Production and N-glycosylation of recombinant human cell adhesion molecule L1 from insect cells using the stable expression system. Effect of dimethyl sulfoxide.
L1 is a cell adhesion molecule that is heavily glycosylated and is essential for normal development of the central nervous system. In this work, we compare the N-glycosylation of the L1 mutant that consists of immunoglobulin domains 5 and 6 (L1/Ig5-6), expressed in insect Spodoptera frugiperda Sf9 and Trichoplusia ni Tn cells, using the stable expression system. L1/Ig5-6 levels of 30 and 8mgl(-1) were obtained from the two cell lines, respectively. The N-glycans were characterized by high-performance anion-exchange-chromatography with pulsed-amperometric-detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The N-glycans from Sf9 cells were more homogeneous and consisted predominantly of the paucimannose-type structure Manalpha6(Manalpha3)Manbeta4GlcNAcbeta4(Fucalpha6)GlcNAc. On the other hand, the N-glycans from Tn cells were more heterogeneous and consisted of paucimannose-type structures with or without terminal N-acetylglucosamine. Allergenic proximal alpha3-linked fucose was only found in Tn cells. Dimethyl sulfoxide at 1.5% concentration has been found to increase the levels of L1/Ig5-6 and the L1 ectodomain in the Sf9 and Tn cells, without affecting cell viability nor protein integrity. Furthermore, the N-glycan composition of L1/Ig5-6 was not affected by dimethyl sulfoxide, with only a slight increase in the percentage of the minor high-mannose-type structures.
Gouveia
Ricardo
R
Instituto de Tecnologia Química e Biológica, Avenida da República, 2780-157 Oeiras, Portugal.
Kandzia
Sebastian
S
Conradt
Harald S
HS
Costa
Júlia
J
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D024861:Genetic Enhancement / Q000379:methods; D006031:Glycosylation; D006801:Humans; D039842:Neural Cell Adhesion Molecule L1 / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D014162:Transfection / Q000379:methods*
D039842:Neural Cell Adhesion Molecule L1; D011994:Recombinant Proteins; D004121:Dimethyl Sulfoxide