Effect of hyperhomocysteinemia on rat cardiac sarcoplasmic reticulum

Molecular and Cellular Biochemistry - Increased concentration of plasma homocysteine (Hcy) is an independent risk factor of cardiovascular disease, yet the mechanism by which hyperhomocysteinemia...     

Identification of B‐cell epitopes of Borrelia burgdorferi outer surface protein C by screening a phage‐displayed gene fragment library

Outer surface protein C (OspC) of Borrelia stimulates remarkable immune responses during early infection and is therefore currently considered a leading diagnostic and vaccine candidate. The sensitivity and specificity of serological tests based on whole protein OspC for diagnosis of Lyme disease are still unsatisfactory. Minimal B‐cell epitopes are key in the development of reliable immunodiagnostic tools. Using OspC fragments displayed on phage particles (phage library) and anti‐OspC antibodies isolated from sera of naturally infected patients, six OspC epitopes capable of distinguishing between LD patient and healthy control sera were identified. Three of these epitopes are located at the N‐terminus (OspC E1 aa19–27, OspC E2 aa38–53, OspC E3 aa62–66) and three at the C‐terminal end (OspC E4 aa155–163, OspC E5 aa184–190 and OspC E6 aa201–207). OspC E1, E4 and E6 were highly conserved among LD related Borreliae. To our knowledge, epitopes OspC E2, E3 and E5 were identified for the first time in this study. Minimal B‐cell epitopes may provide fundamental data for the development of multi‐epitope‐based diagnostic tools for Lyme disease.     

An insight into the ligand-receptor interactions involved in the translocation of pathogens across blood-brain barrier

Traversal of pathogen across the blood-brain barrier (BBB) is an essential step for central nervous system (CNS) invasion. Pathogen traversal can occur paracellularly, transcellularly, and/or in infected phagocytes (Trojan horse mechanism). To trigger the translocation processes, mainly through paracellular and transcellular ways, interactions between protein molecules of pathogen and BBB are inevitable. Simply, it takes two to tango: both host receptors and pathogen ligands. Underlying molecular basis of BBB translocation of various pathogens has been revealed in the last decade, and a plethora of experimental data on protein-protein interactions has been created. This review compiles these data and should give insights into the ligand-receptor interactions that occur during BBB translocation. Further, it sheds light on cell signaling events triggered in response to ligand-receptor interaction. Understanding of the molecular principles of pathogen-host interactions that are involved in traversal of the BBB should contribute to develop new vaccine and drug strategies to prevent CNS infections.     

Variable regions in the sushi domains 6-7 and 19-20 of factor H in animals and human lead to change in the affinity to factor H binding protein of Borrelia

Borrelia binds host's complement regulatory factor H (fH) to evade complement attack. However, binding affinities between fH-binding-proteins (FHBPs) of Borrelia and fH from various hosts are disparate. Experiments performed to unfold the underlying molecular basis of this disparity revealed that recombinant BbCRASP-1 (major FHBP of Borrelia burgdorferi) neither interacted with sushi 6-7, nor with sushi 19-20 domains of fH in cattle and pig, however, showed binding affinity to both sushi domains of human fH, sushi 6-7 of mouse and sushi 19-20 of sheep. Further, peptide-spot assay revealed three major binding sites (sushi 6:(335-346), sushi 7:(399-410) and sushi 20:(1205-1227)) in human fH that can form BbCRASP-1:fH interface, while (337)HENMR(341) residues in sushi 6 are crucial for rigid BbCRASP-1:fH complex formation. Amino acid stretches DTIEFTCRYGYRPRTALHTFRTT in ovine sushi 19-20 and SAYWEKVYVQGQ in mouse sushi 7 were important sites for fH:BbCRASP-1 interaction. Comparative analysis of the amino acid sequences of sushi 6 of cattle, pig and human revealed that bovine and porcine fH lack methionine and arginine in HENMR pocket, that may impede formation of fH:BbCRASP-1 interface. Increasing numbers of FHBPs from animal and human pathogens are being discovered, thus results presented here can be important benchmark for study of other FHBPs:FH interactions.     

Development of simple and rapid elution methods for proteins from various affinity beads for their direct MALDI-TOF downstream application

Commercially available desalting techniques, necessary for downstream MALDI-TOF analysis of proteins, are often costly or time consuming for large-scale analysis. Here, we present techniques to elute proteins from various affinity resins, free from salt and ready for MALDI mass spectrometry. We showed that 0.1% TFA in 50% acetonitrile or 40% ethanol can be used as salt-free eluents for His-tagged proteins from variety of polyhistidine-affinity resins, while washing of resin beads twice with double-distilled water prior to the elution effectively desalted and recovered wide-range-molecular size proteins than commercially available desalting devices. Modified desalting and elution techniques were also applied for Flag- and Myc-tag affinity resins. The technique was further applied in co-precipitation assay, where the maximum recovery of wide-range molecular size proteins is crucial. Further, results showed that simple washing of the beads with double distilled water followed by elution with acetonitrile effectively desalted and recovered 150 kDa factor H protein of the sheep and its binding partner ~30 kDa BbCRASP-1 in co-precipitation assay. In summary, simple modifications in the desalting and elution strategy save time, labor and cost of the protein preparation for MALDI mass spectrometry; and large-scale protein purifications or co-precipitations can be performed with ease.