Toggle Main Menu Toggle Search

Open Access padlockePrints

Expression of mutant and wild-type TIMP3 in primary gingival fibroblasts from Sorsby's fundus dystrophy patients

Lookup NU author(s): Dr Christine Arris, Professor Debra Bevitt, Michael Clarke, Dr Norman Balfour-McKie


Full text for this publication is not currently held within this repository. Alternative links are provided below where available.


Gingival fibroblast cell lines were derived from Sorsby's fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations. These cell lines were grown in culture to study expression of the wild-type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3) alleles from a normal diploid cell type. Firstly, patient cells were found to co-express the wild-type and mutant TIMP3 alleles, S181C TIMP3 or E139X TIMP3, at the mRNA level using restriction fragment length polymorphism (RFLP) analysis. A SpeI RFLP for E139X TIMP3 is described. Low levels of endogenous TIMP3 protein expression were elevated using the natural polysaccharide calcium pentosan polysulfate (CaPPs) in combination with the cytokine IL-1α. Immunoblotting detected protein expression from both wild-type and mutant alleles, S181C TIMP3 or E139X TIMP3. S181C TIMP3 from these cells was found to dimerise and retain MMP2 inhibitory activity. To facilitate studies of the E139X TIMP3 protein, the allele was expressed using HighFive insect cells. In this cell type, the E139X TIMP3 was synthesised as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in gelatin zymography. Expression of mutant E139X or S181C TIMP3 protein from a normal diploid patient-derived fibroblast cell had no effect on either MMP2 or MMP9 expression or activation whilst transcribed from their normal promoter context. © 2003 Elsevier Science B.V. All rights reserved.

Publication metadata

Author(s): Arris CE, Bevitt DJ, Mohamed J, Li Z, Langton KP, Barker MD, Clarke MP, McKie N

Publication type: Article

Publication status: Published

Journal: Biochimica et Biophysica Acta - Molecular Basis of Disease

Year: 2003

Volume: 1638

Issue: 1

Pages: 20-28

ISSN (print): 0925-4439

ISSN (electronic): 0006-3002

Publisher: Elsevier BV


DOI: 10.1016/S0925-4439(03)00036-X

PubMed id: 12757930


Altmetrics provided by Altmetric