Cancer-associated autoantibodies hold promise as sensitive biomarkers for early detection of cancer. Aberrant posttranslational 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.Colorectal cancer develops in a multistep process that arises from genetic or epigenetic alterations.1 Most colorectal cancers can be treated by removal of early malignant lesions, 2,3 but despite this colorectal cancer remains the second most common cause of cancer-related death in the western world. 4 Current screening techniques, which include fecal occult blood, sigmoid and colonoscopy and computed tomographic colonography, are complicated with low compliance and high cost.5 Therefore, there is a need for biomarkers, which can identify colorectal cancer at early stages and aid in the surveillance and identification of high-risk populations.Broad genomic and proteomic approaches for identification of biomarkers have so far failed to develop simple, reliable and noninvasive screening test for early detection of colorectal cancer.6 Current serum assays detecting cancer glycoproteins such as CEA and CA-19-9 have limited use for early stages with low specificity and sensitivity. 7,8 As an alternative, circulating autoantibodies elicited by exposure to aberrant cancer proteins lacking immunological tolerance are emerging as promising biomarkers for the early detection of cancer.9-13 However, relatively few autoantibody epitopes have been identified and characterized to date.14 In a small study, we recently demonstrated the existence of such cancerassociated autoantibodies to the aberrantly O-glycosylated MUC1 mucin in patients with breast, ovarian and prostate cancer at time of diagnosis.15 These results provide basis for further discovery of autoantibody targets with the aim to increase sensitivity and organ specificity of biomarker assays through signatures of autoantibo...

The glucosyltransferase amylosucrase is structurally quite similar to the hydrolase ␣-amylase. How this switch in functionality is achieved is an important and fundamental question. The inactive E328Q amylosucrase variant has been co-crystallized with maltoheptaose, and the structure was determined by x-ray crystallography to 2.2 Å resolution, revealing a maltoheptaose binding site in the B-domain somewhat distant from the active site. Additional soaking of these crystals with maltoheptaose resulted in replacement of Tris in the active site with maltoheptaose, allowing the mapping of the ؊1 to ؉5 binding subsites. Crystals of amylosucrase were soaked with sucrose at different concentrations. The structures at ϳ2.1 Å resolution revealed three new binding sites of different affinity. The highest affinity binding site is close to the active site but is not in the previously identified substrate access channel. Allosteric regulation seems necessary to facilitate access from this binding site. The structures show the pivotal role of the B-domain in the transferase reaction. Based on these observations, an extension of the hydrolase reaction mechanism valid for this enzyme can be proposed. In this mechanism, the glycogen-like polymer is bound in the widest access channel to the active site. The polymer binding introduces structural changes that allow sucrose to migrate from its binding site into the active site and displace the polymer.

Islet-brain 1 (IB1 or JIP-1) is a scaffold protein that interacts with components of the c-Jun N-terminal kinase (JNK) signal-transduction pathway. IB1 is expressed at high levels in neurons and in pancreatic b-cells, where it controls expression of several insulin-secretory components and secretion. IB1 has been shown to homodimerize, but neither the molecular mechanisms nor the function of dimerization have yet been characterized. Here, we show that IB1 homodimerizes through a novel and unique set of Src homology 3 (SH3)-SH3 interactions. X-ray crystallography studies show that the dimer interface covers a region usually engaged in PxxP-mediated ligand recognition, even though the IB1 SH3 domain lacks this motif. The highly stable IB1 homodimer can be significantly destabilized in vitro by three individual point mutations directed against key residues involved in dimerization. Each mutation reduces IB1-dependent basal JNK activity in 293T cells. Impaired dimerization also results in a reduction in glucose transporter type 2 expression and in glucose-dependent insulin secretion in pancreatic b-cells. Taken together, these results indicate that IB1 homodimerization through its SH3 domain has pleiotropic effects including regulation of the insulin secretion process.

IB1 is a mammalian scaffold protein that interacts with components of the c-Jun N-terminal kinase (JNK) signal-transduction pathway mainly via its protein-protein interaction domains. Crystallization of the key Src homology 3 (SH3) domain of IB1 has been achieved. Crystallization experiments with unmodified protein and deliberately oxidized protein have led to different crystal forms. X-ray data have been collected to 3.0 A resolution from a crystal form with rectangular prism morphology. These crystals are orthorhombic (P2(1)2(1)2(1)), with unit-cell parameters a = 45.9, b = 57.0, c = 145.5 A. These are the first crystallographic data on a scaffold molecule such as IB1 to be reported.

No abstract