A. Schematic drawing of cloned human Hp
4.4.6 Evolutionary advantage of deer Hp protein being a tetramer
In addition to the superior binding affinity to hemoglobin, Hp is an anti-inflammatory
molecule and a potent antioxidant [9]. In humans, the large complicated polymers of Hp 2-2
are a risk in the association of diabetic nephropathy [39,40]. One explanation is that the
large polymer dramatically retards penetration of the molecular into the extracellular space
[39]. We have shown in the present study that deer Hp 2-2 was not able to form the
complicated polymers, because the diversity in amino acid sequence between the tandem
repeat of α-chain has produced steric hindrance (Fig. 8) that may be advantageous of deer.
In conclusion, we have shown that deer possess an Hp 2 with a tandem repeat that could
have occurred at least 25 or between 25 and 45 million years ago based on the phylogenetic
analysis. The phenotypic and biochemical structure of their Hp is markedly homogeneous,
with a tetrameric arrangement due to the orientation of the two available –SH groups,
preventing the formation of the complicated Hp polymers found for human Hp 2-2. In terms
of molecular evolution, this steric hindrance may have conferred an advantage on deer Hp that
compensate for the undesired tandem repeat in the α-chain.
Acknowledgements
This work was supported by NSC grant 95-2313-B-009-003-MY2 from the National Science
Council, Taiwan. We especially thank Drs Suen-Chuain Lin (Veterinary Medicine Teaching
Hospital, National Pingtung University of Science and Technology) and Yi-Ping Lu (Pingtung
Livestock Disease Control Center) for kindly providing the animal plasma. We also
acknowledge James Lee of National Chiao Tung University for his scientific critiques and
editorial comments.
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Figure 1. Schematic drawing of the human Hp α-chain and molecular arrangement of Hp phenotypes. (A) Human Hp α1-chain includes two avaiable –SH groups. That at the C-terminus always links to a β-chain to form a basic α1-β unit and the other at N-terminus links either α1-β unit or (α2-β)n units. The sequence of α2 is identical to that of α1 except for a partial repeat insertion of residues 12-70. However, the extra Cys74 means that Hp 2-1 and 2-2 form complicated polymers. (B) Hp 1-1 forms the simplest homodimer (α1-β)2, whereas Hp 2-1 is polymeric in linear form, forming a homodimer (α1-β)2, a trimer (α-β)3, and other polymers. Here, α represents α1- or α2-chains. Hp 2-2 forms cyclic structures:
a trimer (α2-β) and other cyclic polymers.
Figure 2. Hemoglobin-binding patterns of deer and human plasma Hp on 7%
native-PAGE. Lane 1: hemoglobin only. Lanes 2, 3, and 4: human plasma of Hp 1-1, 2-1, and 2-2 with hemoglobin, respectively. Lane 5: deer plasma with hemoglobin.
Figure 3. Isolation of deer Hp using size-exclusion Superose-12 column on an HPLC system. (A) A dialyzed supernatant of 50% saturated ammonium-sulfate fraction from plasma was applied on Superose-12 column (1x30 cm) at a flow-rate of 0.3 mL/min, while using PBS as the mobile phase. The bar represents the pooled fractions corresponding to Hp.
(B) SDS-PAGE and Western blot analyses of eluted Hp fractions. (C) Hemoglobin-binding property of isolated Hp and plasma containing native Hp on 7% native-PAGE. Lane M, molecular markers in kDa (Invitrogen).
Figure 4. SDS-PAGE, Western blot, and molecular mass analyses of isolated deer and human Hp. (A) The isolated proteins were run on 10-15% PAGE under reducing condition.
The Western blot was performed using a human α-chain specific mAb (W1) that cross-reacts with the deer α-chain. Lane M, molecular markers in kDa (Invitrogen). (B) Left panel:
Western blot analysis of the polymeric structure of isolated human and deer Hp under 4%
non-reducing SDS-PAGE using α-chain specific mAb W1. Lane M: molecular markers in kDa (Invitrogen). Lane 1, isolated human Hp 2-2. Lane 2, isolated deer Hp. Right panel:
On the Western blot, mAb W1 only recognizes human polymeric Hp, but not deer tetrameric Hp. (C) Dot-blot analysis of isolated human Hp (hHp) and deer Hp (dHp) using α-chain specific mAb W1 in the presence or absence of the reducing reagent β-ME (143 mM). BSA was used as a negative control.
Figure 5. Putative amino-acid sequence analysis and divergence of mammal Hps. (A, B) Amino-acid sequence alignment of the α- or β-chains of human and deer. Variable regions are shaded in black. The cDNA nucleotide sequence corresponding to deer Hp in this study has been deposited in GenBank under the accession number of EF601928. (C) Divergence of the amino-acid sequence of Hp β-chains among ten mammals. (D) Phylogenetic tree constructed according to the amino-acid sequence of Hp β-chains for ten mammals. The tree was plotted using the MEGALIGN program in the DNASTAR package. Branch lengths (%) are proportional to the level of sequence divergence, while units at the bottom indicate the number of substitution events.
Figure 6. Schematic drawing of tandem repeat region (B and B1) of deer and human α-chain. The most significant feature of human α2 is that it matches the ABC domains of α1 but with an additional insertion of a redundant sequence (B1 region). The repeat unit contains 59 amino acid residues between Asp12 to Ala70. The sequence homology in the repeat region of human is 96% (two amino acids mutated). Deer also have a redundant sequence (B and B1), but the sequence homology between the two repeat units is approximately 68%. The full length of the α-chain contains 142 and 136 residues in human and deer, respectively. The position and number of Cys (total of seven) residues are completely identical between the two species (the one at C-terminal region is not shown).
Divergence of the amino acids within the species is marked in yellow.
Figure 7. SDS-PAGE and native PAGE analyses of renaturation of deer and human Hp polymers. Denaturation of deer Hp using 6M urea under reducing conditions (143 mM β-ME) followed by renaturation resulted in the formation of (α-β)4 and some (α-β)3.
Figure 8. A hypothetical model illustrating the steric hindrance involved in the formation of a deer Hp tetramer. (A) A basic Hp subunit comprising one α- and one β-subunit. The –SH groups that connect the Hp subunits into polymers are assumed to be located with a steric hindrance between the SH binding sites A and B. (B) The two different possible forms of tetramers. (C) The trimeric form of deer Hp is possible to assemble according to this model, but steric hindrance is seen which prevents the –SH groups from linking to some extent. (D) Formation of a pentamer or higher-order polymer is not possible.
Figure 9. Model of formation of human Hp 2-2 polymers. The positioning of the -SH groups involved in polymer formation differs from those in deer Hp. (A) A basic human Hp 2-2 subunit comprised of one α- and one β-subunit. The –SH groups that connect the Hp subunits into polymers are located at the edge of the surface. The hindrance between the –SH binding sites A and B prevents formation of a dimer. (B) A trimer is able to form to some extent with some steric hindrance. (C-E) Polymers of a higher order than tetramers can form without any steric hindrance.
Figure 10. Phylogenetic tree illustrating the molecular evolution of mammals, and phenotyping of human, whale, dolphin and ruminant α-chains. The tree is constructed by assuming all eutherian orders radiated at about the same point in evolutionary time, approximately 75 million years ago. Alternative branching orders give essentially identical results. Within a eutherian order, branch points are assigned using evolutionary times based on fossil records [30]. Western blot analysis of Hp of six mammals (with a branching point before and after deer) was conducted using a 10-15% SDS-PAGE gradient gel under reducing conditions with an α-chain specific mAb (W1) prepared against human Hp.
Chapter 5 Discussion
This thesis presents evidence that neutrophils were associated with the biosynthesis and
release of Hp in milk. It further shows that Hp was significantly elevated in the epithelium
of mammary gland tissue with mastitis and was also expressed in the cultured mammary
epithelial cells. We propose neutrophils and epithelial cells may play an essential role in
elevating milk Hp in addition to previous suggestions that Hp may be derived from mammary
tissues and circulation. During bovine mastitis, activated neutrophils produce significant
amount of reactive oxygen species which may cause tissue damage. Hp is an extremely
potent antioxidant that can directly scavenge the free radicals, it may there effectively utilize
Hp to attenuate such intracellular damage. Using recombinant Hp, we further find the major
antioxidant domain was located in the β chain. The present study provides a potential utility
for the future design of “mini-Hp” in developing a novel potent antioxidant. On the other
hand, we cloned the cDNA of deer Hp showing that the putative amino-acid sequence mimics
that of human Hp 2-2, and that the α-chain of deer also possess a unique tandem repeat. By
phylogenetic analysis, we have shown that deer possess an Hp 2 with a tandem repeat that
could have occurred at least 25 million years ago. The evolved tetrameric structure of deer
Hp might be of a physiologic advantage. We further proposed that a steric hindrance
mechanism is involved in forming Hp tetramers.