Combinatorial evolution of high-affinity peptides that bind to the Thomsen-Friedenreich carcinoma antigen

Thomsen-Friedenreich (TF) antigen occurs on approximately 90% of human carcinomas, is likely involved in carcinoma cell homotypic aggregation, and has clinical value as a prognostic indicator and marker of metastasized cells. Previously, we isolated anti-TF antigen peptides from bacteriophage display libraries. These bound to TF antigen on carcinoma cells but were of low affinity and solubility. We hypothesized that peptide amino acid sequence changes would result in increased affinity and solubility, which would translate into improved carcinoma cell binding and increased inhibition of aggregation. The new peptides were more soluble and exhibited up to fivefold increase in affinity (K_d 60 nM). They bound cultured human breast and prostate carcinoma cells at low concentrations, whereas the earlier peptides did not. Moreover, the new peptides were potent inhibitors of homotypic aggregation. The maturated peptides will have expanded applications in basic studies of the TF antigen and particular utility as clinical carcinoma-targeting agents.     

High degree of conservation of the enzymes synthesizing the laminin-binding glycoepitope of α-dystroglycan

The dystroglycan (DG) complex plays a pivotal role for the stabilization of muscles in Metazoa. It is formed by two subunits, extracellular α-DG and transmembrane β-DG, originating from a unique precursor via a complex post-translational maturation process. The α-DG subunit is extensively glycosylated in sequential steps by several specific enzymes and employs such glycan scaffold to tightly bind basement membrane molecules. Mutations of several of these enzymes cause an alteration of the carbohydrate structure of α-DG, resulting in severe neuromuscular disorders collectively named dystroglycanopathies. Given the fundamental role played by DG in muscle stability, it is biochemically and clinically relevant to investigate these post-translational modifying enzymes from an evolutionary perspective. A first phylogenetic history of the thirteen enzymes involved in the fabrication of the so-called ‘M3 core’ laminin-binding epitope has been traced by an overall sequence comparison approach, and interesting details on the primordial enzyme set have emerged, as well as substantial conservation in Metazoa. The optimization along with the evolution of a well-conserved enzymatic set responsible for the glycosylation of α-DG indicate the importance of the glycosylation shell in modulating the connection between sarcolemma and surrounding basement membranes to increase skeletal muscle stability, and eventually support movement and locomotion.     

Glycoproteins are species-specific markers and major IgE reactants in grass pollens

Grass pollen allergic patients are concomitantly exposed and sensitized to pollens from multiple Pooideae (i.e. common grass) species. As such, they are currently desensitized by allergen‐specific immunotherapy using extracts made from mixes of pollens from Anthoxanthum odoratum, Dactylis glomerata, Lolium perenne, Phleum pratense and Poa pratensis. Herein, we demonstrate that species‐specific glycoprotein patterns are documented by 1D and 2D electrophoresis and Western blotting analysis, which can be used as an identity test for such pollens. Most allergens are glycoproteins bearing complex N‐glycans encompassing β1,2 xylose and α1,3 fucose glycoepitopes. Glycoepitope destruction using periodate oxidation has no impact on seric IgE reactivity in 75% atopic patients (n = 24). The latter have thus no significant IgE responses to carbohydrate‐containing epitopes. In contrast, periodate treatment strongly impairs IgE recognition of glycoallergens in 25% of patients tested, demonstrating the presence of carbohydrate‐specific IgE in those patients. While the clinical impact of carbohydrate‐specific IgE is still a matter of controversy, the presence of these IgE in the serum of many allergic patients illustrates the need for cross‐reacting carbohydrate epitope‐free recombinant allergens to develop relevant diagnostic tests. These data also support the pertinence of mixing multiple grass pollens to desensitize atopic patients, with the aim to broaden the repertoire of glycoepitopes in the vaccine, thus mimicking natural exposure conditions.