The B7-1 Cytoplasmic Tail Enhances Intracellular Transport and Mammalian Cell Surface Display of Chimeric Proteins in the Absence of a Linear ER Export Motif

Abstract

Membrane-tethered proteins (mammalian surface display) are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids) and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

SPLICELECT™: an adaptable cell surface display technology based on alternative splicing allowing the qualitative and quantitative prediction of secreted product at a single-cell level

“…The different elements of the AS construct can be modified in order to modulate the levels of secreted and membranebound antibody. The replacement of the M1M2 domain by B7-1, a murine transmembrane domain often used for the construction of chimeric transmembrane proteins and known to lead to high surface display of chimeric proteins, [65][66][67] did not affect the secretion level, but increased the surface staining. This increased staining may be due to favorable folding characteristics of B7-1 compared to the M1M2 domain or to enhanced stability of the chimeric construct.…”

“…PTCRA was used because the T-cell receptors are structurally similar to immunoglobulins and the murine B7-1 transmembrane domain because it has been described to allow efficient secretion of transmembrane chimeric proteins. [65][66][67] Whereas the secretion level was not improved compared to the initial M1M2 transmembrane domain construct, a substantial impact was seen on the surface staining, with B7-1 allowing the highest level of surface display, with 5x more cells stained and an increase of the mean fluorescent signal of 30x (Figure 2a and b).…”

“…The B7-1 cytosolic tail was shown not to contain any linear nor structural ER exportation signal. 65 In order to assess the impact of the ER exportation signal in the AS constructs, different cytosolic tails with or without the ER exportation signal were used. No significant effect on the staining results, nor on secretion levels, was observed ( Figure 2f), indicating that the ER exportation signal was not contributing in a significant manner.…”

Enhanced efficacy and limited systemic cytokine exposure with membrane-anchored interleukin-12 T-cell therapy in murine tumor models

“…Implementation of these strategies has been facilitated by a fusion protein in which the two IL-12 subunits are linked by a polypeptide connector, permitting expression of IL-12 from a single open reading frame. 18 In humans, tumor-directed therapy has principally been achieved by intratumoral injection [22][23][24][25][26][27] or by T-cell-mediated delivery, as in one clinical trial. 14 Various methods to improve IL-12-based therapy by controlling gene expression have also been proposed, including the use of an NFAT promoter 16 28 or a smallmolecule gene-activation system.…”

Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells

“…To enhance cell surface expression of membrane-tethered proteins, design strategies have focused on the careful selection of the TM, the cytoplasmic tail and the extracellular juxtamembrane linker [15]. Elements such as the transmembrane domain and the cytoplasmic tail of the murine B7-1 antigen and the hinge-CH 2 -CH 3 region of the human immunoglobulin G (IgG 1 ) heavy chain have been established to enhance protein expression on the cell surface [16, 17]. While the minimum sequences required to target specific proteins such as the respiratory syncytial virus F protein [18] and the SNARE protein SNAP-25 [19] have been elucidated in independent studies previously, these specific minimum sequences could differ between target proteins.…”