Jonathan M. Gershoni

Blot analyses of glycoconjugates: Enzyme-hydrazide—a novel reagent for the detection of aldehydes

A procedure for the general staining of glycoproteins and other glycoconjugates on protein blots has been developed. Aldehydes are formed on the sugars of glycoconjugates by periodate oxidation which then react with hydrazide groups of enzyme-hydrazides, a novel reagent designed for aldehyde detection. The bound enzyme-hydrazide is demonstrated histochemically. The new assay is advantageous over periodic-acid Schiff staining of gels as its reagents and signals are stable and the process is simple and expedient, and provides greater sensitivity.

Emerging techniques: Protein blotting: developments and perspectives

Abstract Protein blotting is the method of choice for identifying proteins in complex biological mixtures. This method can be employed in detecting and characterizing glycoproteins, as well as ligand-protein interactions.

Protein blot analysis of virus receptors: identification and characterization of the Sendai virus receptor.

Receptors for Sendai virions in human erythrocyte ghost membranes were identified by virus overlay of protein blots. Among the various erythrocyte polypeptides, only glycophorin was able to bind Sendai virions effectively. The detection of Sendai virions bound to glycophorin was accomplished either by employing anti-Sendai virus antibodies or by autoradiography, when 125I-labeled Sendai virions were used. The binding activity was associated with the viral hemagglutinin/neuraminidase (HN) glycoprotein, as inferred from the observation that the binding pattern of purified HN glycoprotein to human erythrocyte membranes was identical to that of intact Sendai virions. No binding was observed when blots, containing either human erythrocyte membranes or purified glycophorin, were probed with the viral fusion factor (F glycoprotein). Active virions competed effectively with the binding of 125I-labeled Sendai virions (or purified HN glycoprotein), whereas no competition was observed with inactivated Sendai virus. The results of the present work clearly show that protein blotting can be used to identify virus receptors in cell membrane preparations.

Identification of glycoproteins that are receptors for peanut agglutinin on immature (cortical) mouse thymocytes

Binding of peanut agglutinin is being widely used as a marker for immature mouse thymocytes and for the separation of these cells from the mature thymocytes. Two cell surface glycoproteins that bind peanut agglutinin were detected on unfractionated as well as immature thymocytes by lectin overlay and affinity chromatography: one of M r between 170000 and 180000, and the other, a minor component, of M r 110000, both of which are partially sialylated. No receptors for peanut agglutinin were detected on the mature cells, whereas desialylation experiments revealed the presence of a glycoprotein of M r 110000. These findings were corroborated by electrophoretic analysis of cell surface glycoproteins of the isolated thymocyte subpopulations labeled in their carbohydrate moieties.

Identification of cell surface glycoproteins by periodate-alkaline phosphatase hydrazide

A novel method for the detection of cell surface glycoconjugates has been developed. Cells are subjected to mild surface oxidation of vicinal hydroxyls with sodium periodate. Afterward, cellular proteins are resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted, and then probed with alkaline phosphatase hydrazide. The technique is sensitive, reproducible, and inexpensive. It obviates the need for radiolabeled NaBH4 and the subsequent processing of polyacrylamide gels for fluorography. Results are easily obtained in a matter of a few hours.

Identification of peanut agglutinin-binding glycoproteins on immature human thymocytes

Previous studies in our laboratory have shown that peanut agglutinin (PNA), a lectin specific for the disaccharide Gal beta 3GalNAc, binds to immature (cortical) thymocytes of mouse and man and not to the mature (medullary) cells. Using lectin overlay of protein blots and lectin-affinity chromatography, we have found that the major PNA-binding glycoproteins on total as well as on immature (PNA+) human thymocytes correspond to two bands of Mr 170,000 and 180,000. Another glycoprotein, of Mr 110,000, also binds PNA but to a lesser extent. All three glycoproteins contain sialic acid as demonstrated by cell surface labeling with NaIO4-NaB3H4, binding of wheat germ agglutinin, and reaction with alkaline phosphatase-hydrazide. After treatment with sialidase, binding of PNA to these glycoproteins is significantly enhanced.

Localization of azidophencyclidine-binding site on the nicotinic acetylcholine receptor α-subunit

Nicotinic acetylcholine receptors in receptor-rich membranes from Torpedo californica and from T. marmorata electric tissue were photolabeled with the non-competitive inhibitor [3H]azidophencyclidine. The receptor subunits were separated on SDS-polyacrylamide gels and the alpha-subunits recovered from the gel, were subjected to Staphylococcus aureus V8 protease cleavage. The proteolytic fragments were resolved by SDS-polyacrylamide gel electrophoresis and were identified on protein blots by 125I-labeled alpha-bungarotoxin binding and by staining with concanavalin A. The site of specific azidophencyclidine labeling has been localized to the V8-18 kDa fragment which binds toxin. Labeling of the V8-18 kDa fragment was observed in the absence and in the presence of carbamylcholine. This was found for both the species of Torpedo used here.

Synthetic peptides and their antibodies in the analysis of the acetylcholine receptor.

The amino acid sequence of the entire acetylcholine receptor (AChR) of electric fish and of several other species has been established with the advent of gene cloning and recombinant DNA technologies employed by several groups (for review see Patrick et al., this volume). The sequence data have been the basis of predictions concerning the transmembrane orientation of the AChR subunits, as well as the possible location of the acetylcholine ( ACh) binding site, the glycosylation and phosphorylation sites, and the main immunogenic determinants. In our laboratory we are employing synthetic peptides from various regions in the AChR molecule, and their specific antibodies, for structure and function analysis of the AChR. In the following we would like to report on our recent studies on the application of synthetic peptides and their antibodies for (a) mapping the cholinergic binding site of AChR (b) preparation of species-specific anti-AChR antibodies; (c) identification of highly immunogenic regions in the receptor; and (d) analysis and localization of phosphorylation sites in AChR. Synthetic peptides corresponding to sequences from the AChR a-subunit were employed for studying the first three topics (a-c), and synthetic peptides corresponding to sequences from the AChR &subunit were employed in the study of the fourth one (d). The peptides that were synthesized and analyzed for these studies are listed in FIGURE 1.

Towards an Understanding and use of the Cholinergic Binding Site

Systematic analysis of the α-subunit of the nicotinic acetylcholine receptor (AChR) has led to the identification of the cholinergic binding site. By probing protein blots of proteolysed α-subunits with: α-bungarotoxin (BTX), lectins, sequence-specific antibodies and biotinylated SH reagents, the disulfide arrangement of this subunit has been determined (Mosckovitz and Gershoni 1988) and the a-neurotoxin binding domain has been mapped to the region of a 180–200 (Criado et al. 1986, Neumann et al. 1985; 1986, Pedersen et al. 1986, Wilson et al. 1984; 1985). Indeed, synthetic polypeptides corresponding to this area specifically and directly bind BTX (Gotti et al. 1987; 1988, Mulac-Jericevic and Atassi 1986, Neumann et al. 1986, Ralston et al. 1987, Wilson et al. 1985) as do similar recombinant fusion proteins (Aronheim et al. 1988, Barkas et al. 1987, Gershoni 1987). Moreover, competition assays reveal that these sites bind d-tubocurarine as well. Recently, selective T1 NMR relaxation measurements have shown that the sequence α 184–200 can bind both nicotine and acetylcholine specifically, proving that this region is an essential element of the cholinergic binding site (Fraenkel et al. 1988).

The Cholinergic Binding Site: From Sequence to Function

Whereas the sequence of the nicotinic acetylcholine receptor (AChR) has been known since 1983 (Numa et al 1983), a true understanding of how this receptor functions is far from clear. In fact, this situation is quite characteristic of much of today’s molecular biology. The purification of a given protein is readily feasible, N-terminal sequencing has been impressively perfected and the subsequent steps for cloning the desired protein and the’ generation of a postulated sequence have become almost routine. Yet strategies, which allow the efficient correlation between sequence and function, are still being developed and few have been proven effective.