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研究生:朱壯康
研究生(外文):Chuang-Kang Chu
論文名稱:以凝集化學的方法探討從黑桑樹皮純化的凝集素(MornigaM)和醣類結構單位在N-linked醣蛋白的結合性質
論文名稱(外文):Lectinochemical Studies on the Binding Properties of a Lectin from Morus nigra Bark (Morniga M) and Carbohydrate Structural Units in N-linked Glycoproteins
指導教授:吳明道
指導教授(外文):A. M. Wu
學位類別:碩士
校院名稱:長庚大學
系所名稱:天然藥物研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:91
語文別:中文
論文頁數:74
中文關鍵詞:凝集素黑桑樹樹皮甘露醣酵素凝集分析法N-linked醣蛋白
外文關鍵詞:lectinsmulberry (Morus nigra) barkmannoseELLSAN-linked glycoproteinMorniga M
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凝集素(Lectin)是一種可與碳水化合物結合之蛋白或醣蛋白,其重要功能之一是在生命過程中調節細胞和細胞之間的辨識作用。這些應用性凝集素的性質可做為研究生命現象的重要工具。而研究複雜性碳水化合物的生物活性區和抗原決定因子的化學結構及其結合性質,將有助於瞭解這些碳水化合物的分子在生物學和醫學上所代表的價值。這些之事對於疫苗的製造,癌症的診斷,遺傳異常的研究和治療的應用是有相當助益的。同時對於人類血型間的關係、微生物如何附著在宿主細胞和宿主細胞如何防止微生物侵入的過程,都可用分子或原子層次來解釋。基於這些理由,探討凝集素和醣蛋白的結合性質是醣類免疫化學研究室過去數十年來的研究主軸,而本篇論文即是此主題中的一環。我的研究可分成兩個主題來介紹:
甲、首先利用凝集化學法來探討具有甘露醣特異性凝集素(Morniga M)對於辨識碳水化合物上的結構單位的能力。Morniga M是從黑桑樹的樹皮(Morus nigra bark)中所純化出來的凝集素。其乃與Jacalin相關及對甘露醣有特異性。為了瞭解這個新凝集素的功能及應用,所以利用ELLSA及lectin-glycan inhibition assay來分析。與之反應的碳水化合物則是在生物功能上扮演重要角色的哺乳動物血清及母乳中分離出的醣蛋白。
其結果發現N-glycans(如:bovine alpha-1 acid gp和lactoferrin)上的結構單位(如:bovine alpha-1 acid gp和lactoferrin)和凝集素有很好反應性,但是對於O-glycans和多醣體(如:mannan)卻是沒有反應。Morniga M凝集素與各種配醣體的親和力排列順序為:glycoproteins carrying multiple N-glycans with oligomannosyl residues >> N-glycopeptide with a single trimannosyl core > Tri-Man oligomer (Manα1→6(Manα1→3) Man), Penta-Man oligomer (Manα1→6(Manα1→3)Manα1→6(Manα1→3) Man) ≧ Manα1→2, 3 or 6 Man > Man > GlcNAc, Glc >> LFuc, Gal, GalNAc (inactive),因而證實了這個凝集素的專一特異性。
乙、此篇的另一個主題則是對於N-glycans的探討。大多數N-linked醣蛋白上的碳水化合物部分,在每一個醣鏈上都有一個由三個單醣組成的核心區和醯胺天門冬酸(Asn)共價鍵結形成Manβ1→4GlcNAcβ1→4GlcNAcβ1→Asn的結構。N-linked醣蛋白結構現已被鑑別出來並可被分成三類:(1) high mannose type:除了核心部分外還有六個甘露醣,在卵白蛋白、甲狀腺球蛋白、細胞膜上鍵結的醣蛋白均發現有此形式;(2)complex type:除了甘露醣外還有唾液酸、岩藻醣、半乳醣及N-acetylglucosamine;(3)hybrid type:結構包含oligomannose-及N-acetyllactosamine-形式的寡醣;這些複雜性碳水化合物在分子間的辨識,扮演很重要角色。
這裡我利用ELLSA及醣蛋白-凝集素作用抑制分析法來描述N-linked醣蛋白上的碳水化合物單位與不同凝集素的鍵結特性,綜合結果可歸納出以下兩點:(1) N-linked醣蛋白上有Galβ1→4GlcNAcβ1→ (II)特性的接受器;(2)其會和Man complex特異性凝集素(如Morniga M等)作用。這些結果不但幫助我們瞭解細胞表面上配醣體與凝集素的辨識,而且提供給我們生物技術及臨床醫學治療的資訊,尤其是在的偵測與純化上的運用。

Lectins are carbohydrate binding proteins and function as mediators in cell-cell recognition. Some, defined as applied lectins, can be used as tools in glycobiology studies. Grouping of these applied lectins based on their interaction with complex oligosaccharides rather than with monosaccharides, should facilitate their utilization as structural probes for glycan characterization, which may aid interpretation of the distribution and properties of carbohydrates alteration on cell surface. Knowledge of the structures and binding properties of the glyco-bioactive sites and antigenic determinants of carbohydrates should contribute to a better understanding of the functional roles of these complex carbohydrates at the molecular and biological levels, and provide valuable information about the following aspects: (a) an in-depth understanding of life processes and mechanisms of infections and genetic disorders; (b) developing vaccines or drugs to prevent infection & inflammation; (c) providing new reagents to detect cancers; and (d) improving serodiagnosis. For this reason, two goals have been accomplished. (i) Lectinochemical studies on recognition factors of a mannose specific lectin (Morniga M) isolated from the bark of the mulberry (Morus nigra) and (ii) characterization of the binding properties of N-linked glycoproteins from mammalian sera and milk which are playing various important biological functions.
Morniga M is a jacalin-related and mannose-specific lectin. In order to evaluate the function and application of this lectin, the binding properties of Morniga M were studied in detail using an enzyme-linked lectinosorbent assay (ELLSA) and lectin-glycan binding inhibition assay with extended glycan/ligand collection. From the results, it was found that the di-, tri-, and oligomannosyl structural units of N-glycans, such as those of bovine α1-acid gp and lactoferrin, were the most-active glycoproteins, but not the O-glycans or polysaccharides, including mannan from yeast. The binding affinity of MornigaM for ligands can be ranked in decreasing order as follows: glycoproteins carrying multiple N-glycans with oligomannosyl residues >> N-glycopeptide with a single trimannosyl core > Tri-Man oligomer (Manα1→6(Manα1→3) Man), Penta-Man oligomer (Manα1→6(Manα1→3)Manα1→6(Manα1→3) Man) ≧ Manα1→2, 3 or 6 Man > Man > GlcNAc, Glc >> LFuc, Gal, GalNAc (inactive), demonstrating the unique specificity of this lectin that may not only assist in our understanding of cell surface carbohydrate ligand-lectin recognition, but also provide informative guidelines for the application of this structural probe in biotechnological and clinical regimens, especially in the detection and purification of N-linked glycans.
Most carbohydrate moieties of N-linked glycoproteins have the same sequence of three sugars, called the core region, linked to asparagine, Manβ1→4GlcNAcβ1→4GlcNAcβ1→Asn. However, many different N-linked oligosaccharides varying in structural complexity have been identified and can be classified into three different groups. One, the high mannose group, is found in glycoproteins such as ovalbumin, thyroglobulin, and certain cell surface membrane-bound glycoproteins and contains up to six mannose residues in addition to those found in the core region. The second group, called the complex type, contains several different monosaccharides in addition to mannose, including sialic acid, fucose, galactose, and N-acetylglucosamine. The high mannose and complex types of oligosaccharides may have two, three, or four branches and accordingly have been designated as bi-, tri-, or tetraantennary oligosaccharides. Structures containing both oligomannose- and N-acetyllactosamine-type oligosaccharides are designated as hybrid type as third group. These complex carbohydrates play important roles in molecular recognition.
The binding properties of carbohydrate units in N-linked glycoproteins with various lectins have been characterized by enzyme linked lectinosorbent assay (ELLSA), and by inhibition of glycoprotein-lectin interaction. Mapping the interaction and inhibition profiles leads to the conclusion that the carbohydrate moieties of N-linked glycoproteins provide valuable receptors for characterizing Galβ1→4GlcNAcβ1→ (II), and Man complex specific lectins.

目錄..... .............................................i
表目錄... .............................................iv
圖目錄... .............................................v
中文摘要...............................................vi
英文摘要...............................................ix
第一章 緒論............................................1
第一節 前言............................................1
第二節 研究碳水化合物的沿革............................5
第三節 碳水化合物及凝集素之應用........................7
第四節 研究方向........................................8
第五節 Morniga M(Morus nigra) lectin...................8
第六節 乳液醣蛋白......................................10
第七節 血清醣蛋白......................................11
第二章 研究材料........................................16
1. 材料................................................16
2. 血液材料............................................17
3. 試藥................................................17
4. 儀器................................................20
第三章 研究方法........................................21
1. 紅血球凝集活性之測定(Hemagglutination, HA)方法......21
2. 紅血球凝集抑制之測定(Hemagglutinin-inhibition assay,
HIA)..................................................21
3. 去除唾液酸(sialic acid)型式的醣蛋白(Asialo glycoproteins)
純化...................................................22
4. 酵素凝集分析法(Enzyme Linked Lectinosorbent assay, ELLSA).................................................22
第四章 結果............................................25
第一部份 凝集素活性的探討: 以具有Mannose特異性的凝集素
(Morniga M),來探討複雜性碳水化合物和凝集素的
結合性質....................................25
(1) Morniga M凝集素和人類不同血型之間的相互作用關係
(Lectin — human erythrocyte interaction)........25
第二部份 凝集素結合性質的探討: 以具有Mannose 特異性的
凝集素(Morniga M),來探討複雜性碳水化合物和
凝集素的結合性質......... ..................26
(1) Morniga M凝集素和醣蛋白之間的相互作用關係
(Lectin - glycoform interaction).................26
(2) 藉由不同醣蛋白上醣鏈的競爭性抑制來探討Morniga M和
glycoform的相互作用關係(Inhibition of Morniga M- glycoform interaction by various glycans).....27
第三部分 N-linked醣蛋白中碳水化合物結構單位的探討....28
(1) 利用酵素凝集分析法(ELLSA)來探討N-linked醣蛋白和不同
凝集素間的結合性質.............................28
第五章 討論............................................31
參考文獻...............................................36

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