After two reextractions of the wheat-germ agglutinin containing bottom phase, the protein content and the activities of alkaline phosphodiesterase (APDE), 5'-nucleotidase (5'-N), galactosyltransferase (GAL), arylesterase (AE), and N-acetylglucosaminidase (NACG) were measured in the combined top phases and the final bottom phase. Subsequent viral infection of transgenic mice that expressed Cre recombinase only in neuropeptide Y-expressing neurons consequently induced viral replication only in this neuronal population. The carbonyl and amide groups of Neu5Ac are hydrogen-bonded to Ser62 and Glu115, respectively, of WGA. A pseudorabies virus has been generated that requires Cre mediated recombination for its replication. Schematic representation of WGA cross-linked with a branched sialoglycopeptide from glycophorin.1,67 Reproduced from Wright, C. S. J. Mol. In addition, these methods do not allow the labeling of a specific neuronal population and often give rise to nonreproducible results between animals. The lectin has been sequenced (171 amino acids per polypeptide chain) and its X-ray crystallographic structure determined at a 2.0 Å resolution [118,119]. Microinjection of an adeno-associated virus expressing Cre recombinase and GFP, as a marker of viral infection, into the cerebellar cortex or visual cortex induced expression of the WGA protein in the infected areas and allowed its transsynaptic transfer to connected neurons. Specific labeling of a neuronal subset can be achieved via expression of a transgene encoding a transsynaptic tracer fused to a marker molecule, under the control of a neuronal-specific promoter. BL expression has also been used to map sensory input in the olfactory cortex. Breeding mice that express WGA only after excision of a loxP-flanked lacZ gene with mice expressing Cre recombinase under the control of the L7 (Pcp2) promoter generated offspring expressing WGA selectively in Purkinje cells. The carbonyl group is hydrogen-bonded to the hydroxyl of Ser62 and its amide to the carbonyl of Glu115. Unlike legume lectins, aspartic acid or asparagine do not participate in hydrogen bonding. Genetic mapping will also prevent the nonspecific labeling that occurs after tracer injection and circumvents the need for invasive brain surgery that may damage the neural circuit under investigation. WGA–Sepharose is prepared by coupling of WGA to CNBr-activated Sepharose to give a final ratio of 5 mg lectin/ml of swollen gel. As a result, WGA conjugates will label yeast bud scars and the cell membrane of gram+ bacteria and mammalian cells. Transgenic mice expressing the GFP–TeNT HC fusion protein have also been used to investigate neuronal circuitry. WGA is a protein found abundantly in wheat kernels. It has a high binding affinity for N-acetyl-glucosamine (GlcNAc), one of the essential components of PG glycan strands, and thus can be used to probe PG specifically. If so, the dual endowment with R- and Q-type SNAREs, PtSyx3 and PtSyb11, may indicate additional vesicle input, for example, from the Golgi apparatus (Allen, 1988). Bilsland, G. Schiavo, in Encyclopedia of Neuroscience, 2009. Each protomeric unit of wheat germ agglutinin consists of 4 structurally homologous domains with a high degree of amino acid sequence homology among the 4 domains. Fortunately, the lectin–glycoprotein complex can be dissociated by addition of a carbohydrate competing with the lectin binding sites. Sci. Brewer, in Comprehensive Glycoscience, 2007. The mechanism for staining is the binding of the wheat germ agglutinin (WGA) to the exposed n-acetylglucosamine residues of the peptidoglycan layer of the gram-positive bacteria (25). Because of its high binding affinity for N-acetylglucosamine subunits embedded in glycan strands, FWGA enables PG labelling in very low concentrations and short incubations time when compared to traditional Gram staining. The contact amino acids belong to two different subunits of the lectin, a feature not usually observed in lectins. The carboxylate group of sialic acid is within hydrogen-bonding distance of the hydroxyl of Ser114. The most crucial points of this section can be summarized as follows. Several van der Waals contacts are observed with the phenyl ring of Tyr73. Specific SNAREs, H+-ATPase, and actin isoforms are clearly engaged in constitutive endocytosis in ciliates, as we find specific paralogs associated with the established sites, the parasomal sacs. For the Pa dorsal cap cells, labeling was bilateral but slightly greater ipsilaterally. Copyright © 2020 Elsevier B.V. or its licensors or contributors. It can fade following prolonged exposure to light or heat, or exposure to chemicals used for counterstaining. Near the ciliary basis there may occur some particular sites predetermined for vesicle trafficking, as PtSyb10 is enriched there in patches (Schilde et al., 2010). Potentially, this technology may be used in combination with single-neuron electroporation to allow the electrophysiological characterization of synaptic contacts of selected neurons. Jörg Striessnig, Hartmut Glossmann, in Methods in Neurosciences, 1991. A long-standing question regarding bacterial growth is how bacterial cells regulate PG synthesis while precisely maintaining cell morphology. Gerhard Kopperschläger, in International Review of Cytology, 1999. Further manipulation via insertion of additional genes, such as β-galactosidase or WGA, into the glycoprotein-coding region will induce selective protein expression. An aliquot of the solution is saved for determination of the protein concentration. N-acetyl-D-glucosamine in the natural environment of wheat is found in the chitin of insects, and the cell membraneof yeast & bacteria. Similarity in configuration of sialic acid to GlcNAc at their Af-acetamido groups (positions C-5 and C-2, respectively) and adjacent hydroxyl groups (positions C-4 and C-3) account for this recognition by WGA [123,124], Complexes of WGA with sialic-acid-containing oligosaccharides and glycopeptides have been analyzed by Wright [125,126] using X-ray crystallographic analysis. Y.-P. Hsu, ... M.S.
Following replication in transfected cells, the virus could spread transsynaptically, in a retrograde direction only, to monosynaptically connected neurons, identified by enhanced GFP expression, but no further due to the lack of glycoprotein in those neurons.
Between experiments we recommend storage at neutral pH and 4°C in 0.1 M NaCl, 2 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), and 0.05% (w/v) sodium azide. Fluorescent wheat germ agglutinin (FWGA) is another tool that has been used for PG labelling. In spite of their flexibility, these tools also have severe limitations. For long-term storage equilibrate the gel in detergent-free buffer containing 2% (w/v) N-acetyl-d-glucosamine and 0.05% (w/v) sodium azide. FWGA was first used as an alternative to the Gram stain and used to distinguish Gram-negative and Gram-positive bacteria (Sizemore, Caldwell, & Kendrick, 1990). The PG turnover pattern was computationally analysed in order to map the growth, geometry, and cytoskeletal organization at subcellular resolution. This work investigates costaining of muscle, bone, ligament, and tendon tissue sections with fluorescently tagged wheat germ agglutinin (WGA) lectin as a … Before use the resin should be washed extensively with distilled water, 0.5 liter of WGA column buffer (buffer F), and at least 100 ml of solubilization buffer (buffer E). Although there are many new approaches for tracing neuronal connections, the lectin-based wheat germ agglutinin (WGA) tracing approach is still widely used, and it is firmly regarded as a classic method in the field. The TMB chromogen, although most sensitive for anterograde tracing with WGA-HRP, has some disadvantages. The development of multiple anterograde and retrograde tracers, as listed above, has provided extensive information on neuronal projections. Indeed, in the olfactory system, intranasal application of an adenovirus encoding a WGA transgene under the chromogranin A promoter induced a pattern of lectin expression in the OE neurons (first-order), the main olfactory bulbs (second-order), and the main terminal fields of the olfactory cortex (third-order) similar to that obtained with transgenic expression of lectins under the olfactory marker protein promoter. 15). The carboxylate group of the Neu5Ac is within hydrogen-bonding distance of the hydroxyl of Ser114. How is the molecular equipment of the terminal cisternae, that is, the early endosomes, in Paramecium (Allen, 1988)? This solution releases minor amounts of protein from the column not recovered upon biospecific elution. The results revealed that nascent PG insertion occurs in a heterogeneous fashion and is correlated with MreB spatiotemporally. More recently, a viral transcomplementation method was also employed to restrict viral expression to selected neuronal populations. A number of hydrogen bonds stabilize the complex. This labeling was found in the dorsal cap of the paraventricular hypothalamic nucleus (Pa) and in the intermediate zone of the spinal gray.
Co-expression of BL with two distinct odorant receptor genes generated a transsynaptic tracer that could be monitored in localized areas of the OE, through the mitral and tufted relay neurons in the olfactory bulbs, to specific clusters of olfactory cortical neurons.
WGA has been used extensively to unravel both simple and complex neural networks in the central and peripheral nervous systems. One example of membrane affinity partitioning is given in Fig. WGA (100 mg) is dissolved in 50 ml of coupling solution (solution A) containing 3 g of N-acetyl-d-glucosamine.
It has a high binding affinity for N -acetyl-glucosamine (GlcNAc), one of the essential components of PG glycan strands, and … 1990, 215, 635–651, with permission of American Society for Biochemistry & Molecular Biology (ASBMB). Furthermore, transsynaptic transfer permitted the labeling of neurons of the main and accessory olfactory bulbs, which represent the terminal fields of OE and VNO neurons (second-order), and of granule cells of the olfactory cortex (third-order), which are innervated by neurons from the main and accessory olfactory bulbs. Wheat germ agglutinin (WGA) is a carbohydrate-binding lectin that has high affinity for sialic acid and N-acetylglucosamine moieties of glycoproteins. Labeled neurons in the spinal cord were scattered in the intermediate zone near the central canal, predominantly contralaterally.
Typical working concentrations of wheat germ agglutinin conjugate solutions are 1–10 µg/mL.
WGA-HRP may not be taken up equally well by all neurons due to the insufficient number of binding sites; some selectivity has been noted (see Ref. Wheat germ agglutinin (WGA) is one of the most widely used lectins in cell biology. 1993, 18, 221–226, copyright (1993), with permission from Elsevier. We could localize PtSyb11 (Schilde et al., 2010), PtSyx3 (Kissmehl et al., 2007), and H+-ATPase SU-a1 (Wassmer et al., 2005, 2006) to this organelle. Figure 24. Affinity partitioning of rat liver membranes. Potentially viral vectors can also be genetically targeted to specific neuronal populations via insertion of a neuron-type specific promoter into the coding region of the viral glycoprotein gene. However, the precise means by which MreB regulates PG synthesis is not fully understood. A third aromatic side chain, that of Tyr64, interacts through nonpolar contacts with the glycerol tail of the Neu5Ac. Quantitative estimates for polar, nonpolar, and ionic interactions revealed that hydrogen bonding makes the largest contribution to complex stabilization, in agreement with thermodynamic data.66, Structures of WGA complexed with a branched sialoglycopeptide that possesses both (α2-3)- and (α2-6)-linked terminal Neu5Ac moieties show that the glycopeptide cross-links two crystallographically related dimers (Figure 25). Furthermore, this technology can be used to investigate the development of neuronal circuits in utero and may provide functional insights since transsynaptic transfer of tracers will occur only in active circuits. Many of these cells were so weakly labeled as to be at the threshold of detection.