Daniel A. KIRSCHNER - US grants

Overall objectives: To characterize the molecular organization of lipids and proteins in myelin membranes; and to define the molecular interactions that stabilize the membrane packing in normal myelin and in myelin that has been modified by in vitro chemical treatment or by genetic or pathological conditions. A correlation of biophysical and biochemical techniques (including X-ray diffraction, electron microscopy, SDS-polyacrylamide gel electrophoresis, immunobloting, and thin-layer chromatography) applied to different types of specimens (including whole unfixed or fixed tissue, tissue homogenates, and reconstituted model systems of lipids and proteins) will be used to address the following specific questions: (1) Where are specific lipids and proteins localized in the myelin membrane? The effects of metal cations on the structure of intact myelin will be corrected with their binding to isolated myelin lipids and proteins and to reconstituted multi-layers of lipids and proteins. Changes in myelin composition of neurological mutant mice will be correlated with the structure of myelin membranes in these mutants. (2) What is the structural and chemical basis of the stability of membrane packing in myelin? Myelin fron neurological mutant mice will be surveyed for possible correlations between altered composition and changes in membrane packing. The chemical composition of the nerve sheaths from certain invertebrates and phylogenetically-older vertebrates will be related to the ultrastructure of the membrane assemblies in these sheaths. The dependence of membrane packing on lipid and protein composition will be determined from an analysis of multilayer structure in reconstituted model systems. (3) How do the specialized junctions in the myelin sheath relate to its stability? Attempts will be made to detail the organization and stability of paranodal axo-glial junctions by X-ray diffraction from unfixed nerve regions which are enriched in these structural differentiations of myelin. The meridional diffraction from central nervous system myelin will be analyzed with respect to its possible origins from the tight junctions peculiar to this myelin and from the organization of lipids and proteins in the plane of the myelin membrane.

The hallmark morphological abnormalities of Alzheimer's disease (AD) are deposits of amyloid fibers, which constitute neuritic plaques and cerebrovascular amyloid, and paired helical filaments (PHF), which constitute neurofibrillary tangles. The formation and accumulation of these fibrous assemblies profoundly affect memory, language and behavior. Our research is focused on the major protein of AD amyloid, called beta/A4, and on the microtubule-associated protein tau, which is a major cytoskeletal protein and an integral component of PHF. Using a correlation of results from X-ray diffraction, electron microscopy and Fourier-transform infrared spectroscopy, we plan to provide a detailed description of the molecular and macromolecular structures of the fibrous assemblies of AD. In addition, we will determine the forces that promote their formation, the factors that underlie their stability, and the specific residues that promote their folding. To accomplish our objective, this proposal focuses on the following three Specific Aims: (1) To test specific hypotheses regarding the self-assembly and stability of synthetic beta/A4 homologues, and their interactions with tissue components that are thought to promote their formation. The hypotheses are: (i) electrostatic interactions involving the formation of a salt-bridge within the core region of beta/A4 are crucial in stabilizing the beta-pleated sheets of the fibrils; (ii) a single amino acid residue change in the sequence of beta/A4 accounts for the extremely high level of aggregation of amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of the Dutch type; (iii) alpha- anti-chymotrypsin (ACT) binds specifically to and alters the structure of beta/A4, thus affecting its proteolytic processing; and (iv) sulfated proteoglycans of brain extracellular matrix promote the polymerization and/or aggregation of AD amyloid by electrostatic interactions involving the sulfate groups. (2) To investigate the assembly of PHF from modified forms of microtubule-associated tau protein. A microdialysis technique originally developed for crystallizing membraneproteins will be used to manipulate and control the gradual formation of PHF-like filaments. Suitable samples will be subjected to X-ray diffraction, which is expected to provide new details of PHF organization that has so far resisted such analysis. (3) To develop molecular models of these fibrillar assemblies based on constraints imposed by the biophysical and ultrastructural results. We will continue to develop analytical tools that will be used to interpret the fiber diffraction patterns and to model details of fibril organization at the molecular level. In achieving our objective, we hope to develop sufficient understanding for the eventual rational development of diagnostic/therapeutic strategies for AD, for related neurodegenerative diseases, and for amyloidoses in general.

Overall objective: To characterize the molecular organization of lipids and proteins in nerve myelin membranes; and to define the molecular interaction that stabilize the membrane arrays. A correlation of biophysical and biochemical technique (including X-ray diffraction, electron microscopy, SDS polyacrylamide gel electrophoresis, immunoblotting, and thin-layer chromatography) applied to different types of specimens (including whole unfixed or fixed tissue, tissue homogenates, tissue fractions, and model systems of lipids and proteins) will be used to address the following specific questions (1) What is the fine-structure and composition of the interlamellar tight junctions of CNS myelin? Computer image analysis will be carried out on electron micrographs. SDS-PAGE/immunoblotting will be used to identify proteins in tissue fractions that are enriched in the junctions. X-ray patterns will be recorded from intact CNS myelin after contrast enhancement of the junctions, and also from tissue fractions enriched in the junctions. (2) What is the arrangement of lipids and proteins in myelin? Correlation between biochemical and X-ray measurements will be made for selected neuron logical mutants of the mouse, certain phylogenetically-older vertebrates, different nerves from within the peripheral nervous system, and isolated myelin preparations. Structural modifications of myelin induced by specific metal cations will be analyzed. The structure and inter-bilayer interaction of multilayers reconstituted from non-denatured proteolipids protein (PLP) plus myelin lipids, and PLP plus myelin basic protein will be analyzed. (3) What is the structural basis of demyelination? The structural and biochemical effects of calcium-ionophore and complement on CNS myelin will be determined. The integrity of interlamellar tight junctions will be examined in white matter from animals having experimental allergic encephalomyelitis and from autopsy material of humans with multiple sclerosis.

Overall objective: To characterize the molecular organization of lipids and proteins in nerve myelin membranes; and to define the molecular interaction that stabilize the membrane arrays. A correlation of biophysical and biochemical technique (including X-ray diffraction, electron microscopy, SDS polyacrylamide gel electrophoresis, immunoblotting, and thin-layer chromatography) applied to different types of specimens (including whole unfixed or fixed tissue, tissue homogenates, tissue fractions, and model systems of lipids and proteins) will be used to address the following specific questions (1) What is the fine-structure and composition of the interlamellar tight junctions of CNS myelin? Computer image analysis will be carried out on electron micrographs. SDS-PAGE/immunoblotting will be used to identify proteins in tissue fractions that are enriched in the junctions. X-ray patterns will be recorded from intact CNS myelin after contrast enhancement of the junctions, and also from tissue fractions enriched in the junctions. (2) What is the arrangement of lipids and proteins in myelin? Correlation between biochemical and X-ray measurements will be made for selected neuron logical mutants of the mouse, certain phylogenetically-older vertebrates, different nerves from within the peripheral nervous system, and isolated myelin preparations. Structural modifications of myelin induced by specific metal cations will be analyzed. The structure and inter-bilayer interaction of multilayers reconstituted from non-denatured proteolipids protein (PLP) plus myelin lipids, and PLP plus myelin basic protein will be analyzed. (3) What is the structural basis of demyelination? The structural and biochemical effects of calcium-ionophore and complement on CNS myelin will be determined. The integrity of interlamellar tight junctions will be examined in white matter from animals having experimental allergic encephalomyelitis and from autopsy material of humans with multiple sclerosis.

Overall objective: To characterize the molecular organization of lipids and proteins in nerve myelin membranes; and to define the molecular interaction that stabilize the membrane arrays. A correlation of biophysical and biochemical technique (including X-ray diffraction, electron microscopy, SDS polyacrylamide gel electrophoresis, immunoblotting, and thin-layer chromatography) applied to different types of specimens (including whole unfixed or fixed tissue, tissue homogenates, tissue fractions, and model systems of lipids and proteins) will be used to address the following specific questions (1) What is the fine-structure and composition of the interlamellar tight junctions of CNS myelin? Computer image analysis will be carried out on electron micrographs. SDS-PAGE/immunoblotting will be used to identify proteins in tissue fractions that are enriched in the junctions. X-ray patterns will be recorded from intact CNS myelin after contrast enhancement of the junctions, and also from tissue fractions enriched in the junctions. (2) What is the arrangement of lipids and proteins in myelin? Correlation between biochemical and X-ray measurements will be made for selected neuron logical mutants of the mouse, certain phylogenetically-older vertebrates, different nerves from within the peripheral nervous system, and isolated myelin preparations. Structural modifications of myelin induced by specific metal cations will be analyzed. The structure and inter-bilayer interaction of multilayers reconstituted from non-denatured proteolipids protein (PLP) plus myelin lipids, and PLP plus myelin basic protein will be analyzed. (3) What is the structural basis of demyelination? The structural and biochemical effects of calcium-ionophore and complement on CNS myelin will be determined. The integrity of interlamellar tight junctions will be examined in white matter from animals having experimental allergic encephalomyelitis and from autopsy material of humans with multiple sclerosis.

We have established a mass spectrometric differential peptide display method to detect changes of peptides directly in tissue homogenates, after application of a defined physiological stimulus. We employed matrix-assisted laser desorption mass spectrometry (MALDI-TOF MS), using a single reference peptide in combination with careful scanning of the whole crystal rim of the matrix/analyte preparation, to determine in a semi-quantitative way the changes in the levels of peptides in an unfractionated methanol extract of the rat neurointermediate lobe (NIIL) after salt-loading of the animals. In the salt-loaded rat, a considerable decrease in the intensities of 6 molecular species of the NIIL extract was observed as compared to the control situation. These molecular ions corresponded to the masses of vasopressin, oxytocin, the neurophysins, and an unidentified molecule with a protonated mass of 5930 Da. Purification of this unidentified molecule, followed by Edman degradation, yielded the amino acid sequence of the carboxyl terminal glycopeptide of the vasopressin precursor. Using tandem MS, the major carbohydrate on the peptide was determined to consist of HeX3HexNAc5Fuc. To illustrate the potential of an MS-based differential display strategy, vasopressin and oxytocin were structurally characterized by direct tandem MS analysis of the molecular ions in the unfractionated NEL extract. Moreover, by means of a combination of Edman degradation and NLkLDI-MS analysis of the enzymatic digest of purified peptides (i.e., the neurophysin derived from propressophysin as well as two truncated analogs of the neurophysin derived from prooxyphysin) were structurally identified. Finally, using direct NLkLDI mass analysis of n-~cropunches of the neural lobe and of single melanotrope cells, it was possible to assign subsets of peptides to various distinct anatomical compartments of the NI1L..

DESCRIPTION (Applicant's abstract): The overall objective of this application is to define the molecular organization of myelin, the diverse adhesive mechanisms that stabilize its multilamellar sheath, and what defects in its organization and molecular constituents may lead to dysmyelination or demyelination such as occur in certain peripheral neuropathies and in multiple sclerosis. The approach is based on a correlation of results from X-ray crystallography and solution scattering, membrane diffraction, and electron microscopy. The specific aims, which are focused on the structure and role of the major transmembrane protein of peripheral nerve myelin (P0-glycoprotein) are: (1) to determine the three-dimensional structure of P0-glycoprotein for human and Xenopus. The proteins analyzed will include recombinant molecules having the native amino acid sequence, as well as those having specific sequence alterations known to occur in human peripheral neuropathies. These studies will inform about the atomic structure of P0 and about the crystal contacts or adhesion interfaces that may be responsible for the role of this protein in myelin formation and stability. (2) To characterize the protein-protein interactions between nearest-neighbor P0 molecules in a membrane mimetic environment using small-angle X-ray scattering. The membrane protein will be solubilized in aqueous solutions of detergents at very low concentration, and solution scattering will be undertaken using a synchrotron X-ray source. These studies will provide information about the interprotein molecular contacts that P0 molecules make in a milieu that more closely resembles its native environment (the lipid bilayer of the myelin membrane) than does a crystal. (3) To evaluate the membrane-membrane interactions in myelin of Xenopus peripheral nerves. Determining the pH- and ionic strength-dependence of membrane structure and packing in dissected peripheral nerves that have been incubated at different will provide strong constraints for testing hypotheses about the adhesion mechanisms of P0 at both the cytoplasmic and extracellular membrane appositions. This hierarchy of experimental objectives will allow the investigator to uniquely correlate structural data from the atomic to the molecular, to the membrane level, and thus contribute to an understanding of the structural biology of a membrane protein that figures significantly in both health and disease.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Myelin protein zero (P0) is a major protein for the formation and maintenance of myelin. It is an integral membrane glycoprotein containing a single N-glycosylation site. The mutations and deletions in the P0 gene correlate with hereditary peripheral neuropathies. The glycans of P0 play an important role in cell-to-cell adhesion. Studies on bovine, murine and human P0 suggest that P0 exists as tetramers in the membrane. However, the predominant form of P0 in Xenopus is a dimer. A mass spectrometry strategy including SDS-polyacrylamide-gel-electrophoresis (SDS-PAGE) was utilized to determine interactions underlying the P0 dimer and the post-translational-modification profiles of the monomers and oligomers. These results should contribute to understanding of the phylogenetic development of P0's adhesion role in myelin. Xenopus P0 oligomers were purified by SDS-PAGE. Bands of interest were excised and reloaded in the mini-slab gel to study the interaction(s) underlying the stability of the oligomers. In order to determine the glycoform profiles and verify the assignments of the protein present in each fraction, the monomers and oligomers were in-gel deglycosylated with PNGase F, and then in-gel digested with proteases. The peptides and the oligosaccharides were characterized using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS), nano ESI-TOF MS and custom built ESI-qQq-FTMS. The carbohydrates and peptides of interest were sequenced by MS/MS. In previous studies, a number of physical and chemical methods were used in attempts to disrupt the oligomers, including temperature, pH, detergents, and denaturants, but these approaches seemed only to increase the amount of oligomers relative to the monomer. In this study, when the dimer bands separated by SDS-PAGE were reloaded in a gel, it was observed that half of the dimer decomposed into the monomer. Furthermore, it was observed that almost all of the dimer dissociated into the monomer when a Mini-prep-cell" was used to purify the proteins from the mixture. Based on these observations, we propose that the Xenopus PNS P0 dimer is stabilized by non-covalent interaction(s) and there is equilibrium between the non-covalent dimer and the monomer. Using protease in-gel digestion, followed by MALDI-MS and ESI-MS/MS, the initially assigned dimer and the monomer were confirmed to be forms of P0, based on ca. 60% sequence coverage. In order to explore the factors affecting the folding and aggregation of Xenopus P0, the post-translational-modifications (PTM) of the P0 monomers and oligomers were investigated. Specific acylation and glycosylation patterns were observed and the structures of the modifying groups were determined. MALDI-MS showed that the Cys152 was acylated with stearic acid (C18:0). Xenopus P0 was found to contain a series of high mannose, hybrid and complex glycans. The major glycans were determined by ESI FTMS/MS (CAD) with high accuracy. In addition, Asn92 was confirmed as the single fully occupied N-glycosylation site by MALDI MS and ESI MS/MS. The PTMs in Xenopus P0 differ from those reported for other species such as cattle. The unique acylation and glycosylation may underlie the unusual folding and aggregation behaviour of P0 from Xenopus laevis. In addition, P0 dimer and monomer have slightly different glycoforms, and slightly different glycoform distributions;this may contribute to the differences in their aggregation. Our results will aid investigation of atypical adhesion in peripheral myelin and thus should contribute to understanding of the phylogenetic development of P0's adhesion role in myelin. Two manuscripts based on these results were recently published, one appeared in the previous reporting year and the second during the current year.