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1.
Ghaderi D Strauss HM Reinke S Cirak S Reutter W Lucka L Hinderlich S 《Journal of molecular biology》2007,369(3):746-758
The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key enzyme for the biosynthesis of sialic acids, the terminal sugars of glycoconjugates associated with a variety of physiological and pathological processes such as cell adhesion, development, inflammation and cancer. In this study, we characterized rat GNE by different biophysical methods, analytical ultracentrifugation, dynamic light-scattering and size-exclusion chromatography, all revealing the native hydrodynamic behavior and molar mass of the protein. We show that GNE is able to reversibly self-associate into different oligomeric states including monomers, dimers and tetramers. Additionally, it forms non-specific aggregates of high molecular mass, which cannot be unequivocally assigned a distinct size. Our results also indicate that ligands of the epimerase domain of the bifunctional enzyme, namely UDP-N-acetylglucosamine and CMP-N-acetylneuraminic acid, stabilize the protein against aggregation and are capable of modulating the quaternary structure of the protein. The presence of UDP-N-acetylglucosamine strongly favors the tetrameric state, which therefore likely represents the active state of the enzyme in cells. 相似文献
2.
UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase) is the key enzyme in the de novo synthesis pathway of neuraminic acid, which is widely expressed as a terminal carbohydrate residue on glycoconjugates. UDP-GlcNAc 2-epimerase is a bifunctional enzyme and catalyzes the first two steps of neuraminic acid synthesis in the cytosol, the conversion of UDP-N-acetylglucosamine to ManAc and the phosphorylation to ManAc-6-phosphate. So far, regulation of this essential enzyme by posttranslational modification has not been shown. Since UDP-N-acetylglucosamine is a cytosolic protein containing eight conserved motifs for protein kinase C (PKC), we investigated whether its enzymatic activity might be regulated by phosphorylation by PKC. We showed that UDP-GlcNAc 2-epimerase interacts with several isoforms of PKC in mouse liver and is phosphorylated in vivo. Furthermore, PKC phosphorylates UDP-GlcNAc 2-epimerase and this phosphorylation results in an upregulation of the UDP-GlcNAc 2-epimerase enzyme activity. 相似文献
3.
The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme for the biosynthesis of sialic acids, terminal components of glycoconjugates
associated with a variety of physiological and pathological processes. Different protein isoforms of human and mouse GNE,
deriving from splice variants, were predicted recently: GNE1 represents the GNE protein described in several studies before,
GNE2 and GNE3 are proteins with extended and deleted N-termini, respectively. hGNE2, recombinantly expressed in insect and
mamalian cells, displayed selective reduction of UDP-GlcNAc 2-epimerase activity by the loss of its tetrameric state, which
is essential for full enzyme activity. hGNE3, which had to be expressed in Escherichia coli, only possessed kinase activity, whereas mGNE1 and mGNE2 showed no significant differences. Our data therefore suggest a
role of GNE1 in basic supply of cells with sialic acids, whereas GNE2 and GNE3 may have a function in fine-tuning of the sialic
acid pathway. 相似文献
4.
Enhanced sialylation of EPO by overexpression of UDP-GlcNAc 2-epimerase/ManAc kinase containing a sialuria mutation in CHO cells 总被引:1,自引:0,他引:1
Sialylation (e.g. expression of sialic acid) plays a crucial role for function and stability of most glycoproteins. The key enzyme for the biosynthesis of sialic acid is the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE). Mutations in the binding site of the feedback inhibitor CMP-sialic acid of the GNE leads to sialuria, a disease in which patients produce sialic acid in gram scale. Here, we report on the use in biotechnology of sialuria-mutated GNE. Expression of the sialuria-mutated GNE in CHO-cells leads to increased sialylation of recombinant expressed erythropoietin (EPO). Our data show that sialuria-mutated-GNE over-expressing cells are the perfect platform to express highly sialylated therapeutic proteins, such as EPO. 相似文献
5.
The metal ion requirement for both enzymatic activitiesof the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosaminekinase (E.C. 5.1.3.14/ 2.7.1.60), the key enzyme of N-acetylneuraminic acidbiosynthesis in ratliver, was investigated. UDP-N-acetylglucosamine 2-epimerase was active inimida-zole/HCl buffer in the complete absence of any metal ion. 200 mM Na + , K + , Rb + and Cs +activated enzymeactivity up to five-fold, whereas lower concentrations of thesemonovalent metal ions showed only a small effect on UDP-N-acetylglucosamine 2-epimeraseactivity. In sodium phosphate buffer the enzyme activitywas increased by 0.5 mM Mg , Sr , Ba and Mn , while in the presence of 200 mM NaCl UDP-N-acetyl-glucosamine2-epimerase activity showed astronger activation by these divalent metal ions. In imidazole/HClbuffer, UDP-N-acetylglucosamine2-epimerase activity was partially inhibited by 0.5 mM Be , Mg , Ba ,Mn , Sn and Fe , and completely inhibited by 0.5 mM Zn and Cd . Divalent metal ions were essen-tialforN-acetylmannosamine kinase activity, the most effective being Mg , followed byMn and Co .The optimal concentration of these metal ions was 3 mM. Less effective were Ni and Cd , whereas Ca ,Ba , Cu , Fe and Zn showed no effect on enzyme activity. 相似文献
6.
7.
Blume A Ghaderi D Liebich V Hinderlich S Donner P Reutter W Lucka L 《Protein expression and purification》2004,35(2):387-396
UDP-GlcNAc 2-epimerase/ManNAc kinase is the key enzyme of sialic acid biosynthesis in mammals. Its functional expression is a prerequisite for early embryogenesis and for the synthesis of several cell recognition motifs in adult organism. This bifunctional enzyme is involved in the development of different diseases like sialuria or hereditary inclusion body myopathy. For a detailed understanding of the enzyme, large amounts of the pure active protein are needed. Different heterologous cell systems were therefore analyzed for the enzyme, which was found to be functionally expressed in Escherichia coli, the yeast strains Saccharomyces cerevisiae and Pichia pastoris, and insect cells. In all these cell types, the expressed enzyme displayed both epimerase and kinase activities. In E. coli, up to 2mg protein/l cell culture was expressed, in yeast cells only 0.4mg/L, while up to 100mg/L, were detected in insect cells. In all three cell systems, insoluble protein aggregates were also observed. Purification from E. coli resulted in 100microg/L pure and structurally intact protein. For insect cells, purification methods were established which resulted in up to 50mg/L pure, soluble, and active protein. In summary, expression and purification of the UDP-GlcNAc 2-epimerase/ManNAc kinase in Sf-900 cells can yield the milligram amounts of protein required for structural characterization of the enzyme. However, the easier expression in E. coli and yeast provides sufficient quantities for enzymatic and kinetic characterization. 相似文献
8.
Salama I Hinderlich S Shlomai Z Eisenberg I Krause S Yarema K Argov Z Lochmuller H Reutter W Dabby R Sadeh M Ben-Bassat H Mitrani-Rosenbaum S 《Biochemical and biophysical research communications》2005,328(1):221-226
Hereditary inclusion body myopathy (HIBM) is a unique group of neuromuscular disorders characterized by adult-onset, slowly progressive distal and proximal muscle weakness, which is caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the biosynthetic pathway of sialic acid. In order to investigate the consequences of the mutated GNE enzyme in muscle cells, we have established cell cultures from muscle biopsies carrying either kinase or epimerase mutations. While all myoblasts carrying a mutated GNE gene show a reduction in their epimerase activity, only the cells derived from the patient carrying a homozygous epimerase mutation present also a significant reduction in the overall membrane bound sialic acid. These results indicate that although mutations in each of the two GNE domains result in an impaired enzymatic activity and the same HIBM phenotype, they do not equally affect the overall sialylation of muscle cells. This lack of correlation suggests that the pathological mechanism of the disease may not be linked solely to the well-characterized sialic acid pathway. 相似文献
9.
Krause S Hinderlich S Amsili S Horstkorte R Wiendl H Argov Z Mitrani-Rosenbaum S Lochmüller H 《Experimental cell research》2005,304(2):365-379
The bifunctional enzyme UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) is essential for early embryonic development and catalyzes the rate limiting step in sialic acid biosynthesis. Although epimerase and kinase activities have been attributed to GNE, little is known about the regulation, differential expression, and subcellular localization of GNE in vivo. Mutations in GNE cause a rare inherited muscle disorder in humans called hereditary inclusion body myopathy (HIBM). However, the role of GNE in HIBM pathogenesis has not been defined yet. Here, we show that the GNE protein is expressed in various mammalian cells and tissues with highest levels found in cancer cells and liver. In human skeletal muscle, GNE protein is developmentally regulated: high levels are found in immature myoblasts but low levels in mature skeletal muscle. The GNE protein colocalizes with resident proteins of the Golgi compartment in a variety of human cells including muscle. Drug-induced disruption of the Golgi and subsequent recovery reveals co-distribution of GNE along with Golgi-targeted proteins. This subcellular localization of GNE is in good agreement with its established role as the key enzyme of sialic acid biosynthesis, since the sialylation of glycoconjugates takes place in the Golgi complex. Surprisingly, GNE is also detected in the nucleus. Upon nocodazole treatment, GNE redistributes to the cytoplasm suggesting that GNE may act as a nucleocytoplasmic shuttling protein. A regulatory role for GNE shifting between the nuclear and the Golgi compartment is proposed. Further insight into GNE regulation may promote the understanding of HIBM pathogenesis. 相似文献
10.
N-Acetylneuraminic acid, an important component of glycoconjugates with various biological functions, can be produced from N-acetyl-d-glucosamine (GlcNAc) and pyruvate using a one-pot, two-enzyme system consisting of N-acyl-d-glucosamine 2-epimerase (AGE) and N-acetylneuraminate lyase (NAL). In this system, the epimerase catalyzes the conversion of GlcNAc into N-acetyl-d-mannosamine (ManNAc). However, all currently known AGEs have one or more disadvantages, such as a low specific activity, substantial inhibition by pyruvate and strong dependence on allosteric activation by ATP. Therefore, four novel AGEs from the cyanobacteria Acaryochloris marina MBIC 11017, Anabaena variabilis ATCC 29413, Nostoc sp. PCC 7120, and Nostoc punctiforme PCC 73102 were characterized. Among these enzymes, the AGE from the Anabaena strain showed the most beneficial characteristics. It had a high specific activity of 117 ± 2 U mg−1 at 37 °C (pH 7.5) and an up to 10-fold higher inhibition constant for pyruvate as compared to other AGEs indicating a much weaker inhibitory effect. The investigation of the influence of ATP revealed that the nucleotide has a more pronounced effect on the Km for the substrate than on the enzyme activity. At high substrate concentrations (≥200 mM) and without ATP, the enzyme reached up to 32% of the activity measured with ATP in excess. 相似文献
11.
Katharina Schmölzer Christiane Luley-Goedl Tibor Czabany Doris Ribitsch Helmut Schwab Hansjörg Weber Bernd Nidetzky 《FEBS letters》2014
Bacterial sialyltransferases of the glycosyltransferase family GT-80 exhibit pronounced hydrolase activity toward CMP-activated sialyl donor substrates. Using in situ proton NMR, we show that hydrolysis of CMP-Neu5Ac by Pasteurella dagmatis α2,3-sialyltransferase (PdST) occurs with axial-to-equatorial inversion of the configuration at the anomeric center to release the α-Neu5Ac product. We propose a catalytic reaction through a single displacement-like mechanism where water replaces the sugar substrate as a sialyl group acceptor. PdST variants having His284 in the active site replaced by Asn, Asp or Tyr showed up to 104-fold reduced activity, but catalyzed CMP-Neu5Ac hydrolysis with analogous inverting stereochemistry. The proposed catalytic role of His284 in the PdST hydrolase mechanism is to facilitate the departure of the CMP leaving group. 相似文献
12.
N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) catalyzes the reversible epimerization between N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-mannosamine (ManNAc). We report here the 2.0 A resolution crystal structure of the GlcNAc 2-epimerase from Anabaena sp. CH1. The structure demonstrates an (alpha/alpha)(6) barrel fold, which shows structural homology with porcine GlcNAc 2-epimerase as well as a number of glycoside hydrolase enzymes and other sugar-metabolizing enzymes. One side of the barrel structure consists of short loops involved in dimer interactions. The other side of the barrel structure is comprised of long loops containing six short beta-sheets, which enclose a putative central active-site pocket. Site-directed mutagenesis of conserved residues near the N-terminal region of the inner alpha helices shows that R57, H239, E308, and H372 are strictly required for activity. E242 and R375 are also essential in catalysis. Based on the structure and kinetic analysis, H239 and H372 may serve as the key active site acid/base catalysts. These results suggest that the (alpha/alpha)(6) barrel represents a steady fold for presenting active-site residues in a cleft at the N-terminal ends of the inner alpha helices, thus forming a fine-tuned catalytic site in GlcNAc 2-epimerase. 相似文献
13.
Rotavirus infection leads to the death of half a million children annually. The exact specifics of interaction between rotavirus particles and host cells enabling invasion and infection have remained elusive. Host cell oligosaccharides are critical components, and their involvement aids the virus in cell-recognition and attachment, as well as dictation of the remarkable host-specificity that rotaviruses demonstrate. Interaction between the rotavirus spike-protein carbohydrate-binding domain (VP8*) and cell surface oligosaccharides facilitate virus recognition of host cells and attachment. Rotaviruses are considered, controversially, to recognise vastly different carbohydrate structures and either with incorporation of terminal sialic acid or without, as assessed by their ability to infect cells that have been pre-treated with sialidases. Herein, the X-ray crystallographic structures of VP8* from the sialidase insensitive Wa and the sialidase sensitive CRW-8 rotavirus strains that cause debilitating gastroenteritis in human and pig are reported. Striking differences are apparent regarding recognition of the sialic acid derivative methyl alpha-D-N-acetylneuraminide, presenting the first experimental evidence of the inability of the human rotavirus strain to bind this monosaccharide, that correlates with Wa and CRW-8 recognising sialidase-resistant and sialidase-sensitive receptors, respectively. Identified are structural features that provide insight in attainment of substrate specificity exhibited by porcine strains as compared to rhesus rotavirus. Revealed in the CRW-8 VP8* structure is an additional bound ligand that intriguingly, is within a cleft located equivalent to the carbohydrate-binding region of galectins, and is suggestive of a new region for interaction with cell-surface carbohydrates. This novel result and detailed comparison of our representative sialidase-sensitive CRW-8 and insensitive Wa VP8* structures with those reported leads to our hypothesis that this groove is used for binding carbohydrates, and that for the human strains, as for other sialidase-insensitive strains could represent a major oligosaccharide-binding region. 相似文献
14.
Tadanobu Takahashi Asako Hashimoto Mami Maruyama Makoto Kiso Yoshihiro Kawaoka Yasuo Suzuki Takashi Suzuki 《FEBS letters》2009,583(19):3171-3174
To identify a determinant of human H3 hemagglutinin (HA) amino acid residues linked to the recognition of molecular species of sialic acid, we generated six mutant viruses possessing either the wild-type HA gene from A/Memphis/1/71 (H3N2) or a genetically single-mutated HA gene at position 137, 144, 155, 158 or 193 from a genetic backbone of A/WSN/33 (H1N1) by reverse genetics. We evaluated the binding ability with four types of synthetic sialylglycolipids. The results indicate that the amino acid substitutions Thr155 to Tyr and Glu158 to Gly in H3 HA facilitate virus binding to N-glycolylneuraminic acid. 相似文献
15.
The intracellular concentration of sialic acid regulates the polysialylation of the neural cell adhesion molecule 总被引:1,自引:0,他引:1
Sialic acids are expressed as terminal sugars in many glycoconjugates and play an important role during development and regeneration, as they are involved as polysialic acid in a variety of cell-cell interactions mediated by the neural cell adhesion molecule NCAM. The key enzyme for the biosynthesis of sialic acid is the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE). Mutations in the binding site of the feedback inhibitor CMP-sialic acid of the GNE leads to sialuria, a disease in which patients produce sialic acid in gram scale. Here, we report on the consequences after expression of a sialuria-mutated GNE. Expression of the sialuria-mutated GNE leads to a dramatic increase of both cellular sialic acid and polysialic acid on NCAM. This could also be achieved by application of the sialic acid precursor N-acetylmannosamine. Our data suggest that biosynthesis of sialic acid regulates and limits the synthesis of polysialic acid. 相似文献
16.
Smith CA 《Journal of molecular biology》2006,362(4):640-655
For bacteria, the structural integrity of its cell wall is of utmost importance for survival, and to this end, a rigid scaffold called peptidoglycan, comprised of sugar molecules and peptides, is synthesized and located outside the cytoplasmic membrane of the cell. Disruption of this peptidoglycan layer has for many years been a prime target for effective antibiotics, namely the penicillins and cephalosporins. Because this rigid layer is synthesized by a multi-step pathway numerous additional targets also exist that have no counterpart in the animal cell. Central to this pathway are four similar ligase enzymes, which add peptide groups to the sugar molecules, and interrupting these steps would ultimately prove fatal to the bacterial cell. The mechanisms of these ligases are well understood and the structures of all four of these ligases are now known. A detailed comparison of these four enzymes shows that considerable conformational changes are possible and that these changes, along with the recruitment of two different N-terminal binding domains, allows these enzymes to bind a substrate which at one end is identical and at the other has the growing polypeptide tail. Some insights into the structure-function relationships in these enzymes is presented. 相似文献
17.
Ivan Campeotto Amanda H. Bolt Thomas A. Harman Caitriona Dennis Simon E.V. Phillips Arwen R. Pearson 《Journal of molecular biology》2010,404(1):56-69
The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P21 at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another. 相似文献
18.
The Clostridium botulinum type C 16S progenitor toxin contains a neurotoxin and several nontoxic components, designated nontoxic nonhemagglutinin (HA), HA1 (HA-33), HA2 (HA-17), HA3a (HA-22-23), and HA3b (HA-53). The HA3b subcomponent seems to play an important role cooperatively with HA1 in the internalization of the toxin by gastrointestinal epithelial cells via binding of these subcomponents to specific oligosaccharides. In this study, we investigated the sugar-binding specificity of the HA3b subcomponent using recombinant protein fused to glutathione S-transferase and determined the three-dimensional structure of the HA3a-HA3b complex based on X-ray crystallography. The crystal structure was determined at a resolution of 2.6 Å. HA3b contains three domains, domains I to III, and the structure of domain I resembles HA3a. In crystal packing, three HA3a-HA3b molecules are assembled to form a three-leaved propeller-like structure. The three HA3b domain I and three HA3a alternate, forming a trimer of dimers. In a database search, no proteins with high structural homology to any of the domains (Z score > 10) were found. Especially, HA3a and HA3b domain I, mainly composed of β-sheets, reveal a unique fold. In binding assays, HA3b bound sialic acid with high affinity, but did not bind galactose, N-acetylgalactosamine, or N-acetylglucosamine. The electron density of liganded N-acetylneuraminic acid was determined by crystal soaking. In the sugar-complex structure, the N-acetylneuraminic acid-binding site was located in the cleft formed between domains II and III of HA3b. This report provides the first determination of the three-dimensional structure of the HA3a-HA3b complex and its sialic acid binding site. Our results will provide useful information for elucidating the mechanism of assembly of the C16S toxin and for understanding the interactions with oligosaccharides on epithelial cells and internalization of the botulinum toxin complex. 相似文献
19.
Gengxiang Zhao Nantaporn Haskins Zhongmin Jin Norma M. Allewell Mendel Tuchman Dashuang Shi 《Biochemical and biophysical research communications》2013
Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in l-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by l-arginine, although l-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by l-arginine, we have determined the structure of the mmNAGS/K complexed with l-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of l-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the l-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when l-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by l-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism. 相似文献
20.
Sola-Carvajal A Sánchez-Carrón G García-García MI García-Carmona F Sánchez-Ferrer Á 《Biochimie》2012,94(1):222-230
N-Acyl-d-Glucosamine 2-epimerase (AGE) catalyzes the reversible epimerization between N-acetyl-d-mannosamine (ManNAc) and N-acetyl-d-glucosamine (GlcNAc). Bacteroides ovatus ATCC 8483 shows 3 putative genes for AGE activity (BACOVA_00274, BACOVA_01795 and BACOVA_01816). The BACOVA_00274 gene encodes an AGE (BoAGE1) with strong similarity to the AGE previously characterized in Bacteroides fragilis. Interestingly, the BACOVA_01816 gene (BoAGE2) shares 57% identity with Anabaena sp. CH1 AGE, but has an extra 27-amino acid tag sequence in the N-terminal. When cloned and expressed in Escherichia coli Rosetta (DE3)pLys, BACOVA_01816 was able to convert ManNAc into GlcNAc and vice versa. It was stable over a broad range of pHs and its activity was enhanced by ATP (20 μM). The incubation with ATP stabilized its structure, raising its melting temperature by about 8 °C. In addition, the catalytic efficiency for ManNAc synthesis was higher than that for GlcNAc synthesis. These characteristics make BoAGE2 a promising biocatalyst for sialic acid production using cheap GlcNAc as starting material. BoAGE2 could be considered a Renin-binding Protein and its interaction with renin was studied for the first time in a prokaryotic AGE. Surprisingly, renin activated BoAGE2, an effect which is contrary to that described for mammalian AGE and unrelated with the unique N-terminal tag, since a mutant without this tag was also activated by renin. When BoAGE2 sequence was compared with other related (real and putative) AGE described in the databases, it was seen that AGE enzymes can be divided in 3 different groups. The relationship between these groups is also discussed. 相似文献