Glycosylated Threonine but not 4-Hydroxyproline Dominates the Triple Helix Stabilizing Positions in the Sequence of a Hydrothermal Vent Worm Cuticle Collagen |
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| Institution: | 1. Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany;2. Shriners Hospital for Crippled Children, Oregon Health Sciences, Portland, OR 97201, USA;3. Department of Biochemistry and Molecular Biology, Oregon Health Sciences, Portland, OR 97201, USA;4. UPR CNRS 9042, URM 7 Université Pierre et Marie Curie, F-75005, Paris, France;1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA;2. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA;3. Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA;4. Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA;3. From the Department of Life Sciences, Research Center for Cellular Homeostasis, Ewha Womans University, Seoul 120-750, Korea,;4. the Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea, and;5. the College of Medicine, Department of Physiology and Biophysics, Inha University, Incheon 402-751 Korea;1. Department of Surgery, University of Wisconsin, Madison, WI, 53705, USA;2. Department of Biomedical Engineering and Wisconsin Institutes for Discovery, University of Wisconsin–Madison, 1550 Engineering Drive, 3144 Engineering Centers Building, Madison, WI, 53715, USA;3. Department of Materials Science and Engineering, University of Wisconsin–Madison, Madison, WI, 53715, USA;4. University of Texas Health Sciences Center San Anto-Division, 3500 Camp Bowie Blvd, Fort Worth, TX, 76107, USA;5. Department of Surgery, University of Alabama – Birmingham, Birmingham, 1808 7th Avenue South / Suite 502, 35233, AL, USA |
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| Abstract: | The cuticle collagen of the vestimentiferanRiftia pachyptila, an organism which is endemic to deep-sea hydrothermal vents, has several unusual properties including an extraordinary length (1.5 μm), a high thermal stability (37°C) in spite of a low 4-hydroxyproline content and an atypically high threonine content (20 mol%). We have now purified the constituent chain of cuticle collagen and show that it contains about 40% carbohydrate, which is mainly galactose, indicating that the chain has a molecular mass of approximately 750 kDa. Several large (30 to 150 kDa) fragments, which all contained carbohydrate, could be produced by cleavage with endoproteinase Lys-C, bacterial collagenase and cyanogen bromide (CNBr). Edman degradation of these and several smaller fragments was used to determine about 3000 sequence positions comprising 60% of the total triple-helical sequence. This demonstrated mainly typical Gly-X-Y triplet repeats with a few imperfections and a longer N-terminal non-triplet sequence. Most of the 4-hydroxyproline was found in triplet position X, where it decreases the stability of the triple helix. About 40% of the Y positions could not be identified, which correlated with a low abundance of threonine in the sequence and the demonstration of threonine in these positions after deglycosylation of several peptides by treatment with hydrofluoric acid. Matrix-assisted laser desorption ionisation mass spectrometry of selected peptides indicated that the blocked threonine residues are occupied by chains of one, two or three hexoses (presumably galactose). These glycosylated threonine residues in Y positions are therefore likely to replace 4-hydroxyproline as the major contributor to triple helix stabilization. Studies with a synthetic (Gly-Pro-Thr)10oligopeptide demonstrated a low thermal stability of its triple helix which emphasizes a crucial role of glycosylation for stabilization. |
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