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ARTICLES

Journal of Biomolecular Techniques

15:97–106 © 2004 ABRF

RF AB

Comparative Proteomics of Cannabis sativa Plant Tissues

Tri J. Raharjo, a,b Ivy Widjaja, c Sittiruk Roytrakul, b and Robert Verpoorte b

a Department of Chemistry, Gadjah Mada University, Yogyakarta, Indonesia; b Division of Pharmacognosy, Institute of Biology, Leiden University, Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands

Comparative proteomics of leaves, flowers, and glands of Cannabis sativa have been used to identify specific tissue- expressed proteins.These tissues have significantly different levels of cannabinoids. Cannabinoids accumulate primarily in the glands but can also be found in flowers and leaves. Pro- teins extracted from glands, flowers, and leaves were sepa- rated using two-dimensional gel electrophoresis. Over 800 protein spots were reproducibly resolved in the two-dimen- sional gels from leaves and flowers.The patterns of the gels were different and little correlation among the proteins could be observed. Some proteins that were only expressed in flowers were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprint database searching.Flower and gland

ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Robert Ver-

poorte, Division of Pharmacognosy, Institute of Biology, Leiden University, Gorlaeus Laboratories, PO Box 9502, 2300 RA Lei- den,The Netherlands (fax: 31-71-5274511; e-mail: verpoort@ lacdr.leidenuniv.nl).

c Current address: Genome Institute of Singapore, 1 Science Park Road, #05–01,The Capricorn, Singapore Science Park II, Singapore 117528.

proteomes were also compared, with the finding that less then half of the proteins expressed in flowers were also expressed in glands. Some selected gland protein spots were identified:F1D9.26-unknown prot.(Arabidopsis thaliana), phos- pholipase D beta 1 isoform 1a (Gossypium hirsutum),and PG1 (Hordeum vulgare).Western blotting was employed to identify a polyketide synthase, an enzyme believed to be involved in cannabinoid biosynthesis, resulting in detection of a single protein.

KEY WORDS: Cannabis sativa, comparative, mass fingerprint, proteomic.

T he biosynthesis of cannabinoids, a class of ter-

penephenolic compounds found in Cannabis

sativa, is not yet fully known. Cannabinoids

are found in all tissues of the C. sativa plant, but the amount in which they are present differs considerably among the tissues. 1 Cannabinoids are most abundant in flowers, especially in the glands. This raises the question of whether biosynthesis of cannabinoids occurs in all tissues but in different quantities, or only in one tissue and is then transported to the others. In both scenarios it is assumed that the expression level of the genes involved in the cannabinoid biosynthe- sis is different among the tissues. In any case, the dif- ferential expression of cannabinoid biosynthesis may be used to further clarify this pathway by comparing, on the level of proteins or mRNAs, the tissues with varying amounts of cannabinoids with the tissues that do not produce cannabinoids. Gene expression can be studied by measuring

mRNA levels using methods such as microarrays, ser- ial analysis of gene expression, and real-time polym- erase chain reaction. However, studies in yeast revealed the absence of a strong correlation between the abundance of the protein and the corresponding mRNA. 2 Alternative methods of study involve the use of enzyme assays or proteome analysis to identify expressed proteins. The enzymes known to be involved in cannabis biosynthesis are olivetolic acid

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T. J. RAHARJO ET AL.

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1

Molecular Weight and pI of Characterized Enzymes in Cannabinoid Biosynthesis

MW (kDa)

pI

No.

Enzymes

Calculated

Experimental a

Calculated

Experimental a

1

THCA synthase

61.9

74

9.04

6.4

2

CBDA synthase

62.2

75

8.88

6.1

3

CBCA synthase b

71

7.3

a According to Morimoto et al. 4 b Not completely sequenced. THCA, tetrahydrocannabinolic acid; CBDA, cannabidiolic acid; CBCA, cannabichromenic acid.

prenylase, tetrahydrocannabinolic acid synthase (THCA synthase), cannabidiolic acid synthase (CBDA synthase), and cannabichromenic acid synthase (CBCA synthase). 3,4 Though assays are available for several of the enzymatic steps of the cannabinoid biosynthesis, it would be an immense task to purify each of these enzymes for sequencing. Moreover, not all of the steps are known. Thus, proteome analysis (proteomics) seems to be preferable to enzyme assay- ing in obtaining sequence information from all pro- teins connected with cannabinoid biosynthesis. The use of proteomics in the study of secondary metabo- lite biosynthesis has been reviewed by Jacobs et al. 5 Proteomics is a new tool used to identify and characterize all proteins expressed in an organism or cell. 6 Single proteins can be separated using column chromatography or two-dimensional (2D) elec- trophoresis prior to mass spectrometric (MS) analy- sis. 7 Advanced MS allows ionization of macromole- cules such as proteins and peptides. 8 Proteins can be identified by matching peptide mass fingerprinting with database sequences or by sequencing whole- length proteins with tandem MS. Peptide fingerprints can be obtained by ionization of the peptides that result from enzymatic digestion, usually by trypsin. Accurate peptide masses of peptide fingerprints can be used for searching matching proteins in the data- bases resulting in molecular weight search (Mowse) score. 9 The peptides themselves can be fragmented using tandem MS resulting in the amino acid sequences. Thousands of proteins occur in the cell, and to choose and separate the protein responsible for a particular function is not an easy task. Using 2D elec- trophoresis proteins are separated based on pI and molecular weight (MW), which results in a proteome pattern of the cells or tissues under a certain condi- tion. Proteins involved in the production of metabo-

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lites can be studied by comparing producing with nonproducing conditions of the cells or tissues: Pro- teins that are present in the producing conditions but not in the nonproducing conditions might be involved in the production of the compounds of interest. This comparison can be performed more easily with cell cultures, as they tend to have a less complex matrix than plant tissues. Unfortunately, cannabinoids are not produced by cell cultures. Another option is to compare high-producing tissues, such as flowers, with low-producing tissues, such as leaves. The pI and MW of THCA synthase, CBDA synthase, and CBCA synthase are available (Table 1). Therefore, these pro- teins might be identified from the tissues of flowers and glands using 2D electrophoresis and confirmed by MS analysis. Western blot analysis using antibodies against a group of proteins known from other plants, e.g., cytochrome P450 or chalcone synthase, can be com- bined with 2D electrophoresis in order to choose one or more spots for MS analysis. In the case of cannabinoid biosynthesis, a polyketide synthase called stilbene synthase carboxylate-like (STCSL) enzyme is predicted to catalyze the first step of cannabinoid biosynthesis, the reaction between a molecule of n-hexanoyl-CoA with three molecules of malonyl-CoA yielding olivetolic acid. A serum anti- body of chalcone synthase, also a polyketide syn- thase enzyme, was available to try to identify STCSL in C. sativa. Here we report a comparison of the proteomes of leaves, flowers, and glands of C. sativa for analyzing the biosynthesis of cannabinoids. We also tried to identify the polyketide synthase involved in the biosynthesis of cannabinoids, STCSL, by means of a combination of 2D electrophoresis and Western blot analysis followed by MS analysis.

COMPARATIVE PROTEOMICS OF C. SATIVA PLANT TISSUES

MATERIAL AND METHODS

modification of Blum et al. 14 After staining, the gels

Plant material

Plants of the C. sativa “four-way” cultivar (The Sensi

were scanned with a UMAX Powerlook III-scanner. The gel images were analyzed with the software package ImageMaster 2D Elite v3.01 (Amersham Biosciences) in order to calculate the number of spots and for compar-

Seed Bank, Amsterdam The Netherlands) were legal-

ison

analysis.

lly grown from seeds in a protected greenhouse. The flowers were harvested from 18-week-old female plants and stored at –20 C. The glands were isolated from the harvested flowers according to the method

Protein Digestion

developed by Hammond and Mahlberg. 10

Gel

pieces containing the protein of interest were cut

out

from the stained gel. They were cut into small

Protein extraction

pieces (1 mm 2 ) and transferred to 0.5-mL tubes. Sam-

ples were then digested in-gel with trypsin according

The proteins were extracted by trichloroacetic acid/acetone precipitation based on the method devel- oped by Granier. 11 Frozen flowers (0.5 g) cooled with liquid nitrogen were ground in a mortar with a pestle.

to the procedure of Shevchenko et al. 7 with slight modifications. The excised gel pieces were washed with 100 L of 100 mM NH 4 HCO 3 for 5 min and then dehydrated in 100 L of acetonitrile for 10 min. One hundred microliters of reduction buffer (10 mM

Five milliliters of cold ( 20 C) 10% trichloroacetic acid

dithiothreitol in 100 mM NH 4 HCO 3 ) was added and

in

acetone containing 0.07% -mercaptoethanol was

incubated for 30 min at 56 C. After cooling to room

added to the powdered sample. The sample was kept

temperature, 150 L of alkylation buffer containing 55

at

20 C to allow complete precipitation. The sample

mM

iodoacetamide in 100 mM NH 4 HCO 3 was added

was centrifuged (15 min at 4000 rpm) in a Heraeus

concentration was determined according to Peterson. 12

and

incubated at ambient temperature in the dark for

centrifuge, than washed three times with 5 mL acetone

20

min. The gel pieces were washed with 100 L of

containing 0.07% -mercaptoethanol. The precipitate was lyophilized for 1 h and suspended with 2 mL lysis buffer (9.5 M urea, 2% CHAPS (3[(3-cholamido- propyl)dimethylammonio] propanesulfonic acid), 1% dithiothreitol, and 0.8% carrier ampholytes from Amer- sham Biosciences, Uppsala, Sweden). The protein

100 mM NH 4 HCO 3 for 5 min and dehydrated with 100 L of acetonitrile for 10 min; this procedure was repeated a second time. After drying under vacuum, the gel pieces were rehydrated in a digestion buffer containing 50 mM NH 4 HCO 3 , 5 mM CaCl 2 , and 12.5 ng/ L trypsin (Promega, Madison, WI, USA) on ice. After 45 min, the supernatant was removed and

A

smaller amount of the gland (0.1 g) and propor-

replaced with 10–20 L of the same buffer but with-

tionally smaller amounts of solvent were used for pro- tein extraction with the same method as for flowers.

out trypsin, and incubated for 16 h at 37 C. The result- ing tryptic peptides were subsequently extracted for

2D Electrophoresis

Approximately 50 g and 150 g of protein were brought to a total volume of 125 L and 375 g and loaded on 7-cm and 18-cm IPG strips (Amersham Bio- sciences) consecutively. Two types of IPG strips were used, pH gradient 4–7 and 3–10. Isoelectric focusing was performed at 20 C for 80 kVh on an IPGphor (Amersham Biosciences). For SDS-PAGE, first IPG strips were equilibrated for 2 10 min by 6 M urea, 30% glyc- erol, 2% SDS in 0.05 M Tris-HCl containing 1% DTT and 4% iodoacetamide. SDS-PAGE (12% T, 2.6% C) in

Laemmli buffer system 13 was performed at 20 C and 100

V with MINIPROTEAN II system (Bio-Rad, Hercules,

CA, USA) using Power Pac 300 (Bio-Rad) power supply. Low molecular weight marker proteins (Amersham Bio- sciences) were applied on the gel via a small piece of filter paper. The gels were silver-stained according to a

10 min in an ultrasonic bath by addition of 10–15 L

of 25 mM NH 4 HCO 3 , acetonitrile, 5% formic acid, and acetonitrile, respectively. Pooled extracts were dried using a SpeedVac and the extracts were re-dissolved in 10 L of 0.1% trifluoroacetic acid and purified with Zip-Tip C 18 pipette tips (Millipore, Billerica, MA, USA), using the procedure recommended by the manufac- turer. The peptides were eluted from the tip directly onto the matrix-assisted laser desorption/ionization (MALDI) plate with matrix solution of cyano-4- hydroxycinnamic acid saturated in 50% acetonitrile, 0.1% trifluoroacetic acid.

MALDI-ToF-MS Analysis and Database Searching

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) experiments were performed on a Voy- ager-DE STR mass spectrometer (Applied Biosystems,

JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 15, ISSUE 2, JUNE 2004

99

T. J. RAHARJO ET AL.

T. J. RAHARJO ET AL. FIGURE 1 Two-dimensional gel electrophoresis of proteins expressed in flower (a)

FIGURE 1

Two-dimensional gel electrophoresis of proteins expressed in flower (a) and leaves (b). The first dimension was carried out by running 50 g of protein into 18-cm IPG strips,pH 3–10,and second dimension was SDS-PAGE (19 x 15 cm) with three replicates. Numbers 1–10 are selected spots for MALDI-MS analysis.

Framingham, MA, USA) equipped with delay ion extraction. Data were acquired in the delayed ion

extraction mode using a 20-kV bias potential, a 6-kV pulse, and a 150-ns pulsed delay time. Mass spectra were obtained over a mass range of 600–4,000 Da. For identification of proteins, the peptide mass fingerprinting data were used to search in databases NCBInr.20030905 for viridiplantae using Mascott pro- gram (http://www.matrixscience.com/cgi/index.pl?

page=

The peptide mass fingerprinting

of the proteins were scored with the Mowse score.

/home.html).

Western Blot Analysis

Proteins separated by 2D electrophoresis were blotted onto an Immobilon-P transfer membrane (Millipore).

The blotting was performed using a BioRad TransBlot

Electrophoresis Cell Apparatus for 1 h at 4 C at 100 V,

for 2 10 min in PBST solution and for 2 10 min in blocking solution. Detection of the protein on the membrane was done using alkaline phosphatase con- jugated anti-rabbit IgG antibody (Promega) at a 1:5000 dilution in PBST solution. After 30 min incubation, the membrane was washed 2 10 sec. The membrane then was exposed with staining solution until the sig- nal reached the desired contrast. Staining solution con- sists of 200 L NBT/BCIP stock solution [18.75 mg/mL 5-bromo-4-chloroindoxyl phospate (Sigma-Aldrich, St. Louis, MO, USA), 9.4 mg/mL 4-nitro blue tetrazolium (Sigma-Aldrich) in 67% DMSO], and 250 L 1 M MgCl 2 in 10 mL TBS buffer (0.1 M Tris, 0.1 M NaCl, pH 9.5). All the reactions were performed in sealed plastic bags using 5 mL solution.

RESULTS AND DISCUSSION

according to the manufacturer’s instructions. After blot- ting, the dried membrane was incubated for 1 h in blocking solution consisting of 1% BSA in phosphate- buffered saline tween (PBST: 10 mM NaH 2 PO 4 , 150

Comparison of Proteins Expressed in Cannabis Leaves and Flowers

Cannabinoids are more accumulated in flowers than

mM

NaCl adjusted to pH 7.2, and 0.5% Tween-20).

in the leaves. 1 Some proteins are expressed more in

The

chalcone synthase antibody was bound to the

flowers than in the leaves as well. These proteins

membrane by incubation for 1 h using a 1/1000 dilu- tion in PBST solution. The membrane was then washed

might be involved in cannabinoid biosynthesis, and identified by using MS in combination with genome

100

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COMPARATIVE PROTEOMICS OF C. SATIVA PLANT TISSUES

Peptide Mass Fingerprint Database Search of Selected Spots from 2D Electrophoresis of Flower Proteins

Spot No.

pI

MW (kDa)

MASCOT results

1

8.3

80

39 = NADP-dependent sorbitol-6-phosphate dehydrogenase (Prunus emerginata)

 

38

= Protein kinase family (Arabidopsis thaliana)

30

= Extensin precursor (proline-rich glycoprotein)

2

8.3

40

42 = Elicitor-responsive gene 3 (Oriza sativa)

 

39

= OSJNBb0016D16.13 (Oriza sativa)

37

= Putative ribonucleoprotein (Oriza sativa)

3

7.5

40

46 = Unknown (Arabidopsis thaliana)

 

40

= Ribosomal protein S11 (Zea mays)

39

= RTNLB35w (Ceratopteris richardii)

4

7.0

35

47 = Hypothetical protein (Arabidopsis thaliana)

 

41

= GL2-type homeodomain prot. (Oriza sativa)

40

= ORF64c (Pinus koraiensis)

5

5.5

25

47 = Protein F12M16.4 (Arabidopsis thaliana)

 

46

= Map kinase alpha 1(Arabidopsis thaliana)

43

= 36.4-kDa proline-rich protein

6

4.5

15

52 = Putative transcription factor (Oriza sativa)

 

44

= Unnamed protein product (Oriza sativa)

43

= AtpB (Begonia formosana)

7

6.5

15

43 = Putative GAG-POL precursor (Oriza sativa)

 

42

= DNA binding protein MNB1A

40

= AtpB (Begonia formosana)

8

7.0

65

47 = Unknown protein (Arabidopsis thaliana)

 

44

= Expressed protein (Arabidopsis thaliana)

39

= P0410E11.30 (Oriza sativa)

9

6.5

60

50 = Histone H4-alfalfa (fragment)

 

46

= Putative transcription factor (Oriza sativa)

42

= Hypothetical protein (Arabidopsis thaliana)

10

6.5

55

43 = Putative transcription factor (Oriza sativa)

41

= Protein F26F24.22 (Arabidopsis thaliana)

40

= Expressed protein (Arabidopsis thaliana)

database searching. 15,16 No studies have yet been per- formed on the C. sativa genome, but the genomes of the model plants Arabidopsis thaliana and rice (Oriza sativa) have been completely sequenced and could be helpful. The proteins of flowers and leaves were sepa- rated using 2D electrophoresis. We started using 18-cm IPG strips, pH 3–10, for the first dimension and then followed with SDS-PAGE (19 15 cm) for the second dimension. Using this broad pH range we expected to obtain an overall picture of all major proteins. In Figure 1 we observe that the pattern of leaf proteins in the gel is more crowded than that of the flower proteins. Both gels show more than 800 indi- vidual proteins, but it seems that the leaves contain a

greater variety of proteins than do the flowers. We found several protein spots that appear in the flowers but not in the leaves. We checked some of these pro- teins using MALDI-MS and obtained peptide finger- prints of these proteins. For peptide fingerprint database searching we set the following criteria: Coverage of the mature protein by the match must reach a minimum of 15%, at least four independent peptides should match, mass toler- ance is 1 Da, and maximum number of missed tryp- tic cleavages is 1. The modification parameters were oxidation of Met and modification of Cys. Table 2 shows the results of searching the peptide fingerprint database. We can see that none of the analyzed pro- teins is related to known cannabinoid biosynthesis

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101

FIGURE 2 Comparison between 2D gel electrophoresis of proteins expressed in flowers (a) and glands

FIGURE 2

Comparison between 2D gel electrophoresis of proteins expressed in flowers (a) and glands (b).The first dimension was carried out by running 50 g of protein into 7-cm IPG strips, pH 3–10, and second dimension was SDS-PAGE (7.4 x 6.8 cm). C: Expanded 2D gel electrophoresis (pH 4–7) of glands.

T ABLE

3

Peptide Mass Fingerprint Database Search of Gland Proteins

Spot No. a

pI

MW (kDa)

MASCOT results

1

4.8

17

53 = N2,N2-dimethylguanine tRNA methyltransferase-like prot. (Arabidopsis thaliana)

 

52

= T1F9.9 (Arabidopsis thaliana)

44

= Uridylyltransferase-related (Arabidopsis thaliana)

2

5.3

17

57 = P0699H05.2 (Oriza sativa)

 

53

= EMB-1 protein

53

= Exocyst subunit EXO70 family (Arabidopsis thaliana)

3

5.6

21

108 = F1D9.26-unknown prot. (Arabidopsis thaliana)

 

47

= Putative polyprotein (Oriza sativa)

46

= Hypothetical protein (Arabidopsis thaliana)

4

5.0

23

47 = F-box protein family (Arabidopsis thaliana)

 

46

= Probable DNA-binding protein (Arabidopsis thaliana)

42

= Expansin (Fragaria x ananassa)

6

5.3

29

56 = OSJNBa0052O21.1 (Oriza sativa)

 

48

= Hypothetical protein (Arabidopsis thaliana)

48

= Putative guanylate-binding protein (Oriza sativa)

9

7.6

43

53 = CHP-rich zinc finger protein, putative (Arabidopsis thaliana)

 

49

= DNA-directed RNA pol. II 13.6K chain (Arabidopsis thaliana)

47

= OSJNBa0079 M09.4 (Oriza sativa)

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3

(cont.)

Spot No. a

pI

MW (kDa)

MASCOT results

10

7.0

44

56 = CHP-rich zinc finger protein, putative (Arabidopsis thaliana)

 

54

= Hypothetical protein (Oriza sativa)

47

= Proton pump interactor-related (Arabidopsis thaliana)

11

6.9

43

89 = Phospolipase D beta 1 isoform 1a (Gossypium hirsutum)

 

64

= CHP-rich zinc finger protein, putative (Arabidopsis thaliana)

63

= Unnamed protein product (Oriza sativa)

21

6.8

43

67 = PG1 (Hordeum vulgare)

 

55

= HDA1 (Oriza sativa)

50

= CHP-rich zinc finger protein, putative (Arabidopsis thaliana)

22

6.6

27

65 = Expressed protein (Arabidopsis thaliana)

 

55

= PG1 (Hordeum vulgare)

50

= P0552C05.17 (Oriza sativa)

24

5.9

25

63 = PGPD14 protein (Arabidopsis thaliana)

 

59

= Unknown (Arabidopsis thaliana)

58

= Unnamed protein product (Oriza sativa)

30

4.8

54

53 = P0489G09.11 (Oriza sativa)

 

52

= Short-chain dehydrogenase/reductase family prot. (Arabidopsis thaliana)

43

= Putative 60S ribosomal prot. L31 (Picea abies)

41

7.0

43

54 = Glycin G4 precursor (soybean)

 

50

= Protein F9C16.23 (Arabidopsis thaliana)

50

= Unnamed protein product (Oriza sativa)

45

4.2

60

43 = P0436D06.5 (Oriza sativa)

 

43

= Expressed protein (Arabidopsis thaliana)

41

= Ribosomal protein L20 (Abies fabri)

46

6.3

50

50 = Expressed protein (Arabidopsis thaliana)

 

46

= Hypothetical protein F8D20.250 (Arabidopsis thaliana)

45

= WD-40 repeat protein family (Arabidopsis thaliana)

47

6.1

55

44 = Hypothetical prot. chrom. 10 OSJNBa0034L04.23 (Oriza sativa)

 

43

= Putative zinc finger protein, putative (Oriza sativa)

43

= Putative polyprotein (Oriza sativa)

48

6.0

49

49 = OSJNBa0072D21.18 (Oriza sativa)

 

45

= Photosysthem I P700 chlorophyll a apoprotein A2 (Pandorina morum)

42

= Maturase (Ginkgo biloba)

49

6.0

65

47 = Photosysthem I P700 chlorophyll a apoprotein A2 (Pandorina morum)

 

42

= PPR repeat containing protein (Arabidopsis thaliana)

38

= Glycine-rich protein GRP-17 (Arabidopsis thaliana)

50

5.9

64

53 = Squalene synthase (Panax ginseng)

 

51

= Phosphoglycerate kinase, cytosolic

51

= CHP-rich zinc finger protein, putative (Arabidopsis thaliana)

51

6.0

80

49 = Unknown protein (Arabidopsis thaliana)

 

48

= Expressed protein (Arabidopsis thaliana)

44

= Kinase R-like protein (Triticum aestivum)

52

5.8

76

49 = BZIP transcription factor (Phaseolus vulgaris)

 

42

= Invertase/pectin methylesterase inhibitor-related (Arabidopsis thaliana)

40

= ABC transporter family protein (Arabidopsis thaliana)

53

5.7

80

48 = PEP carboxylase-kinase (Mesembryanthemum crystallinum)

 

46

= AtpB (Begonia aptera)

46

= Maturase (Altingia excelsa)

54

5.6

68

52 = P0571D04.4 (Oriza sativa)

 

49

= Hypothetical protein (Arabidopsis thaliana)

41

= Ras-related GTP-binding protein (ARA-2) (Arabidopsis thaliana)

104 104

T. J. RAHARJO ET AL.

104 104 T. J. RAHARJO ET AL. FIGURE 3 We stern blot analysis of C. sativa

FIGURE 3

Western blot analysis of C. sativa leaf proteome fol- lowing 2D gel electrophoresis. The first dimension was carried out by running 50 g of protein in 7-cm IPG strips, pH 4–7, and second dimension in SDS- PAGE (7.4 x 6.8 cm). The antibody is anti-chalcone synthase of Pinus sylvestris.

proteins. Even the Mowse scores are at less than sig- nificant ( 64) levels. Most proteins that have the highest score are proteins found in A. thaliana or O. sativa, two plants for which the genome has been sequenced. This proves that genomic data are essen- tial for protein identification using proteome analysis.

Comparison of Proteins Expressed in Cannabis Flowers and Glands

In our comparison of the proteome of leaves and flowers we did not find proteins involved in cannabinoid biosynthesis. Therefore, we moved to a more specific part of the flower, the glands. This part of the plant has the highest levels of cannabinoids and the biosynthesis of cannabinoids is thought to take place in the glands. We compared the 2D gel pro- teome pattern of flowers with that of the glands. We expected that some proteins in the flowers would overlap with proteins of the glands, as we used flow- ers that included the glands. The number of protein spots in 2D gels of the flowers and the glands are significantly different (Fig. 2). Fewer than 100 proteins were detected in the pI 3–10, 7.4 6.8-cm gel of the glands, compared with more than 300 proteins detected for the flower in the same size gel. The lower number of proteins present

in the glands helped us study the majority by MS. The proteins of the glands primarily have a pI from 4 to 7. Therefore, for MALDI-MS, most proteins were excised from a pI 4–7 gel to provide optimum separation. Fifty-five proteins spots of the gland were tar- geted for identification. In Table 3 we can see the results of peptide fingerprint database searching (only spots with a Mowse score greater than 40 are shown). Only three protein spots have a Mowse score greater than 64 (significant). They are spots 3, 11, and 21, and identify as F1D9.26-unknown prot. (A. thaliana), phospholipase D beta 1 isoform 1a (Gossypium hir- sutum), and PG1 (Hordeum vulgare), respectively. Unfortunately, these proteins are not known to be involved in or related to cannabinoid biosynthesis. Spot numbers 46–55 are located in the pI and MW range of known cannabinoid biosynthesis proteins. Once again, the highest Mowse scores are obtained from proteins of A. thaliana and O. sativa; however, in most cases these are proteins with unknown func- tions. None of the peptide fingerprints of the selected spots matches with peptide fingerprints of THCA syn- thase, CBDA synthase, and CBCA synthase. This result might be caused by the fact that the proteins involved in the cannabinoid biosynthesis are expressed at very low levels even in the glands. Most of the protein spots that we identified are involved in primary metabolism. Studying proteins involved in the sec-

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COMPARATIVE PROTEOMICS OF C. SATIVA PLANT TISSUES

Highest Mowse Score of Peptide Mass Fingerprint Database Searching of Positive Spot in Western Blot

No.

Protein

Mowse Score

1

OSJNBb0040D15 (Oriza sativa)

50

2

RTNLB24w (Gossypium arboreum) Putative glycine-rich protein (Oriza sativa)

44

30

3

Oxidoreductase (Setaria italica) B1078G07.23 (Oriza sativa)

43

29

4

Cytosolic malate dehydrogenase (Cicer arietinum)

43

5

Malate dehydrogenase, cytoplasmic

38

ondary metabolism such as cannabinoid biosynthesis remains a major challenge because of the species- specific enzymes (and genes) involved.

Western Blot-Guided Protein Identification

To determine if any cannabinoid biosynthesis protein is present we decided to detect it by means of an anti- body against chalcone synthase. We expected to iden- tify one or more polyketide synthase proteins in the gels of the leaf, flower, and gland proteomes. The first step of cannabinoid biosynthesis is olive- tolic acid formation. 17 This step is thought to be cat- alyzed by STCSL enzyme, a polyketide synthase. Many sequences of polyketide synthase enzyme have been submitted to the databases both on the gene and protein level, such as chalcone synthase, stilbene syn- thase, 15 and stilbene carboxylate synthase. 18 Among the polyketide synthases, the sequence homology is high (70%). 15 Therefore, the antibody of one polyke- tide synthase might be used as a tool for identifying other polyketide synthases in the 2D gel elec- trophoresis. We used proteins extracted from leaves and a Pinus sylvestris chalcone synthase antibody in order to identify polyketide synthase proteins in C. sativa. We believe that in C. sativa more than one polyketide synthase is present. Therefore we expected to detect more than one spot in the 2D gel electrophoresis with the antibody. Figure 3 shows the 2D gel electrophoresis of leaf protein using IEF pH 4–7 and SDS-PAGE (7.4 6.8 cm). Only one spot gave a positive test in the West- ern blot. We performed the same experiment using proteins extracted from the gland, and again we

detected only the same protein. This protein has a MW of approximately 45 kDa and pI 6.5. The size of this protein is similar to that of other polyketide syn- thases such as chalcone synthase 19 and valerophe- none synthase 20 of Humulus lupulus. The protein spot in the gel was subject to trypsin digestion and the peptides analyzed by MALDI-ToF. Table 4 shows the result of database searching of peptide mass fingerprinting data. There was no polyketide synthase matching with this protein spot. Because we used a very narrow gel (7 cm in width), this result may be due to overlapping with another much more prominent protein. We also divided the spot into four parts prior to trypsin digestion and did MS analysis separately. All parts of the spot gave the same result. Overall, it appears that the cannabinoid biosyn- thesis genes are not easily found by using proteomics. The first difficulty is the lack of the genome sequence of C. sativa. But even with this sequence available, it is not certain whether the levels of the proteins are sufficiently high to be observed. The chalcone syn- thase-reactive protein spot is an indication of this problem, as within that spot no peptides were detected that match with polyketide synthase pro- teins. Most likely it is overlapped with a much more abundant primary metabolism protein.

ACKNOWLEDGMENTS

The authors would like to thank to Prof. J. Schröder (Uni- versity Freiburg, Germany) for his generous gifts of antibod- ies against P. sylvestris chalcone synthase. We thank Mr. W. Snoeijer for growing the cannabis plant. This work was sup- ported financially by the Quality Undergraduate Education project, Chemistry Study Program, Gadjah Mada University, Department of National Education Republic of Indonesia.

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