Isolation of monoclonal antibodies and cDNA and application in the characterisation of the human neuronal and general neuroendocrine marker protein, PGP9.5
Protein gene product 9.5 was discovered by two dimensional electrophoretic comparison of the soluble extracts of various human tissues and is a general marker for almost all neurons and cells of the diffuse neuroendocrine system or 'DNES' (which includes insulin and other hormone producing cells of the pancreatic islet, adrenal chromaffin cells, calcitonin producing cells of the thyroid, parathyroid hormone producing cells, and endocrine cells of the carotid body, gut and lung). These cells are of significance because of their functional similarities, and PGP9.5 is of importance since it represents one of a small set of general specific markers (neuron-specific enolase, chromogranin A, secretogranins I and II and synaptophysin) for neurons and the DNES. As such its characterization should shed light on the general function and development of such cells and it might also be exploited as a diagnostic marker (for instance in serum) during disease. Its abundance and solubility make it a promising candidate for the latter. This thesis describes the derivation of a series of molecular probes to pursue these objectives, along with concomitant information derived about PGP9.5 itself. Chapter 1 introduces the concept of 'molecular anatomy' as applied to establishing markers for neurons, the characteristics defining the DNES, and previous studies of PGP9.5. The aims of the thesis project are broadly outlined. Chapter 2 describes the characterization of the primary structure of about 50% of the protein PGP9.5, including description of amino acid analysis of the protein, fragmentation into peptides and determination of the amino acid sequences of these peptides. One peptide is noted to be homologous with the active site of rat neuron-specific enolase and other enolase isoenzymes. Chapter 3 describes the isolation of complementary DNA clones representing the human messenger RNA for PGP9.5 using oligonucleotide probes synthesized on the basis of information described in Chapter 2. Sequence analysis of these clones generated a complete messenger RNA and hence a complete deduced protein sequence also. Isolation and limited sequencing of human neuron-specific enolase complementary DNA were used to demonstrate that the relationship of these two general markers for neurons and the DNES is not one of evolutionary divergence. Indeed database searches did not identify any protein related to PGP9.5, although in the future its function may become apparent from other homologies as the databases expand. The deduced protein is consistent with the characterized protein, and contains 212 amino acids. The messenger RNA also contains 350 nucleotides of 3' untranslated RNA. The deduced structure has been employed (Chapter 5) to design and interpret experiments to characterize conformational instabilities of PGP9.5 discovered during the course of this work (below). Chapter 4 describes the production of two monoclonal antibodies to different epitopes of the PGP9.5 monomer and their characterization and use to construct a two site immunoradiometric assay for PGP9.5 applicable to body fluids. The apparent elevation in serum in a patient with a head injury is shown. The existence of PGP9.5 in at least two conformations, one immunoreactive and the other not reactive, is demonstrated: the implications are discussed and more detailed examination of the conformation of PGP9.5 is presented in Chapter 5. Chapter 5 utilizes a combination of DNA, protein and antibody techniques and data (Chapters 2, 3 and 4) to define a set of factors influencing the conformation of PGP9.5 in vitro, and perhaps in vivo, and also contains a preliminary description of the secondary structure and cysteine interactions within the protein. The epitope for one monoclonal antibody is delineated, and the generality of the conformational instability to many epitopes of PGP9.5 is shown using a polyclonal antiserum. The phenomenon is discussed in relation to other proteins in vitro and in vivo.