Glycobiology

Enhancement of QD-fluorescence

Although QD fluoresces by itself, its fluorescence is enhanced by addition of the enhancing solution used in the DELFIA methodology (Hemmilä et al., 1984). As soon as QD-labeled samples are mixed with the enhancing solution, its fluorescence is greatly (~500-fold) increased. This instant enhancement is not likely to be caused by dissociation of europium, because QD-labeled proteins in 10 mM DTPA lose only 20% of its original fluorescence after 5 days (Saitoh et al., unpublished observations). The enhancing effect is most likely due to the hydrophobic agents such as trioctylphosphine oxide and Triton X-100 contained in the enhancing solution. However, upon prolonged (days) exposure to the enhancing solution, there is additional increase in europium-fluorescence, which is perhaps due to slow dissociation of europium from the macrocyclic chelator. We used the enhancing solution designed for polycarboxylate chelates (Hemmilä et al., 1984), because the enhancing solution proposed for QD (Adeyiga et al., 1996) did not result in higher sensitivity.

GalT assay

Both QD-SBA and QD-RCA gave linear response to the amount of GalT used, but the apparent sensitivity using QD-RCA was about 5-fold higher than QD-SBA (Figure 3A,B). Since the QD-level in RCA and SBA was comparable, the reason for this difference in the sensitivity is likely to be caused by the difference in their binding affinities. Although the combination of biotin-RCA and europium-streptavidin (method 2) has the advantage of both reagents being commercially available, the response was not linear in the range of 0-500 µU of the enzyme. This is perhaps because the method requires a two-stage reaction, while the QD-lectins involves only one reaction, before the fluorescence measurement is made.

MBP-A binding

Comparison of Figure 4B and C clearly indicates that QD is as effective a tracer as radioiodide. The higher background (30%) in the MBP binding assay using QD-Man₃₁-BSA compared to using ¹²⁵I-Man₂₈-BSA (Figure 4A) is most likely caused by nonspecific binding attributable to the hydrophobicity of macrocyclic QD, because similar levels of counts were observed both in the binding of QD-Man₃₁-BSA to uncoated plates and the binding of a nonligand, QD-Gal₃₄-BSA, to the plates irrespective of the amount of MBP-A coating. However, this level of nonspecific binding is quite manageable in the inhibition assay. The I₅₀ values obtained with QD-Man₃₁-BSA were only slightly higher than values obtained with ¹²⁵I-Man₂₈-BSA (Figure 4).

Assay of Gal/GalNAc receptor in rat liver membrane

The two methods described here for assaying the Gal/GalNAc-specific hepatic lectin using QD-labeled Gal-BSA both demonstrated sugar-specific binding that is dose-dependent. The filtration method is a direct extension of the existing assay method that utilized radiolabeled ligands (Lee and Lee, 1982). Despite the built-in disadvantage of determining fluorescence in a larger well (2.3 cm diameter), this method is quite convenient and its sensitivity is only slightly inferior to the radiolabeled ligand (i.e., assays can be carried out at the ligand concentration in the submicromolar range). However, since the enhancement of bound QD-Gal/Man-BSA (to the filter or to the microtiter well) is an even slower process than that of QD-proteins in solution, assays by both methods required at least 24 h to obtain meaningful data. One would underestimate the amount of sugar-specific binding (the amount of QD-Gal BSA bound over that of QD-Man-BSA bound) considerably, if the initial values instead of the final values are used.

When the 96-well coating assay was used instead of the filtration assay, ~5-fold increase in sensitivity is gained in terms of the amount of plasma membrane required. However, the 96-well method required about 10-fold higher concentration of the ligand (~1 µM) to demonstrate the specific binding (Table I). This may be due to the difference in binding environment of the two assay systems: in the filtration assay, the binding occurs in a well-equilibrated suspension, while in the 96-well assay the lectin is immobilized and therefore physically restrained.

The major drawback of the QD-based assay of hepatic lectin on the plasma membrane is apparent difficulty in obtaining meaningful inhibition data. A very high nonspecific binding of QD-BSA derivatives to filters and microtiter well surface is suspected to be the cause of this difficulty.

Comparison of QD-labels with radioiodide

As mentioned earlier, our results amply demonstrate that QD-labeled samples, like other europium chelates, can be detected as sensitively and reliably as radioiodide. One indisputable advantage of europium label over radioiodide is that, unlike radioiodide, europium labels do not decay. This advantage alleviates the need for frequent labeling of samples required for radioiodides. The stability also leads to more consistent results over a relatively long period of time. Moreover, since fluorescence of europium is measured in 'cps" rather than 'c.p.m." as for radioiodide (compare y-axes of Figure 4B and C), the time required for measurement is considerably shorter than that for radioiodide.

Nonspecific binding of QD-labeled neoglycoproteins

We found that QD-labeled BSA neoglycoproteins in general give a high non-specific binding (background), despite our attempt to keep the number of QD incorporated into the BSA-neoglycoproteins low and to increase hydrophilicity of QD by adding aspartic acid to react with the remaining isothiocyanate group. The high nonspecific binding probably results from strong hydrophobicity of QD itself. In the case of the filtration assay for the hepatic lectin, for which a direct comparison with the ¹²⁵I-labeled ligand is available (Lee and Lee, 1982), the amount of ¹²⁵I-labeled Gal-BSA bound to GF/C filters in the absence of plasma membrane was typically less than 10% of that bound in the presence of plasma membrane. In contrast, this value is as high as 80% in the case of QD-Gal-BSA. In the case of 96-well plates, the nonspecific binding was about 60% for the BSA-blocked wells.

In contrast, the nonspecific binding in the MBP-A assay using QD-Man₃₁-BSA is considerably lower (~30%). This is perhaps due to two factors: (1) the material immobilized is a single protein species; (2) the PVLA-immobilization method allows a much denser coating of MBP compared to the surface adsorption method (Suzuki et al., 1997). In contrast, the plasma membrane contains, in addition to hepatic lectin, many hydrophobic components that are irrelevant in the sugar-specific binding of QD-Gal-BSA. Hydrophobicity of QD did not figure prominently in the GalT assays.

Materials

Quantum dye and HEPES were from Research Organics, Inc. (Cleveland, OH). Borane-pyridine complex and diethylenetriaminepentaacetic acid (DTPA) were from Aldrich Chem. Co. (Milwaukee, WI). Na¹²⁵I was from Amersham (Arlington Heights, IL). Europium-labeled streptavidin (labeled with the Wallac europium-labeling reagent, shown in Figure 1, containing ~7 europium ) was from Wallac, Inc. (Gaithersburg, MD). [beta]-1,4-Galactosyltransferase and UDP-galactose, RCA120 and its biotin conjugate were from Sigma Chemical Co. (St. Louis, MO). Bovine serum albumin derivatives (neoglycoproteins) bearing 28 and 31 Man residues (Man₂₈-BSA and Man₃₁-BSA), 34 Gal residues (Gal₃₄-BSA), or 35 GlcNAc residues, on the average, via amidino linkages, were prepared as described previously (Stowell et al., 1993). Soybean agglutinin (SBA) was prepared essentially by the published method (Gordon et al., 1972), except lactose-immobilized Sepharose CL-6B (divinylsulfone) was used. Fiberglass filter membranes, 934-AH and GF/C, were products of Whatman Inc. (Clinton, NJ). The carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein (MBP-A CRD) was expressed and purified according to the published method (Drickamer, 1989), except that cells were lysed in a French press. Rat hepatic plasma membrane was a gift from Dr. R. L. Schnaar (Department of Pharmacology and Molecular Science, Johns Hopkins Medical School).

The low fluorescence 96-well plates were from Wallac and 12-well cell culture plates were from Costar (Cambridge, MA). Immulon-1, -2, -4 Removawell strips were from Fisher Scientific (Pittsburgh, PA). SealPlate tape was from Elkay Products (Shrewsbury, MA). The method of coating of the microtiter plate with polyvinylbenzyl lactonoylamide (PVLA) and the subsequent activation was as described previously (Suzuki et al., 1997).

Buffers and solutions

The enhancement solution (Hemmilä et al., 1984) (containing 50 mM trioctylphosphine oxide, 15 mM 1-(2-naphthoyl)- 3,3,3-trifluoroacetylacetone, and 0.1% Triton X-100 in 100 mM acetate adjusted to pH 3.2 with potassium hydrogen phthalate) was from Wallac (Gaithersburg, MD).The buffer for coupling QD to proteins contained 81 mM sodium carbonate and 206 mM sodium bicarbonate (pH 9.2). The HEPES buffer consisted of 25 mM HEPES, 1.25 M NaCl, and 25 mM CaCl₂, with the pH adjusted to 8.0 with NaOH. The Tris buffer contained 1.25 M NaCl and 25 mM CaCl₂ in 25 mM Tris-(hydroxymethyl)aminomethane (adjusted to pH 8.0 with HCl). The TBST solution contained 100 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20 at pH 7.4. The substrate buffer for Gal-transferase (GalT) contained 2.5 mM UDP-Gal, 40 mM MnCl₂ and 40 mM sodium cacodylate at pH 6.0. The membrane assay buffer consisted of 50 mM HEPES buffer, pH 7.5, containing 0.5 M NaCl, 25 mM CaCl₂, and 0.1% BSA. The membrane washing buffer consisted of 10 mM Tris buffer, pH 7.5, containing 0.1 M NaCl, 10 mM CaCl₂, and 0.1% BSA.

General methods

Man₂₈-BSA (10 µg) was radioiodinated with 0.5 mCi Na¹²⁵I using the Chloramine T method (Greenwood et al., 1963). Radioactive ¹²⁵I was counted with a Packard Minaxi [gamma]-counter.

During the reaction or development of fluorescence, the 96-well plate was sealed with SealPlate tape. The europium fluorescence was measured in a 96-well microtiter plate or a 12-well plate at certain specified time after addition of 200 µl (for 96-well plates) or 2 ml (for 12-well plates) of the enhancement solution and 1 ml of water, using a model 1420 Victor Multilabel Counter (Wallac, Gaithersburg, MD) in a time-resolved fluorometric mode (Ex = 340nm, Em = 615nm).

Labeling of proteins with QD

Man₃₁-BSA, Gal₃₄-BSA, SBA, and RCA were labeled as follows.

(1) Labeling of Gal₃₄-BSA and Man₃₁-BSA with QD. Gal-BSA or Man-BSA (5 mg) was dissolved in 1 ml of coupling buffer to which 0.25 ml of DMSO was added. QD (1 mg) was dissolved in 250 µl of DMSO just prior to use, and 10 µl of the QD solution was added to each of the neoglycoprotein solution every min over a 10 min period. The mixture was kept for 1 h at room temperature, and the reaction was terminated with 100 ml of 0.28% aspartic acid. The reaction mixture was applied to a Sephadex G-50 column (1 ×14 cm) equilibrated in 0.2 mM Tris-HCl buffer, pH 7.6, and the column was eluted in the same buffer, collecting 0.44 ml fractions. Effluent was monitored by A_280nm as well as by the europium fluorescence. The results are shown in Figure 2. The level of QD labeling was 3.1 mol/mol protein for Gal-BSA, and 3.6 mol/mol protein for Man-BSA..

(2) Labeling of SBA with QD. For labeling of SBA with QD, 2 mg of the lectin was dissolved in 0.5 ml of the coupling buffer and 50 µl of DMSO was added. To the mixture, 10 µl of QD solution (4 mg/ml in DMSO) was added 10 times at 1 min intervals. The reaction mixture was incubated for 15 h at room temperature. After terminating the reaction by addition of 100 ml of 0.28% aspartic acid, QD-labeled SBA was purified with the Sephadex G-50 column as above. About 1 mol QD was found per mol SBA.

(3) Labeling of RCA with QD. To the solution of 270 ml of RCA (Sigma) containing 2 mg protein, 230 µl of 2-fold concentrated coupling solution and 50 ml of DMSO were added. The reaction with QD and the subsequent purification were as descried above. About 0.9 mol QD per mol protein was coupled.

The levels of QD per protein (mol/mol) were calculated from the fluorescent counts of Eu⁺³ (measured 1 h after addition of the enhancing solution) and the protein concentration of the column-purified sample. The standard curve of fluorescence count of QD was used for calibration.

Preparation of MBP-A-CRD immobilized plate

Preparation of PVLA-wells and coupling of protein to the wells have been described previously (Suzuki et al., 1997). Briefly, Immulon-1 removable microplate wells were coated with 40 µl of 2 mg/ml PVLA for 15 h at 4°C, and the coated PVLA was oxidized with 1 mM NaIO₄ for 1 h at room temperature. To each oxidized PVLA well were added 40 µl of MBP-A CRD solution in the HEPES buffer and 5 µl of 2% (v/v) borane-pyridine complex in the same HEPES buffer, and the mixture was incubated at 37°C for 16 h. In order to quench the remaining aldehydo-groups, 10 µl of 1 M glycylglycine was added and the mixture was incubated for 4 h. After removing the liquid, the wells were blocked with 2% BSA in the Tris buffer at 4°C for 2 h, and then washed with the Tris buffer.

Assay for [beta]-1,4-Gal-transferase (GalT) using biotin-labeled RCA and europium-labeled streptavidin

The 96-well microtiter plate was coated with 100 µl of 100 nM GlcNAc₃₅-BSA in PBS for 18 h at 4°C. The wells were washed with 200 µl of the TBST solution 4 times and blocked with 200 µl of 1% BSA in TBST for 1 h at 4°C. After washing with TBST (4 × 200 ml), the wells were incubated with GalT in 100 µl of the substrate buffer for 1 h at 37°C. After removal of the reaction mixture, the wells were washed and 100 µl of 300 nM biotin-labeled RCA was added to each well. After 1 h incubation at 4°C, the wells were washed and then incubated with 100 µl of 500-fold diluted europium-labeled streptavidin (20 ng) at 37°C for 1 h. After washing with TBST 6 times, 200 µl of the enhancement solution was added to the wells. The wells were shaken for 5 min, and fluorescence count was measured.

Assay for GalT using QD-SBA or QD-RCA

The preparation of GlcNAc₃₅-BSA coated wells and the GalT reaction were carried out as described above. After washing the plate with TBST, 100 µl of 300 nM QD-SBA or QD-RCA was added to each well and incubated for 1 h at 4°C. The reaction mixture was removed and the wells were washed with TBST (6 × 200 µl). The enhancement solution (200 µl) was then added to each well, and the fluorescence count was measured after 1 h.

Ligand binding and inhibition assay for MBP-A

A labeled BSA derivative, either ¹²⁵I-Man₂₈-BSA (0.8 ng, ~20,000 c.p.m.), QD-Man₃₁-BSA, or QD-Gal₃₄-BSA (each 1.6 ng, ~80000 cps) in 100 µl of Tris buffer containing 1% BSA and 1 µg of unlabeled Man₂₈-BSA was added into the MBP-A immobilized plate and incubated at 4°C for 20 h. After removal of the liquid, the wells were washed with the Tris buffer six times. When ¹²⁵I-Man₂₈-BSA was used, the well strips were broken into individual wells and counted. When QD-Man₃₁-BSA or QD-Gal₃₄-BSA was used, 200 µl of the enhancement solution was added to each well, and the mixture was incubated at 4°C for 16 h. After the plates were warmed to room temperature, the Eu³⁺-fluorescence was measured.

For inhibition assays, 25 µl each of inhibitor and ¹²⁵I-Man₂₈-BSA or QD-Man₃₁-BSA solution in the Tris buffer containing 1% BSA was added to the MBP-A (2 µg/well) immobilized plate. The mixtures were incubated at 4°C for 20 h, and the amount of bound ligand was measured as above.

Binding of QD-Gal₃₄-BSA by rat liver plasma membrane preparation

Filtration assay. The binding reaction was carried out essentially as described previously (van Lenten and Ashwell, 1972) except for substituting QD-Gal₃₄-BSA for ¹²⁵I-labeled ASOR. Rat liver plasma membrane (25 µg) was shaken at 37°C for 90 min with QD-Gal₃₄-BSA at 30-150 nM in 0.5 ml of the membrane assay buffer. After the reaction, the tubes were cooled on ice and the mixture was filtered through pre-washed Whatman GF/C and washed three times with the membrane washing buffer. Prewashing of GF/C filters involved passing 1 ml of 0.005% DTPA in 50 mM potassium hydrogen phthalate two times through the filters on the filtration assembly, then washing thoroughly with water followed by the membrane washing buffer just prior to use. After filtration, damp filters were placed and pressed to the bottom of the wells of 12-well cell culture plates. Two milliliters of the enhancement solution and 1 ml of water were added to each well, and the plate was gently shaken on a rotary shaker for an hour. The europium fluorescence was measured periodically over the course of 48 h.

In order to assess the extent of nonspecific binding of QD-Gal₃₄-BSA, the incubation mixture without the plasma membrane as well as a mixture containing plasma membrane and QD-Man₃₁-BSA were set up. Since the fluorescent intensity was greatly influenced by the presence of filter in the well (see Results and Discussion), known amounts of QD-Gal₃₄-BSA and QD-Man₃₁-BSA were placed in the 12-well culture plates together with enhancing solution and washed GF/C filters, and the fluorescence was measured periodically as references.

Direct coating assay in a 96-well microtiter plate. A suspension of rat liver plasma membrane (40 µl) in the HEPES buffer was placed in the individual wells of a 96-well plate and incubated overnight at 4°C. BSA (10 µl of 10% solution) was added and the incubation was continued for further 12 h or longer. The content of wells was removed by gentle suction and wells were washed twice with the HEPES buffer (150 µl). To each well was added QD-Gal₃₄-BSA or QD-Man₃₁-BSA, with or without a potential inhibitor, in a modified membrane assay buffer that contained 1.9% instead of 0.1% BSA. After incubation at 37°C for 90 min, the content of the wells was removed and the wells were washed with the HEPES buffer as before. To each well was added 200 µl of the enhancement solution and the plates were shaken gently on a rotary shaker. The fluorescence was measured at intervals for up to 48 h.