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헌터증후군의 효소대체요법에 사용되는 두 재조합 효소의 생화학적 및 물리화학적 비교
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 김용성 | - |
| dc.contributor.author | Chung, Yo-Kyung | - |
| dc.date.accessioned | 2018-11-08T08:17:29Z | - |
| dc.date.available | 2018-11-08T08:17:29Z | - |
| dc.date.issued | 2016-02 | - |
| dc.identifier.other | 21406 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/12423 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :분자과학기술학과,2016. 2 | - |
| dc.description.tableofcontents | CHAPTER 1. General introduction 1 1.1 Mucopolysaccharidosis 1 1.2 Hunter syndrome 5 1.3 Iduronate-2-sulfatase 6 CHAPTER 2. Characterization of two recombinant enzymes 8 2.1 Abstract 8 2.2 Introduction 10 2.3 Materials and Methods 15 2.3.1 Idursulfase and idursulfase beta 15 2.3.2 Amino acid sequencing by peptide mapping 15 2.3.3 Deamidation and methionine oxidation 16 2.3.4 Disulfide bridge analysis 17 2.3.5 N-terminal sequencing 17 2.3.6 Formylglycine content 18 2.3.7 Molecular weight 19 2.3.8 Monosaccharide composition 19 2.3.9 Mannose-6-phosphate content 20 2.3.10 Sialic acid analysis 21 2.3.11 Glycan mapping 21 2.3.12 Glycosylation type 22 2.3.13 Determination of glycosylation sites 23 2.3.14 Higher-order structure 23 2.4 Results 25 2.4.1 Amino acid sequence 25 2.4.2 Deamidation and methionine oxidation 28 2.4.3 Disulfide bridge 31 2.4.4 N-terminal sequence 33 2.4.5 Formylglycine content 35 2.4.6 Molecular weight 37 2.4.7 Monosaccharide composition 37 2.4.8 Mannose-6-phosphate content 39 2.4.9 Sialic acid content 41 2.4.10 Glycan mapping 43 2.4.11 Glycosylation type 47 2.4.12 Determination of glycosylation sites 47 2.4.13 Higher-order structure 49 2.4.14 Summary of structural and glycosylational characterization 51 2.5 Discussion 54 CHAPTER 3. A biochemical and physicochemical comparison of two recombinant enzymes 56 3.1 Abstract 56 3.2 Introduction 58 3.3 Materials and Methods 60 3.3.1 Idursulfase and idursulfase beta 60 3.3.2 Formylglycine content 61 3.3.3 Specific enzyme activity 62 3.3.4 Mannose-6-phosphate content 62 3.3.5 Sialic acid analysis 63 3.3.6 Cellular uptake activity 63 3.3.7 Statistical analysis 64 3.4 Results 65 3.4.1 Formylglycine content 65 3.4.2 Specific enzyme activity 68 3.4.3 Mannose-6-phosphate content 71 3.4.4 Sialic acid content 73 3.4.5 Cellular uptake activity 75 3.5 Discussion 77 CONCLUSION 81 REFERENCES 83 ABSTRACT IN KOREAN 88 PUBLICATIONS AND PATENTS 90 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 헌터증후군의 효소대체요법에 사용되는 두 재조합 효소의 생화학적 및 물리화학적 비교 | - |
| dc.title.alternative | A biochemical and physicochemical comparison of two recombinant enzymes used for enzyme replacement therapies of Hunter syndrome | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | Yo-Kyung Chung | - |
| dc.contributor.department | 일반대학원 분자과학기술학과 | - |
| dc.date.awarded | 2016. 2 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.localId | 739373 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000021406 | - |
| dc.subject.keyword | hunter syndrome | - |
| dc.subject.keyword | recombinant enzyme | - |
| dc.subject.keyword | enzyme replacement therapy | - |
| dc.description.alternativeAbstract | Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase®, Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase®, Green Cross Corporation, Yongin, Korea), are currently available in Korea. The manufacturing process of idursulfase beta was developed for enzyme replacement therapy of Hunter syndrome. The structural and glycosylational differences of two recombinant enzymes were compared. The efficacy and safety of idursulfase beta were evaluated by phase I/II clinical trial. Urine GAGs were significantly reduced in the 0.5 mg/kg and 1.0 mg/kg idursulfase beta groups when compared to the 0.5 mg/kg idursulfase group. Changes in 6MWT (six-minute walk test) were significantly greater in the 0.5 mg/kg and 1.0 mg/kg idursulfase beta groups than in the 0.5 mg/kg idursulfase group. To investigate these clinical differences, the biochemical and physicochemical differences between idursulfase and idursulfase beta were compared. The formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes were examined. The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4±0.9 vs. 68.1±2.2 %, P<0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6±1.1 vs. 27.8±0.9 nmol/min/ug protein, P<0.001). The levels of M6P and sialic acid were not significantly different (2.4±0.1 vs 2.4±0.3 mol/mol protein for M6P and 12.3±0.7 vs. 12.4±0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (Kuptake, 5.09±0.96 vs. 6.50±1.28 nM protein, P=0.017). In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content. | - |
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