hydrophobic form of the small intestinal sucrase-isomaltase complex. Biochim Biophys Acta 1975;406:433-46. 2. Kolinska J, Semenza G. Studies on intestinal sucrase and on intestinal sugar transport. V. Isolation and properties of sucrase-isomaltase from rabbit small intestine. Biochim Biophys Acta 1967;146:181-95. of small-intestinal 3. Semenza G. Molecular pathophysiology sucrase-isomaltase. Clin Gastroenterol 1981;10:691-706. 4. Hauri H, Wacker H, Rickli E, Bigler-Meier B, Quaroni A, Semenza G. Biosynthesis of sucrase-isomaltase. J Biol Chem 1982;257:4522-8. 5. Brunner J, Hauser H, Braun H, Wilson KJ, Wacker H, O’Neill B, Semenza G. The mode of association of the enzyme complex sucrase-isomaltase with the intestinal brush border membrane.J Biol Chem 1979;254:1621-8, 6. Braun H, Cogoli A, Semenza G. Dissociation of small intestinal sucrase-isomaltase complex into enzymatically active subunits. Eur J Biochem 1975;52:475-80. 7. Hauri HP, Quaroni A, Isselbacher KJ. Biogenesis of intestinal plasma membrane. Posttranslational route and cleavage of sucrase-isomaltase. Proc Nat1 Acad Sci USA 1979;76:5183-6. 8. Hauri H, Quaroni A, Isselbacher K. Monoclonal antibodies to sucraseiisomaltase: probes for the study of postnatal development and biogenesis of the intestinal microvillus membrane. Proc Nat1 Acad Sci 1980;77:6629-33. 9. Montgomery RK, Sybicki MA, Forcier AG, Grand RJ. Rat intestinal microvillus membrane sucrase-isomaltase is a single high molecular weight protein and fully active enzyme in the absence of luminal factors. Biochim Biophys Acta 1981; 663:163-8. 10. Cezard J, Conklin KA, Das BC, Gray GM. Incomplete
intracellular forms of intestinal surface membrane sucrase-isomaltase. J Biol Chem 1979;254:8969-75. 11. Skovbjerg H, Sjostrom H, Noren 0. Does sucrase-isomaltase always exist as a complex in human intestine? FEBS Lett 1979;108:399-402, 12 Grand RJ, Chong DA, Isselbacher KJ. Intracellular processing of disaccharaidases: the effect of actinomycin D. Biochim Biophys Acta 1972;261:341-52.
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ET AL.
13. Nilsson OS, Dallner G. Enzyme and phospholipid asymmetry in liver microsomal membranes. J Cell Biol 1977;72:568-83. 14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951:193:265-75. 15. Phillips AH, Landon RG. Hepatic triphosphopyridine nucleotide-cytochrome C reductase: isolation, characterization, and kinetic studies. J Biol Chem 1962;237:2652-60. 16. Kessler M, Acute 0, Storelli C, Murer H, Muller M, Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Biochim Biophys Acta 1978;506:136-54. 17. Cogoli A, Mosimann H, Vock C, von Balthazar A, Semenza G. A simplified procedure for the isolation of the sucraseisomaltase complex from rabbit intestine. Em J Biochem 1972;30:7-14. 18. Campbell DN, Garvey JS, Cremer NE, Sussdorf DH. Methods in immunology. 2nd ed. New York: W.A. Benjamin, 1970: 193-7. 19. Williams CA, Chase MW. Methods in immunology and immunochemistry. Vol. 3. New York: Academic, 1971:146-60, 234-73. 29. Hirsch JG, Fedorko ME. Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and “postfixation” in many1 acetate. J Cell Biol 1968;38:615-27. 21. Spurr AR. A low viscosity epoxy resin embedding medium for electron microscopy. J IJltrastruct Res 1969;26:31-43. 22. Neville DM. Molecular weight determination of protein dodecyl sulphate complex by gel electrophoresis in a discontinuous buffer system. J Biol Chem 1971;246:6328-34. 23. Laskey RA, Mills AD. Quantitative film detection of “H and 14C in polyacrylamide gels by fluorography. Eur J Biochem 1975;56:335-41. 24. Horrocks D. Applications of liquid scintillation counting. New York: Academic, 1974. 25. Forstner GC. (I-‘%)Glucosamine incorporation by subcellular fractions of small intestine mucosa. J Biol Chem 1970; 245:3584-92. 26. Shirley RJ, Chakraborly J, Bridges JW. An improved method of preparing rat small intestine microsomal fractions for studying drug metabolism. Anal Biochem 1979;93:71-81. 27. Beaufay H, Amar-Costesss A, Feytmans E, et al. Analytical study of microsomes and isolated subcellular membranes from rat liver. J Cell Biol 1974;61:188-231. 28. Eriksson LC. Preparation of liver microsomes with a high
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30. 31.
32.
33. 34.
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2
recovery of endoplasmic reticulum and a low grade of contamination. Biochim Biophys Acta 1978:508:155-64. Alpers D. Protein synthesis in intestinal mucosa: the effect of route of administration of precursor amino acids. J Clin Invest 1972;51:167-73. Palade G. Intracellular aspects of the process of protein synthesis. Science 1975;189:347-58. Sjostrom H, Noren 0, Christiansen L, Wacker H, Semenza G. A fully active, two-active-site, single-chain sucrase-isomaltase from pig small intestine. J Biol Chem 1980;255:1132-8. Montgomery RK, Sybicki M, Grand RJ. Autonomous biochemical and morphological differentiation in fetal rat intestine transplanted at 17 and 26 days of gestation. Develop Biol 1981;87:76-84. Blobel G, Dobberstein B. Transfer of proteins across membranes J Cell Biol 1975;67:835-51. Struck DK, Lennarz WJ. The function of saccharide-lipids in synthesis of glycoproteins. In: Lennarz WJ, ed. The biochemistry of glycoproteins and proteoglycans. New York: Plenum, 1980:35-83.
35. Robbins PW, Hubbard SC, Turco SJ, Wirth DF. Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins. Cell 1977;12:893-900. 36. Herscovics A, Bugge B, Quaroni A, Kirsch K. Characterization of glycopeptides labeled from d-(2-,‘H]mannose and L-[6“Hlfucose in intestinal epithelial cell membranes during differentiation. Biochem J 1980;192:145-53. 37. Danielsen EM, Sjostrom H, Noren 0. Biosynthesis of intestinal microvillar proteins. FEBS Lett 1981;127:129-32. 38. Danielson EM, Skovbjerg H, Noren 0, Sjostrom H. Biosynthesis of intestinal microvillar proteins. FEBS Lett 1981: 132:197-200. 39. Kelly JJ, Alpers DH. Properties of human intestinal glucoamylase. J Biol Chem 1973;248:8216-21. 40. Katz FN, Rothman JE, Lingappa VR, Blobel G, Lodish HF. Membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein. Proc Natl Acad Sci USA 1977;74:3278-82. 41. Rothman JE, Lodish HF. Synchronized transmembrane insertion and glycosylation of a nascent membrane protein. Nature 1977;269:775-80, 42. Ahnen DJ, Mircheff AK, Santiago NA. Yoshioka C, Gray GM. Intestinal surface aminooligopeptidase. Distinct molecular forms during assembly on intracellular membranes in vivo. J Biol Chem 1983;258:5960-66.
1985;88:531-8
Synthesis and Intracellular Processing of Sucrase-Isomaltase in Rat Jejunum RICHARD J. GRAND, ROBERT K. MONTGOMERY, and ARTURO PEREZ Division of Gastroenterology and Nutrition, Department of Medicine, The Children’s Hospital Medical Center; Division of Pediatric Gastroenterology and Nutrition, New England Medical Center, Floating Hospital; and Departments of Pediatrics. Harvard Medical School and Tufts Universitv School of Medicine, Boston, Massachusetts
To examine directly the synthesis and intracellular processing of rat intestinal sucrase-isomaltase, [“HIIeucine-labeled enzyme was solubilized from purified microsomal and microvillus fractions, immunoprecipitated, and analyzed. Immunologic identity between the microsomal and microvillus forms of the enzyme was demonstrated. Time-course studies using both intravenous and intraluminal labeling demonstrated an initial peak of microsomal sucrase-isomaltase labeling at 30 min followed by a sharp decline, and a subsequent rise in microvillus sucrase-isomaltase labeling, suggesting a precursorproduct relationship. Fluorography and quantitative analysis of electrophoresed immunoprecipitates confirmed these results, and revealed the presence of two high molecular weight proteins in the microsomes. Susceptibility of the smaller high molecular weight precursor to cleavage with endo-P-N-acetylglucosaminidase H indicated that this was the initially synthesized form of the glycoprotein. These data are consistent with the membrane flow hypoth-
Received September 17. 1981. Accepted August 29, 1984. Address requests for reprints to: Dr. Richard J. Grand, Chief, Division of Pediatric Gastroenterology and Nutrition, New England Medical Center, 171 Harrison Avenue, Box 213, Boston, Massachusetts 02111. This study was supported by U.S. Public Health Service Research Grants HD-14498, AM-14523, and AM-32658, and Training Grants AM-07183, AM-07333. and AM-07471 from the National lnstitute of Arthritis, Digestive and Kidney Diseases. This work was presented in part at a meeting of the American Gastroenterological Association, Las Vegas, Nevada, May 1978 (GASTROENTEROLOGY 1978;74:1126). The authors thank Ezio Merler. Ph.D. for valuable consultations; Mrs. Mariana Sybicki, Ms. Ann Forcier, and Mrs. Stephanie I. Ryan for expert technical assistance; Ms. Cynthia Richardson and Ms. Karen Cox for preparations of electron micrographs; Gabriel Goldberger, M.D. for ovalbumin; and Ms. Lisa LoPresti for preparation of the manuscript. 0 1985 by the American Gastroenterological Association 0016~5085/85/$3.30
esis and the single polypeptide sucrase-isomaltase synthesis.
model suggested for
Sucrase-isomaltase is an intrinsic protein of the intestinal microvillus membrane. This protein can only be removed by detergents (1) or by controlled proteolytic digestion (2). Extensive evidence has demonstrated conclusively that sucrase-isomaltase is located on the luminal side of the microvillus membrane (3,4). When isolated from the microvillus membrane, sucrase-isomaltase is a dimer consisting of two subunits (sucrase-a-glucohydrolase, EC. 3.2.1.48, and isomaltase, E.C. 3.2.1.10) which may be readily separated from one another by reduction (5) or citraconylation (6). The functional dimer is attached to the membrane by a hydrophobic anchor sequence which is part of the isomaltase subunit (4). Although the location and structure of mature sucrase-isomaltase have been well established, the mechanisms by which the enzyme is synthesized and transported to the cell surface remain to be completely elucidated. Most recent evidence demonstrates that sucraseisomaltase is synthesized as a single high molecular weight polypeptide (3,4,7-g), although there are data suggesting other mechanisms (10,ll). The Golgi form of the enzyme has been well studied (7,8), but not much is known of the earlier synthetic forms. Accordingly, the present studies were undertaken to provide direct evidence for microsomal synthesis of sucrase-isomaltase, and to demonstrate transfer of the newly synthesized enzyme from microsomes to microvillus membrane. The data also provide additional evidence in support of the single-polypeptide synthetic mechanism.
Abbreviations used in this paper: Endo H. endo+N-acetylglucosaminidase H; SDS, sodium dodecyl sulfate; OVA, ovalbumin.
532 GRAND ET AL.
GASTROENTEROLOGY Vol. 88,No. 2
Materials and Methods Experimental procedures were similar to those described previously (12). All experiments were performed using adult, male, albino rats (Charles River Breeding Laboratories, Wilmington, Mass.]. The animals weighed 125-150 g and were fed standard laboratory Chow [Ralston Purina Co., St. Louis, MO.) and water ad libitum. Studies were always begun by 9 AM. Radiolabeling of intestinal mucosal proteins was performed with animals maintained under light ether anesthesia. When intravenous labeling was used, 2 &i/g body wt of [“Hlleucine (40-60 Ci/mmol, New England Nuclear, Boston, Mass.) was administered by tail vein injection. When intraluminal labeling was used, the segment of intestine to be used was identified, the proximal end was ligated, and enough warm phosphate-buffered saline was administered via a plastic catheter to fill the required segment. The segment was then filled with -95 &i/g mucosa of [3H]leucine (40-60 mCi/mmol, New England Nuclear] in 0.9% NaCl solution. The radioisotope was allowed to remain in the intestinal lumen for 20 min; there was no observable loss of volume from the lumen during this period. This was followed by a chase of 5 mM cold leucine in phosphate-buffered saline. At appropriate times after labeling, animals were killed, and the jejunum, defined as the middle third of the small intestine (measured from the ligament of Treitz to ileocecal valve), was quickly excised and rinsed with 50 ml of ice-cold 0.9% NaCl solution. Scraped mucosa was subjected to fractionation techniques as described below.
Preparation
of Subcellular
Fractions
Jejunal microsomes were prepared by differential centrifugation according to a modification of previously published techniques (12). Mucosa was homogenized in 6 vol of 0.25 M sucrose containing 1 mM MgClz and 0.5 mM KC1 (pH 7.0). The final microsomal pellet (100,000 g for 1 h, Beckman Ultracentrifuge model L8-55) was suspended in isotonic sucrose (0.25 M), dialyzed overnight against the same solution, and then stored at 4°C for further processing, or frozen (-20°C). To obtain microsomal sucrase-isomaltase free of contamination by other membranous forms of the enzyme, intestinal microsomes were subjected to further purification. As sucrase-isomaltase is known to be located external to the microvillus membrane, it was assumed that the microsomal form of the enzyme would be found exclusively on the inside of the microsomal vesicles. This allowed the use of controlled protease digestion to remove contaminating microvillus forms of the enzyme without affecting the microsomal vesicles (I 3). The total microsomal protein was determined by the method of Lowry et al. (14); 50 pg of elastase (porcine pancreas type III) per milligram of microsomal protein was added and incubated at 30°C for 30 min (13). At the end of the incubation period, the microsomes were suspended in the original volume of isotonic sucrose, and the microsomal pellet was recovered as before and resuspended in
isotonic sucrose (pH 7.0). Enrichment of the microsomal fraction was verified using the specific activity of nicotinamide adenine dinucleotide phosphate, reduced form, cytochrome C reductase as a marker (15), and by electron microscopic demonstration of a lack of contamination by recognizable fragments of other organelles. Microvillus membranes were prepared from mucosal scrapings suspended in 50 mM mannitol and 2.0 mM TrisHCl, pH 7.1, by the method of Kessler et al. (16). Sucrase specific activity (12,14) was used as a marker for microvillus membrane purification. Final preparations had sucrase activities comparable to those reported by other groups using this and other methods of microvillus membrane isolation (1.2-1.5 pmol of glucose per milligram of protein per minute). Immunoprecipitation
Techniques
To measure the incorporation of radioactivity into newly synthesized sucrase-isomaltase in microsomal and microvillus fractions, a quantitative immunoprecipitation assay was developed. Highly purified rat jejunal sucraseisomaltase was prepared by the method of Cogoli et al. (17), which yielded a LOO-to 400-fold purification. Further purification was obtained by polyacrylamide disk gel electrophoresis in a nondenaturing system on which suerase-isomaltase migrated as a single band. The sucraseisomaltase band identified by correlation of enzyme activity and Coomassie Blue staining was sliced from untreated gels using calculated Rf value, and sufficient slices were pooled to yield 60 pg of sucrase-isomaltase. Gel slices were homogenized in equal volumes of complete Freund’s adjuvant, and this preparation was injected into rabbits injections at 3-wk intervals). (three subcutaneous Immunoglobulin G was purified from antisera by ammonium sulfate fractionation and chromatography on Whatman diethylaminoethanol-cellulose (18). The resulting antibody was shown to be monospecific by radial diffusion and by immunoelectrophoresis (data not shown) (19). Quantitative immunoprecipitation using this antibody and purified sucrase-isomaltase showed that, at equivalence, >95% of the sucrase activity was precipitated. This antibody was used throughout the experiments reported. Subcellular fractions were prepared for immunoprecipitation as described previously. Each fraction was sonicated (Ultrasonic I, Plainview, N.Y.) at half-maximal setting for 15 s in 6 bursts [with 15 s between each burst). LubrolPX [Sigma Chemical Co., St. Louis, MO.) was added to a final concentration of 0.6%. After 1 h at 2O"C,fractions were certrifuged at 100,000 g for 1 h. The clarified supernatants were then used in the immunoprecipitation assay. Quantitative immunoprecipitation curves were constructed for each sample using a fixed concentration of antisucrase-isomaltase immunoglobulin G and varying quantities of sample. Equivalence was estimated for these initial curves from the enzyme activity of the sample compared with the activity of purified sucrase precipitated at equivalence (-0.0843 U of sucrase activity per 25 ~1 of antisucrase immunoglobulin G). Reaction mixtures were incubated first at 37°C for 1 h and then at 4°C for at least 2 h. After equivalence was determined, immunoprecipita-
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SUCRASE-ISObIALTASE INTRACELLULAR PROCESSING 533
tion was repeated with larger volumes of sample and antibody to assure sufficient pellet for measurement of radioactivity. Precipitates were recovered by centrifugation at 12,000g for 5 min in an Eppendorf Microfuge (Brinkman Instruments, Inc., Westbury, N.Y.). The supernatants were assayed for sucrase activity, and any remaining activity was subtracted from the original units used. The pellets were washed once in saline, dissolved in 0.1 N NaOH, and aliquots were assayed for radioactivity. Nonspecific precipitation of radioactivity was determined from reaction mixtures containing solubilized microsomal or microvillus fractions, purified ovalbumin (OVA, 0.5 mgiml, Schwartz-Mann, Mannheim, F.R.G.), and rabbit anti-OVA (22.5 mg/ml generously provided by Dr. Gabriel Goldberger and Dr. Harvey Colten, Division of Cell Biology, Children’s Hospital Medical Center, Boston, Mass.). Immunoprecipitates from these mixtures were treated exactly as described previously. Radioactivity precipitated by the OVA-anti-OVA complex was subtracted from the radioactivity of the original precipitate to yield net radioactivity precipitated by antisucrase immunoglobulin G. Correction for nonspecific precipitation of radioactivity was also achieved by reprecipitation through the remaining supernatant, using added unlabeled sucraseisomaltase and antibody at equivalence, which gave the same pattern of labeling as described.
Electron
Microscopy
Microsomal pellets prepared as described previously were fixed (201, processed to Spurr’s plastic (21), cut on the LKB Ultratome III with a diamond knife (sections 800 A), and examined at 60 KV in a Philips 300 electron microscope (Philips Electronic Instruments, Inc., Mahwah, N.J.).
Sodium Dodecyl Electrophoresis
Sulfate-Polyacrylamide
Gel
Immunoprecipitates of labeled microsomal suerase-isomaltase were prepared as described previously. Precipitates were solubilized by heating to 100°C in a boiling water bath for 5 min in 0.056 M Tris buffer containing 1% sodium dodecyl sulfate (SDS) and 5% pmercaptoethanol. All other samples were solubilized in the same manner. Electrophoresis was performed on SDSpolyacrylamide slab gels consisting of a 3% stacking gel and a 7.5% separating gel made according to the method of Neville (22). After running, gels were simultaneously fixed and stained in a 0.006% solution of Coomassie Blue in acetic acid. methanol, and water. Gels were prepared for fluorography according to the method of Laskey and Mills (23), except that En”Hance (New England Nuclear) was used as the scintillant. Fixed and destained gels were soaked in En3Hance, washed in tap water, and dried on a filter paper backing. The dried gels were enclosed in an x-ray cassette against a sheet of preflashed Kodak X-Omat film (Eastman Kodak Co., Rochester, N.Y.) and stored in a -70°C freezer for 4 wk. When quantification of radioactivity on the gels was required,
the appropriate bands were cut from the gels, eluted in 5% Protosol in Econofluor at 37°C overnight [New England Nuclear Applications Note 22), and counted as described previously.
Treatment of Microsomal Preparations Endo-P-N-Acetylglucosaminidase H
With
Equal aliquots of solubilized microsomal fractions were incubated with and without 20 mU of endo+Nacetylglucosaminidase H (Endo H, Miles Laboratories, Elkhart, Ind.) prepared as described by the manufacturer. After 30 min of incubation at 37°C. equal amounts of antibody were added to each sample and immunoprecipitates were prepared as described previously. The resulting immunoprecipitates were solubilized, electrophoresed, and fluorograms were prepared as described for all samples.
Liquid
Scintillation
Counting
Radioactivity of immunoprecipitates was determined in a liquid scintillation spectrometer model LS 8000 (Beckman Instrument Co., Irvine, Calif.). Disintegrations per minute were calculated using a commercial quench series (New England Nuclear) and the Compton edge shift measure of counting efficiency (24). Data presented are mean + SE of at least three triplicate experiments at each time point described in the figure legends.
Results Characterization
of the Microsomal
Fraction
Enrichment of this fraction was confirmed by a lo-fold increase in the specific activity of nicotinamide adenine dinucleotide phosphate, reduced form, cytochrome C reductase, compared with the specific activity in whole mucosal homogenate, as reported also by others (25,26). Similar values have been obtained for hepatic microsomes (27,283. There was no enrichment of sucrase activity in this fraction, which, as reported previously, was 3.7% ? 0.05% of that in the homogenate (12). Examination of the microsomal pellets by electron microscopy revealed rough and smooth endoplasmic reticulum vesicles without evidence of extraneous organelle fragments.
Labeling
of Sucrase-lsomaltase
The incorporation of intravenously administered [3H]leucine into immunoprecipitable sucraseisomaltase from microsomes and microvillus membranes is shown in Figure 1. In the microsomal fractions, there was a peak of labeling of sucraseisomaltase at 30 min, followed by an abrupt fall and plateau at low levels of radioactivity. In the microvil-
534
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GRAND ET AL.
Vol. 88, No. 2
MINUTES
Figure
Figure
I. Specific radioactivity in sucrase-isomaltase immunoprecipitated from microsomal (closed circles) and microvillus (open circles] fractions prepared at the indicated times after intravenous label injection as described in Materials and Methods.
lus fraction, radioactivity of the enzyme increased slowly, with a maximal value established by 240 min. This fraction showed a distinct lag in maximal radioactivity in comparison with that in the microsomal fraction, suggesting a precursor-product relationship between the sucrase-isomaltase in the two fractions. The pattern of labeling obtained when [3H]leucine was introduced intraluminally is shown in Figure 2. In the microsomal fraction, a peak in the incorporation of radioactivity into sucrase-isomaltase was achieved by XI min, with a rapid reduction and plateau thereafter. Labeling of sucrase-isomaltase in the microvillus fraction showed a steady increase over time, with a maximal value occurring at 240 min. Comparing these patterns with those obtained by the intravenous route (Figure l),microsomal sucrase-isomaltase again appeared as a biosynthetic precursor to the microvillus form of enzyme. In addition, as noted previously by Alpers (29), the intraluminal labeling techniques yielded greater than a lo-fold increase in radioactivity in the fractions in comparison with that obtained by the intravenous route. Fractional incorporation of Label Into Sucrase-Isomaltase To compare the labeling of sucrase-isomaltase with the labeling of total proteins in the microsomal and microvillus fractions, relative specific activity or fractional incorporation was calculated according to the following formula: Fractional
incorporation
=
in sucrase-isomaltase immuno2. Specific radioactivity precipitated from microsomal (closed circles) and microvillus (open circles] fractions prepared at the indicated times after intraluminal label injection as described in Materials and Methods.
where MVM is the microvillus membrane and DPM is disintegrations per minute. In the microsomal fraction, sucrase-isomaltase represented 2.4% +-0.5% of the newly labeled proteins, whereas in the microvillus fractions sucraseisomaltase accounted for 9.3% -I- 1.3% of the newly labeled proteins. The difference in the fractional incorporation in microsomal and microvillus fractions is compatible with the concept that numerous rapidly labeled microsomal proteins are not destined for insertion into the microvillus membrane. Hence, the proportion of rapidly labeled microsomal suerase-isomaltase is less than that in the microvilli. Electrophoresis
and Fluorography
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated sucrase-isomaltase from microsomal fractions followed by fluorography revealed two high molecular weight protein bands as the earliest labeled form of sucraseisomaltase (Figure 3, lane 1). On the other hand, immunoprecipitation of sucrase-isomaltase from microvilli labeled for the same length of time, followed by identical electrophoresis and fluorography, showed no label incorporation (data not shown], although, at 120 min, the same procedure revealed label in both microvillus subunits (Figure 3, lane 2). To quantify the distribution of radioactivity in the gels at various times after labeling, stained bands were cut out of the gels and counted as described previously. The data are shown in Table 1. At 30 min, >99% of the radioactivity in microsomal SUerase-isomaltase was in the high molecular weight
milligrams
of lactase
in MVM x DPM/milligram
of lactase
milligrams
of protein
in MVM x DPM/milligram
of protein
x 100,
February
1985
SUCRASE-ISOMALTASE
INTRACELLULAR
specific and characteristic in the fraction.
PROCESSING
535
of the form of the enzyme
Treatment of Microsomal Precursor With Endo-P-N-Acetylglucosaminidase H
Figure
3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated sucrase-isomaltase. Lane 1 shows a fluorogram of sucrase-isomaltase immunoprecipitated from solubilized microsomes that were prepared after a 30-min period of labeling with [3H]leucine. For comparison, lane z shows a fluorogram of sucrase-isomaltase immunoprecipitated from solubilized brush borders that were prepared after a 120-min period of labeling with [3H]leucine. Solubilization, electrophoresis, and fluorography of both were carried out as described in Materials and Methods. Lane 3 shows a sample of papain-purified brush border sucrase-isomaltase run as marker on the same gel system and stained with Coomassie Blue. Whereas the sucrase corn&rates in lanes 2 and 3, isomaltase in the papain-purified sample in lane 3 runs slightly more rapidly than that in the detergentsolubilized sample in lane 2, due to the cleavage of the anchor sequence which is the first step in the purification procedure. All three samples were aligned by internal markers in order to compare relative migration distance, and demonstrate that the only proteins immunoprecipilabeled at 30 min in the microsomal tates were the two high molecular weight forms.
Further characterization of the two high molecular weight proteins in the microsomes was carried out by treating solubilized microsomes with Endo H before immunoprecipitation. As shown in Figure 4A, Endo H digestion did not affect the larger of the two proteins, but increased the mobility of the smaller of the two. Figure 4B demonstrates also that short-term labeling sometimes resulted in incorporation of label into only the smaller of the two high molecular weight forms, suggesting that it is the earliest synthesized protein. The Endo H effect indicates that the smaller band is an incomplete glycoprotein, containing only the core carbohydrate structure that is susceptible to Endo H cleavage, whereas the larger protein band, on the other hand, is not susceptible to such cleavage, and represents a more complete glycoprotein. Since these experiments were performed, Miles Laboratories has advised that their Endo H may be contaminated with protease. Our microsome fraction has, in effect, an internal control, because it displays two protein bands, one with mobility that is changed by Endo H treatment, and one with mobility that is not changed. The differential effect on the two protein bands mitigates against the result being due to nonspecific proteolytic cleavage. Because this initial glycosylation step is known to occur concurrent with or immediately after translation of the protein message, these data also demonstrate that this form
Table
1. Quantification Isomaltase
Fraction Microsomes
form. Subsequently, the counts in the gels fell rapidly, and no other peaks were identifiable. In the microvillus fractions at the time points examined, there were negligible counts in the high molecular weight region, but radioactivity in the sucrase and isomaltase subunits increased over time. These results confirm those from liquid scintillation counting of the total sucrase-isomaltase immunoprecipitates (Figures 1 and 2). In addition, they show that the microsomal sucrase-isomaltase label is actually in two high molecular weight proteins, and that the labeling of sucrase-isomaltase in each fraction is
Microvilli
Time (min) 15 30 60 240 15 30 60 240
of Radioactivity in Fractions
Sucrase-
in Subcellular DPM applied to gel 101
18,167 2,737 971 82 100 229 27.300
DPM distribution
in gel Ab
SI
PSI 0
18,100 2,140 308 o 0 3 351
0
362 84 589 0 26 23 18,830
0
36 58 119 0 3 43 131
As described in Materials and Methods, immunoprecipitates containing comparable quantities of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel bands were cut out and counted as described. PSI refers to high molecular weight forms; SI refers to counts in the sucrase band plus the isomaltase band; Ab is the radioactivity migrating with the antibody: DPM is disintegrations per minute.
536
GRAND
ET AL.
GASTROENTEROLOGY
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88, No. 2
of sucrase-isomaltase is in fact located in the rough endoplasmic reticulum.
Discussion
Figure
4. Treatment
of microsomal sucrase-isomaltase with Endo-P-N-acetylglucosaminidase H (Endo H]. A. Coomassie Blue-stained gel showing the effect of Endo H on the slower (PJ and faster (PI) migrating precursor forms of microsomal sucrase-isomaltase. Absence or presence of Endo H indicated by (-) or (+). Arrows indicate the increase in mobility of the Endo H-treated precursor (P,). Precursor P, is unresponsive to Endo H, suggesting more extensive glycosylation. B. Fluorogram of two samples of microsomal sucrase-isomaltase labeled for 30 min with [“Hlleucine. immunoprecipitated, and electrophoresed after incubation without (-1 and with (+) Endo H. Arrows indicate the change in mobility of the sucrase-isomaltase precursor.
These studies provide information concerning both the time-course and localization of synthesis and intracellular processing of sucrase-isomaltase. The time-course of leucine incorporation shows an early peak in the microsomal form of the enzyme, followed by a rapid decline, and a succeeding progressive rise in labeling of the microvillus form, suggesting a precursor-product relationship. This pattern is consistent with the hypothesis that membrane derived from the microsomes, containing this intrinsic protein, ultimately merges with the microvillus membrane, thereby transporting sucrase-isomaltase to the cell surface. Because the sucraseisomaltase that is immunoprecipitated from the microsomal vesicles is first solubilized by detergent treatment to disrupt the membranes with which it sediments, these data are consistent with an attachment of the newly synthesized enzyme protein to the endoplasmic reticulum membrane, presumably similar to the attachment demonstrated for the microvillus enzyme (5). These data also eliminate the possibility that the early microsomal peak is due to contamination of the microsomal fraction with microvillus fragments because, at the time of maximal microsomal labeling, microvillus labeling is negligible. This is further corroborated by both fluorography and quantification of electrophoresed immunoprecipitates, which demonstrates that only the high molecular weight forms of sucrase-isomaltase are rapidly labeled, whereas the microvillus form is not. These findings are in agreement with the membrane flow hypothesis described by Palade (30). Taken together, our labeling and electrophoretic data indicate that sucrase-isomaltase is initially synthesized in the microsomal fraction as a single high molecular weight protein. This evidence provides direct confirmation of the hypothesis proposed by Semenza’s group that sucrase-isomaltase is originally synthesized as a single polypeptide which is subsequently processed to the dimeric form found on the microvillus membrane (31).Thus, there is accumulating evidence that the intracellular form of sucrase-isomaltase is a single high molecular weight protein (8,9,32). Studies of glycoprotein synthesis in a number of systems indicate that a major class of glycoproteins, which have mannose and N-acetylglucosamine cores attached to the peptide via an N-glycosidic bond between asparagine and N-acetylglucosamine, are first glycosylated with a core sugar structure via a dolichol-linked intermediate located on or in the
February
1985
membrane of the endoplasmic reticulum (33). This core carbohydrate structure is then modified by additional glycosylation in the endoplasmic reticulum and finally undergoes excision of some residues and terminal glycosylation in the Golgi apparatus (34). A uniquely specific probe for this core glycosylation was discovered with the isolation of Endo H. This enzyme specifically cleaves between the two Nacetylglucosamine residues of incomplete glycoproteins, whereas the mature form of the glycoprotein is not affected (35). Susceptibility to Endo H is thus a specific and sensitive probe for a particular stage in the synthesis of this kind of glycoprotein. Our data indicate that the first identifiable precursor form of sucrase-isomaltase is an immature form of glycoprotein of this family, and that the higher molecular weight form of the precursor has had the glycoprotein modified so that it is now insensitive to Endo H. Thus, both are precursor forms at different stages of maturity. This is supported by our finding that the briefest exposure to radioactivity labels predominantly the smaller of the microsomal precursors, showing that it is the initially synthesized form. The sensitivity to Endo H demonstrates that the smaller precursor form is indeed an immature glycoprotein of the high mannose core structure. Our data on Endo H susceptibility of the microsomal sucraseisomaltase precursor are consistent with the findings of other investigators (36-38) that there is a group of mannose-rich glycoproteins in isolated intestinal epithelial cells that are Endo H degradable and, from changes both in this susceptibility and in kinetics of labeling, appear to be precursors of the group of complex glycoproteins. It is also consistent with the data of Kelly and Alpers (39) that rat sucrase-isomaltase has more mannose than any other sugar and the report of Cezard et al. (10) that sucrase-isomaltase binds to concanavalin A, a lectin that specifically binds mannose residues. Glycoproteins of this structure are members of the group synthesized via a dolichol intermediate, suggesting that sucrase-isomaltase is also a member of this group. Finally, as the formation of the core glycoprotein is known to occur in the endoplasmic reticulum (40,41), the Endo H digestion indicates that this early labeled high molecular weight sucrase-isomaltase is in fact located in the endoplasmic reticulum of the enterocytes. It is generally accepted that the subcellular “microsome” fraction contains a spectrum of cellular membrane vesicles that sediment together and includes vesicles of endoplasmic reticulum membrane, as well as vesicles of Golgi apparatus membrane. The presence of two protein bands in our preparation is consistent with a combination of
SUCRASE-ISOMALTASE
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immature high molecular weight endoplasmic reticulum precursor, and a Golgi-derived higher molecuSuch a situation has been lar weight precursor. reported also for aminopeptidase, sucrase-isomaltase, maltase-glucoamylase, and lactase-phlorizin hydrolase from pig intestinal “microsomes” prepared by calcium precipitation (42). These authors also showed that in each case the faster migrating band in electrophoresis was susceptible to Endo H digestion, whereas the slower was not. Our data thus identify the early precursor form of sucrase-isomaltase present in the microsomal fraction of the enterocyte. The structure of this precursor remains to be elucidated in detail, as does the question of whether there is an even earlier precursor form incorporating a subsequently removed signal or whether the anchor protein of isomaltase represents a retained signal sequence.
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