Structure Predictions of Two <i>Bauhinia variegata</i> Lectins Reveal Patterns of C-Terminal Properties in Single Chain Legume Lectins

<div><p><i>Bauhinia variegata</i> lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant <i>Bauhinia variegata</i>. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and –II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from <i>Bauhinia</i> spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.</p> </div

Protein sequence alignment of the studied lectins.

<p>All lectins with defined C-terminal processing have their cleavage spot located 5-8 amino acids after the conserved Leu indicated by the arrow. This spot is just after an acid, polar or hydrophobic amino acid (Ser, Asn and Pro). Moreover, the cleaved peptide starts and ends with hydrophobic/small amino acids (Pro, Leu, Ala, Ile and Met). Ellipses (○), glycosylation sites. Grey highlights, amino acids in the BVL-I sequence which were not detected by Edman sequencing (starts between Ser248 and Ala249), and amino acids cleaved from the C-terminal region of the other lectins. Underlined, conserved pattern of GS-IV quaternary association. Black highlights, conserved pattern of GS-I quaternary association. Open box (□), amino acids from the C-terminal α-helix of BVL-II/BHα, SBA/BH and DBL (PDB entry: 1BJQ, chain A). Asterisk (*), conserved amino acids. Colon (:), amino acids with strong similar properties. Period (.), amino acids with weak similar properties.</p

Constitution of the C-terminal peptide and its structural representation in SBA, DBL and BVL-II.

<p>The underlined C-terminal peptide of SBA (A), DBL (B) and BVL-II (C) has hydrophobic/small amino acids at its start (Pro, Leu and Ala, respectively) and end (Ile, Leu and Met, respectively). Moreover, these three lectins are cleaved in the region afterward 5-8 amino acids of the conserved Leu (red, in positions 232, 233 and 242, respectively) right after Asn240 (SBA), Pro241 (DBL) or Ser248 (BVL-II). The cleaved C-terminal peptide shown underlined has its first Leu (red, positions 242, 242 and 250, respectively) with buried side chain in all three lectins. Since the analysed EcorL and PNA do not show any Leu on their C-terminal peptide, this amino acid may play an important role to stabilize the C-terminal α-helix and thus avoiding its cleavage.</p

Plot of the diffusion coefficient versus biopolymer concentration and the light scattering intensity as a function of the distribution in the relaxation times (DRT) for xanthan.

<p>(A) The mean diffusion coefficient (± SEM) of a range of aqueous biopolymer solutions (5–5.0×10⁻⁴ g.L⁻¹) are presented. The c* value (based on the plateau) and the c** value (equivalent to the maximum diffusion coefficient) are indicated by the dotted lines. The graphs represent solutions at different concentration regimes: (B) concentrated, showing a trimodal distribution, while the (C) semi-diluted and the (D) diluted shown a bimodal and a monomodal distribution, respectively.</p

Plot of the diffusion coefficient versus biopolymer concentration and the light scattering intensity as a function of the distribution in the relaxation times (DRT) for tara gum.

<p>(A) The mean diffusion coefficient (± SEM) of a range of aqueous biopolymer solutions (5–5.0×10⁻⁴ g.L⁻¹) are presented. The c* value (based on the plateau) and the c** value (equivalent to the maximum diffusion coefficient) are indicated by the dotted lines. The graphs represent solutions at different concentration regimes: (B) concentrated, showing a trimodal distribution, while the (C) semi-diluted and the (D) diluted shown a bimodal and a monomodal distribution, respectively.</p

Values for the critical concentrations (c* and c**) for xanthan solutions, reported in the literature.

<p>Values for the critical concentrations (c* and c**) for xanthan solutions, reported in the literature.</p

Quantification of leptospires by qPCR and the IM.

<p>A. Standard curve of the <i>lipL32</i> real-time PCR assay using DNA extracted from ten-fold serial dilutions of an <i>L. interrogans</i> strain Cop culture. Each DNA sample was quantified in duplicate and repeated twice. B. Quantification of the leptospiral load in the rat and hamster models. Rats were infected with 10⁸ leptospires and were euthanized on day 28 pi. Hamsters were inoculated with 500 leptospires (3×LD₅₀) and euthanized eight days pi. The leptospiral load in the kidneys was determined by qPCR (open symbols) and the IM (solid symbols). The leptospiral loads for the qPCR (leptospires per µg kidney DNA) and the IM (leptospires per 10 fields-of-view, ×1000 magnification) for the rat (r) and hamster (h) are presented as a scatter dot plot of the individual values for each animal, the horizontal line represents the mean value and the error bars the SEM. C. Representative examples of the imprint slides using kidney samples from an infected rat, a hamster and a non-infected control animal (magnification 1000×).</p

Comparison of culture isolation (CI), the imprint method (IM) and real-time PCR (qPCR) for the detection of leptospires in animal models simulating chronic (rat) and acute (hamster) infection.

<p>Comparison of culture isolation (CI), the imprint method (IM) and real-time PCR (qPCR) for the detection of leptospires in animal models simulating chronic (rat) and acute (hamster) infection.</p

Schematic of the Lig proteins, expression and purification of rLigB(131–645).

<p>A) The full length amino acid sequences for LigA (1224 amino acids, 128.1 kDa) and LigB (1890 amino acids, 200.8 kDa) are indicated (black line), the square boxes indicate the BIDs and the LigB C-terminal domain is shown (rectangle). The recombinant proteins used as vaccine candidates are indicated: LigB(131–645) (green boxes) includes amino acids 131–645 (53.5 kDa) and is highly identical (97.9% pairwise identity) to the same region in LigA; the LigA(631–1224), also known as LigANI, (red boxes, amino acids 631–1224, 62.8 kDa) and LigB(625–1259), also known as LigBNI, (blues boxes, amino acids 625–1259, 66.2 kDa) fragments are not highly conserved (38.1% pairwise identity). B) Expression and purification of rLigB(131–645) analysed by 10% SDS-PAGE and Coomassie staining. Lanes 1: molecular mass marker (kDa); Expression of rLigB(131–645) in an <i>E</i>. <i>coli</i>(pLigB(131–645)) clone, lane 2: supernatant (soluble) fraction and lane 3: insoluble fraction; lane 4: IMAC purified rLigB(131–645), expected molecular mass of 57.2 kDa. C) Immunoblot analysis of rLigB(131–645), following transfer the nitrocellulose membrane was probed with an anti-His-HRP antibody, lane 1: molecular mass marker (kDa); lane 2: purified rLigB(131–645).</p

Protection conferred by immunization with rLigB(131–645) against lethal challenge in the hamster model of leptospirosis.

<p>Protection conferred by immunization with rLigB(131–645) against lethal challenge in the hamster model of leptospirosis.</p