Browsing by Author "Yang, Ming Chi"

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A unique tetrameric structure of deer plasma haptoglobin - an evolutionary advantage in the Hp 2-2 phenotype with homogeneous structure
(2008-03-01)
Lai, I. H.
;
Lin, Kung-Yu
;
Larsson, Mikael
;
Yang, Ming Chi
;
Shiau, Chuen-Huei
Similar to blood types, human plasma haptoglobin (Hp) is classified into three phenotypes: Hp 1-1, 2-1 and 2-2. They are genetically inherited from two alleles Hp 1 and Hp 2 (represented in bold), but only the Hp 1-1 phenotype is found in almost all animal species. The Hp 2-2 protein consists of complicated large polymers cross-linked by alpha 2-beta subunits or (alpha 2-beta)(n) (where n >= 3, up to 12 or more), and is associated with the risk of the development of diabetic, cardiovascular and inflammatory diseases. In the present study, we found that deer plasma Hp mimics human Hp 2, containing a tandem repeat over the alpha-chain based on our cloned cDNA sequence. Interestingly, the isolated deer Hp is homogeneous and tetrameric, i.e. (alpha-beta)(4), although the locations of -SH groups (responsible for the formation of polymers) are exactly identical to that of human. Denaturation of deer Hp using 6 M urea under reducing conditions (143 mM beta-mercaptoethanol), followed by renaturation, sustained the formation of (alpha-beta)(4), suggesting that the Hp tetramers are not randomly assembled. Interestingly, an alpha-chain monoclonal antibody (W1), known to recognize both human and deer alpha-chains, only binds to intact human Hp polymers, but not to deer Hp tetramers. This implies that the epitope of the deer alpha-chain is no longer exposed on the surface when Hp tetramers are formed. We propose that steric hindrance plays a major role in determining the polymeric formation in human and deer polymers. Phylogenetic and immunochemical analyses revealed that the Hp 2 allele of deer might have arisen at least 25 million years ago. A mechanism involved in forming Hp tetramers is proposed and discussed, and the possibility is raised that the evolved tetrameric structure of deer Hp might confer a physiological advantage.
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
(2009-04-01)
Yang, Ming Chi
;
Chen, Nai Chi
;
Chen, Chun-Jung
;
Wu, Chin Yun
;
Mao, Simon J. T.
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.
Rational Design for Crystallization of beta-Lactoglobulin and Vitamin D(3) Complex: Revealing a Secondary Binding Site
(2008-12-01)
Yang, Ming Chi
;
Guan, Hong-Hsiang
;
Yang, Jirin-Moon
;
Ko, Cheng-Neng
;
Liu, Ming-Yih
beta-Lactoglobulin (LG) is a major milk whey protein containing primarily a calyx for vitamin D(3) binding, although the existence of another site beyond the calyx is controversial. Using fluorescence spectral analyses in the previous study, we showed the binding stoichiometry for vitamin D3 to LG to be 2:1 and a stoichiometry of 1:1 when the calyx was "disrupted" by manipulating the pH and temperature, suggesting that a secondary vitamin D binding site existed. To help localize this secondary site using X-ray crystallography in the present study, we used bioinformatic programs (Insight II, Q-SiteFinder, and GEMDOCK) to identify the potential location of this site. We then optimized the occupancy and enhanced the electron density of vitamin D3 in the complex by altering the pH and initial ratios of vitamin D(3)/LG in the cocrystal preparation. We conclude that GEMDOCK can aid in searching for an extra density map around potential vitamin D binding sites. Both pH (8) and initial ratio of vitamin D(3)/LG (3:1) are crucial to optimize the occupancy and enhance the electron density of vitamin D(3) in the complex for rational-designed crystallization. The strategy in practice may be useful for future identification of a ligand-binding site in a given protein.