Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei

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.

Biochemical and structural characterization of a protein complex containing a hyaluronidase and a CRISP-like protein isolated from the venom of the spider Acanthoscurria natalensis

“…showed maximum activity between 30 and 60 °C. Hyaluronidases from C. salei and V. dubius also showed activity at the same temperature range, but their maximum activity occurred at 50 °C e 35-40 °C, respectively [7,39].…”

“…AnHyal showed specificity to the hyaluronic acid substrate, like the one produced by Brachypelma vagans [55] and H. partita [38]. Conversely, hyaluronidase from L. intermedia [44], V. dubius [39], and several other species of spiders [7], were also able to degrade chondroitin sulfate. Hyaluronic acid, heparan and chondroitin sulfate are the main components of the extracellular matrix of vertebrates [56] and invertebrates [57], respectively.…”

“…[36,37]. Hyaluronidases from the spiders Hippasa partita, Vitalius dubius and Cupiennius salei [7,38,39] have already been purified, but the purification process involved several chromatographic steps.…”

“…However, its primary structure was described only for few species, through the venom gland RNA-seq or partially by Edman degradation [2]. Recently, these strategies have been associated with mass spectrometry, enabling the complete sequencing of a hyaluronidase from Cupenius salei venom [7].…”

Electrophysiological studies of Nephila clavata venom and toxin influence on hippocampal neuronal membranes after pretreatment with Aspergillus oryzae proteases

“…It is known that Arthropods' venoms contain a mixture of various biologically active substances including toxins that are their physiologically important components [6,22,35]. These toxins are used successfully in the contemporary electrophysiology [4,6,7,9,15,26,[27][28][29][30][31][32][33][34][35].…”