Inhibition of collagenolytic cathepsins by beta-lactoglobulin in milk and its suppressive effect on bone resorption.
It is well known that the collagenolytic cathepsins play an important role in the degradation of bone matrix. Therefore, the purpose of this study was to clarify the prevention effect of bone resorption by milk components. Using double-layer reverse zymography, we found a 20 kDa protein in milk which inhibited cysteine proteases. This inhibitory protein was identified as beta-lactoglobulin B. The inhibitory activity of beta-lactoglobulin B against cathepsin K was stronger than that of beta-lactoglobulin A. Beta-lactoglobulin B specifically inhibited papain type cysteine proteases such as cathepsins K and L, but not serine proteases, aspartic proteases or metallo proteases. Beta-lactoglobulin B inhibited cathepsin K non-competitively and the Ki value was 10(-5) M. The formation of osteoclastic pits in the culture system was effectively inhibited by 10(-4)-10(-5) M beta-lactoglobulin B in vitro. Furthermore, we demonstrated that beta-lactoglobulin B inhibited degradation of type I-collagen by collagenolytic cathepsins. Using the everted-sac method in rat small intestines, it was found that beta-lactoglobulin was more easily absorbed from the intestines of young rats (5 wk-old) than from those of older rats (more than 20 wk-old). The digested products of beta-lactoglobulin B with lysyl-endopeptidase showed a similar inhibitory activity against cathepsin K to the intact beta-lactoglobulin B did. Therefore, peroral intake of beta-lactoglobulin in milk and its digested peptides are expected to help protect osteoclastic bone resorption via inhibition of collagenolytic cathepsins K and L.
Ogawa
Naoko
N
Faculty of Life Science, Tokushima Bunri University, Tokushima, Japan. ogawanao@tokushima.bunri-u.ac.jp
Takahashi
Masae
M
Ishidoh
Kazumi
K
Katunuma
Nobuhiko
N
D000375:Aging / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001862:Bone Resorption / Q000517:prevention & control*; D056657:Cathepsin K; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002417:Cattle; D003094:Collagen / Q000378:metabolism*; D024042:Collagen Type I / Q000378:metabolism; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D007408:Intestinal Absorption / Q000502:physiology; D007782:Lactoglobulins / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D008297:Male; D008892:Milk / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
D024042:Collagen Type I; D015853:Cysteine Proteinase Inhibitors; D007782:Lactoglobulins; D010446:Peptide Fragments; D003094:Collagen; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534280:Ctsl protein, rat; D056657:Cathepsin K; C534133:Ctsk protein, rat