Immunoelectrophoretic studies on pig intestinal brush border proteins

Biochimica et Biophysica Acta, 494 (1977) 332-342

© Elsevier/North-Holland Biomedical Press BBA 37768 I M M U N O E L E C T R O P H O R E T I C STUDIES ON PIG INTESTINAL BRUSH BORDER PROTEINS

E. MICHAEL DANIELSEN ~, HANS SJOSTROM", OVE NORI~N" and ERIK DABELSTEENb " Department of Biochemistry C, The Panum Institute, University of Copenhagen, Copenhagen and u Department of Oral Pathology, Royal Dental College, Copenhagen (Denmark)

(Received April 25th, 1977)

SUMMARY Brush borders were prepared from pig intestinal mucosa and the membrane proteins solubilized with either Triton X-100 or papain. Proteins, thus released,were used as antigens to raise antisera in rabbits. The immunoglobulin G fractions were isolated and shown by the double layer immunofluorescence staining technique to react only with the brush border region of the enterocyte. The antibodies obtained were used in immunoelectrophoretic studies on the brush border proteins. Eight hydrolytic activities were identified by the use of histochemical staining methods. These were the microsomal aminopeptidase (EC 3.4.11.2), aspartate aminopeptidase (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.X), lactase (EC 3.2.1.23), glucoamylase (EC 3.2.1.3), sucrase (EC 3.2.1.48), isomaltase (EC 3.2.1.10) and alkaline phosphatase (EC 3.1.3.1). In addition, at least four faint immunoprecipitates were formed but none of these were identified.

INTRODUCTION The epithelial brush border membrane of the small intestine is known to contain a number of hydrolytic enzymes, supposed to participate in the final digestion of nutrients [1]. The brush border proteins can be solubilized by various means. A most efficient way is to treat the brush borders with ionic detergents such as sodium dodecyl sulphate which has been reported to release 90 per cent of the membranebound protein [2]. Sodium dodecyl sulphate, however, is a powerful denaturing agent which, even at low concentration, inactivates many of the enzymes [3]. For this reason nonionic detergents or proteolytic enzymes, releasing the proteins in an enzymatic active state, are often preferred as solubilizing agents. In the study on the protein composition of the enterocyte brush border, polyacrylamide gel electrophoresis has been widely used. The application of this technique has made it possible to estimate the number of protein components and also allowed a suggestion of their molecular weights [4, 5]. More recently, the use of Abbreviation: IgG, immunoglobulin G.

333 gel slicing techniques [3] and histochemical staining methods [6] has attributed enzymic activities to some of the protein bands obtained in polyacrylamide gel electrophoresis. Unfortunately, a good separation of proteins, solubilized with nonionic detergents, is conditioned by the presence of sodium dodecyl sulphate both in gel and buffer which, as mentioned above, causes an inactivation of many of the enzymes. Electrophoresis of brush border proteins, released with proteolytic enzymes, is not hampered by these difficulties as their higher migration-velocities render the addition of sodium dodecyl sulphate superfluous. It has been shown, however, that proteolytic agents are able to solubilize only a limited number of the'membrane proteins [4, 6] in a state, that cannot be considered "native" from a structural point of view [7]. Crossed immunoelectrophoresis is an analytical method that allows high resolution of proteins in the presence of nonionic detergents. The method has previously proven to be useful in studies on erythrocyte-membrane proteins [8]. In the present paper crossed immunoelectrophoresis has been applied t o the separation and enzymatic identification of the brush border proteins. MATERIALS L-Alanyl-fl-naphthylamide, L-aspartyl-fl-naphthylamide, L-lysyl-fl-naphthylamide, L-),-glutamyl-fl-naphthylamide and glycyl-L-prolyl-fl-naphthylamide were purchased from Bachem, Liestal, Switzerland. Naphthol AS MX phosphoric acid disodium salt, MTT tetrazolium bromide, phenazine methosulphate, Fast red TR salt, trehalose and maltose were obtained from Sigma, Saint Louis, U.S.A. Lactose, sucrose, glucose oxidase and papain were delivered by Merck, Darmstadt, Germany. Fast blue B salt was obtained from Gurr, London, Great Britain, Triton X-100 from Roth, Karlsruhe, Germany, agarose from L'Industrie Biologique Francaise, France, fluorescein-conjugated pig anti-rabbit serum IgG from Dakopatts, Copenhagen, Denmark, Freunds incomplete adjuvant from Statens Seruminstitut, Copenhagen, Denmark, and bovine serum albumin from Armour Pharmaceutical Co., Eastbourne, Great Britain. Isomaltose was kindly delivered by Dr. N. G. Asp, Lund, Sweden. All other chemicals used were of analytical grade and the water de-ionized by a Meg-OLite ZD-30.230.00 system (Millipore, Bedford, Mass., U.S.A.). METHODS If not otherwise stated, all preparative steps were performed at 4 °C.

Preparation of brush borders Intact brush borders were prepared from pig small intestine, taken 1-6 m from the pylorus. Immediately after the halothane anesthesized (in nitrous oxide and oxygen) animals were bled to death, the small intestine was removed and cooled on ice. The preparation procedure of Eichholz and Crane [9] was used with minor modifications. The initial homogenization of the mucosal scrapings was performed in a 50 mM Tris.HC1 buffer, pH 7.5, containing 5 mM EDTA and 100 mM sucrose. The contaminating nuclei were lysed by allowing the preparation, suspended in a 5 mM EDTA buffer, pH 7.5, to stand overnight. During this time, DNA from the disrupted nuclei sedimented as a viscous material, and the supernatant, containing

334 the brush borders, was decanted. The preparative steps were followed by phase contrast microscopy and the final preparation judged to consist mainly of brush borders. As brush border membranes tend to disintegrate upon freezing, the solubilization-steps described below were carried out immediately after the brush border preparations had been made.

Preparation of a crude membrane fraction A crude membrane fraction was prepared from pig small intestine. The mucosa was collected by pressing the intestine through rubber rollers [10]. Homogenization of the mucosa was performed in four volumes of a 50 mM Tris. HCI buffer, pH 8.0, by an Ultra Turrax homogenizer (Janke and Kunkel, Staufen, Germany) for 30 s. The homogenate was centrifuged (15 000 x g, 30 min), and the supernatant collected and centrifuged (435 000 x g, 60 min). The resulting pellet, the crude membrane preparation, was stored at --20 °C until use. Solubilization with Triton X-IO0 The brush border and crude membrane preparations were solubilized by 1 Triton X-100 in a 50 mM Tris.HC1 buffer, pH 8.0, for 1 h under frequent stirring. The insoluble material was removed by centrifugation (30 000 x g, 25 min for the brush border preparation, 435 000 × g, 60 min for the crude membrane preparation) and the clear supernatants, in the following denoted "detergent brush border antigen" and "crude membrane antigen" were kept frozen at --20 °C until use. Solubilization with papain The brush border preparation was solubilizedby papain (1 mg/ml) in a 50 mM potassium phosphate buffer, pH 6.0, containing 4 mM cysteine. The incubation at 37 °C lasted for 1 h. The insoluble material was removed by centrifugation (30 000 x g, 25 min), and the clear supernatant, the "papain brush border antigen", was collected and stored at --20 °C until use. Preparation of the antisera Before immunization the rabbits were bled for 25 ml to be used as control sera. Rabbits were immunized with brush border and crude membrane antigens, previously mixed with equal volumes of Freunds incomplete adjuvant. The animals were injected intracutaneously every second week with 200 ffl of the mixture (approximately 200 fig of protein). A week after the fourth injection, they were bled for 25 ml. Boosters were given every sixth week, followed by new bleedings. A total number of 10 rabbits were immunized, 4 with crude membrane antigen, 3 with detergent brush border antigen and 3 with papain brush border antigen. The IgG fractions of the antisera were isolated by ammonium sulphate fractionation and ion-exchange chromatography [11], and the purified fractions, "crude membrane antiserum", "detergent brush border antiserum" and "papain brush border antiserum", were stored at 4 °C in a 154 mM NaC1 solution, containing 15 mM NaNa. Immunoelectrophoresis Crossed immunoelectrophoresis [12] was performed on 1.5 mm thick agarose gels casted on 1.5 x 100 x 100 mm glassplates. When the papain brush border

335 antigen was subjected to electrophoresis, the gel consisted of 1 ~ agarose in a 20 mM sodium barbital buffer, pH 8.6. The first dimension was run for 1 h at l0 V/cm, the second for 20 h at 3 V/cm. The detergent brush border antigen and the crude membrane antigen was electrophoresed for 3 h at 10 V/cm in the first dimension and 20 h at 10 V/cm in the second dimension, using a 37 mM sodium barbital buffer, pH 8.7, containing 0.38 M glycine, 0.79 M Tris.HCl and 0.1~o Triton X-100. In general, 10 #l (about 20 #g of protein) antigen was applied in the application well, and the antibody content of the gel was approximately 10/zg/cm2.

Staining methods After termination of the immunoelectrophoresis, the plates were pressed and then washed in 0.1 M NaCl for 1 h, followed by a washing in distilled water for 15 min. The plates were then dried by a jet of cold air from a hair-drier, and the precipitates stained, either for protein with Coomassie brilliant blue R 250 or for enzymatic activities. The precipitate, corresponding to a particular enzyme, was visualized by carefully pouring the proper reaction mixture over the plate. Incubation at 37 °C in the dark lasted from 15 to 60 min. After the staining, the gels were cautiously washed with buffer and dried in the air. The following reaction mixtures were used to identify the different enzymes [6, 13]: Disaccharidases: l0 mg MTT Tetrazolium bromide, 10 mg Phenazine methosulphate, 5 mg glucoseoxidase together with 100 mg of sucrose, lactose, maltose, trehalose or 10 mg isomaltose, dissolved in 10 ml 10 mM potassium phosphate buffer, pH 6.0. Alkaline phosphatase: 10 mg Naphthol AS MX phosphoric acid disodium salt and 20 mg Fast red TR salt dissolved in 10 ml 50 mM Tris. HC1 buffer, pH 8.0. Peptidases: 10 mg of the fl-naphthylamide of either L-alanine, L-aspartic acid, L-lysine or glycyl-L-proline together with 20 mg Fast blue B salt dissolved in l0 ml 100 mM sodium bicarbonate buffer, pH 8.5. y-Glutamyl transferase: 10 mg ),-glutamyl-fl-naphthylamide, 100 mg glycylglycine and 20 mg Fast blue B salt dissolved in l0 ml 100 mM sodium bicarbonate buffer, pH 9.01

Immunofluorescence The histological localization of the brush border antigen was performed by a double-layer immunofluorescence staining technique [14]. The first layer in the staining was the detergent brush border antiserum and the second layer was a pig anti-rabbit serum IgG conjugated with fluorescein isothiocyanate. Unfixed 4/~m frozen sections of pig small intestine were used as antigen. The tissue sections were airdried for 10 min. The slides were then incubated at room temperature in a moist chamber with diluted brush border antiserum (10/~g/ml) for 40 min and washed three times 5 min in a 15 mM sodium phosphate and 4 mM potassium phosphate buffer, pH 7.3, containing 150 mM NaC1 (phosphate buffered saline). Then they were incubated for 40 minutes with the conjugate and again subjected to three washings in phosphate buffered saline as described above. Finally they were mounted in a 100 mM Tris.HC1 buffer, pH 8.3, containing 20% glycerol. Control experiments were performed with phosphate buffered saline and with the absorbed IgG-fractions [15] and

336 the corresponding control-sera diluted to the same protein concentration as the brush border antiserum.

Microscope The fluorescent microscope was an Ortoplan (Leitz, Wetzlar, Germany) modified with a Tiyoda wide-angle darkfield oil immersion condensor. The light source was an Osram HBO 200 lamp. The primary filter was a F I T C interference filter with a red contrast band (Laboratory for Technical Optics, Lyngby, Denmark). The secondary filter was a 2 mm glass filter (Schott and Gen., Mainz, Germany) matched to fit the primary filter.

Polyacrylamide gel electrophoresis Detergent brush border antigen was electrophoresed in polyacrylamide gels in the presence of sodium dodecyl sulphate [16]. Samples of 150/~g of protein were heated to 100 °C for 3 min in the presence of 1 ~o sodium dodecyl sulphate and 1 2-mercaptoethanol before they were applied to the gels having a monomer concentration of 10 ~ of which 1 ~ was N,N'-methylene bisacrylamide. Electrophoresis was run for 3 hours at 8 mA/gel, and afterwards the protein bands were visualized by staining with Coomassie brilliant blue R 250.

Assays Protein was determined according to Wang et al. [17], using crystalline bovine serum albumin as a standard. Aminopeptidase, ~,-glutamyl transferase and dipeptidyl peptidase IV activities were determined spectrophotometrically at 37 °C by use of the Reaction rate analyzer LKB 8600 (LKB Produkter AB, Stockholm, Sweden). The rate of liberation of pnitro aniline and fl-naphthylamine was measured at 410 nm and 340 nm respectively. Aminopeptidase activity was determined in a 50 mM Tris. HCI buffer, pH 8.0, containing I mM L-alanyl-p-nitroanilide [18], ),-glutamyl transferase activity in a 174 mM Tris. HCI buffer, pH 8.0, containing 90 mM glycylglycine and 3 mM L-~,-glutamylp-nitroanilide [19] and dipeptidyl peptidase IV activity, according to the principle of Lee et al. [20], in a 50 mM Tris.HCl buffer, pH 8.0, containing 1 mM glycyl-Lprolyl-fl-naphthylamide. One unit of enzyme activity is defined as the activity hydrolyzing 1 #mol substrate per min under the conditions stated above using the extinction coefficients 8850 M - l . c m - 1 and 1780 M-1. cm- 1 for the liberated p-nitroaniline and fl-naphthylamine respectively. RESULTS AND DISCUSSION

Characterization of the antigens Solubilization with Triton X-100 was found to release 70-80~o of the peptidase activities studied. The corresponding values for the papain treatment varied between 40--50 ~oThe specific activities of microsomal aminopeptidase, ~,-glutamyl transferase and dipeptidyl peptidase IV for the brush border- and crude membrane antigens are listed in Table I. The degrees of enrichment for these marker enzymes in the brush border preparations are of the same magnitude as reported by Kenny et al. [21].

337 TABLE I PEPTIDASE ACTIVITIES OF THE ANTIGEN PREPARATIONS The activities of microsomal aminopeptidase, 7-glutamyl transferase and dipeptidyl peptidase IV were determined as described in Methods. Antigen preparation

Crude membrane antigen Detergent brush border antigen Papain brush border antigen"

Specificactivity (units/rag) Microsomal aminopeptidase

~,-Glutamyl Dipeptidyl transferase peptidase IV

0.20 2.18 1.05

0.25 0.71 0.36

0.03 0.34 0.15

" The listed values are corrected for the papain added. Crossed immunoelectrophoresis o f the crude membrane antigen against the corresponding antiserum yielded at least 10 precipitates. However, when papain or detergent brush border antigen was run in the same electrophoretic system, only five, clearly distinct precipitates appeared (Fig. 1), also indicating that a considerable purification had been achieved in the brush border preparations. Similar precipitatepatterns were obtained in crossed immunoelectrophoresis of crude membrane or brush border antigen against the brush border antisera. Polyacrylamide gel electrophoresis o f detergent released brush border proteins in the presence of sodium dodecyl sulphate has been reported to yield from 15 to 25 bands [4, 5]. These data could suggest the relative few number of immunoprecipitates as a result of insufficient solubilization o f the brush borders. However, at least 15 bands were observed after polyacrylamide gel electrophoresis of the detergent brush border antigen.

Specificity of the antisera Crossed immunoelectrophoresis of any o f the antigens against the control-sera did not result in the formation of immunoprecipitates. The specificity of the brush border antisera was tested by applying the doublelayer immunofluorescence technique to sections of the pig small intestine. Fluorescence was only conspicuous in the brush border region of the enterocytes, indicating the brush border specificity o f the antisera (Fig. 2). No fluorescence appeared in the control experiments. The brush border specificity o f the antisera was further examined by subjecting the 435 000 x g supernatant from the crude membrane preparation to crossed immunoelectrophoresis against the detergent brush border antiserum. As no precipitates were formed, it was concluded, that the antiserum did not contain antibodies directed towards any soluble cytoplasmic proteins.

Identification of the immunoprecipitates The results of the histochemical stainings of the immunoplates are shown in Table II. Enzymes, corresponding to the five immunoprecipitates, were identified. Only precipitate No. 1 was stained, when alanyl-fl-naphthylamide was used as

338

Fig. 1. Crossed immunoelectrophoresis of the detergent brush border antigen against the crude membrane antiserum. Fig. 2. Section of the small intestine stained by the immunofluorescence technique. The first layer was an anti-brush border protein IgG fraction. L, lumen; C, connective tissue. Arrows indicate the basal lamina between the epithelium and the connective tissue. Positive reaction, seen as a bright band, is only present in the brush border region of the cells. TABLE II IDENTIFICATION OF THE IMMUNOPRECIPITATES After termination of the immunoelectrophoresis, the plates were incubated with each of the substrates listed below according to the procedure outlined in Methods. The numbers refer to the immunoprecipitates in Figs. 1 and 5,A. Substrate

Hydrolyzed by precipitate No.

Naphthol AS MX phosphoric acid Sucrose Maltose Lactose Isomaltose Trehalose y-Glutamyl-fl-naphthylamide Alanyl-fl-naphthylamide Aspar tyl-fl-naphthylamide Lysyl-fl-naphthylamide Glycylprolyl-fl-naphthylamide

6 4 2 and 4 3 4 not hydrolyzed not hydrolyzed 1 5 1 and 5 7

substrate (Fig. 3, A) thus identifying it as the microsomal aminopeptidase (EC 3.4.11.2, ref. 10). The enzyme also showed activity towards lysyl-fl-naphthylamide. Both precipitates Nos. 2 and 4 were capable o f h y d r o l y s i n g maltose (Fig. 3, B). But as opposed to precipitate No. 2, the latter also hydrolyzed sucrose and isomaltose and was thereby shown to represent the sucrase, isomaltase complex (EC 3.2.1.48 and E C 3.2.1.10, ref. 22). Immunoprecipitate No. 2 p r o b a b l y corresponds to the maltaseactivity o f the a-l,4-glucan glucohydrolase (EC 3.2.1.3, ref. 23). Only precipitate No. 3 caused a colour-reaction, when lactose was used as substrate (Fig. 3, C), thus characterizing it as lactase (EC 3.2.1.23). A l t h o u g h the intestinal mucosa is reported to contain three fl-galactosidases, only one has been shown to be present in the brush border region [24]. W h e n aspartyl-fl-naphthylamide was used as substrate, only precipitate No. 5

339

Fig. 3. Crossed immunoelectrophoresis of the papain or detergent brush border antigen against the corresponding antiserum. After termination of the electrophoresis, the plate was incubated with the following substrates: A, alanyl-fl-naphthylamide (detergent system); B, maltose (papain system); C, lactose (detergent system) and D, aspartyl-fl-naphthylamide (detergent system). (The weak, unspecific staining of precipitate No. 1 is due to entrapment-phenomena [28].) Fig. 4. Crossed immunoelectrophoresis of a dipeptidyl peptidase IV preparation against the detergent brush border antiserum. After termination of the electrophoresis, the plate was incubated with glycyl-L-prolyl-fl-naphthylamideas substrate. was stained (Fig. 3, D). It therefore represents the aspartate aminopeptidase (EC 3.4.11.7, ref. 25). Surprisingly this enzyme also hydrolyzed lysyl-fl-naphthylamide, but the intensity of the colour-reaction was very weak compared to the one caused by the microsomal aminopeptidase. A diffuse colour-reaction, not representing any of the numbered immunoprecipitates stained with Coomassie brilliant blue, appeared below the other precipitates when glycylprolyl-fl-naphthylamide was used as substrate. It could not be due to some kind of unspecific staining, because a preparation, enriched in dipeptidyl peptidase IV (EC 3.4.14. X) by chromatographic methods [26], used as antigen gave rise to a regular immunoprecipitate (Fig. 4). This experience clearly demonstrates, that the Coomassie brilliant blue staining-method fails to detect immunoprecipitates below a certain level of intensity, i.e. precipitates formed in systems, where the concentration of the antigen and (or) antiserum is too low. The possible presence of other undetected precipitates could not be excluded. For this reason, a concentrated solution o f papain-released microvillus proteins prepared by the method of Sigrist et al. [27] was used as antigen in a crossed immunoelectrophoresis, where the antibody-content

340

Fig. 5. Crossed immunoelectrophoresis of papain-solubilized microvillus proteins against the papain brush border antiserum. 10 #1 (250/zg protein) of sample was applied in the well, and the antibodycontent of the gel was 150 pg/cm 2. A, the precipitates stained with Coomassie brilliant blue. B, the plate was incubated with Naphthol AS MX phosphoric acid as substrate. (The weak, unspecific staining of some of the other precipitates is due to entrapment-phenomena [28].)

341 of the gel was increased fifteen times. In addition to the five already identified precipitates, at least six faint, but clearly visible precipitates appeared (Fig. 5, A). One of these was shown to represent the dipeptidyl peptidase IV, and another identified as alkaline phosphatase (EC 3.1.3.1. Fig. 5, B). The rest of the precipitates remained unidentified after the substrates, listed in Table II, had been tried out. As the immunoprecipitation of an antigen in general is unlikely to destroy all the biological activity, they probably represent proteins either with unknown enzymatic properties or transport functions. Immunoprecipitates corresponding to ~,-glutamyl transferase (EC 2.3,2.2) and trehalase (EC 3.2.1.28) could not be observed even in the more sensitive electrophoretic system described above. The most obvious explanation is that these enzymes, although present in the antigen-preparations, have been below the threshold-value required to induce a sufficient immuneresponse. As shown in Table I, solubilization with Triton X-100 resulted in specific activities of the brush border peptidases about two times higher than did the treatment with papain. This indicates either a more selective release of the studied enzymes by Triton X-100 or reflects an inactivating effect of papain. Furthermore, the papain antigen/antiserum system gave rise to the same number of immunoprecipitates as the corresponding detergent system. Together these results suggest, that the enzymatic activities of the brush border proteins are more sensitive to the proteolytic effect of papain than are their antigenic properties. The purpose of applying crossed immunoelectrophoresis to studies on the brush border proteins has been to offer an alternative to polyacrylamide gel electrophoresis. The method used in this work, separating the proteins according to their charge and antigenicity, gives good resolution even of proteins solubilized by nonionic detergents and preserves the enzymatic activities at a time, thereby allowing histochemical staining techniques to be performed. The failure to fulfil this requirement is the major obstacle by using polyacrylamide gel electrophoresis. Once a sufficient pool of antiserum has been prepared and the precipitatepatterns have been established, crossed immunoelectrophoresis can be used to follow the preparative purification of the membrane proteins. Because it is also a quantitative method and only requires small amounts of sample, in our laboratory crossed immunoelectrophoresis is now being used in the study on brush border proteins obtained by peroral biopsies on humans. ACKNOWLEDGEMENTS The staff of the Department of Experimental Pathology, Rigshospitalet, Copenhagen, Denmark, is thanked for having donated the pig small intestines used in the work. REFERENCES 1 Crane, R. K. (1968) in Handbook of Physiology,section 6, Alimentary Canal (Code, C. F., ed.), Vol. 5, pp. 1323-1352, American PhysiologicalSoc., Washington D.C. 2 Critchley,D. R., Howell, K. E. and Eichhoiz, A. (1975) Biochim.Biophys. Acta 394, 361-376 3 Maestracci, D., Preiser, H., Hedges, T., Schmitz, J. and Crane, R. K. 0975) Biochim. Biophys. Acta 382, 147-156 4 Alpers, D. H. (1972) J. Clin. Invest. 51, 2621-2630

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