No abstract

The lectin from Helix pomatia (HPA) binds to adenocarcinomas with a metastatic phenotype but the glycoconjugates of cancer cells that bind to the lectin have yet to be characterized in detail. We used a model of metastatic (HT29) and nonmetastatic (SW480) human colorectal cancer cells and a proteomic approach to identify HPA binding glycoproteins. Cell membrane proteins purified by HPA affinity chromatography, were separated by 2-DE and analyzed by MS. Competitive inhibition experiments with N-acetylgalactosamine, N-acetylglucosamine, and sialic acid confirmed that HPA binding was via a glycan-mediated interaction. Western blot analysis showed that HPA binds to proteins not recognized by an antibody against blood group A epitope. The proteomic study showed the main HPA binding partners include integrin alphav/alpha6 and annexin A2/A4. These proteins were found complexed with microfilament proteins alpha and beta tubulin, actin, and cytokeratins 8 and 18. HPA also bound to Hsp70, Hsp90, TRAP-1, and tumor rejection factor 1. This study revealed that the prognostic utility of HPA lies in its ability to bind simultaneously to many glycoproteins involved in cell migration and signaling, in addition, the proteins recognized by HPA are glycosylated with structures distinct from the blood group A epitope.

Atherosclerosis has received wide attention as a primary cause of premature death in developed countries. The retention of low-density lipoprotein (LDL) particles in the intima, the inner layer of the capillaries, has been imputed as the main cause of the development of atherosclerotic plaques. The entrapment of LDL is mainly due to the specific interaction between the lysine-rich site on apolipoprotein B-100 (apoB-100), a major apolipoprotein of LDL, and extracellular matrix (ECM) components such as collagen, proteoglycans, and glycosaminoglycans (GAGs). Although valuable techniques already exist for studies on apoB-100 and ECM interactions, there is continued need for miniaturized tools that can complement the tools already available and even provide totally new data. This work explores the applicability of the quartz crystal microbalance (QCM) for interaction studies between apoB-100 peptide fragments and various components of the ECM. Two positive peptide fragments, PP and PP(2), and two components of the ECM, collagen I and a selected GAG, chondroitin 6-sulfate (C6S), were immobilized on polystyrene and carboxyl sensor chips. C6S was injected as analyte for PP- and PP(2)-coated surfaces, while PP was the analyte for collagen I and C(6)S surfaces. The estimated dissociation constant (K(D)) indicates that the interactions occur via the positive residues, lysine and arginine, of apoB-100. The continuous-flow QCM system employed in this study is shown to be an excellent tool for the elucidation of interactions between these types of biomolecules.

Collagen, the major component of extracellular matrix (ECM) and the most abundant protein in the human body, is implicated in the development of atherosclerosis. Collagen types I and III were immobilized on fused-silica capillary to investigate their shape, size and structure by atomic force microscopy (AFM). For comparison, collagen was also immobilized on a mica surface. Our studies demonstrated that not only does the adsorption pattern on the substrate vary with the type of collagen, but also the substrate material plays an important role in the fibril formation process. Decorin, which promotes the binding of low-density lipoprotein (LDL) particles with collagen, was investigated for its effect on the fibrillogenesis. On both substrate materials, addition of decorin clearly reduced the fibril diameter of collagen surfaces. Moreover, a quartz crystal microbalance (QCM)-based biosensor approach was applied to clarify and evaluate the affinity of different collagen coatings immobilized on a silicon dioxide sensor chip toward apolipoprotein B-100, the major protein of LDL. The results confirmed the importance of collagen type and their fibrillogenesis on the binding of the positive residues of apolipoprotein B-100 on negatively charged collagen surfaces.