Molecular cloning and expression of amadoriase isoenzyme (fructosyl amine:oxygen oxidoreductase, EC 1.5.3) from Aspergillus fumigatus.

Amadoriase is an enzyme catalyzing the oxidative deglycation of Amadori products to yield corresponding amino acids, glucosone, and H2O2. We previously reported the purification and characterization of two amadoriase isozymes from Aspergillus sp. that degrade both glycated low molecular weight amines and amino acids (Takahashi, M., Pischetsrieder, M., and Monnier, V. M. (1997) J. Biol. Chem. 272, 3437-3443). To identify the primary structure of the enzymes, we have prepared a cDNA library from Aspergillus fumigatus induced with fructosyl propylamine and isolated a clone using polyclonal anti-amadoriase II antibody. The primary structure of the enzyme deduced from the nucleotide sequence comprises 438 amino acid residues with a predicted molecular mass of 48,798 Da. The deduced primary structure exhibits the presence of an ADP-binding motif near the NH2 terminus. The identity of the amadoriase II cDNA was further confirmed by expression in Escherichia coli cells with an inducible expression system. Northern blotting analysis revealed that amadoriase II was induced by fructosyl propylamine in a dose-dependent manner.


From the Institute of Pathology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106
Amadoriase is an enzyme catalyzing the oxidative deglycation of Amadori products to yield corresponding amino acids, glucosone, and H 2 O 2 . We previously reported the purification and characterization of two amadoriase isozymes from Aspergillus sp. that degrade both glycated low molecular weight amines and amino acids (Takahashi, M., Pischetsrieder, M., and Monnier, V. M. (1997) J. Biol. Chem. 272, 3437-3443). To identify the primary structure of the enzymes, we have prepared a cDNA library from Aspergillus fumigatus induced with fructosyl propylamine and isolated a clone using polyclonal anti-amadoriase II antibody. The primary structure of the enzyme deduced from the nucleotide sequence comprises 438 amino acid residues with a predicted molecular mass of 48,798 Da. The deduced primary structure exhibits the presence of an ADP-binding motif near the NH 2 terminus. The identity of the amadoriase II cDNA was further confirmed by expression in Escherichia coli cells with an inducible expression system. Northern blotting analysis revealed that amadoriase II was induced by fructosyl propylamine in a dose-dependent manner.
Nonenzymatic glycation of proteins has been implicated in the development of diabetic complications and the aging process (1). This reaction leads to dysfunction of target molecules through formation of sugar adducts and cross-links. Considerable interest has focused on the Amadori product that is the most important "early" modification during nonenzymatic glycation in vitro and in vivo. Amadori products are precursors of protein cross-links, fluorescent and UV-active compounds, and glycoxidation products such as pentosidine and N ⑀ -(carboxymethyl)lysine (2)(3)(4). Furthermore, Amadori products are a potent source of reactive oxygen species (5,6) and can be taken up by specific receptors in macrophages and mesangial and aortic cells (7)(8)(9).
To elucidate the effects of nonenzymatic glycation, we (and other investigators (14 -19)) have searched for novel ways to deglycate proteins enzymatically (10 -13). In a previous paper, we reported the presence of amadoriase isozymes in Aspergillus sp. that could degrade both glycated low molecular weight amines and amino acids (13). Most deglycating enzymes isolated so far (13-15, 18, 19), except for a Pseudomonas enzyme (11,12), have several features in common, such as 1) molecular mass of approximately 50 kDa, 2) FAD as a cofactor, and 3) the release of glucosone and H 2 O 2 during the catalytic deglycation of glycated amino acids. Since these products are potentially highly damaging molecules, we hypothesized that implication of such enzymes in diabetes might contribute to the development of complications (13).
In this paper, we describe the cloning, sequencing, and expression of cDNA of amadoriase II from a cDNA library of Aspergillus fumigatus from soil (13).

EXPERIMENTAL PROCEDURES
Materials-Fructosyl propylamine was prepared as described previously (13). All other materials were analytical grade.
Strains and Growth Conditions-Escherichia coli strains XL-1 Blue MRFЈ and SOLR (Stratagene) were used. A. fumigatus was isolated from soil and cultivated as described previously (13). Batch cultures were grown in nutrient broth containing 1 mg/ml fructosyl propylamine, and incubation was carried out at 37°C for 3 days aerobically.
Preparation of Antiserum-After anesthetizing, a New Zealand White rabbit was immunized by injection into popliteal lymph nodes of 50 g of purified amadoriase II protein (13) emulsified in complete Freund's adjuvant (Sigma). Three weeks later, booster injections were performed subcutaneously with 100 g of enzyme emulsified in incomplete Freund's adjuvant. Antiserum was obtained 10 days after the booster injection. Nonspecific antibody was reduced by incubating with XL1-Blue MRFЈ lysate before use.
Construction of the Aspergillus sp. cDNA Library-Total RNA was extracted from A. fumigatus (1 g) according to Chomczynski and Sacchi (20). Poly(A) ϩ RNA was obtained from total RNA using an oligo(dT)cellulose (Pharmacia Biotech Inc.) column. Five g of poly(A) ϩ RNA were converted to a double-stranded cDNA and ligated to the Uni-ZAP XR vector using a ZAP cDNA synthesis kit (Stratagene) according to the manufacturer's instruction. The resulting DNA was packaged using Gigapack Gold III (Stratagene) and amplified in E. coli XL1-Blue MRFЈ. A library containing 4.0 ϫ 10 6 clones was made, of which approximately 99% were recombinants.
Cloning of Amadoriase II cDNA-Screening of the cDNA library was carried out as follows. After transferring plaques onto nitrocellulose filters (Micron Separations Inc.), plaques presenting amadoriase II epitopes by isopropyl-␤-D-thiogalactopyranoside induction were detected by anti-amadoriase II antiserum and goat anti-rabbit IgG alkaline phosphatase conjugate. Positive clones were converted to phagemids carrying cDNA inserts between EcoRI and XhoI sites of pBluescript SK(Ϫ) by helper phage superinfection as described in the Uni-ZAP XR manufacturer's manual (Stratagene). Nucleotide sequences of both strands were determined by the dideoxy chain termination method (21)  containing 50 mg/ml ampicillin and 40 mg/ml tetracycline for 150 min and then for 6 h in the presence of 10 mM isopropyl-␤-D-thiogalactopyranoside. The cells were harvested by centrifugation and suspended in 1 ml of ice-cold lysis buffer (25 mM Hepes-NaOH, pH 7.6, 100 mM KCl, 0.1 mM EDTA, 12.5 mM MgCl 2 , 0.1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10% (v/v) glycerol). After one freeze-thaw cycle, the cells were disrupted by sonication. The cell debris were removed by centrifugation, and the supernatant was subjected to enzyme assays and immunoblot analysis.
Assays for Enzymatic Activity-The enzyme activity was monitored by the release of glucosone as measured by a colorimetric reaction with o-phenylenediamine as described previously (13). Briefly, the enzyme solution added to the reaction mixture contained 20 mM sodium phosphate, pH 7.4, 10 mM o-phenylenediamine, 10 mM fructosyl propylamine in a final volume of 1 ml. After incubation at 37°C for 2 h, the absorbance at 320 nm was measured. One unit of enzyme activity was defined as the amount of enzyme that produces 1 mol of glucosone/ min. Synthesized glucosone was used as a standard.
Immunoblotting-Crude extracts were subjected to 10% SDS-PAGE, transferred to nitrocellulose (Bio-Rad), and probed using anti-amadoriase II antiserum and goat anti-rabbit IgG alkaline phosphatase conjugate.
Protein Determination-Protein concentration was determined by the method of Bradford (22) with a Bio-Rad protein assay kit using bovine serum albumin as a standard. Protein analysis by SDS-PAGE was carried out according to Laemmli (23).
Northern Blotting-Northern blot analysis was carried out as described (24) with total RNA (10 g) from A. fumigatus cultured in the presence of various concentrations of fructosyl propylamine. Random primer 32 P-labeled amadoriase II cDNA was used as a probe. The blots on the membrane were exposed to Kodak XAR films at Ϫ80°C for 2 days with an intensifying screen.

RESULTS AND DISCUSSION
The library constructed in Uni-ZAP XR was screened with antiserum against amadoriase II of A. fumigatus. By screening 5 ϫ 10 4 plaques, we obtained six positive clones. Sequence analysis of one of these clones is shown in Fig. 1. The amadoriase II coding sequence was considered to have 1314-base pair coding for a novel protein of 438 amino acids (nucleotide positions 174 -1487). The open reading frame is preceded by a 173-base pair 5Ј-noncoding region and followed by a 20-base pair 3Ј-noncoding region and a poly(A) tail. We assumed the ATG codon at positions 174 -177 as the translational initiation codon for the following reasons. First, the sequence of the first 21 deduced amino acid residues was consistent with the NH 2terminal sequence obtained from the purified protein except for the first methionine (13). Second, the molecular mass calculated and corrected for loss of one methionine was 48,798 daltons, which is in agreement with values previously estimated by SDS-PAGE (13). Potential ribosome-binding sites (AGGA) were located 8 bases before the start codon. Close inspection revealed that amino acids present at positions 8 -37 satisfy all of the 11 consensus sequence requirements for an ADP-binding domain (25). Conserved features include three glycine residues (Glu 13 , Glu 15 , and Glu 18 ), which allow for a sharp turn between the first ␤-strand and the ␣-helix, a core of six hydrophobic amino acids (Leu 9 , Leu 11 , Ala 22 , Leu 25 , Val 33 , and Val 35 ), and the presence of an acidic residue (Asp 37 ) that forms a hydrogen bond with the 2Ј-OH group of the FAD adenine ribose. These are capable of forming a structural motif ␤␣␤ fold to interact with the bottom of the ADP moiety of the FAD cofactor (25).
A search for protein homology using BLAST (26) revealed that amadoriase II has no significant similarities with mammalian enzymes reported so far, implying that amadoriase II is an enzyme representing a new family of fructosyl amine:oxygen oxidoreductases.
To confirm the identification of the cDNA, pBluescript II containing amadoriase II cDNA was introduced into E. coli XL1-Blue MRFЈ, and the enzyme was expressed as a ␤-galactosidase fusion protein. Cells were disrupted by sonication, and the extract was assayed for amadoriase activity using fructosyl  propylamine as a substrate. As shown in Table I, the transformant exhibited significant activity, whereas the control cells showed no activity. Immunoblot analysis was performed to examine whether the amadoriase II protein was indeed present in the transformant (Fig. 2). The transformant cell extracts exhibited a band that was not seen in control cells. This band had a molecular size that is slightly larger than the purified enzyme, and this increase in size was consistent with the fusion of the NH 2 -terminal region of ␤-galactosidase to the enzyme. These results confirmed that the cloned cDNA encodes 49-kDa amadoriase II.
Using amadoriase II cDNA as a hybridization probe, Northern blotting analysis was performed for an A. fumigatus that was cultured in the presence of various concentrations of fructosyl propylamine. As shown in Fig. 3, amadoriase II was induced in a dose-dependent manner. This result suggests the existence of a mechanism for up-regulation of amadoriase II by an Amadori compound. To our knowledge, no other protein has been reported that is inducible by an Amadori compound. However, it is unclear yet whether the Amadori product binds to an intracellular receptor or whether it induces gene expression through indirect pathways, such as oxidative stress.
In summary, we have described the primary structure of amadoriase II from A. fumigatus, which was selected by using fructosyl adamantanamine and cultured in the presence of fructosyl propylamine. Although the BLAST search detected no significant homology of amadoriase II with any human protein in the data base, the possible occurrence of amadoriase homologues in human tissue could have considerable implications for the development of diabetic complications. While growing evidence implicates Amadori products in the formation of H 2 O 2 , the mechanism and source of H 2 O 2 formation in vivo in relationship to the Maillard reaction remains to be solved. Although metal-catalyzed oxidation of Amadori products can generate glucosone and H 2 O 2 in vivo (6,27,28), the existence of an enzyme such as amadoriase suggests a possible role of flavin as an electron acceptor in oxidation of Amadori products.