The surface glycoproteins of human skin fibroblasts detected after electrophoresis by the binding of peanut (Arachis hypogaea) agglutinin and Ricinus communis (castor-bean) agglutinin I.

A new methodology was developed to study the cell-surface glycoproteins of cultured human skin fibroblasts. This was based on the binding of a variety of biotinyl-lectins to nitrocellulose electrophoretic transfers of total fibroblast lysates after separation in sodium dodecyl sulphate/polyacrylamide gels, followed by reaction with avidin-biotinyl-peroxidase complexes and detection with 3,3'-diaminobenzidine. The technique proved to be very sensitive and a large number of glycoproteins were detected by binding of concanavalin A and wheat-germ agglutinin. Binding of peanut agglutinin and to a lesser extent of Ricinus communis agglutinin I were found to be dependent on prior removal of sialic acid residues from the glycoproteins. Since by treatment of intact viable cells with neuraminidase only external sialic acid residues were removed, peanut agglutinin and Ricinus communis agglutinin I could thus be utilized for selective detection of cell-surface glycoproteins. Also, because peanut agglutinin was known to bind preferentially to oligosaccharides of the O-glycosidic type, and Ricinus communis agglutinin I to those of the N-glycosidic type, the two lectins were complementary in displaying the surface glycoproteins and in providing information about their oligosaccharide composition.     

Role of sialic acid in bovine sperm-zona pellucida binding

Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.     

Modification of blood-borne arrest properties of lymphoma cells by inhibitors of protein glycosylation suggests the existence of endogenous lectins

The requirement for intact carbohydrates of glycoproteins at the cell surface was investigated after treatment of lymphoma cells with compounds which interfere at different steps in N-linked glycosylation: swainsonine and 1-deoxynojirimycin act at different levels during the processing, so that complex oligosaccharides cannot be formed; 2-deoxyglucose, beta-hydroxynorvaline, and tunicamycin completely prevent the formation of N-linked (high-mannose as well as complex) oligosaccharides. The role of sialic acid was investigated by treating the cells with neuraminidase. These treatments resulted in altered patterns of surface-labelled glycoproteins after SDS-polyacrylamide gel electrophoresis. Blood-borne arrest of lymphoma cells in the spleen was sensitive to neuraminidase and to treatments interfering with the processing of complex N-linked oligosaccharides. It is suggested that carbohydrates are signals for cellular interactions involved in the recirculation and homing behaviour of lymphoid cells and probably interact with endogenous lectins at their site of homing.     

Developmental Changes in the Biosynthesis of Sialoglycoprotein Substrates for Intrinsic Synaptic Sialidase

Abstract: N′-Acetyl-d -[6-³H]mannosamine was administered to 13- and 28-day-old rats by intraventricular injection. At various time intervals following the injection, synaptic membranes were prepared and the incorporation of radiolabel into sialic acid residues released from endogenous glycoproteins and gangliosides by intrinsic sialidase determined. Radiolabel was incorporated into synaptic membrane gangliosides and glycoproteins, and at all times tested, >90% of the label was associated with sialic acid. Sialic acid released from endogenous glycoproteins by intrinsic sialidase present in 28-day membranes incorporated only 20–25% as much radiolabel per nmole as sialic acid released by mild acid hydrolysis or by exogenous neuraminidase. In contrast, sialic acid released from glycoproteins present in 13-day-old membranes by intrinsic sialidase, mild acid hydrolysis, or exogenous neuraminidase all were similarly labelled. At both ages the specific radioactivity (cpm/nmol) of sialic acid released from gangliosides by the intrinsic enzyme was similar to the total ganglioside sialic acid released by mild acid hydrolysis. The results identify glycoprotein substrates for intrinsic synaptic membrane sialidase as a distinct metabolic class in the mature brain and suggest the occurrence of a developmentally related change in the metabolism of these glycoproteins.     

Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin-peroxidase reagents

A rapid and convenient method was established for analysis of the N-linked carbohydrate chains of glycoproteins on nitrocellulose sheets. Proteins were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, reacted with peroxidase-coupled lectins, and detected by color development of the enzyme reaction. Four glycoproteins having N-linked oligosaccharide chains were used as test materials: Taka-amylase A (which has a high-mannose-type chain), ovalbumin (high-mannose-type chains and hybrid-type chains), transferrin (biantennary chains of complex type), and fetuin (triantennary chains of complex type and O-linked-type chains). Concanavalin A interacted with Taka-amylase A, transferrin, and ovalbumin but barely interacted with fetuin. After treatment of the glycoproteins on a nitrocellulose sheet with endo-beta-N-acetylglucosaminidase H, transferrin reacted with concanavalin A but Taka-amylase A and ovalbumin did not. Wheat germ agglutinin interacted with Taka-amylase A but not ovalbumin; therefore, they were distinguishable from each other. Fetuin and transferrin were detected by Ricinus communis agglutinin or peanut agglutinin after removal of sialic acid by treatment with neuraminidase or by weak-acid hydrolysis. Erythroagglutinating Phaseolus vulgaris agglutinin detected fetuin and transferrin. Thus, the combined use of these procedures distinguished the four different types of N-linked glycoproteins. This method was also applied to the analysis of membrane glycoproteins from sheep red blood cells. The terminally positioned sugars of sialic acid, alpha-fucose, alpha-galactose, and alpha-N-acetylgalactosamine were also detected with lectins from Limulus polyphemus, Lotus tetragonolobus, Maclura pomifera, and Dolichos biflorus, respectively.     

Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride.

Low concentrations of sodium metaperiodate induce specific oxidative cleavage of sialic acids between carbon 7 and carbon 8 or carbon 8 and carbon 9. The aldehydes formed can easily be reduced with NaB3H4 to tritiated 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid or 5-acetamido-3,5-dideoxy-L-arabino-2-octulosonic acid. At 0 degrees, the periodate anion penetrates the cell plasma membrane very slowly and only externally exposed sialic acids are oxidized. This was shown by (a) limited labeling of the sialoglycoproteins in a preparation of inside-out erythrocyte vesicles; (b) trapping 14C-labeled fetuin within resealed erythrocyte ghosts; fetuin was then poorly labeled, whereas the erythrocyte sialoglycoproteins were highly labeled; (c) comparison of labeled glycoproteins of mouse lymphoid cells before and after treatment with neuraminidase. This simple method of specifically introducing a radioactive label into cell surface sialic acids is useful in the study of cell surface sialic acid-containing glycoproteins.     

External labeling of human erythrocyte glycoproteins. Studies with galactose oxidase and fluorography.

Glycoproteins of the human erythrocyte membrane were labeled with tritiated sodium borohydride after oxidation of terminal galactosyl and N-acetylgalactosaminyl residues with galactose oxidase. After separation of the polypeptides on polyacrylamide slab gels, a scintillator was introduced into the gel, and the radioactive proteins were visualed by autoradiography (fluorography). The following results were obtained. (a) The erythrocyte membrane contains at least 20 glycoproteins, many of which are minor components. (b) The carbohydrate of all the labeled glycoproteins is exposed only to the outside, since no additional glycoproteins can be labeled in isolated unsealed ghosts. (c) The membrane contains two major groups of glycoproteins. The first group of proteins contains sialic acids linked to the penultimate galactosyl/N-acetylgalactosaminyl residues, which are efficiently labeled only after pretreatment with neuraminidase. The second group has terminal galactosyl/N-acetylgalactosaminyl residues which can be easily labeled without neuraminidase treatment. The glycoproteins from fetal erythrocytes all belong to the first group, whereas only five glycoproteins of erythrocytes from adults belong. (d) Trypsin cleaves the proteins containing sialic acids, and fragments containing carbohydrate remain tightly bound and exposed in the membrane. (e) Pronase cleaves Band 3 in addition to the sialic acid containing glycoproteins, but most of the glycoproteins still remain unmodified in the membrane. (f) No difference is seen between membrane glycoproteins from cells of different ABH blood groups.     

NEURAMINIDASE-RELEASABLE SURFACE SIALIC ACID OF CULTURED ASTROBLASTS EXPOSED TO ETHANOL

Abstract— Chronic exposure of intact cultured primary hamster astroblasts, clonal line NN, to 100 mM ethanol resulted in significant increases (10–52%) in the releasability of sialic acid from the cell by exogenously added Clostridium perfringens neuraminidase. Enzymatically releasable sialic acid was from the glycoprotein but not the ganglioside fraction of the cell. Addition of ethanol to the medium did not affect the enzymatic hydrolysis of sialyllactose nor did ethanol, over a concentration range of 0–200 mM, alter the enzymatic release of sialic acid from intact NN cells. The total cellular sialic acid content was independent of length of cell exposure to ethanol but varied with the age of the cells. These data suggest that exposure of cells for prolonged periods to ethanol results in steric modification of surface glycoproteins.     

Plasmodium berghei: modification of sialic acid on red cells from infected mouse blood

The surface membrane glycoproteins of normal mouse erythrocytes can be labeled by oxidation with either periodate or galactose oxidase in the presence of neuraminidase, followed by reduction with NaB³H₄. Without neuraminidase there is little galactose oxidase-catalyzed labeling of protein. Analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis showed that both methods labeled the same set of glycoproteins. Plasmodium berghei infection dramatically reduced the sialoglycoprotein labeling of red blood cells from infected blood using the periodate/NaB³H₄ method. Provided neuraminidase was present, labeling by the galactose oxidase method gave identical results to normal erythrocytes. We conclude that the glycoprotein sialic acid of uninfected as well as infected red cells is modified during infection such that it is refractory to periodate oxidation. Acylation of the exocyclic hydroxyls of sialic acid is suggested to account for this. Lectin binding and cell agglutination experiments using Limulin, soybean and wheatgerm lectins, and concanavalin A confirmed and extended these observations. The possible implications of these results with regard to anemia induced by malaria are briefly discussed.     

Localization of protein-bound carbohydrate residues on the cytoplasmic surface of rough and smooth microsomes and golgi vesicles from rat liver

Rough and smooth microsomes and Golgi membranes isolated from rat liver were treated with proteolytic enzymes under conditions which removed 30–40% of the surface proteins without seriously disrupting the membrane structure. This treatment also removed 40–60% of protein-bound mannose, galactose and glucosamine. When protease treatment was combined with neuraminidase treatment, 80% of the sialic acid was removed from intact rough microsomal and Golgi vesicles and about half of the sialic acid of smooth microsomes was solubilized. It appears that half, or probably more, of the membrane glycoproteins are associated with the cytoplasmic surface of these membranes.     

Electrostatic repulsion among erythrocytes in tube flow, demonstrated by the thickness of marginal cell-free layer

Electrostatic repulsion among erythrocytes in flow was evaluated through measurement of the thickness of the marginal cell-free layer in narrow glass tubes of 20-50 microns in inner diameter. To reduce the electrostatic repulsive force, due mainly to sialic acid of the membrane glycoproteins, human erythrocytes were treated with neuraminidase. The surface negative charge of the erythrocytes, as determined from the electrophoretic mobility using free-flow electrophoresis, was found to be proportional to the sialic acid content. When erythrocytes with decreased sialic acid content flowed through narrow tubes, the thickness of cell-free layer determined using an image processor increased even in the absence of erythrocyte aggregation in the suspension. The effect was more pronounced at acidic pH. The addition of Dextran T-70 (70,400 Mol. Wt.) further increased the cell-free layer thickness due to erythrocyte aggregation. Thus, reducing the negative charge density on the erythrocyte surface by itself accelerates the axial accumulation of erythrocytes in flow due to the decreased electrostatic repulsive force between the cells, even in the absence of erythrocyte aggregation.     

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.     

Topographic Distribution of Enzymes Synthesizing Phosphatidylcholine and Phosphatidylethanolamine in Chicken Brain Microsomes

Abstract: The localization of phosphatidylethanolamine and phosphatidylcholine biosynthetic enzymes within the transverse plane of chicken brain microsomes was investigated by using proteases (trypsin and pronase) and neuraminidase. Treatment of intact microsomes with the proteases inactivated the phosphocholine transferase completely and the ethanolamine phosphotransferase only slightly. This latter enzyme was, however, completely inactivated when deoxycholate-treated microsomes were exposed to proteases. Treatment of intact microsomes with neuraminidase had no effect on both phosphotransferases, although 65% of the sialic acid of sialoglycoproteins and 37% of that of gangliosides were removed. With deoxycholate-disrupted microsomes nearly all sialic acid from the sialoglycoproteins and about 70% of that of gangliosides were released. In parallel, the phosphoethanolamine transferase was 90% inactivated. It is suggested that phosphocholine transferase is localized on the outer face of the microsomal vesicle, whereas the phosphoethanolamine transferase could be a sialoglycoprotein, possibly situated on the inner face of the vesicle, or perhaps a transmembrane protein.     

A simple and reliable method for the detection of sialylation in complex/hybrid-type carbohydrate chains of glycoproteins by mixed lectins

A practical and convenient method for discriminating between the presence and the absence of sialic acid in carbohydrate chains of glycoproteins was devised using paramagnetic beads and two lectins, Sambucus sieboldiana lectin (SSA) and Ricinus communis agglutinin (RCA120). The glycoproteins of transferrin or thyroglobulin were firstly captured to paramagnetic beads that were previously coated with corresponding antibody, and then the lectins of RCA120-biotin and SSA-FITC were bound to the glycoproteins on the paramagnetic beads. Finally, the fluorescence intensity of the beads was measured to determine the ratios of lectins RCA120-biotin/Cy5-streptavidin to SSA-FITC. The mixed lectins method showed bigger difference of the ratios between the presence and the absence of sialic acid, indicating higher discrimination efficiency than the ordinary non-mixed lectins method. Furthermore, statistical analysis by two-side t-test indicated that the mixed lectins method was more highly reliable than the ordinary non-mixed lectins method in discriminating between the presence and the absence of sialic acid. The reaction with the two lectins can be performed in a single tube and readily automated taking advantage of the use of paramagnetic beads.     

The effects of cell membrane alteration on glucocorticoid uptake by the AtT-20/D-1 target cell

The glucocorticoid-sensitive AtT-20/D-1 cell line was used to study cellular uptake of glucocorticoids. A previous observation that glucocorticoid uptake by these cells was temperature dependent had prompted us to postulate that glucocorticoids entered the cell by a temperature-sensitive transport process located in the cell membrane. Attempts were then made to perturb the membrane mechanism. In some of these experiments, intact cells were treated with neuraminidase or pronase. The release of sialic acid in the case of neuraminidase treatmentand of sialic acid and cell surface peptides in the case of pronase treatment demonstrated that the enzymes were effective. Approx. 60% of total cellular sialic acid was released by a 15 min incubation with 20 μg/ml neuraminidase at 25°C. The treated cells appeared to be viable, in that they continued to produce corticotropin at a normal rate, yet intact cell glucocorticoid binding at both 4 and 25°C was only 20–30% of that of untreated cells. Treatment with pronase also caused steroid uptake at 4 and 25°C to be reduced, although the extent of reduction was less than that seen following neuraminidase treatment.In other experiments, the effect of exposure of AtT-20/D-1 cells to ethanol or dimethyl sulfoxide was determined. The solvent concentrations used (0.5–10%) did not alter cell viability significantly, and the ability of the cytosol receptor to bind steroid in a cell-free preparation was unimpaired. However, incubation of intact cells with 10% (v/v) dimethyl sulfoxide or ethanol resulted in an 80–90% decrease in steroid uptake at 25°C.We conclude that steroid uptake by the intact cell can be perturbed by treatments which do not affect the cytosol receptor or alter cell viability. These results support the postulate that glucocorticoids enter the AtT-20/D-1 cell by a specific membrane-associated mechanism.     

Characterization and comparison of the major glycoprotein from three strains of Rous sarcoma virus.

The major glycoprotein g2 was purified from three strains of Rous sarcoma virus, representing subgroups A, B, and C. Carbohydrate analysis showed that glucosamine, mannose, galactose, fucose and sialic acid constitute 40% of the weight of the subgroup A glycoprotein and 15% of the subgroup B and C glycoproteins. The molar ratios of sugars were very similar and amino acid compositions were similar but not identical for the three glycoproteins. Glycosidase digestions of subgroup A and C glycoproteins suggested the presence of two types of oligosaccharide chains, the complex serum type, with terminal sequences sialic acidα-Galβ-GlcNAcβ- and the high mannose type with terminal α-linked mannosyl residues. After removal of 70% of the carbohydrate by glycosidases, subgroup A glycoprotein contained only glucosamine and mannose, in the molar ratio 2.0:1.3. The sequence of sugar release was consistent with oligosaccharide structures such as those which have been described for other glycoproteins. The plant lectins concanavalin A and wheat germ agglutinin were shown to interact strongly with the g2 glycoprotein from viruses of all three subgroups.     

Identification of differences between the surface proteins and glycoproteins of normal mouse (Balb/c) and human erythrocytes

Summary The topography of the external surface of the Balb/c mouse erythrocyte has been investigated and compared to the human erythrocyte by using a series of protein radiolabeling probes. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the pattern of Coomassie Blue stained proteins was very similar for mouse and human erythrocyte ghosts, as was the distribution of radioactivity in protein bands after lactoperoxidase catalyzed radioiodination. The mouse erythrocyte glycoproteins identified by periodic-acid-Schiff and Stains-All reagents, sialic acid analysis of gel slices, binding of¹²⁵I-wheat germ agglutinin and¹²⁵I-concanavalin A to the gels, and glycoprotein radiolabeling techniques, differed markedly from the sets of proteins labeled by radioiodination, and also differed from the human erythrocyte glycoproteins. Instead of the PAS I to PAS IV series of sialoglycoproteins characteristic of human erythrocytes, the mouse erythrocyte possesses a broad band of sialoglycoproteins with several peaks ranging in mol wt from 65,000 to 32,000. The same group of sialoglycoproteins were labeled by the periodate/B³H₄ ^– technique specific for terminal sialic acid, and the galactose oxidase/B³H₄ ^– method (plus neuraminidase) specific for galactosyl/N-acetylgalactosaminyl residues penultimate to sialic acid. These results emphasize the necessity to employ a variety of protein radiolabeling probes based on different labeling specificities, to study the membrane topography of cells which are poorly understood compared to the human erythrocyte membrane.     

Effect of modification of synaptosomal membrane glycoproteins on adenylate cyclase activity

The effect of the modification of synaptosomal membrane glycoproteins on the activity of adenylate cyclase was studied. It was found that the binding of concanavalin A to unmodified guinea pig cerebral cortex synaptosomal membrane did not change adenylate cyclase activity. Concanavalin A binding to synaptosomal membrane of hypoxic brain cortex resulted in no decrease of enzyme activity. The level of protein-bound sialic acid in these synaptosomal fractions was 20% lower than in the control. Treatment of synaptosomal membranes with neuraminidase resulted in a decrease of sialic acid content by about 70%, but it had no significant effect on adenylate cyclase activity. The modification with concanvalin A of sugar end groups exposed by neuraminidase treatment resulted in significant decrease of both basal and fluoride-stimulated adenylate cyclase activity. These results seem to indicate that some component of the adenylate cyclase complex of brain synaptosomal membranes is closely interacting with a carbohydrate-containing macromolecule on the cell surface.This work was supported by, the Polish Academy of Sciences within the project 10.4.     

Localization of penultimate carbohydrate residues in zona pellucida and acrosomes by means of lectin cytochemistry and enzymatic treatments

Lectins from peanuts (PNA) and soy beans (SBA) bind terminal residues of galactose (Gal) and N-acetyl-galactosamine (GalNAc) respectively. Galactose oxidase oxidizes the hydroxyl group at C-6 of terminal Gal and GalNAc blocking the binding of PNA and SBA. Binding of these lectins to sugar residues is also severely limited by the existence of terminal residues of sialic acid. In the present study, lectin cytochemistry in combination with enzymatic treatments and quantitative analysis has been applied at light and electron microscopical levels to develop a simple methodology allowing the in situ discrimination between penultimate and terminal Gal/GalNAc residues. The areas selected for the demonstration of the method included rat zona pellucida and acrosomes of rat spermatids, which contain abundant glycoproteins with terminal Gal/GalNAc residues. Zona pellucida was labelled by LFA, PNA and SBA. After galactose oxidase treatment, terminal Gal/GalNAc residues are oxidized, and reactivity to PNA/SBA is abolished. The sequential application of galactose oxidase, neuraminidase and PNA/ SBA has the following effects: (i) oxidation of terminal Gal/GalNAc residues; (ii) elimination of terminal sialic acid residues rendering accessible to the lectins preterminal Gal/GalNAc residues; and (iii) binding of the lectins to the sugar residues. Acrosomes were reactive to PNA and SBA. No LFA reactivity was detected, thus indicating the absence of terminal sialic acid residues. Therefore, no labelling was observed after both galactose oxidase--PNA/SBA and galactose oxidase--neuraminidase--PNA/SBA sequences. In conclusion, the combined application of galactose oxidase, neuraminidase and PNA/SBA cytochemistry is a useful technique for the demonstration of penultimate carbohydrate residues with affinity for these lectins. This revised version was published online in November 2006 with corrections to the Cover Date.     

Carbohydrate-mediated Recognition of Larval Mosquito Hosts by Romanomermis culicivorax

Proteases, glycosidases, and lectins were tested and the results supported a role in host recognition for glycoproteins containing β-glucose and α-mannose on the cuticular surface of host and parasite. Carbohydrates containing α-glucose, galactose, fucose, or N-acetylglucosamine residues apparently are not involved in nematode attachment. Chitin or a related N-acetylglucosamine polymer was found in R. culicivorax preparasites. Treatment of preparasites with neuraminidase, which hydrolyzes sialic acids, increased nematode attachment to Anopheles freeborni larvae.