Succinylated Wheat Germ Agglutinin Research Articles

Comparison of brush border membrane glycoproteins and glycoenzymes in the proximal and distal rat small intestine

Brush border membranes isolated from the proximal and distal portions of the rat small intestine were examined to see whether qualitative differences exist in their glycoprotein constituents. After SDS-polyacrylamide gel electrophoresis distinct differences were observed, indicating that the protein and glycoprotein profiles of the distal intestine are less complex. A competitive radioassay of lectin receptors revealed that there are significantly more wheat germ agglutinin and succinylated wheat germ agglutinin receptors present on brush border membranes from proximal intestine as compared to distal intestine. However, binding of Ricinus communis agglutinin I to brush border membranes of distal intestine was 2-times higher than that of proximal intestine. These segmental differences were also reflected in the binding patterns of individual brush border membrane hydrolases to wheat germ agglutinin and R. communis agglutinin I. Carbohydrate analysis demonstrated that the overall sugar content of brush border membranes is higher in distal intestine, with more galactose and sialic acid residues. No difference was found in the content of N-acetylglucosamine between the two segments. When brush border membranes from both segments were used as acceptors for galactosyltransferase, those from proximal intestine were better acceptors. Neuraminidase treatment significantly enhanced galactose oxidase/sodium borotritide labeling of brush border membranes from distal intestine and altered the electrophoretic mobility of dipeptidyl aminopeptidase IV and aminopeptidase N. No significant changes in labeling or enzyme electrophoretic mobility were noted in brush border membranes from proximal intestine after neuraminidase treatment. These studies indicate that the glycoproteins from brush border membranes of proximal and distal intestine are qualitatively different and that the glycoproteins from distal intestine may have more completed oligosaccharide side chains.

Cell surface of mouse blastocysts at the trophectoderm-uterine interface during the adhesive stage of implantation.

The molecular basis for the acquisition of adhesiveness between blastocysts and uterine luminal epithelium is an interesting problem in reproductive biology. It is rather difficult to study implantation-stage blastocysts of mice because during the implantation period each blastocyst becomes lodged within a crypt formed by decidualizing stroma. After trophectoderm adheres to uterine luminal epithelium, it is not possible to flush intact blastocysts from the uterus by standard recovery procedures. By identifying implantation sites with the Evans blue technique and splitting or gently separating the apposed epithelium of finely trimmed sites, it was possible to expose nonadhesive and adhesive trophectoderm to polycationized ferritin (PCF) and a series of ferritin-conjugated lectins. Examination by transmission electron microscopy revealed that both adhesive and nonadhesive trophectoderm bound PCF, concanavalin A, wheat-germ agglutinin, Ricinus communis agglutinin I, and Limulus polyhemus agglutinin, but not Dolicos biflorus agglutinin or peanut agglutinin. Nonadhesive trophectoderm bound succinylated wheat germ agglutinin but adhesive trophectoderm did not. There was no apparent difference in the relative amounts of each lectin bound to adhering and nonadhering cells.

Purification and characterization of two glycoproteins from oligodendroglial plasma membranes

Two major glycoproteins of 99 kDa and 77 kDa have been purified from oligodendroglial plasma membranes. These two glycoproteins exhibit intense binding to the lectin, wheat germ agglutinin. The 99-kDa and 77-kDa glycoproteins were purified by Sephadex LH-60 chromatography, wheat germ agglutinin affinity chromatography and SDS-polyacrylamide pore gradient gel electrophoresis. Re-electrophoresis of excised gel slices containing the two glycoproteins demonstrated their apparent homogeneity. The isoelectric points of the 99-kDa and 77-kDa glycoproteins were 6.15 and 6.00, respectively. Peptide mapping revealed structural differences between the two glycoproteins. Lectin binding studies with radiolabeled succinylated wheat germ agglutinin demonstrated that the binding of the 99-kDa and 77-kDa glycoproteins to wheat germ agglutinin was due to N- acetyl- d-glucosamine residues in the oligosaccharide side-chains.

Stage-specific alterations in the apical membrane glycoproteins of endometrial epithelial cells related to implantation in rabbits.

The rabbit endometrial epithelium undergoes differentiation prior to the time of blastocyst implantation, including loss of surface negativity and a change in glycocalyx morphology. Nonpregnant (estrous) and pseudopregnant rabbits were used to study specific alterations in proteins and saccharide composition of the luminal epithelial membrane and its glycocalyx related to the acquisition of receptivity to implantation. Pregnant animals were used to study further modification of the luminal surface by implanting blastocysts. The apical surface of luminal epithelial cells was solubilized by a 15-min intraluminal incubation of 1% Triton X-100 containing protease inhibitors. Proteins in extract solutions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Three new polypeptides (24 kDa, 42 kDa and 58 kDa) were identified in uteri from receptive rabbits. Binding of succinyl Wheat Germ Agglutinin (sWGA) and Ricinus communis Agglutinin (RCA-I) lectins to the 24 kDa and 42 kDa components on Western blots of extracts separated by SDS-PAGE identified them as glycoproteins. Additionally, other polypeptides (26 kDa, 80-86 kDa and 145 kDa) showed changes in affinity for WGA, RCA-I or concanavalin A (Con A), depending on the hormonal state. Correlating with these findings was an increased binding of these lectins to intact nonciliated cells in uteri of receptive rabbits compared to estrous animals; ciliated cells bound Dolichos biflorus Agglutinin (DBA) specifically, regardless of the hormonal condition. Treatment of uteri from estrous animals, or Western blots of proteins from these animals, with neuraminidase prior to lectin exposure suggested the presence of glycoproteins having a sialic acid-D-galactose terminus in nonreceptive rabbits. Reduced binding of lectin to intact cells at implantation sites and to blots of proteins isolated from these sites, compared to nonimplantation sites, was noted. These results provide evidence for stage-specific alterations in protein and saccharide composition of the apical surface of endometrial epithelium prior to implantation, and indicate that implanting blastocysts further modify the luminal surface.

Identification of chitin as a structural component of Giardia cysts.

The intestinal parasite Giardia lamblia is a significant cause of diarrheal disease, which is perpetuated by the infective cyst form of the parasite. Although a rational approach to the control of giardiasis would be to inhibit cyst formation, nothing is known of the chemical composition of the cyst wall or of its biosynthesis. In these studies, we have shown that chitin is a major structural component of G. lamblia and G. muris cyst walls. This conclusion is based on the finding that chitinase specifically destroys the cyst wall, as revealed by electron microscopy. The presence of chitin was also shown directly by lectin binding studies. Of 12 lectins with diverse carbohydrate recognition specificity, only the N-acetylglucosamine-specific lectins wheat germ agglutinin, succinylated wheat germ agglutinin, and tomato lectin bound to cyst walls, as shown by fluorescence microscopy and cytochemistry. Wheat germ agglutinin binding was completely abolished by treatment of the cysts with purified chitinase. This effect was specific since it could be prevented by incubating the enzyme with chitin before treatment of the cysts. Treatment of cysts with N-acetyl-beta-glucosaminidase partially inhibited wheat germ agglutinin binding, whereas other glycosidases and proteases had no effect. These findings indicate that chitin is a major structural component of Giardia cyst walls and raise the possibility that inhibitors of chitin synthesis may be of use in preventing encystation and thus controlling spread of the disease.

Soluble chondrogenic factors in bone matrix: [formula omitted] and [formula omitted] Dept. of Biology, Case Western Reserve Univ., Cleveland, OH 44106

We examined the effects of NGF on the TGC (trophoblastic giant cell) transformation of the ectoplacental cone (EPC) cells derived from the mouse pregnant uteri on day 7.5 p.c. The addition of NGF strongly accelerated the rate of the TGC transformation of EPC cells in a dose-dependent manner. The promoting action of NGF was abolished by the addition of anti-NGF MAb. Histochemical staining with succinyl wheat germ agglutinin (s-WGA), anin situmarker for the TGCs on day 10.5 p.c., revealed that NGF induced the expression of s-WGA-binding glycoproteins in TGCsin vitro.RNA analysis revealed that NGF mRNA was expressed in the pregnant uterus on day 7.5 p.c., mainly in the decidua, but it could not be detected in the EPC. p75NGFRmRNA was expressed in the EPCs, whereas TrkA mRNA was not detected in the placental tissues throughout day 7.5 to 10.5 p.c. We therefore conclude that maternally derived NGF may play a role in mouse placentation by promoting the giant-cell transformation of trophoblast cells through p75NGFR.

Response of C3H/10T1/2 fibroblasts to an external steady electric field stimulation: Reorientation, shape change, conA receptor and intramembranous particle distribution and cytoskeleton reorganization

C3H/10T1/2 mouse embryo fibroblasts were stimulated by a steady electric field ranging up to 15 V/cm. The percentage of spindle-shaped cells increased with the field strength and duration of the stimulation. These cells oriented preferentially with their long axis perpendicular to the field direction. A small percentage of the cells were found to move slightly toward the cathode during the course of electric stimulation. Although no apparent field-induced redistribution of fluorescent-labelled concanavalin A (conA) receptor along the cell periphery was observed, the bright perinuclear area appeared preferentially on the anode side. Correlative fluorescence and scanning electron microscopy (SEM) revealed no difference in the density of conA-gold microsphere labels on either side of the cell. The density of intramembranous particles on the E-face of the plasma membrane was 54% higher on the anode side than on the cathode side of the cell. The microfilament bundles were observed to be disrupted after 30 min of 10 V/cm stimulation by rhodamine phalloidin labelling of F-actin. The cell sensitivity to electric field-induced reorientation and cell shape changes was reduced by pretreatment with conA, and to a lesser extent, with succinyl conA or wheat germ agglutinin (WGA). ConA pretreatment alone also reduced the prominence of microfilament bundles. However, post-field lectin binding to the cell has no effect on cell recovery. It is possible that the generally flat 10T1/2 cells retract and realign in order to minimize the disruption of their membrane potential. The conA binding-mediated receptor-cytoskeletal linkage temporarily immobilizes the cell and inhibits subsequent field-induced shape changes.

Subsets of Epidermal Langerhans Cells as Defined by Lectin Binding Profiles

In this study we characterize the cell surface glycoconjugate moieties of strain 2 guinea pig epidermal Langerhans cells (LC) in single cell suspension by using a battery of 17 fluorescent lectins. All LC displayed binding sites for concanavalin A, succinylated concanavalin A, Lens culinaris agglutinin, Pisum sativum agglutinin, wheat germ agglutinin, succinylated wheat germ agglutinin, Griffonia simplicifolia agglutinin I, Ricinus communis agglutinin I, Phaseolus vulgaris E agglutinin, and Phaseolus vulgaris L agglutinin, but failed to bind Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA), and Ulex europaeus agglutinin I (UEA I). Neuraminidase pretreatment rendered LC reactive for SJA, but not for DBA and UEA I. The binding profiles of certain lectins point to the existence of LC subpopulations in that Griffonia simplicifolia I-B4 isolectin, peanut agglutinin (PNA), Helix pomatia agglutinin, and soybean agglutinin bound to only 80% (range 70-90%) of Ia-positive epidermal cells; binding sites for these lectins on primarily unreactive Ia-positive cells were unmasked when epidermal cells were treated with neuraminidase prior to lectin labeling. Ultrastructural PNA labeling studies revealed that the vast majority of Birbeck granule-containing LC displayed PNA binding sites, whereas indeterminate cells were consistently PNA-negative. Identification of carbohydrate configurations expressed on LC surfaces by lectin binding may provide a clue for the elucidation of the mechanisms of established LC functions and possibly the discovery of as yet unknown properties of this cell type.

Identical Lectin Binding Patterns of Human Melanocytes and Melanoma Cells In Vitro

Cell surface glycoconjugate patterns of human epidermal cells and of melanoma cells (MC) in primary culture derived from 11 primary and metastatic melanomas were investigated using fluorescent and horseradish peroxidase conjugated lectins for visualization at the light and electron microscopic level. The lectin labeling profiles of human melanocytes (M) and MC were found to be identical. According to their binding patterns, the lectins tested were grouped into three categories: (1) lectins binding to both keratinocytes (K) and M/MC, irrespective of neuraminidase pretreatment (concanavalin-A, wheatgerm agglutinin, succinylated wheatgerm agglutinin); (2) lectins binding to K but not to M/MC, irrespective of neuraminidase pretreatment (Ulex europaeus agglutinin I); (3) lectins binding to K, but to M/MC only after neuraminidase pretreatment (soybean, Helix pomatia, and peanut agglutinins). Untreated M were reactive for soybean and peanut agglutinins only at contact sites with K. Since the lectins from soybean, Helix, and peanut bind specifically to D-galactose and N-acetyl-D-galactosamine residues, we conclude that these particular glycoconjugates are normally masked by sialic acid on M/MC surfaces and can be unmasked by neuraminidase. These features, which have been previously observed in guinea pig M, appear to be interspecies surface markers of melanocytic cells which remain unaltered in the course of malignant transformation.

Perturbations of membrane glycosylation in retinoid-treated epidermis.

Differentiation of keratinizing epithelial results in the formation of an exterior cornified layer that is both sufficiently cohesive to provide a protective barrier and dyshesive enough to permit orderly, apical desquamation. Membrane glycoconjugates, which are of presumed importance for intercellular adhesion and growth control, have recently been visualized in mouse, rat, and human epidermis. Because retinoids are known to profoundly influence epidermal differentiation, the lectin-staining pattern in normal mouse epidermis was compared to that observed in mice treated with etretinate (Ro 10-9359, 50 mg/kg/day). Within 2 days of treatment with 50 mg/kg/day of etretinate, abnormal transepidermal water loss began, followed by mild acanthosis after 4 to 6 days, and focal loss of stratification by 10 days. Despite these dramatic alterations in structure and function, abnormalities in lectin staining first appeared only after 15 days. Both wheat germ agglutinin and succinyl wheat germ agglutinin stained an etretinate-dependent product localized in the granular layer in samples treated for 21 to 38 days. Both the intensity and the distribution of PNA (galactose-N-acetyl-galactosamine) and Ulex ( alpha -L-fucose) staining were affected by retinoid treatment, but the staining pattern with other lectins was not altered until 40 to 45 days, at which time the sugars in some samples were either restricted to isolated nests of cells or greatly diminished. Although etretinate appeared to provoke selective changes in keratinocyte membrane glycoconjugates, these alterations became apparent only after the retinoid had profoundly perturbed epidermal differentiation. These altered patterns may not only reflect retinoid-induced aberrations in cohesion and barrier function, but also may provide clues about the role of certain membrane sugars in the control of normal epidermal differentiation.

Succinylated and acetylated concanavalin A activate the classical complement pathway

Succinylated and acetylated concanavalin A, but not the native lectin, lyse sheep erythrocytes in the presence of guinea pig complement. The effect appears to be specific since succinylated wheat germ agglutinin is inactive and hemolysis is inhibited selectively by α-D-methylglucopyranoside. Hemolytic activity is enhanced by preincubation of succinylated lectin-sensitized cells with complement at 4° and correlates with the number of molecules of the first component of complement bound to cells that were treated with succinylated concanavalin A and then washed.

Cell surface receptors for wheat germ agglutinin and limulin in baby hamster kidney cells and ricin resistant variants

The cell surface glycoconjugates of Baby Hamster Kidney cells and of four ricin resistant variants were investigated by the use of 125I-substituted ricin (Ricinus communis toxin) which binds galactose residues, and by the use of fluorescein labelled lectins which bind N-acetylneuraminic acid and/or N-acetylglucosamine: Limulin (Limulus polyphemus agglutinin), wheat germ agglutinin (Triticum vulgare agglutinin) and succinylated wheat germ agglutinin. Striking differences in the number of lectin and/or ricin receptors were found between the cell surface of wild type cells and that of ricin resistant variants. The results are discussed on the basis of the main glycopeptide structure, and of the specificity of the sugar binding proteins used. The ricin resistance of variant cells is concomitant to modifications of the concentration of certain glycoconjugate structures which are accessible to the sugar binding proteins. Depending on the variants, N-asparaginyl glycopeptide types and/or O-glycosidic glycopeptide types are affected.

Quantitative Fluorimetric Determination of Cell-Surface Glycoconjugates with Fluorescein-Substituted Lectins

Fluorescein-substituted lectins, which can be used to visualize cell surface glycoconjugates, are shown to be usable in the quantitative determination of the number of receptor sites and of their association constant. The fluorescence measurements of the fluorescein-substituted lectins released from the cell surface with the related inhibitor, give quantitative data in a large range of fluorescein-substituted lectin concentration (0.1 to 100 microgram/ml). Using fluorescein-substituted concanavalin A or [3H]acetyl-concanavalin A, it was found that baby hamster kidney cells (BHK 21, wild-type) bind 10 +/- 2 x 10(6) lectin molecules per cell with an apparent association constant of 1.8 or 1.7 x 10(6) 1 x mol-1, respectively. Using the fluoresceinyl and [3H]acetyl-substituted wheat germ agglutinin, we found 40 +/- 5 x 10(6) sites per cell with an apparent binding constant of 1 and 1.3 +/- 0.3 x 10(6) 1 x mol-1, respectively. When fluorescein-substituted succinyl wheat germ agglutinin was used instead of the unsuccinylated wheat germ agglutinin derivatives, the number of binding sites was reduced 7 times, while the binding constant was very slightly lowered. Concanavalin A derivatives gave monotonic Scatchard plots; on the opposite, wheat germ agglutinin derivatives gave biphasic Scatchard plots suggesting that wheat germ agglutinin binds to two classes of receptors.