Hans H. Wandall

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.

Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines

Zinc-finger nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed analysis of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN targeting to truncate the O-glycan elongation pathway in human cells, generating stable SimpleCell lines with homogenous O-glycosylation. Three SimpleCell lines expressing only truncated GalNAcα or NeuAcα2-6GalNAcα O-glycans were produced, allowing straightforward isolation and sequencing of GalNAc O-glycopeptides from total cell lysates using lectin chromatography and nanoflow liquid chromatography–mass spectrometry (nLC-MS/MS) with electron transfer dissociation fragmentation. We identified >100 O-glycoproteins with >350 O-glycan sites (the great majority previously unidentified), including a GalNAc O-glycan linkage to a tyrosine residue. The SimpleCell method should facilitate analyses of important functions of protein glycosylation. The strategy is also applicable to other O-glycoproteomes.

Engineered CHO cells for production of diverse, homogeneous glycoproteins.

Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferase genes controlling N-glycosylation in CHO cells and constructed a design matrix that facilitates the generation of desired glycosylation, such as human-like α2,6-linked sialic acid capping. This engineering approach will aid the production of glycoproteins with improved properties and therapeutic potential.

Serum Galectin-2, -4, and -8 Are Greatly Increased in Colon and Breast Cancer Patients and Promote Cancer Cell Adhesion to Blood Vascular Endothelium

Purpose: Adhesion of disseminating tumor cells to the blood vascular endothelium is a pivotal step in metastasis. Previous investigations have shown that galectin-3 concentrations are increased in the bloodstream of patients with cancer and that galectin-3 promotes adhesion of disseminating tumor cells to vascular endothelium in vitro and experimental metastasis in vivo. This study determined the levels of galectin-1, -2, -3, -4, -8, and -9 in the sera of healthy people and patients with colon and breast cancer and assessed the influence of these galectins on cancer-endothelium adhesion. Experimental Design: Serum galectins and auto–anti-MUC1 antibodies were assessed using ELISA and mucin protein (MUC1) glycan microarrays, and cancer-endothelium adhesion was determined using monolayers of human microvascular lung endothelial cells. Results: The levels of serum galectin-2, -3, -4, and -8 were significantly increased up to 31-fold in patients with cancer and, in particular, those with metastases. As previously shown for galectin-3, the presence of these galectins enhances cancer-endothelium adhesion by interaction with the Thomsen-Friedenreich (TF; Galβ1,3GalNAcα-) disaccharide on cancer-associated MUC1. This causes MUC1 cell surface polarization, thus exposing underlying adhesion molecules that promote cancer-endothelium adhesion. Elevated circulating galectin-2 levels were associated with increased mortality in patients with colorectal cancer, but this association was suppressed when anti-MUC1 antibodies with specificity for the TF epitope of MUC1 were also present in the circulation. Conclusions: Increased circulation of several members of the galectin family is common in patients with cancer and these may, like circulating galectin-3, also be involved in metastasis promotion. Clin Cancer Res; 17(22); 7035–46. ©2011 AACR.

UDP-N-acetyl-α-D-galactosamine:polypeptide N-Acetylgalactosaminyltransferase IDENTIFICATION AND SEPARATION OF TWO DISTINCT TRANSFERASE ACTIVITIES

Using a defined acceptor substrate peptide as an affinity chromatography ligand we have developed a purification scheme for a unique human polypeptide, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) (White, T., Bennett, E. P., Takio, K., Sørensen, T., Bonding, N., and Clausen, H.(1995) J. Biol. Chem. 270, 24156-24165). Here we report detailed studies of the acceptor substrate specificity of GalNAc-transferase purified by this scheme as well as the GalNAc-transferase activity, which, upon repeated affinity chromatography, evaded purification by this affinity ligand. Using a panel of acceptor peptides, a qualitative difference in specificity between these separated transferase preparations was identified. Analysis of GalNAc-transferase activities in four rat organs and two human organs also revealed qualitative differences in specificity. The results support the existence of multiple GalNAc-transferase activities and suggest that these are differentially expressed in different organs. As the number of GalNAc-transferases existing is unknown, as is the specificity of the until now cloned and expressed GalNAc-transferases (T1 and T2), it is as yet impossible to relate the results obtained to specific enzyme proteins. The identification of acceptor peptides that can be used to discriminate GalNAc-transferase activities is an important step toward understanding the molecular basis of GalNAc O-linked glycosylation in cells and organs and in pathological conditions.

Early detection of cancer in the general population: a blinded case-control study of p53 autoantibodies in colorectal cancer.

Background:Recent reports from cancer screening trials in high-risk populations suggest that autoantibodies can be detected before clinical diagnosis. However, there is minimal data on the role of autoantibody signatures in cancer screening in the general population.Methods:Informative p53 peptides were identified in sera from patients with colorectal cancer using an autoantibody microarray with 15-mer overlapping peptides covering the complete p53 sequence. The selected peptides were evaluated in a blinded case–control study using stored serum from the multimodal arm of the United Kingdom Collaborative Trial of Ovarian Cancer Screening where women gave annual blood samples. Cases were postmenopausal women who developed colorectal cancer following recruitment, with 2 or more serum samples preceding diagnosis. Controls were age-matched women with no history of cancer.Results:The 50u2009640 women randomised to the multimodal group were followed up for a median of 6.8 (inter-quartile range 5.9–8.4) years. Colorectal cancer notification was received in 101 women with serial samples of whom 97 (297 samples) had given consent for secondary studies. They were matched 1u2009:u20091 with 97 controls (296 serial samples). The four most informative peptides identified 25.8% of colorectal cancer patients with a specificity of 95%. The median lead time was 1.4 (range 0.12–3.8) years before clinical diagnosis.Conclusion:Our findings suggest that in the general population, autoantibody signatures are detectable during preclinical disease and may be of value in cancer screening. In colorectal cancer screening in particular, where the current need is to improve compliance, it suggests that p53 autoantibodies may contribute towards risk stratification.

Autoantibodies to MUC1 glycopeptides cannot be used as a screening assay for early detection of breast, ovarian, lung or pancreatic cancer.

Background:Autoantibodies have been detected in sera before diagnosis of cancer leading to interest in their potential as screening/early detection biomarkers. As we have found autoantibodies to MUC1 glycopeptides to be elevated in early-stage breast cancer patients, in this study we analysed these autoantibodies in large population cohorts of sera taken before cancer diagnosis.Methods:Serum samples from women who subsequently developed breast cancer, and aged-matched controls, were identified from UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) and Guernsey serum banks to formed discovery and validation sets. These were screened on a microarray platform of 60mer MUC1 glycopeptides and recombinant MUC1 containing 16 tandem repeats. Additional case–control sets comprised of women who subsequently developed ovarian, pancreatic and lung cancer were also screened on the arrays.Results:In the discovery (273 cases, 273 controls) and the two validation sets (UKCTOCS 426 cases, 426 controls; Guernsey 303 cases and 606 controls), no differences were found in autoantibody reactivity to MUC1 tandem repeat peptide or glycoforms between cases and controls. Furthermore, no differences were observed between ovarian, pancreatic and lung cancer cases and controls.Conclusion:This robust, validated study shows autoantibodies to MUC1 peptide or glycopeptides cannot be used for breast, ovarian, lung or pancreatic cancer screening. This has significant implications for research on the use of MUC1 in cancer detection.

Deconstruction of O-glycosylation—GalNAc-T isoforms direct distinct subsets of the O-glycoproteome

GalNAc‐type O‐glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O‐glycoproteome is regulated by up to 20 polypeptide GalNAc‐Ts and the contributions and biological functions of individual GalNAc‐Ts are poorly understood. Here, we used a zinc‐finger nuclease (ZFN)‐directed knockout strategy to probe the contributions of the major GalNAc‐Ts (GalNAc‐T1 and GalNAc‐T2) in liver cells and explore how the GalNAc‐T repertoire quantitatively affects the O‐glycoproteome. We demonstrate that the majority of the O‐glycoproteome is covered by redundancy, whereas distinct subsets of substrates are modified by non‐redundant functions of GalNAc‐T1 and GalNAc‐T2. The non‐redundant O‐glycoproteome subsets and specific transcriptional responses for each isoform are related to different cellular processes; for the GalNAc‐T2 isoform, these support a role in lipid metabolism. The results demonstrate that GalNAc‐Ts have different non‐redundant glycosylation functions, which may affect distinct cellular processes. The data serves as a comprehensive resource for unique GalNAc‐T substrates. Our study provides a new view of the differential regulation of the O‐glycoproteome, suggesting that the plurality of GalNAc‐Ts arose to regulate distinct protein functions and cellular processes.

Loss of Function of GALNT2 Lowers High-Density Lipoproteins in Humans, Nonhuman Primates, and Rodents

Human genetics studies have implicated GALNT2, encoding GalNAc-T2, as a regulator of high-density lipoprotein cholesterol (HDL-C) metabolism, but thexa0mechanisms relating GALNT2 to HDL-C remain unclear. We investigated the impact of homozygous GALNT2 deficiency on HDL-C in humans and mammalian models. We identified two humans homozygous for loss-of-function mutations in GALNT2 who demonstrated low HDL-C. We also found that GALNT2 loss of function in mice, rats, and nonhuman primates decreased HDL-C. O-glycoproteomics studies of a human GALNT2-deficient subject validated ANGPTL3 and ApoC-III as GalNAc-T2 targets. Additional glycoproteomics in rodents identified targets influencing HDL-C, including phospholipid transfer protein (PLTP). GALNT2 deficiency reduced plasma PLTP activity in humans and rodents, and in mice this was rescued by reconstitution of hepatic Galnt2. We also found that GALNT2 GWAS SNPs associated with reduced HDL-C also correlate with lower hepatic GALNT2 expression. These results posit GALNT2 as a direct modulator of HDL metabolism across mammals.

Cancer‐associated autoantibodies to MUC1 and MUC4—A blinded case–control study of colorectal cancer in UK collaborative trial of ovarian cancer screening

Recent reports suggest that autoantibodies directed to aberrantly glycosylated mucins, in particular MUC1 and MUC4, are found in patients with colorectal cancer. There is, however, limited information on the autoantibody levels before clinical diagnosis, and their utility in cancer screening in the general population. In our study, we have generated O‐glycosylated synthetic MUC1 and MUC4 peptides in vitro, to mimic cancer‐associated glycoforms, and displayed these on microarrays. The assays performance was tested through an initial screening of serum samples taken from patients at the time of colorectal cancer diagnosis and healthy controls. Subsequently, the selected biomarkers were evaluated in a blinded nested case–control study using stored serum samples from among the 50,640 women randomized to the multimodal arm of the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS), where women gave annual blood samples for several years. Cases were 97 postmenopausal women who developed colorectal cancer after recruitment and were age‐matched to 97 women without any history of cancer. MUC1‐STn and MUC1‐Core3 IgG autoantibodies identified cases with 8.2 and 13.4% sensitivity, respectively, at 95% specificity. IgA to MUC4 glycoforms were unable to discriminate between cases and controls in the UKCTOCS sera. Additional analysis was undertaken by combining the data of MUC1‐STn and MUC1‐Core3 with previously generated data on autoantibodies to p53 peptides, which increased the sensitivity to 32.0% at 95% specificity. These findings suggest that a combination of antibody signatures may have a role as part of a biomarker panel for the early detection of colorectal cancer.