Component PP3 from bovine milk is a substrate for transglutaminase. Sequence location of putative crosslinking sites

Abstract

As the demands for food products of high quality increase, it will become more important to be able to modify the properties of food proteins. One possibility is to use enzymic modifications to improve functional properties and nutritional values. A highly efficient class of enzymic crosslinkers are transglutaminases (TG, EC 2.3.2.13), calcium-dependent enzymes that catalyse an acyl transfer reaction between protein-bound glutaminyl residues and primary amines (for review, see Aeschlimann & Paulsson, 1994). When protein-bound lysyl residues act as acyl acceptors, the reaction leads to the formation of intramolecular and/or intermolecular isopeptide bonds. Although a wide variety of amines such as putrescine, cadaverine or lysyl residues may act as amine donors in this reaction, only a limited number of glutamine residues in certain proteins will act as amine acceptors (Gorman & Folk, 1980).

Ikura et al. (1981, 1985) reported that TG can be used to introduce methionine into casein and soyabean proteins and lysine into wheat gluten, thereby showing that TG can also be utilized to improve the nutritional value of food proteins. There have been a number of reports concerning the TG-mediated polymerization of food proteins such as α-lactalbumin and β-lactoglobulin (Aboumahmoud & Savello, 1990; Færgemand et al. 1997), soyabean proteins and casein components (Ikura et al. 1980a, b) and pea legumin (Larré et al. 1993). Recently, improved protein foaming capacity and stability have been demonstrated in TG-catalysed polymers of soyabean protein and whey protein isolate (Yildirim et al. 1996).

Bovine PP3 (for review, see Girardet & Linden, 1996) is a phosphorylated glycoprotein isolated from the proteose peptone fraction of milk (Sørensen & Petersen, 1993a). The primary structure of PP3 has been determined (Sørensen & Petersen, 1993b) and comprises a polypeptide backbone of 135 amino acid residues containing five phosphorylated serines, two threonine-linked O-glycosylations, and one N-glycosylation. Immunological studies have shown that PP3 is present in the milk fat globule membrane, and that PP3 forms multimeric aggregates in bovine milk (Sørensen et al. 1997). Several functions have been suggested and investigated for bovine PP3 and fractions enriched with PP3, including emulsification (Shimizu et al. 1989), inhibition of lipolysis (Girardet et al. 1993) and mitogenesis (Mati et al. 1993).

Previously, we have localized the potential TG-reactive glutamines in the four bovine caseins (Christensen et al. 1996) and in milk osteopontin (Sørensen et al. 1994). In the present study we have shown that component PP3 is a substrate for guinea-pig liver TG containing both reactive glutamine and lysine residues. In addition, we have localized the glutamine residues that act as amine acceptors.