Protein Paucimannosylation Is an Enriched N-Glycosylation Signature of Human Cancers.

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.

Context-specific interactions in literature-curated protein interaction databases.

BACKGROUND: Databases of literature-curated protein-protein interactions (PPIs) are often used to interpret high-throughput interactome mapping studies and estimate error rates. These databases combine interactions across thousands of published studies and experimental techniques. Because the tendency for two proteins to interact depends on the local conditions, this heterogeneity of conditions means that only a subset of database PPIs are interacting during any given experiment. A typical use of these databases as gold standards in interactome mapping projects, however, assumes that PPIs included in the database are indeed interacting under the experimental conditions of the study. RESULTS: Using raw data from 20 co-fractionation experiments and six published interactomes, we demonstrate that this assumption is often false, with up to 55% of purported gold standard interactions showing no evidence of interaction, on average. We identify a subset of CORUM database complexes that do show consistent evidence of interaction in co-fractionation studies, and we use this subset as gold standards to dramatically improve interactome mapping as judged by the number of predicted interactions at a given error rate. CONCLUSIONS: We recommend using this CORUM subset as the gold standard set in future co-fractionation studies. More generally, we recommend using the subset of literature-curated PPIs that are specific to the experimental context whenever possible.

Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

Reduced Circulating Insulin Enhances Insulin Sensitivity in Old Mice and Extends Lifespan.

The causal relationships between insulin levels, insulin resistance, and longevity are not fully elucidated. Genetic downregulation of insulin/insulin-like growth factor 1 (Igf1) signaling components can extend invertebrate and mammalian lifespan, but insulin resistance, a natural form of decreased insulin signaling, is associated with greater risk of age-related disease in mammals. We compared Ins2+/- mice to Ins2+/+ littermate controls, on a genetically stable Ins1 null background. Proteomic and transcriptomic analyses of livers from 25-week-old mice suggested potential for healthier aging and altered insulin sensitivity in Ins2+/- mice. Halving Ins2 lowered circulating insulin by 25%-34% in aged female mice, without altering Igf1 or circulating Igf1. Remarkably, decreased insulin led to lower fasting glucose and improved insulin sensitivity in aged mice. Moreover, lowered insulin caused significant lifespan extension, observed across two diverse diets. Our study indicates that elevated insulin contributes to age-dependent insulin resistance and that limiting basal insulin levels can extend lifespan.

Comprehensive glycomics comparison between colon cancer cell cultures and tumours: implications for biomarker studies.

Altered glycosylation is commonly observed in colorectal cancer. In vitro models are frequently used to study this cancer but little is known about the differences that may exist between these model cell systems and tumour tissue. We have compared the membrane protein glycosylation of five colorectal cancer cell lines (SW1116, SW480, SW620, SW837, LS174T) with epithelial cells from colorectal tumours using liquid chromatography tandem mass spectrometry. Remarkably, there were five abundant O-glycans in the tumour cells that were undetected in the low-mucin producing cell lines, although two were found in the mucinous LS174T cells. The O-glycans included the well-known glycan cancer marker, sialyl-Tn, which has been associated with mucins. Using qRT-PCR, sialyl-Tn expression was found to be associated with an increase in α2,6-sialyltransferase gene (ST6GALNAC1) and a decrease in core 1 synthase gene (C1GALT1) in LS174T cells. The expression of a subset of mucins (MUC2, MUC6, MUC5B) was also correlated with sialyl-Tn expression in LS174T cells. Overall, the membrane protein glycosylation of the model cell lines was found to differ from each other and from the epithelial cells of tumour tissue. These findings should be noted in the design of biomarker discovery experiments particularly when cell surface targets are being investigated. BIOLOGICAL SIGNIFICANCE: The extent of protein glycosylation differences between in vitro cell lines and ex vivo tumours in colorectal cancer research is unknown. Our study expands current knowledge by characterising the membrane protein glycosylation profiles of five different colorectal cancer cell lines and of epithelial cells derived from resected colorectal cancer tumour tissue, using liquid chromatography tandem mass spectrometry. The detailed structural differences found in both N- and O-linked glycan structures on the membrane glycoproteins were determined and correlated with the mRNA expression of the relevant proteins in the cell lines. The glycosylation differences found between cultured cancer cell lines and epithelial cells from tumour tissue have important implications for glycan biomarker discovery.