Molecular and biological characterization of the Streptococcal ADP-ribosyltransferase SpyA and ADP-ribosylation of the intermediate filament vimentin

ADP-ribosyltransferases represent an important class of enzymes. These enzymes can be endogenous regulators or toxins, which affect a myriad of cellular processes by the covalent transfer of ADP-ribose, donated from NAD+, onto a substrate protein. SpyA, the first ADP-ribosyltransferase (ADPRT) identified in the human pathogen Streptococcus pyogenes, was found to modify the cytoskeletal protein actin and the intermediate filament vimentin. While ADPRTs have low sequence homology, they share conserved structural motifs, found by mutational analysis to be important to SpyA ADP-ribosylation and general NAD-glycohydrolase activities. Actin is a well-characterized substrate of ADP-ribosylation. Actin-specific bacterial ADPRT toxins universally target a single site on actin, arginine-177. SpyA has a unique ADPRT activity as it targets a novel site of modification of actin, arginine-30. Quantitative comparison of the activity of SpyA on vimentin and actin found vimentin was the preferred substrate for SpyA (k cat, 58.5±3.4 min−1) relative to actin (k cat, 10.1±0.6 min−1) and vimentin was modified at a rate 9.48±1.95 fold faster than actin. Using the novel mass spectrometry method of ADP-ribose marker ion identification, the primary sites of ADP-ribosylation of vimentin were found to be arginine-44, -49 and -63 in the N-terminal regulatory head domain, with several additional secondary sites identified. Because the primary sites are located in the head domain of vimentin, a region responsible for regulation of polymerization via phosphorylation, we investigated the effects of SpyA on vimentin filamentation. Modification of vimentin in vitro was shown to cause a direct defect in polymerization. Additionally, we demonstrated that intoxication of HeLa cells with SpyA resulted in collapse of the vimentin cytoskeleton. We conclude that SpyA modification of vimentin in an important regulatory region has significant functional effects on vimentin assembly. SpyA lacks a traditional binding domain, which mediates the ability of many ADPRTs to translocate across the eukaryotic cell membrane. Though it is currently unknown how SpyA gains entry to the host cell, we present evidence that SpyA associates with the cellular membrane. This interaction appears to be stable when subjected to high ionic conditions but susceptible to treatment with extracellular trypsin suggesting SpyA may be imbedded in the phospholipid bilayer