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Figure 22: Analysis of the lectican-containing fraction F18 from experiment (B). The MALDI-MS spectrum shows the permethylated glycan alditols. Monoisotopic masses corresponding to O-mannosyl glycans are underlined. Glycan fragments without a reduced end are indicated with an asterisk, arrows indicate signals derived by a loss of a methyl group and sodium (-36 Da). Signals marked by CS are a result of unspecific cleavage of chondroitin sulfate chains. The insert shows the Mascot results of protein identification in the respective fraction by ESI-MS/MS. 3 Results 51
3 Results In order to get more information on the O-mannosylation present in calf brain fraction F16 from experiment (B), which contained several O-mannosylated proteins (neurocan, versican, neurofascin and phosphacan (RPTPζ), see Table 5), was analyzed in more detail (in cooperation with Dr. Lefeber from the Radboud University Nijmegen, the Netherlands). The antibody IIH6 recognizes the laminin-binding epitope on α-DG (Ervasti & Campbell, 1993). A Western blot using this antibody revealed many signals in samples originating from muscle cells but no signal was detected in the calf brain derived sample (see Figure 23A). In addition, a laminin overlay assay was performed (see Figure 23B) which shows if the containing proteins bind laminin. But again no signal could be detected in the sample originating from calf brain. These findings indicate that the brain derived O-mannosylated proteins do not bind to laminin and accordingly that they are not modified with the laminin-binding glycan epitope present on α-DG from muscle. Table 5: Protein identification based on Mascot results of protein fraction F16 (experiment B). Protein (bovine) Score Neurocan core protein 1138 Versican core protein 705 Neural cell adhesion molecule 1 431 Neurofascin 301 Myelin-associated glycoprotein 270 Hyaluronate-binding protein 251 Excitatory amino acid transporter 1 227 Receptor-type tyrosine-protein phosphatase ζ 227 Tenascin-R 171 L1 cell adhesion molecule 145 Sei<strong>zu</strong>re protein 6 homolog 138 Sodium/potassium-transporting ATPase subunit α-1 136 Microtubule-associated protein 1B 108 Actin, cytoplasmic 1 97 52
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 and 30: 1 Introduction Together the above m
Page 31 and 32: 1 Introduction 1.4 Aim of this proj
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: 3 Results Subsequentially, the resu
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: Erklärung Ich versichere, dass ich
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