PROTEIN Writuparna Dutta Debpali Sur Presidency University Kolkata Abstract Spiders are predaceous organisms that utilize their venom to overpower their prey Spider venoms are dominated by disulfiderich peptides ID: 786502
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PRESENT SCENARIO OF THERAPEUTIC APPLICATIONS OF SPIDER VENOMPROTEIN
Writuparna
Dutta
,
Debpali
Sur
;
Presidency
University, Kolkata
Slide2AbstractSpiders are predaceous organisms that utilize their venom to
overpower their prey. Spider venoms are dominated by disulfide-rich peptides
having high affinity and
specificity
for particular subtypes of ion channels
and receptors. Spider venoms act as a valuable resource for drug discovery in
case of chronic pains without any ill effects. Here we review the structure and
pharmacology of different spider-venom peptides that are being used for the
development of therapeutics against a wide range of
pathophysiological
conditions including chronic pain, potential neurotoxins and their function on
collagen
fibres.
Keywords
: spider venom; peptide; therapeutics; drug discovery
Slide3Are you Miss Muffet ?
“Little
Miss
Muffet
sat on a tuffetEating her curds and whey,Along came a spider,Who sat down beside herAnd frightened Miss Muffet away”
Despite of the importance of spiders, they are largely neglected mainly due to ignorance, fear, and subsequent disliking
Slide4About 20 fresh venom glands taken in 1 ml ringer saline will be subjected to homogenization followed by centrifugation at 11000xg for 10 minutes. Discarding supernatant pellet is to be collected, that seems to contain the crude venom. Biochemical assay will be done on crude venom to identify its nature.
Following standard protocols PAGE and SDS-PAGE will be done simultaneously for preliminary identification of the entire polypeptides and proteins on the basis of their molecular weight (which are predicted to be present)
Venom isolation protocol
Slide5Obtained bands will be stained coomasie brilliant blue, silver nitrate, and Sudan black to find out any presence of glycoprotein or lipoproteins simultaneously. Gel filtration chromatography will be done to purify the polypeptides according to their sizes. Purified fragments will be subjected to test their LD50 and LC50 on
murine
model.
Ion-exchange chromatography will lead to the purification of proteins according to their charges. Purified fragments carrying different charges are now subjected to test their LD50 and LC50. Differentiated proteins will be treated with mild reducing agents to break, if any disulfide linkage present within it. N-terminal amino acid sequence of differentiated protein fragments will be identified by Sanger’s treatment. .
Slide6Other chromatographic techniques will be applied to differentiate the mixture of obtained protein fragments. When ‘n’ numbers of protein fragments will be isolated, their synergetic toxicity is to be measured on murine model. By Edman
degradation method amino acid sequences of the entire protein can be identified
Slide7Peptides are poor candidates for human therapeutics owing to their susceptibility to
proteolytic
degradation and limited penetration through intestinal mucosa.
Presence of inhibitor
cysteine knot in most of the spider toxin provides these peptides extraordinary stability.The cysteine knot comprises a ring, formed by 2 disulfide and theintervening sections of polypeptide backbone with a third disulfide piercing thering to create a pseudo knot. The compact hydrophobic core of the ICK motifconsists of primarily two central disulfide bridges that emanate from the two β-strands that characterize the
ICK.IMPORTANCE OF ICK MOTIFS IN SPIDER VENOM
Slide8Snaps taken during field work
Funnel Web Spider (
Macrothele
sp
)Poecilotheria sp
Slide9Nephila sp.
WOOD SPIDER (from BUXA TIGER RESERVE)
Slide10RECLUSE SPIDER
(
Loxosceles
sp.)
Medically important spider
Slide11Analgesic Potentials of spider toxins
Common chronic pain drugs have critical role in
pathophysiology
of pain but have different side effects.
Spider venoms act as modulators of ASICS, voltage gated sodium channels and P2X3 receptors which are involved in acute pain and chronic neuropathic pain. Venom peptide isolated from Green velvet tarantula has potential to block human NaV 1.7 channels with an IC50 of 0.3 nM. PT1 venom isolated from Gelycosa
sp act as modulator of P2X3
Slide12Black widow: A boon in disguise…BASIC BODY PLAN
BLACK WIDOW SPIDER
Slide13Potent toxins from different serotypes of Clostridium botulinum
Slide14Effect of Botulinum neurotoxin
Slide15Application of Latrotoxin (LTX) against Botulinum Neurotoxin
LTX
Slide16Spider Diversity in India
Spider Diversity in India
Slide17Gasteracantha sp.
Slide18Signature Spider Designing its Web
Slide19Argiope sp.
Slide20Slide21Present trends in researches on spider venom in India
Recently
Partitagin
, a hemorrhagic
metalloprotease isolated from Hippasa partita, Indian funnel web spider which was the first report on isolation and characterization of spider venom from Indian subcontinent.It showed specificity of action on the components of ECM [ Extra cellular Matrix ] and degraded collagen type IV and
fibronectin but not on collagen type-I.Hippasa agelenoides spider venom glands extract which revealed the presence of Hag-protease II.
Hag-protease II hydrolyzed casein, fibrinogen and fibrin, however it did not hydrolyze gelatin,
fibronectin
and collagen types I and IV.
Slide22Acknowledgement
We express our gratitude to
Dr.Nirmal
Kr.
Sarkar
,
Dr.
Tushar
K.
Mukherjee
,
Dr.
Rina
Rani
Ray and
Mr.
Souryadeep
Mukherjee
PG Department of Zoology and Molecular Biology,
Presidency University, Kolkata.
Slide23THANK YOU…