Notexin

{{Infobox nonhuman protein |Name=Notexin |image=Notexin.png |width= |caption=Schematic diagram of the three-dimensional structure of notexin({{PDB|1AE7}}). |Organism=Notechis scutatus |TaxID=8663 |Symbol=N/A |CAS_number=37223-96-4 |PDB=1AE7 |UniProt=P00608 }} '''Notexin''' is a toxin produced by the tiger snake (''Notechis scutatus''). It is a myotoxic and presynaptic, neurotoxic phospholipase A2 (PLA₂s).<ref name="Test_first">{{Cite book| publisher = Elsevier Health Sciences| isbn = 978-0-7020-7606-0| vauthors = Bersten AD, Handy J | title = Oh's Intensive Care Manual E-Book| date = 2018-08-15}}</ref> These are enzymes that hydrolyze the bond between a fatty acid tail and glycerol in fatty acids on the 2-position.<ref>{{Cite book| publisher = Academic Press| isbn = 978-0-323-90528-2| pages = 137–174| vauthors = Koltai T, Reshkin SJ, Baltazar F, Fliegel L | veditors = Koltai T, Reshkin SJ, Baltazar F, Fliegel L | title = Prostate Cancer Metabolism| chapter = Chapter 5 - Lipid metabolism part II: sphingolipids and ceramides| access-date = 2022-03-15| date = 2021-01-01 | chapter-url = https://www.sciencedirect.com/science/article/pii/B9780323905282000126 | doi = 10.1016/B978-0-323-90528-2.00012-6 | s2cid = 237968682}}</ref>

== History == The name notexin comes from the fact that this toxin was first found to be the major component in the venom of the tiger snake.<ref name="history1">{{Cite journal| doi = 10.1016/0041-0101(72)90066-9| issn = 0041-0101| volume = 10| issue = 4| pages = 405–413| vauthors = Karlsson E, Eaker D, Rydén L | title = Purification of a presynaptic neurotoxin from the venom of the Australian tiger snake Notechis scutatus scutatus| journal = Toxicon| access-date = 2022-03-14| date = 1972-06-01| pmid = 5070579| bibcode = 1972Txcn...10..405K| url = https://dx.doi.org/10.1016/0041-0101%2872%2990066-9| url-access = subscription}}</ref> The name notexin is thus a combination of the genus name ''Notechis'' and the word toxin. The tiger snake was first described by Wilhelm Peters in 1861.<ref>{{cite journal | vauthors = Peters WK |title=Eine zweite Übersicht (vergl. Monatsberichte 1859 p. 269) der von Hrn. F. Jagor auf Malacca, Java, Borneo und den Philippinen gesammelten und dem Kgl. zoologischen Museum übersandten Schlangen. |journal=Monatsberichte der Königlichen Preussischen Akademie der Wissenschaften zu Berlin |date=1861 |pages=683–691}}</ref> The toxin was first purified more than a hundred years later in 1972 by Karlsson ''et al''.<ref name="history1"/><ref name="history2">{{cite journal | vauthors = Tu AT | title = Neurotoxins of animal venoms: snakes | journal = Annual Review of Biochemistry | volume = 42 | issue = 1 | pages = 235–258 | date = 1973 | pmid = 4581225 | doi = 10.1146/annurev.bi.42.070173.001315 }}</ref> This prompted more research into notexin.<ref name="history2"/><ref>{{cite journal | vauthors = Harris JB, Karlsson E, Thesleff S | title = Effects of an isolated toxin from Australian tiger snake (Notechis scutatus scutatus) venom at the mammalian neuromuscular junction | journal = British Journal of Pharmacology | volume = 47 | issue = 1 | pages = 141–146 | date = January 1973 | pmid = 4352085 | doi = 10.1111/j.1476-5381.1973.tb08168.x | pmc = 1776509 }}</ref>

== Structure == Notexin consists of a single molecule. This molecule is a single-peptide chain of 119 amino acid residues that are cross-linked with 7 disulfide-bridges.<ref>{{Cite web | work = RCSB Protein Data Bank | title = RCSB PDB - 1AE7: NOTEXIN, A PRESYNAPTIC NEUROTOXIC PHOSPHOLIPASE A2| access-date = 2022-03-17| url = https://www.rcsb.org/structure/1AE7}}</ref>

X-ray diffraction has been used to determine the crystal structure of notexin and led to the conclusion that notexin belongs to either the P3121 or P3221 space group with lattice parameters a = b = 74.6 Å, c = 49.0 Å with a β of 120⁰. This data was found with a resolution of 2.0 Å and had an R-factor of 16.5%.<ref name="structure1">{{cite journal | vauthors = Westerlund B, Nordlund P, Uhlin U, Eaker D, Eklund H | title = The three-dimensional structure of notexin, a presynaptic neurotoxic phospholipase A2 at 2.0 A resolution | journal = FEBS Letters | volume = 301 | issue = 2 | pages = 159–164 | date = April 1992 | pmid = 1568473 | doi = 10.1016/0014-5793(92)81238-h | bibcode = 1992FEBSL.301..159W | s2cid = 2302749 | doi-access = free }}</ref> For protein data, this R-factor is usually 20%,<ref>{{Cite book| publisher = Elsevier| isbn = 978-0-08-045044-5| pages = 531–550| veditors = Taylor JB, Triggle DJ | vauthors = Laskowski RA, Swaminathan GJ | title = Comprehensive Medicinal Chemistry II| chapter = 3.24 - Problems of Protein Three-Dimensional Structures| location = Oxford| access-date = 2022-03-15| date = 2007-01-01| chapter-url = https://www.sciencedirect.com/science/article/pii/B008045044X000973 | doi = 10.1016/B0-08-045044-X/00097-3 }}</ref> indicating that the crystal structure of notexin is relatively well defined.

The supramolecular structure of notexin is very similar to that of other PLA₂s. Both notexin and many PLA₂s contain four characteristic main helices (the αA, αB, αC and αE helices) and a short carboxyl end helix in their secondary structure. Also the active site seems to be similar enough to that of other PLA₂s in order to use their model building studies when discussing enzymatic properties. Notexin does deviate significantly from other PLA₂s due to different main chain lengths and its conformation in the 69th amino acid residue.<ref name="structure1" />

The active site of notexin contains His-48. This residue is in close contact with the carboxylate oxygens of an Asp-99 residue, which is also present in notexin.<ref name="structure1"/> For most PLA₂s the wall of the active site is covered with hydrophobic residues. When a lone pair on the oxygen of water attacks the ester, the His-48 residue facilitates a proton transfer and the substrates's carbonyl oxygen is possibly fixated and stabilized by the positively charged NH-groups on the PLA₂s.<ref>{{cite journal | vauthors = Dijkstra BW, Drenth J, Kalk KH | title = Active site and catalytic mechanism of phospholipase A2 | journal = Nature | volume = 289 | issue = 5798 | pages = 604–606 | date = February 1981 | pmid = 7464926 | doi = 10.1038/289604a0 | bibcode = 1981Natur.289..604D | s2cid = 4308155 | url = https://pure.rug.nl/ws/files/14867412/1981NatureDijkstra.pdf }}</ref>

== Mechanism of action == Notexin is generally lethal if it enters the bloodstream in rats. This lethal effect is the result of a presynaptic blockade of transmission across neuromuscular junctions of the breathing muscles, causing asphyxiation. It has also been shown to have myotoxic effects upon intravenous injection.<ref name="structure1"/> Myotoxic effects generally entail muscle necrosis.<ref>{{cite journal | vauthors = Gutiérrez JM, Cerdas L | title = [Mechanism of action of myotoxins isolated from snake venoms] | journal = Revista de Biología Tropical | volume = 32 | issue = 2 | pages = 213–222 | date = November 1984 | pmid = 6400184 }}</ref>

It was proposed (Dixon et al., 1996)<ref name="mechanism1">{{cite journal | vauthors = Dixon RW, Harris JB | title = Myotoxic activity of the toxic phospholipase, notexin, from the venom of the Australian tiger snake | journal = Journal of Neuropathology and Experimental Neurology | volume = 55 | issue = 12 | pages = 1230–1237 | date = December 1996 | pmid = 8957446 | doi = 10.1097/00005072-199612000-00006 | s2cid = 9299375 | doi-access = free }}</ref> that this myotoxicity of notexin is the result of notexin binding to the sarcolemma, causing hypercontraction and thereby muscle necrosis as a result of the membrane between places of hypercontraction rupturing.<ref name="mechanism1"/> The presynaptic activity is, however, much more potent, at least in mice.<ref name="structure1"/>

Notexin causes an indirect reduction or complete end of the release of acetylcholine in the affected nerve terminals. This acetylcholine normally causes an action potential and thereby muscle contraction. It was found that this reduction of acetylcholine release was caused by an impaired recycling of synaptic vesicles as a reduction in the content of synaptic vesicles and abnormally large vesicles were observed in the affected tissues. This was followed by shrinking of the nerve terminals and the amount of vesicles in these terminals decreasing.<ref name="structure1"/>

The exact way of interaction with the cell is unknown, but it is suggested that notexin, like other PLA₂s, interacts with high-affinity specific protein receptors or low-affinity lipid domains of muscle cells and motor neurons. Interaction of notexin with the plasma membrane results in the hydrolysis of the phospholipids in the cell membrane.<ref name="mechanism2">{{cite journal | vauthors = Montecucco C, Gutiérrez JM, Lomonte B | title = Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action | journal = Cellular and Molecular Life Sciences | volume = 65 | issue = 18 | pages = 2897–2912 | date = September 2008 | pmid = 18563294 | doi = 10.1007/s00018-008-8113-3 | s2cid = 36666240 | pmc = 11131735 }}</ref> A study <ref>{{cite journal | vauthors = Kao PH, Lin SR, Chang LS | title = Phospholipase A2 activity-independent membrane-damaging effect of notexin | journal = Toxicon | volume = 50 | issue = 7 | pages = 952–959 | date = December 2007 | pmid = 17889218 | doi = 10.1016/j.toxicon.2007.07.011 }}</ref> showed that without the PLA₂ activity, notexin also has membrane damaging effects, suggesting that notexin has multiple mechanisms to damage the cell membrane.

Cell membranes become permeable for ions and cause an influx of Ca²⁺ from the extracellular medium. In muscle cells the influx of Ca²⁺ causes hypercontraction of myofilaments, which can cause mechanical damage to the plasma membrane.<ref name="mechanism1"/><ref name="mechanism2"/> The mitochondria will take up Ca²⁺, eventually leading to a reduced mitochondrial functionality. The high Ca²⁺ concentration in the cytosol activates Ca²⁺-dependent proteinases, calpains,<ref>{{cite journal | vauthors = Bartoli M, Bourg N, Stockholm D, Raynaud F, Delevacque A, Han Y, Borel P, Seddik K, Armande N, Richard I | display-authors = 6 | title = A mouse model for monitoring calpain activity under physiological and pathological conditions | journal = The Journal of Biological Chemistry | volume = 281 | issue = 51 | pages = 39672–39680 | date = December 2006 | pmid = 17056592 | doi = 10.1074/jbc.M608803200 | doi-access = free }}</ref> and the endogenous Ca²⁺-dependent phospholipase A₂. The calpains degrade the cytoskeletal components of the cell and Ca²⁺-dependent phospholipase A₂ hydrolyses the cell membrane, which leads to further cell degradation and bigger influx of Ca²⁺. At a certain point the damage is irreversible and necrosis of the cell occurs.<ref name="mechanism1"/><ref name="mechanism2"/>

In neurons the influx of calcium causes the release of the ready-to-release synaptic vesicles and the reserve pool of synaptic vesicles.<ref name="mechanism2"/><ref name="mechanism3">{{cite journal | vauthors = Harris JB, Grubb BD, Maltin CA, Dixon R | title = The neurotoxicity of the venom phospholipases A(2), notexin and taipoxin | journal = Experimental Neurology | volume = 161 | issue = 2 | pages = 517–526 | date = February 2000 | pmid = 10686073 | doi = 10.1006/exnr.1999.7275 | s2cid = 6714210 }}</ref> Research <ref name="mechanism3"/> showed that neurons, after treatment with notexin, had strongly reduced numbers of synaptic vesicles. These results seem to indicate that notexin inhibits the endocytosis of new synaptic vesicles, besides the exocytosis as a result of the Ca2+ influx.<ref name="mechanism2"/><ref name="mechanism3"/><ref>{{cite journal | vauthors = Rigoni M, Schiavo G, Weston AE, Caccin P, Allegrini F, Pennuto M, Valtorta F, Montecucco C, Rossetto O | display-authors = 6 | title = Snake presynaptic neurotoxins with phospholipase A2 activity induce punctate swellings of neurites and exocytosis of synaptic vesicles | journal = Journal of Cell Science | volume = 117 | issue = Pt 16 | pages = 3561–3570 | date = July 2004 | pmid = 15226375 | doi = 10.1242/jcs.01218 | s2cid = 931111 }}</ref> Like in muscle cells, the Ca2+ influx in neurons also leads to reduced mitochondrial functionality and the activation of calpains and endogenous Ca²⁺-dependent PLA₂s. This leads to the same structural damage as in muscle cells.<ref name="mechanism2"/>

Experimental research also showed that notexinhad nephrotoxic effects on mice. A study<ref>{{cite journal | vauthors = Zimmerman SE, Yong LC | title = Nephrotoxicity of notexin in experimental mice | journal = Experimental and Toxicologic Pathology | volume = 47 | issue = 2–3 | pages = 149–155 | date = May 1995 | pmid = 7580101 | doi = 10.1016/S0940-2993(11)80305-2 | bibcode = 1995EToxP..47..149Z }}</ref> showed that depending on the dose, there was renal tubular and glomerular damage within 24 hours.

== Immune response == Not much about the metabolism of notexin is known. However, studies have shown that the toxin can be made ineffective by specific antibodies.<ref name="immune1">{{cite journal | vauthors = Mollier P, Chwetzoff S, Frachon P, Ménez A | title = Immunological properties of notexin, a potent presynaptic and myotoxic component from venom of the Australian tiger snake Notechis scutatus scutatus | journal = FEBS Letters | volume = 250 | issue = 2 | pages = 479–482 | date = July 1989 | pmid = 2753144 | doi = 10.1016/0014-5793(89)80780-X | bibcode = 1989FEBSL.250..479M | s2cid = 41178189 | doi-access = free }}</ref><ref name="immune2">{{cite journal | vauthors = Middlebrook JL | title = Cross-neutralizations of phospholipase A2 neurotoxins from snake venoms | journal = Toxicon | volume = 29 | issue = 12 | pages = 1481–1487 | date = 1991-01-01 | pmid = 1801325 | doi = 10.1016/0041-0101(91)90004-B }}</ref><ref name="immune3">{{cite journal | vauthors = Yang CC, Chang LS, Wu FS | title = Venom constituents of Notechis scutatus scutatus (Australian tiger snake) from differing geographic regions | journal = Toxicon | volume = 29 | issue = 11 | pages = 1337–1344 | date = 1991-01-01 | pmid = 1814009 | doi = 10.1016/0041-0101(91)90120-G | bibcode = 1991Txcn...29.1337Y }}</ref> In a certain study, mice become resistant to notexin, similar isoforms of the toxin and other venoms from the same origin. This was done by exposing the mice to the non-detoxified notexin. It was found that the C-terminal part of the notexin peptide chain is the binding site for these antibodies and thus it is known that this is the site for an antigenic domain.<ref name="immune1"/> Another study showed that at least some of the antigens that are able to block the effects of notexin do this by cross-neutralizations.<ref name="immune2"/> Certain antibodies have also been shown to have different affinities for different notexin isoforms. These different isoforms occur in snakes that have different geographical locations.<ref name="immune3"/> It is thus not the case that notexin antibodies necessarily bind to all notexin isoforms with the same affinity.

== Symptoms == Notexin causes pain at the site of the bite followed by excessive salivation, weakness, drowsiness, difficulty breathing, decreased blood pressure and paralysis of lips, larynx, tongue and facial muscles. Possibly, blurring of vision, ptosis, headaches and convulsions may also occur.<ref>{{Cite journal| doi = 10.1002/bjs.1800671233| issn = 1365-2168| volume = 67| issue = 12| pages = 900| vauthors = Snook R | title = Handbook of poisoning. R. H. Dreisbach. Tenth edition. 176 × 108 mm. Pp. 578. Illustrated. 1980. Los Altos, Ca: Lange. $9.00| journal = BJS (British Journal of Surgery) | date = 1980 }}</ref><ref name="symptoms">{{Cite web| title = T3DB: Notexin| access-date = 2022-03-17| url = http://www.t3db.ca/toxins/T3D2638}}</ref>

== Toxicity == There has been no research on the reaction of notexin in humans, however it is known that upon injection with the toxin, muscle damage and myoglobinuria will follow.<ref name="mechanism1"/> Data has shown that the tiger snake is one of the major causes of snake bites in Australia leading to being the second most common cause of death from snakebites.<ref>{{Cite book | vauthors = Walter FG, Chase PB, Fernández MC, McNally J | publisher = W.B. Saunders| isbn = 978-0-7216-0693-4| pages = 399–432| veditors = Shannon MW, Borron SW, Burns MJ | title = Haddad and Winchester's Clinical Management of Poisoning and Drug Overdose | edition = Fourth | chapter = Chapter 21 - Venomous Snakes| location = Philadelphia| access-date = 2022-03-15| date = 2007-01-01| chapter-url = https://www.sciencedirect.com/science/article/pii/B9780721606934500268 | doi = 10.1016/B978-0-7216-0693-4.50026-8 }}</ref>

Several results on the toxic effects of notexin in rodents have been reported.<ref name="symptoms"/><ref name="toxicity1">{{Cite book| publisher = Elsevier| isbn = 978-0-323-71317-7| pages = 446–491| veditors = Bertorini TE, Oh SJ | title = Neuromuscular Disorders | edition = Second | vauthors = Oh SJ | chapter = 19 - Treatment and Management of Disorders of the Neuromuscular Junction| location = St. Louis (MO)| access-date = 2022-03-17| date = 2022-01-01| chapter-url = https://www.sciencedirect.com/science/article/pii/B9780323713177000196 | doi = 10.1016/B978-0-323-71317-7.00019-6 }}</ref> When injecting 1 to 2 μg of pure toxin into a rat soleus muscle, it will destroy all of the muscle fibers.<ref name="toxicity1"/> In mice the LD₅₀ is 0.214&nbsp;mg/kg when applied subcutaneously and 0.04&nbsp;mg/kg when applied intravenously.<ref name="symptoms"/><ref name="toxicity1"/> There have also been researches into functional and morphological properties of regrowing mouse extensor digitorum longus (EDL) muscles after a notexin injection. Three days after injection there was complete fiber breakdown and loss of functional capacity. After ten days the muscles were made up entirely of regrowing fibers.<ref>{{cite journal | vauthors = Plant DR, Colarossi FE, Lynch GS | title = Notexin causes greater myotoxic damage and slower functional repair in mouse skeletal muscles than bupivacaine | journal = Muscle & Nerve | volume = 34 | issue = 5 | pages = 577–585 | date = November 2006 | pmid = 16881061 | doi = 10.1002/mus.20616 | s2cid = 25556704 }}</ref>

== Treatment == There is no notexin antivenom available on the market. There are two general tiger snake antivenoms available that potentially could work.<ref>{{Cite web | work = Healthdirect Australia | title = tiger snake antivenom| format = text/html| access-date = 2022-03-15| url = https://www.healthdirect.gov.au/medicines/medicinal-product/aht,20357/tiger-snake-antivenom}}</ref> There are no studies found on the effectiveness of the tiger snake antivenoms on notexin.

== References == {{reflist}}

{{Neurotoxins}} {{Toxins}}

Category:Neurotoxins Category:Snake toxins Category:Acetylcholine release inhibitors