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.
Highlights
Membrane-tethered proteins and peptides are increasingly utilized for basic research, biotechnology and medical applications [1]
We show that the B7-1 cytoplasmic domain enhances the intracellular transport of chimeric proteins, but extensive deletion and mutagenesis studies did not identify the presence of linear endoplasmic reticulum (ER) export motifs in the B7-1 cytoplasmic tail
A single-chain antibody fused to the platelet-derived growth factor (PDGFR) transmembrane domain and first 6 amino acids of the cytoplasmic domain is poorly expressed on cells [35]
Summary
Membrane-tethered proteins and peptides are increasingly utilized for basic research, biotechnology and medical applications [1]. Effective utilization of membrane-tethered proteins benefits from efficient expression of chimeric proteins on the cell surface, which in turn can be limited by slow intracellular transport [17]. Interactions between components of the COPII transport vesicles, in particular the Sec24p subunit, and short linear amino acid sequences in the cytoplasmic domain of membrane-anchored proteins, termed ER export motifs, concentrates cargo proteins at ER exit sites and enhances cargo recruitment into COPII vesicles [25]. We investigated the role of the B7 cytoplasmic domain in accelerated intracellular transport and surface display of chimeric proteins on mammalian cells. We show that the B7-1 cytoplasmic domain enhances the intracellular transport of chimeric proteins, but extensive deletion and mutagenesis studies did not identify the presence of linear ER export motifs in the B7-1 cytoplasmic tail. Our findings may help guide the design of improved fusion proteins for expression on mammalian cells and might help explain the mechanism of certain diseases associated with intracellular protein accumulation
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