Mechanism-based fluorescent labeling of β -galactosidases: An efficient method in proteomics for glycoside hydrolases

(4- N -Dansyl-2-difluoromethylphenyl)- β -D-galactopyranoside was synthesized, and successfully tested on β -galactosidases from Xanthomonas manihotis [Wong-Madden, Glycobiology (1995) 5 , 19-28; Taron, Glycobiology (1995) 5 , 603-610], E. coli [Jacobson, Nature (1994) 369 , 761-766], and Bacillus circulans [Fujimoto, Glycoconjugate J . (1988) 15 , 155-160] for the rapid identification of the catalytic site. Reaction of the irreversible inhibitor with enzymes proceeded to afford a fluorescent labeled protein suitable for further high-throughput characterization by using anti-dansyl antibody and MALDI-TOF/TOF. Specific probing by a fluorescent aglycon greatly facilitated identification of the labeled peptide fragments from β -galactosidases.


INTRODUCTION
The fate of cell surface carbohydrates-bearing proteins and lipids are controlled by the systematic enzymatic reactions. Functional oligosaccharide chains of glycoconjugates such as glycoproteins, proteoglycans, and glycolipids produced by glycosyltransfarases are eventually degraded by the specific individual metabolic systems (1). Glycoside hydrolases (GHs, carbohydrases, glycosidases; EC 3. 2. 1), take part in such degradation of the glycoconjugates (2)(3)(4). In addition, it is well known that glycoside hydrolases for α-D-mannopyranosides (α-mannosidases) have crucial roles as trimming enzymes in the glycoprotein synthetic pathways to control the eventual types of oligosaccharide structures such as high mannose-type, hybrid-type, and complex-type (5,6). Therefore, precise analysis of the structures, functions, and catalytic mechanism of GHs has become one of the most important processes in systematic biosynthesis and metabolism of glycoconjugates. Moreover, it is also evident that fine structural information on the active site of GHs permit rational design and efficient synthesis of potential modulators or inhibitors as sugar-based therapeutic reagents (7)(8)(9). Three-dimensional structures of GHs may be obtained by X ray crystallographic analysis of the crystallized proteins (10)(11)(12)(13). However, large-scale expression and crystallization of proteins often become a bottle neck in the practical protein engineering as well as basic proteomics research by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 5 and structural biology.
Recent progress in mass spectroscopy based on various ionization techniques such as matrix-associated laser desorption ionization (MALDI) and electrospray ionization (ESI) techniques greatly facilitated proteomic analysis useful for post-genomic science and technology. In fact, combined use of mass spectroscopy with high performance liquid chromatography (HPLC) or two-dimensional polyacrylamide gel electrophorasis (2D-PAGE) seems to be the most powerful tools for the high-throughput proteomics research (14-17). In the present paper, we report an efficient method for isolation and characterization of galactosidases based on fluorescence labeling by a suicide substrate. The value of this method for high-throughput proteomics study was demonstrated using Xanthomonas manihotis β-galactosidase as a model GH.
They are distributed widely in a number of microorganisms, plants, and animal tissues (18). Human isozymes of β-galactosidase have been investigated extensively from the standpoint of genetic disorders such as lactose intolerance (19) and mucolipidoses (20)(21)(22)(23)(24). Bacterial isozymes of β-galactosidases served as reporter molecules for the by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 6 evaluation of gene regulation (25). β-Galactosidases are divided into four distinct GH families, GH-1, GH-2, GH-35, and GH-42 based on the similarity in nucleotide and amino acid sequences (26)(27)(28)(29). However, at present, only limited information on three dimensional structure, substrate binding site, catalytic site, and post-translational modifications are available. Considering the difficulties in isolation and characterization as well as expression of various isozymes of β-galactosidases, an alternative method to alleviate these time-consuming and tedious procedures for the functional proteomics of the GHs is highly desired.
In 1990, Danzin et al. developed and reported an irreversible inhibitor (suicide substrate) of β-galactosidases based on a fluoromethylated aryl group as a novel aglycon (30). The reactive moiety generated from an aglycon conveniently labeled active site of the GHs for subsequent characterization, because nucleophilic attack of this aglycon by a nearby amino acid side chain at the active site gives rise to a tagged amino acid residue. Subsequently, applications of this principle have emerged.
Ichikawa et al. developed a novel biotin-conjugated suicide substrates for isolation and characterization of N-acetyl-β-D-glucosaminidase (31). Janda and co-workers have reported a valuable application of this tagging strategy for chemical selection of catalytic antibody having β-galactosidase-like activity from a phage library (32). 7 Plückthun and co-workers have also succeeded in trapping a catalyst with alkaline phosphatase activity by using similar mechanism-based chemical selection strategy (33).
In adopting this principle, we chose fluorescent labeling of GH followed by peptide fingerprinting by mass spectroscopy in the high-throughput proteomics of GHs.
When a glycosidase is expressed and produced using common bacterial systems, identification of the target protein in a cell lysate, a complex mixture of proteins and other biomolecules, requires rather tedious sequence of blotting, digestion and staining procedures prior to mass spectroscopic analyses (17). In addition, complexity of peptide fragments derived by treating the tagged proteins with specific peptidases still makes precise analyses of peptide sequences difficult. Therefore, we thought that irreversible fluorescence-labeling of GHs would facilitate isolation and identification of labeled active-site peptides as well as labeled GHs. We herein demonstrate that a mechanism-based tagging of β-galactosidases with a fluorescent-labeled suicide substrate ( Figure 1  MALDI TOF mass spectrometry (34,35). Samples were desalted and concentrated using 10-µL C18 ZipTips TM (Millipore) according to the manufacturer's instructions.
Typically, samples were dissolved in 1 µL of 90% (v/v) acetonitrile containing trifluoroacetic acid (TFA) and mixed with the same volume of a saturated solution of 2,5-dihydroxy-benzoic acid (DHB) in 33% acetonitrile containing 0.1% TFA. The above mixtures (1 µL) were applied to a stainless steel target MALDI plate and air-dried before analysis in the mass spectrometer. All measurements were performed using an Ultraflex TOF/TOF mass spectrometer equipped with a reflector, and controlled by the
Proteins were blotted to PVDF membranes (Immun-Blot TM PVDF membrane, Bio-Rad), and the dansylated proteins were detected with the Donkey anti-rabbit IgG AP conjugate (Promega. Co. Ltd) and anti-dansyl rabbit IgG complex using Western Blue (Promega. Co. Ltd) as a substrate for alkaline-phosphatase. After blocking using 0.3% BSA in TBS buffer, the primary and secondary anti-body were used at a dilution of 1:5000 (about 0.2 µg/mL) and exposed to the membranes for 30 min at 37°C, and washed with TBS containing 0.01% Tween 20 at each step. The final development with alkaline-phosphatase was performed at 37°C for 20 min.   We also examined efficacy of this inhibitor as a fluorescent labeling reagent for these enzymes. All β-galactosidases used in this study were successfully labeled by this reagent, because enzymes exhibited significant fluorescence emission attributable to dansyl group (λ em = 520 nm) only after mixed with the suicide substrate. Western blotting of these enzymes also indicated covalent labeling by compound 4 as shown in   Characterization of catalytic domain of β-galactosidases from Xanthomonas manihotis, E. coli, and Bacillus circulans. We firstly utilized β-galactosidase from Xanthomonas manihotis (39,40) because this enzyme showed the lowest affinity (K i =38.5 mM, t 1/2 =21.1 min) for the suicide substrate 4 among enzymes used herein as indicated in Figure 2(d). Thus, the labeled protein was treated with trypsin and endoproteinase Glu-C to give complex peptide fragments observed in MALDI-TOF mass spectra. However, the MALDI-TOF mass spectrum of the peptide readily  observed. In addition, the fragmentation from a dansylated arginine was detected, allowing us to conclude that this peptide (the calculated molecular mass; [M+H] 1404.68) was sequence as 56 IPRAYWKD 63 in which Arg-58 was dansylated.
Interestingly, it was found by homology analysis (LALIGN, Local Alignments) of amino acid sequences of three β-galactosidases listed in PDB (10-12) that the sequence from Leu-46 to Tyr-194 including the Arg-58 dansylated sequence of Xanthomonas manihotis β-galactosidase seems to be quite similar to the sequence corresponding to the active site of Thermus thermophilus β-galactosidase (12), the sequence from Met-1 to Tyr-151 (data not shown). Although these sequences of 151 amino acids between Thermus thermophilus and Xanthomonas manihotis β-galactosidases have low homology (25.3%), one may speculate on the commonality that Glu-184 can be the catalytic nucleophile, and Arg-147 and Asn-183 may provide hydrogen bonding with OH groups of the galactose residue. In fact, a plausible model of three-dimensional structure clearly predicts that the Arg-58 residue of Xanthomonas manihotis β-galactosidase seems to locate quite similar position in the active site to the corresponding Lys-13 residue in the TIM barrel structure of the Thermus thermophilus β-galactosidase known as a member of GH-42 family (12). This similarity of the amino acid sequence was not found in other two β-galactosidases from E. coli (10) and hyperthermophilic archeon Sulfolobus solfatataricus (11). 22 MALDI-LIFT-TOF/TOF is a nice tool for the identification of such (post translational) modification sites with unstable functional groups such as hemiacetal and/or acyl related linkages found in this case as well as labile O-glycans (41). As for Bacillus circulans (42), it was suggested that one of the two catalytic amino acids, Glu-259