Haptoglobin在生理與生化上之功能角色

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國

立

交

通

大

學

生化工程研究所

博士論文

Haptoglobin 在生理與生化上之功能角色

Biochemical and physiologic role of haptoglobin

研究生：賴以祥

指導教授：毛仁淡講座教授

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Haptoglobin 在生理與生化上之功能角色

Biochemical and physiologic role of haptoglobin

研究生：賴以祥 Student: I-Hsiang Lai

指導教授：毛仁淡講座教授 Advisor: Chair Prof. Simon JT Mao

國立交通大學

生化工程研究所

博士論文

A Thesis

Submitted to Institute of Biochemical Engineering College of Biological Science and Technology

National Chiao Tung University in partial Fulfillment of the Requirements

for the Degree of PhD in

Biochemical Engineering

June 2009

Hsinchu, Taiwan, Republic of China

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Haptoglobin 在生理與生化上之功能角色

研究生：賴以祥指導教授：毛仁淡講座教授國立交通大學生化工程研究所博士班

摘要

Haptoglobin（Hp）是當動物受到感染或炎症反應時，血液中濃度會大量增加的一種急性反應期蛋白質。其最主要功能是可與血液中游離的血紅素形成複合物，而避免溶血時所造成的氧化壓力及組織傷害。乳牛之乳房因細菌感染而產生炎症反應時，其牛奶中可發現大量體細胞數與Hp 濃度上升。但是牛奶中 Hp 形成機制尚未明瞭。本研究發現牛奶中 Hp 濃度與體細胞數目呈現正相關。進一步經由反轉錄聚合酶連鎖反應，西方轉印法、免疫化學染色法、雙重免疫螢光染色法及Hp release 實驗進行分析，證明牛奶中 Hp 可經由嗜中性球合成與分泌。本研究發現，當牛感染乳房炎時，牛奶中活化的嗜中性球會分泌大量 Hp，而利用 Hp 本身具有之抗氧化能力，可避免自由基形成所造成的細胞組織傷害。關於 Hp 之抗氧化能力，本研究進一步利用Cu2+所誘發的氧化試驗發現各個重組Hp 次單位皆具有抗氧化能力，可保護低密度脂蛋白不被氧化，並且發現β次單位之抗氧化能力明顯高於 native Hp（3.5 X）、α次單位（10X）與臨床上使用藥物 Probucol（15 X）。此研究結果可提供未來設計mini-Hp 作為強抗氧化藥物之參考，並且可進一步了解 Hp 其抗氧化能力之機制。在反芻動物 Hp 研究方面，本研究利用基因選殖分析發現，鹿 Hp 蛋白質之胺基酸 序列與人類 Hp 2 類似，在α次單位內皆具有一段重複序列，並且-SH group 在胺基酸序

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列上的位置與人類 Hp 2 相同，經蛋白質電泳分析顯示鹿 Hp 蛋白質為 tetramer (α-β)4之結構。為了進一步了解鹿Hp tetramer 結構，利用已知可辨認人類與鹿 Hp α次單位之單株抗體（W1）進行分析，結果發現此單株抗體僅能辨認人類 Hp 2 polymers，但卻無法辨認鹿之Hp tetramer，顯示鹿 Hp tetramer 結構形成後此單株抗體位於α次單位上之抗原決定位（epitope）無法完全暴露在表面，而導致此抗體無法辨認鹿之 Hp tetramer，研究結果發現α次單位上胺基酸序列的差異所形成之空間障礙（steric hindrance）在人類與鹿形成Hp polymer 上扮演重要的關鍵。此外，將鹿與其他哺乳類動物之 Hp 進行親緣關係 與免疫化學之分析，發現鹿 Hp 2 基因可能在 2 千 5 百萬年前就存在。 綜合以上所述，本研究發現牛奶中嗜中性球可分泌大量 Hp，其抗氧化能力可保護細胞而避免遭受到氧化性傷害。而Hp 之抗氧化功能區域主要位於β次單位上。Hp 胺基 酸序列經由親緣關係分析，發現鹿 Hp 2 基因可能在 2 千 5 百萬年前就存在，其 Hp tetramer 之演化可能在生理方面佔有一定的優勢。

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Biochemical and physiologic role of haptoglobin

Student: I-Hsiang Lai Advisor: Chair Prof. Simon JT Mao

Institute of Biochemical Engineering

National Chiao Tung University

Abstract

Haptoglobin (Hp) is an acute phase protein responsive to inflammation and infection. One

of the major functions of Hp is to capture released hemoglobin during excessive hemolysis

and to scavenge the hemoglobin-induced free radicals during oxidative stress.

Hp and somatic cell counts (SCC) are sharply elevated in bovine milk following

intramammary administration of endotoxin or bacteria. However, the sources of milk Hp

responsible for such increase are not fully understood. In this study, our results reveal that

milk Hp concentrations were correlated with SCC. Reverse transcriptase-polymerase chain

reaction, Western blot, immunocytochemistry, double immunofluorescence, and Hp releasing

experiment demonstrate that neutrophils were associated with the biosynthesis and release of

Hp in milk. We propose neutrophils 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

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attenuate such intracellular damage.

With respect to the antioxidant activity of Hp, we demonstrated that the antioxidant

activity was found to be associated with both Hp α and β chains when assessed by Cu2+-induced oxidation of low density lipoprotein. Interestingly, the antioxidant activity of

β chain was extremely potent and markedly greater than that of native Hp (3.5 x), α chain (10 x) and probucol (15 x). The present study provides a potential utility for the future design of

“mini-Hp” in developing a novel potent antioxidant. It may also provide a new insight in

understanding the mechanism involved in the antioxidant nature of Hp.

Using ruminant Hp, we found that deer Hp mimics human Hp 2 containing a tandem

repeat over the α-chain based on our cloned cDNA sequence. Interestingly, the isolated deer Hp is homogeneous and tetrameric as a (α-β)4, although the location of –SH groups

(responsible for the formation of polymers) is exactly identical to that of human. Most

interestingly, an α-chain monoclonal antibody (W1) known to recognize both dissociated human and deer α-chains, only binds to the intact human Hp polymers, but not to deer Hp tetramers. It implicates that the epitope of deer α-chain is no longer exposed on the surface when forming tetramers. 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.

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is raised that the evolved tetrameric structure of deer Hp might confer a physiological

advantage.

Collectively, this thesis presents evidence that neutrophils were associated with the

biosynthesis and release of Hp in milk. The major antioxidant domain of Hp was located in

the β chain. Phylogenetic analysis revealed that the Hp 2 allele of deer might have arisen at least 25 million years ago. The evolved tetrameric structure of deer Hp might be of a

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致謝

五年的博士研究生涯，轉眼間就要告一段落了，回想修業期間藉由許

多人的幫忙才得以完成論文。特別感謝指導教授毛仁淡講座教授，不論在

學業或生活上都給予悉心指導與愛護，並且提供一個良好的研究環境，讓

我能無慮的完成學問。感謝中興大學毛嘉洪院長、台灣大學陳明汝教授及

交通大學林志生教授，對於論文的賜教及指正，使學生論文更完善。感謝

屏東科技大學廖明輝教授、李嘉偉教授及屏東縣家畜疾病防治所魯懿萍小

姐，對我在實驗過程中無私的教導與協助，不僅提供許多實驗材料，而且

毫無保留提供我寶貴的技術與經驗，讓我的研究得以順利完成，在此致上

最誠摯的謝意。

感謝嘉義永榮牧場曹榮旭先生全家人的支持，提供我許多寶貴的材料

並且在實驗過程中全力的配合與協助，在此表達我由衷的感激。除此以外，

特別感謝已畢業的博士班學長鄭財木博士、曾繼鋒博士及陳文亮博士，在

研究過程中提供我許多寶貴意見與協助，還有感謝實驗室的夥伴們：明誌、

宏輝、俊瑩、佩真、韶禪、文昭、立品、冠佑、中曦、乃齊、詩璇、筑婷、

威延、怡安、佩宇、佶穎、巾涵、俐穎、柏如、宛伶、沁紜、伊玟等學弟

妹及靜怡、善琦小秘書，大家給予我的鼓勵與幫忙，陪我共同經歷了實驗

過程中的起起伏伏，不僅讓我學習到很多，也充實了我的生活，在此一併

致上我由衷的謝意。

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最後感謝我最親愛的家人，父親賴茂雄先生、母親張碧雲女士、哥哥

賴以恆，大嫂方宜綸，姪兒致成與姪女姿穎，還有非常關心我的大伯父賴

幹雄先生、四叔賴甘霖先生，四嬸陳慧楨女士、堂弟宇凡、宇建與毅桓，

因為有你們的支持與鼓勵，讓我能無後顧之憂的順利完成博士學位。

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Dissertation index

Abstract in Chinese---I Abstract in English---III Acknowledgements---VI Index---VIII Chapter 1: Overview---1

Work objectives of present investigation---1

References---7

Chapter 2: Neutrophils as one of the major haptoglobin sources in mastitis affected milk (Paper 1) ---13

Abstract---13

Introduction---14

Materials and Methods---16

Results---21

Discussion---24

Acknowledgments ---28

References---28

Figures---34

Chapter 3: Cloning and expression of human haptoglobin subunits in Escherichia coli: delineation of a major antioxidant domain (Paper 2) ---41

Abstract---41

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Results and Discussion---50

Acknowledgments---56

References---56

Tables---62

Figures---64

Chapter 4: A unique tetrameric structure of deer plasma haptoglobin: an evolutionary advantage in the Hp 2-2 phenotype with homogeneous structure (Paper 3) ---69

Abstract---69

Introduction---70

Results---78 Discussion---84 Acknowledgments---91 References---91 Figures---98 Chapter 5: Discussion---108 Appendix---109 Curriculum vitae---110 Publications---116

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Chapter 1 Overview

Haptoglobin (Hp) is an acute phase protein, responsive to infection and inflammation,

ubiquitously present in the plasma of all mammals [1-4]. A recent study has found that Hp

also exists in lower vertebrates, bony fish but not in frog and chicken [5]. The most

frequently reported biological functions of the protein are to capture released hemoglobin (Hb)

during excessive hemolysis and to transport Hb to the liver [6]. The captured hemoglobin is

internalized by a macrophage/monocyte receptor, CD163, via endocytosis. Interestingly, the

CD163 receptor only recognizes Hp and hemoglobin in complex, which indicates the

exposure of a receptor-binding neoepitope [6]. Thus, CD163 is identified as a hemoglobin

scavenger receptor. Besides the recycling of Hb-iron, the formation of the Hp-Hb complex

possesses two additional benefits. First, Hp has a bacteriostatic effect by hampering the iron

requiring process of bacterial replication as shown in rats inoculated with pathogenic

Escherichia coli (E. coli) [7]. Second, Hp was assigned an antioxidant role by inhibiting

Hb-driven free radical oxidative tissue damage [8-9]. Recently, we have shown that Hp is an

extremely potent antioxidant, which directly protects low-density-lipoprotein (LDL) from

Cu2+-induced oxidation. The potency is markedly superior to probucol: one of the most

potent antioxidants used in antioxidant therapy [10-12]. Transfection of Hp cDNA into

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Human Hp is originally synthesized as a single αβ polypeptide; following posttranslational cleavage by a protease, α and β chains are formed and then linked by disulfide bridges producing the mature Hp [13]. The gene is characterized by two common

alleles Hp 1 and Hp 2 corresponding to α1β and α2β polypeptide chains, respectively, resulting in three main phenotypes: Hp 1-1, 2-1 and 2-2. DNA encoding for these two

chains are linearly oriented in chromosome 16q22.1 [14] with 5 and 7 introns in Hp 1 and Hp

2, respectively. These introns, however, are exclusively located at the region corresponding

to the α chain [9]. The α2 contains a unique nonhomologous crossing-over in a part of the α1 [15]. Hp 2-1 and 2-2 form polymers due to an additional –SH group at the α2 chain, which determines its phenotype specificity [16]. All the phenotypes share the same β chains,

each containing 245 amino-acid residues. The α1 chain (each with 83 amino acids) is “monovalent” forming one disulfide bridge with α and β subunit resulting in Hp dimer (α1β)2.

In contrast, the α2 chain (each with 142 amino acids) is “divalent” containing one extra free –SH that is able to interact with an additional α2. As such, α2 chains can bind to either α1 or α2 chains to form large polymers [(α1β)2-(α2β)n in Hp2-1 and (α2β)n in Hp2-2].

Because of its weaker binding affinity to hemoglobin and retarded mobility (or

penetration) between the cells, the polymeric structure of Hp 2-2 is dramatically more

prevalent in some groups of patients with certain diseases, such as diabetes and

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originated from Hp 1 about 2 million years ago and then gradually displaced Hp 1 as a

consequence of a nonhomologous crossing-over between the structural alleles (Hp 1) during

meiosis [20-22], and is the first example of partial gene duplication of human plasma proteins

[20,23-24]. Thus, only humans possess additional Hp 2-1 and 2-2 phenotypes.

All mammalian species studied to date have been shown to possess Hp. Analysis of the

electrophoretic patterns of Hp-Hb complexes has suggested that most of mammals Hps are

similar to human Hp 1-1 [25]. Only the Hp found in the plasma of ruminants resemble

polymeric forms of human Hp 2-2 [26]. Structurally, bovine Hp is unique possessing only

Hp 2-2 phenotype [27-28] with molecular weight ranged from 660 to 730 kDa [28]. Their

size and hemoglobin binding complex are significantly larger than that of human Hp 2-2.

The amino-acid sequence deducted from the nucleotide sequence is similar to human Hp 2

containing a tandem repeat over the α chain. Thus, the Hp 2 allele is not unique in human. Recently, we have found that there is one additional -SH group (Cys-97) in bovine α chain

with a total of 8 -SH groups, which may be responsible for the overall polymeric structure

that is markedly different from human Hp 2-2 [28].

1.1 Work objectives of present investigation

There are three objectives of this present dissertation:

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definition of the source of milk Hp from dairy cows with mastitis.

2. Production of the recombinant Hp subunits in Escherichia coli and determination of a

major antioxidant domain of human Hp.

3. Definition of the molecular evolution of mammals Hp and study of the polymeric

structure of deer Hp.

1.2 Neutrophils as one of the major haptoglobin sources in mastitis affected milk (Paper 1)

Bovine Hp, while not abundantly expressed in normal bovine plasma, is considered to be one

of the sensitive acute phase proteins during bacterial infections [28-29]. Several studies

indicate that its concentration increases dramatically in both plasma and milk during clinical

mastitis of dairy cows [30-32]. In experimentally induced mastitis by intramammary

bacterial-challenge with Streptococcus uberis, the observed increase in milk Hp has been

suggested to originate from the circulation [33]. Hiss et al., 2004 also found elevated Hp in

bovine milk after intramammary endotoxin challenge. It was further observed that Hp could

be locally synthesized within the mammary gland [34]. However, the source of milk Hp

found in naturally occurring mastitis is poorly understood

The purpose of the present study was to define the source of milk Hp from dairy cows

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presence of Hp protein using Western blot, Hp mRNA expression using reverse

transcriptase-polymerase chain reaction (RT-PCR), partial cell typing using double confocal

immunofluorescence, and endogenously released Hp using ELISA. Finally, we localized Hp

in normal and mastitic mammary gland tissue and in cultured mammary epithelial cell line

(MAC-T). Our findings shows that the increased Hp was associated with neutrophils, in

which Hp was released into the medium in an ex vivo experiment. In mastitis affected

mammary glands, Hp was mainly localized within the epithelial cells. We also observed for

the first time that cultured epithelial MAC-T cells could synthesize Hp in vitro. In addition

to locally synthesized Hp within the mammary gland and Hp transferred from the circulation,

we propose that milk neutrophils and mammary epithelium also contribute to milk Hp

concentrations.

1.3 Cloning and expression of human haptoglobin subunits in Escherichia coli: delineation of a major antioxidant domain (Paper 2)

Since the purification for human native Hp and its corresponding α or β chain has been difficult, the purpose of the present study was to effectively express each recombinant Hp

subunit and to localize its antioxidant domain(s). Using E. coli, we show that α1, α2, β, and α2β chain was abundantly expressed and primarily present in the inclusion bodies consisting of about 30% of the cell-lysate proteins. In human liver cells, however, the α2β is

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postranscritionally cleaved into α and β chains between the residues Arg and Ile [35-36]. A more recent study suggests that a complement C1r-like protease is responsible for the

cleavage [37]. Our data indicate that this protease is not present in the E. coli. Each cloned

subunit retained its immunoreactivity as confirmed using antibodies specific to α or β chain. By circular dichroism, the structure of each expressed subunit was disordered as compared to

the native Hp. The antioxidant activity was found to be associated with both α and β chains when assessed by Cu2+-induced oxidation of low density lipoprotein (LDL). Of remarkable

interest, the antioxidant activity of β chain was extremely potent and markedly greater than that of native Hp (3.5 x), α chain (10 x) and probucol (15 x). The present study is a clinically proved potent compound used for antioxidant therapy. The “unrestricted”

structure of β subunit may therefore render its availability for free-radical scavenge, which provides a utility for the future design of a “mini-Hp” in antioxidant therapy. It may also

provide a new insight in understanding the mechanism or specific amino acids involved in the

antioxidant nature of Hp.

1.4 A unique tetrameric structure of deer plasma haptoglobin: an evolutionary advantage in the Hp 2-2 phenotype with homogeneous structure (Paper 3)

Similar to blood types, human Hp is classified as three phenotypes: Hp 1-1, 2-1, and 2-2.

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phenotype or Hp 1 in almost all the animal species [25]. The Hp 2-2 protein consists of

complicated large polymers cross-linked by α2-β subunits or (α2-β)n (where n>3, up to 12 or

more) and is associated with the risk of the development of diabetic, cardiovascular, and

inflammatory diseases [17-19]. In the present study, we found that deer plasma Hp mimics

human Hp 2 containing a tandem repeat over the α-chain based on our cloned cDNA sequence. Remarkably interesting, the isolated deer Hp is homogeneous and tetrameric as a

(α-β)4, although the location of –SH groups (responsible for the formation of polymers) is

exactly identical to that of human. Denaturation of deer Hp using 6M urea in reducing

condition (143 mM β-mercaptoethanol) followed by renaturation still sustained the formation of (α-β)4 suggesting that the Hp tetramers are not randomly assembled. Interestingly, an

α-chain monoclonal antibody (W1) known to recognize both human and deer α-chains, only binds to the intact human Hp polymers, but not to deer Hp tetramers. It implicates that the

epitope of deer α-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

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References

[1] González-Ramón N., Hoebe K., Alava M.A., Van Leengoed L., Piñeiro M., Carmona S.,

Iturralde M., Lampreave F., Piñeiro A., Pig MAP/ITIH4 and haptoglobin are

interleukin-6-dependent acute-phase plasma proteins in porcine primary cultured

hepatocytes, Eur. J. Biochem. (2000) 267:1878-1885.

[2] Wang Y., Kinzie E., Berger F.G, Lim S.K., Baumann H., Haptoglobin, an

inflammation-inducible plasma protein, Redox Rep. (2001) 6:379-385.

[3] Raijmakers M.T., Roes E.M., te Morsche R.H., Steegers E.A., Peters W.H., Haptoglobin and

its association with the HELLP syndrome, J. Med. Genet. (2003) 40:214-216.

[4] Gervois P., Kleemann R., Pilon A., Percevault F., Koenig W., Staels B., Kooistra T., Global

suppression of IL-6-induced acute phase response gene expression after chronic in vivo

treatment with the peroxisome proliferator-activated receptor-{alpha} activator fenofibrate, J.

Biol. Chem. (2004) 279:16154-16160.

[5] Wicher K.B., Fries E., Haptoglobin, a hemoglobin-binding plasma protein, is present in bony

fish and mammals but not in frog and chicken, Proc. Natl. Acad. Sci. U. S. A. (2006)

103:4168-4173.

[6] Kristiansen M., Graversen J.H., Jacobsen C., Sonne O., Hoffman H., Law S.K.A., Moestrup

S.K., Identification of the haemoglobin scavenger receptor, Nature (2001) 409:198-201.

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(1982) 215:691-693.

[8] Miller Y.I., Altamentova S.M., Shaklai N., Oxidation of low-density-lipoprotein by

hemoglobin stems from a heme-initiated globin radical: antioxidant role of haptoglobin,

Biochemistry (1997) 36:12189-12198.

[9] Langlois M.R., Delanghe J.R., Biological and clinical significance of haptoglobin

polymorphism in humans, Clin. Chem. (1996) 42:1589-1600.

[10] Mao S.J.T., Yates M.T., Jackson R.L., Antioxidant activity and serum levels of probucol and

probucal metabolites, Methods Enzymol. (1994) 234:505-513.

[11] Tseng C.F., Lin C.C., Huang H.Y., Liu H.C., Mao S.J.T., Antioxidant role of human

haptoglobin, Proteomics (2004) 4:2221-2228.

[12] Lai I.H., Tsai T.I., Lin H.H., Lai W.Y., Mao S.J.T., Cloning and expression of human

haptoglobin subunits in Escherichia coli: Delineation of a major antioxidant domain, Protein

Expr. Purif. (2007) 52:356-362.

[13] Kurosky A., Barnett D.R., Lee T.H., Touchstone B., Hay R.E., Arnott M.S., Bowman B. H.,

Fitch W.M., Covalent structure of human haptoglobin: a serine protease homolog, Proc. Natl.

Acad. Sci. U. S. A. (1980) 77:3388-3392.

[14] McGill J.R., Yang F., Baldwin W.D., Brune J.L., Barnett D.R., Bowman B.H., Moore C.M.,

Localization of the haptoglobin alpha and beta genes (HPA and HPB) to human chromosome

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[15] Bowman B.H., Kurosky A., Haptoglobin: the evolutionary product of duplication, unequal

crossing over, and point mutation, Adv. Hum. Genet. (1982) 12:189-261.

[16] Wejman J.C., Hovsepian D., Wall J.S., Hainfeld J.F., Greer J., Structure and assembly of

haptoglobin polymers by electron microscopy, J. Mol. Biol. (1984) 174:343-368.

[17] Miyoshi H., Ohshiba S., Matsumoto A., Takada K., Umegaki E., Hirata I., Haptoglobin

prevents renal dysfunction associated with intravariceal infusion of ethanolamine oleate, Am.

J. Gastroenterol. (1991) 86:1638-1641.

[18] Engstrom G., Lind P., Hedblad B., Wollmer P., Stavenow L., Janzon L., Lindgarde F., Lung

function and cardiovascular risk: relationship with inflammation-sensitive plasma proteins,

Circulation (2002) 106:2555-2560.

[19] Hochberg I., Roguin A., Nikolsky E., Chanderashekhar P.V., Cohen S., Levy A.P.,

Haptoglobin phenotype and coronary artery collaterals in diabetic patients, Atherosclerosis

(2002) 161:441-446.

[20] Maeda N., Yang F., Barnett D.R., Bowman B.H., Smithies O., Duplication within the

haptoglobin Hp2 gene, Nature (1984) 309:131-135.

[21] Maeda N., Nucleotide sequence of the haptoglobin and haptoglobin-related gene pair. The

haptoglobin-related gene contains a retrovirus-like element, J. Biol. Chem. (1985)

260:6698-6709.

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primates, J. Biol. Chem. (1988) 263:15740-15747.

[23] Smithies O., Walker N.F., Genetic control of some serum proteins in normal humans, Nature

(1955) 176:1265-1266.

[24] Black J.A., Dixon G.H. Amino-acid sequence of alpha chains of human haptoglobins,

Nature (1968) 218:736-741

[25] Javid J., Human haptoglobins, Curr. Top Hematol. (1978) 1:151-192.

[26] Travis J., Sanders B., Haptoglobin evolution: polymeric forms of Hp in the bovidae and

cervidae families, J. Exp. Zool. (1972) 180: 141-148.

[27] Lai I.H., Lin K.Y., Larsson M., Yang M.C., Shiau C.H., Liao M.H., Mao S.J.T., A unique

tetrameric structure of deer plasma haptoglobin: an evolutionary advantage in the Hp 2-2

phenotype with homogeneous structure, FEBS J. (2008) 275:981-993.

[28] Lai Y.A., Lai I.H., Tseng C.F., Lee J., Mao S.J.T., Evidence of tandem repeat and extra

thiol-groups in the polymeric formation of bovine haptoglobin: a unique structure of Hp 2-2

phenotype, J. Biochem. Mol. Biol. (2007) 40:1028-1038.

[29] Petersen H.H., Nielsen J.P., Heegaard P.M., Application of acute phase protein

measurements in veterinary clinical chemistry, Vet. Res. (2004) 35:163-187.

[30] Eckersall P.D., Young F.J., Nolan A.M., Knight C.H., McComb C., Waterston M.M., Hogarth

C.J., Scott E.M., Fitzpatrick J.L., Acute phase proteins in bovine milk in an experimental

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[31] Grönlund U., Hallén Sandgren C., Persson Waller K., Haptoglobin and serum amyloid A in

milk from dairy cows with chronic sub-clinical mastitis, Vet. Res. (2005) 36:191-198.

[32] Nielsen B.H., Jacobsen S., Andersen P.H., Niewold T.A., Heegaard P.M., Acute phase

protein concentrations in serum and milk from healthy cows, cows with clinical mastitis and

cows with extramammary inflammatory conditions, Vet. Rec. (2004) 154:361-365.

[33] Pedersen L.H., Aalbaek B., Røntved C.M., Ingvartsen K.L., Sorensen N.S., Heegaard P.M.,

Jensen H.E., Early pathogenesis and inflammatory response in experimental bovine mastitis

due to Streptococcus uberis, J. Comp. Pathol. (2003) 128:156-164.

[34] Hiss S., Mielenz M., Bruckmaier R.M., Sauerwein H., Haptoglobin concentrations in blood

and milk after endotoxin challenge and quantification of mammary Hp mRNA expression, J.

Dairy Sci. (2004) 87:3778-3784.

[35] Goldstein L.A., Heath E.C., Nucleotide sequence of rat haptoglobin cDNA. Characterization

of the alpha beta-subunit junction region of prohaptoglobin, J. Biol. Chem. (1984)

259:9212-9217.

[36] Hanley J.M., Heath E.C., A novel proteolytic activity in serum processes rat prohaptoglobin,

Arch. Biochem. Biophys. (1985) 239:404-419.

[37] Wicher K.B., Fries E., Prohaptoglobin is proteolytically cleaved in the endoplasmic

reticulum by the complement C1r-like protein, Proc. Natl. Acad. Sci. U. S. A. (2004)

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Chapter 2 Neutrophils as one of the major haptoglobin sources in mastitis affected

milk (Paper 1)

Abstract

The antioxidant haptoglobin (Hp) is an acute-phase protein responsive to infectious and

inflammatory diseases. Hp and somatic cell counts (SCC) are sharply elevated in bovine

milk following intramammary administration of endotoxin or bacteria. However, the

sources of milk Hp responsible for such increase are not fully understood. The purpose of

this study was to define the source of milk Hp from dairy cows with naturally occurring

mastitis. Quarter milk samples selected from 50 dairy cows were separated into four groups

according to SCC as group A: <100 (n=19); B: 100-200 (n=10); C: 201-500 (n=10); and D:

>500 × 103 (n=11) cells/mL. Our results reveal that milk Hp concentrations were correlated

with SCC (r=0.742; P<0.01), and concentrations in group D were ~10 fold higher than in

group A. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicates that

the milk somatic cell cells from group D were not only capable of synthesizing Hp but could

also markedly increase Hp mRNA expression. Western blot, immunocytochemistry, double

confocal immunofluorescence, and Hp releasing experiment demonstrate that neutrophils

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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.

Key words: haptoglobin / neutrophil / mastitis / MAC-T cells / immunocytochemistry

2.1 Introduction

Haptoglobin (Hp) is an acute phase protein responsive to inflammation and infection [1, 2, 3].

One of the major functions of Hp is to capture released hemoglobin during excessive

hemolysis [4] and to scavenge the hemoglobin-induced free radicals during oxidative stress

[5]. We have recently shown that Hp is an extremely potent antioxidant, which directly

prevents low-density-lipoproteins (LDL) from Cu2+- and radical compound-induced oxidation

[6, 7]. Transfection of Hp cDNA into Chinese hamster ovary (CHO) cells protects them

against oxidative stress [7]. Structurally, bovine Hp is unique possessing only Hp 2-2

phenotype [8, 9] with molecular weight ranged from 660 to 730 kDa [9] which is different

from that of human expressed as Hp 1-1, 2-1, or 2-2.

Bovine Hp, while not abundantly expressed in normal bovine plasma, is considered to be

one of the sensitive acute phase proteins during bacterial infections [3, 9]. Several studies

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mastitis of dairy cows [1, 10, 11]. In experimentally induced mastitis by intramammary

bacterial-challenge with Streptococcus uberis, the observed increase in milk Hp has been

suggested to originate from the circulation [12]. Hiss et al., 2004 also found elevated Hp in

bovine milk after intramammary endotoxin challenge [13]. It was further observed that Hp

could be locally synthesized within the mammary gland [13]. The source of milk Hp found

in naturally occurring mastitis is poorly understood.

The purpose of the present study was to define the source of milk Hp from dairy cows

with high somatic cell counts (SCC) (or mastitis). We investigated milk somatic cells for the