View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 68, number 2 FEBS LETTERS October 1976 AFFINITY CYTOCHEMISTRY: THE LOCALIZATION OF LECTIN AND ANTIBODY RECEPTORS ON ERYTHROCYTES VIA THE AVIDIN-BIOTIN COMPLEX Edward A. BAYER, Meir WILCHEK and Ehud SKUTELSKY” Department of Biophysics and *Section of Biological Ultrastructure, The Weizmann Institute of Science, Rehovot, Israel Received 21 July 1976 1. Introduction In the present communication, we describe the further use of ferritin-avidin conjugates for the Ferritin-conjugated proteins, such as lectins and visualization of Con A**, PNA and antibody receptor antibodies, are widely used for localization of cell sites on erythrocytes. The study is based on the surface receptors in the electron microscope [ 1,2] . interaction between ferritin-avidin and biotinylated Procedures designed to effect direct covalent coupling binding proteins and is comprised of the following of ferritin to the binding-protein are, for the most steps: (a) Biotin is covalently attached to the desired part, cumbersome and inefficient. The resultant binding protein. (b) The characterized conjugate is complex is of high molecular weight - often a multi- incubated with appropriate cells. (c) Subsequent mer - thus affecting both the physical and chemical incubation with FAv enables the visualization of the binding characteristics and the biological activity of given cell surface receptor. It is hoped that this the conjugate. approach may unify affinity cytochemical techniques Biotin, derivatized to bacteriophages via the and circumvent some of the problems related to terminal carboxy moiety, has previously been shown ferritin-protein conjugation. to be available for subsequence interaction with avidin, resulting in phage inactivation [3] . A later investigation demonstrated that ferritin-avidin 2. Materials and methods conjugates can be used for the specific labeling of biotin-tagged proteins and membrane preparations 2.1. Chemicals and synthesis [4]. Recently, we have described an improved techni- BNHS was prepared from biotin and N-hydroxy- que for the preparation of ferritin-avidin conjugates succinimide (both purchased from Sigma Chem. Corp.) for use as an ultrastructural marker in the localization in the presence of dicyclohexylcarbodiimide (Fluka) of surface receptor sites on biotin-derivatized whole as described previously [3,7]. Other reagents were of cells [S] . The visualization of tRNA hybridized to the highest commercial grade available. DNA has also been reported [6]. 2.2. Proteins The preparation of ferritin-avidin conjugates **Abbreviations: Con A, concanavalin A; PNA, peanut agglutinin; rAb, goat gamma globulin against rabbit red (FAv) was described previously [S] . Concanavalin A blood cell membranes; mAs, antiserum against mouse (Con A) was purchased from Miles Yeda Ltd. Peanut erythrocyte membranes; B-Con A, B-PNA B-rAb, B-mAs agglutinin (PNA) was kindly provided by N. Sharon - biotinylated-Con A, etc.; FAv, ferritin-avidin conju- [8]. Rabbit antimouse RBC antiserum and goat gates; rRBC, mRBC, hRBC - rabbit, mouse and human erythrocytes, respectively; BNHS, biotinyl-Whyd.roxy- antirabbit RBC antiserum were prepared as previously succinimide ester; PBS, phosphate buffered saline, pH 7.2; described [9]. Goat Ig was separated by ammonium VELS, Verona&acetate buffered saline, pH 7.4. sulfate fractionation. 240 North-Holland Publishing Company - Amsterdam Volume 68, number 2 FEBSLETTERS October 1976 2.3. Preparation of biotin derivatized proteins fixed with 2% glutaraldehyde for 30 min. Processing Con A was treated with various molar ratios of for electron microscopy was carried out as previously BNHS in a solution of 0.1 M sodium bicarbonate reported [S] . containing 1 M sodium chloride (see table 1). The reaction was allowed to proceed for 4 h at room temperature, and the biotinylated samples were 3. Results and discussion dialyzed overnight against PBS with several buffer changes. 3.1. Characterization of derivatized Con A The amount of biotin per conjugate was deter- The successful application of this method requires mined by deamination and subsequent amino acid that the biotinylated protein be subject to unhindered analysis of biotin-protected lysines [lo] . Quantifica- interaction both with its receptor as well as the tion of the extent of exposed biotin moieties/conju- ferritin-avidin conjugate. In other words, the amount gates was accomplished by retention on avidin- of biotin derivatized to the protein should be sufficient Sepharose columns [ 111. Affinity chromatography to react with avidm and, at the same time, should on avidin columns was performed in the presence not be too much so as to disturb the binding of the of 0.2 M glucose. The effect of the attached biotinyl protein to its receptor. The example of biotin-conju- residues on the activity of Con A was analyzed by gated Con A will demonstrate this principle. an agglutination assay of rabbit erythrocytes using a The derivatization of biotin, by means of its Fragiligraph [ 121. N-hydroxysuccinimide ester, to Con A was nearly quantitative as determined by amino acid analysis 2.4. Procedure for labeling cell surface receptors (table 1). In order to check the avidin-binding Rabbit, mouse or human erythrocytes were iso- capacity of these conjugates, samples were adsorbed lated from fresh heparinized blood by sedimentation, to a column of avidin-Sepharose. In a conjugate rinsed twice in VBS and fixed briefly for 2 min at containing an average of about 5 biotin residues per room temperature with 2% glutaraldehyde in VBS. tetramer, 60% was retained on avidinSepharose. An Aliquots of lo7 cells were washed three times in VBS average of 15 biotin molecules/tetramer resulted in and then incubated for 30 min at room temperature 80% binding to such columns. Full binding could not with an appropriate solution of B-lectins or B-rAb at be achieved until attachment of 30 biotins/tetramer. a concentration of 1 mg protein/ml, or in 5% B-mAs. It is apparent that nearly total derivatization is Neuraminidase treatment preceding B-PNA incubation necessary in order to ensure complete interaction was carried out as previously described [8]. Controls between the biotinylated protein and avidinSepharose. were performed in the presence of 0.2 M of the This phenomenon is surprising, considering the extra- competitive sugar or with underivatized lectins and ordinary affinity between biotin and avidin [ 131. antibodies. After the previous step, cells were washed Accordingly, a portion of the derivatized biotin three times with VBS and treated with FAv (1 mg/ml residues are apparently unexposed and not available in ferritin) for 15 min at room temperature. Next, for combination with avidin-Sepharose. It should be cells were rinsed twice with VBS and additionally noted, however, that this does not exclude the Table 1 Analysis of biotin-derivatized Con A saniples Retained on Protected B-Con A Amount (mg) Biotin/dimer avidin column lysines Sample Con A BNHS Molar ratio (%I (per dimer) A 100 2.8 4.4 59 2-3 B 100 5.6 8.8 79 7-9 C 100 11.2 17.6 92 n.d. D 10 5.0 77.8 n.d. total 241 Volume 68, number 2 FEBS LETTERS October 1976 TIME (min) Fig.1. Rate of rRBC agglutination by native (N) and biotinyl-Con A preparations (Samples A-D, table 1). L.T.: Light transmission. possibility of complete interaction between FAv and implies that this sample may have been converted to B-proteins in solution. Binding of the latter to mem- a monovalent form of Con A by heavy biotinylation - brane receptors may cause conformational changes, similar to that obtained by chemical [ 141 or by resulting in the exposure of more biotinyl residues for enzymatic means [ 15,161. The final inadequacy of complex formation with avidin. this sample was observed under the electron micros- In order to determine the effect of biotinylation on cope in the form of extensive aggregates. biological activity, an agglutination assay using In view of these results, it is therefore advisable to rRBC was performed. As can be seen from fig. 1, most derivatize proteins under near-saturating conditions, of the derivatives were still capable of agglutinating but to avoid excessive treatment with BNHS. A sample, the cells. However, supersaturation of Con A with at least 80% of which binds to avidinSepharose, is biotin (sample D) had several undesirable attributes. probably appropriate for the studies described in this First, rRBC agglutination was completely inhibited report. Similar results were also obtained with biotinyl- (fig.1) indicating that the biological activity of this ated PNA and antibodies. sample was significantly altered - but not destroyed, since total retention by affinity chromatography on 3.2. Specific labeling of the cell surface Sephadex G-75 was observed. The combined lack of rRBC, incubated with B-PNA, B-Con A or B-rAb rRBC agglutination versus binding to Sephadex, were specifically labeled with FAv (figs.2 and 3). When Fig.2. rRBC treated with B-Con A and labeled with FAv. Fig.3. rRBC incubated with B-rAb and labeled with FAv. 242 Volume 68, number 2 FEBS LETTERS October 1976 Fig.4. rRBC treated with B-Con A in 0.1 M glucose and labeled with FAv. Fig.5 hRBC treated with B-PNA and FAv. Fig.6. hRBC treated with 50 U/ml neuraminidase, incubated with B-PNA and labeled with FAv. the cells were incubated with B-Con A or B-PNA in membrane-bound, multi-subunit lectin are available the presence of their competitive sugar, no labeling for further interaction with FAv. This explanation is was observed (fig.4). hRBC also lacked labeling when supported by the fact that neither underivatized PNA, incubated with B-PNA followed by FAv, since mem- Ab or As affected FAv labeling of the cell surface. brane receptors for this lectin are not exposed (fig.5).