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. alpha (sl)- and beta -casein. alpha (sl)- or beta -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 0.1-5 units ml(-1) of this stock solution was added to alpha (sl)- or beta -casein in buffer. Samples were taken over a 24 h incubation at 37 degreesC and analysed by urea polyacrylamide gel electrophoresis and high performance liquid chromatography. Isolated peptides were identified by Nterminal sequencing and mass spectrometry. Cathepsin G cleaved alpha (sl)-casein at at least 16 sites and beta -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 alpha (sl)- and beta -casein and it is therefore possible that indigenous cathepsin G in milk may be of significance for the protcolysis of milk proteins. Of particular interest was the production of the small peptide alpha (sl)-casein (fl-23), which also results from cleavage of the Phe(23)-Phe(24) bond by chymosin in cheese, and is hydrolysed rapidly during cheese ripening by the cell envelope-associated proteinase of Lactococcus. (C) 2001 Elsevier Science Ltd. All rights reserved.