Int J of Com Bio and Bioinfo

TITLE

Author 11*, Author 21 and Author3 1

1Affiliations, India

A b s t r a c t

It has been estimated that autoimmune diseases are the top leading causes of death among women in all age groups beyond 65 years. It is the third most common ailment in the developing countries after cancer and heart disease. According to WHO (2014) reports, 1% of world population and 3% of Indian population suffer from Rheumatoid Arthritis (RA). Rheumatoid Arthritis is a type of autoimmune disease characterized by chronic inflammation and fibrosis around joints, leading to permanent joint destruction, deformity and functional disability. The current study explores the causal agents in the activation cascade of the complement system. Under natural conditions CD59 prevents C9 from polymerizing and forming Membrane Attack Complex (MAC). However, under autoimmune conditions, the availability of CD59 is limited, and the concentration of C8 and C9 are higher. Hence, in order to balance, the MAC concentrations, snake venom neurotoxins which resemble the structure (three finger protein) of CD59 could be administered to supplement the availability of CD59. Thus, an attempt has been made to study the molecular level of interactions of these Snake Venom Neurotoxins (natural and modified forms) via docking studies with the terminal Complement Factors C8 and C9. The results indicate that modified neurotoxins bind specifically and irreversibly to the complement factors better, thereby suggesting their efficacious roles as specific therapeutic agents.

Int J of Com Bio and Bioinfo

*Corresponding Author / Keywords / License / Article Info
Name
Affiliation
University
Email: / Rheumatoid Arthritis,
venom neurotoxin, CD59,
C8
C9 / / Received:XXXXX
Revised:XXXX
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Online: XXXX

Int J of Com Bio and Bioinfo

INTRODUCTION

Autoimmune disease is a condition where our self-immune cells recognize host cells as pathogenic, and destroys them. There are around 80 different illnesses associated with autoimmune diseases, which are restricted to a particular organ or a particular tissue. Few of the commonly known autoimmune diseases are Amyotrophic Lateral Sclerosis, Ankylosing Spondylitis, Diabetes Mellitus Type 1, Multiple Sclerosis, Narcolepsy, Juvenile Arthritis, Psoriasis, Rheumatoid Arthritis, Eczema and various others [1-3].

Rheumatoid Arthritis (RA) is a type of autoimmune disease characterized by chronic inflammation and fibrosis around joints, leading to permanent joint destruction, deformity and functional disability [4,6]. It has been estimated that autoimmune diseases are the top leading causes of death among women in all age groups up to 65 years. It is the third most common ailment in the developing countries after cancer and heart disease (NIH2002), and the estimates suggest that it affects approximately 8% of the population. According to WHO 2014 report about 3% of Indian population suffers from RA.

Currently available drugs to treat complement mediated immune system disorders like corticosteroids (prednisone) and nonsteroid drugs such as azathioprine, cyclophosphamide, mycophenolate, sirolimus or tacrolimus are often prescribed to control or reduce the immune system's response (as immunosuppressive agents). Monoclonal antibodies based treatments are expensive and their prolonged usages have proven side effects. Hence there is an immediate need for alternative efficacious and selective therapy.

Complement cascade pathway plays a vital role in autoimmune diseases. Terminal Complement components C8 and C9 help in the formation of Membrane Attack Complex (MAC), which destroys self-cells and lead to Auto Immune disorders. Naturally, CD59 a membrane bound glycoprotein made up of 77 amino acids and having a molecular weight of 18 to 25 kDa, neutralizes the effect of MAC. Because of this property CD59 is also called as MAC inhibitory Protein. CD59 and Three Fingered Proteins like Neurotoxins from many cobra species resemble in their 3D structure. There are hypothesis that these Neurotoxins from reptiles have developed into CD59 during evolution in chordates [5]. Based on this background information, the present work intends to explore the plausible peptide modification efforts towards exploiting these neurotoxins as drugs for effective treatment of RA, thus making the neurotoxin not to interact with its natural target (Acetylcholine Receptor), rather bind to the terminal Complement factors 8 and/or 9. CD59 is insoluble in nature hence we plan to utilize neurotoxin as therapeutic agent since neurotoxin readily gets dissolved in blood [6].

Neurotoxins are the chemical or natural or synthetic agents which disrupt the transmission of signals between Neurons i.e., presynaptic or postsynaptic. Present work is concentrated on the α-neurotoxins from the Indian cobra belonging to Naja genera which are categorized to be postsynaptic neurotoxin, which occurs on the receiving end of a discharge across the synapse. These neurotoxins mimic the shape of the acetylcholine molecule and fit into the acetylcholine receptors, and thus block the flow of acetylcholine, resulting in numbness and paralysis. Neurotoxin residues that contact the acetylcholine receptor lies on the concave face of the toxin where as in CD59 these residues lie in the analogous face [7].

Visachikitsha, the division of Ayurveda deals with the use of venoms to cure diseases through traditional techniques known as Suchikavoron (venom at the tip of a needle) and Shodhono (detoxification of venom) [8]. The venom is a poisonous fluid that some animals like spiders and snakes secrete and introduce into the body of their victims by biting or stinging or spitting mechanisms. The number of venom components in snake, scorpion or cone snail ranges from 50-200 toxin substances.

Arthritis was induced in rats by injecting Complete Freund’s adjuvant (CFA) [9]. The administration of Cobratoxin (CTX) (17.0 μg/kg, ip) significantly reduced paw swelling, mechanical and thermal hyperalgesia. The flow Cytometry analysis has revealed that there is increased expression in the level of CD59 [10].

Since there is growing number of identifying snake venom neurotoxin sequences, it is very difficult to study them by experimentation only. The detailed Bioinformatics analysis offers a convenient methodology for studying the possible function of toxins and their efficacies of binding.

MATERIAL AND METHODS

The protocol followed in the study is illustrated as a flowchart in Fig. 1. Neurotoxins known to have affinities with the terminal Complement Factors were selected (refer Table 1 and Table 2). As detailed in Tables 3& 4, the RMSD between the variants of neurotoxins for C-atom and all atoms indicates that the conformations of the variants are similar.

Figure 1: Work Flow Chart followed in this project

To determine the modes of molecular interactions, docking studies were carried out using online tool ClusPro[11][12][13][14]. The details of interactions are provided in Table 5. The interactions were analyzed around 5Å distances from the target atoms.

Table 1: Details of Neurotoxins used for docking studies where C9 structure was not available in the PDB server so we did obtain the designed structure from ModBase, which was built on template 4EOS_B.

Sl No. / Neurotoxin
(ligand) / PDB ID / Length / Molecular Weight
(Daltons)
1 / Long Neurotoxin 1 (LN1) / 2CTX / 71 / 7847
2 / Weak Neurotoxin 5 (WN5) / 1LN7 / 62 / 6943
3 / Weak Neurotoxin 6 (WN6) / 1LN9 / 65 / 7568
4 / Weak Neurotoxin 7 (WN7) / 1LQ3 / 65 / 7637
5 / Weak Neurotoxin 8 (WN8) / 1LMG / 65 / 7581

Table 2: Details of different receptor targets for Rheumatoid Arthritis and Natural Targets of Neurotoxin

Sl. No / Name of Receptor / PDB ID / Length
(Residues) / Molecular Weight
(in Daltons)
1 / CD59 / 1CDQ / 77 / 14177
2 / C8 / 2QQH / 334 / 65163
3 / C9 / Theoretical Model / 559 / 63173
4 / Acetyl Choline Receptor / 4D01 / 218 / 25997

Table 3: Superimposition studies among Neurotoxins and RMSD Values for C alpha atoms in Angstroms (number of atoms involved are mentioned in brackets)

1LN7-WN5 / 1LN9- N6 / 1LQ3- WN7 / 1LMG- WN8
2CTX- LN1 / 1.17
(49) / 1.25 (59) / 1.31
(49) / 1.29
(51)
1LN7- WN5 / - / 1.09 (61) / 1.21
(59) / 1.08
(57)
1LN9-WN6 / - / - / 1.03
(64) / 1.13
(63)
1LQ3-WN7 / - / - / - / 1.13
(56)

Table 4: Superimposition studies among Neurotoxins and RMSD Values for all atoms in Angstroms (number of atoms involved are mentioned in brackets)

1LN7- WN5 / 1LN9- WN6 / 1LQ3- WN7 / 1LMG- WN8
2CTX- LN1 / 1.28
(196) / 1.27
(196) / 1.26
(192) / 1.32 (204)
1LN7- WN5 / - / 1.1
(248) / 1.24
(236) / 1.11 (228)
1LN9- WN6 / - / - / 1.08
(256) / 1.18 (268)
1LQ3- WN7 / - / - / - / 1.16 (228)

Table 5: Sum total of Hydrogen bonding and Hydrophobic interactions of Acetylcholine Receptor, CD59, C8 and C9, respectively, with the all neurotoxins (NT) under study.

Number of Interactions within 5Å / 2CTX- Long NT / 1LN7- Weak NT5 / 1LN9- Weak NT6 / 1LQ3- Weak NT7 / 1LMG- Weak NT8
Acetylcholine Receptor / 12+8=20 / 2+5=7 / 1+5=6 / 1+6=7 / 2+6=8
CD59 / 8+3=11 / 5+3=8 / 8+4=12 / 12+5=17 / 9+6=15
C8 / 9+5=14 / 10+6=16 / 13+9=22 / 19+13=32 / 16+11=27
C9 / 14+13=27 / 13+7=20 / 10+7=17 / 22+15=37 / 19+14=33

RESULTS AND DISCUSSIONS

It is known that CD59 is structurally similar to the Snake Neurotoxins, and hence docking studies have revealed that small changes in the sequence of neurotoxin can be suitably made to develop the same as a therapeutic agent for the Rheumatoid Arthritis. As observed from table 5, the maximum interaction of Long Neurotoxin with Acetylcholine Receptor was observed when compared to the Weak Neurotoxins and Acetylcholine Receptor. Interestingly, the interaction of Neurotoxins with CD59 is comparatively lower. However, the interaction of C8 with WN6, WN7 and WN8 is higher compared to WN5 and LN. Similarly, C9 interacts relatively stronger with LN, WN5, WN7 and WN8 respectively.

Figure 2: Multiple sequence alignment between neurotoxins (where P29181: Weak Neurotoxin 7, P29182: Weak Neurotoxin 8, P29180: Weak Neurotoxin 6, P29179: Weak Neurotoxin 5 and P25668: Long Neurotoxin 1), depicting the amount of similarity and identity among these neurotoxins. Almost 30% of the sequences were identical and 20% of the additional residues were found to be similar among other sequences.

Figure 3: Multiple sequence alignment among neurotoxins and CD59 where P13987 is CD59 sequence

It was interesting to observe from the sequence alignment (Figs. 2 & 3) and network of interactions with Neurotoxins and Acetylcholine Receptor atoms respectively (Fig. 4) that, residues like Proline 7, Cysteine 17, Glycine 20, Cysteine 24, Arginine 39, and Arginine 41 are well conserved across the Neurotoxins. Hence in order to study their importance in binding to respective Receptors, as a case study, we looked at mutating (deleting) the Proline 7 in the sequence of the neurotoxins and intended to investigate the changes in the binding patterns, if any. So the coordinates of Proline7 was deliberately removed from the PDB structure file of all the neurotoxins and the set of 5 neurotoxins were again docked with acetylcholine receptor, CD59, complement factor 8 and complement factor 9, respectively.

Table 6: Interaction table depicting sum total of Hydrogen bonding and Hydrophobic for modified neurotoxin with respective receptors

Number of Interactions within 5Å / Modified 2CTX –
LN / Modified 1LN7 – WN5 / Modified 1LN9 – WN6 / Modified 1LQ3 –WN7 / Modified 1LMG – WN8
Acetylcholine Receptor / 8+4=12 / 7+4=11 / 9+7=16 / 18+12=30 / 11+8=19
CD59 / 9+7=16 / 8+6=14 / 11+9=20 / 12+10=22 / 10+9=19
C8 / 8+4=12 / 11+6=15 / 12+7=19 / 17+7=24 / 14+9=23
C9 / 17+9=26 / 16+11=27 / 17+9=26 / 15+9=24 / 15+12=27

Figure 4: Ligplot for Neurotoxins with Acetyl Choline Receptor depicting Proline7 being the common amino acid in all the interactions.

As can be appreciated from Table 6 the interactions with Modified Neurotoxins (devoid of Proline 7) and respective targets have drastically changed. Interestingly, it can be observed that, the interaction with C9 has tremendously increased with Weak Neurotoxin 7. Thus, this effort has enabled us to explore the overall effect of preventing the binding of Neurotoxins to its Natural Receptor (Acetylcholine Receptor), and enable its specific binding to complement the systems C8 and C9, respectively, towards orienting their utilities for RA. Consequently, as a hypothesis, we propose that modification in selective residues of Neurotoxins could be programmed to achieve specific binding to the complement systems of interest, for enabling a plausible solution for the treatment of Autoimmune Diseases. With these clues from the computational exercises, the course of future work would be to validate this insilico hypothesis via wet lab exercises.

CONCLUSIONS

The binding interaction studies as described via docking exercisesclearly reveal that the modified neurotoxin from snake Naja exhibits potential binding properties with Complement Factor 8 and/or 9, thus Neurotoxins could be designed as a plausible therapeutic peptide to tackle Rheumatoid Arthritis.

CONFLICT OF INTERESTS

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the generous support and facilities extended by Sir M. Visvesvarya Institute of Technology and Sri Krishnadevaraya Educational Trust, Bangalore, towards this project. I would like to thank Ms. Shrutila and Mr. Jagadeesh Kumar for their support and constant help during the course of this project.

REFERENCES

1.Khan MA, Khan MK (1982). "Diagnostic Value of HLA-B27 Testing in Ankylosing Spondylitis and Reiter's Syndrome". Annals of Internal Medicine 96 (1): 70–76; author reply 76. doi:10.7326/0003-4819-96-1-70.PMID 7053711.

2.Autoimmune Diseases at the US National Library of Medicine Medical Subject Headings (MeSH)