Proteolytic specificity of cathepsin G on bovine αs1- and β-caseins

Mastitis is an inflammation of the mammary gland which results in an increase in numbers of somatic cells, particularly polymorphonuclear leucocytes (PMN), which contain very active proteinases. The objective of this study was to determine the cleavage specificity of cathepsin G, one of the principal PMN proteinases, on αs1- and β-casein. αs1- or β-casein (5 mg ml−1) were dissolved in 0.1 M HEPES buffer, pH 7.5, containing 0.05% NaN3. Cathepsin G was dissolved in 0.1% Brij 35 and 0.5 M NaCl and 0.25 units ml−1 of this stock solution was added to αs1- or β-casein in buffer. Samples were taken over a 24 h incubation at 37 °C and analysed by urea polyacrylamide gel electrophoresis and high performance liquid chromatography. Isolated peptides were identified by N-terminal sequencing and mass spectrometry. Cathepsin G cleaved αs1-casein at at least 16 sites and β-casein at at least 21 sites, some of which were also cleavage sites of chymosin, plasmin, elastase, cathepsin B or the cell envelope-associated proteinase of Lactococcus. Thus, cathepsin G had a broad specificity on αs1- and β-casein and it is therefore possible that indigenous cathepsin G in milk may be of significance for the proteolysis of milk proteins. Of particular interest was the production of the small peptide αs1-casein (f1-23), which also results from cleavage of the Phe23-Phe24 bond by chymosin in cheese, and is hydrolysed rapidly during cheese ripening by the cell envelope-associated proteinase of Lactococcus.