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• W2043485602 abstract "Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities. While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgariserythroagglutinating and leukoagglutinating lectins (E4- and L4-PHA) are unique in recognizing larger structures. E4- and L4-PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a β1,6-linked branch, respectively. However, the detailed mechanisms of molecular recognition are poorly understood. In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E4- and L4-PHA. We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433–445). The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance. These comparisons indicate that the high specificities of E4- and L4-PHA toward bisecting GlcNAc and β1,6-linked branch structures are almost solely attributable to loop B. The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible. Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus. Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E4- and L4-PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a β1,6-linked branch. Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities. While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgariserythroagglutinating and leukoagglutinating lectins (E4- and L4-PHA) are unique in recognizing larger structures. E4- and L4-PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a β1,6-linked branch, respectively. However, the detailed mechanisms of molecular recognition are poorly understood. In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E4- and L4-PHA. We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433–445). The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance. These comparisons indicate that the high specificities of E4- and L4-PHA toward bisecting GlcNAc and β1,6-linked branch structures are almost solely attributable to loop B. The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible. Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus. Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E4- and L4-PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a β1,6-linked branch. The legume lectins are a family of sugar-binding proteins found mainly in the seeds of plants belonging to theLeguminosae family (1.Sharon N. Lis H. FASEB J. 1990; 4: 3198-3208Crossref PubMed Scopus (397) Google Scholar, 2.Loris R. Hamelryck T. Bouckaert J. Wyns L. Biochim. Biophys. Acta. 1998; 1383: 9-36Crossref PubMed Scopus (480) Google Scholar, 3.Etzler M.E. Trends Glycosci. Glycotech. 1998; 10: 247-255Crossref Scopus (13) Google Scholar). Lectins from leguminous plants constitute a large family of homologous proteins displaying remarkable divergence in their carbohydrate specificity. Elucidation of the mechanism by which these lectins can possess such a broad range of binding specificities while maintaining a strikingly similar three-dimensional monomer structure will be key to understanding the essence of carbohydrate-protein interactions. At present, the crystal structures of ∼20 legume lectins have been solved. 1The 3D Lectin Data Bank is available on the World Wide Web at webenligne.cermav.cnrs.fr/databank/lectine/. These various lectin monomers share a common so-called “jellyroll” structure composed primary of a six- and a seven-stranded antiparallel β-sheet. Each monomer binds a manganese and a calcium ion that are both essential for carbohydrate binding. Amino acid sequence comparisons, x-ray crystallographic analysis, and mutagenesis studies revealed that the differences in carbohydrate specificity appear to be due primarily to differences in amino acid residues residing in loops adjacent to the carbohydrate binding site. The primary carbohydrate-binding site of legume lectins is a shallow depression on loops associated with the concave face of the seven-stranded curved β-sheet (5.Young N.M. Oomen R.P. J. Mol. Biol. 1992; 228: 924-934Crossref PubMed Scopus (107) Google Scholar). It is constructed mainly by residues from four sequentially separate regions, which are described by Sharma and Surolia (6.Sharma V. Surolia A. J. Mol. Biol. 1997; 267: 433-445Crossref PubMed Scopus (193) Google Scholar) as loops A, B, C, and D. Side chains of two highly conserved residues, Asp and Asn (contributed by loops A and C, respectively), together with the backbone chain NH of a Gly or Arg residue from loop B, play crucial roles in carbohydrate recognition, since they participate in four key hydrogen bonds with the monosaccharide. The carbohydrate is further stabilized by stacking interactions with hydrophobic residues present in loop C. Gross differences in the size of loop D were shown to be crucial for distinguishing between Man/Glc and Gal/GalNAc (6.Sharma V. Surolia A. J. Mol. Biol. 1997; 267: 433-445Crossref PubMed Scopus (193) Google Scholar). Although legume lectins have been subdivided into categories based on their monosaccharide specificities (1.Sharon N. Lis H. FASEB J. 1990; 4: 3198-3208Crossref PubMed Scopus (397) Google Scholar, 7.Goldstein I.J. Poretz R.D. Liener I.E. Sharon N. Goldstein I.J. The Lectins: Properties, Functions and Applications in Biology and Medicine. Academic Press, Inc., New York1986: 33-247Crossref Google Scholar), there are marked differences in the fine specificities of the lectins within a single category. Despite clear delineation of the primary binding site, the mechanism by which legume lectins expand fine specificities and improve binding affinity by subsite multivalence (8.Rini J.M. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 551-577Crossref PubMed Scopus (430) Google Scholar) is largely unknown. Among legume lectins, E4- and L4-PHA 2The abbreviations used are: E4-PHAP. vulgaris erythroagglutinating lectinL4-PHAP. vulgaris leukoagglutinating lectinAUCarea under the curveAUCaAUC of the association phaseAUCd0→∞AUC of dissociation phase extrapolated over an infinite time intervalE-bLchimera lectin of E4-PHA of which loop B is replaced with that of L4-PHAE-cLchimera lectin of E4-PHA of which the central portion of loop C is replaced with that of L4-PHAE-bLcLchimera lectin of E4-PHA of which loop B and the central portion of loop C are replaced with those of L4-PHAL-bEchimera lectin of L4-PHA of which loop B is replaced with that of E4-PHAL-cEchimera lectin of L4-PHA of which the central portion of loop C is replaced with that of E4-PHAL-bEcEchimera lectin of L4-PHA of which loop B and the central portion of loop C are replaced with those of E4-PHALacNAcN-acetyllactosamineBPH4-(biotinamido) phenylacetylhydrazideSPRsurface plasmon resonanceNA2NA2B, NA3, NA4, and NGA2, see Fig. 1NA4-HDNA4 hendecasaccharide are exceptional in displaying considerably greater degrees of oligosaccharide specificity than had previously been appreciated. Both are isolectins isolated from Phaseolus vulgaris (red kidney bean) seeds (9.Leavitt R.D. Felsted R.L. Bachur N.R. J. Biol. Chem. 1977; 252: 2961-2966Abstract Full Text PDF PubMed Google Scholar). Despite differences in binding specificities and hemagglutinating properties, subunits from E4- and L4-PHA are similar in molecular weight as well as carbohydrate and amino acid compositions. E4- and L4-PHA exhibit greatest affinity toward complex typeN-glycans containing either bisecting GlcNAc or β1,6-linked LacNAc, respectively (10.Cummings R.D. Kornfeld S. J. Biol. Chem. 1982; 257: 11230-11234Abstract Full Text PDF PubMed Google Scholar, 11.Hammarstrom S. Hammarstrom M.L. Sundblad G. Arnarp J. Lonngren J. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1611-1615Crossref PubMed Scopus (128) Google Scholar, 12.Green E.D. Baenziger J.U. J. Biol. Chem. 1987; 262: 12018-12029Abstract Full Text PDF PubMed Google Scholar, 13.Bierhuizen M.F. De Wit M. Govers C.A. Ferwerda W. Koeleman C. Pos O. Van Dijk W. Eur. J. Biochem. 1988; 175: 387-394Crossref PubMed Scopus (117) Google Scholar, 14.Kobata A. Yamashita K. Methods Enzymol. 1989; 179: 46-54Crossref PubMed Scopus (32) Google Scholar). The rather narrow differences between these two lectins make them well suited for the elucidation of subsite multivalence. Mirkov and Chrispeels (15.Mirkov T.E. Chrispeels M.J. Glycobiology. 1993; 3: 581-587Crossref PubMed Scopus (24) Google Scholar) reported that mutation of Asn128 to Asp in loop C of L4-PHA eliminates carbohydrate binding and biological activity, suggesting that this residue plays a key role in primary recognition. However, the mechanism of L4-PHA specificity toward β1,6-linked branch structures is not known. Although the three-dimensional structure of L4-PHA has been recently solved (16.Dao-Thi M.H. Hamelryck T.W. Poortmans F. Voelker T.A. Chrispeels M.J. Wyns L. Proteins. 1996; 24: 134-137Crossref PubMed Scopus (9) Google Scholar, 17.Hamelryck T.W. Dao-Thi M.H. Poortmans F. Chrispeels M.J. Wyns L. Loris R. J. Biol. Chem. 1996; 271: 20479-20485Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), neither the crystal structure of the complex nor the three-dimensional structure of E4-PHA has yet been reported. P. vulgaris erythroagglutinating lectin P. vulgaris leukoagglutinating lectin area under the curve AUC of the association phase AUC of dissociation phase extrapolated over an infinite time interval chimera lectin of E4-PHA of which loop B is replaced with that of L4-PHA chimera lectin of E4-PHA of which the central portion of loop C is replaced with that of L4-PHA chimera lectin of E4-PHA of which loop B and the central portion of loop C are replaced with those of L4-PHA chimera lectin of L4-PHA of which loop B is replaced with that of E4-PHA chimera lectin of L4-PHA of which the central portion of loop C is replaced with that of E4-PHA chimera lectin of L4-PHA of which loop B and the central portion of loop C are replaced with those of E4-PHA N-acetyllactosamine 4-(biotinamido) phenylacetylhydrazide surface plasmon resonance NA2B, NA3, NA4, and NGA2, see Fig. 1 NA4 hendecasaccharide In light of these outstanding questions, we chose to evaluate the effect of loop substitution on the sugar binding specificities for the identification of subsites. To be concrete, the primary sequences of E4- and L4-PHA were compared, and loop B and the central portion of loop C were chosen for swapping, since the biggest differences among the four loops (A–D) were concentrated in these two. Sugar binding specificities of six prepared chimeric lectins were extensively analyzed using a quantitative assay system based on immobilized oligosaccharides. This study clearly suggested that each loop functions to recognize different parts of an oligosaccharide, and the combination of these loops determines both the specificities and affinities of sugar binding. Fetuin (insolubilized on 4% beaded agarose) was purchased from Sigma. BIACORE®(BIACORE AB, Uppsala, Sweden), which are based on surface plasmon resonance (SPR), were used to measure the biomolecular interactions. E4- and L4-PHA were purchased from Honen Corp. (Tokyo, Japan). The abbreviations and structures of the oligosaccharides used in this study are shown in Fig. 1. All of the oligosaccharides, except for NA4-HD, shown in Fig. 1, were purchased from Oxford GlycoSystems (Abingdon, UK). The BIACORE SensorChip SA, Surfactant P20 was obtained from BIACORE AB. The HBS buffer comprised 10 mm HEPES (pH 7.4), 150 mm NaCl, 1 mm CaCl2 and 0.05% Surfactant P20 in distilled water. Lectins were purified by Superdex® 200, PC3.2/30 (Amersham Biosciences) using the HBS buffer as a solvent. Quantitation of the lectin was carried out with ultraviolet absorbance at 280 nm monitored by a SMART® (Amersham Biosciences) UV monitor. 4-(Biotinamido) phenylacetylhydrazide (BPH) was synthesized as previously reported (18.Shinohara Y. Sota H. Gotoh M. Hasebe M. Tosu M. Nakao J. Hasegawa Y. Shiga M. Anal. Chem. 1996; 68: 2573-2579Crossref PubMed Scopus (6) Google Scholar). Total RNA was isolated from midmaturation cotyledons (0.7 g total weight, 7–10 mm in length) of P. vulgaris cv. Tendergreen, according to the procedure reported by Hoffman et al. (19.Hoffman L.M. Ma Y. Barker R.F. Nucleic Acids Res. 1982; 10: 7819-7828Crossref PubMed Scopus (84) Google Scholar), using the QuickPrep® Total RNA Extraction Kit (Amersham Biosciences). RNA quantitation was done by extinctional measurement of 260 nm. First-strand cDNA was synthesized from total RNA described previously, using the First-Strand cDNA Synthesis Kit (Amersham Biosciences). All PCRs used for cloning purposes were carried out with the proofreading enzyme KOD polymerase (Toyobo) according to the manufacturer's instructions. E4- and L4-PHA cDNAs were amplified from the first-strand cDNA via the PCR. For amplifying L4-PHA, we used the oligonucleotide primers 5′-CATGAATTCATCCATGGCTTCCTCCAAGTTCT-3′ and 5′-TGGAGTTTGGATCCTAGAGGATTTTGTTGAGGA-3′, and for E4-PHA, we used the primers 5′-CATGAATTCATCCATGGCTTCCTCCAACTTAC-3′ and 5′-GTGGAGATGGATCCTAGAGGATTTGGTTGAGGA-3′. After digestion withEcoRI, each PCR-generated fragment was inserted into theSmaI and EcoRI site of pBluescript II SK(+) (TOYOBO, Osaka, Japan). From these clones, gene sequences lacking the signal peptide regions (L4-PHA, amino acids 1–20; E4-PHA, amino acids 1–21) (20.Hoffman L.M. Donaldson D.D. EMBO J. 1985; 4: 883-889Crossref PubMed Scopus (130) Google Scholar) were amplified by PCR using oligonucleotide primers 5′- CTCACCCACGCAATCATGAGCAACGATATCTAC-3′ (L-forward), 5′-GGTCGACTCTAGAACTAGTGG-3′ (L-reverse, for L4-PHA), 5′-CTCACCCACGCAACCATGGCCAGCCAAACCTCC-3′ (E-forward), and 5′-GGTCGACTCTAGAACTAGTGG-3′ (E-reverse, for E4-PHA). The two PCR-generated fragments were each cloned separately into theSmaI site of pUC18 using the SureClone™Ligation Kit (Amersham Biosciences). E4-PHA and L4-PHA were compared along their length with respect to hydrophobicity using Grease software based on the algorithms of Kyte and Doolittle (21.Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17296) Google Scholar) and Pearson and Lipman (22.Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9393) Google Scholar). The E4- and L4-PHA genes each contain two EcoO109I sites flanking the region encoding the divergent B loop along with most of the divergent C loop. To facilitate use of these sites for domain swapping, the genes lacking signal peptides were subcloned via SalI and EcoRI from their pUC18 constructs into a pBluescript II SK(+) derivative from which the unique EcoO109I site had been removed. Following restriction digestion, the EcoO109I fragments were exchanged to create E-bLcL and L-bEcE. In order to construct chimeric genes encoding independent variation in loop B and loop C, overlap extension PCR was used for site-directed mutagenesis of the short segment encoding the two variant amino acids (VH or KD) at the center of loop C. In detail, overlapping NH2- and COOH-terminal encoding halves of each construct were amplified using appropriate outer primers (L-forward and L-reverse or E-forward and E-reverse) together with mutagenic overlapping inner primers, and the two amplified halves were combined in the overlap extension step. The overlapping primers for converting PHAE residues to PHAL residues were 5′-TGGGGTCCCAGTCCTTGTTGTAGAGGGTGTC-3′ (L-BND-RV) and 5′-GACACCCTCTACAACAAGGACTGGGACCCCA-3′ (L-BND-FW), while the opposite conversion was accomplished with the overlapping primers 5′-TGGGGTCCCAGTGAACGTTGTAGAGGGTGTC-3′ (E-BND-RV) and 5′-GACACCCTCTACAACGTTCACTGGGACCCCA-3′ (E-BND-FW). In this way, E-cL and L-cE were derived from E4-PHA and L4-PHA, respectively, and E-bL and L-bE were similarly derived from E-bLcL and L-bEcE. Using the BamHI and NcoI sites present in the outer forward and reverse primers, all eight PHA gene sequence variants (including wild types) were introduced individually into pET-11d (Stratagene) for expression. The constructed plasmids were introduced into E. coli BL21(DE3) grown on an LB plate containing 100 μg/ml ampicillin. The BL21(DE3) cells containing the plasmids were grown to the midlog phase at 37 °C in LB medium with 0.1 mg/ml ampicillin and then induced by the addition of isopropyl-β-thiogalactoside to 1 mm. Cells were harvested by centrifugation at 6,000 × gfor 10 min. Extracts of expressed cells were obtained by sonicating cells in phosphate-buffered saline and collecting the supernatant after centrifugation at 20,000 × g for 10 min. The collected supernatant was applied to a fetuin-agarose (Sigma) column, which had been equilibrated with 7.5 volumes of HBS buffer at 4 °C. After washing the column with HBS buffer, protein was eluted with 50 mmH3PO4, followed by desalting using a PD-10 column (Amersham Biosciences). The protein was concentrated by lyophilization and purified by Superdex 200, PC3.2/30 (Amersham Biosciences) using HBS as the running buffer. BPH tagging of oligosaccharides was performed under the previously reported conditions (18.Shinohara Y. Sota H. Gotoh M. Hasebe M. Tosu M. Nakao J. Hasegawa Y. Shiga M. Anal. Chem. 1996; 68: 2573-2579Crossref PubMed Scopus (6) Google Scholar). Briefly, the oligosaccharide dissolved in water was incubated with a 4-fold molar excess of BPH in 30% acetonitrile at 90 °C for 1 h. After the reaction, 50 mm formate buffer (pH 3.5) was added and stored at 4 °C for 12 h to promote tautomerization from the acyclic Schiff base type hydrazone to stable β-glycosides. The reaction mixture was injected directly into reverse phase high pressure liquid chromatography to purify the BPH adducts. Oligosaccharide binding activities of E4-PHA, L4-PHA, and PHA mutants were measured by SPR using a BIACORE instrument. In this system, a BPH-labeled oligosaccharide (10 pmol) was introduced onto a streptavidin sensor surface (SensorChip SA; BIACORE AB, Tokyo, Japan). Lectins (10 μg/ml in HBS) were introduced onto the surface at a flow rate of 20 μl/min. The interaction between lectin and oligosaccharide was monitored as the change in SPR response at 25 °C. After 3 min, flow was switched from sample to HBS buffer in order to initiate dissociation. Sensor surfaces were regenerated with 50 mmH3PO4. The apparent k d was analyzed by fitting the dissociation phase directly to the following equation,Rt=R0 exp(−kdt)Equation 1 using nonlinear least squares analysis (23.O'Shannessy D.J. Brigham-Burke M. Soneson K.K. Hensley P. Brooks I. Anal. Biochem. 1993; 212: 457-468Crossref PubMed Scopus (522) Google Scholar, 24.Karlsson R. Roos H. Fagerstam L. Persson B. Methods. 1994; 6: 99-110Crossref Scopus (285) Google Scholar).R 0 represents the amplitude of the dissociation process. The k a was then analyzed by fitting the association phase directly to the following equation,Rt=CkaRmax/(Cka+kd)[1−exp{−(Cka+kd)t}]Equation 2 where R max represents the maximum lectin-oligosaccharide complex concentration (in resonance units), and C is the constant concentration of injected lectin. The area under the curve (AUC) for both association and dissociation phases was calculated using the trapezoidal rule. AUCd was extrapolated over an infinite time interval using nonlinear regression.AUC=∫t=t1t=t2RtdtSubstituting Equation 1 into Equation 3, and integration fromt 0, when dissociation was initiated, to ∞ gives Equation 4,AUCd0→∞=R0/kdEquation 4 Substituting Equation 2 into Equation 3 and integration from 0 to t 0 gives the following.AUCa=(CkaRmaxt0−R0)/(Cka+kd).Equation 5 The program package SYBYL® version 6.7 (Tripos Inc.) was used for all modeling. All computations have been carried out in a Silicon Graphics OCTANE™ work station provided with two 195-MHz R10000 processors. The molecules to be docked were first built with the correct chirality using the builder module in SYBYL and then minimized (500 cycles) using the MMFF94s force field (25.Mastryukov V.S. Chen K. Yang L.R. Allinger N.L. Theochem. 1993; 99: 199-204Google Scholar). All docking simulations were carried out with the program FlexX™ (26.Rarey M. Kramer B. Lengauer T. Klebe G. J. Mol. Biol. 1996; 261: 470-489Crossref PubMed Scopus (2400) Google Scholar), which is part of the SYBYL package. FlexX uses a fast docking method that allows flexibility in the ligands, keeping the receptor fixed. It places ligands into the active site starting with one fragment and consequently building the whole molecule into the binding site, fragment by fragment. FlexX uses formal charges, which were turned on during the docking simulations. All of the relevant receptor information necessary for the docking simulations is stored in the receptor definition file (rd file or rdf). The protein structure used was the 2.8-Å resolution structure of phytohemagglutinin-L (17.Hamelryck T.W. Dao-Thi M.H. Poortmans F. Chrispeels M.J. Wyns L. Loris R. J. Biol. Chem. 1996; 271: 20479-20485Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) with accession code 1FAT in the Protein Data Bank. The coordinates of chain B were used, since this chain was not broken. Defaults have been used when creating the rd file and no special customizations have been done. The residues belonging to the binding site file in the rd file are Gln-19, Arg-20, Gly-44, Leu-46, Arg-48, Gly-83, Pro-84, Ala-85, Asp-86, Pro-94, Gly-96, Ser-97, Gln-98, Pro-99, Lys-100, Asp-101, Lys-102, Gly-103, Gly-104, Phe-105, Leu-106, Gly-107, Phe-109, Asp-110, Gly-111, Ser-112, Asn-113, Ser-114, Asn-115, Phe-116, His-117, Leu-126, Tyr-127, Asn-128, Lys-129, Asp-130, Trp-131, Asp-132, Asn-143, Ser-144, Ile-145, Arg-146, Thr-210, Thr-211, Gly-212, Ile-213, Asn-214, Lys-215, Gly-216, Asn-217, and Val-218. These residues were chosen because they surround the binding site formed between and around loops B and C. Five oligosaccharides (NGA2, NA2, NA2B, NA3, and NA4) were chosen as model oligosaccharides based on the known sugar binding specificities of E4- and L4-PHA. These were biotinylated with BPH and then immobilized onto streptavidin-coated sensor surfaces. The molar amount of each immobilized oligosaccharide is presumed to be nearly constant, since the amount of streptavidin was constant, and an excess of purified BPH-oligosaccharide was introduced (18.Shinohara Y. Sota H. Gotoh M. Hasebe M. Tosu M. Nakao J. Hasegawa Y. Shiga M. Anal. Chem. 1996; 68: 2573-2579Crossref PubMed Scopus (6) Google Scholar). A solution containing the lectin was passed over the sensor surface, and binding was monitored as changes in the SPR signal. The sensorgrams obtained for native E4- and L4-PHA with these model oligosaccharides are shown in Fig. 2. E4-PHA exhibited the greatest increases in resonance signal with NA2, NA2B, and NA3, and the maximum response with each was nearly equal although the patterns for the association and dissociation phases differed quite markedly. E4-PHA also interacted with NGA2 and NA4, displaying smaller increases in resonance signals. The apparent rate constant values were calculated for NA2, NA2B, and NA3 by fitting their sensorgrams using a simple one-to-one interaction model, and these are summarized in Table I. The differences in apparent k a were rather small, whereas the apparentk d differed by up to an order of magnitude. The other sensorgrams were difficult to fit to this model. In order to obtain quantitative values for comparison among all the sensorgrams, the AUCs for both the association and dissociation phases were calculated (see details under “Experimental Procedures”). As shown in Fig. 3 a, the calculated AUCd0→∞ was extremely high for NA2B, and the other values in descending order were NA3 > NA2 > NGA2 ≥ NA4. This is a reasonable order in view of the previously reported sugar binding specificity of immobilized E4-PHA during affinity chromatography (12.Green E.D. Baenziger J.U. J. Biol. Chem. 1987; 262: 12018-12029Abstract Full Text PDF PubMed Google Scholar, 14.Kobata A. Yamashita K. Methods Enzymol. 1989; 179: 46-54Crossref PubMed Scopus (32) Google Scholar, 27.Kobata A. Endo T. J. Chromatogr. 1992; 597: 111-122Crossref PubMed Scopus (83) Google Scholar).Table IKinetic parameters obtained for the interaction analysis of E4-PHA with immobilized NA2, NA2B, and NA3k a × 105k d× 10−4M −1 s −1s −1NA23.111.5NA2B1.31.2NA31.65.4 Open table in a new tab Figure 3Calculated AUC a (black bar) and AUC d0→∞(white bar) for the interactions of E4-PHA (a) and L4-PHA (b) with immobilized NGA2, NA2 , NA2B, NA3, and NA4. After the injection of each lectin at a concentration of 10 μg/ml, association and dissociation were monitored for 3 min, respectively. The AUCs for both phases was calculated using the trapezoidal rule. AUCd was extrapolated over an infinite time interval using nonlinear regression. RU, response units.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As for L4-PHA, it also bound to all of the oligosaccharides. The peak resonance signals obtained in descending order were NA2 > NA4 > NGA2 > NA2B ≈ NA3. Patterns for association and dissociation phases again varied dramatically. As shown in Fig. 3 b, the calculated AUCd0→∞ was extremely high for NA4 and decreased for the remaining four oligosaccharides in the order NA2 > NA3 > NGA2 ≥ NA2B. This also agreed well with the order previously reported for elution from immobilized L4-PHA during analytical affinity chromatography (10.Cummings R.D. Kornfeld S. J. Biol. Chem. 1982; 257: 11230-11234Abstract Full Text PDF PubMed Google Scholar, 12.Green E.D. Baenziger J.U. J. Biol. Chem. 1987; 262: 12018-12029Abstract Full Text PDF PubMed Google Scholar,28.Bierhuizen M.F.A. Tedzes H. Schiphorst W.C.M. Van den Eijnden D.H. Van Dijk W. Glycoconj. J. 1988; 5: 85-97Crossref Scopus (35) Google Scholar). These results demonstrated the validity of this method as a quantitative procedure for comparing sugar binding specificities of chimeric lectins. The calculated AUCa showed no correlation with the results from affinity chromatography. The other main observation from this study is that the AUCd0→∞ values calculated for E4-PHA with the various oligosaccharides were generally higher than those for L4-PHA. The calculated AUCd0→∞ value of NA2B was 1,400-fold higher for E4-PHA than for L4-PHA. The only exception, NA4, exhibited a value 1.7-fold higher for L4- than for E4-PHA. The calculated AUCd0→∞values for the remaining oligosaccharides were uniformly higher with E4- than L4-PHA (by 70–270-fold). The oligosaccharide binding profiles confirmed that both native lectins can recognize fairly long sequences and that the lectins' binding specificities can differ considerably from each other. Whereas these differences must reflect differences in their structures, the three-dimensional structure of E4-PHA has yet to be solved, so no direct three-dimensional comparison can be made. Therefore, primary structures of four loops thought to play important roles in sugar recognition were compared (Fig. 4). The greatest differences resided in loop B; of 26 amino acid residues composing this loop in E4-PHA, five differed in identity, and two were absent altogether from L4-PHA. To date, the extent of knowledge about the role of loop B in legume lectins is that a Gly or Arg residue therein hydrogen-bonds with monosaccharides (6.Sharma V. Surolia A. J. Mol. Biol. 1997; 267: 433-445Crossref PubMed Scopus (193) Google Scholar). How this longest loop contributes to fine sugar recognition is not known and therefore merits study. Moreover, two of four variant residues in loop C were also chosen. This loop contains calcium-binding sites and a strikingly important Asn residue, whose substitution to Asp eliminates sugar binding activity (15.Mirkov T.E. Chrispeels M.J. Glycobiology. 1993; 3: 581-587Crossref PubMed Scopus (24) Google Scholar, 29.van Eijsden R.R. Hoedemaeker F.J. Diaz C.L. Lugtenberg B.J. de Pater B.S. Kijne J.W. Plant Mol. Biol. 1992; 20: 1049-1058Crossref PubMed Scopus (44) Google Scholar). These amino acid differences between E4- and L4-PHA were clearly reflected in a hydrophobicity scale comparison (Fig. 5). Averaged over an 11-residue window, E4-PHA is markedly less hydrophobic than L4-PHA in the C-terminal portion of loop B, whereas the opposite pattern exists for loop C. Due to the reasons described, we focused on those two loops, and six chimeric lectins were designed for the study of their sugar binding specificities.Figure 5Comparison of hydrophobicity profiles between E4- and L4-PHA. Hydrophobicity values were computed according to Kyte and Doolittle (21.Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17296) Google Scholar) usin" @default.
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• W2043485602 title "The High Specificities of Phaseolus vulgaris Erythro- and Leukoagglutinating Lectins for Bisecting GlcNAc or β1–6-Linked Branch Structures, Respectively, Are Attributable to Loop B" @default.
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