N-Glycosylation and Biological Activity of Recombinant Human Alpha1-Antitrypsin Expressed in a Novel Human Neuronal Cell Line

被引：44

作者：

Blanchard, Veronique ^{[1
]}

Liu, Xi ^{[1
]}

Eigel, Susann ^{[1
]}

Kaup, Matthias ^{[1
]}

Rieck, Silke ^{[1
]}

Janciauskiene, Sabina ^{[2
]}

Sandig, Volker ^{[2
]}

Marx, Uwe ^{[2
]}

Walden, Peter ^{[3
]}

Tauber, Rudolf ^{[1
]}

Berger, Markus ^{[1
]}

机构：

[1] Charite, Cent Inst Lab Med & Pathobiochem, D-10117 Berlin, Germany

[2] ProBioGen AG, Berlin, Germany

[3] Charite, Clin Res Grp Tumor Immunol, Dept Dermatol, D-10117 Berlin, Germany

来源：

BIOTECHNOLOGY AND BIOENGINEERING | 2011年 / 108卷 / 09期

基金：

瑞典研究理事会;

关键词：

N-glycosylation; alpha-1-antitrypsin; human neuronal cell line; Lewis X; anti-inflammatory activity; PERFORMANCE LIQUID-CHROMATOGRAPHY; HUMAN ALPHA-1-PROTEASE INHIBITOR; HAMSTER OVARY CELLS; MASS-SPECTROMETRY; ALPHA(1)-PROTEINASE INHIBITOR; HUMAN ALPHA-1-ANTITRYPSIN; OLIGOSACCHARIDE CHAINS; PROTEINASE-INHIBITORS; CARBOHYDRATE CHAINS; PRION PROTEIN;

D O I：

10.1002/bit.23158

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Human alpha-1-antitrypsin (A1AT) is a protease inhibitor that is involved in the protection of lungs from neutrophil elastase enzyme that drastically modifies tissue functioning. The glycoprotein consists of 394 amino acids and is N-glycosylated at Asn-46, Asn-83, and Asn-247. A1AT deficiency is currently treated with A1AT that is purified from human serum. In view of therapeutic applications, rA1AT was produced using a novel human neuronal cell line (AGE1.HN (R)) and we investigated the N-glycosylation pattern as well as the in vitro anti-inflammatory activity of the recombinant glycoprotein. rA1AT (300 mg/L) was biologically active as analyzed using elastase assay. The N-glycan pool, released by PNGase F digestion, was characterized using 2D-HPLC, MALDI-TOF mass spectrometry, and by exoglycosidase digestions. A total of 28 N-glycan structures were identified, ranging from diantennary to tetraantennary complex-type N-glycans. Most of the N-glycans were found to be (alpha 1-6) core-fucosylated and part of them contain the Lewis X epitope. The two major compounds are a monosialylated diantennary difucosylated glycan and a disialylated diantennary core-fucosylated glycan, representing 25% and 18% of the total N-glycan pool, respectively. Analysis of the site-specificity revealed that Asn-247 was mainly occupied by diantennary N-glycans whereas Asn-46 was occupied by di-, and triantennary N-glycans. Asn-83 was exclusively occupied by sialylated tri- and tetraantennary N-glycans. Next, we evaluated the anti-inflammatory activity of rA1AT using A1AT purified from human serum as a reference. rA1AT was found to inhibit the production of TNF-alpha in neutrophils and monocytes as commercial A1AT does. Biotechnol. Bioeng. 2011; 108: 2118-2128. (C) 2011 Wiley Periodicals, Inc.

引用

页码：2118 / 2128

页数：11

共 41 条

[1] PRIMARY STRUCTURE OF N-LINKED CARBOHYDRATE CHAINS OF A HUMAN CHIMERIC PLASMINOGEN-ACTIVATOR K2TU-PA EXPRESSED IN CHINESE-HAMSTER OVARY CELLS [J].

BERGWERFF, AA ;

VANOOSTRUM, J ;

ASSELBERGS, FAM ;

BURGI, R ;

HOKKE, CH ;

KAMERLING, JP ;

VLIEGENTHART, JFG .

EUROPEAN JOURNAL OF BIOCHEMISTRY, 1993, 212 (03) :639-656

[2] NONSELECTIVE AND EFFICIENT FLUORESCENT LABELING OF GLYCANS USING 2-AMINO BENZAMIDE AND ANTHRANILIC ACID [J].

BIGGE, JC ;

PATEL, TP ;

BRUCE, JA ;

GOULDING, PN ;

CHARLES, SM ;

PAREKH, RB .

ANALYTICAL BIOCHEMISTRY, 1995, 230 (02) :229-238

[3] Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris [J].

Blanchard, Veronique ;

Gadkari, Rupali A. ;

Gerwig, Gerrit J. ;

Leeflang, Bas R. ;

Dighe, Rajan R. ;

Kamerling, Johannis P. .

GLYCOCONJUGATE JOURNAL, 2007, 24 (01) :33-47

[4] Mechanisms of disease -: Alpha1-antitrypsin deficiency -: A model for conformational diseases [J].

Carrell, RW ;

Lomas, DA .

NEW ENGLAND JOURNAL OF MEDICINE, 2002, 346 (01) :45-53

[5] HUMAN ALPHA-1-ANTITRYPSIN - CARBOHYDRATE ATTACHMENT AND SEQUENCE HOMOLOGY [J].

CARRELL, RW ;

JEPPSSON, JO ;

VAUGHAN, L ;

BRENNAN, SO ;

OWEN, MC ;

BOSWELL, DR .

FEBS LETTERS, 1981, 135 (02) :301-303

[6] The use of osazones as matrices for the matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates [J].

Chen, P ;

Baker, AG ;

Novotny, MV .

ANALYTICAL BIOCHEMISTRY, 1997, 244 (01) :144-151

[7] Production, Purification, and Characterization of Human α1 Proteinase Inhibitor From Aspergillus niger [J].

Chill, Liat ;

Trinh, Loc ;

Azadi, Parastoo ;

Ishihara, Mayumi ;

Sonon, Roberto ;

Karnaukhova, Elena ;

Ophir, Yakir ;

Golding, Basil ;

Shiloach, Joseph .

BIOTECHNOLOGY AND BIOENGINEERING, 2009, 102 (03) :828-844

[8]

Conradt HS, 1995, MED CHEM TODAY TOMOR

[9] A pilot study comparing the purity, functionality and isoform composition of alpha-1-proteinase inhibitor (human) products [J].

Cowden, DI ;

Fisher, GE ;

Weeks, RL .

CURRENT MEDICAL RESEARCH AND OPINION, 2005, 21 (06) :877-883

[10]

CUMMING D A, 1991, Glycobiology, V1, P115, DOI 10.1093/glycob/1.2.115

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