2Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovaèiæa 1, 10000 Zagreb, Croatia and 3Biology Department, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA
Received on April 8, 1999; revised on November 8, 1999; accepted on November 8, 1999.
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Abstract |
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Key words: glycoprobe/lectin/photoaffinity/carbohydrate
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Introduction |
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One of the proposed functions of the carbohydrate structures on glycoconjugates is the transfer of information through interaction with specific lectin receptors (Lee, 1993). Relative complexity of these interactions has hindered the search for specific carbohydrate receptors, but during the past years strong evidence has accumulated to render support to this hypothesis in some systems (Varki, 1993
). The best examples are selectins and their interaction with S-Lex oligosaccharides in inflammation (Lasky, 1995
). Though the existence of Ca2+-dependent carbohydrate binding activity on hepatocytes was reported more than two decades ago (Hudgin et al., 1974
), the significance of this discovery manifested a full impact only recently, when the role of selectins in the interpretation of cell-specific carbohydrate information during inflammation was demonstrated (Lasky, 1992
). Understanding of this process has proven to be of vital importance, and it led to the development of promising novel carbohydrate drugs which could prevent damage by excessive inflammation in conditions like septic shock (Wada et al., 1996
).
However, the number of elucidated functional lectincarbohydrate interactions is still relatively small, and it appears that one of the main problems is the lack of effective methods to identify lectin activity in complex biological samples. Various synthetic neoglycoproteins have been used for this purpose (Lee and Lee, 1994), but they can only identify the existence of carbohydrate binding activity in the sample, and can not attribute this activity to specific proteins within the mixture.
An application of small photoreactive monosaccharide probes with biotin (Lauc et al., 1994b) or digoxigenin (Lauc et al., 1995
) has been previously proposed for this purpose. The method was demonstrated to be a sensitive and useful tool in lectin studies (Lauc et al., 1994a
), but its application was somewhat limited due to relatively low affinity of lectins for monosaccharides. A solution to this problem is the use of multivalent oligosaccharide structures which bind more strongly to lectins (Lee and Lee, 1987a
). Such structures have already been used as photoaffinity labels for lectins (Rice et al., 1990
; Rice and Lee, 1993
), and we describe here the development of a novel method which combines simplicity of digoxin detection with high specificity of complex oligosaccharidelectin interactions.
We have designed the synthetic schemes in such a way as not to require laborious purification at each step. Meticulous purification is not practical when the ligands to be used in glycoprobe are precious, and minimal handling is desirable for attaining reasonable yields.
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Results |
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General structure of the glycoprobe is shown in Figure 1 and the concept for its application in Figure 2. There are three vital parts of the glycoprobe: (1) glycan, (2) digoxin tag, and (3) photoreactive crosslinker. When incubated in dark, glycans interact with lectins in the sample and form stable noncovalent complexes. After UV illumination, the azido group in the photoreactive crosslinker becomes a highly reactive nitrene group which nonselectively forms a covalent bond with adjacent structures (Bayley and Knowles, 1977; Wong, 1991
). Consequently, the lectin is tagged by digoxin, which subsequently can be easily identified and isolated with anti-digoxin or anti-digoxigenin antibodies.
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Discussion |
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We have chosen to use nonradioactive labels for identification to avoid complications associated with the use of radioisotopes. Two small organic molecules are frequently used as tags for immunochemical detection: biotin and digoxigenin. Though biotin enables easy detection with avidin, streptavidin and their derivatives, its applicability to biological samples is severely restricted by ubiquitous existence of endogenous biotin in the form of the covalently linked coenzyme. On the other hand, though digoxigenin is not widespread in biological samples, its commercial availability is limited and the application restricted by the existing patents. Thus, we have decided to use a third compound, plant glycoside digoxin from Digitalis purpurea. Because of its widespread therapeutic applications excellent antibodies have been developed against digoxin, and even antibodies against digoxigenin strongly recognize digoxin.
Digoxin has been derivatized by periodate oxidation of vicinal diols at terminal (nonreducing) digitoxose (Takemura et al., 1994; Adamczyk et al., 1995
). However, in our hands, oxidized digoxin was not stable enough to permit successful synthesis of the desired product. Instead of periodate oxidation, we have used CNBr "activation" which was found to yield significantly more stable product (Kohn and Wilchek, 1981
).
One of the major problems with the study of carbohydratelectin interactions is relatively weak binding in most of these interactions. To overcome this problem we have applied a dual strategy: (1) we have used polyvalent oligosaccharides which usually bind more strongly to lectins, and (2) we incorporated photoreactive crosslinker in the probe. With the enhanced binding and formation of a covalent linkage between the probe and neighboring proteins, we were able to, among all proteins in the sample, selectively label only those proteins which were adjacent to the probe at the moment of illumination. With an appropriate concentration of the probe, vast majority of the labeled proteins will be those with affinity for the glycoprobe. By comparing results obtained with glycoprobes containing different glycan structures, proteins which bind nonspecifically, or recognize digoxin and/or other parts of the probe can be identified and differentiated from those that specifically recognize glycans.
Contrary to the frequently used detection methods such as RIA, where receptor-ligand complexes have to survive all subsequent steps (incubation with antibodies, washing, etc.) to be detected, our glycoprobe method establishes covalent bonds by photolabeling, after which the lectin activity of the protein will not be important. This enables the analysis of labeled proteins by SDS-PAGE and permits direct identification of different lectins in the sample, as well as their approximate molecular sizes. With appropriate standardization, even semiquantitative analysis of lectin activity is possible. In addition, digoxin tag enables one-step purification of labeled lectins using affinity columns with immobilized anti-digoxin antibodies. High sensitivity of digoxin to acidic conditions (Gault et al., 1977) provides an easy way to remove bound lectins from the affinity column without the need for harsh conditions which could denature or inactivate the protein.
Despite the usefulness of glycoprobes, some caution is necessary when interpreting obtained results. Under some conditions photolabeling can result in dimerization or even oligomerization of labeled proteins. Furthermore, although digoxin does not exist in humans or animals, several proteins does bind to it to some extent (e.g., ability of digoxin to bind to and inhibit Na+/K+ ATPase is being used to strengthen cardiac activity for centuries), and it is possible that some of the detected bands could be digoxin-binding, and not carbohydrate-binding proteins. Adequate controls, like specific inhibitors, "blank" glycoprobes, or glycoprobes containing noncompatible carbohydrate structures should be used before attributing lectin activity to individual bands on the blot.
Conclusions
Despite rapid growth of glycobiology in the last few years and significant improvements of methods to study oligosaccharide structures, there is still no convenient method to directly identify potential lectin receptors for the defined oligosaccharides. We have hereby described synthesis and application of a novel glycobiological tool which we have named glycoprobe. We have demonstrated its functionality and its applicability by identifying asialoglycoprotein receptor in rat liver membranes and conglutinin in bovine serum. Glycoprobe is expected to be useful in identification of receptors for specific gangliosides confined to distinct population of neurons (Heffer-Lauc et al., 1998), demonstration of lectins binding to stress-induced glycoproteins (Barisic et al., 1996
; Lauc et al., 1999
), or some other cases of physiological carbohydratelectin interaction (Varki, 1993
). Though it was developed for identification of lectins, the strategy for glycoprobe can be adapted for identification of receptors for other small biomolecules such as neuropeptides and hormones.
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Materials and methods |
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General methods
Thin-layer chromatography.
Thin-layer chromatography (TLC) was performed on 0.2 mm Silica Gel 60 F254 precoated on umalinum sheets (Merck, Darmstadt, Germany). Various solvents were used for development and are described for each application. All solvent ratios are expressed as volume ratios (v/v).
High pressure liquid chromatography.
High pressure liquid chromatography (HPLC) was performed on a Gilson system with two model 303 HPLC pumps. Eluate from the column was monitored by ISCO V4 variable absorbance UV detector. A Phenomenex Jupiter 5u C4 300A column (250 x 4.6 mm) was used and eluted with a gradient of acetonitrile in water. TFA (0.01%) was included in both solvents. Exact gradients are refined for each application. When needed, fractions were collected manually for further manipulation.
Mass spectrometry.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was performed using a Kompact MALDI 4 from Kratos Analytical (Manchester, England) located in the Middle Atlantic Mass Spectrometry Laboratories at the Johns Hopkins University Medical School (Baltimore, MD).
Dry samples were resuspended in 510 µl of 50% acetonitrile, 0.3 µl portion was deposited on the target, supplemented with 0.3 µl of saturated solution of 6-aza-2-thiothymine (matrix) and air-dried. Nitrogen laser pulses (337 nm) were used for desorption and ionization, while the extraction voltage was set to 20 kV. Spectra were acquired in both positive and negative linear modes. Each spectrum was the average of 50 laser irradiations.
Electrophoresis and blotting.
Proteins were separated electrophoretically in SDS-polyacrylamide slab gels as described by Laemmli (Laemmli, 1970) and transferred onto Immobilon PVDF (polyvinylidene difluoride) membrane using a wet-blotting system (Bio-Rad) according to Towbin (Towbin et al., 1979
). After blotting, membranes were blocked overnight with 3% BSA, and digoxigenin-conjugated protein bands were visualized by incubation with anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. 5-Bromo-4-chloro-3-indolyl phosphate (0.02 mg/ml) in 50 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2 was used as a substrate, and the color was intensified with addition of 0.04 mg/ml nitro-blue tetrazolium.
Synthesis of the pre-glycoprobe
Activation of digoxin using CNBr.
Digoxin (100 µmol) was added to a final concentration of 33 mM in 33% tetrahydrofuran, 66% 2 M K-phosphate buffer (pH = 12) to form a biphasic mixture. CNBr (20-fold excess) was added to the mixture as 5 M solution in acetonitrile. Reaction mixture was stirred at room temperature for 3060 min, and the formation of the product was monitored by reversephase HPLC (30 min linear gradient 050% acetonitrile in water, 0.01% TFA was included in both solvents). Digoxin (starting material) eluted after 22.4 min, while the more hydrophobic reaction product (CNBr-activated digoxin) eluted after 26 min. The product yield was usually 4060% (as estimated from the A220 nm peaks). If CNBr was excluded from the reaction mixture, neither TLC nor HPLC analysis revealed any change in digoxin within 60 min of the reaction, suggesting unusual stability of the lactone ring to alkaline conditions.
The reaction mixture was evaporated under reduced pressure, and the dried powder was redissolved in 10 ml of chloroform and 10 ml of 1 M NaCl. After vigorous shaking, the phases were separated and the water phase was extracted with additional 10 ml chloroform. Virtually all digoxin derivative was found in the combined chloroform phases. The combined chloroform phases were briefly washed with 5 ml water to remove any residual water-soluble material, and dried under reduced pressure. According to HPLC and/or TLC analysis, the "activated" digoxin could be routinely obtained in ~50% yield. The unreacted digoxin does not interfere with the next reaction step. If stored dry, the ratio of "activateddigoxin" to digoxin remained constant for several weeks at room temperature.
Conjugation of -Fmoc-lysine with digoxin.
-N-(9-Fluorenylmethoxycarbonyl)-L-lysine (Fmoc-Lys, 50 µmol) was dissolved in 66% tetrahydrofuran, 33% 0.1 M Na-phosphate buffer (pH 7) to a final concentration of 40 mM. This mixture was added to a vial containing
50 µmol of dried activated digoxin (100 µmol of total digoxin, molar ratio of
-Fmoc-Lys to "activateddigoxin" was approximately 1 to 1 assuming 50% activation) and reacted for 1824 h at 42°C with vigorous shaking (wrist-action shaker) to prevent separation of phases. The reaction was followed by thin layer chromatography using chloroform:methanol:water (v/v) 80:20:1 as a developing solvent.
-Fmoc-lysine was identified under UV lamp and digoxin by "charring" after spraying with 15% H2SO4 in 95% ethanol. When ca. 60% of
-Fmoc-lysine (RF = 0.13) was converted to Fmoc-Lys-Dig (RF = 0.52) the reaction was stopped by drying.
The dried mixture of starting materials (digoxin, "activated" digoxin, and -Fmoc-lysine) and the reaction product (Fmoc-Lys-Dig) was dissolved in 10 ml of chloroform and 10 ml of 1% TFA in water and vigorously shaken for 3 min. NaCl (0.2 ml of 5 M solution) was added to facilitate separation of phases. After separation of phases, the water phase, containing unreacted
-Fmoc-Lysine and phosphates, was extracted with 2 x 5 ml chloroform and discarded.
To the pooled chloroform solution (20 ml) was slowly added 18 ml hexanes and the mixture was gently mixed for 10 min. During this time, precipitate was formed which consisted mainly of Fmoc-Dig-Lys and some digoxin. The precipitate adhered strongly to glass walls and enabled easy removal of the clear supernatant. After evaporation, the supernatant was dissolved in 5 ml of chloroform and precipitated with 4.5 ml of hexanes. Precipitate from both vials was combined and designated as Fmoc-Lys-Dig.
Preparation of the N-hydroxysuccinimide ester of Fmoc-Lys-Dig (pre-glycoprobe).
The free COOH group in Fmoc-Lys-Dig was activated by esterification with N-hydroxysuccinimide. Approximately 20 µmol of Fmoc-Lys-Dig (final concentration 130 mM) was incubated with N-hydroxysuccinimide (600 mM) and EDAC (250 mM) in distilled dry DMF. Small amount of anhydrous NaH2PO4 was added to the reaction vial to provide sufficient buffering capacity to prevent hydrolysis of both digoxin and Fmoc. After 34 h, virtually all Fmoc-Lys-Dig (RF = 0.52) was converted to RF = 0.92 material (NHS ester) and the reaction was stopped by evaporation of DMF under reduced pressure.
After two extractions with chloroform:water (1:1) to remove free N-hydroxysuccinimide, EDAC and phosphates, the combined chloroform phase was evaporated and dissolved in 30% acetonitrile, 0.01% TFA. A quarter of this mixture was separated by HPLC on a C4-column (4.6 x 25 mm) by a linear gradient of 0.01%TFA in water and 0.01% TFA in acetonitrile (0 min, 40%; 15 min, 70%; 15.1 min, 90%; 20 min, 90%; 20.1 min, 40%; 25 min, 40% acetonitrile).
The major peak absorbing at 254 nm eluted after 14.8 min and according to MALDI-MS analysis (Figure 5) contained -Fmoc-(
-digoxin)-lysine-N-hydroxysuccinimide ester (pre-glycoprobe). TLC analysis using chloroform:methanol:water (80:20:1) revealed existence of one major spot (RF = 0.92) and a trace amount (<5%) of Fmoc-Dig-Lys (RF = 0.52), presumably formed during evaporation of the HPLC fractions. The major contaminant, digoxin, eluted at 5.2 min and was clearly separated from the pre-glycoprobe.
Preparation of the photoaffinity glycoprobe
Incorporation of glycopeptides in the pre-glycoprobe.
Two hundred nanomoles of Man9 or YEE(ahGalNAc)3 was dissolved in 10 µl of 0.5 M Na-phosphate buffer (pH 7.0). Pre-glycoprobe (1 µmol) was dissolved in 10 µl of DMF, mixed with glycopeptides (pre-glycoprobe to glycopeptide ratio 5:1) and incubated at room temperature. Reaction was monitored by TLC using chloroform:methanol:water 65:25:1 solvent system. After 4 h reaction at room temperature, virtually all glycopeptides (RF = 0) were incorporated into a glycoprobe (RF = 0.01 and 0.2 for Man9-glycoprobe and YEE(ahGalNAc)3-glycoprobe, respectively).
The reaction mixture was evaporated and dissolved in a mixture of 100 µl chloroform and 100 µl water. After extraction, all glycoprobes remained in the water phase while unnreacted and hydrolyzed pre-glycoprobe was extracted into chloroform. The released N-hydroxysuccinimide remained in the water phase, but since it did not interfere with the subsequent operation, no further purification steps were performed.
Neat piperidine (25 µl) was added to the water phase and incubated for 10 min at room temperature to deprotect -amino group of lysine. Free fluorenylmethanol and piperidine were removed by toluene extraction while oligosaccharide-containing glycoprobes remained in the water phase.
Attachment of the photoreactive crosslinker.
A photoreactive crosslinker was incorporated into glycoprobes using N-hydroxysuccinimide ester of 6-(4-azido-2-nitrophenylamino)hexanoic acid. All reactions were performed in darkness. One micromole 6-(4-azido-2-nitrophenylamino)hexanoic acid N-hydroxysuccinimide ester was incubated with ~0.2 µmol of YEE(ahGalNAc)3 or Man9 glycoprobes in 200 µl of 50% tetrahydrofuran, 50% 0.1 M Na-phosphate buffer (pH 7.0) for 4 h. Two micromoles benzylamine was added and incubated for 2 h at room temperature to quench residual N-hydroxysuccinimide esters.
Water (100 µl) and chloroform (200 µl) were added to the mixture and after vigorous shaking, layers were separated. Water phase containing final photoaffinity glycoprobe and N-hydroxysuccinimide was collected, while the chloroform phase containing other reactants was discharged. The photoaffinity glycoprobes were divided into small portions (~20 nmol), evaporated and stored in dry state at 20°C.
Application of the photoaffinity glycoprobe: labeling of lectins with the photoaffinity glycoprobe
Protein sample (rat liver membranes or serum) was diluted with PBS supplemented with 1 mM Ca2+ and Mg2+ to a final volume of 10 µl. Dried glycoprobe was dissolved in 50% DMF in 0.1 M Na-phosphate buffer (pH 7.0) and diluted with PBS supplemented with 1 mM Ca2+ and Mg2+ to the final concentration of 10 µM. Ten µl of the probe was added to the sample and incubated in the dark at room temperature for 60 min to allow specific binding of glycans in the glycoprobe by lectins. The probe was then crosslinked to the adjacent proteins by illumination of the reaction mixture with UV (long wavelength) lamp for 10 min.
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Acknowledgments |
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Footnotes |
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