Core fucosylated glycan-dependent inhibitory effect of QSOX1-S on invasion and metastasis of hepatocellular carcinoma

The goal of the present study was to identify glycoproteins associated with the postoperative relapse of hepatocellular carcinoma (HCC) and to investigate their potential role in HCC metastasis. A method for quantitating N-glycoproteome was used to screen for, and identify, recurrence-related N-linked glycoproteins from 100 serum samples taken from patients with early-stage HCC. The prognostic significance of candidate glycoproteins was then validated in 193 HCC tissues using immunohistochemical staining. Serum core fucosylated quiescin sulfhydryl oxidase 1 (cf-QSOX1) was identified as a leading prognostic glycoprotein that significantly correlated with HCC recurrence. Patients with high serum cf-QSOX1 levels had a significantly longer time to recurrence (TTR) as compared with those with low serum cf-QSOX1. As was seen with serum cf-QSOX1, QSOX1 in HCC tissues was further shown to be significantly associated with good patient outcome. Gain-functional and loss-functional analyses of QSOX1-S were performed in vitro and in vivo. QSOX1-S overexpression significantly increased in vitro apoptosis, but decreased the invasive capacity of HCC cells, and reduced lung metastasis in nude mice models bearing human HCC. Furthermore, overexpression of a mutant version of QSOX1-S, which had eliminated the core-fucosylated glycan at Asn-130, showed no demonstrable effect on invasion or metastasis of HCC cells. Our study suggests that serum cf-QSOX1-S and tumor QSOX1 levels are helpful for predicting recurrence in HCC patients, and its core-fucosylated glycan at Asn-130 is critical for the inhibitory effects of QSOX1-S on invasion and metastasis of HCC

After lectin affinity chromatography, the glycoproteins obtained from the samples were desalted using SepPak C18 cartridges, and then dried in a vacuum centrifuge. The glycoproteins were then mixed with immobilized trypsin (20% slurry v/w) for 20 min with gentle shaking, and then lyophilized. The lyophilized peptides were dissolved in 100 μL acetonitrile in 50 mM NH4HCO3 (pH 6.8) (ACN/NH4HCO3, 20% v/v) prepared with H2 16 O or H2 18 O in advance, then incubated at 37°C for 24 h to catalyze the labeling of tryptic peptides at the C-terminus. The immobilized trypsin beads were then removed by MicroSpin columns. A total of 5 μL formic acid was added to further inhibit any possible residual trypsin activity. The peptides were lyophilized and then dissolved in 100 mM NH4HCO3 buffer prepared in H2 16 O or H2 18 O. PNGase F was added at a concentration of 1 μL PNGase-F/mg of crude protein, and the labeling was conducted at 37°C overnight. Finally, the 16 O-and 18 O-labeled peptides were mixed at a ratio of 1:1 for samples from recurrence and non-recurrence group and lyophilized.

Nano LC-electrospray ionization (ESI)-MS/MS
The lyophilized peptides were resuspended with 2% ACN in 0.1% formic acid, separated by nano LC, and then analyzed by online electrospray tandem mass spectrometry. The experiments were performed on a Nano Aquity UPLC system (Waters) connected to an LTQ Orbitrap XL mass spectrometer (Thermo Electron Corp., Bremen, Germany) interfaced with an online nano electrospray ion source (Michrom Bioresources, Auburn, CA). The peptide separation was performed in a Michrom CAPTRAP (500 μm i.d. × 2 mm trap column) and a Michrom C18 (3.5 μm, glycoprotein digests (0.5 μg) were loaded onto the trap column and leached at a flow rate of 20 μL/min for 3 min. The mobile phases included 2% ACN in 0.1% formic acid (phase A and the loading phase) and 95% ACN in 0.1% formic acid (phase B). To achieve sufficient separation, a 60-min (for glycoprotein standards) or 90-min (for serum samples) linear gradient from 5% to 45% at phase B was employed. The flow rate of the mobile phase was set at 500 nL/min, and the electrospray voltage used was 1.6 kV. The linear gradient was adjusted to 90 min for serum samples analyses, while all other parameters remained unchanged. The LTQ Orbitrap XL mass spectrometer was operated in the data-dependent mode with an automatic switch between MS and MS/MS acquisition. The survey full-scan MS spectra with two microscans (m/z 350-1800) was acquired in Orbitrap at a resolution of 100,000 (at m/z 400) followed by eight MS/MS scans in LTQ trap. Dynamic exclusion was set to initiate a 60 s exclusion for ions analyzed twice within a 10 s interval.

Western blot
Western blot was conducted as described previously [2]. Briefly, the tested cells were subjected to lysis with RIPA buffer, and the cell lysates were separated by electrophoresis and transferred to PVDF membrane. After blocking, the membranes were incubated with primary antibodies, and subsequently incubated with horseradish peroxidase-conjugated secondary antibody. The signal was detected by exposure to X-ray film.

Cell Proliferation and Apoptosis Analyses
Cell proliferation was examined using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). According to the manufacturer's instructions for Cell Counting Kit-8, harvested cells were seeded in 96-well plates at 1×103 per well in a final volume of added into each well, and the absorbance at 450 nm was measured after incubation for 2 hours at 37°C to calculate the number of viable cells. Apoptosis analyses were performed using propidium iodide (Keygen, Nanjing, China) and phycoerythrin (PE)-annexin apoptosis detection kit (BD Pharmingen, San Jose, CA, USA) on flow cytometry (Epics Altra, Beckman Coulter，USA). Figure S1. The flowchart of this study.

Methods Purpose
Screening potential glycan structures linked to recurrence of HCC Identifying recurrence-related glycoproteins with the specific glycan structures Confirming the recurrence-related glycoproteins with the specific glycan structures Validating the prognostic significance of QSOX1 in HCC tissue  Note: aa, amino acid residue. *N-linked glycosylation site.