Ｎ端醣基修飾在真核細胞蛋白質表達扮演重要的生理功能，不僅提供蛋白質合成過程與折疊時的關鍵品質管制，也在細胞的內外訊息傳遞，以及細胞與細胞相互作用，扮演重要的生物功能，因此近年來有關醣基修飾的結構與功能關聯研究逐漸受到重視。
蛇毒蛋白亦包含醣基修飾，但是過去探討蛇毒的作用機制，多在不含醣基的小分子量毒蛋白，對於含醣基的高分子量醣蛋白及其醣基的功能較少研究。 本研究乃針對台灣眼鏡蛇毒中的兩種金屬水解酵素 Atragin 和K-like，探討去除部分醣基後的酵素活性功能的變化，以了解這些醣基的可能生物功能。 我們採用Endo F3 去醣水解酵素去除大部分醣基，僅保留與蛋白質共價鍵結的雙醣， 然後比較去醣後酵素功能的變化，並進一步用二色圓光譜及螢光光譜等技術了解結構改變造成活性變化的原因。

結果顯示去除醣基後，對於不同受質蛋白如 fibrinogen, fibronectin, collagen type I, 和 azocasein會有不同程度的影響，因受質蛋白的不同，分別有增加或減少活性的作用，因此醣基應該對於不同的受質蛋白扮演進一步選擇的功能。 就結構而言，去醣後的整體構型並無顯著影響，但是局部的構型會因部分醣基的去除導致動態的變化。 另外一個影響是去除醣基所含的負電荷後，蛋白質的表面電位也跟著改變， 並且曝露出更多的疏水作用區，這些改變，部分反應在肝素與此兩種金屬蛋白水解酵素的結合能力變化以及肝素進一步調控酵素活性的能力。

Background
N-linked glycosylation is one of most important post-translational modification for several physiological processes in all eukaryote cells. There is growing interest in understanding the roles of the attached N-glycans on the structures and stability of the investigated proteins be-cause recent studies have shown that N-linked glycans are not only play a role in the quality control process during protein synthesis, but also affect significantly on their biological func-tions. Snake venom toxins also consist of glycoproteins, but the function of glycosylation re-mains illusive.

Methods
In this study, we investigate two P-III snake venom metalloproteinases (SVMPs), for example, Atragin and K-like from Naja atra, and correlate its enzymatic activities before and after cleaving the N-glycan complexes with Endoglycosydase. We express Endoglycosidase F3 and use it to remove N-glycan complexes of Atragin and K-like SVMPs to understand whether the truncated sugar molecule with one N-acetylglucosamine and a fuccose residue remaining on the asparagine could still maintain their structure and activity. Three different substrates from extracellular matrix, for instance, fibrinogen, fibronectin, collagen type I, and azocasein were tested and the protease activity of Atragin and K-like SVMPs were compared. Finally, their structures were examined by Circular Dichroism and fluorescence spectroscopy.

Results
The P-III SVMPs and its mammalian homologues of ADAMs, and ADAMTSs all consist of at least one N-linked protein glycosylation, but the exact location of the N-glycans appear to vary significantly among different SVMPs and its homologues. Comparison of the enzymatic activities between K-like and Atragin SVMPs indicate that the effect of deglycosylation is substrate dependent. For instance, while there was not a significant change in the enzymatic activity of N-deglycosylated Atragin and – K-like with fibronectin and vitronectin substrates, the fibrinogenolytic and collagenolytic activity of Atragin and K-like, however, were signifi-cantly perturbed after deglycosylating. Moreover, the azocaseinolytic activity with sulfonila-mide-azocasein, a nonspecific substrate, showed a significantial fall off in the N-deglycosylated K-like activity, whereas Atragin retain the same activity. Finally, we also dis-covered that the N-deglycosylated K-like bound to heparin column much more stronger than native K-like, and the observed heparin binding could significantly enhance its enzymatic ac-tivity.
In order to understand the observed change in its enzymatic activity, we examine the possible effect of glycosylation on its structural stability. The N-glycans contributed to a change in negatively charged surface and shifting the pH value of protein toward acidic pI of Atragin and K-like. Since there is no detected conformational change based on Circular Di-chroism result, the N-glycan appears to retain the stability of protein avoided to aggregation and precipitation, likely leading to an decrease in protein stability with high temperatures and denaturant.

CHAPTER 1 Introduction 1
I. Classification and modular organization of snake venom metalloproteinases (SVMPs) 1
II. Biological activites of SVMPs 2
III. Crystal structures of SVMPs 3
IV. Substrates for analysing the biological activity of SVMPs 4
V. Glycosylation and its roles on glycoproteins 5
VI. Glycosylation of SVMPs and ADAMs family 10

CHAPTER 2 Experimental Procedures 12
I. Materials 12
II. Expression and purification of TEV-his tag protease and Endo F3 12
III. Purification of deN-glycosylated Atragin and – K-like treating to Endo F3 13
IV. Measurement of biological activities 14
V. Measurement of proteolytic activity of heparin-binding deN-glycosylated Atragin and – K-like 14
IV. Spectroscopy assays 14
VII. Structure visulization 15

CHAPTER 3 Results 16
I. The P-III snake venom metalloproteinases are N-linked glycosylation proteins 16
II. N-glycosylation stabilizes the enzymatic activity of Atragin and K-like 16
III. N-glycosylation affects local conformation, but not global structure of SVMPs 18
IV. Removing N-glycosylation singificantly increase heparin-binding affinity and proteinolytic activity of K-like 19

CHAPTER 4 Discussions 21

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