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.

Molecular Cloning and Functional Characterization of a Lepidopteran Insect β4-N-Acetylgalactosaminyltransferase with Broad Substrate Specificity, a Functional Role in Glycoprotein Biosynthesis, and a Potential Functional Role in Glycolipid Biosynthesis

“…It is interesting to consider in retrospect that we actually intended to isolate an insect ␤4-galactosyltransferase cDNA in this study. It appears that we did not isolate this cDNA, suggesting that T. ni encodes another ␤4-galactosyltransferase family member, which is responsible for the galactosyltransferase activity observed in cell lysates (30) and the terminal galactose residues observed on some N-glycans produced in these cells (32,38,39). Alternatively, it is possible that the cDNA isolated in this study encodes a bifunctional enzyme that has both ␤4-N-acetylgalactosaminyltransferase and ␤4-galactosyltransferase activities in vivo.…”

“…In addition, several studies had shown that T. ni cells could produce recombinant glycoproteins with terminally galactosylated N-glycans when infected with baculovirus expression vectors (32,35,38,39). Thus, it was theoretically possible to isolate a ␤4-galactosyltransferase family member from this organism.…”

“…However, more recent reports have shown that lepidopteran insect cell lines actually do have low levels of some of the glycosyltransferase activities mediating elongation of N-glycan termini, including N-acetylglucosaminyltransferase I (28,29), N-acetylglucosaminyltransferase II (28), ␤4-galactosyltransferase (30,31), and ␤4-N-acetylgalactosaminyltransferase (30). In addition, various reports have documented the presence of terminal N-acetylglucosamine, galactose, N-acetylgalactosamine, and even sialic acid residues on N-glycans produced by insect cells (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). Finally, putative N-acetylglucosaminyltransferase, galactosyltransferase, and sialyltransferase genes have been identified in the D. melanogaster data bases (44), and the biochemical functions of the putative fly N-acetylglucosaminyltransferase I (45) and sialyltransferase (46) gene products have been experimentally confirmed in published studies.…”

“…This finding was among the first to suggest that insect cells could produce complex N-glycans. It was subsequently extended by demonstrations that a subpopulation of the N-glycans isolated from recombinant glycoproteins produced in T. ni cells have terminal galactose residues (32,38,39) and that some N-glycans isolated from uninfected T. ni have terminal N-acetylgalactosamine residues (43). Our specific aims were to molecularly clone a member of the ␤4-galactosyltransferase gene family from T. ni and examine the function of the gene product.…”

Glycoproteins from Insect Cells: Sialylated or Not?

“…Two families of oligosaccharides were found: one consisted of oligo-mannosidic type glycans (Man g to Man 5 GlcNAc 2 ) and the other consisted of short, partially fucosylated pauciman-nose-type glycans, but none were sialylated. Most recently, Rudd et al (2000) used MALDI-TOF to demonstrate the presence of a subpopulation of terminally galactosylated N-glycans on the third eight-cysteine domain of the latent transforming growth factor-β binding protein-1 expressed in baculovirusinfected High Five cells. However, they detected no sialylated structures.…”