The aims of the current proposal are as follows: 1) to investigate possible in vivo functions of the oligodendrocyte-Schwann cell enzyme 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNP), 2) To continue our studies on the structure of CNP, restricting ourselves primarily to bovine, human and rat CNS CNP, 3) To examine posttranslational modification of the CNP molecule, including phosphorylation and methylation, 4) To further examine immunologically the relationships among central and peripheral nervous system sources of CNP, 5) To prepare monoclonal antibodies to human CNP for use in both structural and immunological studies, 6) To determine the cell specificity, relative binding affinity, titer, antibody class (IgG, IgM) of each of the antibodies produced and apply them to CNP localization and cross-reactivity studies in several differente cell types, myelin, and in brain tissue. The proposed research is a logical extension of our initial proposal on the initial isolation of CNP and the development of whole antisera to the purified enzyme and should add considerably to our knowledge of the structure, properties, distribution, and possible function(s) of CNP. Second, the antibodies produced to specific regions of the CNP molecule will permit more detailed direct immunological studies of the CNP molecule and its fragments not previously possible and may permit the distinction of specific cell types in brain and peripheral nerve. At this point, for example, we only know that isolated peripheral nervous system myelin contains a cross-reacting antigen when using rabbit anti-bovine CNP antisera and immunoblot techniques. Specific monoclonal antibodies to CNP should permit the ready identification of not only oligodendrocytes, but also isolated Schwann cells, Schwann cells in culture, as well as in sections of whole peripheral nerve. We are hopeful that some of the monoclonal antibodies produced may recognize CNS cells (oligodendrocytes) and not PNS (Schwann cells) and vice versa. Third, the use of specific monoclonal antibodies to human brain CNP along with either iodinated Staph A, labeled IgG, or Avidin-biotin complexes should permit the direct examination of the immunological relationships among erythrocyte, platelet and lymphocyte """"""""CNP"""""""" and the CNP(s) of the central and peripheral nervous system. Fourth, this study should result in the successful development of several monoclonal antibodies to CNP that will be made available to other investigators for further biochemical and immunological studies.
