Cancer cell metastasis is a major cause of cancer death. Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches. Nevertheless, the dysregulation of the cancer cell secretome is known to play key roles in tumor transformation and progression. The majority of proteins in the secretome are secretory proteins and membrane-released proteins, and, mostly, the glycosylated proteins. Until recently, few studies have explored protein N-glycosylation changes in the secretome, although protein glycosylation has received increasing attention in the study of tumor development processes. Here, the N-glycoproteins in the secretome of two human hepatocellular carcinoma (HCC) cell lines with low (MHCC97L) or high (HCCLM3) metastatic potential were investigated with a in-depth characterization of the N-glycosites by combining two general glycopeptide enrichment approaches, hydrazide chemistry and zwitterionic hydrophilic interaction chromatography (zic-HILIC), with mass spectrometry analysis. A total of 1,213 unique N-glycosites from 611 N-glycoproteins were confidently identified. These N-glycoproteins were primarily localized to the extracellular space and plasma membrane, supporting the important role of N-glycosylation in the secretory pathway. Coupling label-free quantification with a hierarchical clustering strategy, we determined the differential regulation of several N-glycoproteins that are related to metastasis, among which AFP, DKK1, FN1, CD151 and TGFβ2 were up-regulated in HCCLM3 cells. The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study. Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery. The glycoproteome strategy in this study provides an effective means to explore potential cancer biomarkers.

The traditional Chinese drug Bombyx Batryticatus (BB), which is also named the white stiff silkworm, has been widely used in Chinese clinics for thousands of years. It is famous for its antispasmodic and blood circulation-promoting effects. Cardiomyocyte hypertrophy, interstitial cell hyperplasia, and myocardial fibrosis are closely related to the N-glycosylation of key proteins. To examine the alterations of N-glycosylation that occur in diabetic myocardium during the early stage of the disease, and to clarify the therapeutic effect of 1-Deoxynojirimycin (1-DNJ) extracted from BB, we used the db/db (diabetic) mouse model and an approach based on hydrophilic chromatography solid-phase extraction integrated with an liquid Chromatograph Mass Spectrometer (LC-MS) identification strategy to perform a site-specific N-glycosylation analysis of left ventricular cardiomyocyte proteins. Advanced glycation end products (AGEs), hydroxyproline, connective tissue growth factor (CTGF), and other serum biochemical indicators were measured with enzyme-linked immunosorbent assays (ELISA). In addition, the α-1,6-fucosylation of N-glycans was profiled with lens culinaris agglutinin (LCA) lectin blots and fluorescein isothiocyanate (FITC)-labelled lectin affinity histochemistry. The results indicated that 1-DNJ administration obviously downregulated myocardium protein N-glycosylation in db/db mice. The expression levels of serum indicators and fibrosis-related cytokines were reduced significantly by 1-DNJ in a dose-dependent manner. The glycan α-1,6-fucosylation level of the db/db mouse myocardium was elevated, and the intervention effect of 1-DNJ administration on N-glycan α-1,6-fucosylation was significant. To verify this result, the well-known transforming growth factor-β (TGF-β)/Smad2/3 pathway was selected, and core α-1,6-fucosylated TGF-β receptor II (TGFR-βII) was analysed semi-quantitatively with western blotting. The result supported the conclusions obtained from LCA lectin affinity histochemistry and lectin blot analysis. The expression level of α-1,6-fucosyltransferase (FUT8) mRNA was also detected, and the results showed that 1-DNJ administration did not cause obvious inhibitory effects on FUT8 expression. Therefore, the mechanism of 1-DNJ for relieving diabetic cardiomyopathy (DCM)-associated fibrosis can be concluded as the inhibition of N-acetylglucosamine (N-GlcNAc) formation and the reduction of substrate concentration.

Cancer cell metastasis is a major cause of cancer fatality. But the underlying molecular mechanisms remain incompletely understood, which results in the lack of efficient diagnosis, therapy and prevention approaches. Here, we report a systematic study on the secretory proteins (secretome) and secretory N-glycoproteins (N-glycosecretome) of four human hepatocellular carcinoma (HCC) cell lines with different metastatic potential, to explore the molecular mechanism of metastasis and supply the clues for effective measurement of diagnosis and therapy. Totally, 6242 unique gene products (GPs) and 1637 unique N-glycosites from 635 GPs were confidently identified. About 4000 GPs on average were quantified in each of the cell lines, 1156 of which show differential expression (p<0.05). Ninety-nine percentage of the significantly altered proteins were secretory proteins and proteins correlated to cell movement were significantly activated with the increasing of metastatic potential of the cell lines. Twenty-three GPs increased both in the secretome and the N-glycosecretome were chosen as candidates and verified by western blot analysis, and 10 of them were chosen for immunohistochemistry (IHC) analysis. The cumulative survival rates of the patients with candidate (FAT1, DKK3) suggested that these proteins might be used as biomarkers for HCC diagnosis. In addition, a comparative analysis with the published core human plasma database (1754 GPs) revealed that there were 182 proteins not presented in the human plasma database but identified by our studies, some of which were selected and verified successfully by western blotting in human plasma.