Proteomic Characterization of the Venom Of Five Bombus ... · for venom extraction. For venom...

FULL TITLE: PROTEOMIC CHARACTERIZATION OF THE VENOM OF FIVE BOMBUS

(THORACOBOMBUS) SPECIES

Nezahat Pınar Barkana1, Mustafa Bilal Bayazitb, Duygu Demiralp Özelc2

aHacettepe University, Faculty of Science, Department of Biology, Ankara, Turkey

bUniversity of Lausanne, Department of Fundamental Neurosciences, Lausanne,

Switzerland

cAnkara University, Faculty of Engineering, Biomedical Engineering, Ankara, Turkey

1Current Address: University of Gothenburg, Department of Oncology, Sahlgrenska

Academy, Gothenburg, Sweden

2Corresponding author: [email protected]

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted September 25, 2017. . https://doi.org/10.1101/193524doi: bioRxiv preprint

https://doi.org/10.1101/193524

Abstract

Venomous animals use venom; a complex biofluid composed of unique mixtures of proteins and

peptides, to act on vital systems of the prey or predator. In bees, venom is solely used for

defense against predators. However, the venom composition of bumble bees (Bombus sp.) is

largely unknown. Thoracobombus subgenus of Bombus sp. is a diverse subgenus represented

by 14 members across Turkey. In this study, we sought out to proteomically characterize the

venom of five Thoracobombus species by using bottom-up proteomic techniques. We have

obtained two-dimensional polyacrylamide gel (2D-PAGE) images of each venom sample. We

have subsequently identified the protein spots by using matrix assisted laser desorption

ionization / time of flight mass spectrometry (MALDI-TOF MS). We have identified 47 proteins for

Bombus humilis; 32 for B. pascuorum, 60 for B. ruderarius; 39 for B. sylvarum and 35 for B.

zonatus. Our analyses provide the primary proteomic characterization of five bumble bee

species’ venom composition.

Keywords: Bumble bees, MALDI-TOF MS, Proteomics, 2D-PAGE, Venom


https://doi.org/10.1101/193524

Introduction

In venomous animals, the use of venom or poison is one of the most essential mechanisms

used for either capturing prey or defending oneself against predators. In competing for

resources, venom is an adaptive trait and an example of convergent evolution. It is a complex

biofluid secreted by venom gland and is composed of unique mixture of proteins, enzymes,

peptides and biogenic amines that act on vital systems of the recipient (Calvete, 2009). In real

bees (Apoidea), largest family of bees with over 20.000 members (Fitzgerald, 2013), venom is

solely used for defense against predators (Arbuckle, 2017).

Among Apoidea, the majority of venom studies are focused on honey bee Apis mellifera (A.

mellifera). Bombus sp., which also belong in the same family as A. mellifera, are pollinators for

various native and cultivated plants (Free, 1993). The venom composition of Bombus sp. is

poorly understood.

Previous studies on Bombus sp. venom have mainly focused on characterization and isolation of

highly abundant single molecules using bio-assays and chemical sequencing via Edman

degradation (Choo, Lee, Yoon, Je, et al., 2010; Choo, Lee, Yoon, Kim, et al., 2010; Choo, Lee,

et al., 2012; Choo et al., 2011; Choo, Yoon, & Jin, 2012; Favreau et al., 2006; Kim et al., 2013;

Qiu et al., 2011; Qiu, Choo, Yoon, & Jin, 2012a, 2012b; Qiu et al., 2013; Wan et al., 2014).

Among the described peptides, bombolitins are unique to Bombus sp. and enhance

phospholipase A2 in liposome hydrolysis, in a similar fashion to melittin found in A. mellifera

venom and crabrolin and mastoparan found in vespid Vespa crabro venom (Argiolas & Pisano,

1985). Phospholipase A2 (Hoffman, El-Choufani, Smith, & De Groot, 2001; Hoffman & Jacobson,

1996; Van Vaerenbergh, Debyser, Smagghe, Devreese, & de Graaf, 2015), serine proteases

(Choo, Lee, Yoon, Kim, et al., 2010; Choo et al., 2011; Kim et al., 2013; Qiu et al., 2011; Qiu et

al., 2012b; Van Vaerenbergh et al., 2015) , serine protease inhibitors (Qiu et al., 2013; Wan et


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al., 2014), acid phosphatases (Hoffman et al., 2001; Hoffman & Jacobson, 1996; Van

Vaerenbergh et al., 2015), arginine kinase (N. P. Barkan, Demiralp, & Aytekin, 2015),

hyaluronidase (Hoffman & Jacobson, 1996), putrescine (Tom Piek, 2013), citrate (Fenton et al.,

1995), defensin (Rees, Moniatte, & Bulet, 1997), and acethylcoline (T Piek, Veldsema-Currie,

Spanjer, & Mantel, 1983) have also been isolated from Bombus sp. venom.

Recently, the whole genome sequencing of European large earth bumblebee B. (Bombus)

terrestris has become publically accessible. The accessibility to the genome sequence,

combined with the use of mass spectrometry, paved the way for deeply analyzing the venom

proteome composition of this species. This study has identified 55 new molecules in the B.

terrestris venom. Moreover, 72% of identified molecules had homologs in A. mellifera venom

indicating that while there are some species-specific differences, these two bee species share

major defense mechanisms. B. terrestris is the only member of the Bombus sp. whose venom

proteome composition has been characterized.

In bumble bees, color patterns show similarity among species or are highly variable within

species. Therefore, classification based on coat color can be unreliable (P. Williams, 2007). This

is especially true for the Thoracobombus subgenus which consists of 14 species in Turkey that

are morphologically difficult to separate. To overcome this, we have previously used landmark

based geometric morphometrics on their wing structure (N. P. A. Barkan, A. M., 2013). Other

approaches, use of pheromone analysis (Hovorka, Valterova, Rasmont, & Terzo, 2006; Terzo,

Valterová, & Rasmont, 2007; Terzo, Valterova, Urbanova, & Rasmont, 2003; Urbanová,

Valterová, Rasmont, & Terzo, 2002) and DNA based molecular techniques (S. Cameron, Hines,

& Williams, 2007; S. A. Cameron, Hines, & Williams, 2006; S. A. Cameron & Williams, 2003; Du

et al., 2015; Kawakita et al., 2003; Kawakita et al., 2004; P. H. Williams et al., 2012) have also

been used in differentiating bumble bee species.


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Turkey is regarded as one of the countries with the highest amount of species richness both in

number and diversity in the West-Palaearctic region. The main aim of this study is to determine

and compare the venom protein profiles of different Thoracobombus species of Turkey using

bottom-up proteomic strategies. With the adoption of these approaches, not only will we

characterize the venom proteome of Thoracobombus species for the first time, but we will also

highlight the proteomic differences within the subgenus that may help in differentiating between

the species.

2. Methods

2.1. Venom Extraction

B. humilis (Illiger), B. pascuorum (Scopoli), B. ruderarius (Müller), B. sylvarum (Linnaeus), and

B. zonatus (Smith) were collected from Ankara, Bolu, Çankırı and Kayseri provinces from Middle

Anatolian Region during June, July, August and September in 2013 and 2014 (Table S1), with

the permission from the Republic of Turkey, Ministry of Forestry and Water Affairs Directorate of

Nature Conservation & National Parks, and the Republic of Turkey, Ministry of Food, Agriculture

& Livestock. Alive specimens were transported to Hacettepe University Department of Biology

for venom extraction. For venom extraction, the venom reservoir is removed from the stinger in a

sterile environment with forceps and the membrane is disrupted afterwards. From each

individual, this method yielded 2-5 µl of venom which were collected in a cryotube and

transported in liquid nitrogen. For proteomic analyses venom samples were pooled, while for

spectroscopic analyses, populations were pooled separately. Collected samples were stored at -

80°C until use.

2.2. Proteomic Analysis

2.2.1. Protein Quantification and Two-Dimensional Gel Electrophoresis


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Venom of aforementioned bumble bee species was collected from 20 individuals per species.

Protein concentrations of the venom samples were obtained by using the Bradford assay

(Bradford, 1976). Venom was dissolved in 20 µl rehydration buffer [7M urea, 2 M thiourea, 4%

CHAPS (w/v), 1% ampholytes (pH 3-10), 10 mM DTT and trace amount of bromophenol blue].

2D-PAGE was performed as previously described (Igci & Demiralp, 2012) in three technical

replicates and a total of 50 µg of protein was loaded per gel.

2.2.2. 2D-PAGE Analysis

2D gel electrophoresis (2DE) images from venom samples of 5 different Thoracobombus

species were analyzed using PDQuest Software Advanced 8.0.1 (Bio-Rad, CA, USA). PDQuest

software examines each spot on every gel image and it picks a master gel containing most

spots. All spots in each gel image is then matched onto the master gel. Each post is annotated

with an identification number (SSP). For quantifying spots, four different normalization options

are performed including total density in gel images, local regression, total quantity in valid spot

and mean of log ratios. In the present study, 2DE image analysis is performed for both detecting

common and/ or unique spots between samples and for quantifying spot intensity which

accounts for the amount of protein in that spot. All spots were matched manually and ‘total

density in gel images’ normalization was performed. Two-way ANOVA was performed to detect

significancies in spot intensities (IBM Corp. Released 2010. IBM SPSS Statistics for Windows,

Version 19.0. Armonk, NY: IBM Corp.).

2.2.3. In-gel enzymatic digestion, Matrix-Assisted laser desorption ionization/ time

of flight (MALDI-TOF) mass spectrometry and peptide mass fingerprinting (PMF)

analysis

In gel trypsinization of protein spots was performed as previously described (Igci & Demiralp,

2012). Obtained peptides were loaded on MALDI-TOF plates using ZipTip® Pipette Tips


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(Millipore). Tryptic peptides were dissolved in sample solvent containing 0.1% and 50%

acetonitrile and mixed with an equal volume of matrix solution containing alpha-cyano-4-

hydroxycinnamic acid, 0.1% TFA and 75% acetonitrile. 1.5 µl of these mixtures were spotted

onto a target plate and analyzed with MALDI-TOF mass spectrometer (Water, UK) operated in

positive-ion reflectron mode. Obtained spectra were then examined in MASCOT search engine

(http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=PMF). Organisms

taxonomically related to bumble bees were selected as bumble bees are not present in the

database. “Other Metazoa” was selected as the taxonomical group as it is a diverse group

including all venomous animals and Drosophila sp., the taxonomically closest species to bumble

bees. Both Swissprot and NCBInr databases were used to maximize the number of hits.

Identified proteins were categorized into protein families using Swissprot database.

3. Results

3.1. 2D-PAGE Analysis of Thoracobombus Venom

2DE gel images were obtained from each venom sample (Fig 1).

In Thoracobombus matchset 87 protein spots were manually matched in 2DE images of B.

humilis, B. pascuorum, B. sylvarum, B. ruderarius and B. zonatus venom. Spot quantities were

calculated and significant spots were detected statistically. Among these protein spots, 21 spots

were present in each species. Common and differentially expressed protein spots are

summarized (Table S2).

3.2. Mass-spectrometry of Thoracobombus Venom

From the 2DE gel images, we have spectrometrically characterized 47 proteins for B. humilis; 32

for B. pascuorum; 60 for B. ruderarius; 39 for B. sylvarum and 35 for B. zonatus (Fig. S1 and

Table S3-7). Putative toxins are categorized according to their function and subcellular location

(Van Vaerenbergh et al., 2015) (Figure 1). Toxins previously identified in B. terrestris and A.


http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=PMF

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mellifera venoms are used as references (Table 2) (Van Vaerenbergh, Debyser, Devreese, & de

Graaf, 2014; Van Vaerenbergh et al., 2015). Major putative toxins; phospholipase A2, venom

protease, venom acid phosphatase and hyaluronidase were detected in venom of all five

analyzed Thoracobombus species. Arginine kinase, a known component of spider and parasitoid

wasp venom, was also identified in all studied species. Detection of serine protease snake (B.

ruderarius and B. sylvarum), peptidases (B. humilis and B. sylvarum), and kunitz-type serine

protease inhibitor (B. humilis and B. sylvarum), were limited to certain species.

3.3. Species Specific Expression of Putative Venom Toxins

Next, we compared the intensities of 2DE gel spots corresponding to putative toxins in each

sample (Fig. 2). According to these spot intensities, several toxins display species specific

expression patterns (Fig. 3). The expression of phospholipase A2, is the lowest in B. ruderarius

venom among studied species. B. zonatus venom displays the highest venom acid phosphatase

expression. B. pascuorum and B. ruderarius are characterized by a relatively low venom acid

phosphatase expression. B. humilis and B. pascuorum display the most and the least abundant

arginine kinase profile, respectively. B. humilis displays the highest venom protease expression.

Hyaluronidase 1 expression is more than two folds higher in B. ruderarius venom compared to

the other species.

4. Discussion

Identification of Thoracobombus subgenus represents a challenge as its members are

morphologically similar. In our previous study, we had applied landmark based geometric

morphometrics to distinguish its members from each other more accurately. In this current study,

we provide the primary proteomic characterization of the venom profile of five bumble bee

species belonging to this subgenus. Several putative venom toxins are detected in


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Thoracobombus which were in accordance with B. terrestris and A. mellifera venom.

Furthermore, we outline the species specific expression patterns of putative toxins.

In Hymenoptera, the defensive behavior of social groups is similar. Venom is used for protecting

the colonies rather than for capturing prey. Therefore, the venom composition of these groups

are expected to have common overall pattern. It is recently illustrated that the venom profile of B.

terrestris and A. mellifera largely overlap, while also retaining certain species specific

differences.

Accordingly, in Thoracobombus venom we have identified putative venom toxins collectively

used by several Hymenoptera species to debilitate predators such as; phospholipase A2,

arginine kinase, acid phosphatase (venom acid phosphatase Acph-1), serine protease (serine

protease snake), peptidases (peptidase 1 and aminopeptidase N), hyaluronidase (hyaluronidase

1) and serine protease inhibitors (kunitz-type serine protease inhibitor).

While the Thoracobombus venom composition may be similar to other Hymenoptera species in

terms of major toxins identified, it is important to note that the venom may be tailored for the

individual species’ needs with regards to abundancies of protein families present. Accordingly, a

change in the protein family profile of snake venom has been described upon nutritional shift.

Here, we have compared the 2DE gel images obtained from different Thoracobombus species

and highlighted the species specific expression differences of venom proteins. In particular, our

results indicate that expression levels of putative toxins may be used in differentiating between

species. Within the Thoracobombus subgenus, B. humilis is morphologically very similar to B.

pascuorum; while B. ruderarius and B. sylvarum resemble each other in terms of morphology

and coat color. On the other hand, B. zonatus with its characteristic yellow coat color is easily

distinguished from others. We have collected B. ruderarius and B. sylvarum at the highest

altitudes (>2000m) and B. zonatus at the lowest (~ 1000m). Interestingly, we have found that


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morphologically similar species differentiate in their expression of putative venom toxins, even in

a similar habitat.

In future studies, different populations of same species can be used to assess the direct effect of

altitude, vegetation, and prey population on the venom composition of bees. In this study we

utilized 2DE gel images and MALDI-TOF mass spectrometry for assessing the expression levels

of putative toxins, which may have its disadvantages. For example, we could not detect

bombolitin, unique bumble bee peptides, in our samples. As bombolitins are very small peptides

(10 kilo Daltons), we may have failed to detect these peptides with our equipment. More

sensitive quantitative mass-spectrometry approaches may be necessary to confirm the

correlation between microenvironment and the venom profile of bumble bees.

Profiling bumble bee venom can have pharmaceutical benefits. Venom peptides can be orally

active and transported through the blood-brain barrier and through mammalian cell membranes.

Commercial venom-derived drugs are available for the treatment of major diseases such as

diabetes, stroke, and hypertension (King, 2011). Bumble bee venom may have the potential to

serve as an alternative source of these drugs which are mostly snake venom-derived.

Understanding the species specific expression patterns of toxins can also benefit the specific-

compound oriented pharmacological analyses.

In conclusion, we provide the primary proteomic characterization of a diverse bumble bee

subgenus, and illustrate species specific differences in toxin expression.

Acknowledgments

This study was part of the PhD thesis of Nezahat Pinar Barkan. The experiments of this study

were performed completely in Ankara University Biotechnology Institute. We are grateful to

Selen Peker, Naşit İğci, Beycan Ayhan and Hatice Yıldızhan for their great help during


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laboratory work. We would especially like to thank Seçil Karahisar Turan for her great support

and guidance throughout the study.


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Fig. 1 2DE gel images of Thoracobombus

venom

Representitive images of venom profile of (A) B. humilis, (B) B. pascuorum (C) B.

ruderarius (D) B. sylvarum (E) B. zonatus. Gel analyses were conducted in triplicates.

Putative venom toxins marked with roman numerals: I: Peptidase I*, Aminopeptidase

N**, II: Venom acid phosphatase Acph1-like, III: Hyaluronidase 1, IV: Venom protease,

V: Arginine Kinase, VI: Phospholipase A2, VII: Serine protease snake, VIII: Kunitz-type

serine protease inhibitor. (See: Table 1).


https://doi.org/10.1101/193524

16

Fig. 2 Relative 2DE gel spot intensities of putative toxins in Thoracobombus venom.

B.h

um

ilis

B. p

ascu

oru

m

B. ru

dera

r iu

s

B.s

ylv

aru

m

B.z

on

atu

s

0

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

P h o s p h o lip a s e A 2

Sp

ot

Inte

ns

ity

B.h

um

ilis

B. p

ascu

oru

m

B. ru

dera

r iu

s

B.s

ylv

aru

m

B.z

on

atu

s

0

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

V e n o m A c id P h o s p h a ta s e A c p h 1 - lik e

Sp

ot

Inte

ns

ity

B.h

um

ilis

B. p

ascu

oru

m

B. ru

dera

r iu

s

B.s

ylv

aru

m

B.z

on

atu

s

0

2 0 0

4 0 0

6 0 0

S e r in e P r o te a s e S n a k e

Sp

ot

Inte

ns

ity

B.h

um

ilis

B. p

ascu

oru

m

B. ru

dera

r iu

s

B.s

ylv

aru

m

B.z

on

atu

s

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

V e n o m P ro te a s e

Sp

ot

Inte

ns

ity

B.h

um

ilis

B. p

ascu

oru

m

B. ru

dera

r iu

s

B.s

ylv

aru

m

B.z

on

atu

s

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

P e p t id a s e s

Sp

ot

Inte

ns

ity

A m in o p e p t id a s e N

P e p t id a s e 1

B.h

um

ilis

B. p

ascu

oru

m

B. ru

dera

r iu

s

B.s

ylv

aru

m

B.z

on

atu

s

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

A rg in in e K in a s e

Sp

ot

Inte

ns

ity

B.h

um

ilis

B. p

ascu

oru

m

B. ru

dera

r iu

s

B.s

ylv

aru

m

B.z

on

atu

s

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

K u n itz - ty p e S e r in e P ro te a s e In h ib ito r

Sp

ot

Inte

ns

ity

B.h

um

ilis

B. p

ascu

oru

m

B. ru

dera

r iu

s

B.s

ylv

aru

m

B.z

on

atu

s

0

1 0 0 0

2 0 0 0

3 0 0 0

H y a lu ro n id a s e 1

Sp

ot

Inte

ns

ity

Intensities, as calculated by PDQuest Software, are presented as mean ± standard deviation (3 replicates).


https://doi.org/10.1101/193524

17

Putative Toxin Family Spot Toxin Name B. terrestris A. mellifera

Peptidase/protease

I Peptidase I*,

Aminopeptidase N**

Dipeptidyl peptidase IV,

Serine carboxypeptidase,

Prolylcarboxypeptidase

Dipeptidyl peptidase IV,

Serine carboxypeptidase,

Prolylcarboxypeptidase

IV Venom protease

VII Serine protease snake Serine protease 1-6 CUB

serine protease

Serine protease snake, CLIP serine

protease, CUB serine protease

Esterase

II Venom acid

phosphatase Acph1-like

Acid phosphatase Acid phosphatase 1

V Arginine Kinase

VI Phospholipase A2 Phospholipase A2-1,

Phospholipase A2-2

Phospholipase A2-1,

Phospholipase A2-2

Carbohdyrate metabolism III Hyaluronidase 1 Hyaluronidase Hyaluronidase

Serine protease inhibitor VIII Kunitz-type serine

protease inhibitor

Serpin 1-3 Serpin 1-2

* B. sylvarum, ** B. humilis

Table 1. Putative toxins identified in Thorocabombus venom Toxin names and the corresponding 2DE gel spots are

indicated. The presence of the identified toxin (and/or its homologs/ortholog) in B. terrestris and A. mellifera venom is

presented.


https://doi.org/10.1101/193524

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