Quiescin sulfhydryl oxidase 1 (QSOX1) glycosite mutation perturbs secretion but not Golgi localization

Horowitz, Ben; Javitt, Gabriel; Ilani, Tal; Gat, Yair; Morgenstern, David; Bard, Frédéric; Fass, Deborah

doi:10.1093/glycob/cwy044

Cited by 12 publications

(8 citation statements)

References 50 publications

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“…The remaining supernatant was passed through a 0.45 µ m filter, and protein was purified by Ni‐NTA chromatography. The catalytic region of human QSOX1 was purified in a similar manner using an expression vector previously described [ 38 ]. Escherichia coli Trx was purified from bacteria essentially as described except without detergent [ 39 ].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2021

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“…1d, i). Although it remains unclear how the disulfides in the D′D3 dimer interface would reshuffle or form at ~pH 5.8 in Golgi and various mechanisms have been proposed 7,9 , our cryo-EM structure of a D′D3 dimer complexed with D1D2s shows that the two Cys 1097 are largely buried in the center of the dimeric interface and there is limited space to allow the participation of any bulky redox-regulating enzymes (Supplementary Figs. S7, S8).…”

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confidence: 97%

Structural mechanism of VWF D’D3 dimer formation

Shu

Zeng

et al. 2022

Cell Discov

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factor (VWF) assembly begins in the endoplasmic reticulum of endothelial cells and megakaryocytes where VWF is synthesized as a precursor with multiple domains (Fig. 1a). The polypeptide including the propeptide and mature VWF chain (proVWF) subsequently forms "tail-to-tail" homodimers through their C-terminal cystine knot (CK) domains. These proVWF dimers are then transported to the Golgi where they assemble into large multimers 'head-to-head' through interchain disulfide bonds between D3 domains of two proVWF dimers 1-3 . The Sadler group firstly identified a peptide containing the Cys 1142 -Cys 1142′ disulfide bond from plasma multimeric VWF 4 . Subsequently, through differential alkylation, proteolytic digestion, and mass spectrometry, they identified two free cysteines Cys 1099 and Cys 1142 from D′D3 monomer and proposed their involvement in D′D3 dimer formation 5 . However, the crystal structure of a monomeric D′D3 mutant (C1099A/ C1142A) reported by Dong and Springer revealed that residue Cys 1099 is largely buried inside of the D′D3 domain and it has to undergo a dramatic conformational change to allow forming a disulfide bond with the same cysteine from the other D′D3 molecule 6,7 . Furthermore, the structures of dimeric D3 domains of Mucin 2 (MUC2), a homologous multimeric protein of VWF, showed Cys 1142 -Cys 1142′ and Cys 1097 -Cys 1097′ disulfide linkages (using VWF residue numbering) 8 . Cys 1091

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“…QSOX2 also shares 50 % similarity with QSOX1, which is more studied and has recently been identified as a marker of metastasis in several cancers, including prostate (81, 82), pancreatic (36, 83), and breast (84, 85) cancers. QSOX1 localizes to the ER and Golgi but can be secreted (83, 86–88), in a mechanism that requires its N-linked glycan (89). Here we have demonstrated that QSOX2 carries polySia on both N - and O -linked glycans, and that secreted QSOX2 is polysialylated.…”

Section: Discussionmentioning

confidence: 99%

A new strategy for identifying polysialylated proteins reveals they are secreted from cancer cells as soluble proteins and as part of extracellular vesicles

Hunter

Derksen

Karathra

et al. 2022

Preprint

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Polysialic acid (polySia) is a long homopolymer consisting of α2,8–linked sialic acid with tightly regulated expression in humans. In healthy adults, it occurs on cell surface glycoproteins in neuronal, reproductive, and immune tissues; however, it is aberrantly present in many cancers and its overexpression correlates with significantly increased metastasis and poor prognosis. Prompted by the observation that the MCF–7 breast cancer cell line contains only intracellular polySia, we investigated the secretion of polySia from MCF–7 cells. PolySia was found predominantly on soluble proteins in MCF–7 conditioned media, but also on extracellular vesicles (EVs), secreted from the cells. Since MCF–7 cells do not express known polysialylated proteins, we developed a robust method for purifying polysialylated proteins that uses a metabolic labelling strategy to introduce a bioorthogonal functionality into polySia. Using this method we identified three previously unknown polysialylated proteins, and found that two of these proteins – AGR2 and QSOX2 – were secreted from MCF-7 cells. We confirmed that QSOX2 found in EV–depleted MCF-7 cell conditioned media was polysialylated. Herein we report the secretion of polysialic acid on both soluble and EV-associated proteins from MCF–7 cancer cells and introduce a new method to efficiently identify polysialylated proteins. These findings have exciting implications for understanding the roles of polySia in cancer progression and metastasis and for identifying new cancer biomarkers.

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Quiescin sulfhydryl oxidase 1 (QSOX1) glycosite mutation perturbs secretion but not Golgi localization

Cited by 12 publications

References 50 publications

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

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

Structural mechanism of VWF D’D3 dimer formation

A new strategy for identifying polysialylated proteins reveals they are secreted from cancer cells as soluble proteins and as part of extracellular vesicles

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