Human blood group A-like determinants as marker of the intracellular pools of glycoproteins in secretory and absorbing of A+ rabbit jejunum

Human blood group A antigenicity of glycoproteins is retained on epon-embedded jejunum sections after glutaraldehyde fixation and osmium treatment. The intracellular location of molecules bearing these determinants was visualized in the four types of epithelial cells of A+ rabbit jejunum sections with immuno-colloidal gold labeling. The brush border membrane and in particular the glycocalyx of absorbing cells as well as the secretory granules of goblet and Paneth cells were heavily labeled. In enteroendocrine cells, the membrane of secretory granules and not their content was lightly labeled. The differential labeling of secretory or membrane bound glycoproteins is accompanied by different labels of the Golgi complex as expected if labeling of the Golgi saccules was due to the presence of glycoproteins in transit. In all cases the label is primarily concentrated in only half the cisternae on the trans side of the Golgi stacks. In absorbing cells, structures have been revealed in the terminal web that could be related to the brush border membrane and consequently implicated in its biogenesis. The fibrillar material of the glycocalyx appears as highly labeled tangled structures which apparently proceed from densely stained "carrier" vesicles arising from the Golgi apparatus. Vesicles fusing at the lower part of microvilli could result of integration of this material into the lightly labeled vesicles strictly found in the terminal web. These last vesicles could also contain newly synthesized brush border hydrolases.     

Physicochemical properties of a human glycoprotein bearing blood group A activity

A human erythrocyte glycoprotein was isolated and purified from blood of group A+1 by a procedure involving chloroform-methanol extraction and affinity chromatography on Helix pomatia lectin-Sepharose 6MB, and some of its physicochemical properties were determined. The resulting preparation was homogeneous as indicated by polyacrylamide gel electrophoresis. The glycoprotein contained nearly 60% carbohydrate and 40% protein. It was water-soluble and inhibited the agglutination of A-erythrocytes. Its molecular weight was 41,900 (amino acid analysis) or 55,200 (light scattering), whereas electrophoresis revealed two bands of 43,000 and 76,000 Da. The ORD spectrum was consistent with 30% alpha-helix, 20% beta-sheet, and 50% random coil. Intrinsic viscosity was 14.61 ml.g-1, partial specific volume was roughly 0.66, isoelectric and isoionic points were 6.90 and 6.95, respectively. The glycoprotein differs from glycophorin and appears to be one of the minor glycoproteins of the human erythrocyte membrane.     

α-Glutamyl-β-naphthylamide hydrolase of rabbit small intestine: Localization in the brush border and separation from other brush border peptidases

About 70% of the total mucosal enzymatic activity hydrolyzing α-l-glutamyl-β-naphthylamide in the rabbit small intestine is present in the brush border; the specific activity in this subcellular fraction is 7 times higher than that of the homogenate. Similar results are obtained for l-leucyl-β-naphthylamide hydrolase.The enzyme activity is efficiently solubilized by papain digestion and is clearly separated from l-leucyl-β-naphthylamide hydrolase by chromatography on concanavalin A-Sepharose. It probably represents a digestive peptidase, different from the other known peptide hydrolases of the digestive surface of the small intestine.     

Simultaneous isolation of brush border and basolateral membrane from rabbit enterocytes. Presence of brush border hydrolases in the basolateral membrane of rabbit enterocytes

By a slight modification of the procedure described by Gratecos et al. (Gratecos, D., Knibiehler, M., Benoit, V. and Sémériva, M. (1978) Biochim. Biophys. Acta 512, 508–524), the basolateral and brush border membranes of rabbit enterocytes have been purified concomitantly from the same aliquot of mucosa. The two types of membrane have been obtained with the same yield (15%) and enrichment of specific markers (18-fold).The presence in the basolateral membrane of hydrolases known to be specific of the brush border membrane has been confirmed by using immunological techniques.     

Alpha-Glutamyl-beta-naphthylamide hydrolase of rabbit small intestine. Localization in the brush border and separation from other brush border peptidases.

About 70% of the total mucosal enzymatic activity hydrolyzing beta-L-glutamyl-beta-naphthylamide in the rabbit small intestine is present in the brush border; the specific activity in this subcellular fraction is 7 times higher than that of the homogenate. Similar results are obtained for L-leucyl beta-naphthylamide hydrolase. The enzyme activity is efficiently solubilized by papain digestion and is clearly separated from L-leucyl-beta-naphthylamide hydrolase by chromatography on concanavalin A-Sepharose. It probably represents a digestive peptidase, different from the other known peptide hydrolases of the digestive surface of the small intestine.     

Specificity of the Na+-dependent monocarboxylic acid transport pathway in rabbit renal brush border membranes

The substrate specificity of a Na+-dependent transport pathway for L-lactate was studied in rabbit renal brush border membrane vesicles. Jmax for L-lactate transport was unaffected by the presence of a fixed concentration of two different short-chain monocarboxylic acids, while the apparent Kt(Ka) for L-lactate increased, and this is compatible with competitive inhibition. The inhibitor constants ("Ki"'s) for the transport pathway for the two solutes examined closely corresponded to the respective "Ki"'s derived from a Dixon plot. A broad range of compounds were then tested as potential inhibitors of L-lactate transport, and the "Ki"'s thereby derived yielded specific information regarding optimal substrate recognition by the carrier. A single carboxyl group is an absolute requirement for recognition, and preference is given to 3 to 6 C chain molecules. Addition of ketone, hydroxyl and, particularly, amine groups at any carbon position, diminishes substrate-carrier interaction. Intramolecular forces, notably the inductive effects of halogens, may play a role in enhancing substrate-carrier interaction; however, no correlation was found between pKa and "Ki" for the substrates examined. We conclude that a separate monocarboxylic acid transport pathway, discrete from either the D-glucose, alpha or beta neutral amino-acid, or dicarboxylic acid carriers, exists in the renal brush border, and this handles a broad range of monocarboxylates.     

The production and characterization of a monoclonal antibody specific for the 94,000 dalton enkephalin-degrading peptidase from rabbit kidney brush border

We have prepared a monoclonal antibody specific for a major 94,000 dalton protein from the brush border membrane of rabbit kidney cortex. The monoclonal antibody was used for the immunoaffinity purification of this protein after solubilization of brush border membranes with octylglucoside. The 94,000 dalton protein is a peptidase capable of cleaving the Gly3-Phe4 bond of methionine-enkephalin. Identification of this peptidase as a previously described 94,000 dalton enkephalinase of kidney cortex was confirmed by its sensitivity to EDTA and inhibitors such as thiorphan and phosphoramidon.     

Interaction of cholera toxin and Escherichia coli heat-labile enterotoxin with glycoconjugates from rabbit intestinal brush border membranes: Relationship with ABH blood group determinants

The capacity of cholera toxin (CT) and type I heat-labile enterotoxin produced by Escherichia coli isolated from human intestine (LTh) to interact with glycoconjugates bearing ABH blood group determinants from rabbit intestinal brush border membranes (BBM) was studied. On the basis of the type of intestinal compounds related to the human ABH blood group antigens, rabbits were classified as AB or H. Toxin binding to the intestinal glycolipids and glycoproteins depends on the blood group determinant borne by the glycoconjugate and on the analyzed toxin. LTh was capable of interacting preferentially with several blood group A- and B-active BBM glycolipids compared to those isolated from animals lacking these antigens (H rabbits). Also, LTh preferably bound to several BBM glycoproteins from AB rabbit intestines compared to those from H ones. One of these glycoproteins, the sucrase-isomaltase complex (EC 3.2.1.48-10) isolated from AB and H rabbits showed the same differential LTh binding. Conversely, CT practically did not recognize either blood group A-, B-, or H-active glycolipids and glycoproteins. These results may be relevant for carrying out in vivo experiments in rabbits in order to disclose the role of ABH active-glycoconjugates in the secretory response induced by LTh in rabbit intestine.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Iron binding proteins and influx of irom across the duodenal brush border: Evidence for specific lactotransferrin receptors in the human intestine

The ability of a range of homologous transferrin-like proteins to donate iron to pieces of human duodenal mucosa, was examined with an in vitro incubation technque. In contrast to serum transferrin and ovotransferrin, only lactotransferrin was able to yield its iron to intestinal tissue, but in an autologous system this protein was unable to donate iron to human reticulocyte preparations. Studies with ¹²⁵I-labelled lactotransferrin and lactotransferrin dual-labelled with ⁵⁹Fe and ¹²⁵I, indicated that the intact protein is excluded from entry into the enterocytes. The experiments suggest that iron may be transported across the brush border after delivery to specific protein binding sites at the cell surface.     

Use of percollTM in the isolation and purification of rabbit small intestinal brush border membranes

(1) Intestinal absorption is altered under a variety of circumstances in health and disease and to determine a possible relationship between intestinal absorptive function and intestinal brush border membrane composition, we undertook the isolation and purification of rabbit jejunal and ileal brush borders, to allow further studies of their lipid composition under varied experimental conditions. (2) A modification of an established method (Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J. and Crane, R.K. (1973) Biochim. Biophys. Acta 323, 98-112) utilized CaCl2 aggregation and sequential centrifugation followed by purification of the brush border pellet (P2) at 27,000 X g on a PercollTM (Pharmacia) self-forming gradient. The PercollTM was removed by ultracentrifugation for 30 min at 100 000 X g, utilizing a batch rotor in the Beckman airfugeTM. (3) Pure brush border membrane vesicles were obtained and characterized by specific marker analysis and electron microscopy. Comparative marker analyses performed on P2 and final PercollTM preparations from animals showed that the purification achieved was 8-11-fold greater when compared to the original homogenates. Verification of purity was also demonstrated by the absence of DNA and very low levels of Beta-gluconridase and (Na+ + K+)-ATPase in the PercollTM preparations. (4) Comparative lipid analyses of P2 and final PercollTM preparations showed that levels of total phospholipid and free fatty acids were several-fold higher in the PercollTM preparations on a per mg protein basis. (5) A comparison of the activity of enzyme markers and the levels of total free fatty acids in P2 pellets obtained after Cacl2 and MgCl2 aggregation showed that CaCl2 aggregation gave the more consistently reproducible results. (6) Although standard procedures of membrane preparations not involving density gradient separation provide membranes of reasonable purity for the estimation of lipid components, we consider the final purification step of density gradient separation using PercollTM is essential for determining small quantitative changes which might occur in the membrane lipid composition under experimental conditions were intestinal absorptive function is altered.     

Studies on the turnover of protein and glycoprotein components in rabbit kidney brush borders

The kinetics of incorporation of [³H]lysine and [¹⁴C]glucosamine into kidney brush borders were studied in vivo. The patterns of incorporation and loss of radioactivity from the brush borders were similar for both radioactively labelled precursors. Maximal labelling occurred 15–20h after injection of the precursors, and then the radioactivity declined rapidly until 50h. The radioactivity of brush borders then remained constant until 120h and thereafter declined slowly. These results are interpreted as indicating that two processes contribute to the turnover of brush-border components, pinocytosis and a slower turnover of the components of the microvilli. Studies of the distribution of radioactivity among glycoprotein components of the brush borders, separated by polyacrylamide-gel electrophoresis, indicated that the membrane components turned over in unison.     

Plasmodium vivax interaction with the human Duffy blood group glycoprotein: identification of a parasite receptor-like protein

The interaction between merozoites of the human pathogen, Plasmodium vivax, and the Duffy blood group glycoprotein on the surface of human erythrocytes is essential for the invasion of erythrocytes and the survival of the parasite. We have identified a P. vivax protein of 135 to 140 kDa which binds with receptor-like specificity to the human Duffy blood group glycoprotein. This interaction can be specifically inhibited by purified Duffy glycoprotein and by pretreating erythrocytes with a monoclonal antibody directed against a novel Duffy determinant. A protein with similar specificity for the Duffy glycoprotein from the phylogenetically related simian malaria, P. knowlesi, is shown to be immunologically related by the generation of cross-reactive antibodies. Despite their shared properties, these two Duffy associating proteins from P. vivax and P. knowlesi differ in some aspects of their interaction with the Duffy glycoprotein. The identification of these proteins will help elucidate the molecular mechanisms of erythrocyte invasion by Plasmodium.     

Characterization of monoclonal antibodies specific for the Lewis a human blood group determinant

Four hybridoma cell lines were derived from the spleen cells of mice immunized with the neutral glycolipids of human meconium. The antibodies secreted by these lines were specific for the Lewis a antigen of the human Lewis blood group system as determined by solid phase immunoassay using synthetic carbohydrate antigens and by plate binding assay and thin layer chromatography-autoradiography using natural glycolipid antigens. Coating protein A-bearing Staphylococcus aureus with one of the antibodies yielded a stable reagent that produced rapid agglutination of Lewis a positive human erythrocytes. The fine structural specificity of these antibodies was assessed by competition radioimmunoassay using synthetic structural analogs of Lewis a conjugated to bovine serum albumin. One antibody was specific for the Lewis a trisaccharide (Gal beta 1 leads to 3(Fuc alpha 1 leads to 4) beta GlcNAc), while a second recognized the entire Lea (1 leads to 3) beta Gal tetrasaccharide. The third and fourth were directed at topography largely provided by only the alpha Fuc and beta GlcNAc units. These monoclonal antibodies not only represent potentially useful reagents for detecting the Lewis a antigen but also provide a system for studying precise relationships between anticarbohydrate antibody structure and binding specificity.     

Improving bioreactor production of a recombinant glycoprotein in Escherichia coli: Effect of specific growth rate on protein glycosylation and specific productivity

In the last decades bacterial glycoengineering emerged as a new field as the result of the ability to transfer the Campylobacter jejuni N- glycosylation machinery into Escherichia coli for the production of recombinant glycoproteins that can be used as antigens for diagnosis, vaccines, and therapeutics. However, the identification of critical parameters implicated in the production process and its optimization to jump to a productive scale is still required. In this study, we developed a dual expression glycosylation vector for the production of the recombinant glycoprotein AcrA-O157, a novel antigen that allows the serodiagnosis of the infection with enterohemorrhagic E. coli O157 in humans. Volumetric productivity was studied in different culture media and found that 2xYP had 6.9-fold higher productivity than the extensively used LB. Subsequently, bioreactor batch and exponential-fed-batch cultures were designed to determine the influence of the specific growth rate (μ) on AcrA-O157 glycosylation efficiency, production kinetics, and specific productivity. At μ_max, AcrA glycosylation with O157-polysaccharide and the specific synthesis rate were maximal, constituting the optimal physiological condition for AcrA-O157 production. Our findings should be considered for the design, optimization, and scaling up of AcrA-O157 production and other recombinant glycoproteins attractive for industrial applications.     

Kinetic properties of the fructose influx across the brush border of the rat jejunum. Effects of a diet rich in fructose]

The unidirectional influx (i.e. initial rate of uptake) of D-fructose across the brush border of rat jejunum is a saturable function of concentration, with a Kt of 125 mM, which implicates a carrier mechanism. This mechanism appears to be very specific for fructose in view of the lack of influx inhibition observed in the presence of large concentrations of the sugars or polyols, D-glucose, D-galactose, D-mannose, D-xylose, L-sorbose, D-tagatose, sorbitol or mannitol. D-Fructose uptake is inhibited by incubation, preceded by a 30-min preincubation in the same inhibitory conditions, in the absence of Na, or in the presence of metabolic poisons, NaF, 2,4-dinitrophenol, monoiodoacetate. Phloridzin (10-3 M), with or without preincubation, has no effect on uptake. D-Fructose influx is stimulated by fructose feeding, mainly because the augmentation of the number of active sites of transfer: Jmax is increased two-fold, Kt is more weakly affected.