1  

Molecular basis for recognition of the cancer glycobiomarker, LacdiNAc (GalNAc[β1→4]GlcNAc) by Wisteria floribunda agglutinin*

Omid Haji-Ghassemi‡, Michel Gilbert§, Jenifer Spence‡, Melissa J. Schur§, Matthew J.

Parker‡, Meredith L. Jenkins‡, John E. Burke‡, Henk van Faassen§, N. Martin Young§ & Stephen V. Evans‡1

‡ Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055 STN CSC, Victoria, BC, Canada V8P 3P6

§ Human Health Therapeutics, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON, Canada K1A 0R6

To whom correspondence should be addressed: Stephen V. Evans, Telephone: (250)-472-4548, E-mail: svevans@uvic.ca

Running title: Wisteria floribunda lectin recognition of cancer biomarker Keywords: Agglutinin; lectin; carbohydrate; carbohydrate-binding protein; cancer; glycobiomarker; structural biology; X-ray crystallography.

ABSTRACT Aberrant glycosylation and the overexpression of specific carbohydrate epitopes is a hallmark of many cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy and diagnostics. Wisteria floribunda agglutinin (WFA) is a legume lectin that recognizes terminal N-acetylgalactosaminides with high affinity. WFA preferentially binds the disaccharide LacdiNAc (β-D-GalNAc-[1→4]-D-GlcNAc), which is associated with tumor malignancy in leukemia, prostate, pancreatic, ovarian, and liver cancers, and has shown promise in cancer glycobiomarker detection. The mechanism of specificity for WFA recognition of LacdiNAc is not fully understood. To address this problem, we have determined affinities and structure of WFA in complex with GalNAc and LacdiNAc. Affinities towards Gal, GalNAc, and LacdiNAc were measured via surface plasmon resonance, yielding KD values of 4.67 × 10-4 M, 9.24 × 10-5 M, and 5.45 × 10-6 M respectively. Structures of WFA in complex with LacdiNAc and GalNAc have

been determined to 1.80 Å – 2.32 Å resolution. These high-resolution structures revealed a hydrophobic groove complementary to the GalNAc and, to a minor extent, to the backface of the GlcNAc sugar ring. Remarkably, the contribution of this small hydrophobic surface significantly increases the observed affinity for LacdiNAc over GalNAc. Tandem MS sequencing confirmed presence of two isolectin forms in commercially available WFA differing only in the identities of two amino acids. Finally, the WFA carbohydrate binding site is similar to a homologous lectin isolated from Vatairea macrocarpa in complex with GalNAc which, unlike WFA, binds not only αGalNAc but also terminal Ser/Thr O-linked αGalNAc (Tn antigen).

Neoplastic transformation often results in cells with unusual glycosylation patterns specific to the type and stage of different cancers, providing numerous venues for therapeutic and diagnostic reagents (1,2). Lectins are non-catalytic proteins with strict specificity for individual mono- or oligosaccharides, which

http://www.jbc.org/cgi/doi/10.1074/jbc.M116.750463The latest version is at JBC Papers in Press. Published on September 6, 2016 as Manuscript M116.750463

Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

2  

can make them powerful tools for detecting changes in the carbohydrate structure in glycoproteins and glycolipids. Several lectins are currently used in histochemical analyses to identify malignant or pre-malignant cells (3,4). Wisteria floribunda agglutinin (WFA2) is a legume lectin that binds N-glycans terminating in β-linked N-acetylgalactosaminides, particularly ones with LacdiNAc (β-D-GalNAc-[1→4]-D-GlcNAc) (Fig. 1) termini, and to terminal galactose residues with lower avidity (5-11). WFA’s biological activities in vitro have long been known to include inducing T lymphocyte activation (12) and hemagglutination (13). As with most legume lectins, the biological role of WFA is poorly understood; it may function as a mediator of symbiosis between nitrogen fixing bacteria and the plant’s roots or as a plant defense mechanism (14).

Lectins are known generally to bind di- and oligosaccharides with higher affinity compared to monosaccharides (14). The detailed specificities of lectins can now be determined by using glycan arrays (15,16), and array data obtained for WFA confirmed it preferentially binds glycans with LacdiNAc termini (Consortium for Functional Glycomics3, dataset #2342). At the lowest concentration of added lectin, the top six glycans recognized all had LacdiNAc termini, and were followed by GalNAc α and β ligands which showed from 28% to 20% of the binding of the best ligand.

The LacdiNAc structure is abundant in invertebrates (5,17-19), and some mammalian glycoproteins and lipids contain N- and O-glycans terminating in LacdiNAc, particularly hormones (20). The GalNAc transferase β4GalNAc-T3 is primarily responsible for its biosynthesis (21). Significantly, glycoproteins expressing terminal LacdiNAc appear to become elevated in a variety of human cancers, including prostate, lung, ovarian, colon, and liver cancers (6,8,9,22-25). The tissue-specific expression of the LacdiNAc is a potent diagnostic marker for specific human cancers

(23), and several recent studies have promoted the use of WFA for detection of LacdiNAc or terminal GalNAc overexpressed during cancer progression and growth (26-33). However, the molecular basis for WFA recognition of and specificity toward LacdiNAc is not known.

To date, there is one high-resolution structure of a mushroom lectin (Clitocybe nebularis, CNL) in complex with LacdiNAc (34). However, a hemagglutinin inhibition assay of the CNL protein showed broad specificity to lactose, galactose, glucose, and sucrose, ranging from high to low inhibition concentration respectively (34). Thus, its cross-reactive potential severely limits its use for cancer biomarker detection. WFA is currently considered the most prominent diagnostic lectin against cholangiocarcinoma when compared to other homologous lectins (26,35) and the most discriminatory against altered N-glycans on Mac2-binding protein, a secretory N-glycoprotein with elevated expression levels during viral hepatitis induced liver cirrhosis (36,37).

Recently, Narimatsu’s group successfully produced recombinant WFA and reported its full length primary sequence along with closely related lectins (38). Despite high sequence homology, the lectins derived from W. japonica and W. brachybotrys have unique sugar-binding activities (9). For instance, W. japonica agglutinin (WJA) only binds terminal α- and β-linked N-acetylgalactosamine.

There is some confusion surrounding the nomenclature and quaternary structure of WFA, where initially it was reported as a dimer with a molecular weight of the monomer ranging from 35 kDa to 28 kDa (9-12), the WFA sold commercially is reported as a tetramer with a molecular weight of 116 kDa in an oxidizing environment. An earlier publication also reported a tetrameric hemagglutinin, purified from seeds of W. floribunda, termed WFH (39). The tetrameric WFH does not have mitogen activity but displays strong hemagglutination and leukoagglutination activity, while the

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

3  

dimeric WFA show phytogenic and hemagglutination activity (38). The dimeric lectin is referred to WFM for its mitogenic capability. It is currently unclear whether these lectins represent different isoforms or entirely different lectins.

A better understanding of WFA and the molecular basis for LacdiNAc-lectin recognition is of significant biomedical and immunological interest. Here we report the high-resolution crystal structures of the tetrameric form of WFA from Vector Labs in complex with GalNAc and LacdiNAc together with binding data. EXPERIMENTAL PROCEDURES Synthesis of LacdiNAcβ-pNP – The bacterial β1,4-galactosyltransferase (gene HP0826, construct HP-21) from Helicobacter pylori and the UDP-GlcNAc/Glc 4-epimerase (gene Cj1131c, construct CPG-13) from Campylobacter jejuni were expressed and purified as reported in Namdjou et al. (40) and Bernatchez et al. (41), respectively. The synthesis reaction mix included 5 mM GlcNAcβ-pNP (15 mg), 10 mM UDP-GlcNAc, 10 mM MnCl2, 50 mM Mes pH 6.5, 39 units of HP-21 and 46 units of CPG-13. The reaction was incubated at 37oC. Additions of 12 units of HP-21 and 14 units of CPG-13 were made after 4 h while an addition of 3 mM UDP-GlcNAc was made after 6 h. Capillary electrophoresis analysis (P/ACE MDQ system equipped with diode array detection, Beckman Coulter, Fullerton, CA, USA) showed that the reaction was 85% complete after 23 h. The reaction mix was applied to a solid phase extraction cartridge (SepPak C18). Unbound material was removed by washing with water and then 20% methanol. The mix of GlcNAcβ-pNP / LacdiNAcβ-pNP was eluted with 50% methanol. Incubation at 4oC in 50% methanol resulted in the selective precipitation of the LacdiNAcβ-pNP. The LacdiNAcβ-pNP was recovered by centrifugation, washed with 100% methanol and solubilized in water.

Surface Plasmon Resonance – The interactions of sugar ligands with immobilized WFA were

measured by SPR using a Biacore T200 instrument (GE Healthcare). Immobilizations were carried out on research grade CM5 sensorchips at a protein concentration of 50 µg/ml in 10 mM sodium acetate buffer pH 4.0 using the manufacturer’s amine coupling kit. The amount of immobilized WFA was 3928 RU, and the reference surface was blocked with ethanolamine alone. The binding analyses were carried out at 25°C in 20 mM HEPES buffer pH 7.5 containing 50 mM NaCl, 0.1 mM CaCl2 and 0.1 mM MnCl2, at a flow rate of 20 µl/min. The data were analyzed with the T200 evaluation software, version 3.0.

Purification & Crystallization – Lyophilized WFA (Vector Laboratories Ltd, Burlington, ON, Canada) was diluted to 1 mg/mL in 20 mM Tris-HCl pH 8.0, 0.1 mM Ca2+, and 0.1 mM Mn2+, and further purified using size exclusion chromatography BioSep SEC-s3000 column (Phenomenex, Torrance, CA, USA) with the same buffer. Samples were concentrated to 15 mg/mL in presence of either 5 mM LacdiNAc or 10 mM GalNAc for crystallization trials. Sitting drops were set up in a 16°C room with 96-well plates using a Gryphon Xtallization Robot (Art Robbins Instruments, San Jose, CA, USA). Crystals of WFA in presence of LacdiNAc appeared in over 10% all screens tested.

The best crystal conditions were obtained in condition 22, 9, and 6 of PEG I, PEG II, and JCSG+ crystal screens (Qiagen, Toronto, ON, Canada) with the following formulas respectively: 0.1 M Tris-HCl pH 8.5, 25% (v/v) PEG 550 MME; 0.1 M NaAc, 0.1 M MES pH 6.5, 30% (w/v) PEG 400; and 0.2 M Li2SO4, 0.1 M phosphate-citrate pH 4.2, 20% (w/v) PEG 1000, referred to as condition I, II, and III respectively (Table 1). Crystals of WFA in presence of GalNAc were grown in Index crystal screen (Hampton Research, Aliso Viejo, CA, USA) condition 48, with the following components: 0.2 M CaCl2, 0.1 M Bis-Tris pH 5.5, 45% (v/v) MPD.

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

4  

Data Collection, Molecular Replacement, & Structure Refinement – All crystals of WFA in presence of GalNAc and LacdiNAc contained appropriate amounts of cryoprotectant, and were flash-frozen in liquid nitrogen batch directly. X-ray diffraction data sets were collected at the Canadian Macromolecular Crystallography Facility on beamline 08ID-1 (CMCF-ID) of the Canadian Light Source (Saskatoon, Saskatchewan, Canada) at 0.979 Å wavelength, with a MarMosaic CCD300 detector, and processed using HKL2000 (HKL Research Inc. Charlottesville, VA, USA).

The structure of WFA in complex with GalNAc was solved by molecular replacement using Phaser (42) with a monomer of a seed lectin from Vatairea macrocarpa (43) (PDB: 4XTM) as a search model. Subsequently, structures I, II, and III in complex with LacdiNAcβ-pNP were solved using the WFA-GalNAc as a search model. Manual fitting of σA-weighted Fo_Fc and 2Fo_Fc electron density maps was carried out with Coot (44). Restrained refinement was carried out using REFMAC5 (45). All stereo figures and r.m.s.d. (root mean square deviation) calculations presented in this paper were made using SetoRibbon (available upon request from author S. V. E.). Electrostatic surface potential figures were made using Chimera molecular visualization software (46). Marvin version 5.7.0 from ChemAxon was used for drawing chemical structures and make models for LacdiNAcβ-pNP used in this study. Geometric restraints and final model generation were obtained from the PRODRG server (47). Buried surface area was calculated with AreaIMol (48) in CCP4 suite (49) using 1.4 Å probe radius and standard van der Waals radii. All solvent molecules were excluded for the calculations. Calculations were averaged to account for small differences between the buried surfaces in each monomer. Tryptic tandem mass spectrometric analysis – Purified WFA was diluted to 1 mg/mL in 20 mM Tris-HCl prior to MS analysis. WFA was

first digested using trypsin (Promega, Madison, WI, USA) for 18 h at room temperature, and subsequently acidified with formic acid and stored in -80 ºC. The sample was subsequently eluted through a zip tip into an Eppendorf tube with 5 µl 0.1% TFA and 50% acetonitrile. Tryptic peptides were subjected to mass spectrometric analysis using a nano-HPLC system (Easy-nLC II, Thermo Fisher Scientific, Mississauga, ON, Canada), coupled to the ESI-source of an LTQ Orbitrap Velos (Thermo Fisher Scientific), using conditions described in (50).

Briefly, samples were injected onto a 100 µm ID, 360 µm OD trap column packed with Magic C18AQ (Bruker-Michrom, Auburn, CA, USA), 100 Å, 5 µm pore size (prepared in-house) and desalted by washing with Solvent A (2% acetonitrile:98% water, 0.1% formic acid (FA)). Peptides were separated with a 30 min gradient (0-20 min: 5-40% solvent B (90% acetonitrile, 10% water, 0.1% FA), 20-22 min: 40-100% B, 22-30 min: 100% B), on a 15 cm long, 75 µm ID, 360 µm OD analytical column packed with Magic C18AQ 100 Å, 5 µm pore size (prepared in-house), with IntegraFrit (New Objective Inc., Woburn, MA, USA) and equilibrated with solvent A. MS data were acquired using a data dependent method, while utilizing dynamic exclusion, with a 10 ppm exclusion window and over a duration of 15 seconds. MS and MS/MS events used 60000 and 15000 resolution FTMS scans, respectively, with a scan range of 400-2000 m/z in the MS. For MS/MS, CID collision energy was set to 35%. The resulting MS/MS datasets were analyzed using PEAKS7 program (Bioinformatics Solutions Inc, Waterloo, ON, Canada). A database containing sixteen possible sequences of WFA, and a known set of possible contaminants including trypsin was used. False discovery rate was set at 0.1% with a cut-off –10log10(P-value) score of 62.0. Peptic tandem mass spectrometric analysis – WFA (1 mg/mL) in Tris buffer was reduced with 10 mM tris[2-carboxyethyl]phosphine for

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

5  

4 hrs at room temperature. 50 pmol of WFA was subsequently diluted into 10 mM HEPES pH 7.5, 50 mM NaCl, and acidified to give a concentration of 0.6 M guanidine-HCl, 0.8% formic acid. Samples were rapidly frozen in liquid nitrogen and stored at −80°C until mass analysis. Protein samples were rapidly thawed and injected onto a UPLC system kept in a cold box at 2 ºC. The sample was run over two porosyme immobilized pepsin columns (Applied Biosystems; porosyme, 2‐3131‐00) in series, one at 10 ºC and the other at 2 ºC using 200 µL/min for 3 min and peptides were collected onto a VanGuard precolumn trap (Waters Ltd., Mississauga, ON, Canada). The trap was subsequently eluted in line with an Acquity 1.7 µm particle, 100 × 1 mm2 C18 UPLC column (Waters), using a gradient of 5-36% B (buffer A 0.1% formic acid, buffer B 100% acetonitrile) over 20 min. Mass spectrometry experiments were performed on an Impact II TOF (Bruker) acquiring over a mass range from 350 to 1500 m/z for 30 min, using an electrospray ionization source operated at 200 °C and a spray voltage of 4.5 kV (Impact).

MS/MS was run in a data-dependent acquisition mode with a 0.5 s precursor scan from 200-2000 m/z followed by 12 fragment scans from 150-2000 m/z of 0.25 s (Impact). The resulting MS/MS datasets were analyzed using PEAKS7 (Bioinformatics Solutions Inc). A database containing a number of possible sequences from WFA, and a known set of possible contaminants was used including pepsin. False discovery rate was set at 0.1% with a cut-off –10log10(P-value) score of 29.9. RESULTS Synthesis of LacdiNAcβ-pNP – The H. pylori β-1,4-galactosyltransferase (HP0826) was able to use UDP-GalNAc as a donor, although its natural donor is UDP-Gal. Analytical test syntheses showed that HP0826 could achieve >99% conversion of a fluorescent GlcNAcβ- derivative to LacdiNAc- when using a large excess (5X) of UDP-GalNAc and extended incubation times (data not shown). The

synthesis of LacdiNAc could be performed at preparative scale by coupling the reaction with the UDP-GlcNAc/Glc 4-epimerase (Cj1131c) from C. jejuni in order to use UDP-GlcNAc as a precursor. Starting with 15 mg of p-nitrophenyl GlcNAc, we synthesized 20 mg of LacdiNAcβ-pNP with a recovery yield >85%. The structure and purity of the product were confirmed by thin layer chromatography (data not shown), capillary electrophoresis (data not shown), mass spectrometry (data not shown) and NMR (Table S1, Figure S1).

Surface Plasmon Resonance – On the basis of the glycan array data, three compounds were chosen for SPR analyses (Figure S2), representing high, medium and low affinity structures (Table 2). In steady state analyses, the KD of WFA for LacdiNAcβ-pNP was 5.45 µM, for GalNAcα-pNP, 92.4 µM, and for Galβ-pNP, 0.47 mM. Kinetic values obtained for LacdiNAcβ-pNP were ka 1.33 × 104 M-1sec-1and kd 0.07 sec-1 resulting in a KD of 5.3 × 10-6 M, in good agreement with the value from the steady state analysis.

X-ray Diffraction Data & Overall Structure – Data collection and refinement statistics for WFA structures are given in Table 1. Data were collected for WFA crystals in complex with GalNAc were collected to 2.34 Å, and solved in the space group P21212, with an Rpim of 3.50%. The structure contained one tetrameric lectin in the asymmetric unit, displaying the characteristic legume fold. (Fig 2A). Like other L-type lectins, WFA possess a conserved metal binding site for the divalent Ca2+ and Mn2+ ions. Their presence allow for the formation of a cis peptide bond between Ala116 and Asp117 (numbering scheme shown in Table 3), a common feature of legume lectins which is essential for proper folding and carbohydrate binding (51).

Excellent electron density is observed for the GalNAc (Fig. 2B) in all four binding sites and for the polypeptide chain all monomers, with exception of solvent exposed residues 68-71, 91-94, 141-148. C-terminal residues 269-

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

6  

273, including the Cys residue were disordered, and thus excluded from the final model.

Data for WFA in complex with LacdiNAcβ-pNP were collected to 1.80, 1.95, and 2.09 Å resolutions for conditions I (pH 8.5), II (pH 6.5), and III (pH 4.2) respectively. All three structures contained one homotetramer in the asymmetric unit and were solved in space group P212121 with an Rpim of 4.30%, 8.90%, and 5.70% respectably. Excellent electron density is observed for the LacdiNAcβ-pNP (Fig. 2C–E) and polypeptides in three structures, with exception of solvent exposed residues in identical regions as the WFA-GalNAc complex. Condition III displayed poor density and high B-factors for the nitrophenyl moiety of LacdiNAcβ-pNP (Fig. 2E).

All structures also showed good density for N-linked GlcNAc on Asn146, while a few of the monomers showed unambiguous density for a bi-antennary N-glycan up to seven ordered carbohydrate residues, including a β(1→2)Xyl residue typically found in plant N-glycans. The most ordered N-glycan structure (lowest mean isotropic temperature factor) is observed for one of the monomers in condition III and is stabilized by crystal contacts via terminal α(1→3)Man (Fig. 2F).

WFA-GalNAc Interactions & Buried Surface Area – There are seven hydrogen bonds formed between WFA and GalNAc (Fig. 2G), summarized in Table 4. The total buried surface area for the GalNAc residue is ~146 Å2. There are multiple hydrophobic interactions between the hydrophobic face of sugar ring and Phe159, in addition to van der Waals contacts from β carbon and C4’ of His249 side chain to C6’ carbon of GalNAc (Fig. 2G). WFA forms a hydrophobic groove via Gly134, Gly135, Trp163, and Leu245 surrounding the acetyl group of GalNAc. There is also an indirect hydrogen bond formed via a water molecule and the carbonyl group of Pro133. The carbonyl group of the acetamide moiety is oriented toward the binding site, forming a weak a hydrogen bond (3.00-3.15 Å) with the amide

atom of Gly134 (Fig. 2G). The methyl moiety of GalNAc forms van der Waals contacts (3.8-4.0 Å) with Trp163 residue. WFA-LacdiNAc Interactions & Buried Surface Area – In the three structures of WFA in complex with LacdiNAc, all direct hydrogen bonds were directed to the GalNAc moiety (Fig. 2G). An additional water mediated hydrogen bond is observed between the amido group of GlcNAc and carbonyl group of Leu245 (Fig. 2H). There were also hydrophobic interactions between Leu245 and the hydrophobic face of the GlcNAc residue (Fig. 2H). The additional buried surface area for GlcNAc is ~ 37 Å2, of which 23 Å2 and 14 Å2 are contributions of Leu245 and Ser246 respectively.

Tandem MS Sequencing & Alignment – During refinement and modelling, we discovered significant differences in the 2Fo−Fc and Fo−Fc electron density maps around Thr183 and Gly190 (Fig 3A) in all structures when compared to sequence published in the patent by Sato et al., (38). Based on their electron density and surrounding environment, we predicted that Thr183 should be an Ile and Gly190 is either an Asn or an Asp (Fig 3B). To confirm sequence and presence of isolectins in sample, we performed tandem mass spec analysis on WFA. We achieved 91% and 94% protein coverage by cleavage with trypsin (Fig. 3C) in native condition and pepsin (Fig. 3D) in reduced condition respectively. Combined, we obtained sequence coverage of 98%, excluding the N and C-terminal cleaved residues that are cleaved post-translationally. Residues ranging 181-204 could not be confirmed using trypsin digest alone, but were confirmed using pepsin digest in conjunction with the electron density. The presence of Ile183 and Asp190 was confirmed via multiple fragments from the pepsin digest. The best one showed a –10log10(P-value) score of 48.5 and a ppm error of 0.7, corresponding to sequence S.IVSRKTISWDLEND.E (Fig. 3E). Interestingly, the original residues Thr183 and Gly190 (not observed in the electron density),

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

7  

were observed in one of the fragments (of sequence S.IVSRKTTSWDLENGEVAN.V) with a –10log10(P-value) score of 57.4 and a ppm error of –2.3 (Fig. 3F). Only five residues could not be confirmed using either method, which were Arg144, His145, Lys146, Trp258, and Thr259. The sequence obtained from the mass spectrometry analysis and with electron density analysis is shown in Table 5, along with related plant lectins.

DISCUSSION Synthesis of LacdiNAcβ-pNP – Palcic and Hindsgaul (52) reported that the bovine N-acetylglucosamine β1,4-galactosyltransferase (B4GalT1) displayed flexibility in the donor substrate specificity. They were able to synthesize GalNAcβ1,4-GlcNAc (LacdiNAc) terminating structures on the mg scale, using UDP-GalNAc as donor. Bojarová et al. (53) introduced a mutation (Y284L) in the human placental B4GalT1 which changed the donor preference from UDP-Gal to UDP-GalNAc. The B4GalTY284L was used to efficiently synthesize mono and bivalent LacdiNAc glycomimetics with good yields. The bacterial β1,4-galactosyltransferase from H. pylori (HP0826) is involved in the biosynthesis of the LPS O-chain backbone. Namdjou et al. (40) reported that HP0826 can be expressed in high yields in Escherichia coli and is an efficient biocatalyst to synthesize compounds with terminal β1,4-linked Gal residues. Similarly to the bovine B4GalT1, HP0826 could achieve >99% conversion of GlcNAcβ derivatives to LacdiNAc derivatives when using a large excess of UDP-GalNAc and extended incubation times. This work reports an efficient protocol that should be applicable for the synthesis of other LacdiNAc derivatives from GlcNAc precursors, using bacterial enzymes that are easy to produce at large scale. WFA Recognition of LacdiNAc and GalNAc – WFA displays one of the highest affinities observed for a legume lectin to a disaccharide, with a KD of 5.45 µM against LacdiNAc. The 17-fold difference in affinity between

LacdiNAc and GalNAc is likely due to the displacement of water around Leu245 and hydrophobic interactions between Leu245 and the hydrophobic face of GlcNAc (Fig. 2H). In contrast, the 5-fold higher affinity to GalNAc compared to Gal is likely due to a combination of an additional hydrogen bond to the acetamido group and hydrophobic interactions to the methyl group. Hydrophobic residues surrounding acetamides are common in carbohydrate binding proteins and other homologous legume lectins.

Comparison to Other Legume Lectins – Most leguminous lectin monomers adopt the “jelly-roll” (a type of β-sandwich) topology characterized by three β-sheets: a six-stranded back sheet, a small top sheet, and a seven-stranded curved front sheet, and finally a series of beta turns and loops which hold the two large sheets together (54,55). The front sheet together with a few of the loops form the carbohydrate binding site. Despite observed structural conservation between leguminous lectins, their specificities, loops, and quaternary structures vary widely (54-56). There are many plant lectins in the literature with sequence identity of 60-70% to WFA, with a subset of these shown in Table 5. The structures of lectins from Robina pseudoacacia and Vatairea macrocarpa have been determined in complex with GalNAc (43,57) (PDB: 1FNZ and 4XTM respectively), and with Tn antigen in the latter case (PDB: 4XTP).

Like WFA, these lectins form a stable tetramer even over a wide range of pH, however WFA oligomerization is further facilitated via disulfide bonds between the C-terminal ends of each monomer (9). This finding was the basis for a patent (38) where a mutation of C-terminal Cys to a Ala residue produced monomeric recombinant WFA. Cys residues in legume lectins are rare; one other GalNAc-specific lectin isolated from Sophora japonica tree bark has disulfide bonds cross-linking monomers (58). The C-terminal residues were

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

8  

disordered in all of the structures of WFA and excluded from the final model.

Many of these lectins use the same amino acid residues in binding and metal coordination (Table 5). The V. macrocarpa lectin shares the highest sequence homology and all residues that are observed to make contact with GalNAc and Tn antigen in those structures are also found in WFA. Secondary structure alignment of WFA with V. macrocarpa lectin shows a near perfect overlap between the bound GalNAc, and the binding site residues (including the underlying residues) with a r.m.s.d. of 0.35 Å. Due to the high structural and sequence homology with V. macrocarpa lectin, WFA would be expected to bind Tn-antigen, but glycan array experiments have shown GalNAcα compounds have much lower affinity. Furthermore, in an earlier study (8), WFA staining did not overlap with an anti-Tn antibody.

In contrast, the lectin isolated from R. pseudoacacia uses an Asp instead of the shorter Ser246 (Ser257 in Table 5), which would form steric clashes with the GlcNAc, hindering the recognition of LacdiNAc. All other residues contacting GalNAc were conservative mutations or identical between R. pseudoacacia and WFA.

To date, only one other lectin structure has been determined in complex with LacdiNAc, and like WFA, Clitocybe nebularis lectin (CNL) forms hydrogen bonds exclusively to the GalNAc moiety (34). There are six hydrogen bonds formed between the CNL and GalNAc compared to the seven of WFA, with the latter also forming a larger buried surface area to the GlcNAc moiety than CNL. This would be consistent with the use of WFA as a more sensitive vehicle for detection of low levels of LacdiNAc expressed on surfaces of cancerous cells. The C. nebularis lectin also differs significantly in its fold and quaternary structure compared to WFA. The CNL fold is similar to the β-chain of ricin (hence referred to as a “ricin-like or R-type lectin”) forming primarily a dimer in its native state, and exhibits

low sequence and structural homology with WFA.

Tandem MS Sequencing & Presence of Isolectins – Due to the discrepancy observed between the electron density and the sequence published in the patent by Sato et al., (38), we performed both tryptic and peptic digest of WFA coupled with mass spectrometric analysis. We obtained a total of 98% sequence coverage. We detected two forms of the lectin present in solution, with multiple polypeptide fragments containing the residues Ile183 and Asp190 while one fragment confirmed the presence of an isolectin with the previously reported sequence of Thr183 and Gly190. However, the 2Fo−Fc and Fo−Fc electron density maps were more appropriate for Ile183 and Asp190 sidechains, possibly due to higher abundance of this isoform.

In addition, we did not observe polypeptide fragments containing Cys272 (Fig. 3C) under native condition, while pepsin digest under reducing condition (Fig. 3D) confirmed the presence of Cys272 and its participation in disulfide bond formation. The disulfide bond between the C-terminal tails of neighboring monomers is likely involved in further stabilization of the tetramer and its flexibility inhibits  access of large molecules to the central channel, which in turn could destabilize oligomerization (43). Sato et al., (38) also postulated that the C-terminal end is processed and cleaved between Asn274 and Asn275 by an enzyme, and our digest under reducing condition corroborated this finding with coverage up to Asn274. Asn-specific cleavage of C-terminal peptides of legume lectins is a common phenomenon (59,60).

Effects of pH on Structure & Binding – Three different structures of WFA were obtained in three different conditions at pH 8.5, 6.5 and 4.2 (condition I, II, and III, respectively). Though no significant differences were observed between the main chain polypeptide structures; i.e. no conformational changes observed, acidic pH had a destabilizing effect on ligand. The

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

9  

respective mean B-factors of the ligand averaged across all four monomers were 44.0, 34.3, and 53.4 for condition I, II, and III. This is also evident in the electron density maps, with poor density observed for the GlcNAc (in some monomers) and nitrophenyl moieties in condition III (Fig. 2E). Conclusion – Recent studies of WFA-reactive colony-stimulating factor 1 receptor and Mac-2-binding protein (27,33), emphasize the clinical importance of this lectin as a tool for diagnosis and detections of cancers, particularly early hepatocellular carcinoma. Until now, the molecular basis for binding of WFA to LacdiNAc was unknown. Our study shows how WFA achieves high affinity to LacdiNAc biomarker, paving the way for rational engineering of the carbohydrate binding site for further improvements. Further, structures of W. floribunda lectin in complex with GalNAc and LacdiNAc in conjunction with dissociation constants, provide the first molecular explanation for the preference of WFA for LacdiNAc over GalNAc. WFA achieves higher affinity for LacdiNAc via additional van der Waals interactions and buried surface area around the hydrophobic face of the GlcNAc sugar ring. Additionally, the carbohydrate binding site of WFA is identical to a structurally related seed lectin derived from V. macrocarpa. This lectin binds both α and β GalNAc residues, and it has been proposed as a means of detecting the Tn-antigen (43). However, glycan array experiments have shown WFA recognizes the Tn-antigen with a much lower affinity than for LacdiNAc, and WFA staining did not overlap that of an anti-Tn antibody (8). Finally, our findings confirm the presence of disulfide bonds between the WFA monomers, and at least one other isolectin from commercial source. In combination with avidity effects, this tetrameric WFA achieves high degree of specificity towards LacdiNAc required for detection of cancerous cells coated with this biomarker.

ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Nicholas I. Brodie and Evgeniy V. Petrotchenko at the Genome British Columbia Proteomics Centre, Vancouver Island Technology Park. We thank Dr. Greg Hussack for help with the SPR analysis, Dr. Evgeny Vinogradov for NMR analysis and Jacek Stupak for mass spectrometry analysis of the LacdiNAcβ-pNP. The glycan array facility of the CFG was supported by NIGMS grant GM62116. Research described in this paper was performed at the Canadian Light Source, which is supported by the Canada Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, the University of Saskatchewan, the Government of Saskatchewan, Western Economic Diversification Canada, the National Research Council Canada, and the Canadian Institutes of Health Research.

CONFLICT OF INTEREST The authors have no conflict of interest to declare. AUTHOR CONTRIBUTIONS O.H.G. performed the majority of the experiments, prepared all figures and tables, analyzed the data, and wrote a major part of the paper. M.G. analyzed the data and contributed to writing the paper, J.S. conducted the initial crystallization trials and contributed to writing the paper. M.J.S. performed the synthesis of LacdiNAc. M.J.P. initiated the project and designed some of the experiments. M.L.J. conducted the peptic digest experiment. J.E.B. helped in analysis of the mass spectrometric results and edited the paper. H.v.F. helped with analysis of the SPR experiment. N.M.Y. conceived some of the experiments and contributed to writing the paper. S.V.E. conceived many of the experiments and wrote a major part of the paper. All authors reviewed the results and approved the final version of the manuscript.  

 

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

10  

REFERENCES 1. Xu, Y. F., Sette, A., Sidney, J., Gendler, S. J., and Franco, A. (2005) Tumor-associated carbohydrate

antigens: A possible avenue for cancer prevention. Immunol Cell Biol 83, 440-448 2. Schietinger, A., Philip, M., and Schreiber, H. (2008) Specificity in cancer immunotherapy. Semin

Immunol 20, 276-285 3. Mody, R., Joshi, S., and Chaney, W. (1995) Use of lectins as diagnostic and therapeutic tools for

cancer. J Pharmacol Toxicol 33, 1-10 4. Yuan, M., Itzkowitz, S. H., Boland, C. R., Kim, Y. D., Tomita, J. T., Palekar, A., Bennington, J. L.,

Trump, B. F., and Kim, Y. S. (1986) Comparison of T-antigen expression in normal, premalignant, and malignant human colonic tissue using lectin and antibody immunohistochemistry. Cancer Res 46, 4841-4847

5. Do, K. Y., Do, S. I., and Cummings, R. D. (1997) Differential expression of LacdiNAc sequences (GalNAc β1-4GlcNAc-R) in glycoproteins synthesized by Chinese hamster ovary and human 293 cells. Glycobiology 7, 183-194

6. Kitamura, N., Guo, S. C., Sato, T., Hiraizumi, S., Taka, J., Ikeita, M., Sawada, S., Fujisawa, H., and Furukawa, K. (2003) Prognostic significance of reduced expression of β-N-acetylgalactosaminylated N-linked oligosaccharides in human breast cancer. Int J Cancer 105, 533-541

7. Nyame, K., Smith, D. F., Damian, R. T., and Cummings, R. D. (1989) Complex-type asparagine-linked oligosaccharides in glycoproteins synthesized by Schistosoma-Mansoni adult males contain terminal beta-linked N-acetylgalactosamine. J Biol Chem 264, 3235-3243

8. Ikehara, Y., Sato, T., Niwa, T., Nakamura, S., Gotoh, M., Ikehara, S. K., Kiyohara, K., Aoki, C., Iwai, T., Nakanishi, H., Hirabayashi, J., Tatematsu, M., and Narimatsu, H. (2006) Apical golgi localization of N,N'-diacetyllactosediamine synthase, β4GalNAc-T3, is responsible for LacdiNAc expression on gastric mucosa. Glycobiology 16, 777-785

9. Soga, K., Teruya, F., Tateno, H., Hirabayashi, J., and Yamamoto, K. (2013) Terminal N-acetylgalactosamine-specific leguminous lectin from Wisteria japonica as a probe for human lung squamous cell carcinoma. Plos One 8

10. Sugii, S., and Kabat, E. A. (1980) Immunochemical specificity of the combining site of Wistaria floribunda hemagglutinin. Biochemistry-Us 19, 1192-1199

11. Kurokawa, T., Tsuda, M., and Sugino, Y. (1976) Purification and characterization of a lectin from Wistaria floribunda seeds. J Biol Chem 251, 5686-5693

12. Toyoshima, S., Akiyama, Y., Nakano, K., Tonomura, A., and Osawa, T. (1971) Phytomitogen from Wistaria floribunda seeds and its interaction with human peripheral lymphocytes. Biochemistry-Us 10, 4457-4463

13. Baker, D. A., Sugii, S., Kabat, E. A., Ratcliffe, R. M., Hermentin, P., and Lemieux, R. U. (1983) Immunochemical studies on the combining sites of Forssman hapten reactive hemagglutinins from Dolichos biflorus, Helix pomatia, and Wistaria floribunda. Biochemistry-Us 22, 2741-2750

14. De Hoff, P. L., Brill, L. M., and Hirsch, A. M. (2009) Plant lectins: the ties that bind in root symbiosis and plant defense. Mol Genet Genomics 282, 1-15

15. Drickamer, K., and Taylor, M. E. (2002) Glycan arrays for functional glycomics. Genome Biol 3, reviews1034.1031

16. Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M. E., Alvarez, R., Bryan, M. C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D. J., Skehel, J. J., van Die, I., Burton, D. R., et al. (2004) Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. P Natl Acad Sci 101, 17033-17038

17. Sasaki, N., Yoshida, H., Fuwa, T. J., Kinoshita-Toyoda, A., Toyoda, H., Hirabayashi, Y., Ishida, H., Ueda, R., and Nishihara, S. (2007) Drosophila beta 1,4-N-acetylgalactosaminyltransferase A synthesizes the lacdiNAc structures on several glycoproteins and glycosphingolipids. Biochem Bioph Res Co 354, 522-527

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

11  

18. Srivatsan, J., Smith, D. F., and Cummings, R. D. (1994) Demonstration of a novel UDP GalNAc-GlcNAc β-1-4 N-acetylgalactosaminyltransferase in extracts of Schistosoma mansoni. J Parasitol 80, 884-890

19. Frederiksen, R. F., Yoshimura, Y., Storgaard, B. G., Paspaliari, D. K., Petersen, B. O., Chen, K., Larsen, T., Duus, J. O., Ingmer, H., Bovin, N. V., Westerlind, U., Blixt, O., Palcic, M. M., and Leisner, J. J. (2015) A Diverse Range of Bacterial and Eukaryotic Chitinases Hydrolyzes the LacNAc (Gal beta 1-4GlcNAc) and LacdiNAc (GalNAc beta 1-4GlcNAc) Motifs Found on Vertebrate and Insect Cells. J Biol Chem 290, 5354-5366

20. Van den Eijnden, D. H., Neeleman, A. P., Vanderknaap, W. P. W., Bakker, H., Agterberg, M., and Vandie, I. (1995) Novel glycosylation routes for glycoproteins. The LacdiNAc pathway. Biochem Soc T 23, 175-179

21. Sato, T., Gotoh, M., Kiyohara, K., Kameyama, A., Kubota, T., Kikuchi, N., Ishizuka, Y., Iwasaki, H., Togayachi, A., Kudo, T., Ohkura, T., Nakanishi, H., and Narimatsu, H. (2003) Molecular cloning and characterization of a novel human β1,4-N-acetylgalactosaminyltransferase, β4GalNAc-T3, responsible for the synthesis of N,N '-diacetyllactosediamine, GalNAcβ1-4GlcNAc. J Biol Chem 278, 47534-47544

22. Machado, E., Kandzia, S., Carilho, R., Altevogt, P., Conradt, H. S., and Costa, J. (2011) N-Glycosylation of total cellular glycoproteins from the human ovarian carcinoma SKOV3 cell line and of recombinantly expressed human erythropoietin. Glycobiology 21, 376-386

23. Hirano, K., Matsuda, A., Shirai, T., and Furukawa, K. (2014) Expression of LacdiNAc groups on N-glycans among human tumors is complex. Biomed Res Int, doi: 10.1155/2014/981627

24. Che, M. I., Huang, J., Hung, J. S., Lin, Y. C., Huang, M. J., Lai, H. S., Hsu, W. M., Liang, J. T., and Huang, M. C. (2014) β1, 4-N-acetylgalactosaminyltransferase III modulates cancer stemness through EGFR signaling pathway in colon cancer cells. Oncotarget 5, 3673-3684

25. Huang, J., Liang, J. T., Huang, H. C., Shen, T. L., Chen, H. Y., Lin, N. Y., Che, M. I., Lin, W. C., and Huang, M. C. (2007) β1,4-N-acetylgalactosaminyltransferase III enhances malignant phenotypes of colon cancer cells. Mol Cancer Res 5, 543-552

26. Yamaguchi, T., Yokoyama, Y., Ebata, T., Matsuda, A., Kuno, A., Ikehara, Y., Shoda, J., Narimatsu, H., and Nagino, M. (2016) Verification of WFA-sialylated MUC1 as a sensitive biliary biomarker for human biliary tract cancer. Ann Surg Oncol 23, 671-677

27. Iio, E., Ocho, M., Togayachi, A., Nojima, M., Kuno, A., Ikehara, Y., Hasegawa, I., Yatsuhashi, H., Yamasaki, K., Shimada, N., Ide, T., Shinkai, N., Nojiri, S., Fujiwara, K., Joh, T., et al. (2016) A novel glycobiomarker, Wisteria floribunda agglutinin macrophage colony-stimulating factor receptor, for predicting carcinogenesis of liver cirrhosis. Int J Cancer 138, 1462-1471

28. Narimatsu, H. (2015) Development of M2BPGi: a novel fibrosis serum glyco-biomarker for chronic hepatitis/cirrhosis diagnostics. Expert Rev Proteomic 12, 683-693

29. Sugahara, D., Tomioka, A., Sato, T., Narimatsu, H., and Kaji, H. (2015) Large-scale identification of secretome glycoproteins recognized by Wisteria floribunda agglutinin: A glycoproteomic approach to biomarker discovery. Proteomics 15, 2921-2933

30. Tamaki, N., Kurosaki, M., Kuno, A., Korenaga, M., Togayachi, A., Gotoh, M., Nakakuki, N., Takada, H., Matsuda, S., Hattori, N., Yasui, Y., Suzuki, S., Hosokawa, T., Tsuchiya, K., Nakanishi, H., et al. (2015) Wisteria floribunda agglutinin positive human Mac-2-binding protein as a predictor of hepatocellular carcinoma development in chronic hepatitis C patients. Hepatol Res 45, E82-E88

31. Matsuda, A., Kuno, A., Nakagawa, T., Ikehara, Y., Irimura, T., Yamamoto, M., Nakanuma, Y., Miyoshi, E., Nakamori, S., Nakanishi, H., Viwatthanasittiphong, C., Srivatanakul, P., Miwa, M., Shoda, J., and Narimatsu, H. (2015) Lectin microarray-based sero-biomarker verification targeting aberrant O-Linked glycosylation on mucin 1. Anal Chem 87, 7274-7281

32. Sogabe, M., Nozaki, H., Tanaka, N., Kubota, T., Kaji, H., Kuno, A., Togayachi, A., Gotoh, M., Nakanishi, H., Nakanish, T., Mikami, M., Suzuki, N., Kiguchi, K., Ikehara, Y., and Narimatsu, H. (2014) Novel glycobiomarker for ovarian cancer that detects clear cell carcinoma. J Proteome Res 13, 1624-1635

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

12  

33. Toyoda, H., Kumada, T., Tada, T., Kaneoka, Y., Maeda, A., Korenaga, M., Mizokami, M., and Narimatsu, H. (2016) Serum WFA(+)-M2BP levels as a prognostic factor in patients with early hepatocellular carcinoma undergoing curative resection. Liver Int 36, 293-301

34. Pohleven, J., Renko, M., Magister, S., Smith, D. F., Kunzler, M., Strukelj, B., Turk, D., Kos, J., and Sabotic, J. (2012) Bivalent carbohydrate binding is required for biological activity of Clitocybe nebularis Lectin (CNL), the N,N '-diacetyllactosediamine (GalNAc β1-4GlcNAc, LacdiNAc)-specific lectin from Basidiomycete C. nebularis. J Biol Chem 287, 10602-10612

35. Matsuda, A., Kuno, A., Kawamoto, T., Matsuzaki, H., Irimura, T., Ikehara, Y., Zen, Y., Nakanuma, Y., Yamamoto, M., Ohkohchi, N., Shoda, J., Hirabayashi, J., and Narimatsu, H. (2010) Wisteria floribunda agglutinin-positive mucin 1 is a sensitive biliary marker for human cholangiocarcinoma. Hepatology 52, 174-182

36. Kuno, A., Ikehara, Y., Tanaka, Y., Ito, K., Matsuda, A., Sekiya, S., Hige, S., Sakamoto, M., Kage, M., Mizokami, M., and Narimatsu, H. (2013) A serum "sweet-doughnut" protein facilitates fibrosis evaluation and therapy assessment in patients with viral hepatitis. Sci Rep-Uk 3, 1065

37. Fujiyoshi, M., Kuno, A., Gotoh, M., Fukai, M., Yokoo, H., Kamachi, H., Kamiyama, T., Korenaga, M., Mizokami, M., Narimatsu, H., Taketomi, A., and Group, H. G.-b. S. (2015) Clinicopathological characteristics and diagnostic performance of Wisteria floribunda agglutinin positive Mac-2-binding protein as a preoperative serum marker of liver fibrosis in hepatocellular carcinoma. J Gastroenterol 50, 1134-1144

38. Sato, T., Chiba, Y., Tateno, H., Kaji, H., Goto, M., and Narimatsu, H. (2014) Modified lectin derived from Wisteria floribunda. in Google Patents (Technology, N. I. O. A. I. S. A. ed., Japan

39. Toyoshima, S., and Osawa, T. (1975) Lectins from Wistaria floribunda seeds and their effect on membrane fluidity of human peripheral lymphocytes. J Biol Chem 250, 1655-1660

40. Namdjou, D. J., Chen, H. M., Vinogradov, E., Brochu, D., Withers, S. G., and Wakarchuk, W. W. (2008) A β-1,4-galactosyltransferase from Helicobacter pylori is an efficient and versatile biocatalyst displaying a novel activity for thioglycoside synthesis. Chembiochem 9, 1632-1640

41. Bernatchez, S., Szymanski, C. M., Ishiyama, N., Li, J. J., Jarrell, H. C., Lau, P. C., Berghuis, A. M., Young, N. M., and Wakarchuk, W. W. (2005) Single bifunctional UDP-GlcNAc/Glc 4-epimerase supports the synthesis of three cell surface glycoconjugates in Campylobacter jejuni. J Biol Chem 280, 4792-4802

42. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software. J Appl Crystallogr 40, 658-674

43. Sousa, B. L., Silva, J. C., Kumar, P., Graewert, M. A., Pereira, R. I., Cunha, R. M. S., Nascimento, K. S., Bezerra, G. A., Delatorre, P., Djinovic-Carugo, K., Nagano, C. S., Gruber, K., and Cavada, B. S. (2016) Structural characterization of a Vatairea macrocarpa lectin in complex with a tumor-associated antigen: A new tool for cancer research. Int J Biochem Cell B 72, 27-39

44. Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D 60, 2126-2132

45. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D 53, 240-255

46. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and Ferrin, T. E. (2004) UCSF chimera - A visualization system for exploratory research and analysis. J Comput Chem 25, 1605-1612

47. Schuttelkopf, A. W., and van Aalten, D. M. F. (2004) PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr D 60, 1355-1363

48. Lee, B., and Richards, F. M. (1971) Interpretation of protein structures: Estimation of static accessibility. J Mol Biol 55, 379-400

49. Bailey, S. (1994) The ccp4 suite: Programs for protein crystallography. Acta Crystallogr D 50, 760-763

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

13  

50. Petrotchenko, E. V., Makepeace, K. A. T., Serpa, J. J., and Borchers, C. H. (2014) Analysis of protein structure by cross-linking combined with mass spectrometry. Methods Mol Biol 1156, 447-463

51. Garcia-Pino, A., Buts, L., Wyns, L., and Loris, R. (2006) Interplay between metal binding and cis/trans isomerization in legume lectins: Structural and thermodynamic study of P. angolensis lectin. J Mol Biol 361, 153-167

52. Palcic, M. M., and Hindsgaul, O. (1991) Flexibility in the donor substrate specificity of beta 1,4-galactosyltransferase: application in the synthesis of complex carbohydrates. Glycobiology 1, 205-209

53. Bojarová, P., Krenek, K., Wetjen, K., Adamiak, K., Pelantova, H., Bezouska, K., Elling, L., and Kren, V. (2009) Synthesis of LacdiNAc-terminated glycoconjugates by mutant galactosyltransferase - A way to new glycodrugs and materials. Glycobiology 19, 509-517

54. Loris, R., Hamelryck, T., Bouckaert, J., and Wyns, L. (1998) Legume lectin structure. Bba-Protein Struct M 1383, 9-36

55. Chandra, N. R., Prabu, M. M., Suguna, K., and Vijayan, M. (2001) Structural similarity and functional diversity in proteins containing the legume lectin fold. Protein Eng 14, 857-866

56. Sharon, N., and Lis, H. (1990) Legume lectins - a large family of homologous proteins. Faseb J 4, 3198-3208

57. Rabijns, A., Verboven, C., Rouge, P., Barre, A., Van Damme, E. J. M., Peumans, W. J., and De Ranter, C. J. (2001) Structure of a legume lectin from the bark of Robinia pseudoacacia and its complex with N-acetylgalactosamine. Proteins 44, 470-478

58. Hankins, C. N., Kindinger, J. I., and Shannon, L. M. (1988) The lectins of Sophora japonica. II. Purification, properties, and N-terminal amino-acid sequences of 5 lectins from bark. Plant Physiol 86, 67-70

59. Young, N. M., Watson, D. C., Yaguchi, M., Adar, R., Arango, R., Rodriguez-Arango, E., Sharon, N., Blay, P. K., and Thibault, P. (1995) C-terminal post-translational proteolysis of plant lectins and their recombinant forms expressed in Escherichia coli. Characterization of "ragged ends" by mass spectrometry. Jf Biol Chem 270, 2563-2570

60. Young, N. M., Watson, D. C., and Thibault, P. (1996) Post-translational proteolytic processing and the isolectins of lentil and other Viciae seed lectins. Glycoconjugate J 13, 575-583

61. Karplus, P. A., and Diederichs, K. (2015) Assessing and maximizing data quality in macromolecular crystallography. Curr Opin Struc Biol 34, 60-68

62. Vaguine, A. A., Richelle, J., and Wodak, S. J. (1999) SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. Acta Crystallogr D 55, 191-205

63. Yoshida, K., and Tazaki, K. (1999) Expression patterns of the genes that encode lectin or lectin-related polypeptides in Robinia pseudoacacia. Aust J Plant Physiol 26, 495-502

64. Van Damme, E. J. M., Barre, A., Bemer, V., Rouge, P., VanLeuven, F., and Peumans, W. J. (1995) A lectin and a lectin-related protein are the two most prominent proteins in the bark of yellow wood (Cladrastis lutea). Plant Mol Biol 29, 579-598

65. Van Damme, E. J. M., Barre, A., Rouge, P., and Peumans, W. J. (1997) Molecular cloning of the bark and seed lectins from the Japanese pagoda tree (Sophora japonica). Plant Mol Biol 33, 523-536

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

14  

FOOTNOTES

* This work was supported by the Natural Sciences and Engineering Research Council of Canada (to S.V.E.). The authors declare that they have no conflicts of interest with the contents of this article. † Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055 STN CSC, Victoria, BC, Canada V8P 3P6 § Human Health Therapeutics, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON, Canada K1A 0R6 The atomic coordinates and structure factors (codes 5KXB, 5KXC, 5KXD, 5KXE) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) 1 To whom correspondence should be addressed: E-mail: svevans@uvic.ca. 2 The abbreviations used are: WFA, Wisteria floribunda agglutinin; LacdiNAc, β-D-GalNAc-(1→4)-D-GlcNAc; pNP, p-nitrophenyl; r.m.s.d., root mean square deviation. 3 Http://www.functionalglycomics.org/glycomics/publicdata/selectedScreens.jsp.

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

15  

Tables and Figures Table 1: Data collection and refinement statistics for W. floribunda agglutinin in complex with GalNAc and LacdiNAcβ-pNP (GalNAc[β1→4]GlcNAc-nitrophenyl).

Ligand GalNAc LacdiNAc-pNP Condition I

LacdiNAc –pNP Condition II

LacdiNAc –pNP Condition III

PDB codes: 5KXB 5KXC 5KXD 5KXE Resolution (Å)

30.0-2.33 (2.39-2.33)a

30.0-1.80 (1.85-1.80)

25.0-1.95 (2.01-1.95)

30.0-2.09 (2.15-2.09)

Space group P21212 P212121 P212121 P212121 a (Å) 121.2 79.7 79.1 76.6 b (Å) 157.3 103.4 104.3 97.9 c (Å) 55.7 148.9 148.8 149.2 α ,β,γ (°) 90 90 90 90 Volume Å3 1.06 × 106 1.23 × 106 1.23 × 106 1.12 × 106 Wavelength (Å) 0.979 0.979 0.979 0.979 Mean B-factor (Å2) 38.5 30.8 22.8 28.5 Z 4 4 4 4 Unique reflections 45331 114060 88386 67193 Redundancy 7.2 (7.3) 7.3 (7.4) 7.5 (7.6) 7.5 (7.5) 〈I/σ(I)〉 23.7 (2.7) 17.9 (1.54) 15.7 (3.46) 17.5 (3.67) CC1/2 (%)b (88.8) (72.2) (78.2) (86.0) Rpim (%)c 3.50 (44.4) 4.30 (65.5) 8.90 (50.8) 5.70 (30.5) Completeness (%) 99.8 (100) 100 (100) 99.3 (98.6) 99.2 (100) Protein atoms 7404 7404 7404 7404 Solvent atoms 259 659 669 607 N-Glycan atoms 167 111 140 188 Ligand atoms 60 152 152 152 Ramachandran outliers 4 4 4 4 Solvent content (%)d 48.5 52.9 52.7 48.2 Refinement Rwork (%) 18.6 20.3 18.5 17.8 Rfree (%)e 22.2 22.4 20.4 21.1 rms bond lengths (Å) 0.010 0.010 0.010 0.011 rms bond angles (°) 1.49 1.54 1.53 1.61

a Values in the parentheses refer to the highest resolution shell. b CC1/2 = σi

2 / σi 2 + σj

2 where σj donates mean error for half-dataset (61). c Formula ∑hkl (1/n−1)1/2 ∑i |Ihkl, i – [Ihkl]| / ∑hkl ∑i Ihkl, i where [Ihkl] is the average of Friedel-related observations (i) of a unique reflection (hkl). d Estimated using sfcheck (62). e 5 % of reflections were omitted for Rfree calculations.  

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

16  

Table 2: Kinetic and binding constants of WFA for immobilized ligands determined by SPR.

KD (M) Observed Rmax (RU)

Theoretical Rmax (RU)

Chi2 (RU2)

LacdiNAc-pNP 5.45 × 10-6 59.7 80 0.154 GalNAc-α-pNP 9.24 × 10-5 42.5 50 0.0653 Gal-β-pNP 4.67 × 10-4 37.7 44 0.0322

Table 3: Sequence of W. floribunda agglutinin as published in the patent by Sato et al., (38). Grey highlighted residues were originally assigned to Thr183 and Asp190, but our data shows that Ile183 and Asp190 (highlighted in grey) are more abundant in solution. Asn146 is glycosylated and Cys272 forms a disulphide bond between monomers of the tetramer. Underlined residues are processed and are not part of the secreted lectin. Residues in contact with LacdiNAc and GalNAc either through water bridges or direct hydrogen bond/van der Waals interactions are shown in bold. Leu245 is the only residue involved in recognition of LacdiNAc.

Lectin Amino acids   10 20 30 40 50

W. floribunda

MASSQTQNSF SVLLSISLTL FLLLLNKVNS KETTSFVFTR FSPDPQNLLL 60 70 80 90 100 QGDTVVTSSG HLQLTQVKDG EPVYSSLGRA LYYAPIHIWD SNTDTVANFV 110 120 130 140 150 TSFSFVIDA PNKAKAADGLA FFLAPVDTEP QKPGGLLGLF HDDRHNKSNH 160 170 180 190 200 IVAVEFDTF KNSWDPEGTHI GINVNSIVSR KTISWDLEND EVANVVISYQ 210 220 230 240 250 ASTKTLTAS LVYPSSSTSYI LNDVVDLKQI LPEYVRVGFT AASGLSKDHV 260 270 280 286 ETHDVLAWT FDSDLPDPSSD DCNNLHLSSN VLRGSI

Table 4: H-bond interactions between W. floribunda agglutinin residues and carbohydrate ligand in GalNAc and GalNAc(β1→4)GlcNAc-para-nitrophenyl (LacdiNAc-pNP) in condition I, II, and III complexed structures. The distance cut-off for hydrogen bond assignment was 3.3 Å. Hydrogen bond distances are averaged over the four molecules in the tetramer in the asymmetric unit. No hydrogen bonds were observed between WFA and GlcNAc in LacdiNAc ligand.

Ligand Residue Distance (Å)

residue atom(s) residue atom GalNAc LacdiNAc I LacdiNAc II LacdiNAc III

GalNAc

O7 Gly135 N 3.22 3.08 3.04 3.10

O6 Ser246 OG 2.58 2.68 2.71 2.75

O4 Asp117 OD2 2.72 2.59 2.59 2.57

Leu245 N 3.02 3.02 3.00 2.99

O3

Asp117 OD1 2.65 2.58 2.57 2.59

Gly135 N 3.15 3.04 3.07 3.03

Asn161 ND2 2.94 2.85 2.87 2.87

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

17  

Table 5: Amino acid alignment of W. floribunda agglutinin along with closely related lectins, including Robina pseudoacacia (63), Cladrastis kentukea (64), Sophora japonica (65), Vatairea macrocarpa (43). Structures of R. pseudoacacia and V. macrocarpa have been determined in complex with GalNAc with PDB codes 1FNZ and 4XTM, respectively (43,57). Residues in contact with GalNAc either directly or via a water bridges are shown in bold and residues involved in metal coordination are underlined.

Lectin Amino acids   10 20 30 40 50 60 70 80

W. floribunda MASSQ----- --TQNSFSVL LSISLTLFLL LL-NKVNSKE TTSFVFTRFS PDPQNLLLQG DTVVTSSGHL QLTQVK-DGE

R. pseudoacacia MASYN----- FKTQNSFPLL LSISF-FFLL LL-NKVNSTG SLSFSFPKFA PNQPYLILQG DALVTSTGVL QLTNVV-NGV

C. kentukea -ANSNSRPHL LQTQKPFSVV LAISITFYLL LL-NKVNSEE ALSFTFTKFV SNQDELLLQG DALVSSKGEL QLTRVE-NGQ

S. japonica MATSNSRPHL LQTHKPFSVV LAISITFFLL LL-NKVNSAE ILSFSFPKFA SNQEDLLLQG DALVSSKGEL QLTTVE-NGV

V. macrocarpa ---------- ---------- ---------- ------MASE VVSFSFTKFN PNPKDIILQG DALVTSKGKL QLTKVK-DGK

90 100 110 120 130 140 150 160

W. floribunda PVYSSLGRAL YYAPIHIWDS NTDTVANFVT SFSFVIDAPN KAKAADGLAF FLAPVDTEPQ KPGG-LLGLF HDDRHNKSNH

R. pseudoacacia PSRKSLGRAL YAAPFQIWDS TTGNVASFVT SFSFIIQAPN PATTADGLAF FLAPVDTQPL DLGG-MLGIF KNGYFNKSNQ

C. kentukea PIPHSVGRAL YSDPVHIWDS STGSVASFVT SFTFVVEAPN ENKTADGIAF FLAPPDTQVQ SLGG-FLGLF NSSVYNSSNQ

S. japonica PIWNSTGRAL YYAPVHIWDK STGRVASFAT SFSFVVKAPV ASKSADGIAF FLAPPNNQIQ GPGGGHLGLF HSSGYNSSYQ

V. macrocarpa PVDHSLGRAL YAAPIHIWDD STDRVASFAT SFSFVVEAPD ESKTADGIAF FLAPPDTQPQ KDGG-FLGLF NDS--NKSIQ   170 180 190 200 210 220 230 240

W. floribunda IVAVEFDTFK N-SWDPEGTH IGINVNSIVS RKTISWDLEN DEVANVVISY QASTKTLTAS LVYPSSSTSY ILNDVVDLKQ

R. pseudoacacia IVAVEFDTFS NRHWDPTGRH LGINVNSIKS VRTVPWNWTN GEVANVFISY EASTKSLTAS LVYPSLETSF IVHAIVDVKD

C. kentukea ILAVEFDTFS N-SWDPTARH IGIDVNSIES TRTATWGWRN GEVAIVLITY VAPAETLIAS LTYPSSQTSY ILSAAVDLKS

S. japonica IIAVDFDTHI N-AWDPNTRH IGIDVNSINS TKTVTWGWQN GEVANVLISY QAATETLTVS LTYPSSQTSY ILSAAVDLKS

V. macrocarpa TVAVEFDTFS N-TWDPSARH IGINVNSIES MKYVKWGWEN GKVANVYISY EASTKTLTAS LTYPSNATSY IVSANVDLKS

250 260 270 280 290 298

W. floribunda ILPEYVRVGF TAASGLSKDH VETHDVLAWT FDSDLPDPSS –DDCNNLHLS SNVLRGSI

R. pseudoacacia VLPEWVRFGF SATTGIDKGY VQTNDVLSWS FESNLPGGNS VASVKNAGLS TYAA----

C. kentukea ILPEWVRVGF SAATGRSAGY VETHDVLSWS FTSTLETGNS GAKQNNAHLA SYALI---

S. japonica ILPEWVRVGF TAATGLTTQY VETHDVLSWS FTSTLETGDC GAKDDNVHLV SYAFI---

V. macrocarpa ALPEWVRVGF SATSGLSRDH VETHDVLDWS FTSTL----- QAPSDDSNLE  

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

18  

Figure legends Figure 1: Chemical diagram of β-D-GalNAc-(1→4)-D-GlcNAc (LacdiNAc). Figure 2: (A) Stereo ribbon diagram of tetrameric WFA in complex with βGalNAc (green). Each monomer of WFA is in a different color, (cyan, red, orange, and blue). Divalent cations Ca++ and Mn++ are shown as spheres (yellow, purple). (B) Stereo diagram showing 2Fo−Fc electron density map (blue) contoured at 1.0 σ for the GalNAc observed in the carbohydrate binding sites of WFA. (C) Stereo diagram showing 2Fo−Fc electron density map (blue) contoured at 1.0 σ for N,N’-diacetyllactosediamine-p-nitrophenyl (LacdiNAc-pNP) observed in the carbohydrate binding sites of WFA in condition I, (pH 8.5), (D) II (pH 6.5), and (E) III (pH 4.2). (F) 2Fo−Fc electron density map for N-glycans observed in one of the monomers of condition III WFA structure. Map contoured to 0.9 σ. βGlcNAc, β-D-N-acetylglucosamine (light blue); αFuc, α-L-fucose (magenta); βXyl, β-D-xylose (orange); βMan, β-D-mannose (green); αMan, α-D-mannose (green). (G) Stereo view of WFA in complex with βGalNAc (green) and (H) LacdiNAc, showing hydrogen bonds (purple dashed spheres), van der Waals contacts (black dashes), and water (blue spheres) bridges between WFA and ligand. Interactions between WFA and GalNAc are identical to Fig. 2G, and thus not shown in Fig. 2H. Figure 3: (A) Stereo diagram 2Fo−Fc (blue) and Fo−Fc (magenta) density maps contoured at 1.0 σ and 3.00 σ observed around Thr183 and Gly190 after three cycles of refinement. (B) Stereo diagram 2Fo−Fc (blue) and Fo−Fc (magenta) density maps contoured at 1.0 σ and 3.00 σ observed after mutating Thr183 to Ile and Gly190 to Asp, following three cycles of refinement. (C) Tandem mass spectrometric coverage obtained after trypsin digest of WFA under native condition. (D) Tandem mass spectrometric coverage obtained after pepsin digest of WFA under reducing condition (10 mM TCEP). (E) Mass spectra from LC-MS/MS analysis showing annotated polypeptide fragments corresponding to electron density observed in Fig. 3B, and (F) sequence published by Sato et al. (38).

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

19  

Figure 1:

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

20  

Figure 2

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

21  

Figure 3

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

EvansMeredith L. Jenkins, John E. Burke, Henk van Faassen, N. Martin Young and Stephen V.

Omid Haji-Ghassemi, Michel Gilbert, Jenifer Spence, Melissa J. Schur, Matthew J. Parker,1-4)GlcNAc) by Wisteria floribunda agglutinin

βMolecular basis for recognition of the cancer glycobiomarker LacdiNAc (GalNAc(

published online September 6, 2016J. Biol. Chem. 

  10.1074/jbc.M116.750463Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

  http://www.jbc.org/content/suppl/2016/09/06/M116.750463.DC1

by guest on May 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from