Evidence for beta-lactoglobulin involvement in vitamin D transport in vivo--role of the gamma-turn (Leu-Pro-Met) of beta-lactoglobulin in vitamin D binding.
Beta-lactoglobulin (LG) is a major bovine milk protein, containing a central calyx and a second exosite beyond the calyx to bind vitamin D; however, the biological function of LG in transporting vitamin D remains elusive. Crystallographic findings from our previous study showed the exosite to be located at the pocket between the alpha-helix and beta-strand I. In the present study, using site-directed mutagenesis, we demonstrate that residues Leu143, Pro144 and Met145 in the gamma-turn loop play a crucial role in the binding. Further evidence is provided by the ability of vitamin D(3) to block the binding of a specific mAb in the gamma-turn loop. Using the mouse (n = 95) as an animal model, we initially demonstrated that LG is a major fraction of milk proteins responsible for uptake of vitamin D. Most interestingly, dosing mice with LG supplemented with vitamin D(3) revealed that native LG containing two binding sites gave a saturated concentration of plasma 25-hydroxyvitamin D at a dose ratio of 2 : 1 (vitamin D(3)/LG), whereas heated LG containing one exosite (lacking a central calyx) gave a ratio of 1 : 1. We have demonstrated for the first time that LG has a functional advantage in the transport of vitamin D, indicating that supplementing milk with vitamin D effectively enhances its uptake.
Yang
Ming Chi
MC
Department and College of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan.
Chen
Nai Chi
NC
Chen
Chun-Jung
CJ
Wu
Chin Yun
CY
Mao
Simon J T
SJ
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D018360:Crystallography, X-Ray; D005260:Female; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014807:Vitamin D / Q000737:chemistry* / Q000378:metabolism*
D007782:Lactoglobulins; D009842:Oligopeptides; D014807:Vitamin D