The role of the C1 and C2 a-domains in type VI collagen assembly.
Constructs of each of the three chains of type VI collagen were generated and examined in an in vitro transcription/translation assay supplemented with semipermeabilized cells. Each of the constructs when used in the in vitro system was shown to be glycosylated and to undergo intracellular assembly, the extent of which was determined by the nature of the C-terminal globular domains. All three chains containing the C1 domain formed monomers; however, the C2 domain was required for dimer and tetramer formation. In the case of the full-length alpha2(VI) chain, monomers, dimers, and tetramers formed in a time-dependent manner. Although the splice variant alpha2(VI)C2a could form monomers, it was unable to form dimers and tetramers. Similar results to the alpha2(VI) chain were found for the full-length alpha1(VI) chain, although assembly was at a slower rate. In the case of the alpha3(VI) chain containing both C1 and C2 domains only monomers were observed. Addition of the C3, C4, and C5 did not change this pattern. Homology modeling suggested that a 10-amino acid insertion in the C2 domain of the alpha3(VI) chain may interfere with dimer formation. A near full-length construct of the alpha3(VI) chain only formed monomers but was shown to facilitate tetramer formation in cotranslation experiments.
Ball
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SG
University of Manchester, School of Biological Sciences, Wellcome Trust Centre for Cell/Matrix Research, 2.205 Stopford, Manchester M13 9PT, United Kingdom.
Baldock
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C
Kielty
C M
CM
Shuttleworth
C A
CA
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D011994:Recombinant Proteins; D003094:Collagen; D006026:Glycoside Hydrolases; D010434:Pepsin A