Pseudopod-like basal cell processes in intestinal cholecystokinin cells.

Cholecystokinin (CCK) is secreted by neuroendocrine cells comprising 0.1%-0.5% of the mucosal cells in the upper small intestine. Using CCK promoter-driven green fluorescent protein (GFP) expression in transgenic mice, we have applied immunofluorescence techniques to analyze the morphology of CCK cells. GFP and CCK colocalize in neuroendocrine cells with little aberrant GFP expression. CCK-containing cells are either flask- or spindle-shaped, and in some cells, we have found dendritic processes similar to pseudopods demonstrated for gut somatostatin-containing D cells. Most pseudopods are short, the longest process visualized extending across three cells. Pseudopods usually extend to adjacent cells but some weave between neighboring cells. Dual processes have also been observed. Three-dimensional reconstructions suggest that processes are not unidirectional and thus are unlikely to be involved in migration of CCK cells from the crypt up the villus. Abundant CCK immunostaining is present in the pseudopods, suggesting that they release CCK onto the target cell. In order to identify the type of cells being targeted, we have co-stained sections with antibodies to chromogranin A, trefoil factor-3, and sucrase-isomaltase. CCK cell processes almost exclusively extend to sucrase-isomaltase-positive enterocytes. Thus, CCK cells have cellular processes possibly involved in paracrine secretion.

Localization of intestinal sucrase-isomaltase complex on the microvillous membrane by electron microscopy using nonlabeled antibodies.

Microvillous vesicles isolated from rabbit small intestine showed a trilaminar membrane with a rather smooth surface, which was apparently not affected by papain solubilizing sucrase-isomaltase complex or by trypsin unable to solubilize it. When microvilous vesicles or trysinized ones were incubated with immunoglobulin G against the sucrase-isomaltase complex or monovalent fragments therefrom, an apparently continuous electron-opaque layer approximately 180 A in width appeared around the external surface of vesicles. Such a layer was not formed on papainized vesicles. Microvillous vesicles and trypsinized ones negatively stained with phosphotungate showed a great number of particles protruding approximately 150 A from the membrane surface, but papainized vesicles did not. The particles existed close to one another and appeared to form a particulate layer 150 A in width on the surface. The antibodies, whether they were divalent or monovalent, increased the width of the layer to approximately 200 A and obscured the fine particulate structure of intact and trypsinized vesicles. Papainized vesicles retained their smooth surface upon interaction with antibodies. These results, together with those with the Triton-solubilized sucrase- isomaltase complex (Nishi and Takesue, 1978), J. Ultra-struct. Res., 62:1- 12), indicate not only that sucrase-isomaltase complexes are located close to one another on the membrane, but also that they or at least their protein portions protrude approximately 150 A from the surface of the trilaminar membrane.

A and H blood group antigens as markers of sucrase-isomaltase from the enterocyte-like differentiated human colon carcinoma cell lines HT-29 and Caco-2.

The purpose of this work was to investigate whether sucrase-isomaltase from enterocyte-like differentiated human colon carcinoma cell lines carries blood group antigens of the ABH system. Six cultured lines of blood group A (HT-29, SW-480, Co-115) or O phenotype (Caco-2, HRT-18, HCT-8R) were studied. Only HT-29 cells grown in the absence of glucose (HT-29 Glc-) and Caco-2 cells express an enterocytic differentiation with the presence of sucrase-isomaltase on the apical surface of the cells. Binding of anti-A antibodies to HT-29 Glc- and of UEA-I to Caco-2 cells gave the same apical immunofluorescence pattern of staining as did anti-sucrase-isomaltase antibodies, whereas only a membrane binding was observed in nondifferentiated cells. Sucrase-isomaltase immunoisolated from HT-29 Glc- and Caco-2 cells reacted with anti-A antibodies and Ulex europaeus agglutinin-I (UEA-I), respectively, at sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot. Immunoprecipitation of solubilized brush border-enriched fractions from the same cells with UEA-I or anti-A antibodies resulted in an inhibition of sucrase activity which reached congruent to 80% for Caco-2 cells with UEA-I and approximately equal to 50% for HT-29 cells with anti-A antibodies. Similar results were obtained in the corresponding tumors in nude mice: anti-A antibodies in HT-29 and UEA-I in Caco-2 tumors bound to the same apical structures as did anti-sucrase-isomaltase antibodies; sucrase-isomaltase immunoisolated from the tumors bound anti-A antibodies (HT-29) or UEA-I (Caco-2). These results support the hypothesis that sucrase-isomaltase from enterocyte-like differentiated human colon cancer cells carries blood group antigens of the ABH system. These findings suggest that colon cancers which have been shown to display an apical pattern of expression of ABH antigens should be screened for their possible enterocytic differentiation.

Purification and characterization of alpha-glucosidase complex from the intestine of the frog, Rana japonica.

The enzymes responsible for much of the isomaltase and maltase activities in the intestine of the frog, Rana japonica, were purified by detergent solubilization and affinity chromatography on Sephadex G-200 gel. The two activities paralleled each other during purification. The isomaltase, maltase and glucoamylase activities eluted in the same pattern on Sepharose 4B gel filtration as well as on Sephadex G-200 gel affinity chromatography. Anti-rabbit sucrase-isomaltase antibody inhibited the isomaltase activity but not the maltase or glucoamylase activity of the purified enzyme preparation, while the three activities were precipitated in parallel by the antibody. The isomaltase activity was more stable at 55 degrees C than the maltase and glucoamylase activities. On SDS-polyacrylamide gel electrophoresis under nondissociating conditions the purified enzyme preparation showed only one major band of 330 kDa, while under dissociating conditions it showed two bands of 116 and 212 kDa. These results suggest that isomaltase (apparently with no or minor maltase activity) is due to a protein domain (or protein) different from one which is responsible for maltase and glucoamylase activities. This implies that isomaltase is associated with maltase/glucoamylase to form alpha-glucosidase complex in the brush border membrane of the frog intestine.

Antigenic cross-reactions among human intestinal brush-border enzymes revealed by the immunoblotting method and rabbit anti-enzyme sera.

Antibodies against 3 purified human small intestinal brush-border hydrolases (sucrase-isomaltase, maltase-glucoamylase and neutral aminopeptidase) were produced in rabbits. By double immunodiffusion and crossed immunoelectrophoresis the antisera appeared to be monospecific. However, with the immunoblotting method antibodies reacting with many brush-border proteins were detected. In one case, the cross-reactions were due to the presence of anti-A antibodies. In other cases absorption of the antisera with absorbants with blood group A and H activities did not eliminate these reactions. As the main brush-border proteins are glycosylated and share common lectin reactivities, it is possible that these antibodies are directed against carbohydrate antigens. The use of such antibodies for immunocytochemical methods and for immunoassays must be undertaken with caution. These principles are applicable to other antigen systems.

Use of a monoclonal antibody to sucrase-isomaltase for evaluation of the columnar cuff after stapled restorative proctocolectomy.

PURPOSE: Restorative proctocolectomy with a double-stapled pouch-anal anastomosis retains a cuff of diseased columnar mucosa (columnar cuff) in the upper anal canal that may require biopsy. Biopsying this can be difficult and colonic phenotypic change in the pouch can lead to errors interpreting the histology. This study was designed to investigate the use of a monoclonal antibody to sucrase-isomaltase for differentiating ileal pouch from columnar cuff mucosa. Then, by using this antibody, the ability to accurately take and report biopsies from the anal canal was examined. METHODS: The technique of staining for sucrase-isomaltase was developed. From 113 patients who had a double-stapled pouch-anal anastomosis, 467 formalin-fixed biopsies and 177 fresh-frozen biopsies were taken from the ileal pouch, columnar cuff, or anal transitional zone. Biopsies were stained with a monoclonal antibody to sucrase-isomaltase, and fixed biopsies were routinely reported after staining with hematoxylin and eosin. RESULTS: A monoclonal antibody to sucrase-isomaltase reliably discriminated between ileal from rectal mucosa. A biopsy of columnar cuff mucosa as reported by routine histology was obtained during 72 percent of attempted outpatient examinations. Sucrase-isomaltase staining of reported columnar cuff biopsies showed that biopsies were of pouch rather than columnar cuff in 4.4 percent (95 percent confidence interval, 2-8) of outpatient examinations. CONCLUSIONS: The monoclonal antibody to sucrase-isomaltase used in this study may have a clinical role when interpreting columnar cuff biopsies from patients being investigated for pouch dysfunction, or in patients having surveillance biopsies to exclude neoplasia in the columnar cuff.

Immunoblotting with monoclonal antibodies: importance of the blocking solution.

Four commonly used blocking agents, i.e., fetal calf serum, mammalian gelatin-Nonidet-P40, fish gelatin-Nonidet-P40, and defatted powdered milk were compared with respect to their efficiency to block the nonspecific background and to promote maximal immunoreactivity of monoclonal antibodies against human intestinal sucrase-isomaltase during immunoblotting. Two of five monoclonal antibodies were found to react with the electroblotted enzyme. However, one of the reacting antibodies gave optimal results with fish gelatin-Nonidet-P40 and the other with defatted powdered milk, while fetal calf serum lead to unacceptably high backgrounds. The results suggest that some of the difficulties encountered with monoclonal antibodies in immunoblotting may be due to inappropriate blocking conditions.

Studies on the intestinal disaccharidases of the pigeon IV. Immunochemical properties of sucrase . isomaltase and maltase . glucoamylase.

Antisera against purified pigeon small intestinal sucrase-isomaltase (S-I) and maltase-glucoamylase (M-G) were prepared from rabbits. Both sera showed cross-reactivity. It was demonstrated that the sucrase . isomaltase was purified to homogeneity, supporting our earlier results of SDS-PAGE of pigeon intestinal disaccharidases. Both the sucrase- isomaltase and maltase-glucoamylase activities were not inhibited by either specific or cross-reacting antibodies even when a several fold of either antibody was present. It is inferred from these immunochemical results that the two complexes in the pigeon intestine share many structural identities, and that their catalytic site(s) may not be involved in their antigenic domains.

Monoclonal antibodies to sucrase/isomaltase: probes for the study of postnatal development and biogenesis of the intestinal microvillus membrane.

Two monoclonal antibodies, designated BB 3/34/12 and BB 5/8/40/90, have been produced to rat intestinal sucrase/isomaltase (SI) by the hybridoma technique using microvillus membranes as antigen. The BB 3/34/12 antibody was shown to be specific for the sucrase subunit. These antibodies provided new information regarding the biosynthesis and postnatal development of SI. In rat intestinal fetal transplants, SI was found exclusively in the form of an enzymatically active high molecular weight precursor, confirming our previous observations concerning the role of luminal proteases for the processing of SI in the microvillus membrane. The SI precursor, purified by affinity chromatography using the BB 3/34/12 antibody, had both sucrase and isomaltase activities, suggesting that a single precursor protein generates both sucrase and isomaltase subunits by proteolytic cleavage. The initial appearance of SI during normal postnatal development in the rat intestine was found to be confined to the cells present at the base of the villi. The same localization was observed after precocious induction of SI by cortisone acetate. In both cases, no immunofluorescence was observed in the crypts, suggesting that only the differentiated enterocyte is capable of synthesizing this enzyme. Even at the earliest times of appearance, newly synthesized SI was found almost completely split into its subunits, suggesting that the protease(s) responsible for the processing of the precursor in the microvillus membrane develop(s) in parallel with SI or earlier.

Sucrase-isomaltase and cystic fibrosis.

The intestinal microvillar enzyme complex sucrase-isomaltase has been studied in cystic fibrosis and control ileum. A number of biochemical parameters of the enzyme in ileum homogenates have been determined. Both solubilized as well as membrane-bound sucrase-isomaltase were analyzed with respect to their reaction with monoclonal antibodies against human sucrase-isomaltase. Finally the subcellular localization of sucrase-isomaltase was verified by immunoelectronmicroscopy or via the analysis of purified brush-border membrane preparations. At all levels no significant differences could be detected between sucrase-isomaltase of cystic fibrosis and control ileum. It is concluded that an abnormal subcellular localization and/or abnormal enzymatic activity of sucrase-isomaltase in cystic fibrosis intestine cannot explain the markedly decreased disaccharidase activities in amniotic fluids from pregnancies resulting in a child affected with cystic fibrosis.

The brush border membrane in hereditary sucrase-isomaltase deficiency: abnormal protein pattern and presence of immunoreactive enzyme.

In a child with hereditary sucrase-isomaltase deficiency immunoreactive enzyme was present in the intact duodenal mucosa. Polyacrylamide gel electrophoresis carried out with membrane fragments of an intestinal biopsy showed an abnormal protein band without enzyme activity. The mucosa had a relatively high residual isomaltase activity which was recovered from the gel in a position suggesting higher than normal molecular weight. The results indicated that in this patient the primary structural defect was in the sucrase moiety which was enzymatically inactive. The isomaltase subunits may have aggregated into a large molecular weight complex because of unavailability of their partners. The observation also provided evidence for separate biosynthesis of the two moieties of the sucrase-isomaltase complex.

Evidence of degradation process of sucrase-isomaltase in jejunum of adult rats.

To evaluate degradation processes of sucrase-isomaltase in adult rat jejunum, we determined enzymic activity of sucrase and isomaltase and compared it with the amount of immunoreactive sucrase-isomaltase. In rats fed or starved for 18h, killed at 10:00 h or 22:00 h, sucrase activity (expressed on the basis of total protein or of immunoreactive sucrase-isomaltase) was significantly (P less than 0.02) lower in the lower jejunum than in the upper jejunum; isomaltase activity was similar in both segments. Crossed immunoelectrophoresis demonstrated the existence of a second sucrase-isomaltase antigen reacting with anti-(sucrase-isomaltase) serum. This antigen was present in larger amounts in the lower jejunum than in the upper jejunum, exhibited immunological partial identity with the intact sucrase-isomaltase, and had isomaltase activity but no sucrase activity. Results suggest that this antigen is a degradation product of sucrase-isomaltase in which the sucrase active site has been broken down. To examine the role of pancreatic enzymes in degradation of sucrase-isomaltase, common pancreatico-biliary ducts were ligated. Within 18 h after the operation, the difference of sucrase activity between the upper and the lower jejunum disappeared and the amount of the second sucrase-isomaltase antigen markedly decreased in the lower jejunum. Our results indicate that, during the degradation of intestinal sucrase-isomaltase by the pancreatic proteinases, degradation of the sucrase active site precedes that of the isomaltase active site.

Sucrase-isomaltase is an adenosine 3',5'-cyclic monophosphate-dependent epithelial chloride channel.

BACKGROUND & AIMS: We previously isolated a monoclonal antibody against a Necturus gallbladder epitope that blocks native adenosine 3',5'-cyclic monophosphate (cAMP)-dependent chloride channels in intestine, gallbladder, urinary bladder, and airway epithelia in various animals. METHODS: Using this antibody, we purified a 200-kilodalton protein that, when reconstituted in lipid bilayers, forms 9-pS chloride channels that are blocked by the antibody. RESULTS: Amino acid sequencing of the purified protein showed strong homology to rabbit sucrase-isomaltase, an abundant intestinal enzyme. Western blot analysis of the in vitro-translated sucrase-isomaltase was indistinguishable from that of the protein used in the lipid bilayer studies. Expression of this protein in Chinese hamster ovary cells and in Xenopus laevis oocytes yielded cAMP-dependent chloride currents that in the latter system were blocked by the antibody. CONCLUSIONS: Because the monoclonal antibody blocks cAMP-dependent currents in epithelia as well as those produced both by the reconstituted and by the heterologously expressed protein, sucrase-isomaltase is a cAMP-dependent epithelial chloride channel. Thus an enzyme that can also function as an ion channel has been described for the first time.