CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8␣ and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8␣, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.The cholesterol-dependent cytolysins (CDCs) 4 are a family of pore-forming toxins produced by a diverse group of Grampositive pathogens. Recently crystal structures of the membrane attack complex/perforin proteins complement C8␣, a C9-like protein from Photorhabdus luminescens, and mouse perforin (1-4) suggested that they are structurally and mechanistically related the CDCs (5-7) and may be ancient ancestors (8). Interestingly, this relationship between the CDCs and membrane attack complex/perforin proteins extends to other features of the complement system. Two members of the CDC family, intermedilysin (ILY) secreted by Streptococcus intermedius and vaginolysin (VLY) secreted by Gardnerella vaginalis, specifically bind to the human form of CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-anchored terminal inhibitor of the mammalian complement membrane attack complex (MAC) (9), rather than cholesterol (10, 11). These CDCs bind to hCD59 to initiate the assembly of their oligomeric pore complex on the membrane of human cells (10), whereas the main function of CD59 is to block the assembly of the MAC pore on host cells, thereby protecting them from the lytic effects of activated complement MAC. Domain 4 of the CDCs mediates its interaction with cholesterol-rich membranes (12)(13)(14). Most CDCs use cholesterol as their receptor and specifically recognize its 3-␤-hydroxy group. Recently Farrand et al. (15) defined the cholesterol recognition motif as a threonine/leucine pair located in loop 1 that is conserved in all members of the CDC family, including ILY and VLY. However, ILY initiates its interaction with the cell by binding to hCD59 (10) rather than chole...

Gamma-glutamyl compounds include antioxidants, inflammatory molecules, drug metabolites and neuroactive compounds. Two cell surface enzymes have been identified that metabolize gamma-glutamyl compounds, gamma-glutamyl transpeptidase (GGT1) and gamma-glutamyl leukotrienase (GGT5). There is controversy in the literature regarding the substrate specificity of these enzymes. To address this issue, we have developed a method for comprehensive kinetics analysis of compounds as substrates for GGT enzymes. Our assay is sensitive, quantitative and is conducted at physiologic pH. We evaluated a series of gamma-glutamyl compounds as substrates for human GGT1 and human GGT5. The Kms for reduced glutathione were 11μM for both GGT1 and GGT5. However, the Km for oxidized glutathione was 9μM for GGT1 and 43μM for GGT5. Our data show that the Kms for leukotriene C4 are equivalent for GGT1 and GGT5 at 10.8μM and 10.2μM, respectively. This assay was also used to evaluate serine-borate, a well-known inhibitor of GGT1, which was 8-fold more potent in inhibiting GGT1 than inhibiting GGT5. These data provide essential information regarding the target enzymes for developing treatments for inflammatory diseases such as asthma and cardiovascular disease in humans. This assay is invaluable for studies of oxidative stress, drug metabolism and other pathways that involve gamma-glutamyl compounds.

Background: Human ␥-glutamyltranspeptidase 1 (hGGT1) is a key enzyme in cysteine metabolism and several diseases. Results: We obtained the high resolution crystal structure of hGGT1. Conclusion:The structure reveals the molecular basis for differences between the human and bacterial enzymes in autoprocessing and catalytic activity. Significance: The structure provides a template for the structure-based design of therapeutic inhibitors of hGGT1.

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␥-Glutamyl transpeptidase (GGT) is a heterodimeric membrane enzyme that catalyzes the cleavage of extracellular glutathione and other ␥-glutamyl-containing compounds. GGT is synthesized as a single polypeptide (propeptide) that undergoes autocatalytic cleavage, which results in the formation of the large and small subunits that compose the mature enzyme. GGT is extensively N-glycosylated, yet the functional consequences of this modification are unclear. We investigated the effect of N-glycosylation on the kinetic behavior, stability, and functional maturation of GGT. Using site-directed mutagenesis, we confirmed that all seven N-glycosylation sites on human GGT are modified by N-glycans. Comparative enzyme kinetic analyses revealed that single substitutions are functionally tolerated, although the N95Q mutation resulted in a marked decrease in the cleavage efficiency of the propeptide. However, each of the single site mutants exhibited decreased thermal stability relative to wild-type GGT. Combined mutagenesis of all N-glycosylation sites resulted in the accumulation of the inactive propeptide form of the enzyme. Use of N-glycosylation inhibitors demonstrated that binding of the core N-glycans, not their subsequent processing, is the critical glycosylation event governing the autocleavage of GGT. Although N-glycosylation is necessary for maturation of the propeptide, enzymatic deglycosylation of the mature wild-type GGT does not substantially impact either the kinetic behavior or thermal stability of the fully processed human enzyme. These findings are the first to establish that co-translational N-glycosylation of human GGT is required for the proper folding and subsequent cleavage of the nascent propeptide, although retention of these N-glycans is not necessary for maintaining either the function or structural stability of the mature enzyme.