Abstract
Structure of Cupiennius salei venom hyaluronidaseHyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40–60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis.Function of venom hyaluronidasesBesides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.
Highlights
Hyaluronidases (Hyals) (EC 3.2.1.35) are ubiquitously distributed enzymes found in many organisms ranging from bacteria to humans
dermatan sulfate (DS) is composed of repeating iduronic acid (IdoA)-GalNAc units, whereas IdoA is mainly sulfated at carbon atom 2 and GalNAc at carbon atom 4
Araneus diadematus, Argiope bruennichi, Atypus piceus, Callobius claustrarius, Larinioides sclopetarius, Pisaura mirabilis, Meta menardi and Tegenaria atrica were collected in Switzerland, Filistata insidiatrix in Italy, Sericopelma rubronitens in Panama, Geolycosa vultuosa in Hungary, Nephila pilipes, and Oxyopes sp. in Taiwan, Eusparassus dufouri, Hogna radiata, Lycosa hispanica in Spain, Araneus angulatus, Eresus walckenaerius, Lycosa praegrandis, Uroctea durandi in Greece, Drassodes lapidosus in Austria, and Macrothele calpeiana in Portugal
Summary
Hyaluronidases (Hyals) (EC 3.2.1.35) are ubiquitously distributed enzymes found in many organisms ranging from bacteria to humans. Eukaryotic enzymes are hydrolases that hydrolyze cellular and extracellular matrix (ECM) polysaccharides from the glycosaminoglycan (GAG) family such as hyaluronan (HA), non-sulfated chondroitin (Ch), and chondroitin sulfate (CS), heparan sulfate (HS) and dermatan sulfate (DS). HA forms very long polymeric structures, which can reach molecular masses up to 6–8 MDa. Chemically, HA consists of repeating glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) units [2], whereas Ch is composed of repeating disaccharides of GlcA and N-acetylgalactosamine (GalNAc). DS is composed of repeating iduronic acid (IdoA)-GalNAc units, whereas IdoA is mainly sulfated at carbon atom 2 and GalNAc at carbon atom 4. HS is a complex polysaccharide consisting of repeating disaccharides of GlcA or IdoA with glucosamines, which can be sulfated at various positions.[3]
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