New Insights into Structure and Function of Type I Collagen Der Fakultät Energie-, Verfahrens- und Biotechnik der Universität Stuttgart zur Erlangung der Würde eines Doktor der Naturwissenschaften (Dr. rer. nat) Genehmigte Abhandlung Vorgelegt von Xin Xiong aus V. R. China Hauptberichter: Prof. Dr. H. Brunner Mitberichter: Prof. Dr. R. Ghosh Tag der mündlichen Prüfung: 29. August. 2008 Institut für Grenzflächenverfahrenstechnik der Universität Stuttgart 2008 New Insights into Structure and Function of Type I Collagen Der Fakultät Energie-, Verfahrens- und Biotechnik der Universität Stuttgart zur Erlangung der Würde eines Doktor der Naturwissenschaften (Dr. rer. nat) Genehmigte Abhandlung Vorgelegt von Xin Xiong aus V. R. China Hauptberichter: Prof. Dr. H. Brunner Mitberichter: Prof. Dr. R. Ghosh Tag der mündlichen Prüfung: 29. August. 2008 Institut für Grenzflächenverfahrenstechnik der Universität Stuttgart 2008 Contents Contents ....................................................................................................................... I Zusammenfassung ................................................................................................... IV Abstract .................................................................................................................... VI Abbreviations ........................................................................................................ VIII 1. Introduction ........................................................................................................... 1 1.1 Collagen ................................................................................................................ 1 1.1.1 Major component of extracellular matrix ............................................................ 1 1.1.2 Type I collagen and its structure ........................................................................... 3 1.1.3 Post-translational modifications of collagen and fibril formation ...................... 6 1.1.4 Self-assembly of collagen Type I and its aggregation .......................................... 7 1.1.5 Cell-ECM interaction and role of collagen ......................................................... 10 1.1.6 Collagen as 3D-biomaterial/biomatrix for tissue engineering ........................... 14 1.1.7 Isolation and purification of collagens ................................................................ 17 1.1.8 Rat tail tendon and Type I collagen ..................................................................... 18 1.1.9 Recombinant collagen ........................................................................................... 19 1.1.10 Applications of Type I collagen .......................................................................... 20 1.2 Mass spectrometry ............................................................................................ 21 1.2.1 Introduction to mass spectrometry (MS) ............................................................ 21 1.2.2 Tandem MS and protein identification ............................................................... 21 1.2.3 Mass spectrometry of collagen ............................................................................. 24 1.3 Goals of this study ............................................................................................. 25 2. Materials and Methods ....................................................................................... 26 2.1 Chemicals ........................................................................................................... 26 2.2 Buffers, solutions and Media. ........................................................................... 26 2.2.1 Commonly used buffers: Phosphate buffered saline (PBS), Tris HCl buffer. 26 2.2.2 Laemmli electrophoresis buffer ........................................................................... 26 2.2.3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ..... 27 2.2.4 Western blot buffers .............................................................................................. 27 2.3 Protein isolation from rat tail tendons (RTT) ................................................ 27 2.3.1 Isolation and gel-filtration .................................................................................... 27 2.3.2 Productivity determination .................................................................................. 28 2.4 Gel electrophoresis and Western blot ............................................................. 28 2.4.1 SDS-PAGE analysis ............................................................................................... 28 I Contents 2.4.2 Gel staining ............................................................................................................ 29 2.4.3 Western blot ........................................................................................................... 29 2.5 Chemical modifications of the collagen ........................................................... 30 2.5.1 Biotinylation ........................................................................................................... 30 2.5.2 Purification and concentration of biotinylated protein ..................................... 30 2.5.3 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) determination of free thiols in collagen ............................................................................................................................ 30 2.6 Cleavage of protein ........................................................................................... 31 2.6.1 Cyanogen bromide cleavage ................................................................................. 31 2.6.2 Trypsin digestion ................................................................................................... 31 2.6.3 Chymotrypsin and chymotrypsin/trypsin digestion ........................................... 32 2.6.4 Carboxypeptidase Y digestion .............................................................................. 32 2.7 Mass determination ........................................................................................... 32 2.8 Tandem Mass spectrometric (MS/MS) analysis ............................................. 33 2.8.1 Electrospray ionization MS/MS ........................................................................... 33 2.8.2 MALDI-TOF/TOF ................................................................................................ 33 2.9 Spectroscopic analysis ....................................................................................... 34 2.9.1 UV-spectroscopy .................................................................................................... 34 2.9.2 UV-CD-spectroscopy ............................................................................................. 34 2.9.3 Gaussian analysis of UV-VIS spectra .................................................................. 34 2.9.4 Histidine determination using UV-spectroscopy ................................................ 34 2.10 Reverse-phase-HPLC (RP-HPLC) ................................................................ 35 2.11 Atomic force microscopy. ............................................................................... 35 2.12 Scanning electron microscopy (SEM) ........................................................... 35 2.12.1 SEM of collagen ................................................................................................... 35 2.12.2 SEM of cells grown on different materials ........................................................ 35 2.13 Cell culture ....................................................................................................... 36 2.14. Real Time-PCR (RT-PCR) ............................................................................ 36 2.15 Type I collagen expression .............................................................................. 38 2.15.1 Cell culture and transfection .............................................................................. 38 2.15.2 Expression and purification ............................................................................... 39 3. Results .................................................................................................................. 40 3.1 Collagen extraction and SDS-PAGE analysis. ....................................................... 40 II Contents 3.2 Superose 12 gel filtration and SDS-PAGE analysis .............................................. 41 3.3 Mass determination and tandem MS/MS .............................................................. 43 3.4 UV-CD spectroscopy ................................................................................................ 50 3.5 UV-VIS spectroscopy of urea-extracted collagen .................................................. 52 3.6 Carboxypeptidase and chymotrypsin digestion .................................................... 53 3.7 Biotinylation of collagen .......................................................................................... 55 3.8 AFM and SEM of collagen ...................................................................................... 56 3.9 DTNB reaction and histidine measurement ........................................................... 57 3.10 Chromatographic analysis of cleaved collagen .................................................... 58 3.11 Phenotypic comparison of AC and UC upon cell cultures ................................