Intestinal Brush Border Membrane Proteins Research Articles

Impact of glycosylation and detergent-resistant membranes on the function of intestinal sucrase-isomaltase

Sucrase-isomaltase (SI) is a highly N- and O-glycosylated intestinal brush border membrane protein. SI is sorted with high fidelity to the apical membrane via O-linked glycans that mediate its association with lipid rafts or detergent-resistant membranes (DRMs). Here, we show that N- and O-glycosylation and DRMs are implicated in the regulation of the function of SI in intestinal Caco-2 cells. The activities of sucrase (SUC) and isomaltase (IM) increase substantially in DRMs when N- and O-glycosylation patterns are intact. Disruption of DRMs by solubilization with Triton X-100 at 37 degrees C substantially reduces the activities of SUC and IM. Furthermore, modulation of O-glycosylation by benzyl-2-acetamido-2-deoxy-alpha-d-galactopyranoside and N-glycosylation by deoxymannojirimycin is linked to a decreased capacity of SI to associate with DRMs, with a subsequent reduction of the enzymatic activities of SUC and IM. This is the first report that reveals a direct role of N- and O-glycans in association with DRMs in regulating the function of a membrane glycoprotein.

Binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins.

Transmissible gastroenteritis coronavirus (TGEV) is a porcine pathogen causing enteric infections that are lethal for suckling piglets. The enterotropism of TGEV is connected with the sialic acid binding activity of the viral surface protein S. Here we show that, among porcine intestinal brush border membrane proteins, TGEV recognizes a mucin-type glycoprotein designated MGP in a sialic acid-dependent fashion. Virus binding assays with cryosections of the small intestine from a suckling piglet revealed the binding of TGEV to mucin-producing goblet cells. A nonenteropathogenic mutant virus that lacked a sialic acid binding activity was unable to bind to MGP and to attach to goblet cells. Our results suggest a role of MGP in the enteropathogenicity of TGEV.

The effect of exogenously administered recombinant bovine somatotropin on intestinal phytase activity andin vivophytate hydrolysis in hybrid striped bassMorone chrysops × M. saxatilis

It has been recently demonstrated that hybrid striped bass Morone chrysops × M. saxatilis exhibit intestinal phytase activity, an enzyme capable of liberating inorganic orthophosphate from the phytate molecule, thereby making phytate phosphorus (PP) available for absorption and utilization by the fish. However, it was also determined that hybrid bass were able to digest only 1–2% of dietary PP. Therefore, an experiment was conducted to determine if exogenously administered recombinant bovine somatotropin (rbST) would increase intestinal phytase activity in hybrid striped bass to a level that would improve PP availability. The results indicate that exogenous rbST treatment, administered either through intraperitoneal injection or orally, does increase intestinal brush border membrane protein concentration. While injection with rbST led to significant increases in both specific brush border membrane and total intestinal phytase activity, oral rbST administration did not cause similar increases. Total P and PP digestibility were not influenced by rbST administration. While treatment with rbST does have the potential to enhance intestinal phytase activity in hybrid striped bass, it does not appear to increase PP digestibility. This observation limits the hormone's utility as an effective technique for improving PP bioavailability to this species.

Hibernation Induces Expression of Moesin in Intestinal Epithelial Cells

Identification of proteins that are differentially expressed in mammals that hibernate can provide insight into mechanisms that preserve cellular function at low temperatures. A candidate protein was identified in intestinal brush border membranes of 13-lined ground squirrels. Intestinal brush border membrane proteins were separated using SDS–PAGE and gels were stained with Coomassie blue. We observed a ∼75-kDa band that was specifically increased in brush border membranes isolated from torpid squirrels compared with summer active squirrels. The 75-kDa band was cut from one-dimensional gels and sequenced. A 17 amino acid sequence was identified of which amino acids 2–17 matched exactly a portion of moesin, a membrane–cytoskeletal linking protein and member of the ERM (ezrin/radixin/moesin) family. The sequence results were confirmed using anti-moesin antibodies that detected strong bands at ∼75 kDa on Western blots of brush border membranes in torpid squirrels (Tb∼7°C) and only faint signals in summer squirrels (Tb∼37°C) or aroused hibernators (Tb∼37°C). In contrast, signals obtained using anti-ezrin antibodies were uniformly strong in all squirrels, regardless of activity state. Intestinal brush borders of mice and rats expressed ezrin but not moesin. These results provide evidence for the physiological induction of an ERM protein in intestinal epithelial cells of torpid hibernators and support the idea that hibernation involves differential expression of gene products that may facilitate viability of cells at low temperatures.

Diet Influences Development of the Pig (Sus scrofa) Intestine during the First 6 Hours after Birth

Structural and functional responses of the intestine to colostrum, milk replacer, oral electrolyte solution and food deprivation were examined during the first 6 h after birth in pigs. Total intestinal weight, surface area and mucosal mass were highest (P < 0.05) in pigs fed colostrum. The other diet groups did not differ, except that food-deprived pigs had lower surface area than the other groups. Feeding colostrum was associated with higher mucosal protein content (P < 0.05). Total intestinal brush border membrane protein content of pigs fed milk replacer, oral electrolyte solution and food-deprived pigs were 61, 44 and 56%, respectively, of those fed colostrum (P < 0.05). Pigs fed colostrum had higher total mucosal maltase activities than those that were food deprived, and total brush border membrane activities were higher than in those fed oral electrolyte solution. Total intestinal brush border membrane aminooligopeptidase activity was higher in pigs fed colostrum than in those given oral electrolyte solution or deprived of food, but total intestinal homogenate activities did not differ among groups. Diet influenced lactase activity only in the mid-region, and sucrase was not responsive to diet. Intestinal glucose transport capacity by intact intestinal tissues did not differ among diet groups. The ability of brush border membrane vesicles to actively accumulate glucose was lost when pigs were fed colostrum and milk replacer, but not when fed oral electrolyte solution or deprived of food. Our findings reveal how diet during the first 6 h after birth influences the structure and functional characteristics of the intestine. The responses vary between brush border membrane proteins and intestinal regions, and appear to differ from those described for older animals.

Hormonal effects on the sulfhydryl groups associated with intestinal brush border membrane proteins

Previous studies demonstrated that the administration of 1,25-dihydroxycholecalciferol (1,25(OH) 2D 3) to cholecalciferol-deficient chicks rapidly increases the reactivity and amount of the sulfhydryl (HS −) groups in intestinal brush border membranes (BBM). In the present study, the tissue and hormonal specificity of this effect was investigated. The HS − groups of intestinal and renal BBM were enhanced by vitamin D-3 and / or 1,25(OH) 2D 3, but no change was noted in isolated intestinal mitochondria and purified intestinal basolateral membranes, cardiac sarcolemma and erythrocyte membranes. Other steroid hormones including estradiol, testosterone, aldosterone, cortisol, dexamethasone and progesterone, yielded a response similar to 1,25(OH) 2D 3 on BBM HS − groups. Triiodothyronine and retinoic acid also resulted in an increase in intestinal BBM HS − groups. In a kinetic approach, using a specific sulfhydryl fluorescent probe ( N-7-dimethylamino-4-coumarin-3-yl-maleimide, DACM), the reactivity of the BBM HS − groups was increased by estrogen and testosterone, as was previously shown for 1,25(OH) 2D 3. Intestinal BBM proteins, labeled with DACM, were separated by gel electrophoresis. Fluorescence scans of the gel showed two heavily labeled bands, one of 110 kDa, putatively brush border myosin I, and one of 43 kDa, putatively actin. Labeling of the 110 kDa protein was increased by 1,25(OH) 2D 3 and estradiol. Further studies are required to elucidate the physiological meaning of these hormone-mediated increases in reactivity and amount of the BBM sulfhydryl groups, as well as the nature of the intermediate biochemical reactions involved in this response.

Use of monoclonal antibodies in the study of intestinal structure and function.

The hybridoma technique, originally developed by G. Kohler & C. Milstein, is a powerful new experimental approach for analysis of complex biological systems, and is particularly suited for identification and study of surface-membrane antigens. This technique has been used for the production of monoclonal antibodies to intestinal brush border membrane proteins. Spleen cells, obtained from BALB/c mice immunized with purified brush border membranes, were fused with NSI mouse myeloma cells, and hybrids were selected with a culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Hybridoma cultures were screened for production of specific antibodies by radio-immunobinding assays and by immunofluorescent staining of intestinal frozen sections. Selected hybridoma cultures were cloned twice and used for the production of large amounts of antibodies, which were characterized. Nineteen monoclonal antibodies have been prepared to date, about half of them specifically staining the brush border membrane of mature enterocytes. Ten of the antibodies specifically immunoprecipitate surface-membrane proteins, which were analysed by sodium dodecyl sulphate slab-gel electrophoresis, by two-dimensional slab-gel electrophoresis, and by specific enzyme assays. Two antibodies were found to be specific for sucrase-isomaltase, one for an aminopeptidase, two for an isoenzyme of alkaline phosphatase that is present exclusively in the proximal small intestine, and one for maltase-glucoamylase. These monoclonal antibodies, and others prepared by similar techniques from mice immunized with a wide variety of intestinal subcellular fractions, should prove invaluable tools for the study of the biosynthesis of cell-surface proteins, the fetal and postnatal development of specific intestinal functions, and the process of cell differentiation in the intestinal epithelium.

Turnover of mouse intestinal brush border membrane proteins and enzymes in organ culture. A direct evaluation from studies on the evolution of enzyme activities during the culture.

The turnover of mouse intestinal brush border membrane enzymes has been studied by kinetic analysis of the evolution of enzyme activities during organ culture. By comparing the results obtained in these studies with the predictions from a mathematical model of enzyme synthesis and degradation in organ cultures, it has been possible to reach the following conclusions: (1) There is no degradation of brush border membrane enzymes during culture and the rate of synthesis of each enzyme is directly measurable from the kinetics of total enzyme accumulation (tissue + media). (2) Brush border membrane enzymes are released in culture media by two complementary processes. The first one involves a differential solubilization of enzymes but its exact nature cannot be exactly stated. The second one involves a microvesiculation of brush border membranes, the importance of which in vivo is seen in the possible conciliation between urinary membrane synthesis and heterogeneous turnover of membrane components.

Effect of massive proximal small bowel resection on intestinal brush border membrane proteins in the dog.

Dog enterocyte brush border proteins have been studied after a 75% proximal resection of the small bowel. This study was carried on microvillar membrane preparations purified from ileal mucosa sampled before and after regeneration on neighbouring intestinal segments, each animal acting as its own control. After six weeks of regeneration a statistically significant decrease of the following enzyme specific activities was observed: lactase, cellobiase, maltase, sucrase, palatinase, dextranase, trehalase, alkaline phosphatase, aminopeptidase and gamma-glutamyl transferase. Analysis of brush border proteins by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate have shown after regeneration a decreased rate for the proteins with a molecular weight higher than 100,000 daltons. Modifications of electrophoretic patterns seem to be related to the specific activity decreases observed for brush border enzymes after regeneration, since the molecular weight of these enzymes were found between 116,000 and 285,000 daltons, after gel filtration.

Development of intestinal brush border membrane proteins in the rat

1. 1. The proteins of the intestinal microvillus membrane have been studied during post-natal development in the rat (days 12–37). 2. 2. In suckling animals (up to age 20 days), the majority of alkaline phosphatase, glucoamylase and lactase activities in the distal half of the intestine were located in the supernatant fraction ( 100 000 × g, 60 min ). These enzymes were attached to the membrane from the proximal intestine at all ages. 3. 3. Alkaline phosphatase, maltase and lactase activities in the supernatant fractions chromatographed in Sephadex G-200 in positions similar to the corresponding membrane enzyme. Corresponding activities for lysosomal counterparts of maltase and lactase present in the supernatant fraction chromatographed differently. Moreover, pH optimum of the soluble enzymes was 9.2 for phosphatase and 5.5–6.0 for glycoamylase and lactase. The soluble lactase and alkaline phosphatase were inhibited minimally by p- chloromercuribenzoate , and sodium fluoride respectively. L-Phenylalanine (20 mM) did inhibit the soluble phosphatase by 90%. Thus, the soluble enzymes are not mainly of lysosomal origin, but have characteristics of membrane-bound enzymes. 4. 4. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed 18 protein bands which were present in adult membranes. Two other proteins were unique for membranes of distal intestine in suckling rats. The proteins corresponding to known enzyme activity changed as expected with age (e.g. sucrase, maltase increased, lactase decreased). Most of the other proteins were also altered in amount during development. Thus, the changes in the microvillus membrane during development in the rat are not limited to specific enzymes.

Stimulation of Proteolytic Digestion by Intestinal Goblet Cell Mucus

Intestinal goblet cell mucus (GCM) was added to incubations of casein and trypsin (or chymotrypsin) to discover whether mucus could inhibit proteolysis. Contrary to expectation, GCM stimulated casein hydrolysis, reaching a maximum effect at a GCM to casein ratio (w/w) of 0.083. GCM did not contain proteolytic enzymes or proenzymes as contaminants, nor did GCM serve as a substrate for trypsin. Stimulation was not reduced by removing 85% of the sialic acid from GCM. Harsh physical treatment (boiling and freezing) of casein decreased (50%) the GCM effect, as did partial predigestion of casein by trypsin, and elevation of trypsin concentration beyond 3 mug per ml. Thus the undegraded structure of casein appeared to be important for the stimulation of proteolysis by GCM. GCM also enhanced the hydrolysis by trypsin of intestinal brush border membrane protein, but had no effect on the hydrolysis of hemoglobin, albumin, or benzoyl arginine ethyl ester. These results suggest that GCM reacts with specific substrates, in a fashion which promotes their digestion by trypsin or chymotrypsin.