Title :
Hybrid and complex glycans are linked to the conserved N-glycosylation
site of the third eight-
cysteine domain of
LTBP-1 in insect cells
Abstract :
- Covalent association of LTBP-1 (latent TGF-beta binding protein-1 ) to latent TGF-beta is mediated by the third eight- cysteine (also referred to as TB) module of LTBP-1 , a domain designated as CR3
- Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3 , and we show here that another lepidopteran cell line, Trichoplusia niTN-5B1-4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3
- CR3 contains an N-glycosylation site , which is conserved in all forms of LTBP known to date
- When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells
- Sf9 cells mainly processed the fucosylated paucomannosidic structure (GlcNAc)(2)(Mannose)(3)Fucose, although hybrid and complex N-glycosylations were also detected
- In High-Five cells, the peptide was found to be modified with a wide variety of hybrid and complex sugars in addition to paucomanosidic oligosaccharides
- Most glycans had one or two fucose residues bound through alpha1,3 and alpha1,6 linkages to the innermost GlcNAc.
- On the basis of these results and on the structure of an eight- cysteine domain from fibrillin-1 , we present a model of glycosylated CR3 and discuss the role of glycosylation in eight- cysteine domain protein-protein interactions