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A novel folate binding protein in Arabidopsis expressed during salicylic acid-induced folate accumulation Bijesh Puthusseri, Peethambaran Divya, Veeresh Lokesh, Gyanendra Kumar, Mohammed Azharuddin Savanur, and Bhagyalakshmi Neelwarne J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04236 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 12, 2017

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Journal of Agricultural and Food Chemistry

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A novel folate binding protein in Arabidopsis expressed during salicylic

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acid-induced folate accumulation

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Bijesh Puthusseri,1 Peethambaran Divya,1,# Veeresh Lokesh,1 Gyanendra Kumar,1

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Mohammed Azharuddin Savanur,2,# and Bhagyalakshmi Neelwarne1,* 1

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CSIR-Central Food Technological Research Institute, Mysore-570020, India

6

2

7 #

8 9 10 11

Plant Cell Biotechnology Department

Department of Biochemistry, Karnatak University, Dharwad, India

Present address: Indian Institute of Science, Banaglore-560012, India

Running Title: A novel folate binding protein in Arabidopsis *

Corresponding author: E-mail: [email protected]; [email protected] Telephone: 91-821-2516501

Telefax: 91-821-2517233

12

ACS Paragon Plus Environment


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ABSTRACT: Increasing the quantity of natural folates in plant foods is recently

14

gaining significant interest, owing to their acute deficiencies in various populations. This

15

study observed that foliar salicylic acid treatment enhanced the accumulation of folates

16

in Arabidopsis, which correlated with the increase in a folate binding protein (FBP) and

17

the expression of mRNA of a putative folate binding protein At5G27830. A protein band

18

corresponding to ~43 kDa was observed after resolving the affinity-purified protein on

19

SDS-PAGE, and the partial amino acid sequence indicated that the protein is indeed

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At5G27830. Docking studies performed with At5G27830, confirmed specific binding of

21

folic acid to predicted site. Heterologous expression of At5G27830 in the yeast resulted

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in significant uptake and accumulation of folic acid in cells. This novel study of a plant

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FBP will be useful for folate metabolic engineering of a wide range of crops.

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KEYWORDS: Folate binding protein · Folate receptor · At5G27830 · Plant folate ·

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Metabolic engineering

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1. INTRODUCTION

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Importance of folates in nutrition and health has received newer dimensions ever since

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the discovery of the key roles played by folates as mobile co-factors in intracellular

30

transfer and utilization of one-carbon moieties, transfer of methyl groups, in the

31

synthesis of nucleic acids, methionine replenishment and as co-substrates.1 Folate

32

deficiency is linked to anaemia, poor health and infant mortality in several populations

33

globally. Therefore, efforts are being made to find novel approaches that ensure the

34

delivery of adequate levels of natural folates through plant foods. In plant cells, folate

35

turnover rate is very high owing to a number of key roles in various metabolic

36

processes, in addition to those stated above. This means that folate is utilised very

37

rapidly and needs to be replenished in the same pace. Increasing the quantity of folates

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by metabolic engineering of folate biosynthetic genes has discreetly been successful in

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tomato, fruits2,3 and in rice seeds.4 Transgenic expression of a bovine FBP gene resulted

40

in 150-fold folate increase in rice,5 implying the need to understand the genes involved

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in folate enhancement in plants and to target them for folate metabolic engineering.

42

Researchers have pointed at the need for better understanding of the regulation of the

43

folate biosynthetic pathway in order to explore an engineering strategy applicable to a

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wide range of crops.6–8 In addition to over-expressing biosynthetic genes, folate

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enhancement can also be strategically achieved by improving the stability of the

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synthesised folate via :1) down-regulating folate catabolic enzymes, 2) transforming

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folates into stable derivatives, 3) increasing the abundance of folate binding protein

48

(FBP), 4) enhancing cellular antioxidants, and 5) promoting folate sequestration into the

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vacuole.6,9 In a previous study we have shown that treatment of coriander with aqueous



50

solution of salicylic acid (SA, 250 µM) resulted in two-fold increase in total folates

51

(from 1330 in

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methyltetrahydrofolate (5-MTHF) was the folate derivative present in abundance (60%).

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SA treatment also imparted better folate stability in coriander foliage during post-harvest

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storage,10 indicating that stabilizing factors might be playing a major role in enhancing

55

the accumulation of folates. Other than the biosynthetic genes, the folate binding

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proteins (FBPs) are vital gene-targets for folate metabolic engineering. The

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tetrahydrofolates, which are the highly recommended form of the folate derivatives, are

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significantly stabilised by the presence of the FBPs in milk.11 Transgenic expression of a

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bovine FBP gene in Arabidopsis resulted in the enhancement folates in rice, implying

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that FBPs are indeed present in plant foliage.12 The folate levels were positively

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correlated with the upregulation in levels of FBPs, indicating the importance of FBPs in

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folate enhancement.12 The present study was undertaken to identify and characterize

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FBP at functional level, employing affinity purification, amino acid sequencing, in silico

64

modelling and docking, and heterologous expression of the novel FBP identified in

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Arabidopsis.

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2. MATERIALS AND METHODS

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Extraction and purification of folate binding proteins from foliage

68

Extraction and purification of folate binding proteins (FBPs) involves the principle of

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affinity chromatography, where methotrexate or folic acid bound to a gel matrix acts as

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ligand for binding and retaining the FBPs present in the sample. Such bound FBPs may

71

be eluted by changing the buffer to acidic pH. Experiments were conducted at 4 °C

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unless otherwise stated. For the preparation of the protein samples, an earlier reported

control to 2867 µg/100g DW in SA-treated), where 5-


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method with minor modifications was followed.13 Foliage of the Arabidopsis plants (3

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g) was extracted in 0.05 M mannitol (10 mL), in the presence of protease inhibitor

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phenylmethylsulfonyl fluoride (PMSF). The homogenates were centrifuged at 10000 × g

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for 10 min, and the supernatant was saved. The protein was precipitated with the

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addition of solid CaCl2 to a final concentration of 10 mM, stirred for 20 min and

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centrifuged at 10000 × g for 15 min. To remove the folates that remain bound tightly

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with FBPs and are co-extracted along with the proteins, the pellet was solubilised in 30

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mL 0.05 M mannitol, and acidified with acetic acid to a final pH of 3.5. The proteins

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were precipitated and clarified with CaCl2 method as described above. The final protein

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pellet obtained was suspended in 30 mL 0.05 mannitol and the pH was adjusted to 7.4

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with 5 N NaOH. To this solution, triton-X 100 was added dropwise to a final

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concentration of 2%. The mixture was stirred for 48 h and pelleted by centrifugation at

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10000 × g and the supernatant was collected in a separate tube.

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Methotrexate-sepharose 4B affinity columns were used for the purification of FBPs.14

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CNBr-activated sepharose 4B gel was washed with cold 0.1 M NaHCO3 (3 L), and re-

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suspended in 400 mL of 0.1 M NaHCO3 containing 1.8 g of 1,6-diaminohexane (pH 9).

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The suspension was stirred for 48 h, and then washed with 2 L of 0.1 M NaHCO3. The

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gel was re-suspended in equal volume of 0.1 M NaHCO3 containing 2.4 g methotrexate,

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and the pH was adjusted to 8.5. To this suspension, 2.4 g of 1-ethyl-3(3-

92

dimethylaminopropyl) carbodiimide-HCl was added over a time of 30 min. The

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suspension was stirred for 2 h at room temperature, and then at 4 °C for 24 h. The gel

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was transferred into a coarse sintered glass funnel, and was washed with 3 L of 2 M

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NaCl in 0.05 M potassium phosphate buffer, pH 7, followed by 2 L of water, 2 L of 1 M



96

acetic acid, and then by 2 L of water. The gel was stored at 4 °C after mixing with

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0.03% sodium azide.

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For purification of the FBPs, the total protein extract was dissolved in phosphate

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buffer (final pH 6.4 and 0.5 M). The column with the activated methotrexate-sepharose

100

4B gel was washed with 0.5 M phosphate buffer (pH 6.4). The protein solution was

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passed through the column, followed by washing with 2 M NaCl in 0.05 M sodium

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borate buffer (pH 8). Further washing was done with 0.01 M sodium borate buffer (pH

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8). The FBPs retained in the column were eluted with 0.05 M acetic acid and the acidic

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fraction was collected as eluent. The FBP fraction was precipitated by adding equal

105

quantity of 80% ice-cold acetone. After overnight-storage at -20 °C, the solution was

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centrifuged at 10000 × g for 10 min to recover the protein pellet, which was re-dissolved

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in 0.05 M mannitol solution for protein quantification. For the quantification of protein,

108

Qubit® protein assay kit and Qubit® fluorometer (Life technologies, Gaithersburg, MD)

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were used. To obtain internal sequence data, the purified FBP fraction was run on 10%

110

SDS-PAGE, and protein bands were detected with Coomassie Brilliant Blue R-250.

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The~43 kDa band was excised from the SDS-PAGE gel, homogenized in 100 mM Tris,

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pH 8.0, and digested with trypsin at an estimated enzyme:substrate ratio of 1:20 for 16 h

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at 37 °C. The purified samples were sequenced by an automatic peptide sequencer (ABI

114

491, Procise, Applied Biosystems). Protein with similar sequences in Arabidopsis was

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searched in GenBank databank using the BLAST program. The search returned a

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putative folate binding protein At5G27830.

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In silico analysis of the putative folate binding protein AtFBP1 (At5G27830)


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Search for the conserved domains present in the putative folate binding protein was

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conducted

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(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).15 The best fit protein template,

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human folate receptor alpha (4km7.A) identified by the Swiss-Model template search

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(http://swissmodel.expasy.org/)16

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(https://www.ebi.ac.uk/Tools/msa/clustalw2/).17

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conducted using the AtFBP1 amino acid sequences in NCBI BLAST interface,18 and the

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filtered results were used for generating an unrooted phylogenetic tree by MEGA 6

126

software,19 using neighbour joining algorithm. The significantly related proteins were

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further analysed by ClustalW2.

using

NCBI

was

conserved

aligned

with A

domain

AtFBP1

protein

database

using

BLAST

ClustalW2 search

was

128

Protein secondary structure prediction was done in the ITASSER server

129

(http://zhanglab.ccmb.med.umich.edu/I-TASSER/)20 using the default parameters.

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Ligand

131

(http://zhanglab.ccmb.med.umich.edu/COACH/).21

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structure of the folic acid molecule (zinc_18456289) was obtained from the Zinc

133

database (http://zinc.docking.org/).22 The molecules were prepared for docking using

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Autodock Tools (http://autodock.scripps.edu/resources/adt), where polar hydrogens

135

were added and the files were saved as PDBQT. Docking simulation was conducted

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using Autodock Vina23 using PyRx interface.24 The docking results were analysed using

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the molecular visualization tool UCSF Chimera (https://www.cgl.ucsf.edu/chimera/).25

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LigPlot+26 was employed for the generation of 2D plots of the result and to find the

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interaction of surrounding molecules with the ligand.

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Functional analysis of the putative folate binding protein AtFBP1 (At5G27830)

binding

sites

were

predicted


using

COACH

server

For docking studies, crystal


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Full-length cDNA of the At5G27830 gene was amplified using the cDNA prepared from

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Arabidopsis mRNA. Previous researchers have cloned and overexpressed the AtRBP1 in

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Arabidopsis plants in a study that meant to identify proteins protecting Arabidopsis from

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oxidative stress.27 Out of the 23 proteins with obscure features analysed in this work,

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AtFBP1 did not show a significant protection during oxidative stress when compared

146

with many other proteins. An open reading frame was used, leaving 51 bases of the

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coding sequences (CDS) in the TAIR database at the 5’ end to clone as the full-length

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gene. BLAST search, followed by CLUSTAL alignment of the At5G27830 mRNA in

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the NCBI database pointed towards fact that the initial 51 bases reported in the TAIR

150

database might be of non-coding region as it was absent in the other mRNA sequences

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(Figure 1). PCR reactions for the amplification of the full length CDS with the start

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codon as provided in the TAIR database also failed repeatedly even after trials with

153

different primers, which indicate that the 51 bases at the 5’ end might not be even part of

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the mRNA generated. Hence, the same gene specific-primer sequences used for full

155

length cloning27 were used for the present study as well.

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A forward primer (CGGGGTACCAGATGGGAAGATGTTTAACG) with an added

157

restriction

site

for

Kpn1

and

158

(CCGCTCGAGTTAATCAGAGCTTGTTCTTCT) with an added restriction site for

159

Xho1, in frame with the N-terminal 6His-tag of the pYES2/NTA plasmid were

160

designed. The gene was amplified from the cDNA by PCR reaction with the mixture

161

consisting of 100 ng template, 10 pmol primer, 0.2 mM dNTPs and 1 U of Taq DNA

162

polymerase in 1× reaction buffer. The gene amplification was carried out under the

163

following conditions: initial denaturation of the template at 95 °C for 4 min followed by


a

reverse

primer

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35 cycles at 95 °C for 1 min (denaturation), 60 °C for 1 min (annealing) and 72 °C for 1

165

min (extension). The final extension was done at 72 °C for 10 min. Both the amplified

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DNA and the pYES2/NTA vector were cut using restriction enzymes. The DNA and

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plasmid (1 µg), and restriction enzymes (Kpn1 and Xho1 1 unit each) were digested in

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1× Cut Smart buffer at 37 °C overnight. The digested DNA and plasmid were purified

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by gel extraction kit (Sigma-Aldrich, St. Louis, USA) as per the manufacturer’s

170

instructions. The DNA and the plasmid (50 ng each) were ligated using T4 DNA ligase

171

(1 U) in 1× reaction buffer at room temperature for 15 min and chilled on ice. For

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amplifying the ligated plasmid, the Escherichia coli (E. coli) strain DH5α was used.

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The transformed cells were selected by growing on SOB agar containing nalidixic acid

174

(15 µg/mL) and ampicillin (50 µg/mL). The plates were incubated at 37 °C for 12–16 h.

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The plasmids were isolated by Mini plasmid preparation kit (Sigma-Aldrich, St. Louis,

176

USA) and the restriction enzyme-digested plasmids were run on 0.8% agarose for the

177

confirmation of the presence of insert. The presence of the AtFBP1 gene in frame with

178

the 6His tag was confirmed by sequencing with a T7 forward primer.

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The Saccharomyces cerevisiae wild type (BY4741; Mat a; his3D1; leu2D0;

180

met15D0; ura3D0; YPL268w::kanMX4) strain was used for the protein expression.

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Lithium acetate (LiAc) method28 was used for the yeast transformation. After

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transformation, cells (100 µL) were seeded on the selective media, synthetic defined

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(SD) agar media lacking uracil (SD/−Ura) for the selection of the transformants. The

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presence of the AtFBP1-gene insert in transformants was confirmed by colony PCR and

185

sequencing of the isolated plasmid.



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The yeast cultures with the AtFBP1 insert were grown overnight in SD/−Urabroth

187

with dextrose as the carbon source at 30 °C. For the protein induction, the pellets of

188

cultures were transferred into SD/−Ura broth with the protein-inducing agent,

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galactose, for 32 h. A yeast-lysis buffer was prepared with Tris (pH 8, 100 mM),

190

MgCl2, NaCl (300 mM), PMSF (1 mM) and glycerol along with appropriate quantity of

191

protein inhibitor cocktail. The pellet obtained after centrifugation (3000 × g, 5 min) was

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re-suspended in 500 µL of lysis buffer in a 2 mL microfuge tube and 50 µL of glass

193

beads were added. A vortexing cycle with 30 s vortex followed by 1 min incubation on

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ice for 30 cycles was done to lyse the cells. The cells were pelleted by centrifuging at

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10000 × g for 5 min, and the supernatant was separated from the pellet. The extracted

196

proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis

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(SDS-PAGE).29 The expressed protein was analysed for the presence the 6His tag by

198

Western blotting using the monoclonal anti-polyHistidine antibody. For the Western

199

blotting, the proteins were transferred from the gel to a nitrocellulose membrane using

200

semi-dry

201

California). The transfer to the membrane was carried out at 15 V for 30 min. For the

202

detection of the expressed protein, the procedure provided with the monoclonal anti-

203

polyHistidine antibody produced in mice (Sigma-Aldrich, St. Louis, USA) was

204

followed. After treatment with the primary antibody, followed by washing, the

205

membrane was treated with anti-mouse IgG-alkaline phosphatase as the secondary

206

antibody (Sigma-Aldrich, St. Louis, USA). The membrane was treated with BCIP/NBT

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until the bands developed to optimum intensity. The membrane was washed with

208

distilled water for three times, air dried and documented.

electrophoretic

transfer

instrument

(Transblot,


BioRad,

Richmond,

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209

Uptake of FA-EDA-FITC conjugate by AtFBP1-transformed yeast

210

For determining the binding affinity of the AtFBP1 to the folate, the yeast, expressing

211

the AtFBP1 were grown in the presence of the folate tagged with the fluorescent

212

molecule fluorescein isothiocyanate (FA-EDA-FITC). In brief, the wild type and the

213

transformant were grown overnight in a 1 mL culture at 200 g and 30 °C. The wild type

214

was supplied with a complete SD medium with glucose as the carbon source, and

215

transformant was supplied with SD/-Ura medium with galactose as the carbon source.

216

The cells were centrifuged at 3000 × g for 5 min, and to the pellet fresh medium was

217

added. FA-EDA-FITC was added at 0.5 mg/mL to the culture, vortexed thoroughly to

218

mix, and incubated for 6 h at 100 × g and 30 °C. The cells were pelleted by

219

centrifugation at 3000 × g for 5 min and the pellet was washed twice with distilled water

220

and analysed under a fluorescent microscope (Olympus BX51) having a dichroic mirror

221

DM500 with excitation filter BP450-480, where a barrier filter BA515 was used for

222

observing the FA-EDA-FITC fluorescence. Relative quantitation of folate uptake was

223

analysed by ImageJ software (W.S. Rasband, ImageJ, NIH, Bethesda, MD). For

224

estimating the actual folate uptake by the transformed yeast cells, the yeast cells were

225

harvested and subjected for vortex extraction using glass beads for 3 min in ice cold

226

extraction buffer. The folate content in the samples were determined by microbiological

227

assay using Lactobacillus rhamnosus (ATCC 7469), as reported previously.12

228

Statistical analysis



229

Treatment values in triplicate were compared with the control values to arrive at

230

significantly different values by Student's t-test using SPSS 17 software (SPSS Inc.,

231

Chicago, USA).

232

3. Results and discussion

233

Presence of folate binding proteins in the Arabidopsis foliage

234

Affinity purification of FBPs showed three protein bands upon silver staining. The

235

prominent band was observed at ~43 kDa (Figure 2a). The other two faint bands were of

236

~90 kDa and ~29 kDa. Partial sequences obtained from the amino acid sequencing of

237

the tryptic digested ~43 kDa (Figure 2b) were used to conduct a blast search in NCBI

238

for matches in Arabidopsis genome (taxid:3701). The search tallied with At5G27830, a

239

protein with a folate receptor domain. Although FBPs are known to play a major role in

240

folate enhancement in plants,5,9 hitherto no FBP has been functionally characterized

241

from plants.

242

In silico analysis of the putative folate binding protein AtFBP1 (At5G27830)

243

Conserved domain search revealed that the AtFBP1 does in fact contain the folate-

244

binding domain pfam03024. This domain is characterised by the presence of disulphide

245

bonds, where sixteen cysteine residues have been identified to contribute in many

246

characterised folate binding proteins. Pfam03024 domain is present in the well-studied

247

folate receptors such as α, β, γ and δ forms in humans. This domain search confirmed

248

that ten conserved cysteines from AtFBP1 align perfectly with the Pfam03024 domain

249

(Figure 3a). The template search showed that the PDB structure of the α-folate receptor

250

of humans is structurally the most related one with the AtFBP1. Clustal alignment of


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AtFBP1 with 4km7.A indicated the alignment of six cysteines (Figure 3b). Since normal

252

folate binding proteins, so far characterised only in animals, have sixteen cysteines, the

253

absence of one cysteine in Arabidopsis may be an evolutionary change. For further

254

insights into this, the phylogenetic analysis of this protein was conducted with BLAST

255

results of flowering plant protein sequences (showing query coverage >75 and identity

256

>50), sequences of those excluding flowering plants (query coverage >45 and identity

257

>45) and of the four forms of human folate receptors. All the proteins retrieved by

258

BLAST search were uncharacterised. The phylogenetic tree (Figure 3c) constructed with

259

neighbour joining algorithm and with boot strapping (2000 replicates) clearly depicted

260

two proteins with significant similarity (>80 bootstrap value) with AtFBP1 in one

261

cluster (Figure 3c, indicated by red arrow). Further, all the three proteins (of Capsella

262

rubella, Eutrema salsagineum and AtFBP1) have only fifteen cysteines and the clustal

263

alignment of these three proteins clearly depicted that the cysteine residues are

264

conserved (Figure 3d).

265

The model of the AtFBP1 was predicted using the ITASSER server, and was

266

downloaded as PDB file (C-Score = −2.88, TM-Score = 0.39, RMSD = 13.3 Å). The

267

TM-score was in the median range of reliability, which ranged between 0.17 and 0.5.

268

Binding site prediction by COACH server showed the region with amino acids at 128,

269

131, 168, 172 and 201 may constitute a possible folic acid binding site. This has brought

270

forth a protein receptor model with folic acid as a ligand (C-score 0.01), which was

271

obtained by comparing with the human folate binding protein FOLR1 (4LRHA). The

272

locations of the conserved cysteines in the model were compared with that of 4km7.A,

273

because in case of folate binding proteins the reliability of a predicted structure is



274

largely influenced by the prevalence of cysteine-cysteine interactions.30 Out of the

275

fifteen cysteines in AtFBP1, fourteen were found to contribute towards the formation of

276

the binding site (Figure 4a). The bovine FBP DN512948, which is used to increase

277

folate content in Arabidopsis by heterologous expression, was also found to have similar

278

sequence and structural characteristics of the AtFBP1. The bovine FBP had the same

279

folate binding pfam03024 as the conserved domain. However, DN512948 have

280

seventeen cysteines instead of fifteen cysteines present in AtFBP1. Structural

281

overlapping of the 4km7.A (Figure 4b) on AtFBP1 showed that the binding sites do

282

align with each other (Figure 4c). The folic acid molecule was docked on the predicted

283

binding site of the AtFBP1 (Figure 4d) with a free energy change (∆G) of −9.3. Whereas

284

docking of the folic acid with the human folate binding protein FOLR1 crystal structure

285

4km7.A showed a free energy change of (∆G) of −8. The 2D plot of the docked PDB

286

structure was generated with the LigPlot+, which revealed the formation of five

287

hydrogen bonds between the folic acid molecule and the amino acid residues (Gly 71,

288

Asn 134, Leu 168 and Arg 170) of the protein (Figure 4e).

289

Uptake and accumulation of the FA-EDA-FITC conjugates by AtFBP1 expressing

290

yeast cells

291

Microscopic observations revealed that the yeast cells expressing the AtFBP1 gene had

292

taken up significantly higher quantities FA-EDA-FITC, resulting in emitting bright

293

cellular fluorescence (Figure 5). The intense fluorescence of the transformed cells also

294

indicates significantly higher quantity (relative fold change of 81) of the folate

295

accumulation by the yeast cells over-expressing the AtFBP (Figure 5g). Actual


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296

estimation in transformed yeast cells showed a 352 fold increase in total folates,

297

confirming high folate binding ability of the AtFBP1 protein.

298

Since our earlier studies10,31 demonstrated that the accumulation of higher levels of

299

folates in foliage after SA treatment is by folate stabilising factors rather than folate

300

biosynthesis factors, it appears feasible to target genes of folate-binding protein for

301

folate metabolic engineering. As in the human counterparts, the AtFBP1 might be

302

actively expressed by cells undergoing rapid cell division. The AtFBP1 might be playing

303

a role in the transport of the folates from the site of production (leaves) to the site of

304

storage (fruits and seeds) and utilization (roots) as well.

305

The present study demonstrates for the first time that the genes coding for proteins

306

that help in binding of folates, render higher stability to folates in plant foods. This

307

study also provides a new gene-target for folate metabolic engineering, and the over

308

expression of this gene (AtFBP1) not only increase folate contents but may also

309

enhance the post-harvest stability of this vitamin. Therefore, this approach of targeting

310

folate-binding proteins appears more important for folate-metabolic engineering in

311

leafy vegetables rather than targeting the folate biosynthetic pathway. The major

312

problem with the folate-rich crops is the post-harvest degradation of the folates,

313

resulting in inadequate dietary supply of folate even after consuming large quantity of

314

the food materials.6 Other studies also demonstrate that FBPs render stability to folates

315

present in food sources such as milk11,32 and that the transgenic expression of bovine

316

FBP is known to increase folate content in Arabidopsis,5 which may be applicable to

317

other plants as well.

318

ABBREVIATIONS USED



319

SA, salicylic acid; FBP, Folate binding protein; 5-MTHF, 5-methyltetrahydrofolate; FA-

320

EDA-FITC, Folic acid conjugated with fluorescein isothiocyanate; DW, Dry weight

321

AUTHOR CONTRIBUTION STATEMENT

322

BP and BN conceived the study. BP, PD, LV, GK, MAS and BN designed, conducted

323

the experiments and analysed the data. BP, MAS and BN wrote the manuscript. The

324

authors have no competing financial interests to declare.

325

ACKNOWLEDGMENTS

326

We thank Mr. Avinash Kumar of Plant Cell Biotechnology Department, CSIR-CFTRI,

327

for providing the Arabidopsis seeds, as well as for assisting in conducting PCR analyses.

328

REFERENCES

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420 421



422

Figure legends

423

Figure 1 Clustal alignment of the 5’ end of complete AtFBP1 mRNAs available in the

424

NCBI database. The first-highlighted ATG is that of the reported start codon in TAIR

425

database for AtFBP1 gene. The second-highlighted ATG is the perfectly aligning one

426

and was used as the start codon by previous workers for the AtFBP1 full-length gene

427

cloning (Luhua et al. 2008), which is also used as the start codon for the present study.

428

Figure 2 Folate binding proteins (FBPs) in Arabidopsis foliage. The FBPs were

429

obtained from total protein extract by affinity purification through methotrexate-FBP

430

binding column. The FBPs were separated ona 10% SDS-PAGE gel, followed by

431

detection with silver staining. a A major band of the size ~43 kDa was observed and is

432

denoted with arrow mark. b Amino acid sequences obtained after digestion of protein

433

band ~43 kDa with trypsin.

434

Figure 3 Sequence and Phylogenetic analysis of the AtFBP1 protein. a Conserved

435

domain analysis, using the NCBI conserved domain search interface of AtFBP1protein.

436

Conserved cysteines are blocked and 10 cysteines are shown conserved in this domain

437

search output. b Clustal alignment of AtFBP1 against 4km7.A (human folate receptor

438

alpha).Six cysteines are conserved and are shown in blocks. c An un-rooted neighbour

439

joining-phylogenetic tree constructed with 2000 bootstrap replicates. AtFBP1 is

440

indicated as AraFBP1 (red arrow) and is clustered with the uncharacterised proteins of

441

Capsella rubella and Eutrema salsugineum. d Clustal alignment of proteins from

442

Capsella rubella and Eutrema salsugineum with AtFBP1 showing the alignment of 15

443

conserved cysteines (bracketed).


Page 20 of 27

Page 21 of 27


444

Figure 4 Modelling and docking analysis of AtFBP1 protein. a Homology model of

445

AtFBP1 protein modelled using the I-TASSER server, based on multiple-threading

446

alignments and iterative template fragment assembly simulations, showing cysteines in

447

red colour. b Structure of 4km7.A (human folate receptor alpha) obtained from PDB

448

protein data bank showing cysteines in red colour. c Superposition of AtFBP1 on

449

4km7.A showing the overlapping of folate binding domains of the two proteins. d

450

AtFBP1 with a folic acid molecule docked at the predicted binding site (amino acid

451

residues 128,131,168,172 and 201). e 2D plot of the folic acid docked region of the

452

AtFBP1 with hydrogen bonding shown in dotted lines.

453

Figure 5 Uptake of the fluorescent-tagged folic acid (FA-EDA-FITC) by the yeast cells

454

expressing the AtFBP1 gene analysed by confocal microscopy. Figures a–c are those of

455

the wild type yeast cells and d–f are of the yeast cells expressing the AtFBP1 gene under

456

the galactose inducible promoter. Figure e shows the fluorescent image of the yeast cells

457

expressing the AtFBP1 gene upon transfer to the galactose containing medium

458

supplemented with FITC tagged folates and are brightly fluorescing upon the uptake and

459

accumulation of the FA-EDA-FITC molecules as these cells are expressing AtFBP1

460

gene that binds to the folates, resulting in increased uptake and accumulation of folates.

461

Figure g shows the relative quantification of folates up taken by the yeast cells

462

transformed with AtFBP1 in relation to the wild type. Significances of the mean values

463

were estimated by Student’s t-test.



Page 22 of 27

Figure 1

NM_001203483.1 NM_122665.3 AY039962.1 BT000985.1 AT5G27830 NM_001203484.1

---------------------TCGTCTTTGTCTTTTGTCATTTTTTGCTTCGAGCTAAGA ---------------------TCGTCTTTGTCTTTTGTCATTTTTTGCTTCGAGCTAAGA ----------------------TTTTTTTGTCTTTTGTCATTTTTTGCTTCGAGCTAAGA --------------------------------------------------------------------------------------ATGCAATTGGGTAATC---GGGTCGACCCA--GTTCTAAAACCATAATTGACGGAGTCTATGCAATTGGGTAATC---GGGTCGACCCA---

39 39 38

NM_001203483.1 NM_122665.3 AY039962.1 BT000985.1 AT5G27830 NM_001203484.1

TCAGTGAAGAGAAAAGATGGGAAGATGTTTAACGAAGAAGGTGTTTTTGATTCAGAGTCC TCAGTGAAGAGAAAAGATGGGAAGATGTTTAACGAAGAAGGTGTTTTTGATTCAGAGTCC TCAGTGAAGAGAAAAGATGGGAAGATGTTTAACGAAGAAGGTGTTTTTGATTCAGAGTCC ----------------ATGGGAAGATGTTTAACGAAGAAGGTGTTTTTGATTCAGAGTCC TCAGTGAAGAGAAAAGATGGGAAGATGTTTAACGAAGAAGGTGTTTTTGATTCAGAGTCC TCAGTGAAGAGAAAAGATGGGAAGATGTTTAACGAAGAAGGTGTTTTTGATTCAGAGTCC ********************************************

99 99 98 44 87 114

NM_001203483.1 NM_122665.3 AY039962.1 BT000985.1 AT5G27830 NM_001203484.1

GATCTTGTTTCTTCATCTCCTGATCTCTTTATCGTCCG--------------GTGCAGTG GATCTTGTTTCTTCATCTCCTGATCTCTTTATCGTCCG--------------GTGCAGTG GATCTTGTTTCTTCATCTCCTGATCTCTTTATCGTCCG--------------GTGCAGTG GATCTTGTTTCTTCATCTCCTGATCTCTTTATCGTCCG--------------GTGCAGTG GATCTTGTTTCTTCATCTCCTGATCTCTTTATCGTCCGAACAATGTTTTCAGGTGCAGTG GATCTTGTTTCTTCATCTCCTGATCTCTTTATCGTCCGAACAATGTTTTCAGGTGCAGTG ************************************** ********

145 145 144 90 147 174


27 54

Page 23 of 27


Figure 2 a Ruler

FBP fraction

kDa 97.4 66 43 29 20.1

14.3

b

KASNWESN ELLECSIC KTCCSSV



Page 24 of 27

Figure 3 a c

AtFBP1 Cdd: pfam03024

Populus trichocarpa

97 43

Ricinus communis

29

Citrus sinensis

9

Glycine max

9

Cucumis sativus Vitis vinifera

28


Theobroma cacao Prunus persica

9643 99 40


Fragaria vesca Solanum lycopersicum

100

56

Solanum tuberosum Picea sitchensis Selaginella moellendorffii

Eutrema salsugineum

b

AraFBP1

99

Capsella rubella

81

4km7A AtFBP1

FOLRd FOLR1

100

FOLR2

95 76

4km7A AtFBP1

0.2

d

Capsella rubella Eutrema salsugineum AtFBP1

4km7A AtFBP1


4km7A AtFBP1


4km7A AtFBP1

Capsella rubella Eutrema salsugineum AtFBP1 Capsella rubella Eutrema salsugineum AtFBP1

4km7A AtFBP1



FOLR3

Page 25 of 27


Figure 4 a

b

e c

d Folic acid



Page 26 of 27

Figure 5 DIC

Fluorescent B

A

Merged C

G

10 µm

D

10 µm

10 µm

10 µm

60 20 0.10 0.08 0.05 0.03

Wild type

Transformant 10 µm

P

Novel Folate Binding Protein in - American Chemical Society

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