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Fall 12-11-2020

Functional Investigations of Proteins and Enzymatic Toxins from Functional Investigations of Proteins and Enzymatic Toxins from

Full-Length-Enriched cDNA of Timber Rattlesnake (Crotalus Full-Length-Enriched cDNA of Timber Rattlesnake (Crotalus

Horridus) Horridus)

Joshua Osula josula@stu.jsu.edu

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Candidate:

Major:

Thesis Title:

ApprQval:

Christopher A. Murdock

Professor of Biology

Major Professor

George Cline

Professor of Biology

Professor of Biology

Channing R. Ford

Senior Director, Graduate Studies

THESIS APPROVAL

Joshua Osula

Biology

Functional Investigation of Proteins and Enzymatic Toxins

From Full-Length-Enriched cDNA ofTimber Rattlesnake

(Crotalus Horridus)

Date

Date

Date

Date

FUNCTIONAL INVESTIGATION OF PROTEINS AND ENZYMATIC TOXINS FROM FULL-LENGTH-ENRICHED cDNA OF TIMBER RATTLESNAKE (CROTALUS HORRIDUS)

A Thesis Submitted to the

Graduate Faulty

of Jacksonville State University

in Partial Fulfillment of the

Requirements for the Degree of

Master of Science

with a Major

in Biology

By

JOSHUA OSULA

Jacksonville, Alabama

December 11, 2020

iv

Abstract

Snake Venom is a highly modified form of saliva, contains hundreds of zootoxins,

necrotoxins, cytotoxins, neurotoxins, and mytotoxins, all of which are primarily made up of

mostly proteins, polypeptides, and other components such as enzymes, lipids, and

carbohydrates. Although snake venom is known for its harmful effects, it has also had a positive

impact in scientific discovery and medicine. Some toxins from snakes’ function to inhibit pain,

and this means they can be very effective analgesics. For instance, crotalphine is considered a

14-amino-acid-peptide that has a disulfide bond and shows analgesic properties through TRPA1

desensitization. This is the reason why snake venom peptides are at our center of interest; there

are a plethora of new compounds to discover. Venomous snakes represent a large pool of

potentially bioactive proteins, and despite recent computational studies and discovery efforts of

over 2200 sequences, most of the snake venom toxins’ functions are still uncharacterized.

Using RNA pooled from the venom gland tissues of our sample, we constructed a cDNA

library. In order to generate full-length transcripts, we used the SMARTTM technique for first-

strand cDNA synthesis combined with duplex-specific nuclease enzyme from Kamchatka crab for

cDNA normalization. Five hundred plaques were successfully screened, and 50 of them were

sequenced. The plaques that we screened, amplified, and analyzed using agarose gel

electrophoresis and then sequenced mostly coded for ribosomal proteins. Crotamine, C-type

lectin (CLEC), phospholipase (PLA2), and serine Protease were the components we were able to

identify when we proceeded to annotate our 4 selected sequences with the venom-associated

v

genes. Using predictive modeling, we determined specific locations, coding regions, variation

information, exons, introns, and functions.

Crotamine functions to inhibit pain, and this means it can be a very effective analgesic, it

is a cell-penetrating peptide and a suitable means for transporting macromolecules through cell

membranes. Proteins with C-type lectin possess functions such as cell-to-cell adhesion, immune

response to pathogens, and apoptosis and have also shown anticoagulant and procoagulant

properties. PLA2 enzymes can bind to an array of proteins and target various types of tissues

and organs, and they tend to cause myonecrosis and spastic hind-leg paralysis. Serine proteases

play important roles in the diagnosis of hemorrhagic or thrombotic problems, and they stay in

the human system for a clinically useful length of time.

The four identified components of the full-length-enriched cDNA of timber rattlesnake

(Crotalus horridus) venom and their biochemical properties show promising applications in the

field of pharmacology and medical sciences.

ix., 44 pages

vi

Acknowledgments

I am deeply grateful to my amazing supervisor, Dr. Chris Murdock. I could not have

completed this work without his patience and support. I am also extremely appreciative of Dr.

Benjamin Blair, the little time he spent with me had the most impact. I am thankful to him for

being a scientific role model.

I am also very grateful to my committee members, Dr. James Rayburn and Dr. George

Cline, for dedicating a part of their professional time and knowledge to this project.

To my parents, Mr. & Mrs. G. Osula; my uncle, Dr. Graham O. Osula; my brother and

sister; my girlfriend, Yvonne; and other family and close friends, I could never be the person and

scientist I am now without your belief in me and your continuous support. I just want you to

know I am thankful.

Finally, I am deeply indebted to God, for ordering my steps and giving me an important

direction for my journey so far.

Joshua Osula

vii

Table of Contents

Page

TABLE OF CONTENTS ………………………………………………………………………………………………. vii

LIST OF TABLES …………………………………………………………………………………………………………ix

LIST OF FIGURES ………………………………………………………………………………………………………. x

I. INTRODUCTION ……………………………………………………………………………………………………. 1

II. MATERIALS AND METHODS …………………………………………………………………………………. 7

Venom Samples ……………………………………………………………………………………………………….7

cDNA Normalization …………………………………………………………………………………………………8

cDNA Synthesis ………………………………………………………………………………………………………. 9

cDNA Amplification ………………………………………………………………………………………………… 10

Sfil Digestion and Ligation of cDNA to λTriplEx2 Vector ………………………………………….. 11

Titrating the Unamplified Library ……………………………………………………………................. 12

Library Amplification and Titrating ……………………………………………………….………………… 13

Screening and Sequencing ………………………………………………………………………………………15

Data Analysis …………………………………………………………………………………………………………..15

Gene expression (Real time quantitative PCR)………………………………………………………… 16

III. RESULTS ……………………………………………………………………………………………………………….18

Pre/Post PCR …………………………………………………………………………………………………………..18

Data Analysis …………………………………………………………………………………………………………..19

IV. DISCUSSION …………………………………………………………………………………………………………26

Crotamine ……………………………………………………………………………………………………………….26

C-Type Lectin (CLEC) ………………………………………………………………………………………………..28

Phospholipase A2 (PLA2) …………………………………………………………………………………………28

Serine Protease ……………………………………………………………………………………………………….29

V. CONCLUSION ………………………………………………………………………………………………………..31

viii

CITED REFERENCES ……………………………………………………………………………………………………33

APPENDICIES …………………………………………………………………………………………………………….37

APPENDIX A ………………………………………………………………………………………………………………38

Tables and Figures …………………………………………………………………………………………………..38

APPENDIX B ………………………………………………………………………………………………………………41

THESIS OPTION FORM …………………………………………………………………………………………….42

APPENDIX C ………………………………………………………………………………………………………………43

PROSPECTUS FOR THESIS FORM ……………………………………………………………………………..44

ix

LIST OF TABLES

1. Table 1. Normalization using 3 different DSN enzyme concentrations.........……… 8

2. Table 2. Ligations using 3 different ratios of cDNA to phage vector ……..………….. 14

3. Table 3. Plating dilutions for titrating an amplified library ……………………………..…14

4. Table 4. BLAST search to identify transcripts of 4 selected sequences from

normalized libraries ………………………………………………………………………………………….20

5. Table 5. Biochemical properties for Crotamine, C-type lectin 2,

Phospholipase A2, Serine protease …………………………………………………………………. 24

x

LIST OF FIGURES

1. Figure 1. List of Primers used for first-strand cDNA synthesis……………………..…………….. 11

2. Figure 2. List of primers used for cDNA amplification ………………………….……………….…… 11

3. Figure 3. List of primers used for real-time PCR (reverse transcriptase reaction) ……....16

4. Figure 4. DNA sequences and translated protein sequences for crotamine …………….…21

5. Figure 5. DNA sequences and translated protein sequences for C-type lectin 2 ....…….21

6. Figure 6. DNA sequences and translated protein sequences for phospholipase A2 ……22

7. Figure 7. DNA sequences and translated protein sequences for serine protease ……….23

8. Figure 8 shows Real-time PCR amplification for C. horridus that resulted in the

amplification of 198 bp amplicon ………………………………………………………………………………25

9. Figure 9. Nucleotide sequences for selected venom components ……………………………. 38

a) VG 4 – Crotamine ...…………………………………………………………………………………………….36

b) VG 4 – C-Type Lectin (CLEC) ……………………………………………………………………………….36

c) VG 4 – Phospholipase A2 (PLA2) .……………………………………………………………………….37

d) VG 4 – Serine protease ………..…………………………………………………………………………….37

10. Figure 10. Amino acid sequences for selected venom components ..…………..……….…. 40

a) VG 4 – Crotamine ...…………………………………………………………………………………………….38

b) VG 4 – C-Type Lectin (CLEC) ……………………………………………………………………………….38

c) VG 4 – Phospholipase A2 (PLA2) .……………………………………………………………………….38

d) VG 4 – Serine protease ………..…………………………………………………………………………….38

xi

1

INTRODUCTION

Snakes possess a highly modified form of saliva generally known as venom. Their glands,

which are modified parotid salivary glands, store this venom in the alveoli. Upon release, they

secret an array of zootoxins, necrotoxins, cytotoxins, neurotoxins, and myotoxins, all of which

have building blocks of proteins, polypeptides, and other components such as enzymes, lipids,

and carbohydrates. Most snake venom toxins participate in envenomation through a diverse array

of bioactivities (Ojeda et al. 2017), such as bleeding, inflammation, and pain, as well as cytotoxic,

cardiotoxic, or neurotoxic effects. The functions of these components depend on an extensive

variety and positions of nucleotides and nucleoside triphosphates in the genetic sequence of

these components. Although comparative venom-gland transcriptomics have revealed several

harmful effects of snake venom—such as renal failure, toxins that induce defibrination

coagulopathy, and microangiopathic hemolytic anemia (Chippaux et al. 2009) —it has also had a

positive impact in scientific discovery and medicine. For example, the most famous example of

snake-derived medicine is captopril (Capoten), which was developed by Bristol Myers Squibb and

is now used as a generic medicine for treating hypertension and congestive heart failure. It is a

small-molecule inhibitor of the angiotensin-converting enzyme (ACE) and is derived from

bradykinin-potentiating peptides found in the venom of the South American snake Bothrops

jararaca. It has been very forthcoming in the development of molecular scalpels that play a major

role in elucidation of ion channel function, vasoconstriction, platelet aggregation, signal

transduction, blood pressure regulation, and much more (McCleary and Kini 2013). Thus, it has

provided insight into cardiovascular physiology and is now being used worldwide to treat

2

thrombosis, arthritis, cancer, and strokes. Despite recent computational studies and discovery

efforts of over 2200 sequences, most of the snake venom toxins’ functions are still

uncharacterized. Analyzing these venoms provides a more detailed understanding of venoms and

toxins’ mechanisms of actions and an array of new drug possibilities (Melani et al. 2015). This

project uses modern bioinformatics methods such as pre- and post-PCR (polymerase chain

reaction) techniques to further study, identify, and characterize snake venom components and

their functions.

In rattlesnakes, 4 venom motifs have been identified: neurotoxic Type A, synonymous to

the south; hemorrhagic and proteolytic Type B, synonymous to the north and parts of the

southeast; Type A + B synonymous to southwestern Arkansas and northern Louisiana; and finally

Type C, which is not as strong as the other venoms (Norris 2004). Multiple transcripts occur in

most genes, which can be as a result of alternative splicing, a variety of encoded protein

products that may be either similar, dissimilar, or may even possess functions that are entirely

different within and across species. Ultimately, the purpose of proteomics is to identify and

compare structure-function relationships of proteins across venoms of a number of species.

However, proteomics falls short when it comes to analysis on a molecular level, so we used a

combination of transcriptomics and proteomics for qualitative and quantitative assessments in

order to decrypt our data avalanche.

Whole transcriptome analysis is instrumental in the initial step to functional

characterization and gene annotation previously revealed by DNA sequencing; it identifies

cellular genetic networks and biological, physiological, and biochemical systems that are

3

instrumental to molecular evolutionary analysis and collations that result in libraries of full-

length-enriched cDNA, new gene discoveries. It is advantageous when dealing with organisms

that have no prior sequencing information. Methods used for setting up cDNA libraries come

with their own shortcomings (Gubler and Hoffman 1983). The premature stop of reverse

transcription leads to cDNA clones that are not full length, resulting in inadequate

representation of cDNA 5’ ends (Maruyama and Sugano 1994). Regular DNA methods are

dependent on methylation, which is often not efficient in conserving internal restriction sites

and cloning (McClelland et al. 1994). This is the reason for the SMARTTM method (switching

mechanism at the 5’ end of the RNA transcript) Zhu et al. (2001). It used to increase the length

of cDNA clones by mirroring reverse transcription and switching the template of Moloney

murine leukemia virus (MMLV) (Kulpa et al. 1997). The Sfil sites accommodate the 5’ and 3’ ends

of the first-strand DNA; the 5’end is hybridized to a modified oligo(dT) primer that has a SfilB

restriction site, and the 3’ end is hybridized to a modified oligo(dT) primer that has a SfilA

restriction site (Zhu et al. 2001). As the process progresses, the oligonucleotides that host the

oligo(dT) sequences at the 3’ end bind to the extending template tail of the C-rich cDNA for

reverse transcriptase. Enzymes switch templates when reverse transcriptase gets to the 5’ end

of the mRNA enzyme and continues to replicate up to the oligonucleotide end (Zhumabayeva et

al. 2001). The result is a complete 5’ end at the first-strand cDNA due to premature termination

of cDNA that do not possess a SfilA site (Chenchik et al. 1998). The disadvantage of this

technique is that it is not strand specific and it is only applicable to poly(A)+ RNA. on the plus

side, the advantages are: (I) anonymous mRNA sequencing can be used; (II) starting material as

4

little as 50pg can be used: (III) there is improved coverage across transcripts; and (IV) there is a

high level of mappable reads. Due to its high versatility, this method is integrated into multiple

sequencing techniques.

Duplex-specific nuclease (DSN) is instrumental in the normalization of full-length-

enriched cDNA (Zhulidov et al. 2004). It minimizes the frequency of high-abundance transcripts

and balances transcript concentration levels in a cDNA component, leading to more effective

sequencing outputs and discovery of rare genes that may be transcribed at relatively small levels

or for functional screenings. Recently, a very effective normalization technique was developed.

It is normalization technique (DSN normalization), which is based on the unique properties of

duplex-specific nuclease (DSN) from Kamchatka crab (Bogdanova et al. 2008). DSN is stable at

high temperatures of about 60 - 65°C, which enables the effective removal of ds-DNA from

complex nucleic acids at hybridization temperatures. In this technique, the cDNA is denatured

and then rehybridized. There is more of a transition of abundant transcripts to the ds-form, and

the ss-cDNA fraction is equalized. Post rehybridization, DSN hydrolyzes ds-cDNA, but the ss-

cDNA fraction does not change. PCR is used to amplify the latter and used for construction of

normalized cDNA libraries.

A high-throughput, computer-automated sequencing using the vintage Sanger method

is applied in sequencing of clones from the cDNA library. A single-stranded primer will anneal to

the denatured DNA template in a reaction tube also containing 4 deoxynucleoside triphosphates

(dNTPs) and dideoxyribonucleotides (ddNTPs). Since a number of snake venom peptides are

about 100 residues long, the Sanger sequencing of expressed sequence tags (ESTs) tends to

5

produce the complete DNA sequence of a peptide. The samples are then separated on a single-

lane capillary gel that is scanned with a laser beam. The amount of sequences that are retrieved

is quite smaller than that generated by next-generation sequencing (NGS).

Today we are dealing with an explosion of DNA sequence data; there are a plethora of

“tools” available on the Web to predict homologies, such as BLAST (Basic Local Alignment Search

Tool), Benchling, Clustal Omega, EMBL-EBI (The European Bioinformatics Institute), ExPASy (the

Expert Protein Analysis System), RCSB (the Research Collaboratory for Structural Bioinformatics),

and SMART™. The tools are selected based on the type of questions to which one is trying to

find answers. Alignment, identity, similarity, and expectation value (E-value) can be retrieved

using these tools. Identity is the percentage of similar components in the comparing sequences.

Similarity accounts for a quantitative measurement of the number of components that align at

certain positions—for instance, a leucine–leucine pairing would be more similar than a leucine–

cytosine pairing. The sum of the sequence similarity with respect to the length of the query

sequence that matched the sequence that was found by the tool is known as score; therefore, a

high similarity and a longer stretch will result in a high score. The E-value is the expected

number of sequences that will have that similarity score from the result of a query in the

database. However, the disadvantage of using these tools is that not everything that is added to

databases is correct; anyone can upload their sequences, leaving room for erroneous

annotations. These types of errors become alive in the database because new homologous

sequences may adopt the errors, and even after these mistakes have been noticed and

6

corrected, the erroneous ones will still be in some part of the database. Backtracing the source

of the annotation is the only way to be assured that the results are accurate.

Some toxins from snakes function to inhibit pain, and this means they can be very

effective analgesics. For instance, crotalphine is considered a 14-amino-acid peptide that has a

disulfide bond and shows analgesic properties through TRPA1 desensitization. This is the reason

why snake venom peptides are at our center of interest; there are a plethora of new compounds

to discover. We annotate the selected sequences with the venom-associated genes using

predictive modeling to determine their specific locations, coding regions, variation information,

exons, introns, and functions. Gene expression levels for the select genes are confirmed, and

finally, the gene product is produced.

7

MATERIALS AND METHODS

Venom Samples

The biological sample (venom gland) was extracted from 3 adult timber rattlesnake (Crotalus

horridus) that were captured in the wild and kept in controlled environmental conditions in the

laboratory. The snakes were fed mice once a week in order to maintain a healthy diet. In order

to obtain tissue with high levels of toxin expression, their venom glands were extracted 72 hours

after milking and at the time of milking. In 1974, it was confirmed that epithelial cells become

shorter and more quiescent when venom glands are full compared to when they are empty, and

they are larger and more active (De Lucca et al. 1974). The next step with the aim of isolating

the total RNA involved in placing and homogenizing the venom gland in TRI Reagent® solution.

The reagent solution is built up of guanidine thiocyanate and phenol in a monophasic solution

that restricts RNase activity and gives room to isolation of high yield and total RNA of high

quality. Once a high-quality total mRNA was obtained, an RNA isolation kit (QIAGEN, Tokyo,

Japan) was used to isolate the mRNA. 1.5% agarose/EtBr gel run in 1x TAE buffer is used to make

a confirmation on the integrity of the total RNA.

8

cDNA Normalization

DSN enzyme is purified from the hepatopancreas of Kamchatka crab, and it is also

available commercially. After successfully synthesizing first-strand DNA at 98°C for 2 minutes,

we denatured the reaction mixture, which comprised 1000mg of cDNA, 4µl of 4x hybridization

buffer (20mM HEPES, pH 7.5, 2 M NaCl), and Milli-Q® water making a total volume of 16µl.

Then it was allowed to hybridize at 68°C for 6 hours. Then 5µl of DSN master buffer (100 mM

Tris–HCl pH 8.0, 10 mM MgCl2, 2 mM dithiothreitol) that was already at 68°C was added to the

cDNA samples for a 10-minute time span. Next, at 68°C for 20 minutes, three different

concentrations of the DSN enzyme (Table 1) (Evrogen , Russia) were added to the reaction

mixtures, which were rendered inactive by the addition of 10µl of 5 mM EDTA solution for 5

minutes at the same temperature and then placed on ice.

Table 1. Normalization using 3 different DSN enzyme concentrations.

DSN solution Tube 1 (1/20 DSN)

Tube 2 (1/40 DSN)

Tube 3 (1/80 DSN)

Tube 4 (control)

1/20 DSN solution (0.05U/µl)

1 µl - - -

1/40 DSN solution (0.025U/µl)

- 1 µl - -

1/80 DSN solution (0.0125U/µl)

- - 1 µl -

DSN master buffer - - - 1 µl

9

cDNA Synthesis

A SMARTTM cDNA Library Construction Kit (BD Biosciences Clontech, San Jose, CA, USA)

was used for first-strand cDNA synthesis. For the samples, we used 1–3µl RNA samples (0.5 to

1µg of total RNA) in line with the manufacturer’s instruction. 1µl each of the BD Biosciences

oligonucleotides SMARTTM Oligo VI and CDS-3M that contained the SfilA and SfilB recognition

sequence were used (Figure 1).

cDNA amplification was done by long-distance PCR (QIAGEN, Tokyo, Japan); PCR Primer

M1 was used to amplify the first strand (Figure 1). The PCR master mix for all reaction tubes was

prepared, and it comprised of 80µl Milli-Q® water, 10µl of 10x Advantage 2 PCR buffer, 2µl OF

50X dNTP mixture (10mM each), 4µl of 5’ PCR Primer M1, 2µl of first-strand cDNA, and 2µl of

Advantage 2 polymerase mix. The SMARTTM provided guidelines for the optimal number of

thermal cycles in correspondence to a given amount of poly A+ RNA used in the first-strand

synthesis. Modifying the guidelines provided by SMARTTM, we initiated 21 cycles, and the

thermal cycler was preheated to 95°C. Thermal cycling; the Initial denaturation occurred at 95°C

for 1 minute. The other cycles were 95°C for 15 seconds, 66°C for 20 seconds, and 72°C for 3

minutes, and the final extensions at 66°C for 20 seconds; 72°C for 3 minutes were performed.

After amplification, cDNA purification was carried out using QIAquick PCR Purification Kit

(QIAGEN, Tokyo, Japan) with the aim of removing the excess primer, dNTPS, and salts. After the

purification step, and after precipitation with ethanol, the cDNA was dissolved in Milli-Q® water

to a final concentration at 100 ng/µl.

10

cDNA Amplification

Amplifying normalized cDNA and determining the optimal number of PCR cycles was

achieved with 40.5 µl of Milli-Q® water, 5µl of 10x PCR buffer, 1µl dNTP mix, 1.5µl of PCR

Primer-M1 (Figure 2), 1µl of normalized cDNA, and 1mL of PCR mix. Next, it was put through the

process of cycling at settings: 95°C for 1 minute for initial denaturation 17 PCR cycles at 95°C for

15 seconds, 66°C for 20 seconds, and 72°C for 3 minutes 10µl was collected from the 17-cycle

PCR control tube into a clean tube for further PCR cycling, collecting 12-µl aliquots after 18, 20,

22, 24 and 26 PCR cycles. 5µl of the aliquots of each PCR was analyzed after 17, 19, 21, 23, and

25 cycles alongside of 1kb DNA size markers on a 1.5% agarose/EtBr, run in 1x TAE buffer, to

confirm the optimal number of cycles required for amplification.

When the optimal number of cycles were acquired, 2µl of normalized cDNA was diluted

into 20µl of Milli-Q® water. For the second PCR amplification, the following steps were followed:

80µl of Milli-Q® water, 10µl of 10x PCR buffer, 2µl of diluted normalized cDNA and 2µl of dNTP

mix (10mM), 4µl of PCR Primer M2 (Figure 2), and 2µl of diluted normalized cDNA and 2µl of

polymerase mixture. Then it underwent PCR cycling using the following settings: initial

denaturation at 95°C for 1 minute; 12 PCR cycles at 95°C for 15 seconds; 64°C for 20 seconds;

and heat at 72°C for 3 minutes; and a final extension at 64°C for 15 seconds and 72°C for 3

minutes. After the cDNA amplification cycles were completed, 5µl of each sample were loaded

on a 1.5% (w/v) agarose/EtBr gel in 1x TAE buffer and electrophoresed to observe the PCR

quality from the characteristic gel profile.

11

Figure 1. List of Primers used for first-strand cDNA synthesis.

5’-oligonucleotide adapter for the template switching reaction:

5’- AAGCAGTGGTATCAACGCAGAGTGGCCATTAC GGCCGGG-3’

CDS-3M adapter:

5’-AAGCAGTGGTATCAACGCAGAGTGGCCGAGGCG GCC(T)20 VN-3’

(N=A, C, G, or T; primers V=A, G, OR C)

Figure 2. List of primers used for cDNA amplification:

PCR Primer M1 (10mM): 5’ – AAGCAGTGGTATCAACGCAGAGT-3’

PCR Primer M2 (10mM): 5’ – AAGCAGTGGTATCAACGCAG-3’

Sfil Digestion and Ligation of cDNA to λTriplEx2 Vector

The λTriplEx2 Vector used was provided by Clontech Laboratories, Inc. (now Takara Bio

USA, Inc.) (SMARTTM cDNA Library Construction Kit). Normalized cDNA samples digested by Sfil

were ligated and packaged into a λTriplEx2 vector. Three separate ligation reactions with

different ratios of cDNA to phage vector were set up with varying ratios of cDNA to phage vector

(Table 2). The reaction tube was incubated at 16°C overnight. The packaging protocol was done

according to the manufacturer’s instructions, and MaxPlaxTM Lambda Packaging Extracts for

λTriplEx2 reactions with an efficiency of 3 x 109 pfu/µg DNA were used.

12

Titrating the Unamplified Library

The titrating protocol was instrumental in determining the titer of the unamplified

library and the percentage of recombinant clones. The primary stock plate was prepared by

recovering frozen cells of Escherichia coli XL1-Blue competent cells provided with the SMARTTM

cDNA Library Construction Kit (BD Biosciences Clontech) and streaking about 5µl of it on an

LB/tet plate. The stock plate was then incubated overnight at 37°C.

To ascertain the titer of unamplified library from the initial prepared culture, a single

isolated colony was selected from the working stock plate and used to inoculate 15ml of

LB/MgSO4/maltose broth in a 50-ml test tube. It was then incubated overnight at 37°C while

vibrating at 140 rpm until the culture’s OD600 reached 2.0. Dilutions of the packaging extracts at

1:5, 1:10, 1:15, and 1:20 were made in 1X Lambda dilution buffer (NaCl, MgSO4·7H2O Tris-HCI

[pH 7.5]). 1µl of the ligation reaction was added to E. coli XL1-Blue competent cells overnight;

the phage was allowed to adsorb at 37°C for 15 minutes. In the next step, 2ml of melted

LB/MgSO4 agar was added and poured onto LB/MgSO4 plates. The cooled plates were incubated

at 37°C for 18 hours. The titer of the phage was calculated using the formula

( 𝑝𝑝𝑝𝑝𝑝𝑝𝑚𝑚𝑚𝑚

=𝑛𝑛𝑝𝑝𝑚𝑚𝑛𝑛𝑛𝑛𝑛𝑛 𝑜𝑜𝑝𝑝 𝑝𝑝𝑚𝑚𝑝𝑝𝑝𝑝𝑝𝑝𝑛𝑛𝑝𝑝 𝑥𝑥 𝑑𝑑𝑑𝑑𝑚𝑚𝑝𝑝𝑑𝑑𝑑𝑑𝑜𝑜𝑛𝑛 𝑝𝑝𝑝𝑝𝑓𝑓𝑑𝑑𝑜𝑜𝑛𝑛 𝑥𝑥 103 µ l

mlµl of diluted phage plated

) .

In order to identify the percentage of recombinant clones, blue-white screening was

applied using E. coli XL1-Blue. The lacZ α-complementation was then used to identify a phage-

13

containing insert on a medium with IPTG and X-Gal added to top agar, and this was achieved by

embedding the λTriplEx2 vector cloning site in the coding sequence for LacZ.

Library Amplification and Titrating

200µl of the overnight bacteria culture and 50µl of 1:20 diluted lysate were added to 20

separate 10-ml tubes. It was incubated at 37°C for 15 minutes. Next, 5ml of melted LB/MgSO4

soft top agar is added and poured into the LB/MgSO4 plates. The top agar hardened for 10

minutes. The plates were inverted and placed in the incubator at 37°C for 18 hours. After the

incubation, 12ml of 1X Lambda dilution buffer was added to each plate and kept at 4°C

overnight. Plates were placed on a shaker at 50rpm for an hour at room temperature, prior to

pooling 1X Lambda dilution buffer on each plate we poured λ phage lysate into a sterile 50-ml

screw-cap tube. 10ml of chloroform was added and then vortexed for 2 minutes and centrifuged

at 7000 rpm for 10 minutes to clear the phage lysate of cell debris. The supernatant was then

collected and stored at 4°C.

In order to determine the titer of the amplified library of the prepared dilution’s lysate,

10µl of the lysate (Dilution 1 = 1:100) was added into 1ml of the 1X Lambda dilution buffer. 10µl

of the lysate (Dilution 2 = 1:10000) was added into the second tube. Next, 4 10-ml tubes using

the overnight culture and Phage Dilution 2 were prepared (Table 3), and 5ml of melted

LB/MgSO4 soft top agar was added and poured into LB/MgSO4 agar plates. The top agar

hardened for 10 minutes. The plates were inverted and placed in the incubator at 37°C for 18

14

hours. After the incubation, plaques were counted and the titer was calculated using the

formula ( 𝑝𝑝𝑝𝑝𝑝𝑝/𝑚𝑚𝑚𝑚 =𝑛𝑛𝑝𝑝𝑚𝑚𝑛𝑛𝑛𝑛𝑛𝑛 𝑜𝑜𝑝𝑝 𝑝𝑝𝑚𝑚𝑝𝑝𝑝𝑝𝑝𝑝𝑛𝑛𝑝𝑝 𝑥𝑥 𝑑𝑑𝑑𝑑𝑚𝑚𝑝𝑝𝑑𝑑𝑑𝑑𝑜𝑜𝑛𝑛 𝑝𝑝𝑝𝑝𝑓𝑓𝑑𝑑𝑜𝑜𝑛𝑛 𝑥𝑥 103 µ l

mlµl of diluted phage plated

).

Table 2. Ligations using 3 different ratios of cDNA to phage vector.

Component 1st ligation (µl) 2nd ligation (µl) 3rd ligation (µl) cDNA

Vector (500 ng/µl) 10X ligation buffer

ATP (10mM) T4 DNA ligase

Deionized H2O*

0.5 1.0 0.5 0.5 0.5 2.0

1.0 1.0 0.5 0.5 0.5 1.5

1.5 1.0 0.5 0.5 0.5 1.0

Total volume (µl) 5.0 5.0 5.0

Table 3. Plating dilutions for titrating an amplified library

1X lambda tube 1X lambda dilution buffer

bacterial overnight culture

phage dilution 2

1 2 3 4 (control)

100 µl 100 µl 100 µl 100 µl

200 µl 200 µl 200 µl 200 µl

5µl 10µl 20µl 0µl

15

Screening and Sequencing

Plaques from the plates that were previously prepared to determine titer of amplified

library were randomly selected and assigned an identification number. The pipette was used to

carefully transfer the plaque into PCR tubes containing MgCl, dNTP, Taq polymerase, PCR buffer,

Primer F (5’ primer), Primer R (3’ primer) and Milli-Q® water. PCR protocols were initiated as

follows: initial denaturation at 95°C for 5 minutes, 29°C for 20 seconds, 60°C for 20 seconds,

72°C for 30 seconds, and a final extension of 72°C for 2 minutes. Using another tip, we gently

touched the same plaque and then touched the LB/MgSO4 agar plate with solidified LB/MgSO4

top agar and overnight culture in quadrant that matched the assigned identification number. It

was then incubated at 37°C for 18 hours.

Agarose gel electrophoresis was used to ascertain the integrity of the amplified cDNA

transcripts, and the promising VG clones that showed bands based on size separation were sent

off to BD Biosciences for sequencing.

Data Analysis

The sequencing results received-VG 4 – Crotamine, VG 36 – C-Type Lectin, VG 141 –

Phospholipase A2, and VG 172 – Serine Protease—were uploaded to the Basic Local Alignment

Search Tool (BLAST) to annotate the selected sequences with the venom-associated genes. The

nucleotide BLAST identified regions of similarities between biological sequences (alignments),

the identity, the expectation value (E-value) of the query sequence, and the sum of the

16

sequence similarities with respect to the length of the query sequence that matched the

sequence that was found (score). The matching sequences with the highest similarities and the

longest stretch and with the lowest E-values were then selected. The sequence was identified as

well as the start and stop codons and the poly(A) tail region.

After identifying the nucleotide sequences from the BLAST search, the translation tool

ExPASy, which allows the translation of a nucleotide (DNA/RNA) sequence to a protein

sequence, was used to retrieve the encoded protein sequences from the nucleotide sequence.

Next, the amino acids sequence, frequencies, and net charge were retrieved from the Benchling

cloud .

Gene expression (Real time quantitative PCR)

In order to address issue of the best time to collect tissue for library construction, real time

quantitative PCR was used to measure the relative abundance of crotamine mRNA levels from

tissue taken prior to venom collection (n=3) and those taken 72 hours later (n=3). Reverse

transcriptase reactions were conducted on all six samples, followed by amplification of

crotamine cDNA using the following primers:

Figure 3. List of primers used for real-time PCR (reverse transcriptase reaction):

Forward primer – 5’ CCCAAAACCAGTCTCACCAT 3’

Reverse primer – 5’ TTTGCAGCATTTTCCTTTCC 3’

17

SYBR green intercalating dye was used as the fluorescent reporter for quantification. Abundance

of crotamine cDNA in each sample was cross referenced with a standard curve of amplification

for crotamine levels were compared using Student’s t-test

18

RESULTS

PRE/POST PCR

The adult timber rattlesnake was captured in northeast Alabama. It was 97 cm long and

weighed 800 g. It was kept in controlled conditions in the laboratory for 6 months. The venom

gland was extracted after 72 hours, 24 hours after milking, and at the time of milking. The

collection apparatus used for milking was assembled using rubber gloves, a glass funnel and a

50-ml tube. The venom was collected in the amount of 1.1 ml by making the snake bite into the

rubber gloves; then it was preserved at -70°C. Next, the snake was anesthetized and sacrificed

by decapitation, and 600 mg of venom glands were successfully extracted. These venom gland

tissues were kept in a 50-ml tube and homogenized in TRI Reagent® solution using tissue

blender frozen at -70°C.

Following TRI Reagent® protocol, extraction of total RNA from homogenized tissues was

carried out; agarose gel electrophoresis was then used to confirm its integrity. mRNA was

isolated from the total RNA using the mRNA isolation kit. Using the spectrometer, the

concentration of mRNA was found to be 866ng/µl. First-strand cDNA was synthesized from 2µl

of mRNA using the SMARTTM cDNA Library Construction Kit, and then it was amplified with LD

PCR and confirmed with agarose gel electrophoresis. The amplified cDNA was then purified and

subjected to DSN normalization. The optimal concentration of DSN enzyme for normalization

was observed to be 0,025U/µl. Normalization revealed that the optimal number of cycles for

PCR is 24. The normalized and purified cDNA was Sfil digested and packaged into a vector. Titer

19

of unamplified library was 1X10^6PFU, and after performing blue-white screening, the

percentage of recombinant clones was 100%. Calculation showed the amplified library titer was

1X10^8pfu.

Random selections from 500 plaques were screened, amplified, and analyzed using

agarose gel electrophoresis. 50 of them were selected and sequenced. These sequences were

analyzed using BLAST, and the closest similar match was identified for each one of them. A good

number of the sequences represented transcripts for ribosomal proteins. From the 50 selected

sequences, we further selected 4 sequences that stood out due to their integrity on the agarose

gel and their pharmacological benefits.

DATA ANALYSIS

From the BLAST, search, we confirmed the sequence IDs by finding the matching

sequence with high BLAST percentage and the lowest E-values, and the sequences identified

were VG 4 – Crotamine, VG 36 – C-Type Lectin, VG 141 – Phospholipase A2, and VG 172 – Serine

Protease (Table 4).

These four venom components were translated from nucleotide sequences to protein

sequences with ExPASy and from these 4 protein sequences, we selected VG 4 – Crotamine for

gene expression (Figures 3, 4, 5, 6). The biochemical properties, frequencies, and net charge of

the amino acid sequence for crotamine were identified with Benchling (Figure 7).

20

Table 4. BLAST search to identify transcripts of 4 selected sequences from normalized libraries

(BLAST)

cDNA Closest

match/sequence ID

Score Length Expect ratio Identities Gaps

VG 4 C. horridus crotamine

mRNA,

ID: JF895768.1

412 bits (456) 358 7e-111 279/313

(89%)

0/313(0%)

VG 36 C.horridus C-type lectin

2 mRNA,

ID: HQ414090.1

696 bits (771) 697 0.0 534/630

(85%)

6/630 (0%)

VG 141 C. horridus

phospholipase A2

mRNA,

ID: GQ168369.1

720 bits (798) 445 0.0 428/445

(96%)

3/445 (0%)

VG 172 C. horridus_SVSP-5

serine protease mRNA,

ID: MF974519.1

858 bits (951) 2539 0.0 822/1028

(80%)

35/1028(3%)

21

Figure 4. DNA sequences and translated protein sequences for crotamine (ExPASy).

This figure shows the DNA sequences for 4 component of the timber rattlesnake (C. horridus) venom and their corresponding translated protein sequences, showing the start and stop proteins, Poly(A) signals and Poly(A) tails for VG 4 – C. horridus_crotamine

cggccggaaaaatttggcccaaaaccagtctcacc ATG aaa atc ctt tat ctg M K I L Y L

ctg ttc gct ttt ctt ttc ctt gct ttc ctg tct gaa cca gga aat gcc tat aaa L F A F L F L A F L S E P G N A Y K cgg ggt ctt aaa aaa gga gga ccc tgt ttt ccc aaa acc gaa ata tgt ctt cct R G L K K G G P C F P K T E I C L P cca tct tct gat ttt gga aaa agg gac tgt caa tgg aaa gga aaa tgc tgc aaa P S S D F G K R D C Q W K G K C C K aag gaa agg gaa aat aat gcc ttc ccc atc TAG gaccagggatatcttcaaaatttggcca K E R E N N A F P I *

aggacctgaaagggccccctgctatccctgtatctttcttt aaataaatcaaattgctaccc Poly A Signal aaaaaaaaaaaaaaaaa

Poly A tail

Figure 5. DNA sequences and translated protein sequences for C-type lectin 2 (ExPASy).

This figure shows the DNA sequences for 4 component of the timber rattlesnake (C. horridus) venom and their corresponding translated protein sequences, showing the start and stop proteins, Poly(A) signals and Poly(A) tails for VG 36 – C. horridus_C-type lectin 2

cggccggaaagcaggggtttcaccgcaaaggggccgaggaaaaggaaaggaaaaaaaacc

atg ggg gga ttc ttc ttc ggg agt ttt ggt ttg ctg gcc ggg tcc ctc tcc ctg agg gaa

M G G F F F G S F G L L A G S L S L R E

ACT gca gct gat tgc ccc tcg ggt ggg tct tcc tat gaa ggg ctt tgc tac aac ccc ttc

T A A D C P S G G S S Y E G L C Y N P F

aat gac cca aaa acc ggg gat gat gca aaa aac ttc tgc cca caa cag cac aca ggg gga

N D P K T G D D A K N F C P Q Q H T G G

ctt ttg gtc tcc tcc cac agc act gaa aaa gca aat ttt ggg agc aag ctg gcc ttc caa

22

L L V S S H S T E K A N F G S K L A F Q

acc ttt ggc caa aga att ttc tgg ata gga ctg acc aat gtc tgg aat aaa tgc act tgg

T F G Q R I F W I G L T N V W N K C T W

aaa ggg acc aag ggg gcc ttg ctc aaa tac aaa aac ggg gtt gaa aaa tct tat tgg gtc

K G T K G A L L K Y K N G V E K S Y W V

ttt ttc aag tca cct aat aac aaa tgg agg agt aaa ccc tgc aaa atg ttg gca cat ttc

F F K S P N N K W R S K P C K M L A H F

gtc tgc gag ttc cgg gca TAA tttgaaaatgcggttgacctgaaaaaattctgcagaggcaaggaagcc

V C E F R A *

cccccccccagacccccccactttgctcaagggatgctctctggggcggaatcgggttctgcggctccggaggga

ccaaaaggtccaataaattctgcctaccttgaaaaaaaaaaaaaa

Poly A Signal Poly A tail

Figure 6. DNA sequences and translated protein sequences for phospholipase A2 (ExPASy).

This figure shows the DNA sequences for 4 component of the timber rattlesnake (C. horridus) venom and their corresponding translated protein sequences, showing the start and stop proteins, Poly(A) signals and Poly(A) tails for VG 141 – C. horridus_phospholipase A2

cggccggaaagcaggggtatcaacgcaaagtcattacggccgggggcagaggagcaaagggagcctgccaggtgtgaa tctttgccattttcccctgcctggtttctcctgatccttgcctacaaattatccttgacttacaaccgtttgtttagt gaccgttctaagggcctttttccaaacttcaccagcggaggcaattaacggggtctgcttattcccaagtctggattc gggagg

ATG agg act ctc tgg ata ggg gcc gta ttg ctg ctg ggc gtc aag ggg agc ctg ggg caa

M R T L W I G A V L L L G V K G S L G Q

ttt gaa atg atg atc atg gaa ggg gcg aaa aaa agc ggt ttg ctt tgg tac agc gct tac

F E M M I M E G A K K S G L L W Y S A Y

gga tgc tac tgc ggt tgg ggg ggc cat ggc cgg cca cag gac gcc act gac cgc tgc tgc

G C Y C G W G G H G R P Q D A T D R C C

ttt gtg cac aac tgc tgt tac gga aaa gcg acc gac tgc aac ccc aaa agg gtc agc tat

F V H N C C Y G K A T D C N P K R V S Y

acc tac agc gag gaa aac ggg gaa ttc gtc tgc gga ggg gac gac ccg tgc ggg aca cag

23

T Y S E E N G E F V C G G D D P C G T Q

att tgt gag tgc gac aag gcc gca cca atc tgc ttc cga aac aat ata ccc tca tac gac

I C E C D K A A P I C F R N N I P S Y D

aac aaa tat tgg ctg ttc ccg ccc aaa att gcc agg agg aac caa cca tgc TAA

N K Y W L F P P K I A R R N Q P C *

Gtctctgcaggccgggaaaaacccctcaaattacacaatcgtattgggttactctattattctgatgcatactg

aataataaacaggtgccacttttgcactcaaaaaaaaaaaaaaaaa

Poly A Signal Poly A tail

Figure 7. DNA sequences and translated protein sequences for serine protease (ExPASy).

This figure shows the DNA sequences for 4 component of the timber rattlesnake (C. horridus) venom and their corresponding translated protein sequences, showing the start and stop proteins, Poly(A) signals and Poly(A) tails for VG 172 – C. horridus_SVSP-5 serine protease.

tttcggaagggccccttggttttgttcccggggatccgcccttagggcggggggggaacttcgggattcattctgccag

tgtcccaaattgttggccccccacctgtttattttgatcaaataaagggctttggatcaaaaaacccccgcttggttta

tctaataaaactgatacggaatctcatttttaagttgggaacgggaatcttacaaacaaacggcttaccgggcaaagct

gaagtt

ATG ggg ctg atc aaa ggg cta cca aac ctt ctg ata cta cag ctt tct tac ccc caa

M G L I K G L P N L L I L Q L S Y P Q

aat tct tct gac ctg gcc ttt gga ggg gat aaa tgt aac ata aat gaa ctt cgt ccc ctt

N S S D L A F G G D K C N I N E L R P L

gcc ccc ggg tat atc act aaa ggt ttt ccc tgc cct gga act ttg atc aac aag aaa ggg

A P G Y I T K G F P C P G T L I N K K G

ggg ctg acc gct gcc ccc tgc aac ggg gaa aat ttt ccc ttt tta ctc ggg ggg ctt acc

G L T A A P C N G E N F P F L L G G L T

cta agg gaa cta att aag gat gtg caa aaa aaa gcc cca aag gaa aat ttc ttt tgt ccc

L R E L I K D V Q K K A P K E N F F C P

att agg aaa aaa att gat gaa aag gac agg gac ttc ttg ttt atc agg ttc aac agc cct

I R K K I D E K D R D F L F I R F N S P

gtt acc agc agt aca cac ttc gcg cct ctc agt tgg cct tcc aac cct ccc agg gtg ggc

V T S S T H F A P L S W P S N P P R V G

cca gtt tgc cgt att atg gaa ggg ggc gca atc cca ccc cca aag ggg act ttg ccc ggg

24

P V C R I M E G G A I P P P K G T L P G

gcc cct ctt ttg ggt tac ttt aac ata ctc aat tat aag ggg ggt caa cca gct tac gca

A P L L G Y F N I L N Y K G G Q P A Y A

agg ttg cca caa aca aga aaa aaa ttg ggg tgc agg atc ccg aaa aga gga aaa agt tct

R L P Q T R K K L G C R I P K R G K S S

tgt aag ggg gac tct ggg gga ccc ctc ctc TGA atg gaa aaa tcc agg gta ttg atc tgg

C K G D S G G P L L *

Ggggggaaattttggcccaaccgggagacctgtgcttacccagggctctatttcctgttggatccaaatttttggagga

atacaacgcactgcccccataaatttcgaaaactaaaagaaaaatacactcctctattcccaaccttccacttcttaaa

Poly A Signal Poly A tail

tttagcccgatttctcctacacaatattataaatgcgccc

Table 5. Biochemical properties for Crotamine, C-type lectin 2, Phospholipase A2, Serine protease (Benchling).

Position Molecular weight

Isoelectric point

pH range Net charge

Crotamine 1-70 AAs 7991.41 Da 9.40 4 – 10 11.27 – -4.57

C-type lectin 2 1-146 AAs 16232.43 Da 9.41 4 – 10 18.04 – -6.49

Phospholipase A2 1-137 AAs 15320.45 Da 7.92 4 – 10 12.37 – -18.89

Serine protease 1-1209 AAs 22645.34 Da 9.84 4 – 10 26.38 – -2.47

25

Figure 8 shows Real-time PCR amplification for C. horridus that resulted in the amplification of 198 bp amplicon. Levels were determined to be 18.5 fg/1 ug total RNA for venom glands collected at 0 hr post venom collection; Levels were determined to be 69.5 fg/1 ug total RNA for venom glands collected at 72 hr post venom collection. ( p= 0.12)

26

DISSCUSSION

Since the times of ancient Greece, substances have been extracted from snakes and

used as medicinal remedies. The discovery of snake venom proteins and enzymatic toxins has

had a large part to play in the technological development of proteomics and transcriptomics.

Snake venom, which is a highly modified form of saliva, is made of several proteins and peptides

that are pharmacologically active and specialized in various biological processes. Due to its

analgesic properties, snake venom components have been applied in research and medicine

(Stocker 1990). In this study, we were able to create a cDNA library, annotate our resulting

sequences, and identify 4 of several venom component proteins that can possibly be used as

therapeutic agents. They are: crotamine, phospholipase A2, C-type lectin and serine protease.

Crotamine

In our study, we confirmed crotamine in our cDNA library; its biochemical properties

include 1-70 AAs in position, a molecular weight of 7991.41Da, isoelectric Point (pI) 9.40, pH

range of 4 to 10, and net charge range of 11.27 to -4.57. Real-time PCR amplification for C.

horridus that resulted in the amplification of 198 bp amplicon. Levels were determined to be

18.5 fg/1 ug total RNA for venom glands collected at 0 hr post venom collection; Levels were

determined to be 69.5 fg/1 ug total RNA for venom glands collected at 72 hr post venom

collection. Data suggests that crotamine expression was elevated at 72 hours post venom

collection, however this was not significantly higher than at 0 hour ( p= 0.12). This small, basic

27

myotoxin was one of the identified protein components, and it was the first gene to ever be

mapped on a snake chromosome (Oguiura et al. 2005). It a cationic polypeptide with high

affinity for negatively charged excitable membranes (Santoro et al. 1999). It is made up of short

polypeptides about 42 residues long with 11 basic components (9 lysines, 6 cysteines, and 2

arginines) (Massey et al. 2012). There have been previous studies (Kerkis et al. 2004, Fusco et al.

2020) carried out on the crotamine extracted from the venom of the South American

rattlesnake C. durissus terrificus. Crotamine amino acid sequences were found to have high

similarities; they caused skeletal muscle contractions, which led to paralysis of the hind limbs of

mice through interaction with sodium channels on muscle cells (Oguiura et al. 2005). Nicastro et

al. (2003) used nuclear magnetic resonance (NMR) spectroscopy to show the structure of

crotamine. From their findings, the topology of crotamine was unlike anything previously seen in

active toxins that target ion channels. It comprised a short N-terminal alpha helix protein

formation and a small, antiparallel triple-stranded beta-sheet protein formation. Oguiura et al.

(2005) showed protein formations containing a ab1b2b3 topology arrangement; they found

identical disulfide bridges and structural fold conformations which are analogous to the human

beta-defensin family. Kerkis et al. (2004) found that crotamine specialized in proliferating

different types of cells from in vitro—such as murine embryonic stem cells, human primary

fibroblasts, lymphoblastic cells, and endothelial cells—to in vivo in cells that were isolated from

mice. Crotamine can be delivered to different cells because it forms a complex with plasmid

DNA. Its properties make it a cell-penetrating protein (CPP) comprising nuclear localization with

an ability to terminate active tumor cells (Hayashi et al. 2008). Crotamine being a CPP makes it a

28

suitable means for transporting macromolecules through the cell membranes. All these

biochemical properties make it an effective means of achieving therapeutic routes in

pharmacology and medicine.

C-Type Lectin (CLEC)

We confirmed C-Type lectin in our cDNA library; its biochemical properties include 1-146

AAs in position, molecular weight of 16232.43 Da, isoelectric point (pI) 9.41, pH range of 4 to 10,

and a charge range of 18.04 to -6.49. Popular types of CLEC are galactose or mannose, usually

with homo- or heterodimer structures (Viala et al. 2015). CLEC is a protein domain that binds to

carbohydrate; it requires calcium to perform this binding. Generally, proteins with CLEC possess

functions such as cell-to-cell adhesion, an immune response to pathogens, and apoptosis (Cambi

and Figdor 2009). In recent studies, CLEC from Bothrops moojeni showed anticoagulant and

procoagulant properties; they are affected by platelet aggregation, insulin secretion, and

antibacterial activity (Barbosa et al. 2010).

Phospholipase A2 (PLA2)

We confirmed phospholipase in our cDNA library; its biochemical properties include 1-

137 AAs in position, molecular weight of 15320.45 Da, isoelectric point (pI) 7.92, pH range of 4

to 10, and a charge range of 12.37 to -18.89. These proteins are ubiquitous in nature; they

require Ca2+ for activity, they possess 6 conserved disulfide bonds with 1 or 2 variable disulfide

bonds (Burke and Dennis 2009), and they play a digestive role in hydrolysis of the sn-2 ester

bond of phospholipids (Six and Dennis 2000). Myotoxicity, neurotoxicity, and edema-inducing

29

activities are the usual pharmacological effects of a snake bite (Fernandes et al. 2014). Some

PLA2s have been found to have apparent procoagulant activity (Armugam et al., 2004). Snake

populations with reasonable amounts of Type II venom tend to cause myonecrosis and spastic

hind-leg paralysis (Massy et al. 2012). Venom phospholipase A2 and mammalian phospholipase

A2 enzymes share similarities in both structure and catalytic function although venom PLA2s are

more toxic and have a wider range of pharmacological effects. PLA2 enzymes can bind to an

array of proteins and target various types of tissues and organs.

Serine Protease

We confirmed serine protease in our cDNA library; its biochemical properties include 1-

1209 AAs in position, molecular weight of 22645.34 Da, isoelectric point (pI) 9.84, pH range of 4

to 10, and a charge range of 26.38 to -2.47. Serine proteases get their name because they have

essential serine residues present at their active sites; and they are sometimes referred to as

serine endopeptidases, they serve as nucleophilic amino acids at enzyme active sites and

function to split peptide bonds in proteins (Hedstrom 2002). Based on their structure, they are

divided in 2 categories: trypsin-like/chymotrypsin-like or subtilisin-like (Madala et al. 2010).

Trypsin-like serine proteases have specificity that is driven by negatively charged aspartic acid or

glutamic acid; they cut peptide bonds at amino acids that are positively charged such as lysine or

arginine (Evnin et al. 1990). Chymotrypsin-like proteases are more hydrophobic than trypsin-like

proteases. Subtilisin-like serine protease is found in prokaryotes, and they are known to produce

nucleophilic serine. For serine proteases, coagulation factor levels are necessary for the

diagnosis of hemorrhagic or thrombotic problems. They play major roles in extracellular

30

proteolysis. Due to their long half-life, they stay in the human system for a clinically useful

length of time.

The 4 identified components of the full-length-enriched cDNA of timber rattlesnake (C.

horridus) venom have all shown application possibilities in pharmacology and medical sciences.

λTriplEx2 vectors were very instrumental in this study in producing these 4 components using

cell tissue culture. The various data bases—BLAST Blenching, Clustal Omega, ExPASy, and

RCSB—were very instrumental in retrieving the amino acid sequences and PDB files, getting

multiple sequence alignments, and also identifying their biochemical properties.

31

CONCLUSION

We studied the components of timber rattlesnake (C. horridus) venom using modern

bioinformatics methods such as pre- and post-PCR techniques to further study and identify

snake venom components and determine their functions. Combining transcriptomics and

proteomics, we were able to identify the 4 components from our cDNA library; crotamine,

phospholipase A2, C-type lectin, and serine protease. λTriplEx2 vectors were very instrumental

in this study in producing these 4 components using cell tissue culture. We were able to

annotate the selected sequences with the venom-associated genes determining their

biochemical properties, coding regions, exons, introns, and functions. Gene expression levels for

the select genes were confirmed.

Crotamine functions to inhibit pain, and this means it can be a very effective analgesic. It

can be delivered to different cells because it forms a complex with plasmid DNA. Crotamine,

being a CPP makes it a suitable means for transporting macromolecules through the cell

membranes. Gene expression levels for crotamine was confirmed but showed no significant

different at between 0hr and 72hr (at p=.12). Proteins with C-type lectin possess functions such

as cell-to-cell adhesion, an immune response to pathogens, and apoptosis and have also shown

anticoagulant and procoagulant properties. PLA2 enzymes can bind to an array of proteins and

target various types of tissues and organs, and they tend to cause myonecrosis and spastic hind-

leg paralysis. Serine proteases play important roles in the diagnosis of hemorrhagic or

thrombotic problems, and they stay in the human system for a clinically useful length of time.

32

The 4 identified components of the full-length-enriched cDNA of timber rattlesnake (C.

horridus) venom and their biochemical properties show promising applications in the fields of

pharmacology and medical sciences.

33

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APPENDICIES

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APPENDIX A

Tables and Figures

Figure 9. Nucleotide sequences for selected venom components

a) VG 4 – Crotamine

CGGCCGGAAAAATTTGGCCCAAAACCAGTCTCACCATGAAAATCCTTTATCTGCTGTTCGCTTTTCTTTTCCTTGCTTTCCTGTCTGAACCAGGAAATGCCTATAAACGGGGTCTTAAAAAAGGAGGACCCTGTTTTCCCAAAACCGAAATATGTCTTCCTCCATCTTCTGATTTTGGAAAAAGGGACTGTCAATGGAAAGGAAAATGCTGCAAAAAGGAAAGGGAAAATAATGCCTTCCCCATCTAGGACCAGGGATATCTTCAAAATTTGGCCAAGGACCTGAAAGGGCCCCCTGCTATCCCTGTATCTTTCTTTAAAAAAAAAAAAAAAAA

b) VG 36 – C-type lectin)

CGGCCGGAAAGCAGGGGTTTCACCGCAAAGGGGCCGAGGAAAAGGAAAGGAAAAAAAACCATGGGGGGATTCTTCTTCGGGAGTTTTGGTTTGCTGGCCGGGTCCCTCTCCCTGAGGGAAACTGCAGCTGATTGCCCCTCGGGTGGGTCTTCCTATGAAGGGCTTTGCTACAACCCCTTCAATGACCCAAAAACCGGGGATGATGCAAAAAACTTCTGCCCACAACAGCACACAGGGGGACTTTTGGTCTCCTCCCACAGCACTGAAAAAGCAAATTTTGGGAGCAAGCTGGCCTTCCAAACCTTTGGCCAAAGAATTTTCTGGATAGGACTGACCAATGTCTGGAATAAATGCACTTGGAAAGGGACCAAGGGGGCCTTGCTCAAATACAAAAACGGGGTTGAAAAATCTTATTGGGTCTTTTTCAAGTCACCTAATAACAAATGGAGGAGTAAACCCTGCAAAATGTTGGCACATTTCGTCTGCGAGTTCCGGGCATAATTTGAAAATGCGGTTGACCTGAAAAAATTCTGCAGAGGCAAGGAAGCCCCCCCCCCCAGACCCCCCCACTTTGCTCAAGGGATGCTCTCTGGGGCGGAATCGGGTTCTGCGGCTCCGGAGGGACCAAAAGGTCCAATAAATTCTGCCTACCTTGAAAAAAAAAAAAAAAA

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c) VG 141 – Phospholipase A2

CGGCCGGAAAGCAGGGGTATCAACGCAAAGTCATTACGGCCGGGGGCAGAGGAGCAAAGGGAGCCTGCCAGGTGTGAATCTTTGCCATTTTCCCCTGCCTGGTTTCTCCTGATCCTTGCCTACAAATTATCCTTGACTTACAACCGTTTGTTTAGTGACCGTTCTAAGGGCCTTTTTCCAAACTTCACCAGCGGAGGCAATTAACGGGGTCTGCTTATTCCCAAGTCTGGATTCGGGAGGATGAGGACTCTCTGGATAGGGGCCGTATTGCTGCTGGGCGTCAAGGGGAGCCTGGGGCAATTTGAAATGATGATCATGGAAGGGGCGAAAAAAAGCGGTTTGCTTTGGTACAGCGCTTACGGATGCTACTGCGGTTGGGGGGGCCATGGCCGGCCACAGGACGCCACTGACCGCTGCTGCTTTGTGCACAACTGCTGTTACGGAAAAGCGACCGACTGCAACCCCAAAAGGGTCAGCTATACCTACAGCGAGGAAAACGGGGAATTCGTCTGCGGAGGGGACGACCCGTGCGGGACACAGATTTGTGAGTGCGACAAGGCCGCACCAATCTGCTTCCGAAACAATATACCCTCATACGACAACAAATATTGGCTGTTCCCGCCCAAAATTGCCAGGAGGAACCAACCATGCTAAGTCTCTGCAGGCCGGGAAAAACCCCTCAAATTACACAATCGTATTGGGTTACTCTATTATTCTGATGCATACTGAATAATAAACAGGTGCCACTTTTGCACTCAAAAAAAAAAAAAAAAAA

d) VG 172 – Serine protease

TTTCGGAAGGGCCCCTTGGTTTTGTTCCCGGGGATCCGCCCTTAGGGCGGGGGGGGAACTTCGGGATTCATTCTGCCAGTGTCCCAAATTGTTGGCCCCCCACCTGTTTATTTTGATCAAATAAAGGGCTGTTGGATCAAAAAACCCCCGCTTGGTTTATCTAATAAAACTGATACGGAATCTCATTTTTAAGTTGGGAACGGGAATCTTACAAACAAACGGCTTACCGGGCAAAGCTGAAGTTATGGGGCTGATCAAAGGGCTACCAAACCTTCTGATACTACAGCTTTCTTACCCCCAAAATTCTTCTGACCTGGCCTTTGGAGGGGATAAATGTAACATAAATGAACTTCGTCCCCTTGCCCCCGGGTATATCACTAAAGGTTTTCCCTGCCCTGGAACTTTGATCAACAAGAAAGGGGGGCTGACCGCTGCCCCCTGCAACGGGGAAAATTTTCCCTTTTTACTCGGGGGGCTTACCCTAAGGGAACTAATTAAGGATGTGCAAAAAAAAGCCCCAAAGGAAAATTTCTTTTGTCCCATTAGGAAAAAAATTGATGAAAAGGACAGGGACTTCTTGTTTATCAGGTTCAACAGCCCTGTTACCAGCAGTACACACTTCGCGCCTCTCAGTTGGCCTTCCAACCCTCCCAGGGTGGGCCCAGTTTGCCGTATTATGGAAGGGGGCGCAATCCCACCCCCAAAGGGGACTTTGCCCGGGGCCCCTCTTTTGGGTTACTTTAACATACTCAATTATAAGGGGGGTCAACCAGCTTACGCAAGGTTGCCACAAACAAGAAAAAAATTGGGGTGCAGGATCCCGAAAAGAGGAAAAAGTTCTTGTAAGGGGGACTCTGGGGGACCCCTCCTCTGAATGGAAAAATCCAGGGTATTGATCTGGGGGGGGAAATTTTGGCCCAACCGGGAGACCTGTGCTTACCCAGGGCTCTATTTCCTGTTGGATCCAAATTTTTGGAGGAATACAACGCACTGCCCCCATAAATTTCGAAAACTAAAAGAAAAATACACTCCTCTATTCCCAACCTTCCACTTCTTAAATTTAGCCCGATTTCTCCTACACAATATTATAAATGCGCCC

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Figure 10. Amino acid sequences for selected venom components

a) VG 4 – Crotamine

MKILYLLFAFLFLAFLSEPGNAYKRGLKKGGPCFPKTEICLPPSSDFGKRDCQWKGKCCKKERENNAFPI

b) VG 36 – C-type lectin (CLEC)

MGGFFFGSFGLLAGSLSLRETAADCPSGGSSYEGLCYNPFNDPKTGDDAKNFCPQQHTGGLLVSSHSTEKANFGSKLAFQTFGQRIFWIGLTNVWNKCTWKGTKGALLKYKNGVEKSYWVFFKSPNNKWRSKPCKMLAHFVCEFRA

c) VG 141 – Phospholipase A2 (PLA2)

MRTLWIGAVLLLGVKGSLGQFEMMIMEGAKKSGLLWYSAYGCYCGWGGHGRPQDATDRCCFVHNCCYGKATDCNPKRVSYTYSEENGEFVCGGDDPCGTQICECDKAAPICFRNNIPSYDNKYWLFPPKIARRNQPC

d) VG 172 – Serine protease

MGLIKGLPNLLILQLSYPQNSSDLAFGGDKCNINELRPLAPGYITKGFPCPGTLINKKGGLTAAPCNGENFPFLLGGLTLRELIKDVQKKAPKENFFCPIRKKIDEKDRDFLFIRFNSPVTSSTHFAPLSWPSNPPRVGPVCRIMEGGAIPPPKGTLPGAPLLGYFNILNYKGGQPAYARLPQTRKKLGCRIPKRGKSSCKGDSGGPLL

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APPENDIX B

THESIS OPTION FORM

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APPENDIX C

PROSPECTUS FOR THESIS FORM