Abstract
Cross-linking of proteins catalyzed by tissue transglutaminase has been suggested to play key roles in a variety of cellular events, including cell apoptosis and human pathogenesis (e.g. polyglutamine and Alzheimer diseases). It has often been suggested that tissue transglutaminase enhances aggregation and precipitation of damaged or pathogenic proteins. To ascertain whether this is accurate, we investigated the effects of tissue transglutaminase-catalyzed modulation on the aggregation of structurally damaged and unfolded proteins. Our results indicated that the aggregation and precipitation of some unfolded proteins were inhibited by transglutaminasecatalyzed reaction, although the effect was strongly dependent upon the target protein species. To elucidate the molecular events underlying the inhibitory effect, extensive analysis was performed with regard to reduced beta-lactoglobulin using a number of techniques, including chromatography and spectroscopy. The results indicated that cross-linking yields high molecular weight soluble polymers but inhibits the growth of insoluble aggregates. The cross-linked beta-lactoglobulin retained stable secondary structures with a hydrophobic core. We concluded that the transglutaminase-catalyzed intermolecular cross-linking did not necessarily enhance protein aggregation but could sometimes have a suppressive effect. The results of the present study suggested that tissue transglutaminase modifies aggregation and deposition of damaged or pathogenic proteins in vivo in a wide variety of manners depending on the target protein species and solution conditions.
Highlights
Protein clustering occurs in the highly concentrated intra- and extracellular solutions of the living body [1, 2]
Our results indicated that the aggregation and precipitation of some unfolded proteins were inhibited by transglutaminasecatalyzed reaction, the effect was strongly dependent upon the target protein species
To assess this problem from a general viewpoint, we examined four structurally unrelated proteins as substrates of type transglutaminase (tTG): bovine -lactoglobulin A (LG; -sheet-type), bovine serum albumin (BSA; ␣-helix-type), human ␣-lactalbumin (␣LA; ␣/-type), and bovine ␣-casein. LG, ␣LA, and BSA were unfolded by reducing the disulfide bonds, which might be the simplest model for biological protein damage
Summary
Protein clustering occurs in the highly concentrated intra- and extracellular solutions of the living body [1, 2]. Investigation of the factors that affect the intermolecular interactions of proteins are of primary importance from a biological viewpoint. Such information is essential for clinical control of abnormal protein aggregation, such as amyloid formation in patients with neurodegenerative diseases (8 –12). We examined whether the intermolecular cross-linking of damaged and structurally unfolded proteins enhances or inhibits their aggregation and precipitation To assess this problem from a general viewpoint, we examined four structurally unrelated proteins as substrates of tTG: bovine -lactoglobulin A (LG; -sheet-type), bovine serum albumin (BSA; ␣-helix-type), human ␣-lactalbumin (␣LA; ␣/-type), and bovine ␣-casein (natively unfolded type). This was followed by extensive analysis for LG as a typical case using biochemical and biophysical techniques
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