University of Oulu

Type XIII collagen : organization of the mouse gene, generation of three genetically engineered mouse lines by homologous recombination, and biochemical studies on the molecular properties of the type XIII collagen protein

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Author: Latvanlehto, Anne
Organizations: University of Oulu, Faculty of Medicine, Department of Medical Biochemistry and Molecular Biology
University of Oulu, Biocenter Oulu
Format: eBook
Online Access: PDF Full Text (PDF, 0.9 MB)
Persistent link: http://urn.fi/urn:isbn:9514275934
Language: English
Published: 2004
Publish Date: 2004-11-23
Thesis type: Doctoral Dissertation
Defence Note: Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium of the Department of Pharmacology and Toxicology, on December 2nd, 2004, at 12 noon.
Reviewer: Professor Donald Gullberg
Doctor Manuel Koch
Description:

Abstract

Genomic clones covering the entire mouse type XIII collagen gene (Col13a1) were isolated, and the complete exon-intron organization was characterized. The gene was found to be about 135 kb in size and to locate in the mouse chromosome 10. Comparison of gene structures and promoter regions between man and mouse indicated high conservation between the two species.

In order to understand the biological function of type XIII collagen, a mouse line that expresses type XIII collagen with replacement of the cytosolic and transmembrane domains by a short, non-descript sequence was generated using homologous recombination. Expression of this aminoterminally altered type XIII collagen led to mild but progressive muscular atrophy in mice. The integrity of muscle cells was disturbed and the basement membrane showed areas of detachment from the sarcolemma as well as clearly altered structure at myotendinous junctions. These phenotypical changes were, nevertheless, local, since the majority of the muscle was intact. The results show the importance of the membrane anchorage of the type XIII collagen protein in adhesion and, consequently in the maintenance of muscle integrity.

To study the significance of various regions of type XIII collagen, wild-type and mutant forms of the protein were produced recombinantly in insect cells. The transmembrane domain and the adjacent region of ectodomain were found to be crucial for the formation of type XIII collagen molecules with all of the three collagenous domains in trimeric conformation. A previously characterized conserved membrane-proximal region of the ectodomain was predicted to harbour a coiled-coil conformation. This was suggested to begin in the transmembrane domain of type XIII collagen and in several other collagenous transmembrane proteins. Type XIII collagen lacking this coiled-coil sequence was correctly folded with respect to its central COL2 and carboxylterminal COL3 domains. Between them, in the NC3 domain, a second coiled-coil sequence was found, and this was suggested to function as a second association region. The second coiled-coil sequence was found to be conserved in the two other type XIII collagen-like molecules as well.

To obtain precise information about the location and level of type XIII collagen expression, a reporter mouse line synthesizing a recombinant protein with the cytoplasmic and transmembrane portions of type XIII collagen linked in-frame with the β-galactosidase enzyme was generated. The reporter mice showed high expression of type XIII collagen at neuromuscular junctions and in the periosteum of bone. Interestingly, the growth of the reporter mice was reduced at puberty. Their long bones showed a decreased diameter and impaired mechanical properties. In addition, their peripheral nerves showed areas of detachment from muscle cells at neuromuscular junctions. These results provide further evidence for the role of type XIII collagen in cell adhesion. They also show the importance of proper adhesion conducted by type XIII collagen in signaling between the extracellular matrix and cells and in the cellular response.


Series: Acta Universitatis Ouluensis. D, Medica
ISSN-E: 1796-2234
ISBN: 951-42-7593-4
ISBN Print: 951-42-7592-6
Issue: 811
Subjects:
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