Wang Yun-feng, Ding Jie, Bu Ding-fang, Wang Fang
Department of Pediatrics, Peking University First Hospital, Beijing 100034, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2004 Apr;36(2):185-9.
To analyze the protein structure encoded by COL4A5 gene with a missense mutation and to discuss the effect of gene mutation on basic structure and predicted secondary structure of the encoded protein.
A fragment of cDNA with a g.3246G T mutation resulting in p.G1015V from an X-linked Alport patient, and that of corresponding cDNA from a control were expressed in E.coli. The recombinant and mutant polypeptide was a fragment of COL4A5, containing 158 amino acid residues with a glycine to valine substitution mutation in it. The secondary structure of the two recombinant proteins was analyzed using circular dichroism(CD) spectroscopy.
CD spectra of the control exhibited a negative peak near 200 nm whereas that of the patient exhibited a negative peak near 220 nm. The magnitude of the negative peak of the patient decreased as compared with that of the control. Furthermore, secondary structure of the control polypeptide was mainly composed of beta-sheet and random coil without alpha-helix, whereas that of the patient presented 12.9% alpha-helix.
Not only local structure of the substitution site but also folding kinetics of the entire alpha5 chain may be changed due to Gly-->Val substitution in Alport syndrome. We speculate that the abnormally folded polypeptide chain may not be assembled into the triple helix and the network of type IV collagen, or may be assembled into loosen triple-helix then degraded easily, resulting in the pathognomonic ultrastructural changes of the glomerular basement membrane.
