N-acetylgalactosaminyl transferase-3 is a potential new marker for non-small cell lung cancers

N-acetylgalactosaminyl transferase-3 (GalNAc-T3) is an enzyme involved in the initial glycosylation of mucin-type O-linked proteins. In the present study, we used immunohistochemistry to examine GalNAc-T3 expression in 215 surgically resected non-small cell lung cancers. We analysed the biological and clinical importance of GalNAc-T3 expression, especially with regard to its potential as a prognostic factor. We found that normal bronchial epithelial cells, bronchial gland cells, and alveolar pneumocytes showed cytoplasmic immunostaining for GalNAc-T3. Low expression of GalNAc-T3, observed in 93 of 215 tumours (43.4%), was found more frequently in tumours from smokers than those from nonsmokers (P=0.001), in squamous cell carcinomas than nonsquamous cell carcinomas (P<0.0001), and in moderately and poorly differentiated tumours than well differentiated tumours (P=0.0002). Multivariate logistic regression analysis showed that an association of low GalNAc-T3 expression with squamous cell carcinomas was the only one significant relationship of GalNAc-T3 expression with various factors (P<0.0001). Moreover, tumours losing GalNAc-T3 expression had a significantly higher Ki-67 labelling index than tumours retaining GalNAc-T3 expression (P=0.0003). Patients with low GalNAc-T3 expression survived a significantly shorter time than patients with high GalNAc-T3 expression in 103 pStage I non-small cell lung cancers (5-year survival rates, 58% and 78%, respectively; P=0.02 by log-rank test) as well as in 61 pStage I nonsquamous cell carcinomas (5-year survival rates, 63% and 85%, respectively; P=0.03). Low GalNAc-T3 expression was an unfavourable prognostic factor in pStage I non-small cell lung cancers (hazards ratio, 2.04; P=0.03), and in pStage I nonsquamous cell carcinomas (hazards ratio, 2.70; P=0.03). These results suggest that GalNAc-T3 is a new marker of non-small cell lung cancers with specificity for histology and prognosis

Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc-type O-glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc-transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O-glycosylation (SimpleCells) that enables proteome-wide discovery of O-glycan sites using 'bottom-up' ETD-based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O-glycoproteome with almost 3000 glycosites in over 600 O-glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O-glycosylation. The finding of unique subsets of O-glycoproteins in each cell line provides evidence that the O-glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O-glycoproteome should facilitate the exploration of how site-specific O-glycosylation regulates protein function