RESEARCH ARTICLE Changes in albumin precursor and heat shock protein 70 expression and their potential role in response to corneal epithelial wound repair Shamim Mushtaq 1 , Zulfiqar A. Naqvi 1 , Anwar A. Siddiqui 2 , Carina Palmberg 3 , Jawed Shafqat 3 and Nikhat Ahmed 1 1 Neurochemistry Research Unit, Department of Biochemistry, University of Karachi, Karachi, Pakistan 2 Department of Biological and Biomedical Sciences, Juma Research Laboratory , Aga Khan University, Karachi, Pakistan 3 Department of Medical Biophysics and Biochemistry, Karolinska Institute, Stockholm, Sweden Many proteins displayed differential expression (either up- or down-regulation) when proteome of migrating and non-migrating epithelium was assessed using 2-DE and ESI-Q-TOF MS/MS. From the up-regulated set, we have identified for the first time a 69-kDa albumin precursor pro- tein with four peptides sequences and 70-kDa heat shock protein (hsp70) with one peptide in the active phase of cell migration (48 h) during the healing process. Western blot analysis was used to further characterize these proteins at different phases (24, 48 and 72 h) of healing. An increase in the mRNA expression (measured using RT-PCR) in the active migration phase (48 h) for albumin precursor and hsp70 was also observed. Furthermore, co-immunoprecipitation studies with anti-albumin precursor and anti-hsp70 antibodies, followed by immunoblotting with anti- fibronectin antibody demonstrated a novel and biologically relevant interaction between albumin precursor protein and fibronectin in corneal epithelial wound healing but not with hsp70. The increased gene and protein expression of albumin and hsp70 during the active phase of cell migration (48 h) in the corneal epithelium suggests their possible role in corneal wound healing. These findings may have broader implications for developing therapeutic strategies for treating wound healing disorders. Received: June 19, 2006 Revised: October 11, 2006 Accepted: October 17, 2006 Keywords: Albumin / Cornea / Heat shock protein 70 / Mass spectrometry / Wound healing Proteomics 2007, 7, 463–468 463 1 Introduction The role of proteins such as fibronectin, tenascin [1] lumican [2] and vinculin [3] in corneal re-epithelialization is well documented. Rapid regeneration of epithelium following injury is important as any alteration in the normal healing process results in detrimental outcomes ranging from cor- neal haze to ulceration and perforation. The molecular mechanisms that induce cell migration and modulate cell proliferation during wound healing are not yet fully under- stood. Several studies have implicated various proteins with the healing process involving a modulation of cellular be- havior such as cell migration and proliferation during nor- mal physiological state, tissue repair etc [4]. Albumin is the most abundant water-soluble protein in the human cornea [5, 6] and an important component of tear fluid [7]. In normal (unwounded) mouse corneas, albumin alone represents 13% of the water-soluble proteins whereas the corneal epithelium contains less than 1% [6]. These Correspondence: Professor Nikhat Ahmed, Department of Bio- chemistry, University of Karachi, Karachi-75270 Pakistan E-mail: nikhat_ahmed14@yahoo.co.uk Fax: 192-21-926-1340 Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydro- genase; HBSS, Hanks balanced salt solution; hsp70, heat shock protein70; RTase, RNA-dependent DNA polymerase; TBST , Tris buffered saline Tween 20 DOI 10.1002/pmic.200600446  2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com