Venom characterization and structural insights into phospholipase D isoforms from the spider Loxosceles aff. variegata.

Brown spider envenomation (loxoscelism) represents a significant public health concern in South America, yet most studies focus on a few medically recognized species. Here, we provide the first molecular and functional characterization of the venom gland extract from Loxosceles aff. variegata (LafvVGE), a brown spider collected in synanthropic habitats in Ituiutaba, Minas Gerais, Brazil. SDS-PAGE and immunoblot analyses revealed prominent protein bands consistent with phospholipase D (PLD) toxins, the main agents of loxoscelism symptoms. ELISA assays demonstrated that LafvVGE is effectively recognized by Brazilian therapeutic antivenoms, indicating immunological cross-reactivity. Enzymatic assays confirmed sphingomyelinase and collagenase/gelatinase activities comparable to those of Loxosceles gaucho (a species of acknowledged medical relevance), although LafvVGE from female individuals showed higher activities than male derived pools under our experimental conditions. Neutralization assays showed complete inhibition of sphingomyelinase activity but only partial inhibition of gelatinase activity by the anti-loxoscelic antivenom, highlighting differential susceptibility of venom components to antivenom-mediated neutralization under in vitro conditions. Molecular analysis of venom gland transcripts identified eight distinct PLD isoforms (LafvPLD1-8), all containing conserved catalytic and metal-binding residues characteristic of class II Loxosceles PLDs. Structural modeling revealed isoform-specific variations in the aromatic cage motif and electrostatic surface, suggesting potential effects on membrane interactions and substrate specificity. Collectively, these findings place L. aff. variegata within the biochemical and structural spectrum of medically relevant Loxosceles species, expanding comparative knowledge of PLD diversity and function. While clinical relevance remains to be established in vivo, this study underscores the value of integrating biochemical, immunological, and structural analyses to identify emerging venom phenotypes with potential implications for surveillance and antivenom coverage.