Multiple forms of equine α-lactalbumin: evidence for N-glycosylated and deamidated forms
Introduction
The physical, chemical and functional properties of α-lactalbumin (α-LA) and lysozyme (LZ) have been extensively compared. The two proteins have striking structural homology in spite of marked differences in their functions (Acharya, Stuart, Phillips, McKenzie, & Teahan, 1994; Qasba & Kumar, 1997). α-LA is a part of the lactose synthetase complex that promotes the synthesis of lactose in the lactating mammary gland in modifying the substrate specificity of the UDP-galactosyltransferase (Kuhn, Carrick, & Wilde, 1980). Human α-LA has a strong Ca2+ binding site and a weaker secondary binding site (Chandra, Brew, & Acharya, 1998). Calcium binding strongly influences the molecular stability of α-LA and is required for refolding and disulphide bond formation in the reduced, denatured protein. α-LA can also bind different metal ions in addition to calcium (Permyakov & Berliner, 2000). LZ in equine milk has the essential features of a chicken-type (c-type) LZ (McKenzie & Shaw, 1985) that catalyses the hydrolysis of glycosidic bonds of mucopolysaccharides in bacterial cell walls. Equine milk LZ is a calcium binding protein (Lyster, 1992), although bovine and hen egg white LZs have no calcium. Therefore, on the basis of metal binding properties, the equine LZ is more similar to bovine α-LA than to bovine and hen LZs (Sugai, Ikeguchi, & Shimizu, 1999).
The refolding of bovine α-LA (e.g., Dolgikh et al., 1981) and equine LZ (Sugai et al., 1999; Morozova-Roche, Jones, Noppe, & Dobson, 1999) has been extensively studied in order to elucidate the mechanisms by which proteins fold. The two proteins show an intermediate, partially unfolded state termed “molten globule” state that does not bind calcium any more.
In the mare, three genetic variants of α-LA have been identified (Kaminogawa, McKenzie, & Shaw, 1984; Godovac-Zimmermann, Shaw, Conti, & McKenzie, 1987). Donkey's α-LA presents two isoforms A and B which show an isoelectric point (IEP) difference of 0.23 pH units (Giuffrida, Cantisani, Napolitano, Conti, & Godovac-Zimmermann, 1992). Their aminoacid sequences reveal, however, no differences. This apparent heterogeneity of α-LA would be due to binding of Ca2+ (Giuffrida et al., 1992).
Among the different species investigated, α-LA has been reported to be glycosylated either totally (Prasad, Hudson, Butkowsky, Hamilton, & Ebner, 1979; Cantisani, Napolitano, Giuffrida, & Conti, 1990), or partially (Brew, Castellino, Vanaman, & Hill, 1970; MacGillivray, Brew, & Barnes, 1979; Hopp & Woods, 1979; Halliday, Bell, McKenzie, & Shaw, 1990; Giuffrida et al., 1997). No glycosylation has been reported up to now for horse's and donkey's α-LAs. Moreover, no glycoform of LZ has been found, except the hen egg white LZ, for which only 0.3% of the total protein is glycosylated (Trudel & Asselin, 1995).
The aims of this study were (i) to evidence the existence of minor glycosylated forms of equine α-LA and (ii) to show that the existence of two major forms of equine α-LA was not the result of a modulation by Ca2+ as suggested in the case of the donkey's α-LAs, but was the consequence of a protein degradation process identified as the deamidation of one amidated side-chain. This degradation process was identified by in vitro study as non-enzymatic deamidation and its effects on the Ca2+ binding and on the secondary and tertiary structures of the equine α-LA were discussed.
Section snippets
Preparation of equine whey soluble proteins
Milk samples were collected from a herd of Haflinger mares and immediately stored at −20°C until used. The milk was skimmed by centrifugation (2100×g at 32°C for 30 min) and the whole casein was precipitated at pH 4.2 with 1 m HCl (the minimal solubility of the equine caseins was reached for pH 4.2 instead of pH 4.6 for bovine caseins). Milk whey was neutralised with 1 m NaOH, dialysed against distilled water in the presence of thymol at 4°C for 72 h, and the whey soluble proteins were lyophilised.
Anion-exchange FPLC
Chromatography of equine whey proteins
The whey soluble proteins were separated in four fractions by anion-exchange FPLC (Fig. 1). Each of the four fractions was checked for purity by anion-exchange FPLC and was submitted to amino-terminal microsequencing (Fig. 1). LZ was not retained on the Mono Q column and was entirely recovered in F1. Its amino-terminal region was identical to that reported for equine LZ (accession number P11376; McKenzie & Shaw, 1985). α-LA displayed two isoforms recovered in F2 and F3, respectively. The two
Acknowledgments
We thank Dr. G. Humbert and F. Saulnier (SCSP, UHP-Nancy 1, France) for the peptide sequencing, Dr. D. Mollé (Laboratoire de Recherches de Technologie Laitière, INRA, Rennes, France) for the mass spectrometry, Dr. L. Miclo (Laboratoire des BioSciences de l’Aliment, UHP-Nancy 1, France) and Dr. C. Barbey (Laboratoire de Chimie Structurale, Université Paris 13, France) for good advices and Fanny Bassora for technical assistance.
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