BBA - General Subjects (v.1850, #7)
Editorial Board (i).
Micellization parameters (number average, aggregation number and critical micellar concentration) of bile salt 3 and 7 ethylidene derivatives: Role of the steroidal skeleton II by Mihalj Poša; Srđan Bjedov; Dušan Škorić; Marija Sakač (1345-1353).
Bile salts are steroidal biosurfactants. Micellar systems of bile salts are not only important for solubilization of cholesterol, but they also interact with certain drugs thus changing their bioavailability.The number-average aggregation numbers ( n ¯ ) are determined using the Moroi–Matsuoka–Sugioka thermodynamic method. Critical micellar concentrations were determined by spectrofluorometric method using pyren and by surface tension measurements.Micelles of ethylidene derivatives possess the following values for n ¯ : 7-Eth-D ( n ¯ = 11 (50 mM)– n ¯ = 14.8 (100 mM)); 12-Ox-7-Eth-L ( n ¯ ≈ 8.8 , without concentration dependence) and 7,12-diOx-3-Eth-Ch ( n ¯ ≈ 2.9 , without concentration dependence). In the planes n ¯ –ln k and ln CMC–ln k derivative 7-Eth-D is outlier in respect to hydrophobic linear congeneric groups.Gibbs energy of formation for 7-Eth-D anion micelles in addition to the Gibbs energy of hydrophobic interactions consists excess Gibbs energy (GE ) from hydrogen bond formation between building blocks of micelles. Gibbs energy of formation for 7,12-diOx-3-Eth-Ch and 12-Ox-7-Eth-L anion micelle is determined by the Gibbs energy of hydrophobic interactions. Relative increase in hydrophobicity and aggregation number for ethylidene derivatives is larger when ethylidene group is introduced from the C7 lateral side of steroidal skeleton then it is when ethylidene group is on C3 carbon.Position of outlier towards hydrophobic congeneric groups from n ¯ –ln k and ln CMC–ln k planes indicates the existence of excess Gibbs energy (GE ) which is not of hydrophobic nature (formation of hydrogen bonds). For the bile salt micelles to have GE (formation of secondary micelles) it is necessary that steroidal skeleton possesses C3-α-(e)-OH and C12-α-(a)-OH groups.Display Omitted
Keywords: Micelle of bile salts; Aggregation number; Steroidal skeleton; Hydrophobic linear congeneric group; Excess Gibbs energy;
IMP–GMP specific cytosolic 5′-nucleotidase regulates nucleotide pool and prodrug metabolism by Federico Cividini; Daniela Nicole Filoni; Rossana Pesi; Simone Allegrini; Marcella Camici; Maria Grazia Tozzi (1354-1361).
Type II cytosolic 5′-nucleotidase (cN-II) catalyzes the hydrolysis of purine and, to some extent, of pyrimidine monophosphates. Recently, a number of papers demonstrated the involvement of cN-II in the mechanisms of resistance to antitumor drugs such as cytarabine, gemcitabine and fludarabine. Furthermore, cN-II is involved in drug resistance in patients affected by hematological malignancies influencing the clinical outcome. Although the implication of cN-II expression and/or activity appears to be correlated with drug resistance and poor prognosis, the molecular mechanism by which cN-II mediates drug resistance is still unknown.HEK 293 cells carrying an expression vector coding for cN-II linked to green fluorescent protein (GFP) and a control vector without cN-II were utilized. A highly sensitive capillary electrophoresis method was applied for nucleotide pool determination and cytotoxicity exerted by drugs was determined with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay.Over-expression of cN-II causes a drop of nucleoside triphosphate concentration and a general disturbance of nucleotide pool. Over-expressing cells were resistant to fludarabine, gemcitabine and cytarabine independently of cN-II ability to hydrolyze their monophosphates.An increase of cN-II expression is sufficient to cause both a general disturbance of nucleotide pool and an increase of half maximal inhibitory concentration (IC50) of the drugs. Since the monophosphates of cytarabine and gemcitabine are not substrates of cN-II, the protection observed cannot be directly ascribed to drug inactivation.Our results indicate that cN-II exerts a relevant role in nucleotide and drug metabolism through not only enzyme activity but also a mechanism involving a protein–protein interaction, thus playing a general regulatory role in cell survival.Resistance to fludarabine, gemcitabine and cytarabine can be determined by an increase of cN-II both through dephosphorylation of active drugs and perturbation of nucleotide pool.
Keywords: Chemotherapy; Resistance; cN-II; Fludarabine; Gemcitabine; Cytarabine;
The repressor Rgt1 and the cAMP-dependent protein kinases control the expression of the SUC2 gene in Saccharomyces cerevisiae by Juana M. Gancedo; Carmen-Lisset Flores; Carlos Gancedo (1362-1367).
A low level of glucose is required for maximal transcription of the SUC2 gene in Saccharomyces cerevisiae. Although the repressor Rgt1 binds the SUC2 promoter in gel-shift assays, it has been reported that Rgt1 has only minimal effects on SUC2 expression. Rgt1 acts together with Mth1 to repress the HXT genes encoding glucose transporters, and the release of Rgt1 from some HXT promoters requires cAMP-dependent protein kinase (PKA) activity.The genes RGT1 and MTH1 have been disrupted and the SUC2 promoter modified in several S. cerevisiae backgrounds. Yeast cells were grown in different carbon sources in the presence or absence of 0.1 or 2% glucose, and invertase was assayed in whole cells.Galactose, glycerol or ethanol hindered invertase induction by low glucose, but lactate did not. During growth in lactate, deletion of RGT1 or MTH1 caused a marked increase in invertase levels, and elimination of the Rgt1-binding site in the SUC2 promoter caused also invertase induction. PKA activity decreased invertase levels in cells growing in lactate, and increased them during growth in lactate + 0.1% glucose.The low level of expression of SUC2 in the absence of glucose is mainly due to repression by the Rgt1-Mth1 complex. Repression is dependent on PKA activity, but not on any specific Tpk isoenzyme.The results show that previously overlooked regulatory elements, such as Rgt1 and Tpks, participate in the control of SUC2 expression in S.cerevisiae.
Keywords: Glucose; Invertase; PKA; Rgt1; SUC2; Yeast;
ADAMTS13 deficiency in mice does not affect adipose tissue development by Lotte Geys; Ilse Scroyen; Elien Roose; Karen Vanhoorelbeke; Henri Roger Lijnen (1368-1374).
BMI and ADAMTS13 levels are positively correlated in man. Development of obesity is associated with angiogenesis and inflammation, and increased ADAMTS13 synthesis in the liver.Male wild-type (WT) and ADAMTS13 deficient (Adamts13−/− ) mice were kept on normal chow (SFD) or high fat diet (HFD) for 15 weeks.HFD feeding of WT mice resulted in significantly enhanced levels of ADAMTS13 antigen and activity as compared to SFD feeding. ADAMTS13 deficiency had no significant effect on body weight gain, subcutaneous (SC) or gonadal (GN) adipose tissue mass, or on adipocyte size. In GN fat of obese (HFD) Adamts13−/− mice, adipocyte density was higher and blood vessel density lower as compared to obese WT mice. No marked effects of genotype were observed on mRNA expression of adipogenic, endothelial, inflammatory or oxidative stress markers in adipose tissue. Analysis of metabolic parameters and of glucose and insulin tolerance did not reveal significant differences between both obese genotypes, except for higher adiponectin and cholesterol levels in obese Adamts13−/− as compared to WT mice.Our data do not support a functional role of ADAMTS13 in adiposity nor in associated angiogenesis or inflammation in mice.ADAMTS13 deficiency may cause thrombotic thrombocytopenic purpura (TTP). Obesity, which is associated with enhanced ADAMTS13 levels is nevertheless considered to be an independent risk factor for TTP. To resolve this apparent contradiction, we show that ADAMTS13 does not directly promote development of adipose tissue in a mouse model.
Keywords: ADAMTS13; Obesity; Adipose tissue; Mice;
Membrane-permeable tastants amplify β2-adrenergic receptor signaling and delay receptor desensitization via intracellular inhibition of GRK2’s kinase activity by Einav Malach; Merav E. Shaul; Irena Peri; Liquan Huang; Andrew I. Spielman; Rony Seger; Michael Naim (1375-1388).
Amphipathic sweet and bitter tastants inhibit purified forms of the protein kinases GRK2, GRK5 and PKA activities. Here we tested whether membrane-permeable tastants may intracellularly interfere with GPCR desensitization at the whole cell context.β2AR-transfected cells and cells containing endogenous β2AR were preincubated with membrane-permeable or impermeable tastants and then stimulated with isoproterenol (ISO). cAMP formation, β2AR phosphorylation and β2AR internalization were monitored in response to ISO stimulation. IBMX and H89 inhibitors and GRK2 silencing were used to explore possible roles of PDE, PKA, and GRK2 in the tastants-mediated amplification of cAMP formation and the tastant delay of β2AR phosphorylation and internalization.Membrane-permeable but not impermeable tastants amplified the ISO-stimulated cAMP formation in a concentration- and time-dependent manner. Without ISO stimulation, amphipathic tastants, except caffeine, had no effect on cAMP formation. The amplification of ISO-stimulated cAMP formation by the amphipathic tastants was not affected by PDE and PKA activities, but was completely abolished by GRK2 silencing. Amphipathic tastants delayed the ISO-induced GRK-mediated phosphorylation of β2ARs and GRK2 silencing abolished it. Further, tastants also delayed the ISO-stimulated β2AR internalization.Amphipathic tastants significantly amplify β2AR signaling and delay its desensitization via their intracellular inhibition of GRK2.Commonly used amphipathic tastants may potentially affect similar GPCR pathways whose desensitization depends on GRK2’s kinase activity. Because GRK2 also modulates phosphorylation of non-receptor components in multiple cellular pathways, these gut-absorbable tastants may permeate into various cells, and potentially affect GRK2-dependent phosphorylation processes in these cells as well.
Keywords: Membrane-permeable; Tastants; β2AR; GRK2; Signaling; Desensitization;
Inhibitory effect of ephedrannins A and B from roots of Ephedra sinica STAPF on melanogenesis by Ik-Soo Kim; Sung-Jin Yoon; Young-Jun Park; Hee-Bong Lee (1389-1396).
Melanogenesis, a process producing the pigment melanin in human skin, eyes and hair, is a major physiological response against various environmental stresses, in particular exposure to ultraviolet radiation, and its pathway is regulated by a key enzyme, tyrosinase. In this study, we evaluated the effects of ephedrannins A and B, which are polyphenols from the roots of Ephedra sinica, commonly used in herbalism in oriental countries, on mushroom tyrosinase and melanogenesis in B16F10 melanoma cells.Their effects on mushroom tyrosinase were determined via kinetic studies using a spectrophotometric analysis and those on melanin and tyrosinase production in melanoma cells treated with α-MSH (melanin stimulating hormone) were examined using PCR and ELISA.Both ephedrannins A and B exhibited concentration-dependent inhibitory effects on l-tyrosine oxidation by mushroom tyrosinase, and the inhibition mechanism was competitive and reversible with l-tyrosine as the substrate. In addition, melanin production in melanoma cells was also suppressed in a concentration-dependent manner by ephedrannins A and B without significant effects on cell proliferation at the concentrations tested. Both compounds showed inhibitory effects on melanin production by suppressing the transcription of tyrosinase in the cells.Both compounds exhibited significant inhibitory effects, but the inhibition by ephedrannin B was much more effective than that by ephedrannin A. Both ephedrannins A and B may be good candidates for a whitening agent for skin.This is the first report that describes effective inhibition of melanin production by ephedrannins A and B isolated from Ephedra roots.Display Omitted
Keywords: Epehdra sinica; Ephedrannin A; Ephedrannin B; Tyrosinase activity; Anti-melanogenesis; Competitive inhibition;
Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells by Martín Rossotti; Sofía Tabares; Lucía Alfaya; Carmen Leizagoyen; Gabriel Moron; Gualberto González-Sapienza (1397-1404).
Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domains (nanobodies) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle.Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct use for pull-down/MS target identification.The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells.This strategy streamlines the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents.This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.
Keywords: In vivo biotinylation; Flow cytometry; Immunoprecipitation; Nanobody; Phage display; Cell receptor;
Active site cleft mutants of Os9BGlu31 transglucosidase modify acceptor substrate specificity and allow production of multiple kaempferol glycosides by Juthamath Komvongsa; Sukanya Luang; Joaquim V. Marques; Kannika Phasai; Laurence B. Davin; Norman G. Lewis; James R. Ketudat Cairns (1405-1414).
Rice Os9BGlu31 is a transglucosidase that can transfer glucose to phenolic acids, flavonoids, and phytohormones. Os9BGlu31 displays a broad specificity with phenolic 1-O-β-d-glucose esters acting as better glucose donors than glucosides, whereas the free phenolic acids of these esters are also excellent acceptor substrates.Based on homology modeling of this enzyme, we made single point mutations of residues surrounding the acceptor binding region of the Os9BGlu31 active site. Products of the wild type and mutant enzymes in transglycosylation of phenolic acceptors from 4-nitrophenyl β-d-glucopyranoside donor were identified and measured by UPLC and negative ion electrospray ionization tandem mass spectrometry (LCMSMS).The most active variant produced was W243N, while I172T and L183Q mutations decreased the activity, and other mutations at W243 (A, D, M, N, F and Y) had variable effects, depending on the acceptor substrate. The Os9BGlu31 W243N mutant activity was higher than that of wild type on phenolic acids and kaempferol, a flavonol containing 4 hydroxyl groups, and the wild type Os9BGlu31 produced only a single product from each of these acceptors in significant amounts, while W243 variants produced multiple glucoconjugates. Fragmentation analysis provisionally identified the kaempferol transglycosylation products as kaempferol 3-O, 7-O, and 4'-O glucosides and 3,7-O, 4′,7-O, and 3,4′-O bis-O-glucosides. The Os9BGlu31 W243 mutants were also better able to use kaempferol 3-O-glucoside as a donor substrate.The W243 residue was found to be critical to the substrate and product specificity of Os9BGlu31 transglucosidase and mutation of this residue allows production of a range of glucoconjugates.
Keywords: Rice (Oryza sativa L.); Transglucosidase; Site-directed mutagenesis; Glycoside hydrolase family GH1; Glycosylation; Electrospray ionization mass spectrometry;
Endothelial protective genes induced by statin are mimicked by ERK5 activation as triggered by a drug combination of FTI-277 and GGTI-298 by Uyen B. Chu; Tyler Duellman; Sara J. Weaver; Yunting Tao; Jay Yang (1415-1425).
Statins are potent inhibitors of cholesterol biosynthesis and are clinically beneficial in preventing cardiovascular diseases, however, the therapeutic utility of these drugs is limited by myotoxicity. Here, we explored the mechanism of statin-mediated activation of ERK5 in the human endothelium with the goal of identifying compounds that confer endothelial protection but are nontoxic to muscle.An ERK5-one hybrid luciferase reporter transfected into COS-7 cells with pharmacological and molecular manipulations dissected the signaling pathway leading to statin activation of ERK5. qRT-PCR of HUVEC cells documented the transcriptional activation of endothelial-protective genes. Lastly, morphological and cellular ATP analysis, and induction of atrogin-1 in C2C12 myotubes were used to assess statin-induced myopathy.Statin activation of ERK5 is dependent on the cellular reduction of GGPPs. Furthermore, we found that the combination of FTI-277 (inhibitor of farnesyl transferase) and GGTI-298 (inhibitor of geranylgeranyl transferase I) mimicked the statin-mediated activation of ERK5. FTI-277 and GGTI-298 together recapitulated the beneficial effects of statins by transcriptionally upregulating anti-inflammatory mediators such as eNOS, THBD, and KLF2. Finally, C2C12 skeletal myotubes treated with both FTI-277 and GGTI-298 evoked less morphological and cellular changes recognized as biomarkers of statin-associated myopathy.Statin-induced endothelial protection and myopathy are mediated by distinct metabolic intermediates and co-inhibition of farnesyl transferase and geranylgeranyl transferase I confer endothelial protection without myopathy.The combinatorial FTI-277 and GGTI-298 drug regimen provides a promising alternative avenue for endothelial protection without myopathy.
Keywords: Pitavastatin; ERK5; GGTI-298; FTI-277; Myopathy; Endothelial protection;
Expression in yeast, new substrates, and construction of a first 3D model of human orphan cytochrome P450 2U1: Interpretation of substrate hydroxylation regioselectivity from docking studies by Lionel Ducassou; Gabriella Jonasson; Laura Dhers; Nicolas Pietrancosta; Booma Ramassamy; Yun Xu-Li; Marie-Anne Loriot; Philippe Beaune; Gildas Bertho; Murielle Lombard; Daniel Mansuy; François André; Jean-Luc Boucher (1426-1437).
Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. In humans, it has been found to be predominantly expressed in the thymus and in the brain. CYP2U1 is considered as an “orphan” enzyme as few data are available on its physiological function(s) and active site topology. Its only substrates reported so far were unsaturated fatty acids such as arachidonic acid, and, much more recently, N-arachidonoylserotonin.We expressed CYP2U1 in yeast Saccharomyces cerevisiae, built a 3D homology model of CYP2U1, screened a library of compounds known to be substrates of CYP2 family with metabolite detection by high performance liquid chromatography–mass spectrometry, and performed docking experiments to explain the observed regioselectivity of the reactions.We show that drug-related compounds, debrisoquine and terfenadine derivatives, subtrates of CYP2D6 and CYP2J2, are hydroxylated by recombinant CYP2U1 with regioselectivities different from those reported for CYP2D6 and 2J2. Docking experiments of those compounds and of arachidonic acid allow us to explain the regioselectivity of the hydroxylations on the basis of their interactions with key residues of CYP2U1 active site.Our results show for the first time that human orphan CYP2U1 can oxidize several exogenous molecules including drugs, and describe a first CYP2U1 3D model.These results could have consequences for the metabolism of drugs particularly in the brain. The described 3D model should be useful to identify other substrates of CYP2U1 and help in understanding its physiologic roles.Display Omitted
Keywords: Orphan cytochrome P450; Expression in yeast; Debrisoquine; Terfenadone analogue; Active site topology; Docking experiment;
Hedgehog signaling through GLI1 and GLI2 is required for epithelial–mesenchymal transition in human trophoblasts by Chao Tang; Liu Mei; Liyu Pan; Wenyi Xiong; Haibin Zhu; Hongfeng Ruan; Chaochun Zou; Lanfang Tang; Takuma Iguchi; Ximei Wu (1438-1448).
Epithelial to mesenchymal transition (EMT) is critical for human placental development, trophoblastic differentiation, and pregnancy-associated diseases. Here, we investigated the effects of hedgehog (HH) signaling on EMT in human trophoblasts, and further explored the underlying mechanism.Human primary cytotrophoblasts and trophoblast-like JEG-3 cells were used as in vitro models. Quantitative real-time RT-PCR and Western blot analysis were performed to examine mRNA and protein levels, respectively. Lentiviruses expressing short hairpin RNA were used to knock down the target genes. Reporter assays and chromatin immunoprecipitation were performed to determine the transactivity. Cell migration, invasion and colony formation were accessed by wound healing, Matrigel-coated transwell, and colony formation assays, respectively.Activation of HH signaling induced the transdifferentiation of cytotrophoblasts and trophoblast-like JEG-3 cells from epithelial to mesenchymal phenotypes, exhibiting the decreases in E-Cadherin expression as well as the increases in vimentin expression, invasion, migration and colony formation. Knockdown of GLI1 and GLI2 but not GLI3 attenuated HH-induced transdifferentiation, whereas GLI1 was responsible for the expression of HH-induced key EMT regulators including Snail1, Slug, and Twist, and both GLI1 and GLI2 acted directly as transcriptional repressor of CDH1 gene encoding E-Cadherin.HH through GLI1 and GLI2 acts as critical signals in supporting the physiological function of mature placenta.HH signaling through GLI1 and GLI2 could be required for the maintenance of human pregnancy.
Keywords: Hedgehog signaling; Trophoblast; Epithelial–mesenchymal transition;
Gelatinase A (MMP-2) promotes murine adipogenesis by Dries Bauters; Ilse Scroyen; Matthias Van Hul; H. Roger Lijnen (1449-1456).
Expansion of adipose tissue is dependent on adipogenesis, angiogenesis and extracellular matrix remodeling. A functional role in these processes was suggested for the gelatinase subfamily of the matrix metalloproteinases. Here, we have evaluated a potential role of gelatinase A (MMP-2) in adipogenesis.Murine embryonic fibroblasts (MEF) were derived from wild-type or MMP-2 deficient mice. Genetic manipulation of Mmp2 (shRNA-knockdown or overexpression) was performed in 3T3-F442A preadipocytes. Cell cultures were subjected to an adipogenic medium. As an in vivo model for de novo adipogenesis, 3T3-F442A preadipocytes with or without knockdown were injected subcutaneously in Nude BALB/c mice kept on high fat diet. Mmp2 deficient MEF, as compared to controls, showed significantly impaired differentiation into mature adipocytes, as demonstrated by 90% reduced intracellular lipid content and reduced expression of pro-adipogenic markers. Moreover, selective Mmp2 knockdown in 3T3-F442A preadipocytes resulted in significantly reduced differentiation. In contrast, overexpression of Mmp2 resulted in markedly enhanced differentiation. In de novo formed fat pads resulting from preadipocytes with Mmp2 knockdown expression of aP2, Ppar-γ and adiponectin was significantly lower, and collagen was more preserved. The fat pad weights as well as size and density of adipocytes or blood vessels were, however, not significantly different from controls.Our data directly support a functional role of MMP-2 in adipogenesis in vitro, and suggest a potential role in in vivo adipogenesis.Selective modulation of MMP-2 levels affects adipogenesis.
Keywords: Preadipocyte; Adipogenesis; Obesity; Matrix metalloproteinase; Gelatinase;
Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis by Hiromi Moriuchi; Hideaki Unno; Shuichiro Goda; Hiroaki Tateno; Jun Hirabayashi; Tomomitsu Hatakeyama (1457-1465).
CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin.Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis.Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17 × 103 M− 1 at 25 °C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose.Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs.Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins.
Keywords: Carbohydrate; C-type lectin; Site-directed mutagenesis; X-ray crystallography;