ABSTRACT
Lectin affinity chromatography (LAC) offers a tool that aids purification of cell surface glycoconjugates in sufficient quantities so that studies addressing their structural elucidation could be carried out. It has several advantages over the conventional biochemical methods, such as immunoprecipitation and/or immunoaffinity chromatography, used for the purification of various glycoconjugates. Serial LAC (SLAC) not only helps establish the identity of a glycoprotein or allows purification of a glycoprotein to homogeneity from among a mixture of glycoproteins, but it also successfully resolves the microheterogeneity in these glycoproteins, which is an otherwise impracticable problem to address. Specific cases of the altered expression and maintenance of microheterogeneity of some of the glycoproteins in pathological conditions vis a vis during normal biology are presented. The application of LAC in (i) itself, (ii) a serial fashion, and (iii) conjunction with other techniques such as two-dimensional electrophoresis, capillary electrophoresis, mass spectrometry, etc. in the diagnosis of certain pathological conditions, and the possibility of using this knowledge in designing treatments for various diseases, is discussed.
Subject(s)
Chromatography, Affinity/methods , Diagnostic Techniques and Procedures , Lectins , Cell Membrane/chemistry , Glycoconjugates/isolation & purification , Glycoproteins/isolation & purification , HumansABSTRACT
The thermodynamics of interaction of two model peptides melittin and mastoparan with bovine brain calmodulin (CAM) and a smaller CAM analogue, a calcium binding protein from Entamoeba histolytica (CaBP) in 10 mM MOPS buffer (pH 7.0) was examined using isothermal titration calorimetry (ITC). These data show that CAM binds to both the peptides and the enthalpy of binding is endothermic for melittin and exothermic for mastoparan at 25 degrees C. CaBP binds to the longer peptide melittin, but does not bind to mastoparan, the binding enthalpy being endothermic in nature. Concurrently, we also observe a larger increase in alpha-helicity upon the binding of melittin to CAM when compared to CaBP. The role of hydrophobic interactions in the binding process has also been examined using 8-anilino-1-naphthalene-sulphonic acid (ANS) binding monitored by ITC. These results have been employed to rationalize the energetic consequences of the binding reaction.
Subject(s)
Calcium-Binding Proteins/metabolism , Calmodulin/analogs & derivatives , Calmodulin/metabolism , Peptides/metabolism , Amino Acid Sequence , Anilino Naphthalenesulfonates/metabolism , Animals , Calorimetry , Circular Dichroism , Entamoeba histolytica/metabolism , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Intercellular Signaling Peptides and Proteins , Melitten/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Recombinant Proteins , Sequence Homology, Amino Acid , Thermodynamics , Wasp Venoms/metabolismABSTRACT
Mycobacterium leprae, the causative agent of leprosy, specifically invades and destroys the peripheral nerve, which results in the main clinical manifestation of the disease. Little is known about the bacteria-nerve protein interaction. We show in the present work that M leprae binds to a 25 kDa glycoprotein from human peripheral nerve. This protein is phosphorylatable and it binds to lectins which have alpha-mannose specificity. This M leprae-protein interaction could be of importance in the pathogenesis of leprosy.
Subject(s)
Glycoproteins/metabolism , Mycobacterium leprae/metabolism , Nerve Tissue Proteins/metabolism , Tibial Nerve/metabolism , Autoradiography , Bacterial Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Glycoproteins/chemistry , Humans , Lectins/metabolism , Molecular Weight , Mycobacterium bovis/metabolism , Nerve Tissue Proteins/chemistry , Phosphorylation , Protein BindingABSTRACT
To understand Mycobacterium leprae-peripheral nerve interaction, we have investigated the binding of M. leprae to rat peripheral nerve proteins in an in vitro model using 32P-phosphorylated proteins of the peripheral nerve. Intact M. leprae binds to a major phosphorylated protein of 28-30 kDa and, to a minor extent, to a few proteins of molecular weight 45-55 kDa. This binding was more specific for M. leprae since only insignificant binding was observed with other bacteria, such as M. bovis or Escherichia coli. M. leprae did not show binding to several phosphorylated proteins of the rat brain. The 28-30-kDa binding protein of the rat peripheral nerve was found to be a glycoprotein by concanavalin A-Sepharose column chromatography.
