title: Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.
abstract: Hyaluronidases 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.
  Besides 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Olivier
authors.initials: O
authors.lastname: Biner
authors.affiliation: Functional Genomics Center Zürich, University of Zürich/ETH Zürich, Zürich, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Trachsel
authors.affiliation: Institute of Ecology and Evolution, University of Bern, Bern, Switzerland.
authors.forename: Aline
authors.initials: A
authors.lastname: Moser
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Lukas
authors.initials: L
authors.lastname: Kopp
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Langenegger
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Urs
authors.initials: U
authors.lastname: Kämpfer
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: von Ballmoos
authors.affiliation: Institute of Ecology and Evolution, University of Bern, Bern, Switzerland.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Nentwig
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schürch
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Johann
authors.initials: J
authors.lastname: Schaller
authors.affiliation: Institute of Ecology and Evolution, University of Bern, Bern, Switzerland.
authors.forename: Lucia
authors.initials: L
authors.lastname: Kuhn-Nentwig
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006031:Glycosylation; D006821:Hyaluronoglucosaminidase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013111:Spider Venoms / Q000201:enzymology*; D013112:Spiders; D053719:Tandem Mass Spectrometry
chemical_list: D011994:Recombinant Proteins; D013111:Spider Venoms; D006821:Hyaluronoglucosaminidase