Haptoglobin在生理與生化上之功能角色

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次單位之抗氧化能力明顯高於native Hp（3.5 X）、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次單位內皆具有一段重複序列，並且-SH group在胺基酸序列上的位置與人類Hp 2相同，經蛋白質電泳分析顯示鹿Hp蛋白質為tetramer (-)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 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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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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。

關鍵字

血紅素結合蛋白；嗜中性球；體細胞數；乳房炎；抗氧化物；單株抗體；表現型；演化

並列摘要

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 antioxidant that can directly scavenge the free radicals, it may there effectively utilize Hp to 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 alpha and beta chains when assessed by Cu2+-induced oxidation of low density lipoprotein. Interestingly, the antioxidant activity of betachain was extremely potent and markedly greater than that of native Hp (3.5 x), alpha 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 alphachain based on our cloned cDNA sequence. Interestingly, the isolated deer Hp is homogeneous and tetrameric, although the location of –SH groups (responsible for the formation of polymers) is exactly identical to that of human. Most interestingly, an alphachain monoclonal antibody (W1) known to recognize both dissociated human and deer alphachains, only binds to the intact human Hp polymers, but not to deer Hp tetramers. It implicates that the epitope of deer alpha 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. 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. 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 beta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 physiologic advantage.

並列關鍵字

Haptoglobin ； Neutrophil ； Somatic cell counts ； Mastitis ； antioxidant ； Monoclonal antibody ； Phenotype ； evolution

參考文獻

[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.

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Haptoglobin在生理與生化上之功能角色

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