Research ArticleSTRUCTURAL BIOLOGY

Membrane pore architecture of the CslF6 protein controls (1-3,1-4)-β-glucan structure

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Science Advances  12 Jun 2015:
Vol. 1, no. 5, e1500069
DOI: 10.1126/sciadv.1500069
  • Fig. 1 Characterization of (1-3,1-4)-β-glucan structure in Nicotiana benthamiana leaves.

    (A) Structure of (1-3,1-4)-β-glucan. Glucose molecules are represented by black dots with β1-4 linkages (straight lines) or β1-3 linkages (angled lines). The bacterial enzyme lichenase specifically cleaves (1-3,1-4)-β-glucan at a β1-4 linkage after a β1-3 linkage as indicated by dashed red lines, producing short oligosaccharides with a DP from DP3 to DP9. (B) Oligosaccharides released by lichenase digestion of betaglucan were fluorescently labeled with APTS and separated by capillary electrophoresis. (C) The source of the CslF6 gene affects the DP3/DP4 ratio of the (1-3,1-4)-β-glucan produced in N. benthamiana leaves. The CslF6 proteins can be classified into two groups: those that produce a (1-3,1-4)-β-glucan with a high (>1.3) DP3/DP4 ratio (Bd, Brachypodum distachyon; Ta, Triticum aestivum; Hv, Hordeum vulgare; shown in blue) or a low (<1.1) DP3/DP4 ratio (Zm, Zea mays; As, Avena sativum; Os, Oryza sativa; Sb, Sorghum bicolor; shown in red). T7 is an 11–amino acid epitope tag at the N terminus of some of the CslF6 proteins—it has no effect on the amount or structure of the (1-3,1-4)-β-glucan. Results are averages ± SD from replicate measurements of between 2 and 11 independent experiments with each gene. (D) The DP3/DP4 ratio of the (1-3,1-4)-β-glucan produced by the HvCslF6 or ZmCslF6 proteins is very stable over a wide range of (1-3,1-4)-β-glucan concentrations in independent experiments. There is no correlation between the DP3/DP4 ratio and the amount of (1-3,1-4)-β-glucan produced.

  • Fig. 2 The CslF proteins are found in the membrane fraction.

    (A and B) Proteins from membrane preparations from barley leaf or N. benthamiana leaf expressing the indicated CslF protein probed with (A) multiepitope HvCslF6 antibody or (B) T7 tag antibody. Molecular mass markers (Mr) and the positions of CslF6 and CslF4 are shown at the side.

  • Fig. 3 The effect of chimeric HvCslF6/ZmCslF6 genes on (1-3,1-4)-β-glucan structure.

    Restriction sites used in cloning are shown as vertical lines. The HvCslF6 gene is represented by blue rectangle, and the ZmCslF6-2 gene as a yellow rectangle. (Right) DP3/DP4 ratio of the lichenase digested (1-3,1-4)-β-glucan averaged from the indicated number of independent transformation experiments with SDs shown.

  • Fig. 4 The Bgl IIEco RI fragment of CslF6 genes controls (1-3,1-4)-β-glucan structure.

    Restriction sites used in cloning are shown as vertical lines. The blue or red rectangles represent the CslF6 genes that produce (1-3,1-4)-β-glucan with a high DP3/DP4 ratio, and the yellow or gray rectangles represent those that produce a (1-3,1-4)-β-glucan with a low DP3/DP4 ratio as indicated on the right. All chimeric constructs show a high (>1.4) or a low (<1.2) DP3/DP4 ratio depending on the source of the Bgl II–Eco RI fragment. SDs were less than 0.014 for all samples. The amount of (1-3,1-4)-β-glucan (BG) produced by each construct is shown in the last column as a percentage of dry weight of the freeze-dried leaf.

  • Fig. 5 The predicted TMH4 of CslF6 controls (1-3,1-4)-β-glucan structure.

    (A) Diagrammatic representation of the full-length CslF6 protein sequence (gray rectangle; 1 to 947 amino acids; numbers refer to the HvCslF6 protein), with predicted TMHs shown as thin dark barrels and the approximate positions of the conserved amino acids of the active site in single-letter code. Red lines indicate approximate positions of restriction sites used in construction of chimeric genes shown below as blue or yellow rectangles (representing barley HvCslF6 and maize ZmCslF6, respectively). The numbers within the rectangles are the DP3/DP4 ratios of the (1-3,1-4)-β-glucan produced by the corresponding chimeric proteins in N. benthamiana leaf, and the boxed area indicates the region controlling this ratio (that is, blue area has a high and yellow area has a low DP3/DP4 ratio). Genes were fused at the Bgl II site. (B) The region controlling (1-3,1-4)-β-glucan structure was further defined to be between the Bgl II and Xba I sites encompassing TMH3 to TMH6 of the CslF6 protein. (C) Expanded view of this region with the protein shown as a gray rectangle; numbers refer to the boundaries of the predicted TMHs of the HvCslF6 protein; and the positions of the Bgl II, Pst I, and Xba I sites are shown on top. The hash marks (#) represent the position of the 13 amino acid differences between the HvCslF6 and ZmCslF6 proteins in this region. By comparing the four Bgl II–Xba I chimeras and the single Pst I chimera, the region controlling the DP3/DP4 ratio is further limited to the four amino acid differences in TMH4 of the CslF6 protein.

  • Fig. 6 Single amino acid differences in the predicted TMH4 of CslF6 control (1-3,1-4)-β-glucan structure.

    The amino acid sequence of the TMH4 region of HvCslF6 and ZmCslF6 proteins is shown. Each of the four different single amino acids (underlined) in the HvCslF6 protein was changed to the corresponding ZmCslF6 amino acid, and the effect on the DP3/DP4 ratio and the amount (% dry weight of leaf) of the (1-3,1-4)-β-glucan are shown at the right. Results are means ± SD from replicates of at least two transformation experiments.

  • Fig. 7 The effect of amino acid changes in predicted TMH4 on (1-3,1-4)-β-glucan structure across species.

    The DP3/DP4 ratio of (1-3,1-4)-β-glucan produced in N. benthamiana leaf from wild-type CslF6 proteins with an isoleucine in predicted TMH4 is shown as black bars. When the indicated isoleucine is changed to a leucine, the DP3/DP4 ratio of the (1-3,1-4)-β-glucan is decreased (light gray bars). A further decrease in the DP3/DP4 ratio is observed when a second amino acid substitution is made in the HvCslF6 TMH4 region (HvCslF6S752G,I757L). Shown are averages and SDs of replicates from at least two transformation experiments. The amount of (1-3,1-4)-β-glucan produced for the respective constructs was 1.8, 2.2, 0.4, 0.6, 1.8, 2.4, and 1.8% of the dry weight of the freeze-dried leaf tissue.

  • Fig. 8 The isoleucine-to-leucine change alters the fine structure of (1-3,1-4)-β-glucan.

    (A) Changing the native isoleucine in TMH4 of CslF6 protein (dark blue bars) to a leucine (red bars) increases the proportion of β1-4 bonds in (1-3,1-4)-β-glucan, similar to that produced from those native CslF6 proteins that have a leucine at the same position (light blue bars). (B) The increase in β1-4 bond frequency changes the (1-3,1-4)-β-glucan structure, decreasing the proportion of DP3 and DP4 oligosaccharides. (C) The increase in longer-chain oligosaccharides is largely explained by an increase in DP5 and DP6. Dashed lines are shown to indicate the trends in change of oligosaccharide profiles between the independent experimental measurements. Error bars from duplicate measurements are indicated.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/5/e1500069/DC1

    Fig. S1. Plasmid map of the Agrobacterium transformation vector pSJ226 and pSJ195 used for transient expression studies in N. benthamiana.

    Fig. S2. Amino acid sequence alignment of the C-terminal region of the CslF6 proteins.

    Fig. S3. The Leu-Ile amino acid change in ZmCslF6 TMH4 increases DP3/DP4 ratio.

    Fig. S4. Sequence alignment of the C-terminal region of Rhodobacter sphaeroides BcsA and Hordeum vulgare CslF6 with Oryza sativa CesA and CslD proteins.

    Table S1. Comparison of (1-3,1-4)-β-glucan abundance and structure in N. benthamiana leaf and in cereal wholegrain.

    Table S2. Abundance of DP3-DP9 from Fig. 8.

    Table S3. Primers.

    References (3032)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Plasmid map of the Agrobacterium transformation vector pSJ226 and pSJ195 used for transient expression studies in N. benthamiana.
    • Fig. S2. Amino acid sequence alignment of the C-terminal region of the CslF6 proteins.
    • Fig. S3. The Leu-Ile amino acid change in ZmCslF6 TMH4 increases DP3/DP4 ratio.
    • Fig. S4. Sequence alignment of the C-terminal region of Rhodobacter sphaeroides BcsA and Hordeum vulgare CslF6 with Oryza sativa CesA and CslD proteins.
    • Table S1. Comparison of (1-3,1-4)-β-glucan abundance and structure in N. benthamiana leaf and in cereal wholegrain.
    • Table S2. Abundance of DP3-DP9 from Fig. 8.
    • Table S3. Primers.
    • References (30–32)

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