Conformational switches and redox properties of the colon cancer‐associated human lectin ZG16

Abstract

Zymogen granule membrane protein 16 (ZG16) is produced in organs that secrete large quantities of enzymes and other proteins into the digestive tract. ZG16 binds microbial pathogens, and lower ZG16 expression levels correlate with colorectal cancer, but the physiological function of the protein is poorly understood. One prominent attribute of ZG16 is its ability to bind glycans, but other aspects of the protein may also contribute to activity. An intriguing feature of ZG16 is a CXXC motif at the carboxy terminus. Here, we describe crystal structures and biochemical studies showing that the CXXC motif is on a flexible tail, where it contributes little to structure or stability but is available to engage in redox reactions. Specifically, we demonstrate that the ZG16 cysteine thiols can be oxidized to a disulfide by quiescin sulfhydryl oxidase 1, which is a sulfhydryl oxidase present together with ZG16 in the Golgi apparatus and in mucus, as well as by protein disulfide isomerase. ZG16 crystal structures also draw attention to a nonproline cis peptide bond that can isomerize within the protein and to the mobility of glycine-rich loops in the glycan-binding site. An understanding of the properties of the ZG16 CXXC motif and the discovery of internal conformational switches extend existing knowledge relating to the glycan-binding activity of the protein.

Flagella at the Host-Microbe Interface: Key Functions Intersect With Redundant Responses

“…Similar to the effects of removing LYPD8, the absence of ZG16 also leads to bacteria localizing closer to the epithelium ( 92 ). However, in contrast to both LYPD8 and HD6, which appear to bind to protein ligands, ZG16 i ) contains a CXXC motif on its flexible carboxy terminus that may serve a redox switch to alter its protein conformation ( 93 ), and ii ) is a lectin that specifically binds to the peptidoglycan of Gram-positive bacteria ( 92 ). After binding, ZG16 aggregates bacteria, thereby limiting their motility ( 92 ).…”

Disulfide bond formation and redox regulation in the Golgi apparatus

“…The identity of the substrate proteins on which QSOX1 acts has been a long-standing question. QSOX1 oxidizes a range of reduced proteins in vitro [49,50], including some that are localized to cell types and environments expressing high levels of QSOX1 [51]. Nevertheless, the conclusion that any given protein is a physiological substrate of QSOX1 requires a demonstration that cysteines that are normally disulfide bonded in the protein remain reduced, or the function of the protein in cells or in organisms is compromised, in the absence of QSOX1 activity.…”

“…As described below, no evidence was found that QSOX1 introduces disulfide bonds in vivo into the major glycoproteins that make up mucus. In vitro , QSOX1 oxidizes an intestinal mucus‐associated lectin protein, ZG16 [51], but it is not yet known whether ZG16 is a physiological substrate of QSOX1 in vivo or how the ZG16 disulfide might contribute to mucus properties [59]. In contrast, QSOX1 was found to introduce disulfides into certain Golgi glycosyltransferases in the intestinal epithelium, with clear physiological consequences [21].…”

Reliable energy-based antibody humanization and stabilization

“…QSOX1 activity on Zymogen granule membrane protein 16 (ZG16) for initial antibody screening: ZG16 contains two cysteines in a flexible, accessible CXXC motif that can be oxidized in vitro by QSOX1 49 . Prior to the assay, ZG16 was reduced by incubation with DTT, excess DTT was removed on a PD-10 column (GE Healthcare), and reactions of the reduced ZG16 and QSOX1 were initiated in the presence of the antibody variants.…”

“…QSOX1 activity on Zymogen granule membrane protein 16 (ZG16) for initial antibody screening: ZG16 contains two cysteines in a flexible, accessible CXXC motif that can be oxidized in vitro by QSOX1 49 . ZG16 fused to maltose binding protein (MBP, ~45 kDa, no cysteines) was used to assay QSOX1 activity in the presence of supernatants from antibody-producing HEK293F cells.…”