Engineering β1,4-galactosyltransferase I to reduce secretion and enhance N-glycan elongation in insect cells

Abstract

β1,4-galactosyltransferase (B4GALT1) is a Golgi-resident enzyme that elongates glycoprotein glycans, but a subpopulation of this enzyme is secreted following proteolytic cleavage in its stem domain. We hypothesized that engineering B4GALT1 to block cleavage and secretion would enhance its retention and, therefore, its function. To test this hypothesis, we replaced the cytoplasmic/transmembrane/stem (CTS) domains of B4GALT1 with those from human α1,3-fucosyltransferase 7 (FUT7), which is not cleaved and secreted. Expression of FUT7-CTS-B4GALT1 in insect cells produced lower levels of secreted and higher levels of intracellular B4GALT1 activity than the native enzyme. We also noted that the B4GALT1 used in our study had a leucine at position 282, whereas all other animal B4GALT1 sequences have an aromatic amino acid at this position. Thus, we examined the combined impact of changing the CTS domains and the amino acid at position 282 on intracellular B4GALT1 activity levels and N-glycan processing in insect cells. The results demonstrated a correlation between the levels of intracellular B4GALT1 activity and terminally galactosylated N-glycans, N-glycan branching, the appearance of hybrid structures, and reduced core fucosylation. Thus, engineering B4GALT1 to reduce its cleavage and secretion is an approach that can be used to enhance N-glycan elongation in insect cells.

Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system

“…Fifty mL shake flask cultures of Sf-RVN and Sf9 cells were infected with Sf-rhabdovirus-negative stocks of AcP(−)p6.9hEPO and hEPO was affinity purified from the cell- and virus-free supernatants using Ni-NTA resin (ThermoFisher), as described previously [23]. N -glycans were enzymatically released from the purified hEPO preparations by digestion with PNGaseF (New England Biolabs), and the released N -glycans were purified, derivatized, and analyzed by MALDI-TOF-MS as described previously [23].…”

“…N -glycans were enzymatically released from the purified hEPO preparations by digestion with PNGaseF (New England Biolabs), and the released N -glycans were purified, derivatized, and analyzed by MALDI-TOF-MS as described previously [23]. Structures were assigned to peaks based on predicted masses and knowledge of the N -glycans produced in Sf cells.…”

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

“…typhimurium (Manuel et al 2011), S. lividans (Dubeau et al 2009), wheat (Mihálik et al 2015), transplastomic tobacco plants (chloroplast translation system) (Occhialini et al 2015), P. berghei (Singer et al 2015), S. frugiperda (Geisler et al 2015), S. pneumoniae (Overkamp et al 2013), A.…”

“…Many of the codon optimization software lack citations. Of these software packages, citations have been found for JCat and OPTIMIZER for expression systems such as E. coli (Fahimi et al, 2016;Guo et al, 2016;Karkhah and Amani, 2016;Zhao et al, 2016), S. cerevisiae (Ask et al, 2013;Guadalupe-Medina et al, 2013;Li et al, 2015;Milne et al, 2015;Solis-Escalante et al, 2013), N. benthamiana (Binder et al, 2014), HEK293 cells (Shah et al, 2015), B. subtilis (Reilman et al, 2014), Caulobacter crescentus (Ko et al, 2013), Pseudomonas putida (Dammeyer et al, 2011(Dammeyer et al, , 2013, Salmonella typhimurium (Manuel et al, 2011), S. lividans (Dubeau et al, 2009), wheat (Mih alik et al, 2015, transplastomic tobacco plants (chloroplast translation system) (Occhialini et al, 2015), Plasmodium berghei (Singer et al, 2015), Spodoptera frugiperda (Geisler et al, 2015), S. pneumoniae (Overkamp et al, 2013), A. marginale (Pierl e et al, 2013), R. pomeroyi (Green et al, 2013), L. acidophilus (Askelson et al, 2014), C. reinhardtir (Erpel et al, 2016), Y. lipolytica (Matthaus et al, 2014), S. elongates (van der Woude et al, 2016), and baculovirus (Maghodia et al, 2016). While it is encouraging that codon optimization software programs are used for a variety of species and purposes, most of the papers do not compare yields of native and codon optimized sequences, so yield comparisons cannot be made.…”

Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells

“…We can potentially attribute a reduction in bigalactosylated and biantennary‐glycans following the removal of core fucose and sialyltransferase to a possible change in the interaction between galactosyltransferase or other transferases and certain mutant IgGs with specific amino acid mutations (F241A and F243A). Interestingly, Geisler, Mabashi‐Asazuma, Kuo, Khoo, & Jarvis () reported that overexpression of B4GALT1 can increase galactosylation but also decrease the core fucosylation, which suggests a competition or inhibition of fucosyltransferase following elevation of the galactosyltransferase activity.…”