| Lectins are proteins that bind to the carbohydrate portion of glycoconjugates. The lectin IB4 binds to a subset of small and medium-sized neurons in the rat dorsal root ganglia (DRG). Cytotoxic targeting of IB4-binding neurons results in decreased responses to noxious heat and mechanical stimuli, strongly suggesting IB4-binding neurons play a role in nociception. However, the protein targets of IB4 are not known. Using one and two-dimensional gel electrophoresis, the laminin beta2 chain and the medium and light subunits of neurofilaments were identified as targets of the lectin IB4. Antisera were raised against alpha(1,3) galactosyltransferase (1,3GT) and isoglobotriaosylceramide synthase (iGb3S), the enzymes that synthesize the lectin IB4. Both of the enzymes were expressed in virtually all neurons that bind IB4, but were also found in a significant number of neurons that did not bind IB4. Virtually all neurons that expressed substance P also expressed iGb3S while about one half of the neurons that expressed substance P expressed 1,3GT. A subset of satellite cells also expressed 1,3GT while a subset of large DRG neurons expressed iGb3S. Changes in expression of these enzymes in models of nerve injury and development may help unravel the function of the IB4-binding epitope in DRG. Part of the proteome of the IB4-positive neurons was determined using a subtractive approach enabled by cytotoxic targeting of IB4-binding neurons followed by homogenization of DRG, isotope coded-affinity tagging labeling of the proteins in the resulting homogenate, and mass spectrometric analysis. Methodological modifications of chromatographic separation, mass spectrometry, and delaying the time of sacrifice after injection of the cytotoxin may help greatly enhance the number of proteins labeled with these methods. Knowledge of the differential expression of proteins in IB4-binding neurons compared to those that do not bind IB4 may help reveal functional differences between these neurons. |
