Mads Agervig Tarp

Polypeptide GalNAc-transferase T3 and Familial Tumoral Calcinosis SECRETION OF FIBROBLAST GROWTH FACTOR 23 REQUIRES O-GLYCOSYLATION

Mutations in the gene encoding the glycosyltransferase polypeptide GalNAc-T3, which is involved in initiation of O-glycosylation, were recently identified as a cause of the rare autosomal recessive metabolic disorder familial tumoral calcinosis (OMIM 211900). Familial tumoral calcinosis is associated with hyperphosphatemia and massive ectopic calcifications. Here, we demonstrate that the secretion of the phosphaturic factor fibroblast growth factor 23 (FGF23) requires O-glycosylation, and that GalNAc-T3 selectively directs O-glycosylation in a subtilisin-like proprotein convertase recognition sequence motif, which blocks processing of FGF23. The study suggests a novel posttranslational regulatory model of FGF23 involving competing O-glycosylation and protease processing to produce intact FGF23.

Cancer Biomarkers Defined by Autoantibody Signatures to Aberrant O-Glycopeptide Epitopes

Autoantibodies to cancer antigens hold promise as biomarkers for early detection of cancer. Proteins that are aberrantly processed in cancer cells are likely to present autoantibody targets. The extracellular mucin MUC1 is overexpressed and aberrantly glycosylated in many cancers; thus, we evaluated whether autoantibodies generated to aberrant O-glycoforms of MUC1 might serve as sensitive diagnostic biomarkers for cancer. Using an antibody-based glycoprofiling ELISA assay, we documented that aberrant truncated glycoforms were not detected in sera of cancer patients. An O-glycopeptide microarray was developed that detected IgG antibodies to aberrant O-glycopeptide epitopes in patients vaccinated with a keyhole limpet hemocyanin-conjugated truncated MUC1 peptide. We detected cancer-associated IgG autoantibodies in sera from breast, ovarian, and prostate cancer patients against different aberrent O-glycopeptide epitopes derived from MUC1. These autoantibodies represent a previously unaddressed source of sensitive biomarkers for early detection of cancer. The methods we have developed for chemoenzymatic synthesis of O-glycopeptides on microarrays may allow for broader mining of the entire cancer O-glycopeptidome.

Tumor-Associated Tn-MUC1 Glycoform Is Internalized through the Macrophage Galactose-Type C-Type Lectin and Delivered to the HLA Class I and II Compartments in Dendritic Cells

The type of interaction between tumor-associated antigens and specialized antigen-presenting cells such as dendritic cells (DCs) is critical for the type of immunity that will be generated. MUC1, a highly O-glycosylated mucin, is overexpressed and aberrantly glycosylated in several tumor histotypes. This results in the expression of tumor-associated glycoforms and in MUC1 carrying the tumor-specific glycan Tn (GalNAcalpha1-O-Ser/Thr). Glycopeptides corresponding to three tandem repeats of MUC1, enzymatically glycosylated with 9 or 15 mol of GalNAc, were shown to specifically bind and to be internalized by immature monocyte-derived DCs (iDCs). Binding required calcium and the GalNAc residue and was competed out by GalNAc polymer and Tn-MUC1 or Tn-MUC2 glycopeptides. The macrophage galactose-type C-type lectin (MGL) receptor expressed on iDCs was shown to be responsible for the binding. Confocal analysis and ELISA done on subcellular fractions of iDCs showed that the Tn-MUC1 glycopeptides colocalized with HLA class I and II compartments after internalization. Importantly, although Tn-MUC1 recombinant protein was bound and internalized by MGL, the glycoprotein entered the HLA class II compartment, but not the HLA class I pathway. These data indicate that MGL expressed on iDCs is an optimal receptor for the internalization of short GalNAcs carrying immunogens to be delivered into HLA class I and II compartments. Such glycopeptides therefore represent a new way of targeting the HLA class I and II pathways of DCs. These results have possible implications in designing cancer vaccines.

Seromic profiling of colorectal cancer patients with novel glycopeptide microarray

Cancer‐associated autoantibodies hold promise as sensitive biomarkers for early detection of cancer. Aberrant post‐translational variants of proteins are likely to induce autoantibodies, and changes in O‐linked glycosylation represent one of the most important cancer‐associated post‐translational modifications (PTMs). Short aberrant O‐glycans on proteins may introduce novel glycopeptide epitopes that can elicit autoantibodies because of lack of tolerance. Technical barriers, however, have hampered detection of such glycopeptide‐specific autoantibodies. Here, we have constructed an expanded glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from a panel of human mucins (MUC1, MUC2, MUC4, MUC5AC, MUC6 and MUC7) known to have altered glycosylation and expression in cancer. Seromic profiling of patients with colorectal cancer identified cancer‐associated autoantibodies to a set of aberrant glycopeptides derived from MUC1 and MUC4. The cumulative sensitivity of the array analysis was 79% with a specificity of 92%. The most prevalent of the identified autoantibody targets were validated as authentic cancer immunogens by showing expression of the epitopes in cancer using novel monoclonal antibodies. Our study provides evidence for the value of glycopeptides and other PTM‐peptide arrays in diagnostic measures.

Sialyl-Tn vaccine induces antibody-mediated tumour protection in a relevant murine model

Changes in the composition of glycans added to glycoproteins and glycolipids are characteristic of the change to malignancy. Sialyl-Tn (STn) is expressed by 25–30% of breast carcinomas but its expression on normal tissue is highly restricted. Sialyl-Tn is an O-linked disaccharide that can be carried on various glycoproteins. One such glycoprotein MUC1 is expressed by the vast majority of breast carcinomas. Both STn and MUC1 have been considered as targets for immunotherapy of breast cancer patients. Here we used different immunogens to target STn in an MUC1 transgenic mouse model of tumour challenge. We show that synthetic STn coupled to keyhole limpet haemocyanin (Theratope), induced antibodies to STn that recognised the glycan carried on a number of glycoproteins and in these mice a significant delay in tumour growth was observed. The protection was dependant on STn being expressed by the tumour and was antibody mediated. Affinity chromatography of the STn-expressing tumour cell line, followed by mass spectrometry, identified osteopontin as a novel STn-carrying glycoprotein which was highly expressed by the tumours. These results suggest that if antibodies can be induced to a number of targets expressed by the tumour cells, a humoral response can be effective in controlling tumour growth.

Cancer specific Mucin-1 glycoforms are expressed on multiple myeloma

Present therapies cannot cure the large majority of patients with multiple myeloma (MM) and therefore new treatment strategies are imperative. This study analysed the different glycosylation profiles of Mucin‐1 (MUC1) on MM and acute myeloid leukaemia (AML) cells using a series of anti‐MUC1 antibodies. Seventy‐three per cent of the MM patients had plasma cells that expressed the fully glycosylated forms of MUC1. In contrast to controls, normal bone marrow cells and AML cells, the differentiation‐dependent and cancer‐associated glycoforms of MUC1 were present on 59% and 36% MM tumour cells respectively. This indicated that aberrantly glycosylated MUC1 is a potential immunotherapeutic target in MM patients.

Characterization of Binding Epitopes of CA125 Monoclonal Antibodies

The most used cancer serum biomarker is the CA125 immunoassay for ovarian cancer that detects the mucin glycoprotein MUC16. Several monoclonal antibodies (mAbs) including OC125 and M11 are used in CA125 assays. However, despite considerable efforts, our knowledge of the molecular characteristics of the recognized epitopes and the role played by glycosylation has remained elusive. Here a comprehensive set of recombinant MUC16 tandem repeats (TRs) expressed in glycoengineered mammalian cells and E. coli, together with overlapping peptides, was used to probe antigen-binding epitopes. We present a complete analysis of N- and O-glycosylation sites of a MUC16 TR expressed in CHO cells and demonstrate that neither N- nor O-glycosylation appear to substantially influence binding of OC125 and M11 mAbs. A series of successive N- and C-terminal truncations of a MUC16 TR construct expressed in E. coli narrowed down the epitopes for OC125 and M11 to a segment containing parts of two consecutive SEA domains with a linker. Thus, a complete SEA domain is not required. These findings suggest that binding epitopes of mAbs OC125 and M11 are dependent on conformation but not on glycosylation. The availability of recombinant TR constructs with and without aberrant glycosylation now opens the way for vaccine studies.