Mannosamine supplementation extends the N-acetylglucosaminylation of recombinant human secreted alkaline phosphatase produced in Trichoplusia ni (cabbage looper) insect cell cultures.
A major limitation of the insect cell-baculovirus expression vector system is its poor capacity to perform complex glycosylation, since the glycoproteins produced are usually only of the high-mannose and paucimannose types. Nonetheless, recent evidence indicates that, under various conditions, some insect cell lines are capable of producing complex-type glycans. In the present study, we assessed the effects of supplementation with ManN (mannosamine) ManNAc (N-acetylmannosamine) and Cyt (cytidine) on the glycosylation of recombinant human secreted alkaline phosphatase produced by suspension cultures of Trichoplusia ni (cabbage looper) BTI-Tn5B1-4 cells. Addition of ManN in the range 5-20 mM resulted in a 10-fold increase of the terminal GlcNAc (N-acetylglucosamine) associated with the recombinant protein produced after baculovirus infection. Such an increase yielded a maximum of 12.5% hybrid glycans having terminal GlcNAc with respect to total N-linked glycans. In contrast, no changes in the glycan composition associated with recombinant human secreted alkaline phosphatase were observed on supplementation with up to 20 mM ManNAc or up to 1.5 mM Cyt. N-acetyl-beta-glucosaminidase activity in cell extracts was decreased on incubation with 20 mM ManN, but not with 20 mM ManNAc or 1.5 mM Cyt, indicating that the increased proportion of hybrid glycans obtained on the addition of ManN could be a result of N-acetyl-beta-glucosaminidase inhibition.
Estrada-Mondaca
Sandino
S
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Apdo. Postal 510-3, 62250 Cuernavaca, Morelos, Mexico.
Delgado-Bustos
Luz Adrián
LA
Ramírez
Octavio T
OT
D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002478:Cells, Cultured; D003562:Cytidine / Q000378:metabolism*; D006031:Glycosylation; D006595:Hexosamines / Q000378:metabolism*; D006801:Humans; D009036:Moths / Q000254:growth & development* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis
D006595:Hexosamines; D011994:Recombinant Proteins; C008322:mannosamine; D003562:Cytidine; D000469:Alkaline Phosphatase; C002022:N-acetylmannosamine