Formation of amyloid-like fibrils upon limited proteolysis of bovine α-lactalbumin
Introduction
We have recently discovered that α-lactalbumin (α-LA), the second most abundant protein in the whey fraction of bovine milk, self-assembles into long, uniform, tubular strands, about 20 nm in diameter, upon limited proteolysis by a protease from Bacillus licheniformis (BLP). The tubular strands were building a gel network when hydrolysis was performed with solutions containing 100 g L−1 of α-LA (Ipsen, Otte, & Qvist, 2001). Transmission electron micrographs of these tubular strands show similarities to negatively stained electron migrographs of amyloid fibrils, which raised the question whether the tubular strands resulting from α-LA hydrolysis actually had an amyloid character. ‘Amyloid’ is the fibrillar protein deposit associated with diseases such as Alzheimer's disease, Parkinson's disease, the prion diseases and other neurological disorders (Morozova–Roche et al., 2000; Rochet & Lansbury, 2000). Amyloid fibrils are a few nm thick, branched or unbranched, often twisted, fibrils characterised by a high content of β-sheet and binding of the dyes Thioflavin T and Congo Red (Dobson, 1999; Morozova-Roche et al., 2000; Rochet & Lansbury, 2000; Couzin, 2002; Khurana et al., 2003). Understanding how they are formed, and subsequently how their formation can be prevented, would have significant medical implications.
A large number of recent studies have been aimed at improving our understanding of the mechanisms of fibril assembly, and it seems that partial unfolding of the native protein structure is a key step in formation of protofibrils and fibrils. It is not known what triggers the first conformational change in vivo. However, in vitro studies have shown that partial unfolding and fibril formation may be induced by lowering of pH or addition of alcohols (Krebs et al., 2000; Goers, Permyakov, Permyakov, Uversky, & Fink, 2002). Within the last few years it has become clear that not only proteins and peptides involved in the above-mentioned diseases, but many quite different proteins, under appropriate conditions, form amyloid fibrils (Krebs et al., 2000; Khurana et al., 2003). Milk proteins, such as β-Lg (β-lactoglobulin), immunoglobulins, insulin, and even α-LA have been shown to form fibrils at low pH (Carotta, Bauer, Waninge, & Rischel, 2001; Goers et al., 2002; Khurana et al., 2003). Fibril formation of both proteins and peptides has also been observed in vitro under more physiological conditions (Blackley et al., 2000; Rochet & Lansbury, 2000; Olesen & Dagø, 2000; Morozova-Roche et al., 2000; Shtilerman, Ding, & Lansbury, 2002; Reches, Porat, & Gazit, 2002; Vauthey, Santoso, Gong, Watson, & Zhang, 2002).
In order to understand the formation of tubular fibrils from α-LA by limited hydrolysis at neutral pH, the limiting conditions for formation of the α-LA-derived gels consisting of tubular strands were determined with respect to pH and critical gelation concentration (Ipsen, Otte, & Qvist, 2003; Ipsen & Otte, 2003). Furthermore, recently Otte and co-workers (Otte, Ipsen, Ladefoged, & Sørensen, 2004) identified a large fragment of 8.8 kDa as the primary fragment in aggregates formed from α-LA upon hydrolysis by BLP of α-LA at 10 g L−1, which is below the gelation concentration of this system. However, it is not known whether the fragments and aggregates formed upon hydrolysis of α-La at sub-gelation concentrations are identical to the precursors and tubular strands in gels resulting from hydrolysis of α-La at higher concentrations.
The purpose of this study was to characterise the aggregates formed upon partial hydrolysis of α-LA by BLP at an α-LA concentration of 10 g L−1 with respect to morphology, composition and amyloid character, and to identify the intermediates in the aggregation reaction. The intermediates and aggregates were characterised by size-exclusion chromatography with light scattering and refractive index detection and by liquid chromatography with mass spectrometric detection, and the morphology of the final aggregates was studied by cryo-transmission electron microscopy. Finally, the amyloid character of the aggregates was assayed by thioflavin-binding studies and secondary structure assessment using circular dichroism measurements.
Section snippets
Materials
Two preparations of bovine α-LA were used as substrate. One preparation was isolated from bovine milk whey as described by Kristiansen, Otte, Ipsen, and Qvist (1998) here denoted as (P). This substrate contained 94% α-LA in dry matter and 0.73% Ca, corresponding to a calcium to α-LA ratio of 2.9 on a molar basis (see Otte et al., 2004). The other substrate was a commercial preparation from Sigma (L-5385; Sigma-Aldrich Denmark A/S, Vallensbæk Strand, Denmark) containing 85% α-LA saturated with
Hydrolysis of α-LA and aggregation of hydrolysis products measured by SEC-MALS
The SEC profiles (refractive index signals) of the reaction mixture containing 10 g L−1 of α-LA, after various times of proteolysis by BLP, are shown in Fig. 1. All profiles are normalised to give the same total area upon integration between 0 and 20 min. The peak occurring at 15.9 min has a molar mass of 14.3 kDa, derived from light scattering, and does not move upon changing the protein concentration, as expected for intact α-LA (for all molar mass determinations by SEC-MALS we estimate an
Conclusion
Together with the work of Goers et al. (2002) showing that α-LA can be induced to form fibrils at low pH, the present work showing that partial hydrolysis at neutral pH can also induce fibril formation from α-LA adds the mammary-derived protein α-LA to the list of proteins able to assemble into fibrils under specified conditions.
Furthermore, this work suggests that the initial concentration of α-LA might have a major influence on the morphology of the fibrils formed, since simple 5-nm thick
Acknowledgements
We thank Marianne Lund Jensen and Mila Zakora for their excellent help with laboratory work. Christian Rischel, Department of Natural Sciences, The Royal Veterinary Agricultural University, is thanked for performing the thioflavin binding studies. The study was supported financially by the Centre for Advanced Food Studies in Denmark.
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Deceased.