(Received for publication, March 4, 1997)
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 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.
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 (2-4).
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 (7-9).
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 H2O2 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).
Fructosyl propylamine was prepared as described previously (13). All other materials were analytical grade.
Strains and Growth ConditionsEscherichia coli
strains XL-1 Blue MRF 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.
After anesthetizing, a New Zealand
White rabbit was immunized by injection into popliteal lymph nodes of
50 µg of purified amadoriase II protein (13) emulsified in complete
Freund's adjuvant (Sigma). Three weeks later, booster injections were
performed subcutaneously with 100 µg 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 MRF lysate before use.
Total
RNA was extracted from A. fumigatus (1 g) according to
Chomczynski and Sacchi (20). Poly(A)+ RNA was obtained from
total RNA using an oligo(dT)-cellulose (Pharmacia Biotech Inc.) column.
Five µg of poly(A)+ 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 MRF. A library containing 4.0 × 106 clones
was made, of which approximately 99% were recombinants.
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(
) 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) using Sequenase version 2.0 (U. S. Biochemical Corp.) and
[35S]dATP
S1 (Amersham Life
Science, Inc.) and by ALF DNA Sequence with Auto CycleTM
sequencing kit (Pharmacia).
The
plasmid carrying amadoriase II cDNA was transformed into E. coli XL1-Blue MRF. The transformant was precultured in 10 ml of
LB medium 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 MgCl2, 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.
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 µmol of glucosone/min. Synthesized glucosone was used as a standard.
ImmunoblottingCrude 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 DeterminationProtein 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 BlottingNorthern blot analysis was carried out as
described (24) with total RNA (10 µg) from A. fumigatus
cultured in the presence of various concentrations of fructosyl
propylamine. Random primer 32P-labeled amadoriase II
cDNA was used as a probe. The blots on the membrane were exposed to
Kodak XAR films at 80 °C for 2 days with an intensifying
screen.
The library constructed in Uni-ZAP XR was screened with antiserum
against amadoriase II of A. fumigatus. By screening 5 × 104 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
5-noncoding region and followed by a 20-base pair 3
-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 NH2-terminal 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 (Glu13,
Glu15, and Glu18), which allow for a sharp turn
between the first
-strand and the
-helix, a core of six
hydrophobic amino acids (Leu9, Leu11,
Ala22, Leu25, Val33, and
Val35), and the presence of an acidic residue
(Asp37) that forms a hydrogen bond with the 2
-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 MRF, 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
NH2-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 H2O2, the mechanism and source of H2O2 formation in vivo in relationship to the Maillard reaction remains to be solved. Although metal-catalyzed oxidation of Amadori products can generate glucosone and H2O2 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U82830[GenBank].
We thank Drs. Robert B. Petersen and Matthew C. Weber for helpful discussions and assistance with computerized sequence analysis and John Forgarty for technical assistance with the surgical immunization of rabbits. Sequencing was done in part by automated instruments at the Molecular Biology Core Laboratory, Department of Biochemistry at Case Western Reserve University.
While this publication was in preparation, Yoshida et al. (29) published two homologous sequences. Sequence comparison revealed that amadoriase II from A. fumigatus exhibits 82% identity and 92% similarity with fructosyl amino acid oxidase from Aspergillus terreus (accession number Y09020[GenBank]) and 36% identity and 65% similarity with the same enzyme from Penicillium janthinellum (Y09021[GenBank]), respectively, when calculated without gaps.