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1 Introduction 1.4.2 Neurofascin Neurofascin (NF) is a cell surface protein of the immunoglobulin superfamily which is only expressed in nervous tissue (Kriebel et al., 2012). Several splice variants of NF have been identified with NF155 and NF186 being the predominant ones. The 155 kDa isoform is expressed by glia cells while NF186 is produced by neurons (Kriebel et al., 2011). They both consist of a short cytoplasmic tail, a single transmembrane intersection and a large extracellular part (see Figure 8) which is mainly composed of immunglobulin-like (Ig) and fibronectintype-III domains (Liu et al., 2011). The main difference between the two isoforms is an additional mucin-like domain present in NF186. Both variants play crucial roles in the compartmentalization of axonal proteins. While NF155 is required for paranodal junction assembly at the glial interface, NF186 is essential for the accumulation of the nodal protein complex needed for axonal conduction at nodes of Ranvier (Susuki & Rasband, 2008). In addition NF186 assembles the ECM of the AIS and links it to the cytoskeleton. From their experiments Hedstrom et al. assume that NF186 directly binds to brevican and thereby assembles a specialized brevican-based ECM at the AIS (Hedstrom et al., 2007). Intracellularly, only ankyrin G was identified as an interaction partner of NF186 so far (Susuki & Rasband, 2008) which in turn recruits specialized sodium channels to the nodes of Ranvier. Figure 8: Schematic view of the domain composition of neurofascin. NF harbors six immunoglobulin-like domains (Ig) which form a horseshoe-like structure and four fibronectin type-III domains (Fn). In addition, NF186 comprises a highly glycosylated mucin-like domain (Liu et al., 2011). 17
1 Introduction 1.4 Aim of this project Protein O-mannosylation is a rare posttranslational modification in mammals in general but was found in about one third of all O-glycosidically bound glycans in the brain. So far, only very few proteins have been identified to carry this modification and their O-mannosyl glycans alone cannot account for the high amount present in nervous tissues. In addition, patients suffering from genetic mutations of enzymes involved in the O-mannosylation pathway exhibit a severe neurological phenotype which indicates a still unknown but important role for O-mannosylation in the brain. Therefore, the aim of this study was the identification of new O-mannosylated proteins of the mammalian brain leading to new insights into the mechanism of initiation and the pathology of O-mannosylation defects. The isolation of O-mannosylated proteins ought to be accomplished from mouse and calf brains by means of general proteomics. Since the exposed glycan epitopes of mannose-based glycans are very similar to other N- and O-glycan epitopes immune affinity purification of O-mannosylated proteins was not possible. Therefore, a broad, unbiased proteomics approach was chosen in which mouse or calf brain lysates were to be fractionated via a newly developed sequence of preparative fractionation methods. The generated protein fractions should be analyzed regarding their O-glycan content by MALDI mass spectrometry of released O-glycans and proteins ought to be identified by LC-MS of tryptic peptides. Further information about the O-mannosylation sites was to be gained from glycopeptide analysis via LC-ESI-MS. 18
Page 1 and 2: Identification and Characterization
Page 3 and 4: I Index I Index of Contents I INDEX
Page 5 and 6: I Index FIGURE 13: WESTERN BLOT OF
Page 7 and 8: I Index I.III List of Abbreviations
Page 9 and 10: I Index PBS PMT PNN Phosphate Buffe
Page 11 and 12: I Index I.III.II Amino acids - thre
Page 13 and 14: I Index II Zusammenfassung Die O-Ma
Page 15 and 16: 1 Introduction glycosidically linke
Page 17 and 18: 1 Introduction glycans, complex-typ
Page 19 and 20: 1 Introduction O-glycosylation (typ
Page 21 and 22: 1 Introduction Figure 3: Schematic
Page 23 and 24: 1 Introduction the sites and functi
Page 25 and 26: 1 Introduction posttranslational pr
Page 27 and 28: 1 Introduction transfer of mannose
Page 29: 1 Introduction Together the above m
Page 33 and 34: 2 Materials and methods Lysis and W
Page 35 and 36: 2 Materials and methods 2.1.3.2 Sec
Page 37 and 38: 2 Materials and methods 2.2.1.7 Fra
Page 39 and 40: 2 Materials and methods (see 2.2.2.
Page 41 and 42: Figure 9: O-Glycome of mouse brain.
Page 43 and 44: 3 Results Table 1: Sequential extra
Page 45 and 46: 3 Results that this protein might b
Page 47 and 48: 3 Results 3.2 O-Mannosylation on ne
Page 49 and 50: 3 Results identified by several 361
Page 51 and 52: 3 Results Figure 15: Fragmentation
Page 53 and 54: 3 Results Table 2: Protein identifi
Page 55 and 56: 3 Results Three other O-mannosyl gl
Page 57 and 58: 3 Results 3.3 O-Mannosylation of le
Page 59 and 60: 3 Results 3.3.2 Recombinant express
Page 61 and 62: 3 Results identified just as the gl
Page 63 and 64: 3 Results Subsequentially, the resu
Page 65 and 66: 3 Results In order to get more info
Page 67 and 68: 3 Results brain-specific protein ne
Page 69 and 70: 4 Summary and outlook malformations
Page 71 and 72: 4 Summary and outlook O-mannosylati
Page 73 and 74: 4 Summary and outlook Figure 24: Sc
Page 75 and 76: 5 References Bleckmann, C., Geyer,
Page 77 and 78: 5 References Frischknecht, R. & Sei
Page 79 and 80: 5 References Kriebel, M., Metzger,
Page 81 and 82:
5 References Nizet, V. & Esko, J. (
Page 83 and 84:
5 References Susuki, K. & Rasband,
Page 85 and 86:
6 Appendix 6 Appendix Table 6: Summ
Page 87 and 88:
6 Appendix Figure 27: Comparison be
Page 89 and 90:
6 Appendix Table 8: Protein identif
Page 91 and 92:
6 Appendix Figure 32: O-Glycan anal
Page 93 and 94:
6 Appendix Figure 34: ESI-MS/MS of
Page 95 and 96:
6 Appendix Figure 38: Analysis of t
Page 97 and 98:
6 Appendix Figure 40: Calf brain-de
Page 99 and 100:
6 Appendix Table 13: Protein identi
Page 101 and 102:
6 Appendix Table 14: Summary of hum
Page 103:
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