The calcium-dependent protease of Loxosceles gaucho venom acts preferentially upon red cell band 3 transmembrane protein

Abstract

Eighty micrograms red blood cell (RBC) ghosts from patients who had previously exhibited the cutaneous form of loxoscelism (presenting localized dermonecrosis) and the viscerocutaneous form of loxoscelism (presenting dermonecrosis, hemoglobinuria, hematuria, and jaundice) and from controls were incubated with 2.5 µg crude Loxosceles gaucho venom in 5 mM phosphate buffer, pH 7.4, at 37ºC. Among all membrane proteins, quantitative proteolysis of the important integral transmembrane protein 3 increased with venom dose and with incubation time from 30 to 120 min, as demonstrated by gel densitometry. Similar quantitative data were obtained for RBC ghosts from patients and from control subjects, a fact that argues against the possibility of genetic factors favoring the hemolytic viscerocutaneous form. These data suggest that the clinical forms may be different types of the same disease, with the viscerocutaneous form being the result of large amounts of intravascularly injected venom and the superficial form being the result of in situ venom action. Since protein 3 is a housekeeping integral membrane protein, whose genetic deficiency leads to hemolytic anemia, it is reasonable to relate it to the hemolysis which occurs in the viscerocutaneous form of loxoscelism. The venom protease responsible for the process was not inhibited after 120-min incubation by 0.2 mM paramethylsulfonyl fluoride or by 0.2 mM Nethylmaleimide but was inhibited by 25 mM ethylenediaminetetraacetic acid (a calcium-chelating agent) in 5 mM phosphate buffer at pH 7.4, which suggests that the enzyme is a calcium-dependent metalloprotease.

Brown spider venom toxins: what are the functions of astacins, serine proteases, hyaluronidases, allergens, TCTP, serpins and knottins?

“…The presence of metalloproteases in the venoms of brown spiders were also demonstrated by other authors, who showed metalloprotease-dependent proteolytic activities in L. intermedia venom on the substrates entactin and on heparan-sulfate proteoglycan protein core synthesized by endothelial cells [ 33 , 34 ], in addition to proteolytic activity on fibrinogen [ 35 ]. In addition, it was found that a metalloprotease of L. gaucho venom acts upon red cell band 3 transmembrane protein [ 36 ]. Then, two important descriptions were made: the first showing the biological conservation of these metalloproteases in venoms of different species of Loxosceles spiders, such as L. rufescens [ 37 ], L. gaucho, L. deserta and L. laeta [ 38 ], indicating the importance of metalloproteases in the biology of these animals.…”